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Taxonomic status of putative Verticillum alboatrum isolates
FEMS Microbiology Letters 170 (1999) 211^219
Taxonomic status of putative Verticillum alboatrum isolates John H. Carder *, Derek J. Barbara Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK Received 16 October 1998; accepted 6 November 1998
Abstract Two fungal isolates, formerly classified as Verticillium alboatrum and proposed as forming the basis of a new sub-group (`Group 2') within the species, have been shown to be non-pathogenic to known hosts of V. alboatrum and, on the basis of molecular evidence, to be closely related to Verticillium psalliotae and Verticillium fungicola. We propose that the taxon V. alboatrum be confined to those closely related isolates, usually plant pathogenic and usually producing dark resting mycelium, referred to by other authors as Group 1. The only sub-specific groupings which appear valid (based on pathological and molecular evidence) comprise: (1) host-adapted isolates from lucerne; and (2) all other isolates. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Vascular wilts; Verticillium alboatrum ; Verticillium psalliotae; Verticillium fungicola
1. Introduction Modern molecular methods provide powerful adjuncts to conventional morphological and other methods of classifying fungi. However, if a plethora of apparently new and confusing sub-taxa are to be avoided, it is imperative ¢rstly, that it should be considered that isolates found to be distinct may be more correctly placed in existing taxa, and secondly, that isolates must be maintained pure and true to type. In the case of the wilt pathogen Verticillium alboatrum, it appears that the suggestion that two low pathogenicity isolates be considered as a distinct `Group 2' of this species [1,2] is now, pending further
* Corresponding author. Fax +44 (1789) 470552; E-mail: [email protected]
research, found to be incorrect and that these isolates should be placed in, or taxonomically close to, Verticillium psalliotae and/or Verticillium fungicola. Fungal isolations made from wilted hop (Humulus lupulus) plants from the West Midlands region of the UK in 1978 produced four strains, all identi¢ed then as V. alboatrum using morphological characters, principally the production of dark resting mycelium and verticillate conidiophores. The pathogenicity of these isolates was assessed by hop plant inoculation experiments [3,4] and three were used in a later study [1] in which a single repeat from within the ribosomal RNA gene (rRNA) complex of one of them was cloned and characterised. The resulting restriction map con£icted markedly with RFLP data which had been reported earlier [5] and so we elected to carry out further morphological, pathological and molecular studies on all four isolates to try to clarify their identity and taxonomic status.
0378-1097 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 3 4 - 5
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2. Materials and methods 2.1. Isolates Four of the isolates (619, 621, 629 and 690) used in this study were from wilting hop plants in the West Midlands area in 1978 [3,4]. The ¢fth (1974) also came from hops, but in 1975, and from Kent, UK [3,4]. Following their isolation, these strains were maintained in a collection at HRI-East Malling and supplied to Gri¡en et al. [1] for use in their study. 2.2. Morphological studies and pathogenicity tests Mycelium and conidia produced by all of the unpreserved preparations of living isolates on prunelactose-yeast extract (PLY) agar [6] were observed using light microscopes at 20U and 200U magni¢cations. The lengths of 50 conidia from each isolate were measured using a micrometer eyepiece calibrated with a stage graticule. Conidial suspensions (1U106 conidia ml31 ) were prepared from 14-day-old PLY agar cultures by washing the surface of plates with sterile water. Twenty ml of inoculum was applied to the compost of 4-week-old pot-grown cuttings of hops (cv. Northdown) and to eggplant (Solanum melongena var. Black Beauty) seedlings. Symptoms were recorded after 8 weeks by noting the number of plants showing severe symptoms (extensive foliar chlorosis and/ or necrosis and pronounced stunting), mild symptoms (some foliar chlorosis and/or partial necrosis usually restricted to the lower leaves and slight stunting) or no symptoms. Also at 8 weeks after inoculation, petiole samples were excised from the oldest attached leaves of all plants. These tissue samples were surface sterilised, placed on water agar (20 g l31 agar in water) and incubated at 22³C for 21 days. Microscopic examination of the samples (20Umagni¢cation) determined the presence or absence in the xylem of fungal mycelium indistinguishable morphologically from the inoculated isolate. 2.3. DNA hybridisation studies Seventeen clones selected from a partial genomic library of V. alboatrum isolate 1974 [5] were labelled
by nick-translation using [32 P]dCTP and used to probe Southern blots of EcoRI digested fungal genomic DNA. DNA extraction, preparation of Southern blots and conditions for hybridisation and autoradiography were as described previously [5] except that a hybridisation oven was used instead of polythene envelopes. 2.4. Cloning and molecular analysis of rRNA gene complexes Putative full-length rRNA repeat units from isolates 621, 690 and 1974 were cloned into the PstI site of pUC18 by excising strongly £uorescent bands (ca. 7^8 kbp) from PstI digests of total DNA, which had been separated by electrophoresis through an agarose gel, and then ligation of the DNA into prepared plasmid. Restriction endonuclease digests of these clones were made using a combination of PstI and each of nine other endonucleases. The fragments generated were separated in an agarose gel (15 g l31 in TBE) at 7.5 V cm31 for 1 h. The separated DNA was visualised, using ethidium bromide, photographed under UV light (302 nm) and fragment sizes calculated from relative mobilities by comparison with a series of marker fragments (Stepladder 1018, NBL Gene Sciences). PCR primers from conserved regions of fungal rRNA genes (ITS5, 2, 3 and 4; [7]) were used under conditions described by Morton et al. [8] to amplify all of each of the internal transcribed spacer (ITS) regions together with the 5.8S and parts of the large and small sub-unit rRNA coding regions. This was done for each of the three isolates 621, 690 and 1974. Some products were puri¢ed using Prep-A-Gene (Bio-Rad) and cloned using the pMOSBlue T-vector kit (Amersham Life Science). The cloned PCR products were sequenced using a commercial service (Sequiserve, Germany). The products ampli¢ed by primers ITS1 and 4 were digested using restriction enzymes SacII and BglI. Sequences corresponding to the whole of ITS1 and the 5.8S rRNA gene were identi¢ed and the sequences from isolate 621 were compared with those from the same regions of isolates 690, 1974 and of other fungi retrieved from the EMBL database. The GCG (Genetics Computer Group) program PILEUP was used to compare sequences and produce multiple se-
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quence alignment ¢les. From the PHYLIP (Phylogeny Inferencing Package) suite of programs [9]: DNADIST was used to produce a distance matrix, then NEIGHBOR (Neighbor-joining/UPGMA version 3.52a) was used to draw dendrograms.
