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Molecular variation and sub-specific groupings within Verticillium dahliae
Mycal. Res.
97 (2): 233-239 (1993)
233
Printed in Great Britain
Molecular variation and sub-specific groupings within Verticillium dahliae
C. A. N. OKOLI, J. H. CARDER AND D. J. BARBARN Crop Protection Department, Horticulture Research International, East MaIling, West MaIling, Kent ME19 6B],
u.K.
On the basis of molecular variation, as revealed by RFLPs, isolates of both Verticillium dahliae and V. alboatrum were divided into two major groups, designated A and B in V. dahliae and Land NL in V. alboatrum. There was very limited variation within the groups. A few isolates of V. dahliae did not fall consistently into either of the two groups; when tested with a range of the probe/enzyme pairs which distinguished the groups, these isolates showed combinations of the polymorphisms which distinguished A and B (i.e. an isolate of this type appeared like group A isolates with some pairs and like group B isolates with others). No isolates sharing characters from the Land NL groups were seen in V. alboatrum. In V. alboatrum the groups correlated with original hosts of the isolates (all L isolates being originally from lucerne; isolates from all other hosts were in NL). There was no dear correlation between grouping and original host in V. dahliae but the relative frequency of occurrence of isolates in the groups did vary with host. Cellulase isozyme pattern was correlated with fungal species and in V. dahliae with isolate group; in V. alboatrum most isolates (whether from the L or NL group) gave an identical pattern, but two of the four L group isolates tested gave a second, distinct pattern.
Verticillium dahliae Klebahn and V. alboatrum Reinke & Berthold are both important plant pathogens, causing vascular wilts in a wide range of crops. A recent study (Carder & Barbara, 1991) of molecular variation, as revealed by restriction fragment length polymorphisms (RFLPs) (Michelmore & Hulbert, 1987), within and between six species of Verticillium showed that isolates of V. alboatrum could be divided into two distinct groups. These groups correlated with the original host; isolates obtained from lucerne formed one group (here designated L) and those from all other hosts the second (NL) group. No molecular differences between the three L isolates were detected and, compared to the differences between the groups, only limited variation was found between the seven NL-isolates. In V. dahliae, there was apparently greater variation between isolates than between those in either of the V. alboatrum groups but there was only limited indication of sub-specific groups (of six isolates studied, three were unique whilst the other three were identical to each other) (Carder & Barbara, 1991). As part of our overall objectives of developing reliable diagnostic tools and of identifying polymorphisms suitable for molecular mapping and for examining population structures, a greater range of both V. dahliae and V. alboatrum isolates have been studied. These further studies show that V. dahliae may also be divided into two major groups and that three of the four groups comprising the two species may be sub-divided.
J
Corresponding author.
MATERIALS AND METHODS Fungal isolates and culture conditions The isolates of V. dahliae obtained from established culture collections are listed in Table 1, together with their geographic and temporal origins and the host from which the isolation was made. These isolates were obtained from the personal culture collections of Drs D. C. Harris, G. W. F. Sewell and P. W. Talboys (HRI, East Mailing). A further 13 isolates of V. dahliae collected, in 1990, directly from field soils in which a variety of crops had been grown, were also supplied by Dr D. C. Harris. Five of these isolates came from one field but the rest were from separate sites in the south-east of the United Kingdom. The isolates of V. alboatrum used here but not reported previously (Carder & Barbara, 1991) came either from hop (Humulus lupulu5 L.), grown at various locations in the U.K., or from lucerne. The species identity of all isolates was established by their suppliers. Except where noted, isolates were maintained by mass transfer on prune-lactoseyeast extract (PLY) agar (Talboys, 1960).
DNA extraction and construdion of a partial genomic library Mycelium for DNA extraction was grown as described by Carder & Barbara (1991) and DNA extracted using the procedures described by Murray & Thompson (1980) as modified by Manicom et al. (1987). Further DNA manipulations
Sub-specific groupings in V. dahliae were performed using standard procedures (Sambrook, Fritsch & Maniatis, 1989). A partial genomic library from a strawberry isolate of V. dahliae (327) was constructed in pUC18 as described for V. alboatrum (Carder & Barbara, 1991). Approximately 250 putative recombinants were produced. Insert sizes, as revealed by electrophoresis in agarose gels following restriction endonuclease digestion of DNA produced by a bOiling mini· prep method, ranged from < 0'5 kbp to > 10 kbp. Clones from this new library are referred to with the prefix pVD, those from the previously constructed library derived from V. alboafrum isolate 1974P with the prefix pVA.
Southern blotting and preparation of probes Southern blots of DNA, digested with either feoR I or Hind III and separated by electrophoresis in agarose gels (usually 7 g 1-1 agarose), were prepared by alkaline capillary blotting (Reed & Mann, 1985) onto Hybond-N (Amersham International PLC). Recombinants were selected at random and the plasmids extracted and labelled by nick translation using 32p dCTP. These procedures and conditions for hybridization, autoradiography and for stripping and reprobing blots were all as described by Carder & Barbara (1991).
Cellulase isoenzyme patterns Selected isolates were cultured in a medium with acetone extracted hop bine tissue as a carbon source (Carder, Hignett & Swinburne, 1987) and cellulase isoenzymes separated by isoelectric focusing in vertical polyacrylamide slab gels. Cellulases were detected using a gel overlay technique with carboxymethyl cellulose as substrate (Carder, 1989).
RESULTS
Grouping of V. dahliae isolates using probes from V. dahliae Southern-blotted DNA, digested separately with the restriction enzymes feaR I and Hind III, from 17 isolates of V. dahliae (Table la; isolates 64A to 333 inclusive) were probed with 7I randomly chosen clones from the partial genomic library from V. dahliae. In some cases digestion with one or other enzyme was judged to be incomplete when the ethidium bromide stained gel was examined (and confirmed by the majority of the hybridization occurring at a position corresponding to the highest molecular weight DNA in the gel) and these results were discarded. Clear results were obtained with 53 probes with both enzymes; a further 13 probes gave results with Hind III digested DNA only and five with feaR I digestion only. As with probes derived from V. alboatrum (Carder & Barbara, 1991) all probes tested gave Simple hybridization patterns of one to three bands, again suggesting a low content of repetitive DNA in Verfieillium species. While 75 % of the probe/enzyme combinations did not reveal polymorphisms, 31 of 124 probe/enzyme combinations did so. Eight probes revealed polymorphisms with both
234 enzymes; five of these eight did so by not hybridizing to DNA from some isolates. Six probes revealed polymorphisms with Hind III only and nine with feaR I only. Fifteen of the 17 isolates tested fell clearly into two groups, referred to as RFLP groups A (10 isolates) and B (five isolates); all isolates aSSigned to each of these two groups reacted identically (Fig. I) and 25 of the 3 I polymorphisms identified discriminated between these groups. All isolates to which five of the probes did not hybridize were in group B. It is not known whether this reflects a large single deletion in the Bisolates relative to the A-isolates or smaller scattered deletions.
