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Hybridization of verticillium albo-atrum and Verticillium dahliae
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Trans. Br, mycol. Soc. 60 (3), 5II-523 (1973) Printed in Great Britain
HYBRIDIZATION OF VERTIGILLIUM ALBO-ATRUM AND VERTIGILLIUM DAHLIAE By A. C. HASTIE Department oj Biological Sciences, The University, Dundee (With Plate 47) Four monoconidial haploid wild-type Verticillium isolates obtained from different geographical locations were studied. Two of these had dark mycelium (Verticillium albo-atrum Reinke & Berth.), and two had microsclerotia (Verticillium dahliae Kleb). Auxotrophic mutants were obtained from each isolate, and complementary auxotrophs were combined in attempts to synthesize heterozygous diploids. No diploids were recovered from combinations of auxotrophs derived from the two V. dahliaestrains, but frequent diploidization occurred in combinations of auxotrophs derived from the two V. albo-atrum strains and also in combinations of auxotrophs derived from the V. dahliae and V. albo-atrum strains. The intraspecific diploids (V. albo-atrumx V. albo-atrum) showed frequent haploidization and unrestricted genetic recombination. Interspecific diploids showed infrequent haploidization and restricted genetic recombination. The frequency ofhaploidization in these was apparently increased by growing them on medium containing the fungicide benlate, but these induced haploids also had a limited range of genotypes. The restricted genetic recombination in the interspecific diploids is interpreted as resulting from non-homology between the V. albo-atrum and V. dahliae genomes, and the results are discussed in relation to the taxonomic status of dark mycelial and microsclerotial Verticillium strains.
Most Verticillium strains causing wilt diseases produce either dark resting mycelium or microsclerotia, and the taxonomic status of these morphological types has been the subject of much controversy. The species V. alboatrum has priority (Reinke & Berthold, 1879). The original description has been interpreted by some authors to include both dark resting mycelium and microsclerotia, but Klebahn (1913) obviously excluded microsclerotia from that description and assigned specific rank to a purely microsclerotial Verticillium from dahlia (V. dahliae Kleb.). More recently many authors have maintained this specific ranking for microsclerotial isolates from many different host species (Isaac, 1949; Robinson, Larson & Walker, 1957; Smith, 1965), but others consider that dark mycelial and microsclerotial strains are components ofone polymorphic species (Presley, 1941; Fordyce & Green, 1964). Isaac (1967) has reviewed this controversy and concluded that' both V. albo-atrum and V. dahliae should be accorded specific rank since lumping them together has caused (and continues to cause) needless confusion and inconvenience to plant pathologists'. Specific status is assumed for these two forms in this paper, and the justification for this assumption will be found in the results presented.
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Transactions British Mycological Society Table
I.
The relationships of auxotrophic strains
Wild types
Origin
Species
VP2
England
V. albo-atrum
VI4
England
V. albo-atrum
VI6
England
V. dahliae
VI7
Canada
V. dahliae
Mutant strains
Auxotrophic requirements*
Inositol, p-amino benzoic acid 108 Inositol, nicotinic acid, pyridoxine 49 Lysine 176 Choline, methionine 152 Nicotinamide, methionine 16/I/4 Lysine, tryptophane 16/8/6 Adenine, aneurin 17/8/ 6 17/3/ 14 Arginine, histidine
* Some of these auxotrophic requirements are abbreviated as follows in later parts of the text: inos = inositol; meth = methionine; nic = nicotinic acid; paba = p-aminobenzoic acid.
An attempt is made to resolve these conflicting views by the application of genetical techniques. The concept of a species is that of a group of organisms which can share a common pool of genes through hybridization. If two gene pools are isolated from one another they may diverge by mutation and selection and ultimately become both qualitatively and quantitatively distinct. The genomes of the two isolated populations may then be non-homologous. The significant question in the present context is whether non-homology of the V. albo-atrum and V. dahliae genomes can be demonstrated. Sexual reproduction has never been recorded in either V. albo-atrum or V. dahliae, but parasexual genetic recombination has been extensively studied in V. albo-atrum (Hastie, 1962, 1964, 1968). It is also known that the same system operates when V. dahliae strains are hybridized (A. C. Hastie unpublished). Wild strains of both species are generally haploid, but heterozygous diploid strains can be synthesized and these readily form new haploid and diploid genotypes. This investigation is an attempt to determine whether V. albo-atrum and V. dahliae haploids can be used to synthesize hybrid diploids, and whether these interspecific diploids segregate and recombine as frequently as intraspecific diploids (Hastie, 1964). MATERIALS AND METHODS
Strains
The wild-types used are all potato pathogens supplied by Professor I. Isaac, University College of Wales, Swansea. Each wild-type strain was purified by subculturing from a single uninucleate conidium. The mutant strains derived from each wild-type are therefore near isogenic (Table I). The auxotrophic mutants derived from VP2 were obtained by ultraviolet irradiation. Those from the other wild types were obtained after treatments with N-methyl-N-nitroso-N'-nitroguanidine. The ease with which these recessive mutants were detected indicates that all the wildtypes were haploid (Buxton & Hastie, 1962).