3. Results 3.1. Morphology and pathology All of the isolates produced some aerial hyphae and conidia on PLY agar plates. Cultures of isolates 621 and 690 were morphologically very similar to each other and di¡ered from the other three by producing profuse cottony aerial mycelium and usually solitary, rarely verticillate, phialides bearing ellipsoid conidia of shorter mean length (1974, 7.13 þ 0.68 Wm; 619, 7.52 þ 0.54 Wm; 629, 7.52 þ 0.60 Wm; 621, 5.60 þ 0.40 Wm; 690, 5.68 þ 0.36 Wm). Only isolates 619 and 629 developed submerged dark resting mycelium typical of V. alboatrum [6]. Table 1 shows the results of inoculation tests on hop and eggplant in terms of symptoms (severe, mild or none) and re-isolations of fungus from the xylem of excised petioles. None of the plants of either species inoculated with 621 or 690 developed any wilt symptoms nor could the fungus be re-isolated from them. Conversely, with the other three isolates, all eggplants showed severe wilt and between two and ¢ve hop plants showed symptoms and colonisation of the xylem after inoculation with 619, 629 or 1974.
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3.2. DNA hybridisation Thirteen of the 17 genomic probes derived from V. alboatrum isolate 1974 [5], and which all hybridised with EcoRI digested DNA of 619 and 629 on Southern blots, did not hybridise with DNA from 621 and 690. The remaining four probes hybridised to all ¢ve DNAs tested. Two of these probes (pVA75 and pVARNA12) are known to originate from the DNA coding for rRNA genes [8] and they, like the other two probes (pVA2 and pVA18), hybridised to DNA from a wide range of Verticillium species [5]. 3.3. Molecular analysis of cloned rRNA gene regions When digested with nine restriction endonucleases, cloned PstI fragments of ca. 7.5 kbp from 621, 690 and 1974 (which had been shown to hybridise strongly to the probes pVA75 and pVARNA12; data not shown) revealed striking di¡erences between 1974 and the other two isolates in terms of the size of fragments generated and the number of sites present (Table 2). The estimated overall size of the gene repeat, based on the average of the sums of the fragment sizes, di¡ered between 1974 (average 7.28 kbp) and the other two isolates (average 7.55 kbp) by 270 bp (Table 2). ITS primers 5 and 2 (which £ank the ITS1 region) ampli¢ed fragments of ca. 260 bp from 621 and 690 DNA and ca. 230 bp from 1974. Primers 3 and 4 (which £ank the ITS2 region) ampli¢ed similar sized fragments from all 3 isolates (ca. 350 bp). When several of the PCR products had been cloned and
Table 1 Symptoms and re-isolations from hops and eggplants inoculated with ¢ve putative V. alboatrum isolates Isolate
Hop
Eggplant
Symptoms
621 690 619 629 1974 Uninoculated a b
Isolation
Severe
Mild
None
0a 0 1 0 2 0
0 0 2 2 3 0
6 6 3 4 1 6
0 0 3b 2b 5b 0
Symptoms
Isolation
Severe
Mild
None
0 0 6 6 6 0
0 0 0 0 0 0
6 6 0 0 0 6
0 0 6 6 6 0
In all cases, six test plants inoculated per isolate and isolations attempted from all six. In all cases, successful isolations were from plants showing symptoms; fungus was never isolated from plants without symptoms.
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a
200, 210, 2300, 4600 3000, 4300 400, 480, 1200, 2200, 3000 210, 360, 6700 1800, 2000, 3500 900, 1200, 1800, 3300 7300 320, 450, 6500 2700, 4600
Sizes of restriction fragmentsa
1974
3 1 4 2 2 3 0 2 1
Number of restriction sites 300, 420, 480, 1400, 5000 1400, 2200, 3700 750, 850, 2900, 3100 200, 450, 7000 2900, 4600 300, 1300, 1800, 1850, 2200 1300, 6300 1750, 5900 7600
Sizes of restriction fragments
621 and 690
Fragment sizes (bp) were estimated by comparison with markers in agarose gel electrophoresis.
BglI BglII EcoRI PvuII SacI SacII SalI XbaI XhoI
Restriction enzyme
4 2 3 2 1 4 1 1 0
Number of restriction sites
300, 800, 1300, 5000, 200, 2500, 5000 700, 1200, 2600, 2900 200, 400, 6900 2700, 4800 1500, 2300, 3700 1300, 6200 1600, 5900 Enzyme not used
Sizes of restriction fragments
621 (data from Gri¡en et al. [1])
Table 2 Sizes of restriction fragments produced and numbers of sites present in cloned rRNA gene complexes from isolates 621, 690 and 1974
3 2 3 2 1 2 1 1 ^
Number of restriction sites
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sequenced it was found that there were only four or ¢ve positions within the 607 bp sequence at which 621 and 690 di¡ered. For both these isolates, the ITS1/5.8S/ITS2 regions were clearly di¡erent in sequence as well as overall length when compared to that from V. alboatrum 1974. For further comparisons, 1974 was used as the reference V. alboatrum isolate and 621 as the representative of the 690 and 621 pair. 3.4. Sequence comparisons Published DNA sequences corresponding to the
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internal transcribed spacer region (ITS1) between the end of the small sub-unit rRNA gene and the start of the 5.8S gene for a number of fungi were retrieved from databases and compared. Similarly, sequences equivalent to the 5.8S gene for each of these organisms where available were also retrieved. The species used are shown in Table 3. Sequences for 621 were from the current work. Multiple sequence alignment ¢les were generated for these two sets of sequences by using progressive pairwise alignments. A distance matrix was drawn up and then NEIGHBOR (Neighbor-joining/UPGMA version 3.52a) used to draw unrooted trees
Table 3 Fungi used for comparisons of ITS1 and 5.8S sequences, together with Genbank or EMBL accession numbers Fungus
Abbreviations used in Figs. 1 and 2
Database accession no.