Table 1. Original host, geographical source, year of isolation and RFLP group of isolates of V. dahliae Isolate number
Original host
Year of isolation
Geographical origin
RFLP' group
(a) Isolates not used in Carder &. Barbara (I991) 64A 68 318 319 320 321 322 323 324 325 327 325 329 330 331 332 333 1507 1508 1856 1867 1574 1875 1893 1900 1914 1922 1925 1931
Acer Hop Strawberry Strawberry Strawberry Strawberry Strawberry Phlox Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Quince Melon Quince Potato Raspberry Strawberry Hop Chrysanthemum Potato Rose Tomato Raspberry
1957 1957 1955 19&5 1985 1955 1955 1986 1956 1986 1986 19&7 1957 1989 1989 1989 1989 1957 1957 1963 1964 1965 1965 NK 1965 1969 1970 1971 1971
HRI" HRI HRI HRI HRI HRI HRI Kent HRI Kent HRI Kent Kent Norfolk Kent Kent Shropshire HRI Kent NKd HRI HRI Norfolk NK Avon Canada South Africa Dorset Dorset
A A B B B A A A A I' A A A I B A B A A A A A B A A A B I A
W. Midlands Hampshire Scotland NK Kent Kent
I A
(b) Isolates used in Carder & Barbara (I991) 1764 1571 1877 2341 DC59 Omega
Hop Strawberry Strawberry Hop Hop Hop
1975 1965 1966 NK 1985 1985
A' A A B'
• RFLP groups as determined in this paper, see results for explanation. to HRI. experimental farm at Horticulture Research International, East Mailing, Kent, U.K.; other names are U.K. counties or districts except for country names. , Isolates which react inconsistently with the probes which differentiate RFLP groups A and B and are categorized as intermediate. d NK, not known. , Differentiated from other B-group isolates by pVA28 and designated A 2• f Differentiated from other B-group isolates by pVA76 and pVD104 and designated B2 •
C. A. N. Okoli,
J. H. Carder and D. J. Barbara
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Figs 1 & 2. Autoradiograms of blots of DNA from representative isolates of the A and B groups of V. dahliae probed with clones which (Fig. 1) distinguish (pVD22/EcoR I) and (Fig. 2) do not distinguish (pVD126/EcoR I) the groups. Letters in parentheses indicate RFLP group of isolate (see text for explanation).
The probes appeared to be independent as they differed in size and gave distinct hybridization patterns with A-isolates. The remaining two isolates showed some characteristics of both groups, giving A-type polymorphisms with some probe/ enzyme combinations revealing A/B differences and B-type polymorphisms with others. Isolate 325 showed 24 A-group and one B-group polymorphisms while 330 showed II Agroup and 14 B-group polymorphisms (including all those revealed by the five probes which do not hybridize to DNA from B group isolates). These, and other isolates giving both A and B polymorphisms with different probe/enzyme combinations, are designated intermediate (I) to reflect their having characteristics of both groups. Only the two I-isolates showed polymorphisms not associated with the major A/B differentiation. One probe (which did not distinguish A/B), in conjunction with Hind III digests, differentiated isolate 325 from all other isolates. Similarly five probes (four with fcoR I digests, one with Hind III) each uniquely distinguished isolate 330 from all other isolates. Of the initial set of 17 isolates, 14 were from strawberry and 15 from Kent. To determine whether the division of V. dahliae isolates into two major groups would also occur amongst isolates from a greater range of hosts and a wider geographic area a second set of isolates was examined (Table la; isolates 1807 to 1931 inclusive). In this second group of tests a further selection of probes from the same V. dahliae derived library were tested against the V. dahliae isolates. In order to be able to compare these results with the previous ones, most (16) of the first 17 isolates were incorporated into the blots along with the second set of isolates and hybridized with some of the first set of probes; no inconsistencies were noted between the original results and the repeats. Unfortunately for this second group of isolates, DNA extracted from the majority of them failed to digest with fcoR I and so most additional information was derived from Hind III digests.
Five of 20 newly selected probes, when hybridized to fcoR I digested DNA from previously tested isolates placed them into the same groups as before. With this enzyme, one isolate from the new group was placed unequivocally in each of the two groups. One isolate (1928) was intermediate (one probe/enzyme showed an A-group polymorphism and four B-group polymorphisms). One probe also uniquely distinguished isolate 1928 from all other isolates. The remaining 14 probes revealed no polymorphisms with fcoR I digests. The second set of 20 probes was used successfully with Hind III digests of DNA from 11 new isolates. Only four probes revealed polymorphisms; three distinguished groups A and B amongst the isolates classified in the first experiment. These three, together with previously used probes, placed eight of the second set of isolates unequivocally in group A and two in B (including the two assigned to groups by probing fcoR I digests). Isolate 1928 was again shown to be intermediate (one group-A and two group-B polymorphisms) and the probe which showed this isolate to be unique when used with fcoR I digests again revealed a unique polymorphism. Incomplete data for isolate 1867 suggested that this isolate was also in group A (three probe/enzyme combinations which differentiated A/B groupings put it into group A; seven did not distinguish A/B/1867). The RFLP groups to which the V. dahliae isolates were assigned are given in Table 1. Illustrative blots are shown in Figs 1-4. Soil isolates Thirteen isolates of V. dahliae taken directly from soils (see Materials and Methods) in which a variety of crops had been grown were classified by probing with 10 V. dahliae derived probes, selected because they had shown clear A/B discrimination in previous tests. Nine isolates fell into group A,
236
Sub-specific groupings in V. dahliae
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Figs 3 & 4. Autoradiograms of blots of DNA from two isolates intermediate between V. dahliae groups with probes which reveal unique polymorphisms; (Fig. 3) pVD204/EcoR I; (Fig. 4) pVD39/EcoR I. Letters in parentheses indicate RFLP group of isolate (see text for explanation).
Table 2. Proportions of probe/enzyme combinations differentiating subspecific groups of the two species of Veriicillium studied' Species/ group"
Vd.A
Vd.A V.d.B VaaL V.aa.NL
30'6%' 29'5% 60'7% 53'6%
Vd.B
V.aa.L
V.aa.NL
22/72 cd 23/78"
17/28c
15/28"
16/28·
15/28·
57'1% 53'6%
11/28· 39'3%
" Only results from experiments using probes chosen at random are included. " Vd.A and B = V. dah/iae RFLP groups A and B; Vaa.L and NL = V a/boairum RFLP groups L and NL; , Number of probe-enzyme combinations differentiating groups/number of probes tested. d Data from current work, upper figures EeaR I digested DNA. lower Hind III. c Data from Carder & Barbara (1991) and all from EeaR I digested DNA. r Proportion of combinations tested which differentiated pairs, source of data as top right.
the other four into B. However, five of the nine A-group isolates came from a single field; the remaining four A-group isolates and all the B-group isolates all came from separate sites. There were no I-isolates and no unique polymorphisms.