Verticillium hybrids. A. C. Hastie Culture conditions Stock cultures of all strains were maintained on the modified prune extract agar medium (PE) described by Talboys (1960). Czapek-Dox agar medium (MM) was used as a minimal medium and supplemented as required for the classification of auxotrophic segregants. A glucose minimal medium (G M M ) containing 1'5 % (w/ v) glucose, and the salts in MM, was used for the synthesis and selection of diploid strains. This medium (GMM) was found by M. Ingle & A. C. Hastie (unpublished) to give a better yield of diploids than sucrose based media. All incubation was at 25 "C . Synthesis and selection of diploids The technique used to synthesize diploids has been described in detail elsewhere (Hastie, 1973). It is a modification of that used previously (Hastie, 1962), and employs the principle originally used by Roper (1952). The parent haploid strains must have complementary auxotrophic nutritional requirements. Conidial suspensions of the parents (I06/ml) were prepared in supplemented GMM, and equal volumes of these suspensions were mixed. Droplets of th e mixed suspensions wer e suspended from th e lids of Petri dishes in a humid atmosphere and incubated for about 30 h. During incubation the conidia in the hanging-drops sedimented and germinated, and each hanging-drop ultimately yielded a pellet of mycelium. The pellets were removed, washed in sterile distilled water, and transferred to solid GMM on which they were then incubated for 4 weeks. The pellets initially grew only very slowly on this medium, but many later formed prototrophic sectors with relatively large conidia which were subsequently proved to be diploid. Diploids selected in this way were subcultured either using their prototrophic conidia or by mass hyphal tip transfers. The fertility ofindividual crosses is expressed as the proportion of pellets which formed diploid sectors. Distinction between haploids, diploids and aneuploids Ne arly all the conidia of Verticillium are uninucleate (H astie, 1962; MacGarvie & Isaac, 1966), and those which are binucleate are apparently homokaryotic (Hastie, 1962). Diploid cultures were progeny-tested by taking random samples of conidia from them, and classifying these conidia according to whether they contained a haploid, diploid or aneuploid nucleus. The criteria applied to distinguish between these types were primarily growth rate of the conidium, and the dimensions of the conidia on the culture it produced (Hastie, 1964, 1968). Haploid and diploid conidia both formed mycelia with normal and similar growth rates: Conidium containing a diploid nucleus was however relatively large and formed a mycelium which also formed large conidia. Conidia described as aneuploid formed a mycelium which initially grew very slowly, but later formed faster growing sectors. Each sector had the characteristics of either a haploid or a diploid.
Transactions British Mycological Society Table 2. N umbers of diploid sectors formed by crosses offirst block No. of pellets giving di ploids
P arents
108 x 16/1/4 108 x 17/8/6 176 x 16/1/4 176 x 17/8/6 176 x 108 16/1/4 x 17/8/6
32 60 4 0
18 0
114
Total no. pellets tested go go 87 87 go 75 5 19
RESULTS
Synthesis
of diploids
Crosses were made in blocks of six, and about ninety pellets were tested for each cross. Each block of crosses represents all possible combinations of four complementary auxotrophs; one auxotroph being derived from each of the wild-types listed in Table I. The yield of diploids was scored as the number of pellets which formed diploid sectors. Heterokaryons of Verticillium are difficult to detect and recover (Hastie, Ig62 ), but when nuclear fusion occurs in them the diploid mycelium grows vigorously and soon overgrows the heterokaryon. The yields of diploids recorded are therefore more closely related to the frequency of diploidization than to the frequency with which heterokaryons are formed. Three blocks of crosses were examined, and the yields of diploids are recorded in Tables 2-4. The first diploids appeared after about I week's incubation, and the final yields were scored after 4 weeks . There was obviously great heterogeneity between the final yields of diploids from the individual crosses, and especially from those in the first block (Table 2). This heterogeneity does not arise from a failure of V. albo-atrum and V. dahliae to form hybrid diploids. Indeed when the data in Table 2 are summed over appropriate crosses it appears the V. albo-atrum diploidized V. dahliae (g6/354 = 27 %),just as readily as two V. albo-atrum strains formed diploids (18/go = 20 %). Much of the heterogeneity in Table 2 is apparently due to the influence ofstrain ro8. It was the only strain in that block to hybridize each of the other three strains, and each of the other strains gave its maximum yield of diploids when crossed to strain ro8. Only two crosses are repeated in the three blocks of crosses. These are ro8 x 17/8 /6 (T ables 2 and 4), and 152 x 16/8 /6 (T ables 3 and 4). These repeated attempts of th e same cross give consistent results, but this consistency is not always matched wh en different auxotrophs derived from the same wild-type culture (e.g. 176 and 152) are each crossed to a third strain (e.g. ro8 ). The yields of diploids from these two crosses are shown in Tables 2 and 4, and on comparison by a contingency X2 test show a significant deviation (P < 0'01) . Similarly, strain 176 yielded no diploids when crossed to 17/8/6 (Table 2), but 152 x 17/8/6 gave 16/86 diploids
Verticillium hybrids. A. C. Hastie Table 3. Numbers of diploid sectors formed by crosses of second block
Parents 4gx 16/8/6 4gx 17/3/ 14 152 x 16/8/6 152 X I7/3/ 14 152 x49 16/8/6 XI 7/3/14
No. of pellets giving diploids
Total no. pellets tested
14 10 8 0 10 0 42
83 86 88 go go 88 5 25
Table 4. Numbers of diploid sectors formed by crosses of third block
Parents 108 X 16/8/6 108 XI 7/8/6 152 x 16/8/6 152 x 17/8/6 152 x 108 16/8/6 x 17/8/6
No. of pellets giving diploids
Total no. pellets tested
13 55 12 16 5
go go go 84 go go
°
101
534
(Table 4). It is concluded that there can be significant variations in the fertilities of auxotrophs derived from the same monoconidial wild-type culture, and that this intra-strain variation of fertility may be due to pleiotropic effects of the auxotrophic mutations.
