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Department of Plant Pathology, The Royal Horticultural Society, Wisley, Woking, Surrey GU23 6QB, United Kingdom
Alastair Culham
Department of Botany, School of Plant Sciences, Reading University, Reading RG6 6AS, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A leaf and twig blight disease of Buxus spp. was found to be associated with a new species of Cylindrocladium. The novel species status was confirmed using morphological characters, sequencing of the ribosomal 5.8S RNA gene and the flanking internal transcribed spacers (ITS), the ß-tubulin gene, and the high mobility group (HMG) of the MAT2 mating type gene. Cylindrocladium buxicola is proposed as a new name. Fifteen isolates from the UK and one isolate from New Zealand were paired in all combinations but no fertile perithecia were obtained suggesting that C. buxicola is heterothallic and all isolates belonged to one mating type. AFLP analysis showed that the isolates collected in the UK and New Zealand are genetically homogenous. Phylogenetic analyses indicated that this species falls within a new lineage.
Key words: AFLP, ß-tubulin, Buxus, Calonectria, Cylindrocladium, ITS, MAT2
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The genus Buxus contains more than 70 species in Europe, the West Indies, East Asia and Central America. In the UK, the most commonly planted species is the native Buxus sempervirens L. (Stace 1997, Wigginton 1999). During its long time of cultivation, many variants of B. sempervirens have been produced, such as the dwarf cultivar B. sempervirens ‘Suffruticosa’.
In late 1994, a new blight disease of boxwood was discovered in a nursery in Hampshire (UK). No additional new cases were reported until 1997 when a sudden outbreak of the disease was noticed (Henricot et al 2000). The symptoms of the disease are dark brown spots on the leaves, black streaks on the stems and severe defoliation. A fungus was isolated and initially identified as Cylindrocladium scoparium (R. Cook pers comm). Cylindrocladium scoparium is one of the most pathogenic species of this genus causing disease on a wide range of hosts distributed worldwide (Domsch et al 1980, Peerally 1991). The disease symptoms include damping-off, root rot and blight, stem lesions, stem canker and stem dieback, stunting, leaf spots, wilt, defoliation and fruit rot (Domsch et al 1980, Peerally 1991). It is also the most commonly misidentified species of the genus because of the variability of some of the morphological characters used for its identification (Polizzi and Crous 1999, Schoch et al 1999). In 1998, Cylindrocladium was isolated from a Buxus sp. showing leaf and twig blight symptoms in New Zealand and the species was identified as Cylindrocladium spathulatum (Ridley 1998), more commonly known as a pathogen of Eucalyptus (Crous and Wingfield 1994). It was not ruled out by Ridley that this species may be C. ilicicola given that both species are very similar morphologically and C. ilicicola has a wider host range than C. spathulatum including Buxus (Brayford and Chapman 1987, Crous and Wingfield 1994).
The phylogeny of the genus Cylindrocladium has been the subject of several recent studies in which molecular techniques have been used to supplement morphological descriptions in order to establish the relationship between species, and to identify unknown strains (Crous et al 1993a, b, c, 1999, Victor et al 1997, Schoch and Crous 1999, Schoch et al 2000a, b). Species of the genus Cylindrocladium are differentiated morphologically mainly on the basis of the shape of their vesicle, and the size and septation of the conidia (Crous and Wingfield 1994). However some of these characters are known to be influenced by cultural conditions (Crous et al 1992) and also are not well differentiated between species. This has encouraged the use of molecular techniques such as protein profiles, RAPD and RFLPs to resolve taxonomic disputes (Crous et al 1993a, b, c, 1995, 1997, 1998, 1999, El-Gholl et al 1997, Victor et al 1997, Schoch et al 1999). The ITS region has proven particularly useful for the separation of fungal taxa at the species and genus level (Bruns et al 1991). However, the sequence variation of the ITS region of the Cylindrocladium spp. sequenced so far is insufficient to distinguish all the biological species of the genus (Crous et al 1999, Schoch et al 1999, 2000a). This is why the authors sequenced more variable regions, including parts of the ß-tubulin and MAT2 genes, thus giving a better resolution of the phylogeny of Cylindrocladium (Crous et al 1999, Schoch et al 2000a).