Classification of six previously studied isolates of V. dahliae and sub-division of the groups In the light of the above differentiation into clear groups, the patterns of hybridization of DNA from the six isolates of V. dahliae previously studied (Carder &. Barbara, 1991; Table Ib) were re-examined and compared, on the same blot, with known A and B isolates. Probing with six V. dahliae derived probes, all capable of distinguishing A and B groups, and four
V. alboatrum derived probes showed that isolates 1871, 2341 and DC59 (which were identical in the earlier studies when probed only with V. alboatrum derived probes) were not different from group A members as described above. Isolate 1877 was also identical to group A members except when probed with pVA28, which did not hybridize to DNA from this isolate. pVA28 was previously shown (Carder &. Barbara, 1991) to differentiate the V. alboatrum NL group isolates into two sub-groups, here designated NL 1 (pVA28 hybridized to DNA) and NL 2 (pVA28 did not hybridize to DNA). This sub-division of NL was confirmed by probing DNA from a further 23 NL group isolates from hop with pVA28; 10 isolates were classified as NL 1 and 13 as NL 2 . By analogy we now designate isolate 1877 as sub-group A 2 and all other group A members as Ai' pVA28 does not hybridize to any isolate belonging to either V. alboatrum group L (Carder &. Barbara, 1991) or V. dahliae group B. Of the other two V. dahliae isolates previously examined, Omega corresponded most closely to group B, being distinguished only by polymorphism revealed by pVA76. Subsequent tests showed Omega to be unique in being distinguished by this probe from other group B isolates so far tested (data not shown). These later tests also identified a second probe, pVD104, which distinguished Omega, by a polymorphism, from other B group isolates. Unlike pVA76, pVDI04 differentiated groups A and B. We now designate Omega as being in subgroup B2 while all other isolates are B1 . The two sub-groupings, A 2 and B2 , are represented by only single examples and further isolates need to be sought that conform to the same patterns. The remaining isolate tested previously, 1764, was intermediate between the groups (seven group-A polymorphisms and one group-B) using the eight probes from this set of 10 which distinguish A from B. Previously (Carder &. Barbara, 1991), of the 10 V. alboatrum derived probes which distinguished what we now know to be A and B group
C. A. N. Okoli,
J. H. Carder and D. J. Barbara
237 NL
A
Table 3. Correlation of RFLP group (and sub-group) with isoenzyme pattern in V. dahliae RFLP group Isoenzyme group
A l/2
I"
22
2
a
3
0
B,
B,
a
a a
10
a
3
1
a
1
a Isoenzyme group 1 is equivalent to isoenzyme group B as described by Carder (1989) and isoenzyme group 2 to isoenzyme group A.
E::! ...
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ge
B
V"l
N
r')
L Fig. 5. Inter-relationship of the groups of V. dahliae and V. alboatrum. Lengths of lines are proportional to the relative distances between groups, based on the data in Table 2.
members, six classified 1764 as A and four as B. In the earlier work (Carder & Barbara, 1991) one probe (pVA35) uniquely differentiated 1764 from the other five isolates and this was confirmed. pVA35 hybridized, without polymorphism, to representative group A and B isolates but again not to isolate 1764. Similarly, pVA28 did not hybridize to 1764.
Interrelationship of groups Based on the proportion of probe/enzyme combinations differentiating the species and groups (Table 2), using the data obtained here and that reported in Carder & Barbara (1991), it was possible to estimate the genetic interrelationship of the four groups. A dendrogram was constructed using the standard genetic distance and an unweighted pair group method of clustering (Nei, 1975) (Fig. 5). This analysis was intended only to measure the interrelationships of the major groups and hence the minority of V. dahliae isolates not falling clearly in those groups (the I-isolates) were excluded from the calculation.
Correlation of groups with other characters As previously reported (Carder & Barbara, 1991) the two major RFLP groups of V. alboafrum, Land NL, were correlated with original host. The subgroups NL I and NL z were not obviously correlated with host as both have been isolated from hop. In V. dahliae there was no apparent correlation between the major RFLP groups (A and B) and original host of the isolates (Table I). Nor were the sub-groups Al and A z host-related as both had been isolated from strawberry. The only B2 isolate yet identified, Omega, is the only B group example as yet identified from hop and it is possible that the B sub-groups may be host-related but more B-group hop
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Fig. 6. Cellulase isoenzyme patterns of V. dahliae isolates. Diagram represents the more acidic portion (pH 3'8 to 4'7) of an isoeledric focusing polyacrylamide gel (overall range pH 3'8 to 6'0). In parentheses are the RFLP group and sub-group to which isolates belong and the cellulase isoenzyme pattern (see text for details). V. alboatrum isolate 1974 was included for comparison.
isolates need to be tested before any such conclusion can be drawn. There was also no obvious geographic association in the V. dahliae groupings. Many of the isolates came from the HRI experimental farm or other local farms but these included representatives of both groups. Only two isolates originated from outside the U.K., one was group-A, the other group-B. In V. dahliae, RFLP group was correlated with cellulase isoenzyme pattern (Table 3). The isozyme pattern displayed by the only B2 isolate available (Omega) appeared to be a variant of the pattern associated with the Bl isolates (Fig. 6). Little correlation between cellulase isoenzyme pattern and RFLP group was seen in V. alboafrum. Ten isolates gave identical patterns, including six representatives of the NL j , two of the NL 2 - and two of the L-groups. Two isolates from lucerne gave a unique pattern. Six of these isolates had not previously been assigned to RFLP groups but were tested here using at least four probes capable of distinguishing the groups and with pVA28 to assign to their sub-group.