Sequence usedc
Aspergillus awamori Aspergillus nidulans Colletotrichum acutatum Colletotrichum coccodes Colletotrichum fragariae Colletotrichum fragariae Colletotrichum gloeosporoides Colletotrichum kahawae Colletotrichum linicola Colletotrichum linicola Colletotrichum musae Colletotrichum trichellum Heterobasidion annosum Penicillium clavigerum Penicillium dendriticum Penicillium islandicum Penicillium minioluteum Penicillium vulpinum Talaromyces gossypii Talaromyces minosinus Talaromyces stipitatus Talaromyces thermophilus Talaromyces wortmanii Verticillium alboatrum (NL) Verticillium alboatrum (L) Verticillium alboatrum (Luc 2) Verticillium dahliae Verticillium fungicola var. fungicola Verticillium psalliotae Verticillium tricorpus
Aa An Ca Cc Cf1 Cf2 Cg Ck Cl1 Cl2 Cm Ct Ha Pc Pd Pi Pm Pv Tg Tm Ts Tt Tw VaNL VaL Va2 Vd Vf Vp Vt
UO3518 UO3520 X73810 X73815 X73812 Z32944 X73811 Z32983 Z32984 Z32989 Z32992 Z33001 Z70021 L14533 L14502 L14504 L14505 L14535 L14523 L14526 L14514 L14515 L14532 Z29509 Z29508 L19499 Z29511 ^b ^b L28679
ITS1 ITS1 and 5.8S (part) 5.8S 5.8S 5.8S ITS1 5.8S ITS1 ITS1 ITS1 ITS1 ITS1 ITS1d +5.8Sd 5.8S ITS1 ITS1 ITS1 ITS1 ITS1+5.8S 5.8S ITS1 ITS1 ITS1+5.8S ITS1+5.8S ITS1+5.8S ITS1+5.8S ITS1+5.8S ITS1 and 5.8Sa ITS1 and 5.8Sa ITS1+5.8S
a
Only part of 5.8S sequence available (A. nidulans, 125 bp; V. fungicola, 105 bp; V. psalliotae, 105 bp). Sequences supplied by Drs P.R. Mills and S. Muthumeenakshi (personal communication). c For clarity, where several spp. have identical ITS1 or 5.8S sequences, only one is shown in Figs. 1 and 2 (except for Verticillium spp. where all are shown). d Sequences used as outgroups. b
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Fig. 1. Dendrogram showing the relative similarity between the 5.8S sequences of 18 fungal isolates. Lines are drawn to scale to represent relative genetic distances between isolates. 1 mm=0.00075 length units (as de¢ned by NEIGHBOR). a Sequence used as an outgroup.
Fig. 2. Dendrogram showing the relative similarity between the ITS1 sequences of 25 fungal isolates. Lines are drawn to scale to represent relative genetic distances between isolates. 1 mm=0.0125 length units (as de¢ned by NEIGHBOR). a Sequence used as an outgroup.
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(Figs. 1 and 2). Sequences from Heterobasidion annosum, a basidiomycete, were used as outgroups. Even for the two most dissimilar species, there were few base di¡erences between any two of the 5.8S gene sequences (maximum of 23 in 159) (Fig. 1). In contrast, as the data in Fig. 2 for ITS1 indicates, in many cases, there were large sequence di¡erences with the two most dissimilar sequences exhibiting almost no matching regions. The 5.8S gene sequence data placed isolate 621 in a group comprising all of the Colletotrichum species selected. All the plant pathogenic Verticillum species were also closely grouped together, but quite remote from the Colletotrichum cluster. A third group of isolates contained all the selected examples of Penicillium and Talaromyces (some Talaromyces species have Penicillium anamorphs [10]). The isolate of H. annosum was only very distantly related to the various clusters. The relationships of the isolates of Aspergillus nidulans and V. fungicola are less certain than for the other isolates since only incomplete 5.8S nucleotide sequences were available for these fungi and PILEUP was unable to make suitable allowance. In fact the available V. fungicola 5.8S sequence di¡ers from that of 621 and Colletotrichum coccodes by a single base. Similarly that of A. nidulans is more similar to the sequences of Talaromyces gossypii and Penicillium clavigerum than its position in Fig. 1 would suggest. When ITS1 data are examined (Fig. 2), a similar pattern of clusters to those in Fig. 1 is found and, broadly speaking, the same genus or genera groupings are found. For instance, the plant pathogenic Verticillium species are distinct and remote from other groups; the Talaromyces, Penicillum, Aspergillus cluster is still evident and the Colletotrichum species also form a loose group. The fungicolous species V. fungicola and V. psalliotae here form a distinct cluster with 621, but separate from Colletotrichum species (cf. Figs. 1 and 2).
4. Discussion The morphological studies showed that isolates 621 and 690 were very similar to each other, but displayed several slight di¡erences when compared to 619, 629 and 1974. Dark resting mycelium was
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only produced by 619 and 629, but isolates of V. alboatrum have been known to lose the ability to display this character, particularly after extended periods in culture [11]. Isolates 619 and 629 appear to be typical V. alboatrum isolates and will not be considered further, but the results clearly suggest 621 and 690 are distinct as shown by Gri¡en et al. [1]. The pathogenicity tests demonstrated very clearly that neither 621 nor 690 was able to cause wilt disease symptoms in either of the two host species inoculated with their spores, even when one of these species (eggplant) is known to be extremely susceptible. It is also very likely that these two isolates were unable to infect and colonise the plants, since no reisolations of them were possible from petioles of inoculated plants. Less than a quarter of the probes tested, which were originally derived from V. alboatrum 1974, hybridised with DNA from 621 or 690 indicating large di¡erences between the genomes of these two isolates and those of the other three tested. The results obtained from DNA hybridisation studies indicated signi¢cant di¡erences between the genomes of 621 and 690 when compared to the other three isolates. Despite the relatively low stringency conditions employed, of the probes showing no hybridisation to 621 and 690, two have previously been shown to hybridise with DNA from Verticillum dahliae, Verticillium tricorpus, Verticillum nubilum, Verticillum nigrescens and the entomopathogenic Verticillum lecanii [5]. A further ¢ve of the 17 probes hybridised with all of the above except Verticillum nigrescens DNA whilst the remainder only hybridise with DNA from V. dahliae and V. alboatrum [5]. These results suggest that the genomes of 621 and 690 are less like V. alboatrum than are any of the plant pathogenic species or even V. lecanii. Examination of the cloned rRNA gene regions further emphasised the di¡erences between 621 and 690 and V. alboatrum. The numbers of restriction sites present in the entire ribosomal repeats of 621 and 690 are di¡erent from V. alboatrum 1974 for all but one of the nine enzymes used. The sizes of the fragments produced from most digests of 621 and 690 DNA are very similar to those expected from an examination of the restriction map presented by Gri¡en et al. [1]. Our results showed the presence of at least four BglI and SacII sites, whereas Gri¡en et