Morphology of interspecific hybrids Subcultures of all the interspecific hybrids listed in Tables 2 and 3 were made both by taking mass hyphal-tip transfers from the growing edge of the original diploid sectors, and also by making monoconidial cultures. Both types of subculture were grown on PE. All hybrid diploids grew at about the same linear rate as intra-specific diploids and haploid strains. Hybrid diploids differed from other strains in two principal morphological features; conidial production and the morphology of dark resting structures. They generally formed few conidiophores with verticillate branching, and most conidia were borne in slimy masses resemblingpoinnotes. The individual conidia of hybrid diploids were uninucleate and about the same length as those from intra-specific diploids (Hastie, 1964). Hybrid diploids grown on PE formed less black mycelium than other strains, and their black structures were unlike those of either V. alboatrum or V. dahliae (PI. 47). The black torulose hyphae of V. albo-atrum do not proliferate laterally and the blackening is often continuous along a hypha. The microsclerotia of V. dahliae develop from single hyphae by cell divisions in all planes (Isaac, 1949), and blackening is restricted to themicrosclerotia.
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Table 5. Classification of random conidia from 3 week old diploid cultures derivedfrom crosses in the first block (Table 2) , Cross
108x 16/1/4 108 x 17/8/6 176 x 16/1/4 I76x 108
Types of conidia
,
,
Haploid
Diploid
Aneuploid
Total
0 0 0 237
113 84 123
27 16 28 22
140 100 15 1 270
II
The black structures on hybrid diploids were intermediate in that they showed very limited lateral proliferation, and generally the blackened regions of the mycelium were discrete. Random conidial analysis
Diploid sectors from all fertile crosses of the first block (Table 2) were subcultured on PE by mass hyphal tip transfers. Three independent diploid sectors of each cross were subcultured in this way, and four replicate cultures were made from each of these diploid sectors. Conidial suspensions were prepared from each of these cultures after 3 weeks growth, and incubated on PE plates. It was assumed that each colony had grown from a single conidium, and on inspection of each colony it was decided whether the original conidium had contained a diploid, haploid or aneuploid nucleus by applying the criteria given earlier. The results given in Table 5 are the averages of the progenies from the replicate cultures. The results from cross 176 x roS are obviously different from those of the other three crosses. The diploids from this cross readily yielded conidia containing haploid nuclei as has previously been reported for crosses between strains of V. albo-atrum (Hastie, 1964, 1970, 1973). Crosses between strains of V. dahliae gave diploids which equally readily form haploid segregants (A. C. Hastie, unpublished), and it is therefore concluded that an absence of haploid conidia from samples taken under the above conditions is peculiar to V. albo-atrum x V. dahliae crosses. This conclusion was supported by similar observations made on progenies from the crosses in blocks 2 and 3. The euploid (both haploid and diploid) isolates recorded in Table 5, together with further samples of conidial progeny from these crosses, were replicated on appropriately supplemented media to determine their nutritional requirements. More than rooo random monoconidial isolates from the V. albo-atrum x V. dahliae hybrids were examined, and only one was auxotrophic. It was from cross roS x 17/S/6 and required inositol andp-aminobenzoic acid; the nutritional requirements ofstrain roS. Also, only one of these isolates was haploid. Both the haploid and diploid isolates from cross 176 x roS, the intraspecific cross, showed segregation and recombination for all genetic markers as expected from earlier investigations (Hastie, 1964) and unpublished work with crosses between strains of V. dahliae. The rare occurrence of diploid auxotrophic segregants from
Verticillium hybrids. A. C. Hastie Table 6. Phenotypes of random monoconidial progeny from sectors on colonies grownfrom selected aneuploid conidia
Parental diploid
w8x 16/1/4
No. of aneuploids with diploid sectors
No. of aneuploids with haploid sectors
Phenotypes of random progeny. from haploid sectors
o
12 12
o
II
I
II
I
12
o
II
I
II
I
II II
I
12 12
o o
10
2
26 haploid DM inos paba (not sampled) 26 haploid MS nic 7 haploid DM inos paba, { 19 diploid H prototrophs 26 haploid DM inos paba (not sampled) 26 haploid MS prototrophs 14 haploid MS nic meth, { 12 diploid H prototrophsf
DM = dark mycelial; MS = microsc1erotial; H = hyaline. * Twenty-six conidia were taken randomly from each haploid sector. t These 26 progeny had much smaller microsc1erotia than all other microsc1erotial strains.
crosses of V. albo-atrum x V. dahliae indicates that mitotic recombination is much less frequent in them than in intraspecific diploids (Hastie, 1968).
Segregation in selected spontaneous aneuploids The very rare recovery of haploid segregants in random samples of conidia from interspecific diploid cultures prompted a search for a selective device which would faciliate the recovery of spontaneous haploid segregants from these strains. It is known that haploid nuclei are formed from diploid nuclei through intermediate aneuploid stages in parasexual systems. An attempt was therefore made to find aneuploid coni dia from interspecific diploids which would grow to form colonies with haploid sectors. Random samples of conidia from eight interspecific diploid cultures of cross I76 x 16/ I /4 and eight diploids of cross I76 x 16/ I /4 were spread on PE. Slow-growing colonies which were assumed to be aneuploids were selected after 2 days incubation. Inocula of the selected slow growing colonies (aneuploids) were transferred to further dishes of PE and incubated for up to 5 weeks. All these aneuploids formed fast-growing sectors, and conidia from these sectors were examined to decide whether they were haploid or diploid. The results are given in Table 6. Most aneuploids formed sectors with large diploid conidia and the mycelium had the morphology of an interspecific diploid. Other aneuploids formed sectors with small conidia, and these sectors generally had the mycelial characters of either V. albo-atrum or V. dahliae. Most haploid sectors were progeny tested, and it was shown that conidia from most of them had parental genotypes. One haploid sector was exceptional in that it was prototrophic, and also formed very small microsclerotia.