Sub-specific groupings in V. dahliae
DISCUSSION Previously, it had been shown that V. alboafrum isolates can be placed into one of two clear sub-specific groupings when variation at the molecular level was examined (Carder & Barbara, 1991). The data presented here similarly show that the isolates of V. dahliae studied may be divided into two groups when studied in this way. The data in the earlier report (Carder & Barbara, 1991) support this division of V. dahliae when re-interpreted in the light of a wider range of isolates and the use of probes from V. dahliae. Other groups may become apparent when further isolates are tested. A few isolates intermediate between the two groups of V. dahliae were seen but no such intermediates between the two V. alboafrum groups have been found either here or elsewhere (Carder & Barbara, 1991, and unpublished data). The relative statuses of the V. dahliae groups and the I-isolates are not certain. It might be argued that if sufficient probe I enzyme combinations were examined all isolates would show intermediate characteristics. Whilst this cannot be disproved it seems unlikely as most of the intermediates were identified when tested with only a few AlB discriminating probes. Also, the I-isolates appeared to be qualitatively different from other isolates by all exhibiting polymorphisms unique within this collection of isolates; these probably represent low frequency allelles not associated with the major AlB differentiation. In this study only three such polymorphisms were associated with isolates of V. dahliae placed in the two groups. Of the probes concerned only pVDI04 has not been shown to give similar polymorphisms in other circumstances. pVA76 distinguished the sole sub-group B2 isolate, Omega, but preliminary results show that this probe, which is probably derived from the ribosomal RNA gene region, also distinguishes amongst diploid isolates of V. dahliae in the same way (Okoli, Tabrett, Morton, Carder & Barbara; unpublished results). Similarly, although pVA28 differentiates the sole subgroup A 2 isolate of V. dahliae it also discriminates V. alboatrum sub-groups NL 1 and NL 2 . V. dahliae isolate 327 fell into group Al but no probe equivalent to pVA28 (Le. identifying subgroups within V. dahliae) was found amongst the probes selected from the library derived from this isolate even though a larger number of these were tested than were from the V. alboatrum derived library which was the source of pVA28. The two sub-divisions of V. dahliae both involve single isolates and clearly other members of these small groupings need to be identified before their significance can be assessed. That ca 90 % of the V. dahliae isolates fell into the two groups suggests that they are at some selective advantage relative to the less frequent I-isolates (at least in the U.K., the origin of most of the isolates used). In view of the relatively limited variation within the groups (compared to that between the groups) it seems likely that members of each group have a clonal origin. Whether the I-isolates have origins separate from the groups or result from some parasexual event (Hastie, 1964; Hastie & Heale, 1984) between isolates from different groups is not known. However, the infrequent occurrence of I-isolates suggests that in practice there is little gene flow between the groups. Consideration of the genetic distance between the major
238 groups of the two species (Fig. 5) generally reiterates the earlier conclusion (Carder & Barbara, 1991) that molecular variation supports the retention of V. alboafrum and V. dahliae as separate species (ca 50-60% of probes distinguish NL/L from A/B whilst ca 30% distinguish A from Band ca 40% L from NL). Several host specific so-called 'physiological races' of V. dahliae, not represented amongst the isolates tested here, have been reported. Those from peppermint (Homer, 1954; Fordyce & Green, 1960) (in these reports the isolates were described as V. alboatrum but are now included in V. dahliae (e.g. Horiuchi, Hagiwara & Takeuchi, 1990)) are presumably haploids (like all the isolates tested here) while some isolates from Brassica spp. are natural, stable diploids (Jackson & Heale, 1985). Representative isolates of these two races have now been obtained and will be compared with the isolates grouped here. In V. alboatrum, host specificity appears to be the basis of the L/NL differentiation. Isolates originally from lucerne (presumably equivalent to our L group isolates) and those from other hosts (our NL group) have been reported to exist as separate vegetative compatibility groups (Correll, Gordon & McCain, 1988); once groups were established by host preference, vegetative incompatibility would help maintain them and perhaps explain the lack of intermediate isolates seen in our studies. The biological significance of the V. dahliae groupings is more obscure. The correlation of cellulase isoenzyme pattern with RFLP group in V. dahliae supports the existence of the distinct A and B groups. Preliminary work at HRI suggests that other biological characters may also be correlated with these groups and that, as with the V. alboatrum groups, the two groups of V. dahliae form separate compatibility groups (D. C Harris & Jiarong Yang, pers comm.). In V. dahliae there was no clear correlation of RFLP group with original host species and in earlier studies no correlation of cellulase groups with pathogenicity was found (Carder, Hignett & Swinburne, 1987; Carder, 1989). However, further preliminary results at HRI showed that among the 13 soil-derived isolates the average pathogenicity for strawberry was different for the two RFLP groups (D. C Harris & Jiarong Yang, pers. comm.). Also, B group isolates are relatively more common amongst strawberry (9:6/A:B) (Table 1) and soil isolates (9:4/A:B) than amongst those from other hosts (14: 2/A: B) (Table 1). These results suggest that the groups are biologically significant in relation to their degree of pathogenicity and are possibly differentiated by varying levels of host susceptibility. In neither species was any obvious biological basis for the subgroupings seen. We are grateful to the Association of Commonwealth Universities for supporting CAN.O. and to Dr S. A Archer, University of London, for his continued interest in this project.
REFERENCES Carder. J. H. (1989). Distinctions between cellulase isoenzyme patterns of five plant-pathogenic Verlicillium species. Mycological Research 92. 297-301.
C. A. N. Okoli,
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Carder. 1. H. & Barbara, D. j. (1991). Molecular variation and restriction fragment length polymorphisms (RFLPs) within and between six species of Verticil/ium. Mycological Research 95, 935-942. Carder, 1. H. Hignett, R. C. & Swinburne, T. R. (1987). Relationship between the virulence of hop isolates of Ver/icil/ium albo-a/rum and their in vitro secretion of cell wall degrading enzymes. Physiological and Molecular Plant Pathology 31. 441-452. Correll, 1. c.. Gordon, T. R. & McCain, A H. (1988). Vegetative compatibility and pathogenicity of Verticillium alboalrnm. Phytopathology 78, 1017-1021. Fordyce, C. & Green, R. j. (1960). Studies of the host specificity of Verticil/ium albo-atrum var. menthae. Phytopathology 50, 635. Hastie, A C. (1964). The parasexual cycle in Verticillium albo-atrum. Genetical Research 5, 305-315. Hastie, A C. & Heale, j. B. (1984). Genetics of Verticil/ium. Phytopathology Mediterraneae 23, 130-162. Horiuchi,S., Hagiwara, H. & Takeuchi, S. (1990). Host specificity of isolates of Verticil/ium dahliae towards cruciferous and solanaceous plants. In Biological Control of Soil-borne Plant Pathogens (ed. D. Hornby), pp. 285-298. C.AB. International: Wallingford. U.K. Horner, C. E. (1954). Pathogenicity of Verticillium isolates to peppermint. Phytopathology 44, 239-242.
(Accepted 21 July 1992)
239 jackson, C. W. & Heale,}. B. (1985). Relationship between DNA content and spore volume in sixteen isolates of Ver/icil/ium leeanii and two new diploids of V. dahliae (= V. dahliae var. longisporum Stark). Journal of General Microbiology 131, 3229-3236. Manicom, B. Q.. Bar-joseph, M., Rosner, A, Vigodsky-Haas, H. & Kotze, j. M. (1987). Potential applications of random DNA length polymorphisms in the taxonomy of the fusaria. Phytopathology 77, 669-.Q72. Michelmore. R. W. & Hulbert. S. H. (1987). Molecular markers for genetic analysis of phytopathogenic fungi. Annual Review of Phytopathology 25, 383-404. Murray, M. G. & Thompson. W. F. (1980). Rapid isolation of high molecular weight plant DNA Nucleic Acids Research 8, 4321-4325. Nei, M. (1975). Molecular population genetics and evolution. In Frontiers of Biology 40 (ed. A Neuberger & E. L. Tatum). North-Holland Publishing Co.: Amsterdam. Reed, K. C. & Mann, D. A (1985). Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Research 13, 7207-7221. Sambrook, ).. Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning, 2nd edn, Cold Spring Harbor Laboratory Press: New York. Talboys, P. W. (1960). A culture medium aiding identification of Verticil/ium alboatmm and Verticil/ium dahliae. Pla"t Pathology 9, 57-58.