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al. [1] reported only three and two, respectively. The discrepancy may have resulted from incomplete digestion in the Gri¡en et al. [1] digests or may re£ect di¡erences between the two cloned versions used by us and Gri¡en et al. [1]. PCR ampli¢cation of the two internal transcribed spacer regions and the 5.8S gene also served to distance 621 and 690 from V. alboatrum. The larger PCR product from 621 and 690 with ITS primers 5 and 2 indicated a longer internal transcribed spacer region between the end of the small sub-unit ribosomal RNA and the 5.8S sub-unit RNA coding regions for these isolates when compared to V. alboatrum 1974. This observation was con¢rmed by sequencing. Sequence comparisons of 5.8S and ITS1 regions con¢rmed the dissimilarity of these parts of the ribosomal gene repeats of 621 and 690 from those of the V. alboatrum isolates or indeed from other plant pathogenic Verticillium species. As expected the quantitative di¡erences are greater in ITS1 comparisons than when 5.8S sequences are examined. This is not surprising given the highly conserved nature of 5.8S gene sequences across fungal genera and, by comparison, the much more variable nature of internal transcribed spacer regions. All of the pathological and molecular data presented in this study emphasise the lack of similarity between isolates 621 and 690 and other V. alboatrum isolates. Gri¡en et al. [1] stated that the cloned copy of rDNA from isolate 621 may not be typical of copies from other isolates and alluded to our unpublished data regarding its atypical lack of pathogenicity. We now believe that the cultures of 621 and 690 now available to us and used by Gri¡en et al. [1] are not V. alboatrum isolates and suggest that of the fungi examined here they are most closely related to V. fungicola and V. psalliotae. The key to the four sections of the genus Verticillum described by Gams and Van Zaagen [12] con¢rms the lack of signi¢cant morphological di¡erences between the sections when coloured structures are absent or excluded. The published work [1] illustrates how morphology alone can lead to misclassi¢cation of Verticillum isolates because this gave no reason to exclude 621 and 690 from V. alboatrum using only this criterion. The molecular data presented here has ¢nally resolved the taxonomic relationship between V. albo-
atrum and these two isolates. Furthermore the phenograms show how relatively closely related all the plant pathogenic Verticillium species are and how remote from them are the two fungicolous species V. fungicola and V. psalliotae. It is probable that the original cultures of 621 and 690 were typical V. alboatrum, as evidenced by their former pathogenicity [3,4], but that they became contaminated with other closely related fungi during routine subculturing which outgrew, and replaced, the original V. alboatrum or mycoparasitised it with the same outcome. An organism, morphologically very similar to the present cultures of 621 and 690 and with an almost identical ITS2 sequence (maximum of 2 bases di¡erent; data not presented), was recently received from a commercial culture collection, ostensibly as V. dahliae var. longisporum. This suggests that this particular contaminant of fungal cultures may be more common and widespread than at ¢rst thought. Recently, two con£icting proposals have been made to place isolates classi¢ed as V. alboatrum into `groups' [2,13]. Robb et al. [13] proposed placing the isolate Luc 2 and potato isolates similar to it into a Group 2 (with all other isolates as Group 1); their Group 2 is therefore equivalent to the Group 3 of Gri¡en et al. [2]. We have already stated [8] that as molecular evidence [8,13] suggests that these isolates are more distant from the majority of V. alboatrum isolates than is V. tricorpus, these would be far better placed as a new species. As shown here, the isolates placed by Gri¡en et al. [2] in their `Group 2' are not V. alboatrum, but, in fact, V. psalliotae or V. fungicola (or very similar to them). We suggest, therefore, that, based on genomic RFLP studies [5,14], RFLPs in mitochondrial DNA [5,2], RAPD [15] and earlier pathogenicity studies (see [16] for a review) only two clear sub-speci¢c groups within V. alboatrum have been demonstrated; isolates pathogenic to lucerne (referred to as either the L [5] or alfalfa [15] group) and those from all other hosts (referred to as either the NL [5] or potato [15] group). These correspond to the sub-groups 1A and 1B of Gri¡en et al. [2]. While the taxonomic level to be ascribed to these two groups, for which we prefer the terms L and NL, at least temporarily, is undecided and there is uncertainty as to whether isolates from lucerne are clonal [2] or not [15] and
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the possible existence of some outlying isolates of uncertain a¤nities [15], we feel this simple scheme much better re£ects the classi¢cation and true a¤nities of those isolates which should be included in V. alboatrum.
Acknowledgments This study was supported by the Biotechnology and Biological Sciences Research Council.
References [1] Gri¡en, A.M., Heale, J.B. and Bainbridge, B.W. (1994) Cloning and characterization of the ribosomal RNA gene complex from the plant pathogen Verticillium albo-atrum. FEMS Microbiol. Lett. 118, 291^296. [2] Gri¡en, A.M., Bainbridge, B.W. and Heale, J.B. (1997) Ribosomal, mitochondrial and ampli¢ed DNA polymorphisms in Verticillium albo-atrum pathogenic to hops, lucerne and other plants. Mycol. Res. 101, 1085^1091. [3] Sewell, G.W.F. and Wilson, J.F. (1978) Virulence of hop Verticillium isolates. East Malling Research Station Report for 1977, pp. 86^87. [4] Sewell, G.W.F. and Wilson, J.F. (1979) Virulence of hop Verticillium isolates. East Malling Research Station Report for 1978, pp. 96^97. [5] Carder, J.H. and Barbara, D.J. (1991) Molecular variation and restriction fragment length polymorphisms (RFLPs) within and between six species of Verticillium. Mycol. Res. 95, 935^942. [6] Talboys, P.A. (1960). A culture medium aiding identi¢cation of Verticillium albo-atrum and V. dahliae. Plant Pathol. 9, 57^ 58.
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[7] White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Ampli¢cation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols. A Guide to Methods and Application (Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J. Eds.), pp. 315^322. Academic Press, San Diego, CA. [8] Morton, A., Carder, J.H. and Barbara, D.J. (1995) Sequences of the internal transcribed spacers of the ribosomal RNA genes and relationships between isolates of Verticillium alboatrum and V. dahliae. Plant Pathol. 44, 183^190. [9] Felsenstein, J. (1993) PHYLIP (Phylogeny Inferencing Package) version 3.5c. University of Washington, Seattle, WA. [10] Taylor, J.W., Pitt, J.I. and Hocking, A.D. (1990) Ribosomal DNA restriction studies of Talaromyces species with Paecilomyces and Penicillium anamorphs. In: Modern Concepts in Penicillium and Aspergillus Classi¢cation (Samson, R.A. and Pitt, J.I. Eds.), pp. 357^370. Plenum Press, New York. [11] Heale, J.B. and Isaac, I. (1965) Environmental factors in the production of dark resting structures in Verticillium alboatrum, V. dahliae and V. tricorpus. Trans. Br. Myc. Soc. 48, 39^50. [12] Gams, W. and Van Zaagen, A. (1982) Contribution to the taxonomy and pathogenicity of fungicolous Verticillium species. I. Taxonomy. Neth. J. Plant Pathol. 88, 57^78. [13] Robb, J., Moukhamedou, R., Hu, X., Platt, H. and Nazar, R.N. (1994) Putative sub-groups of Verticillium albo atrum distinguishable by PCR-based assay. Phys. Mol. Plant Pathol. 43, 423^436. [14] Okoli, C.A.N., Carder, J.H. and Barbara, D.J. (1993) Molecular variation and sub-speci¢c groupings within Verticillium dahliae. Mycol. Res. 97, 233^239. [15] Barasubiye, T., Parent, J.-G., Hamelin, R.C., LaBerge, S., Richard, C. and Dostaler, D. (1995) Discrimination between alfalfa and potato isolates of Verticillium albo-atrum using RAPD markers. Mycol. Res. 99, 1507^1512. [16] Heale, J.B. (1985) Verticillium wilt of alfalfa, background and current research. Can. J. Plant Pathol. 7, 191^198.