Transactions British Mycological Society Table 7. Phenotypes of random monoconidial progeny taken from cultures of the interspecific diploid 108 x 16/ I /4 grown in presence and absence if benlate Progeny from benlate containing medium Parental diploid 16/1 16/IA 16/2 16/2A
16/3
16/4
,
Haploids 1 prototroph 23 nic meth 2 prototrophs 1 nic meth 24 inospaba 43 inospaba 1 inospaba nic 3 nic meth 1 methpaba 42 nic meth 143
Progeny from benlate free medium
Diploids
Haploids
51 prototrophs 52 prototrophs 28 prototrophs 24 prototrophs 1 inos
1 prototroph
1 prototroph 2 nic meth 8 prototrophs 16 7
Diploids 51 52 52 52
---.
prototrophs prototrophs prototrophs prototrophs
52 prototrophs
52 prototrophs 3 11
These results show that haploid segregants can be recovered from some aneuploids. Presumably these are aneuploids with near haploid genomes, whereas the aneuploids which formed diploid sectors had near diploid genomes. Effectofbenlate ongrowthandsegregation ofhybrid diploids Benlate is known to cause haploidization in diploid strains of Aspergillus nidulans when incorporated in the culture medium (Hastie, 1970a). Attempts to demonstrate benlate-induced haploidization in diploid strains of V. albo-atrum have been unsuccessful apparently because the frequency of spontaneous haploidization is already high in these diploids (A. C. Hastie, unpublished). The effect of benlate on the growth and segregation of hybrid diploids was investigated after inoculating small pieces of hybrid cultures (about I mms) on PE containing various concentrations of the fungicide. A concentration of 0·5 mg/l benlate in PE greatly restricted growth of the hybrid mycelium, but after 3 weeks incubation on this medium some growth, sectoring and conidiation occurred. Random samples of about 50 conidia were taken from six cultures of the hybrid diploid 108 x 16/1/4 which had been grown on PE for 3 weeks, and also from six parallel cultures which had been grown on PE containing 0·5 mgjl benlate. Cultures from these conidia were examined to decide their ploidy and nutritional requirements. The results are given in Table 7. Nearly half the conidia from cultures grown on PE containing benlate were haploid, but in the absence of benlate only one out of 3 I 2 conidia was haploid. These are the proportions of haploids recovered, and they may not show the true frequencies with which haploids are formed in the presence and absence of benlate. Haploids with the same genotypes recovered from the same culture may be members of a clone arising from one haploidization event. The minimum number of haploidization events detected in the six cultures grown on benlate was ten, and only one
Verticillium hybrids. A. C. Hastie haploidization was detected in the six cultures grown without benlate. This indicates that haploidization is at least ten times more frequent on medium containing benlate. This estimate is of course very approximate. Besides the possible errors arising from clonal multiplication after haploidization, it takes no account of differences in growth rate in the presence and absence of benlate. The recovery of some prototrophic and triauxotrophic haploids shows that some genetic recombination occurred between the V. albo-atrum and V. dahliae genomes, but most of the haploids obtained had parental requirements and morphologies (Table 7). Most haploids either required nicotinic acid and methionine, and formed microsclerotia, or they required inositol and p-aminobenzoic acid, and formed dark mycelia. Although the inositol and p-aminobenozic acid mutants were only regularly recovered in parental combinations of this diploid, and also from other hybrid diploids, these same genes recombined freely in the diploid 176 x 108. The latter is of course derived from two strains of V. albo-atrum. The implications of these variations ofrecombination frequencies in hybrid and selfed diploids will be discussed. DISCUSSION
Heterokaryotic mycelia of Verticillium are very unstable (Hastie, 1962), and if a heterozygous diploid nucleus is formed by nuclear fusion then the heterokaryon is soon overgrown by the diploid mycelium. No attempt was made in this work to estimate the frequencies of hyphal anastomoses and heterokaryon formation. The technique detected only those heterokaryons which formed heterozygous diploids. The formation of these diploids does of course imply the occurrence of hyphal anastomosis, followed by some nuclear migration, and the establishment of at least one heterokaryotic cell. Nuclear fusion (diploidization) was detected by the emergence of a sector ofprototrophic mycelium from an inoculum ofmixed complementary auxotrophs. The diploid nature of these prototrophic sectors was confirmed by examining for relatively large uninucleate prototrophic conidia, and usually by progeny-testing. The results presented indicate that the frequency with which diploids are formed on particular crosses depends upon intrinsic properties of the haploid parent strains involved. Two haploid auxotrophs derived from the same mono-conidial wild-type culture may show significantly different fertilities when each is crossed to a third strain (e.g. 152 or 176 x ro8). This difference may be due to either pleiotropic effects ofthe auxotrophic markers or to differences resulting from the mutagen treatments. These alternatives are now being resolved by a backcross programme. Auxotrophic strains vary in the amounts ofgrowth they make on minimal media, but the effect referred to above is not simply related to the variation in the amount of growth made by the auxotrophs on the selective medium. Diploidization readily occurs between V. albo-atrum auxotrophs derived from the same wild-type strain (Hastie, 1964), and similarly it is shown here and elsewhere (Hastie, 1962) that there is no restriction preventing hyphal anastomsis and diploidization between different wild-type strains of V. albo-atrum. Auxotrophs of V. albo-atrum and V. dahliae equally readily