97 (2): 233-239 (1993)
233
Printed in Great Britain
Molecular variation and sub-specific groupings within Verticillium dahliae
C. A. N. OKOLI, J. H. CARDER AND D. J. BARBARN Crop Protection Department, Horticulture Research International, East MaIling, West MaIling, Kent ME19 6B],
u.K.
On the basis of molecular variation, as revealed by RFLPs, isolates of both Verticillium dahliae and V. alboatrum were divided into two major groups, designated A and B in V. dahliae and Land NL in V. alboatrum. There was very limited variation within the groups. A few isolates of V. dahliae did not fall consistently into either of the two groups; when tested with a range of the probe/enzyme pairs which distinguished the groups, these isolates showed combinations of the polymorphisms which distinguished A and B (i.e. an isolate of this type appeared like group A isolates with some pairs and like group B isolates with others). No isolates sharing characters from the Land NL groups were seen in V. alboatrum. In V. alboatrum the groups correlated with original hosts of the isolates (all L isolates being originally from lucerne; isolates from all other hosts were in NL). There was no dear correlation between grouping and original host in V. dahliae but the relative frequency of occurrence of isolates in the groups did vary with host. Cellulase isozyme pattern was correlated with fungal species and in V. dahliae with isolate group; in V. alboatrum most isolates (whether from the L or NL group) gave an identical pattern, but two of the four L group isolates tested gave a second, distinct pattern.
Verticillium dahliae Klebahn and V. alboatrum Reinke & Berthold are both important plant pathogens, causing vascular wilts in a wide range of crops. A recent study (Carder & Barbara, 1991) of molecular variation, as revealed by restriction fragment length polymorphisms (RFLPs) (Michelmore & Hulbert, 1987), within and between six species of Verticillium showed that isolates of V. alboatrum could be divided into two distinct groups. These groups correlated with the original host; isolates obtained from lucerne formed one group (here designated L) and those from all other hosts the second (NL) group. No molecular differences between the three L isolates were detected and, compared to the differences between the groups, only limited variation was found between the seven NL-isolates. In V. dahliae, there was apparently greater variation between isolates than between those in either of the V. alboatrum groups but there was only limited indication of sub-specific groups (of six isolates studied, three were unique whilst the other three were identical to each other) (Carder & Barbara, 1991). As part of our overall objectives of developing reliable diagnostic tools and of identifying polymorphisms suitable for molecular mapping and for examining population structures, a greater range of both V. dahliae and V. alboatrum isolates have been studied. These further studies show that V. dahliae may also be divided into two major groups and that three of the four groups comprising the two species may be sub-divided.
J
Corresponding author.
MATERIALS AND METHODS Fungal isolates and culture conditions The isolates of V. dahliae obtained from established culture collections are listed in Table 1, together with their geographic and temporal origins and the host from which the isolation was made. These isolates were obtained from the personal culture collections of Drs D. C. Harris, G. W. F. Sewell and P. W. Talboys (HRI, East Mailing). A further 13 isolates of V. dahliae collected, in 1990, directly from field soils in which a variety of crops had been grown, were also supplied by Dr D. C. Harris. Five of these isolates came from one field but the rest were from separate sites in the south-east of the United Kingdom. The isolates of V. alboatrum used here but not reported previously (Carder & Barbara, 1991) came either from hop (Humulus lupulu5 L.), grown at various locations in the U.K., or from lucerne. The species identity of all isolates was established by their suppliers. Except where noted, isolates were maintained by mass transfer on prune-lactoseyeast extract (PLY) agar (Talboys, 1960).
DNA extraction and construdion of a partial genomic library Mycelium for DNA extraction was grown as described by Carder & Barbara (1991) and DNA extracted using the procedures described by Murray & Thompson (1980) as modified by Manicom et al. (1987). Further DNA manipulations
Sub-specific groupings in V. dahliae were performed using standard procedures (Sambrook, Fritsch & Maniatis, 1989). A partial genomic library from a strawberry isolate of V. dahliae (327) was constructed in pUC18 as described for V. alboatrum (Carder & Barbara, 1991). Approximately 250 putative recombinants were produced. Insert sizes, as revealed by electrophoresis in agarose gels following restriction endonuclease digestion of DNA produced by a bOiling mini· prep method, ranged from < 0'5 kbp to > 10 kbp. Clones from this new library are referred to with the prefix pVD, those from the previously constructed library derived from V. alboafrum isolate 1974P with the prefix pVA.
Southern blotting and preparation of probes Southern blots of DNA, digested with either feoR I or Hind III and separated by electrophoresis in agarose gels (usually 7 g 1-1 agarose), were prepared by alkaline capillary blotting (Reed & Mann, 1985) onto Hybond-N (Amersham International PLC). Recombinants were selected at random and the plasmids extracted and labelled by nick translation using 32p dCTP. These procedures and conditions for hybridization, autoradiography and for stripping and reprobing blots were all as described by Carder & Barbara (1991).
Cellulase isoenzyme patterns Selected isolates were cultured in a medium with acetone extracted hop bine tissue as a carbon source (Carder, Hignett & Swinburne, 1987) and cellulase isoenzymes separated by isoelectric focusing in vertical polyacrylamide slab gels. Cellulases were detected using a gel overlay technique with carboxymethyl cellulose as substrate (Carder, 1989).
RESULTS
Grouping of V. dahliae isolates using probes from V. dahliae Southern-blotted DNA, digested separately with the restriction enzymes feaR I and Hind III, from 17 isolates of V. dahliae (Table la; isolates 64A to 333 inclusive) were probed with 7I randomly chosen clones from the partial genomic library from V. dahliae. In some cases digestion with one or other enzyme was judged to be incomplete when the ethidium bromide stained gel was examined (and confirmed by the majority of the hybridization occurring at a position corresponding to the highest molecular weight DNA in the gel) and these results were discarded. Clear results were obtained with 53 probes with both enzymes; a further 13 probes gave results with Hind III digested DNA only and five with feaR I digestion only. As with probes derived from V. alboatrum (Carder & Barbara, 1991) all probes tested gave Simple hybridization patterns of one to three bands, again suggesting a low content of repetitive DNA in Verfieillium species. While 75 % of the probe/enzyme combinations did not reveal polymorphisms, 31 of 124 probe/enzyme combinations did so. Eight probes revealed polymorphisms with both
234 enzymes; five of these eight did so by not hybridizing to DNA from some isolates. Six probes revealed polymorphisms with Hind III only and nine with feaR I only. Fifteen of the 17 isolates tested fell clearly into two groups, referred to as RFLP groups A (10 isolates) and B (five isolates); all isolates aSSigned to each of these two groups reacted identically (Fig. I) and 25 of the 3 I polymorphisms identified discriminated between these groups. All isolates to which five of the probes did not hybridize were in group B. It is not known whether this reflects a large single deletion in the Bisolates relative to the A-isolates or smaller scattered deletions.