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Taxonomic status of putative Verticillum alboatrum isolates John H. Carder *, Derek J. Barbara Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK Received 16 October 1998; accepted 6 November 1998
Abstract Two fungal isolates, formerly classified as Verticillium alboatrum and proposed as forming the basis of a new sub-group (`Group 2') within the species, have been shown to be non-pathogenic to known hosts of V. alboatrum and, on the basis of molecular evidence, to be closely related to Verticillium psalliotae and Verticillium fungicola. We propose that the taxon V. alboatrum be confined to those closely related isolates, usually plant pathogenic and usually producing dark resting mycelium, referred to by other authors as Group 1. The only sub-specific groupings which appear valid (based on pathological and molecular evidence) comprise: (1) host-adapted isolates from lucerne; and (2) all other isolates. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Vascular wilts; Verticillium alboatrum ; Verticillium psalliotae; Verticillium fungicola
1. Introduction Modern molecular methods provide powerful adjuncts to conventional morphological and other methods of classifying fungi. However, if a plethora of apparently new and confusing sub-taxa are to be avoided, it is imperative ¢rstly, that it should be considered that isolates found to be distinct may be more correctly placed in existing taxa, and secondly, that isolates must be maintained pure and true to type. In the case of the wilt pathogen Verticillium alboatrum, it appears that the suggestion that two low pathogenicity isolates be considered as a distinct `Group 2' of this species [1,2] is now, pending further
* Corresponding author. Fax +44 (1789) 470552; E-mail: [email protected]
research, found to be incorrect and that these isolates should be placed in, or taxonomically close to, Verticillium psalliotae and/or Verticillium fungicola. Fungal isolations made from wilted hop (Humulus lupulus) plants from the West Midlands region of the UK in 1978 produced four strains, all identi¢ed then as V. alboatrum using morphological characters, principally the production of dark resting mycelium and verticillate conidiophores. The pathogenicity of these isolates was assessed by hop plant inoculation experiments [3,4] and three were used in a later study [1] in which a single repeat from within the ribosomal RNA gene (rRNA) complex of one of them was cloned and characterised. The resulting restriction map con£icted markedly with RFLP data which had been reported earlier [5] and so we elected to carry out further morphological, pathological and molecular studies on all four isolates to try to clarify their identity and taxonomic status.
0378-1097 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 3 4 - 5
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2. Materials and methods 2.1. Isolates Four of the isolates (619, 621, 629 and 690) used in this study were from wilting hop plants in the West Midlands area in 1978 [3,4]. The ¢fth (1974) also came from hops, but in 1975, and from Kent, UK [3,4]. Following their isolation, these strains were maintained in a collection at HRI-East Malling and supplied to Gri¡en et al. [1] for use in their study. 2.2. Morphological studies and pathogenicity tests Mycelium and conidia produced by all of the unpreserved preparations of living isolates on prunelactose-yeast extract (PLY) agar [6] were observed using light microscopes at 20U and 200U magni¢cations. The lengths of 50 conidia from each isolate were measured using a micrometer eyepiece calibrated with a stage graticule. Conidial suspensions (1U106 conidia ml31 ) were prepared from 14-day-old PLY agar cultures by washing the surface of plates with sterile water. Twenty ml of inoculum was applied to the compost of 4-week-old pot-grown cuttings of hops (cv. Northdown) and to eggplant (Solanum melongena var. Black Beauty) seedlings. Symptoms were recorded after 8 weeks by noting the number of plants showing severe symptoms (extensive foliar chlorosis and/ or necrosis and pronounced stunting), mild symptoms (some foliar chlorosis and/or partial necrosis usually restricted to the lower leaves and slight stunting) or no symptoms. Also at 8 weeks after inoculation, petiole samples were excised from the oldest attached leaves of all plants. These tissue samples were surface sterilised, placed on water agar (20 g l31 agar in water) and incubated at 22³C for 21 days. Microscopic examination of the samples (20Umagni¢cation) determined the presence or absence in the xylem of fungal mycelium indistinguishable morphologically from the inoculated isolate. 2.3. DNA hybridisation studies Seventeen clones selected from a partial genomic library of V. alboatrum isolate 1974 [5] were labelled
by nick-translation using [32 P]dCTP and used to probe Southern blots of EcoRI digested fungal genomic DNA. DNA extraction, preparation of Southern blots and conditions for hybridisation and autoradiography were as described previously [5] except that a hybridisation oven was used instead of polythene envelopes. 2.4. Cloning and molecular analysis of rRNA gene complexes Putative full-length rRNA repeat units from isolates 621, 690 and 1974 were cloned into the PstI site of pUC18 by excising strongly £uorescent bands (ca. 7^8 kbp) from PstI digests of total DNA, which had been separated by electrophoresis through an agarose gel, and then ligation of the DNA into prepared plasmid. Restriction endonuclease digests of these clones were made using a combination of PstI and each of nine other endonucleases. The fragments generated were separated in an agarose gel (15 g l31 in TBE) at 7.5 V cm31 for 1 h. The separated DNA was visualised, using ethidium bromide, photographed under UV light (302 nm) and fragment sizes calculated from relative mobilities by comparison with a series of marker fragments (Stepladder 1018, NBL Gene Sciences). PCR primers from conserved regions of fungal rRNA genes (ITS5, 2, 3 and 4; [7]) were used under conditions described by Morton et al. [8] to amplify all of each of the internal transcribed spacer (ITS) regions together with the 5.8S and parts of the large and small sub-unit rRNA coding regions. This was done for each of the three isolates 621, 690 and 1974. Some products were puri¢ed using Prep-A-Gene (Bio-Rad) and cloned using the pMOSBlue T-vector kit (Amersham Life Science). The cloned PCR products were sequenced using a commercial service (Sequiserve, Germany). The products ampli¢ed by primers ITS1 and 4 were digested using restriction enzymes SacII and BglI. Sequences corresponding to the whole of ITS1 and the 5.8S rRNA gene were identi¢ed and the sequences from isolate 621 were compared with those from the same regions of isolates 690, 1974 and of other fungi retrieved from the EMBL database. The GCG (Genetics Computer Group) program PILEUP was used to compare sequences and produce multiple se-
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quence alignment ¢les. From the PHYLIP (Phylogeny Inferencing Package) suite of programs [9]: DNADIST was used to produce a distance matrix, then NEIGHBOR (Neighbor-joining/UPGMA version 3.52a) was used to draw dendrograms.