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formed diploids, and it is assumed that the failure to recover diploids from V I 4 x V I 7 in the first two blocks of crosses was due to the particular auxotrophs involved (Tables 2 and 3). This assumption seems justified by the successful diploidization OfV14 and V17 auxotrophs in the third block of crosses. Relatively few crosses of V. dahliae have been attempted, and the only fertile crosses have involved auxotrophs derived from the same wildtype strain (intra-strain crosses) (A. C. Hastie, unpublished). The failure to recover diploids from the interstrain crosses of V. dahliae (V I 6 x V I 7) requires further investigation. It may be due to properties of the specific auxotrophs used, or to the existence of incompatibility between V. dahliae strains from different countries. Incompatibility could affect hyphal anastomosis, nuclear migration or nuclear fusion. The existence of heterokaryon incompatibility is of course well known in other fungi (Caten & Jinks, 1966). The similarity of the frequencies with which hybrid diploids (V. albaatrum x V. dahliae) and selfed diploids (V. albo-atrum selfed) are recovered is markedly different from the behaviour of hybrid and selfed diploids when subcultured and progeny-tested (Table 5). Both form conidia containing aneuploid nuclei in similar proportions, and this indicates that aneuploids are formed with the same frequency in both. However, hybrid diploids form spontaneous haploid segregants very rarely. It has been inferred that spontaneous haploidization of V. albo-atrum diploids occurs through intermediate aneuploid stages (Hastie, 1973), as in Aspergillus nidulans (Pontecorvo, 1959). Because aneuploid nuclei are readily formed from hybrid diploid nuclei, it follows that the processes required for spontaneous haploidization occur frequently. It is therefore necessary to formulate some hypothesis which will explain why spontaneous haploidization is rarely realized in these diploids. This hypothesis should also explain why most haploids recovered from hybrid diploids are parental. One hypothesis is that the haploid genomes of V. albo-atrum and V. dahliae are not homologous and differ in both the arrangement and the structure of their genes. Random loss of chromosomes from the corresponding hybrid diploid may only rarely yield balanced and functional haploid nuclei. The haploid chromosome number of V. albo-atrum is probably four (Hastie, 1967; Heale, Gafoor & Rajasingham, 1968). V. albo-atrum diploids would then have four pairs of homologous chromosomes. A haploid segregant derived from such a diploid would contain one set of homologous chromosomes. If we ignore mitotic crossing-over, the number of different haploid genomes which can be derived is 2 4 = 16. These haploid genomes will occur with equal frequency only if the individual chromosomes are lost at random during haploid formation. There would be no restriction on the recombination of genes on non-homologous chromosomes. There would similarly be no restriction on the formation of haploid nuclei and recombination in V. dahliae diploids. However, if the haploid genomes of V. albo-atrum and V. dahliae are not homologous, then hybrid diploids would not necessarily contain pairs of homologous chromosomes. This would impose restrictions on the number of different types of haploids which could be derived from them. The maximum restriction would be that the haploid segregants were only viable if they contained either the V. alba-
Verticillium hybrids. A. C. Hastie
52 1
atrum genome or the V. dahliae genome. In these circumstances there would
be no genetic recombination between the genes introduced into the diploid from the V. albo-atrum and V. dahliae parents. In fact the haploids recovered from such diploids are predominantly parental both regarding morphology and the auxotrophic markers (Tables 6 and 7). The free recombination of the inositol and p-aminobenzoic acid genes (inos-4 paba-7) in V. albo-atrum diploids (108 x 176 and 108 x 152) together with their linked segregation in the hybrid diploids shows that there is restricted recombination between the V. albo-atrum and V. dahliae genomes, although some recombination did occur (Tables 6 and 7). This restricted recombination in diploids synthesized from V. albo-atrum and V. dahliae, together with their rare spontaneous production of haploids, is interpreted as an indication of non-homology between the V. albo-atrum and V. dahliae genomes and justifies maintaining them as separate species. This may seem illogical as hybrid diploids can be synthesized with ease under the highly selective conditions used (Tables 2-4). I find the limited genetic recombination in the hybrid diploids a useful basis on which to erect and maintain the species limits. Given certain specific conditions even man-mouse hybrid cells can be made (Harris & Watkins, 1965). Caution is of course required in drawing conclusions about species limits from the results of a few laboratory crosses. Nevertheless, the results clearly show that the microsclerotial and dark mycelial strains show very limited genetic recombination, unlike selfed diploids, and it seems unwise to follow Fordyce & Green (1964) and rank them as one species. Fordyce & Green (1964) detected genetic recombination between auxotrophic mutants derived from dark mycelial and 'pseudosclerotial' strains of Verticillium. The' pseudosclerotial ' strains were considered identical to V. dahliae Kleb., and they therefore interpreted their results to indicate that both dark mycelial and 'pseudosclerotial' strains should be included in the species V. albo-atrum. However, they reported only the results of crosses between dark mycelial and 'pseudosclerotial' strains, and they required selective techniques to recover segregants and recombinant progeny. I believe V. albo-atrum and V. dahliae should be ranked as distinct species partly because selective techniques are necessary to recover interspecific recombinants. The relationship proposed for V. albo-atrum and V. dahliae is analogous to that known to exist within many genera of flowering plants (Stebbins, 1971). Interspecific hybrids can be formed but they are relatively sterile because of chromosomal non-homology. Extension of this analogy indicates a crucial test for the suggested non-homology in the Verticillium genomes. Sterile interspecific hybrids of flowering plants can be made fertile because, as a result of doubling the chromosome complement, they have two homologous sets of chromosomes. By analogy one may predict that doubling the chromosome complements of V. albo-atrum x V. dahliae hybrids would give allotetraploids. These would readily haploidize but continue to show only limited recombination between the V. albo-atrum and V. dahliae genomes. Attempts to create Verticillium allotetraploids by colchicine treatments have failed.