Table 1. Original host, geographical source, year of isolation and RFLP group of isolates of V. dahliae Isolate number
Original host
Year of isolation
Geographical origin
RFLP' group
(a) Isolates not used in Carder &. Barbara (I991) 64A 68 318 319 320 321 322 323 324 325 327 325 329 330 331 332 333 1507 1508 1856 1867 1574 1875 1893 1900 1914 1922 1925 1931
Acer Hop Strawberry Strawberry Strawberry Strawberry Strawberry Phlox Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Quince Melon Quince Potato Raspberry Strawberry Hop Chrysanthemum Potato Rose Tomato Raspberry
1957 1957 1955 19&5 1985 1955 1955 1986 1956 1986 1986 19&7 1957 1989 1989 1989 1989 1957 1957 1963 1964 1965 1965 NK 1965 1969 1970 1971 1971
HRI" HRI HRI HRI HRI HRI HRI Kent HRI Kent HRI Kent Kent Norfolk Kent Kent Shropshire HRI Kent NKd HRI HRI Norfolk NK Avon Canada South Africa Dorset Dorset
A A B B B A A A A I' A A A I B A B A A A A A B A A A B I A
W. Midlands Hampshire Scotland NK Kent Kent
I A
(b) Isolates used in Carder & Barbara (I991) 1764 1571 1877 2341 DC59 Omega
Hop Strawberry Strawberry Hop Hop Hop
1975 1965 1966 NK 1985 1985
A' A A B'
• RFLP groups as determined in this paper, see results for explanation. to HRI. experimental farm at Horticulture Research International, East Mailing, Kent, U.K.; other names are U.K. counties or districts except for country names. , Isolates which react inconsistently with the probes which differentiate RFLP groups A and B and are categorized as intermediate. d NK, not known. , Differentiated from other B-group isolates by pVA28 and designated A 2• f Differentiated from other B-group isolates by pVA76 and pVD104 and designated B2 •
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Figs 1 & 2. Autoradiograms of blots of DNA from representative isolates of the A and B groups of V. dahliae probed with clones which (Fig. 1) distinguish (pVD22/EcoR I) and (Fig. 2) do not distinguish (pVD126/EcoR I) the groups. Letters in parentheses indicate RFLP group of isolate (see text for explanation).
The probes appeared to be independent as they differed in size and gave distinct hybridization patterns with A-isolates. The remaining two isolates showed some characteristics of both groups, giving A-type polymorphisms with some probe/ enzyme combinations revealing A/B differences and B-type polymorphisms with others. Isolate 325 showed 24 A-group and one B-group polymorphisms while 330 showed II Agroup and 14 B-group polymorphisms (including all those revealed by the five probes which do not hybridize to DNA from B group isolates). These, and other isolates giving both A and B polymorphisms with different probe/enzyme combinations, are designated intermediate (I) to reflect their having characteristics of both groups. Only the two I-isolates showed polymorphisms not associated with the major A/B differentiation. One probe (which did not distinguish A/B), in conjunction with Hind III digests, differentiated isolate 325 from all other isolates. Similarly five probes (four with fcoR I digests, one with Hind III) each uniquely distinguished isolate 330 from all other isolates. Of the initial set of 17 isolates, 14 were from strawberry and 15 from Kent. To determine whether the division of V. dahliae isolates into two major groups would also occur amongst isolates from a greater range of hosts and a wider geographic area a second set of isolates was examined (Table la; isolates 1807 to 1931 inclusive). In this second group of tests a further selection of probes from the same V. dahliae derived library were tested against the V. dahliae isolates. In order to be able to compare these results with the previous ones, most (16) of the first 17 isolates were incorporated into the blots along with the second set of isolates and hybridized with some of the first set of probes; no inconsistencies were noted between the original results and the repeats. Unfortunately for this second group of isolates, DNA extracted from the majority of them failed to digest with fcoR I and so most additional information was derived from Hind III digests.
Five of 20 newly selected probes, when hybridized to fcoR I digested DNA from previously tested isolates placed them into the same groups as before. With this enzyme, one isolate from the new group was placed unequivocally in each of the two groups. One isolate (1928) was intermediate (one probe/enzyme showed an A-group polymorphism and four B-group polymorphisms). One probe also uniquely distinguished isolate 1928 from all other isolates. The remaining 14 probes revealed no polymorphisms with fcoR I digests. The second set of 20 probes was used successfully with Hind III digests of DNA from 11 new isolates. Only four probes revealed polymorphisms; three distinguished groups A and B amongst the isolates classified in the first experiment. These three, together with previously used probes, placed eight of the second set of isolates unequivocally in group A and two in B (including the two assigned to groups by probing fcoR I digests). Isolate 1928 was again shown to be intermediate (one group-A and two group-B polymorphisms) and the probe which showed this isolate to be unique when used with fcoR I digests again revealed a unique polymorphism. Incomplete data for isolate 1867 suggested that this isolate was also in group A (three probe/enzyme combinations which differentiated A/B groupings put it into group A; seven did not distinguish A/B/1867). The RFLP groups to which the V. dahliae isolates were assigned are given in Table 1. Illustrative blots are shown in Figs 1-4. Soil isolates Thirteen isolates of V. dahliae taken directly from soils (see Materials and Methods) in which a variety of crops had been grown were classified by probing with 10 V. dahliae derived probes, selected because they had shown clear A/B discrimination in previous tests. Nine isolates fell into group A,
236
Sub-specific groupings in V. dahliae
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Figs 3 & 4. Autoradiograms of blots of DNA from two isolates intermediate between V. dahliae groups with probes which reveal unique polymorphisms; (Fig. 3) pVD204/EcoR I; (Fig. 4) pVD39/EcoR I. Letters in parentheses indicate RFLP group of isolate (see text for explanation).
Table 2. Proportions of probe/enzyme combinations differentiating subspecific groups of the two species of Veriicillium studied' Species/ group"
Vd.A
Vd.A V.d.B VaaL V.aa.NL
30'6%' 29'5% 60'7% 53'6%
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11/28· 39'3%
" Only results from experiments using probes chosen at random are included. " Vd.A and B = V. dah/iae RFLP groups A and B; Vaa.L and NL = V a/boairum RFLP groups L and NL; , Number of probe-enzyme combinations differentiating groups/number of probes tested. d Data from current work, upper figures EeaR I digested DNA. lower Hind III. c Data from Carder & Barbara (1991) and all from EeaR I digested DNA. r Proportion of combinations tested which differentiated pairs, source of data as top right.
the other four into B. However, five of the nine A-group isolates came from a single field; the remaining four A-group isolates and all the B-group isolates all came from separate sites. There were no I-isolates and no unique polymorphisms.