3. Results 3.1. Morphology and pathology All of the isolates produced some aerial hyphae and conidia on PLY agar plates. Cultures of isolates 621 and 690 were morphologically very similar to each other and di¡ered from the other three by producing profuse cottony aerial mycelium and usually solitary, rarely verticillate, phialides bearing ellipsoid conidia of shorter mean length (1974, 7.13 þ 0.68 Wm; 619, 7.52 þ 0.54 Wm; 629, 7.52 þ 0.60 Wm; 621, 5.60 þ 0.40 Wm; 690, 5.68 þ 0.36 Wm). Only isolates 619 and 629 developed submerged dark resting mycelium typical of V. alboatrum [6]. Table 1 shows the results of inoculation tests on hop and eggplant in terms of symptoms (severe, mild or none) and re-isolations of fungus from the xylem of excised petioles. None of the plants of either species inoculated with 621 or 690 developed any wilt symptoms nor could the fungus be re-isolated from them. Conversely, with the other three isolates, all eggplants showed severe wilt and between two and ¢ve hop plants showed symptoms and colonisation of the xylem after inoculation with 619, 629 or 1974.
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3.2. DNA hybridisation Thirteen of the 17 genomic probes derived from V. alboatrum isolate 1974 [5], and which all hybridised with EcoRI digested DNA of 619 and 629 on Southern blots, did not hybridise with DNA from 621 and 690. The remaining four probes hybridised to all ¢ve DNAs tested. Two of these probes (pVA75 and pVARNA12) are known to originate from the DNA coding for rRNA genes [8] and they, like the other two probes (pVA2 and pVA18), hybridised to DNA from a wide range of Verticillium species [5]. 3.3. Molecular analysis of cloned rRNA gene regions When digested with nine restriction endonucleases, cloned PstI fragments of ca. 7.5 kbp from 621, 690 and 1974 (which had been shown to hybridise strongly to the probes pVA75 and pVARNA12; data not shown) revealed striking di¡erences between 1974 and the other two isolates in terms of the size of fragments generated and the number of sites present (Table 2). The estimated overall size of the gene repeat, based on the average of the sums of the fragment sizes, di¡ered between 1974 (average 7.28 kbp) and the other two isolates (average 7.55 kbp) by 270 bp (Table 2). ITS primers 5 and 2 (which £ank the ITS1 region) ampli¢ed fragments of ca. 260 bp from 621 and 690 DNA and ca. 230 bp from 1974. Primers 3 and 4 (which £ank the ITS2 region) ampli¢ed similar sized fragments from all 3 isolates (ca. 350 bp). When several of the PCR products had been cloned and
Table 1 Symptoms and re-isolations from hops and eggplants inoculated with ¢ve putative V. alboatrum isolates Isolate
Hop
Eggplant
Symptoms
621 690 619 629 1974 Uninoculated a b
Isolation
Severe
Mild
None
0a 0 1 0 2 0
0 0 2 2 3 0
6 6 3 4 1 6
0 0 3b 2b 5b 0
Symptoms
Isolation
Severe
Mild
None
0 0 6 6 6 0
0 0 0 0 0 0
6 6 0 0 0 6
0 0 6 6 6 0
In all cases, six test plants inoculated per isolate and isolations attempted from all six. In all cases, successful isolations were from plants showing symptoms; fungus was never isolated from plants without symptoms.
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a
200, 210, 2300, 4600 3000, 4300 400, 480, 1200, 2200, 3000 210, 360, 6700 1800, 2000, 3500 900, 1200, 1800, 3300 7300 320, 450, 6500 2700, 4600
Sizes of restriction fragmentsa
1974
3 1 4 2 2 3 0 2 1
Number of restriction sites 300, 420, 480, 1400, 5000 1400, 2200, 3700 750, 850, 2900, 3100 200, 450, 7000 2900, 4600 300, 1300, 1800, 1850, 2200 1300, 6300 1750, 5900 7600
Sizes of restriction fragments
621 and 690
Fragment sizes (bp) were estimated by comparison with markers in agarose gel electrophoresis.
BglI BglII EcoRI PvuII SacI SacII SalI XbaI XhoI
Restriction enzyme
4 2 3 2 1 4 1 1 0
Number of restriction sites
300, 800, 1300, 5000, 200, 2500, 5000 700, 1200, 2600, 2900 200, 400, 6900 2700, 4800 1500, 2300, 3700 1300, 6200 1600, 5900 Enzyme not used
Sizes of restriction fragments
621 (data from Gri¡en et al. [1])
Table 2 Sizes of restriction fragments produced and numbers of sites present in cloned rRNA gene complexes from isolates 621, 690 and 1974
3 2 3 2 1 2 1 1 ^
Number of restriction sites
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sequenced it was found that there were only four or ¢ve positions within the 607 bp sequence at which 621 and 690 di¡ered. For both these isolates, the ITS1/5.8S/ITS2 regions were clearly di¡erent in sequence as well as overall length when compared to that from V. alboatrum 1974. For further comparisons, 1974 was used as the reference V. alboatrum isolate and 621 as the representative of the 690 and 621 pair. 3.4. Sequence comparisons Published DNA sequences corresponding to the
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internal transcribed spacer region (ITS1) between the end of the small sub-unit rRNA gene and the start of the 5.8S gene for a number of fungi were retrieved from databases and compared. Similarly, sequences equivalent to the 5.8S gene for each of these organisms where available were also retrieved. The species used are shown in Table 3. Sequences for 621 were from the current work. Multiple sequence alignment ¢les were generated for these two sets of sequences by using progressive pairwise alignments. A distance matrix was drawn up and then NEIGHBOR (Neighbor-joining/UPGMA version 3.52a) used to draw unrooted trees
Table 3 Fungi used for comparisons of ITS1 and 5.8S sequences, together with Genbank or EMBL accession numbers Fungus
Abbreviations used in Figs. 1 and 2
Database accession no.