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I thank Mrs 1. Blair for her excellent technical assistance, and I acknowledge the support of the Science Research Council (B/SR/3474). REFERENCES
BUXTON, E. W. & HAsTIE, A. C. (1962). Spontaneous and utra-violet irradiationinduced mutants of Verticillium albo-atrum. Journal of General Microbiology 28, 62563 2. CATEN, C. E. & JINKS, J. L. (1966). Heterokaryosis: its significance in wild homothallic ascomycetes and fungi imperfecti. Transactions of the British Mycological Society 49, 81-93· FORDYCE, C. & GREEN, R • .J. (1964). Mechanisms of variation in Verticillium albo-atrum. Phytopathology 54, 795-798. HARRIS, H. & WATKINS,J. F. (1965). Hybrid cells derived from mouse and man: artificial heterokaryons of mammalian cells from different species. Nature, London 205, 640-646. HASTIE, A. C. (1962). Genetic recombination in the hop-wilt fungus Verticillium alboatrum. Journal of General Microbiology 27, 373-382. HASTIE, A. C. (1964). The parasexual cycle in Verticillium albo-atrum, Genetical Research 5, 3 05-3 15. HAsTIE, A. C. (1967). Mitotic recombination in conidiophores of Verticillium albo-atrum. Nature, London 214, 249-252. HAsTIE, A. C. (1968). Phialide analysis of mitotic recombination in Verticillium. Molecular and General Genetics 102, 232-240. HASTIE, A. C. (1970). The genetics of asexual phytopathogenic fungi with special reference to Verticillium. Root diseases and soil-borne pathogens (ed. T. A. Toussoun, R. V. Bega and P. E. Nelson). Berkeley: University of California Press. HASTIE, A. C. (1970a). Benlate-induced instability of Aspergillus diploids. Nature, London 226, 771. HASTIE, A. C. (1973). Genetic analysis in Verticillium. Pathological wilting of plants (ed. J. S. Sadasivan, C. V. Subramanian, R. Kalyanasundaram and L. SaraswathiDevi), University of Madras. HEALE, J. B., GAFOOR, A. & RAJASINGHAM, K. C. (1968). Nuclear division in conidia and hyphae of Verticillium albo-atrum. Canadian Journal of Genetics and Cytology 10, 3 2 1-340. ISAAC, I. (1949). A comparative study of pathogenic isolates of Verticillium. Transactions of the British Mycological Society 32, 137-157. ISAAC, I. (1967). Speciation in Verticillium. Annual Review of Phytopathology 5,201-221. KLEBAHN, H. (1913). Beitrage zur kenntnis der fungi imperfecti I. Eine VerticilliumKrankheit auf dahlien. Mykologisches Zentralblatt 3, 49-66. MACGARVIE, Q.D. & ISAAC, I. (1966). Structure and behaviour of the nuclei of Verticillium spp. Transactions of the British Mycological Society 49, 687-693. PONTECORVO, G. (1959). Trends in genetic analysis. London: Oxford University Press. PRESLEY, J. T. (1941). Saltants from a monosporic culture of Verticillium albo-atrum. Phytopathology 31, 1135-1139. REINKE, J. & BERTHOLD, G. (1879). Die zeresetzung der kartoffel durch pilse. Untersuchungen aus dem Botanishen Laboratorium der Universitiit Gottingen I, 1-100. ROBINSON, D. B., LARSON, R. H. & WALKER, J. C. (1957). Verticillium wilt of potato in relation to symptoms, epidemiology and variably of the pathogen. Wisconsin Agricultural Experiment Station Bulletin no. 202, 1-49. ROPER, J. A. (1952). Production of heterozygous diploids in filamentous fungi. Experimentia 8, 14-15. SMITH, H. C. (1965). The morphology of Verticillium albo-atrum, V. dahliae and V. tricorpus. New Zealand Journal of Agricultural Research 8, 450-478. STEBBINS, G. L. (1971). Chromosomal evolution in higher plants. London: Edward Arnold Ltd. TALBOYS, P. W. (1960). A culture medium aiding the identification of Verticillium alboatrum and V. dahliae. Plant Pathology 9, 57-58.
Trans. Br, mycol. Soc.
Vol. 60. Plate 47
,
2
(Facing p. 523)
Verticillium hybrids. A. C. Hastie EXPLANATION OF PLATE
47
All figures , x 400. Fig. I. Dark mycelium of V. albo-atrum, Fig. 2. Microsc1erotia of V. dahliae. Fig. 3. Dark structures of interspecific hyb rids. (V. albo-atrum x V. dahliae).