Classification of six previously studied isolates of V. dahliae and sub-division of the groups In the light of the above differentiation into clear groups, the patterns of hybridization of DNA from the six isolates of V. dahliae previously studied (Carder &. Barbara, 1991; Table Ib) were re-examined and compared, on the same blot, with known A and B isolates. Probing with six V. dahliae derived probes, all capable of distinguishing A and B groups, and four
V. alboatrum derived probes showed that isolates 1871, 2341 and DC59 (which were identical in the earlier studies when probed only with V. alboatrum derived probes) were not different from group A members as described above. Isolate 1877 was also identical to group A members except when probed with pVA28, which did not hybridize to DNA from this isolate. pVA28 was previously shown (Carder &. Barbara, 1991) to differentiate the V. alboatrum NL group isolates into two sub-groups, here designated NL 1 (pVA28 hybridized to DNA) and NL 2 (pVA28 did not hybridize to DNA). This sub-division of NL was confirmed by probing DNA from a further 23 NL group isolates from hop with pVA28; 10 isolates were classified as NL 1 and 13 as NL 2 . By analogy we now designate isolate 1877 as sub-group A 2 and all other group A members as Ai' pVA28 does not hybridize to any isolate belonging to either V. alboatrum group L (Carder &. Barbara, 1991) or V. dahliae group B. Of the other two V. dahliae isolates previously examined, Omega corresponded most closely to group B, being distinguished only by polymorphism revealed by pVA76. Subsequent tests showed Omega to be unique in being distinguished by this probe from other group B isolates so far tested (data not shown). These later tests also identified a second probe, pVD104, which distinguished Omega, by a polymorphism, from other B group isolates. Unlike pVA76, pVDI04 differentiated groups A and B. We now designate Omega as being in subgroup B2 while all other isolates are B1 . The two sub-groupings, A 2 and B2 , are represented by only single examples and further isolates need to be sought that conform to the same patterns. The remaining isolate tested previously, 1764, was intermediate between the groups (seven group-A polymorphisms and one group-B) using the eight probes from this set of 10 which distinguish A from B. Previously (Carder &. Barbara, 1991), of the 10 V. alboatrum derived probes which distinguished what we now know to be A and B group
C. A. N. Okoli,
J. H. Carder and D. J. Barbara
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Table 3. Correlation of RFLP group (and sub-group) with isoenzyme pattern in V. dahliae RFLP group Isoenzyme group
A l/2
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a
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a
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members, six classified 1764 as A and four as B. In the earlier work (Carder & Barbara, 1991) one probe (pVA35) uniquely differentiated 1764 from the other five isolates and this was confirmed. pVA35 hybridized, without polymorphism, to representative group A and B isolates but again not to isolate 1764. Similarly, pVA28 did not hybridize to 1764.
Interrelationship of groups Based on the proportion of probe/enzyme combinations differentiating the species and groups (Table 2), using the data obtained here and that reported in Carder & Barbara (1991), it was possible to estimate the genetic interrelationship of the four groups. A dendrogram was constructed using the standard genetic distance and an unweighted pair group method of clustering (Nei, 1975) (Fig. 5). This analysis was intended only to measure the interrelationships of the major groups and hence the minority of V. dahliae isolates not falling clearly in those groups (the I-isolates) were excluded from the calculation.
Correlation of groups with other characters As previously reported (Carder & Barbara, 1991) the two major RFLP groups of V. alboafrum, Land NL, were correlated with original host. The subgroups NL I and NL z were not obviously correlated with host as both have been isolated from hop. In V. dahliae there was no apparent correlation between the major RFLP groups (A and B) and original host of the isolates (Table I). Nor were the sub-groups Al and A z host-related as both had been isolated from strawberry. The only B2 isolate yet identified, Omega, is the only B group example as yet identified from hop and it is possible that the B sub-groups may be host-related but more B-group hop
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Fig. 6. Cellulase isoenzyme patterns of V. dahliae isolates. Diagram represents the more acidic portion (pH 3'8 to 4'7) of an isoeledric focusing polyacrylamide gel (overall range pH 3'8 to 6'0). In parentheses are the RFLP group and sub-group to which isolates belong and the cellulase isoenzyme pattern (see text for details). V. alboatrum isolate 1974 was included for comparison.
isolates need to be tested before any such conclusion can be drawn. There was also no obvious geographic association in the V. dahliae groupings. Many of the isolates came from the HRI experimental farm or other local farms but these included representatives of both groups. Only two isolates originated from outside the U.K., one was group-A, the other group-B. In V. dahliae, RFLP group was correlated with cellulase isoenzyme pattern (Table 3). The isozyme pattern displayed by the only B2 isolate available (Omega) appeared to be a variant of the pattern associated with the Bl isolates (Fig. 6). Little correlation between cellulase isoenzyme pattern and RFLP group was seen in V. alboafrum. Ten isolates gave identical patterns, including six representatives of the NL j , two of the NL 2 - and two of the L-groups. Two isolates from lucerne gave a unique pattern. Six of these isolates had not previously been assigned to RFLP groups but were tested here using at least four probes capable of distinguishing the groups and with pVA28 to assign to their sub-group.