In this work, we describe the species of Cylindrocladium that causes blight disease on Buxus spp. using morphological characters, and sequencing of the ribosomal 5.8S RNA gene and the flanking internal transcribed spacers (ITS), the ß-tubulin gene, and the high mobility group (HMG) of the MAT2 mating type gene. Amplified fragment length polymorphism (AFLP) is used to explore genetic differences between isolates collected in the UK and New Zealand.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cylindrocladium strains used for comparisons were obtained from CABI Bioscience (IMI, Egham, UK), Forest Research in New Zealand (FS) and the Dutch Plant Protection Service (PD, Plantenziektenkundige Diest, Wageningen). All isolates used in this work are listed in Table I.
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, Crous et al 1992) and incubated at 25 C under near-ultraviolet light. The plates were examined after 7 d or until sporulation occurred (no later than 9 d) and only conidiophores on the carnation leaves were examined. For each isolate, mounts were prepared in lactic acid with aniline blue (0.2 g/100 mL), and measurements of at least 30 conidia, vesicles, stipes, branches and phialides were made at 1000x magnification with an optical microscope (Zeiss).
Growth on agar –
Growth rates of 10 isolates from Buxus collected in the UK and one from New Zealand (Table I) were examined at 5, 8, 10, 15, 20, 25, 27.5, 30, 33, and 35 C. Agar plugs (about 3 mm diam) from 6-d-old colonies of each isolate were placed centrally on 2% malt extract agar (MEA) and three replicates representing three different single spore cultures of each isolate were assessed. The plates were incubated in the dark without Parafilm. Diameters of each colony were measured in two directions (at right angles) on days 2 and 7, and the mean daily radial increment calculated. The experiment was repeated once. The plates at temperature(s) where growth was arrested were then incubated at room temperature to determine the lethal temperature. The effect of temperature on observed growth rates of isolates was analyzed by linear mixed models using the method of residual maximum likelihood (REML) and Tukey's test (SAS/STAT 1989).
Sexual compatibility – Nineteen isolates (Table I) were mated in all possible combinations. Single conidial isolates were grown on PCA for 7 d in the dark at room temperature. Agar plugs about 3 mm diam were taken from the periphery of the colonies and placed on opposite sides of a piece of a sterile carnation leaf on a CLA plate. Plates were sealed with Parafilm and put in a plastic bag. Plates were then incubated under nuv light at 25 C and examined monthly over three months.
Cryomicroscopy – To observe surface features of conidia and conidiophores, pieces of carnation leaf on 0.5 cm square agar from a 7-d-old culture were examined using a scanning electron microscope (SEM, Hitachi S570) fitted with an Emscope SP2000 cryopreparation unit. The specimens were plunged into liquid nitrogen in the absence of vacuum and then put into the microscope via a vacuum transfer device. The temperature of the SEM was then increased to around -60 C to sublime any ice that may have accumulated on the surface. The specimen was then transferred to the preparation unit for gold coating before return to the SEM for observation.
DNA extraction and amplification – Single conidia of 8 isolates were grown on cellophane placed on PCA media and grown at room temperature in the dark. After 7 to 10 d, the mycelium was scraped from the plates and stored at -80 C until DNA extraction. The mycelium was ground in liquid nitrogen using a pestle and mortar and the DNA extracted using the Plant DNAeasyTM mini kit (Qiagen, Germany) according to the manufacturer's instructions.
All PCR reactions (25 µL total volume) were done using PCR beads (Amersham Pharmacia Biotech, UK) according to the manufacturer's instructions, 10 pmol of each primer and approximately 10 to 50 ng of fungal genomic DNA. Reactions were performed on a Progene thermocycler (Techne, Cambridge, UK).
For the ITS region, DNA was amplified using the primers ITS1 and ITS4 (White et al 1990). Reaction conditions were as follows: an initial denaturation for 2 min at 96 C, followed by 35 cycles of 1 min at 96 C, 1 min at 60 C and 2 min at 72 C. A final elongation step of 10 min at 72 C was included.
The 5' end of the ß-tubulin gene was amplified using the primer T1 (O'Donnell and Cigelnik 1997) and the primer Bt2b (Glass and Donaldson 1995). The reaction conditions were the same as those used by Crous et al (1999).
The HMG box of the MAT2 gene was amplified using the primers ColHMG1 and ColHMG2 as described by Schoch et al (2000a).
Cloning of the HMG box of the MAT2 gene – PCR products were cloned into the pGEM-T easy vector (Promega, Madison, Wisconsin, USA) and plasmid DNA was purified using a Wizard® Plus SV Miniprep DNA purification system (Promega, Madison, Wisconsin, USA) both according to the manufacturer's instructions.