(Accepted for publication
34
20
October 1972)
MYC 60
]
Trans. Br, mycol. Soc. 60 (3), 5II-523 (1973) Printed in Great Britain
HYBRIDIZATION OF VERTIGILLIUM ALBO-ATRUM AND VERTIGILLIUM DAHLIAE By A. C. HASTIE Department oj Biological Sciences, The University, Dundee (With Plate 47) Four monoconidial haploid wild-type Verticillium isolates obtained from different geographical locations were studied. Two of these had dark mycelium (Verticillium albo-atrum Reinke & Berth.), and two had microsclerotia (Verticillium dahliae Kleb). Auxotrophic mutants were obtained from each isolate, and complementary auxotrophs were combined in attempts to synthesize heterozygous diploids. No diploids were recovered from combinations of auxotrophs derived from the two V. dahliaestrains, but frequent diploidization occurred in combinations of auxotrophs derived from the two V. albo-atrum strains and also in combinations of auxotrophs derived from the V. dahliae and V. albo-atrum strains. The intraspecific diploids (V. albo-atrumx V. albo-atrum) showed frequent haploidization and unrestricted genetic recombination. Interspecific diploids showed infrequent haploidization and restricted genetic recombination. The frequency ofhaploidization in these was apparently increased by growing them on medium containing the fungicide benlate, but these induced haploids also had a limited range of genotypes. The restricted genetic recombination in the interspecific diploids is interpreted as resulting from non-homology between the V. albo-atrum and V. dahliae genomes, and the results are discussed in relation to the taxonomic status of dark mycelial and microsclerotial Verticillium strains.
Most Verticillium strains causing wilt diseases produce either dark resting mycelium or microsclerotia, and the taxonomic status of these morphological types has been the subject of much controversy. The species V. alboatrum has priority (Reinke & Berthold, 1879). The original description has been interpreted by some authors to include both dark resting mycelium and microsclerotia, but Klebahn (1913) obviously excluded microsclerotia from that description and assigned specific rank to a purely microsclerotial Verticillium from dahlia (V. dahliae Kleb.). More recently many authors have maintained this specific ranking for microsclerotial isolates from many different host species (Isaac, 1949; Robinson, Larson & Walker, 1957; Smith, 1965), but others consider that dark mycelial and microsclerotial strains are components ofone polymorphic species (Presley, 1941; Fordyce & Green, 1964). Isaac (1967) has reviewed this controversy and concluded that' both V. albo-atrum and V. dahliae should be accorded specific rank since lumping them together has caused (and continues to cause) needless confusion and inconvenience to plant pathologists'. Specific status is assumed for these two forms in this paper, and the justification for this assumption will be found in the results presented.
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Transactions British Mycological Society Table
I.
The relationships of auxotrophic strains
Wild types
Origin
Species
VP2
England
V. albo-atrum
VI4
England
V. albo-atrum
VI6
England
V. dahliae
VI7
Canada
V. dahliae
Mutant strains
Auxotrophic requirements*
Inositol, p-amino benzoic acid 108 Inositol, nicotinic acid, pyridoxine 49 Lysine 176 Choline, methionine 152 Nicotinamide, methionine 16/I/4 Lysine, tryptophane 16/8/6 Adenine, aneurin 17/8/ 6 17/3/ 14 Arginine, histidine
* Some of these auxotrophic requirements are abbreviated as follows in later parts of the text: inos = inositol; meth = methionine; nic = nicotinic acid; paba = p-aminobenzoic acid.
An attempt is made to resolve these conflicting views by the application of genetical techniques. The concept of a species is that of a group of organisms which can share a common pool of genes through hybridization. If two gene pools are isolated from one another they may diverge by mutation and selection and ultimately become both qualitatively and quantitatively distinct. The genomes of the two isolated populations may then be non-homologous. The significant question in the present context is whether non-homology of the V. albo-atrum and V. dahliae genomes can be demonstrated. Sexual reproduction has never been recorded in either V. albo-atrum or V. dahliae, but parasexual genetic recombination has been extensively studied in V. albo-atrum (Hastie, 1962, 1964, 1968). It is also known that the same system operates when V. dahliae strains are hybridized (A. C. Hastie unpublished). Wild strains of both species are generally haploid, but heterozygous diploid strains can be synthesized and these readily form new haploid and diploid genotypes. This investigation is an attempt to determine whether V. albo-atrum and V. dahliae haploids can be used to synthesize hybrid diploids, and whether these interspecific diploids segregate and recombine as frequently as intraspecific diploids (Hastie, 1964). MATERIALS AND METHODS
Strains
The wild-types used are all potato pathogens supplied by Professor I. Isaac, University College of Wales, Swansea. Each wild-type strain was purified by subculturing from a single uninucleate conidium. The mutant strains derived from each wild-type are therefore near isogenic (Table I). The auxotrophic mutants derived from VP2 were obtained by ultraviolet irradiation. Those from the other wild types were obtained after treatments with N-methyl-N-nitroso-N'-nitroguanidine. The ease with which these recessive mutants were detected indicates that all the wildtypes were haploid (Buxton & Hastie, 1962).