Sequence usedc
Aspergillus awamori Aspergillus nidulans Colletotrichum acutatum Colletotrichum coccodes Colletotrichum fragariae Colletotrichum fragariae Colletotrichum gloeosporoides Colletotrichum kahawae Colletotrichum linicola Colletotrichum linicola Colletotrichum musae Colletotrichum trichellum Heterobasidion annosum Penicillium clavigerum Penicillium dendriticum Penicillium islandicum Penicillium minioluteum Penicillium vulpinum Talaromyces gossypii Talaromyces minosinus Talaromyces stipitatus Talaromyces thermophilus Talaromyces wortmanii Verticillium alboatrum (NL) Verticillium alboatrum (L) Verticillium alboatrum (Luc 2) Verticillium dahliae Verticillium fungicola var. fungicola Verticillium psalliotae Verticillium tricorpus
Aa An Ca Cc Cf1 Cf2 Cg Ck Cl1 Cl2 Cm Ct Ha Pc Pd Pi Pm Pv Tg Tm Ts Tt Tw VaNL VaL Va2 Vd Vf Vp Vt
UO3518 UO3520 X73810 X73815 X73812 Z32944 X73811 Z32983 Z32984 Z32989 Z32992 Z33001 Z70021 L14533 L14502 L14504 L14505 L14535 L14523 L14526 L14514 L14515 L14532 Z29509 Z29508 L19499 Z29511 ^b ^b L28679
ITS1 ITS1 and 5.8S (part) 5.8S 5.8S 5.8S ITS1 5.8S ITS1 ITS1 ITS1 ITS1 ITS1 ITS1d +5.8Sd 5.8S ITS1 ITS1 ITS1 ITS1 ITS1+5.8S 5.8S ITS1 ITS1 ITS1+5.8S ITS1+5.8S ITS1+5.8S ITS1+5.8S ITS1+5.8S ITS1 and 5.8Sa ITS1 and 5.8Sa ITS1+5.8S
a
Only part of 5.8S sequence available (A. nidulans, 125 bp; V. fungicola, 105 bp; V. psalliotae, 105 bp). Sequences supplied by Drs P.R. Mills and S. Muthumeenakshi (personal communication). c For clarity, where several spp. have identical ITS1 or 5.8S sequences, only one is shown in Figs. 1 and 2 (except for Verticillium spp. where all are shown). d Sequences used as outgroups. b
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Fig. 1. Dendrogram showing the relative similarity between the 5.8S sequences of 18 fungal isolates. Lines are drawn to scale to represent relative genetic distances between isolates. 1 mm=0.00075 length units (as de¢ned by NEIGHBOR). a Sequence used as an outgroup.
Fig. 2. Dendrogram showing the relative similarity between the ITS1 sequences of 25 fungal isolates. Lines are drawn to scale to represent relative genetic distances between isolates. 1 mm=0.0125 length units (as de¢ned by NEIGHBOR). a Sequence used as an outgroup.
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(Figs. 1 and 2). Sequences from Heterobasidion annosum, a basidiomycete, were used as outgroups. Even for the two most dissimilar species, there were few base di¡erences between any two of the 5.8S gene sequences (maximum of 23 in 159) (Fig. 1). In contrast, as the data in Fig. 2 for ITS1 indicates, in many cases, there were large sequence di¡erences with the two most dissimilar sequences exhibiting almost no matching regions. The 5.8S gene sequence data placed isolate 621 in a group comprising all of the Colletotrichum species selected. All the plant pathogenic Verticillum species were also closely grouped together, but quite remote from the Colletotrichum cluster. A third group of isolates contained all the selected examples of Penicillium and Talaromyces (some Talaromyces species have Penicillium anamorphs [10]). The isolate of H. annosum was only very distantly related to the various clusters. The relationships of the isolates of Aspergillus nidulans and V. fungicola are less certain than for the other isolates since only incomplete 5.8S nucleotide sequences were available for these fungi and PILEUP was unable to make suitable allowance. In fact the available V. fungicola 5.8S sequence di¡ers from that of 621 and Colletotrichum coccodes by a single base. Similarly that of A. nidulans is more similar to the sequences of Talaromyces gossypii and Penicillium clavigerum than its position in Fig. 1 would suggest. When ITS1 data are examined (Fig. 2), a similar pattern of clusters to those in Fig. 1 is found and, broadly speaking, the same genus or genera groupings are found. For instance, the plant pathogenic Verticillium species are distinct and remote from other groups; the Talaromyces, Penicillum, Aspergillus cluster is still evident and the Colletotrichum species also form a loose group. The fungicolous species V. fungicola and V. psalliotae here form a distinct cluster with 621, but separate from Colletotrichum species (cf. Figs. 1 and 2).
4. Discussion The morphological studies showed that isolates 621 and 690 were very similar to each other, but displayed several slight di¡erences when compared to 619, 629 and 1974. Dark resting mycelium was
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only produced by 619 and 629, but isolates of V. alboatrum have been known to lose the ability to display this character, particularly after extended periods in culture [11]. Isolates 619 and 629 appear to be typical V. alboatrum isolates and will not be considered further, but the results clearly suggest 621 and 690 are distinct as shown by Gri¡en et al. [1]. The pathogenicity tests demonstrated very clearly that neither 621 nor 690 was able to cause wilt disease symptoms in either of the two host species inoculated with their spores, even when one of these species (eggplant) is known to be extremely susceptible. It is also very likely that these two isolates were unable to infect and colonise the plants, since no reisolations of them were possible from petioles of inoculated plants. Less than a quarter of the probes tested, which were originally derived from V. alboatrum 1974, hybridised with DNA from 621 or 690 indicating large di¡erences between the genomes of these two isolates and those of the other three tested. The results obtained from DNA hybridisation studies indicated signi¢cant di¡erences between the genomes of 621 and 690 when compared to the other three isolates. Despite the relatively low stringency conditions employed, of the probes showing no hybridisation to 621 and 690, two have previously been shown to hybridise with DNA from Verticillum dahliae, Verticillium tricorpus, Verticillum nubilum, Verticillum nigrescens and the entomopathogenic Verticillum lecanii [5]. A further ¢ve of the 17 probes hybridised with all of the above except Verticillum nigrescens DNA whilst the remainder only hybridise with DNA from V. dahliae and V. alboatrum [5]. These results suggest that the genomes of 621 and 690 are less like V. alboatrum than are any of the plant pathogenic species or even V. lecanii. Examination of the cloned rRNA gene regions further emphasised the di¡erences between 621 and 690 and V. alboatrum. The numbers of restriction sites present in the entire ribosomal repeats of 621 and 690 are di¡erent from V. alboatrum 1974 for all but one of the nine enzymes used. The sizes of the fragments produced from most digests of 621 and 690 DNA are very similar to those expected from an examination of the restriction map presented by Gri¡en et al. [1]. Our results showed the presence of at least four BglI and SacII sites, whereas Gri¡en et