Sequencing – All the sequencing was done by MWG BIOTECH AG Ebersberg (Germany). The HMG of the MAT2 gene was sequenced using the T7 and SP6 primers (Promega, Madison, Wisconsin, USA). The ITS regions and the 5' end of the ß-tubulin were sequenced directly from the PCR products with the primers used for their amplifications (as described above). All the DNA fragments were sequenced for both strands.
Sequence alignment and phylogenetic analyses –
The programs EditSeq and MegAlign of Lasergene (DNASTAR 2000) software for Macintosh, were used for editing and aligning the sequence files. Additional sequences were gained from GeneBank (Table II
). The alignments were initially constructed using the CLUSTAL option in Megalign and adjusted manually. Indels (insertions/deletions) were coded as extra character states using MacClade (Maddison and Maddison 1992). The indels were treated as single events and coded as binary characters in subsequent analyses.
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Phylogenetic analyses were performed using PAUP version 4.0b4a (Swofford 1998). Heuristic searches using maximum parsimony with 10 random addition sequence replicates, TBR branch swapping, MULPARS, and steepest descent options were conducted in PAUP for all data sets except the MAT2 HMG box sequence data. Because fewer taxa were included, the MAT2 HMG box data were analyzed using the branch and bound search options. To evaluate clade support, Jackknife analysis (in PAUP) with 30% deletion and fast stepwise addition was calculated for 10 000 replicates. Groups were retained that appeared in 50% or more of the trees.
AFLP fingerprinting –
AFLP fingerprints were generated according to Mueller et al (1996) with the following modifications: for the restriction ligation step, 100 ng of genomic DNA was used instead of 2 µg. The adapters were amplified with PCR beads (Amersham Pharmacia Biotech, UK), 150 ng of primer and approximately 1 ng of DNA. Reactions were performed using a Progene thermocycler (Techne, Cambridge, UK) as described in Mueller et al (1996). The sequence of the primers was:
5'-GACTGCGTACATGCAXX-3' with two base pair extensions (indicated by XX in the sequence) of GA, GT, GC, AC, AG, CG. The primers are referred to in the text by their 2-bp extension.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Growth studies – Using the REML method, no significant differences in the growth rate were detected amongst isolates from Buxus spp. (C. buxicola grouping as proposed below). A mean growth rate was therefore calculated for the whole group at each temperature (Fig. 1). The optimum temperature for linear growth for the C. buxicola group was 25 C (growth rate 2.8 mm/d). The growth rate was drastically reduced at 27.5 C (0.13 mm/d) and there was no growth at 30 C. The lethal temperature was 33 C for all the isolates of the C. buxicola group.
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AFLP analysis – Eighteen isolates of Cylindrocladium from Buxus spp. (Table I) were used to determine the genetic variation among them. The 6 primers yielded profiles that were monomorphic in all isolates except RHS 729 (Fig. 2). Isolate RHS 729 showed polymorphisms with 3 of the 6 primers used (CG, AG, AC). A band of approximately 950 bp using primer CG was unique to this isolate. A band of approximately 870 bp with primer AG and of approximately 510 bp with primer AC was missing from this isolate but present in all the other seventeen isolates. The experiment was repeated with 10 different single spore cultures of isolate RHS 729 and identical results were obtained (data not shown). Thus, apart from isolate RHS 729, the isolates from the UK and New Zealand were identical in banding pattern.
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The ITS sequences of three isolates from UK and one from New Zealand (Table I) were identical. The ITS region yielded 503 characters of which 99 were informative (20%). A maximum of 40 000 trees was fixed for the heuristic search and a strict consensus calculated (Fig. 3). The strict consensus tree was mainly unresolved because the ITS region has few informative sites and could differentiate only a few members of the genus Cylindrocladium. The ITS2 region of the isolates from Buxus showed the most variation having a 7 bp insertion shared only with some accessions of C. floridanum (ATCC 18882, 18834, 22677, CBS 413.67, STE-U 500), C. canadense and C. pacificum and an accession of C. scoparium (IMI 299391).
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Heuristic search yielded 13 120 parsimonious trees from which a strict consensus was calculated (Fig. 4). The alignment generated 464 characters, 189 of which were parsimony-informative (41%). There were enough informative sites in the ß-tubulin to separate all species of Cylindrocladium. The isolates from Buxus form a clade that includes C. penicilloides and C. naviculatum. However, this grouping is not supported by a Jackknife value and therefore the relationship of C. buxicola with these two species remains uncertain. C. buxicola, C. penicilloides and C. naviculatum may represent three different lineages within the genus.