Sub-specific groupings in V. dahliae
DISCUSSION Previously, it had been shown that V. alboafrum isolates can be placed into one of two clear sub-specific groupings when variation at the molecular level was examined (Carder & Barbara, 1991). The data presented here similarly show that the isolates of V. dahliae studied may be divided into two groups when studied in this way. The data in the earlier report (Carder & Barbara, 1991) support this division of V. dahliae when re-interpreted in the light of a wider range of isolates and the use of probes from V. dahliae. Other groups may become apparent when further isolates are tested. A few isolates intermediate between the two groups of V. dahliae were seen but no such intermediates between the two V. alboafrum groups have been found either here or elsewhere (Carder & Barbara, 1991, and unpublished data). The relative statuses of the V. dahliae groups and the I-isolates are not certain. It might be argued that if sufficient probe I enzyme combinations were examined all isolates would show intermediate characteristics. Whilst this cannot be disproved it seems unlikely as most of the intermediates were identified when tested with only a few AlB discriminating probes. Also, the I-isolates appeared to be qualitatively different from other isolates by all exhibiting polymorphisms unique within this collection of isolates; these probably represent low frequency allelles not associated with the major AlB differentiation. In this study only three such polymorphisms were associated with isolates of V. dahliae placed in the two groups. Of the probes concerned only pVDI04 has not been shown to give similar polymorphisms in other circumstances. pVA76 distinguished the sole sub-group B2 isolate, Omega, but preliminary results show that this probe, which is probably derived from the ribosomal RNA gene region, also distinguishes amongst diploid isolates of V. dahliae in the same way (Okoli, Tabrett, Morton, Carder & Barbara; unpublished results). Similarly, although pVA28 differentiates the sole subgroup A 2 isolate of V. dahliae it also discriminates V. alboatrum sub-groups NL 1 and NL 2 . V. dahliae isolate 327 fell into group Al but no probe equivalent to pVA28 (Le. identifying subgroups within V. dahliae) was found amongst the probes selected from the library derived from this isolate even though a larger number of these were tested than were from the V. alboatrum derived library which was the source of pVA28. The two sub-divisions of V. dahliae both involve single isolates and clearly other members of these small groupings need to be identified before their significance can be assessed. That ca 90 % of the V. dahliae isolates fell into the two groups suggests that they are at some selective advantage relative to the less frequent I-isolates (at least in the U.K., the origin of most of the isolates used). In view of the relatively limited variation within the groups (compared to that between the groups) it seems likely that members of each group have a clonal origin. Whether the I-isolates have origins separate from the groups or result from some parasexual event (Hastie, 1964; Hastie & Heale, 1984) between isolates from different groups is not known. However, the infrequent occurrence of I-isolates suggests that in practice there is little gene flow between the groups. Consideration of the genetic distance between the major
238 groups of the two species (Fig. 5) generally reiterates the earlier conclusion (Carder & Barbara, 1991) that molecular variation supports the retention of V. alboafrum and V. dahliae as separate species (ca 50-60% of probes distinguish NL/L from A/B whilst ca 30% distinguish A from Band ca 40% L from NL). Several host specific so-called 'physiological races' of V. dahliae, not represented amongst the isolates tested here, have been reported. Those from peppermint (Homer, 1954; Fordyce & Green, 1960) (in these reports the isolates were described as V. alboatrum but are now included in V. dahliae (e.g. Horiuchi, Hagiwara & Takeuchi, 1990)) are presumably haploids (like all the isolates tested here) while some isolates from Brassica spp. are natural, stable diploids (Jackson & Heale, 1985). Representative isolates of these two races have now been obtained and will be compared with the isolates grouped here. In V. alboatrum, host specificity appears to be the basis of the L/NL differentiation. Isolates originally from lucerne (presumably equivalent to our L group isolates) and those from other hosts (our NL group) have been reported to exist as separate vegetative compatibility groups (Correll, Gordon & McCain, 1988); once groups were established by host preference, vegetative incompatibility would help maintain them and perhaps explain the lack of intermediate isolates seen in our studies. The biological significance of the V. dahliae groupings is more obscure. The correlation of cellulase isoenzyme pattern with RFLP group in V. dahliae supports the existence of the distinct A and B groups. Preliminary work at HRI suggests that other biological characters may also be correlated with these groups and that, as with the V. alboatrum groups, the two groups of V. dahliae form separate compatibility groups (D. C Harris & Jiarong Yang, pers comm.). In V. dahliae there was no clear correlation of RFLP group with original host species and in earlier studies no correlation of cellulase groups with pathogenicity was found (Carder, Hignett & Swinburne, 1987; Carder, 1989). However, further preliminary results at HRI showed that among the 13 soil-derived isolates the average pathogenicity for strawberry was different for the two RFLP groups (D. C Harris & Jiarong Yang, pers. comm.). Also, B group isolates are relatively more common amongst strawberry (9:6/A:B) (Table 1) and soil isolates (9:4/A:B) than amongst those from other hosts (14: 2/A: B) (Table 1). These results suggest that the groups are biologically significant in relation to their degree of pathogenicity and are possibly differentiated by varying levels of host susceptibility. In neither species was any obvious biological basis for the subgroupings seen. We are grateful to the Association of Commonwealth Universities for supporting CAN.O. and to Dr S. A Archer, University of London, for his continued interest in this project.
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Carder. 1. H. & Barbara, D. j. (1991). Molecular variation and restriction fragment length polymorphisms (RFLPs) within and between six species of Verticil/ium. Mycological Research 95, 935-942. Carder, 1. H. Hignett, R. C. & Swinburne, T. R. (1987). Relationship between the virulence of hop isolates of Ver/icil/ium albo-a/rum and their in vitro secretion of cell wall degrading enzymes. Physiological and Molecular Plant Pathology 31. 441-452. Correll, 1. c.. Gordon, T. R. & McCain, A H. (1988). Vegetative compatibility and pathogenicity of Verticillium alboalrnm. Phytopathology 78, 1017-1021. Fordyce, C. & Green, R. j. (1960). Studies of the host specificity of Verticil/ium albo-atrum var. menthae. Phytopathology 50, 635. Hastie, A C. (1964). The parasexual cycle in Verticillium albo-atrum. Genetical Research 5, 305-315. Hastie, A C. & Heale, j. B. (1984). Genetics of Verticil/ium. Phytopathology Mediterraneae 23, 130-162. Horiuchi,S., Hagiwara, H. & Takeuchi, S. (1990). Host specificity of isolates of Verticil/ium dahliae towards cruciferous and solanaceous plants. In Biological Control of Soil-borne Plant Pathogens (ed. D. Hornby), pp. 285-298. C.AB. International: Wallingford. U.K. Horner, C. E. (1954). Pathogenicity of Verticillium isolates to peppermint. Phytopathology 44, 239-242.
(Accepted 21 July 1992)
239 jackson, C. W. & Heale,}. B. (1985). Relationship between DNA content and spore volume in sixteen isolates of Ver/icil/ium leeanii and two new diploids of V. dahliae (= V. dahliae var. longisporum Stark). Journal of General Microbiology 131, 3229-3236. Manicom, B. Q.. Bar-joseph, M., Rosner, A, Vigodsky-Haas, H. & Kotze, j. M. (1987). Potential applications of random DNA length polymorphisms in the taxonomy of the fusaria. Phytopathology 77, 669-.Q72. Michelmore. R. W. & Hulbert. S. H. (1987). Molecular markers for genetic analysis of phytopathogenic fungi. Annual Review of Phytopathology 25, 383-404. Murray, M. G. & Thompson. W. F. (1980). Rapid isolation of high molecular weight plant DNA Nucleic Acids Research 8, 4321-4325. Nei, M. (1975). Molecular population genetics and evolution. In Frontiers of Biology 40 (ed. A Neuberger & E. L. Tatum). North-Holland Publishing Co.: Amsterdam. Reed, K. C. & Mann, D. A (1985). Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Research 13, 7207-7221. Sambrook, ).. Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning, 2nd edn, Cold Spring Harbor Laboratory Press: New York. Talboys, P. W. (1960). A culture medium aiding identification of Verticil/ium alboatmm and Verticil/ium dahliae. Pla"t Pathology 9, 57-58.