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al. [1] reported only three and two, respectively. The discrepancy may have resulted from incomplete digestion in the Gri¡en et al. [1] digests or may re£ect di¡erences between the two cloned versions used by us and Gri¡en et al. [1]. PCR ampli¢cation of the two internal transcribed spacer regions and the 5.8S gene also served to distance 621 and 690 from V. alboatrum. The larger PCR product from 621 and 690 with ITS primers 5 and 2 indicated a longer internal transcribed spacer region between the end of the small sub-unit ribosomal RNA and the 5.8S sub-unit RNA coding regions for these isolates when compared to V. alboatrum 1974. This observation was con¢rmed by sequencing. Sequence comparisons of 5.8S and ITS1 regions con¢rmed the dissimilarity of these parts of the ribosomal gene repeats of 621 and 690 from those of the V. alboatrum isolates or indeed from other plant pathogenic Verticillium species. As expected the quantitative di¡erences are greater in ITS1 comparisons than when 5.8S sequences are examined. This is not surprising given the highly conserved nature of 5.8S gene sequences across fungal genera and, by comparison, the much more variable nature of internal transcribed spacer regions. All of the pathological and molecular data presented in this study emphasise the lack of similarity between isolates 621 and 690 and other V. alboatrum isolates. Gri¡en et al. [1] stated that the cloned copy of rDNA from isolate 621 may not be typical of copies from other isolates and alluded to our unpublished data regarding its atypical lack of pathogenicity. We now believe that the cultures of 621 and 690 now available to us and used by Gri¡en et al. [1] are not V. alboatrum isolates and suggest that of the fungi examined here they are most closely related to V. fungicola and V. psalliotae. The key to the four sections of the genus Verticillum described by Gams and Van Zaagen [12] con¢rms the lack of signi¢cant morphological di¡erences between the sections when coloured structures are absent or excluded. The published work [1] illustrates how morphology alone can lead to misclassi¢cation of Verticillum isolates because this gave no reason to exclude 621 and 690 from V. alboatrum using only this criterion. The molecular data presented here has ¢nally resolved the taxonomic relationship between V. albo-
atrum and these two isolates. Furthermore the phenograms show how relatively closely related all the plant pathogenic Verticillium species are and how remote from them are the two fungicolous species V. fungicola and V. psalliotae. It is probable that the original cultures of 621 and 690 were typical V. alboatrum, as evidenced by their former pathogenicity [3,4], but that they became contaminated with other closely related fungi during routine subculturing which outgrew, and replaced, the original V. alboatrum or mycoparasitised it with the same outcome. An organism, morphologically very similar to the present cultures of 621 and 690 and with an almost identical ITS2 sequence (maximum of 2 bases di¡erent; data not presented), was recently received from a commercial culture collection, ostensibly as V. dahliae var. longisporum. This suggests that this particular contaminant of fungal cultures may be more common and widespread than at ¢rst thought. Recently, two con£icting proposals have been made to place isolates classi¢ed as V. alboatrum into `groups' [2,13]. Robb et al. [13] proposed placing the isolate Luc 2 and potato isolates similar to it into a Group 2 (with all other isolates as Group 1); their Group 2 is therefore equivalent to the Group 3 of Gri¡en et al. [2]. We have already stated [8] that as molecular evidence [8,13] suggests that these isolates are more distant from the majority of V. alboatrum isolates than is V. tricorpus, these would be far better placed as a new species. As shown here, the isolates placed by Gri¡en et al. [2] in their `Group 2' are not V. alboatrum, but, in fact, V. psalliotae or V. fungicola (or very similar to them). We suggest, therefore, that, based on genomic RFLP studies [5,14], RFLPs in mitochondrial DNA [5,2], RAPD [15] and earlier pathogenicity studies (see [16] for a review) only two clear sub-speci¢c groups within V. alboatrum have been demonstrated; isolates pathogenic to lucerne (referred to as either the L [5] or alfalfa [15] group) and those from all other hosts (referred to as either the NL [5] or potato [15] group). These correspond to the sub-groups 1A and 1B of Gri¡en et al. [2]. While the taxonomic level to be ascribed to these two groups, for which we prefer the terms L and NL, at least temporarily, is undecided and there is uncertainty as to whether isolates from lucerne are clonal [2] or not [15] and
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the possible existence of some outlying isolates of uncertain a¤nities [15], we feel this simple scheme much better re£ects the classi¢cation and true a¤nities of those isolates which should be included in V. alboatrum.
Acknowledgments This study was supported by the Biotechnology and Biological Sciences Research Council.
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[7] White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Ampli¢cation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols. A Guide to Methods and Application (Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J. Eds.), pp. 315^322. Academic Press, San Diego, CA. [8] Morton, A., Carder, J.H. and Barbara, D.J. (1995) Sequences of the internal transcribed spacers of the ribosomal RNA genes and relationships between isolates of Verticillium alboatrum and V. dahliae. Plant Pathol. 44, 183^190. [9] Felsenstein, J. (1993) PHYLIP (Phylogeny Inferencing Package) version 3.5c. University of Washington, Seattle, WA. [10] Taylor, J.W., Pitt, J.I. and Hocking, A.D. (1990) Ribosomal DNA restriction studies of Talaromyces species with Paecilomyces and Penicillium anamorphs. In: Modern Concepts in Penicillium and Aspergillus Classi¢cation (Samson, R.A. and Pitt, J.I. Eds.), pp. 357^370. Plenum Press, New York. [11] Heale, J.B. and Isaac, I. (1965) Environmental factors in the production of dark resting structures in Verticillium alboatrum, V. dahliae and V. tricorpus. Trans. Br. Myc. Soc. 48, 39^50. [12] Gams, W. and Van Zaagen, A. (1982) Contribution to the taxonomy and pathogenicity of fungicolous Verticillium species. I. Taxonomy. Neth. J. Plant Pathol. 88, 57^78. [13] Robb, J., Moukhamedou, R., Hu, X., Platt, H. and Nazar, R.N. (1994) Putative sub-groups of Verticillium albo atrum distinguishable by PCR-based assay. Phys. Mol. Plant Pathol. 43, 423^436. [14] Okoli, C.A.N., Carder, J.H. and Barbara, D.J. (1993) Molecular variation and sub-speci¢c groupings within Verticillium dahliae. Mycol. Res. 97, 233^239. [15] Barasubiye, T., Parent, J.-G., Hamelin, R.C., LaBerge, S., Richard, C. and Dostaler, D. (1995) Discrimination between alfalfa and potato isolates of Verticillium albo-atrum using RAPD markers. Mycol. Res. 99, 1507^1512. [16] Heale, J.B. (1985) Verticillium wilt of alfalfa, background and current research. Can. J. Plant Pathol. 7, 191^198.
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