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). The other characteristics such as conidia length (40–52 µm) and width (4–5 µm), conidia septation (1 septum) and vesicle width (5–7 µm) fit both C. scoparium and C. pauciramosum. Similarly the isolate of so-called C. scoparium IMI 299391 did not group with the other isolates of C. scoparium but grouped well with C. ilicicola. The Jackknife value is 97% and strongly supports this grouping (Fig. 5). Although the ß-tubulin sequences of both isolates were identical, the ITS sequences showed clear differences. For example, the 7 bp insertion was present in the isolate IMI 299391 but not in C. ilicicola. As neither of the isolates sporulates using the methodology described above, it was not possible to determine if they shared similar morphological characters.
Morphology –
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HOLOTYPE UNITED KINGDOM. LINCOLNSHIRE: Boston. Buxus sempervirens var ‘Suffruticosa’. Dec 1999, B. Henricot, Kew K(M) 68477, culture ex-type IMI 388262.
Etymology. Named after its host.
Perithecia not found. Macroconidiophores comprised of a stipe, a sterile elongation and a penicillate arrangement of fertile branches. Stipe septate 95–155 µm long, hyaline, terminating in a broadly ellipsoidal vesicle with a pointed or papillate apex, 6.5–11 µm diam. In 97% (168/174) of the vesicles measured, the widest part is above the middle. Primary branches 1 septate or aseptate, (5–) 15–41 (–66) x 3–5 µm, secondary branches aseptate (11–) 13–25 (–35) x 3–5 µm, tertiary branches rare, each terminal branch producing 2–5 phialides; phialides reniform, hyaline, non-septate, (10–) 13–18 (–21) x 2.5–5 µm. Conidia cylindrical, rounded at both ends, straight, hyaline, 42–68 x 4–6 µm, 1 septate held in cylindrical clusters by colorless slime. Microconidiophores not observed. Chlamydospores dark, brown, thickened, formed in moderate numbers on carnation leaves, not on agar, aggregated to form microsclerotia.
Cultural characteristics.
Colony color (reverse) on MEA after 7 d at 25 C is fuscous black at the centre fading through sienna with a pale luteous halo (Rayner 1970). The growing mycelium at the margin is white. Cardinal temperatures. Minimum temperature above 5 C; maximum temperature below 30 C; optimum temperature 25 C. This is a low temperature species. C. buxicola is killed when left for 7 d at 33 C.
Substrate. Living leaves and shoots of Buxus spp.
Distribution. Western Europe, New Zealand.
Additional cultures examined. See Table I.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) rather than vice versa.
Morphologically, C. buxicola is characterized by having one-septate conidia and ellipsoidal vesicles with pointed or papillate apices. Based on this observation, it is different from C. scoparium that lacks pointed vesicles. The width of the vesicles of C. buxicola [(6.5)–8.75–(11) µm] is also larger than the one published of C. scoparium [(6)–7–(8) µm; Crous and Wingfield 1994]. In addition, C. scoparium is an eurythermal species able to grow up to 35 C (Crous and Wingfield 1994) while C. buxicola is a low temperature species and its growth is inhibited at 30 C. Cylindrocladium buxicola is also morphologically different to C. spathulatum and C. ilicicola which initially were thought to be the cause of box blight in New Zealand (Ridley 1998). Cylindrocladium ilicicola and C. spathulatum both have spathulate to clavate vesicles (Crous and Wingfield 1994) while C. buxicola has a broadly ellipsoidal vesicle.
Another species morphologically similar to C. buxicola is C. pauciramosum. Its vesicle is obpyriform to broadly ellipsoidal similarly to C. buxicola (Schoch et al 1999). Cylindrocladium buxicola and C. pauciramosum also have similar vesicle width and conidia size. However, the vesicle of C. pauciramosum distinctly lacks the pointed apex which is characteristic to C. buxicola. Also, C. pauciramosum is able to grow at 30 C unlike C. buxicola. Another morphologically similar species is C. insulare, which has a similar vesicle shape and size to C. buxicola but no pointed vesicles (Schoch et al 1999). The only other species published so far which shares similar pointed vesicles is C. mexicanum (Schoch et al 1999). Cylindrocladium mexicanum has also similar conidia and vesicle sizes (Schoch et al 1999). However, C. mexicanum is a low and high temperature species and is able to grow at a temperature of 35 C (Schoch et al 1999).
The DNA sequences of the ITS, ß-tubulin, and the MAT2 genes of C. buxicola were unique compared to the other Cylindrocladium species published so far, and this supported C. buxicola as a new species.
The ITS region has few informative sites and can differentiate only a few members of the genus Cylindrocladium (Crous et al 1999, Schoch et al 1999, 2000a). However, as the changes in the sequences in all the isolates from Buxus were consistent, they can be used as one of the criteria to differentiate this new species. Most of the sequence variation was found in the ITS2 sequence with one major indel represented by a 7 bp insertion. An identical insertion was found in some isolates of C. floridanum (Jeng et al 1997, Kang et al 2001a, Risède and Simoneau 2001), C. canadense, C. pacificum (Kang et al 2001a) and the isolate IMI 299391 originally identified as C. scoparium. C. pacificum and C. canadense were previously considered to reflect different populations of C. floridanum occurring in Hawaii and Canada respectively (Kang et al 2001a).
The ß-tubulin and the MAT2 HMG box possessed enough informative sites to differentiate all species of Cylindrocladium so far described. In the ß-tubulin-based tree that includes the highest number of Cylindrocladium species, there is no support for the clade formed by C. buxicola with C. penicilloides and C. naviculatum. In the ß-tubulin and ITS combined tree, C. buxicola forms a distinct clade within the genus. This rules out the hypothesis that this species could be the result of cross hybridization between different Cylindrocladium species. A more intermediate grouping in this case would be expected. In the MAT2 HMG box based tree, the new species is grouped with C. ovatum but the phylogenetic stability of this clade must be viewed with caution as the Jackknife value is very low (52%).
The phylogeny of the whole Cylindrocladium group has been recently reviewed by Schoch et al (2001a) based on the sequencing of the ß-tubulin gene. Using a different tree-building method (parsimony analysis), we did not find any major conflict with the distance tree built (neighbor-joining analysis) by Schoch et al (2001a). The authors found that vesicle shape is the morphological character that has the greatest congruence with clade specialization. We did not find this was true in the case of C. buxicola as it was not grouped with the species that is the closest morphologically, i.e., C. mexicanum. We found as well that C. ilicicola and Cylindrocladium sp (IMI 299391) were grouped together in the same clade as C. mexicanum (Jackknife value 88%, Fig. 4). Cylindrocladium ilicicola and C. mexicanum have also different vesicle shapes, and they are therefore one of the several taxa where morphology and DNA analysis are not congruent.
The fact that C. scoparium is regularly confused with other morphologically similar species such as C. pauciramosum is highlighted again in this work. From our molecular data and the morphological characteristics of the so-called C. scoparium PD 88/6 and IMI 299281, it is quite clear that these isolates were initially misidentified and both are likely to be C. pauciramosum. Cylindrocladium scoparium and C. pauciramosum resemble each other morphologically (Crous and Wingfield 1994, Crous et al 1999) and misidentification has been reported before (Polizzi and Crous 1999). If so, this is first record of C. pauciramosum occurring on rose.
The AFLPs show that there is little variation between the isolates of C. buxicola collected in different geographical locations. On the basis of these results, the isolates from boxwood appear to derive from one clone.
The mating experiments support the view that this new species has a heterothallic mating system. It cannot however be ruled out that fertile perithecia may be produced if different environmental conditions are used. If this new species arose through interspecific hybridization, the sexual sterility could be explained.
In conclusion, we have described with morphological and molecular data a new species of Cylindrocladium that causes a leaf and twig blight on Buxus spp. No close relationship has been found with other described Cylindrocladium species. It may be that this species has been introduced recently to Europe, where Buxus plants are widely grown, from a geographically isolated area where it has evolved on one or more Buxus spp. This spread from a very narrow genetic base would also explain why the population of isolates is homogenous.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication April 4, 2002.
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LITERATURE CITED |
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indicates C. scoparium (IMI 299391); 
C. scoparium IMI 299981; + C. buxicola; x C. scoparium (PD 88/6); 
C. ilicicola IMI 299915
HindIII (20). Lanes 2 to 18 are isolates collected at different locations in the UK and are, respectively, RHS 9934, 9961, 10082, 10183, 10428, 10868c, 10868g, 47, 615, 634, 729, 732, 1064, 1180, 1995, 2426, 2517. Lane 19 is the one isolate from New Zealand (FS384). The arrows indicate the polymorphisms of isolate RHS 729.