Verticillium hybrids. A. C. Hastie Culture conditions Stock cultures of all strains were maintained on the modified prune extract agar medium (PE) described by Talboys (1960). Czapek-Dox agar medium (MM) was used as a minimal medium and supplemented as required for the classification of auxotrophic segregants. A glucose minimal medium (G M M ) containing 1'5 % (w/ v) glucose, and the salts in MM, was used for the synthesis and selection of diploid strains. This medium (GMM) was found by M. Ingle & A. C. Hastie (unpublished) to give a better yield of diploids than sucrose based media. All incubation was at 25 "C . Synthesis and selection of diploids The technique used to synthesize diploids has been described in detail elsewhere (Hastie, 1973). It is a modification of that used previously (Hastie, 1962), and employs the principle originally used by Roper (1952). The parent haploid strains must have complementary auxotrophic nutritional requirements. Conidial suspensions of the parents (I06/ml) were prepared in supplemented GMM, and equal volumes of these suspensions were mixed. Droplets of th e mixed suspensions wer e suspended from th e lids of Petri dishes in a humid atmosphere and incubated for about 30 h. During incubation the conidia in the hanging-drops sedimented and germinated, and each hanging-drop ultimately yielded a pellet of mycelium. The pellets were removed, washed in sterile distilled water, and transferred to solid GMM on which they were then incubated for 4 weeks. The pellets initially grew only very slowly on this medium, but many later formed prototrophic sectors with relatively large conidia which were subsequently proved to be diploid. Diploids selected in this way were subcultured either using their prototrophic conidia or by mass hyphal tip transfers. The fertility ofindividual crosses is expressed as the proportion of pellets which formed diploid sectors. Distinction between haploids, diploids and aneuploids Ne arly all the conidia of Verticillium are uninucleate (H astie, 1962; MacGarvie & Isaac, 1966), and those which are binucleate are apparently homokaryotic (Hastie, 1962). Diploid cultures were progeny-tested by taking random samples of conidia from them, and classifying these conidia according to whether they contained a haploid, diploid or aneuploid nucleus. The criteria applied to distinguish between these types were primarily growth rate of the conidium, and the dimensions of the conidia on the culture it produced (Hastie, 1964, 1968). Haploid and diploid conidia both formed mycelia with normal and similar growth rates: Conidium containing a diploid nucleus was however relatively large and formed a mycelium which also formed large conidia. Conidia described as aneuploid formed a mycelium which initially grew very slowly, but later formed faster growing sectors. Each sector had the characteristics of either a haploid or a diploid.
Transactions British Mycological Society Table 2. N umbers of diploid sectors formed by crosses offirst block No. of pellets giving di ploids
P arents
108 x 16/1/4 108 x 17/8/6 176 x 16/1/4 176 x 17/8/6 176 x 108 16/1/4 x 17/8/6
32 60 4 0
18 0
114
Total no. pellets tested go go 87 87 go 75 5 19
RESULTS
Synthesis
of diploids
Crosses were made in blocks of six, and about ninety pellets were tested for each cross. Each block of crosses represents all possible combinations of four complementary auxotrophs; one auxotroph being derived from each of the wild-types listed in Table I. The yield of diploids was scored as the number of pellets which formed diploid sectors. Heterokaryons of Verticillium are difficult to detect and recover (Hastie, Ig62 ), but when nuclear fusion occurs in them the diploid mycelium grows vigorously and soon overgrows the heterokaryon. The yields of diploids recorded are therefore more closely related to the frequency of diploidization than to the frequency with which heterokaryons are formed. Three blocks of crosses were examined, and the yields of diploids are recorded in Tables 2-4. The first diploids appeared after about I week's incubation, and the final yields were scored after 4 weeks . There was obviously great heterogeneity between the final yields of diploids from the individual crosses, and especially from those in the first block (Table 2). This heterogeneity does not arise from a failure of V. albo-atrum and V. dahliae to form hybrid diploids. Indeed when the data in Table 2 are summed over appropriate crosses it appears the V. albo-atrum diploidized V. dahliae (g6/354 = 27 %),just as readily as two V. albo-atrum strains formed diploids (18/go = 20 %). Much of the heterogeneity in Table 2 is apparently due to the influence ofstrain ro8. It was the only strain in that block to hybridize each of the other three strains, and each of the other strains gave its maximum yield of diploids when crossed to strain ro8. Only two crosses are repeated in the three blocks of crosses. These are ro8 x 17/8 /6 (T ables 2 and 4), and 152 x 16/8 /6 (T ables 3 and 4). These repeated attempts of th e same cross give consistent results, but this consistency is not always matched wh en different auxotrophs derived from the same wild-type culture (e.g. 176 and 152) are each crossed to a third strain (e.g. ro8 ). The yields of diploids from these two crosses are shown in Tables 2 and 4, and on comparison by a contingency X2 test show a significant deviation (P < 0'01) . Similarly, strain 176 yielded no diploids when crossed to 17/8/6 (Table 2), but 152 x 17/8/6 gave 16/86 diploids
Verticillium hybrids. A. C. Hastie Table 3. Numbers of diploid sectors formed by crosses of second block
Parents 4gx 16/8/6 4gx 17/3/ 14 152 x 16/8/6 152 X I7/3/ 14 152 x49 16/8/6 XI 7/3/14
No. of pellets giving diploids
Total no. pellets tested
14 10 8 0 10 0 42
83 86 88 go go 88 5 25
Table 4. Numbers of diploid sectors formed by crosses of third block
Parents 108 X 16/8/6 108 XI 7/8/6 152 x 16/8/6 152 x 17/8/6 152 x 108 16/8/6 x 17/8/6
No. of pellets giving diploids
Total no. pellets tested
13 55 12 16 5
go go go 84 go go
°
101
534
(Table 4). It is concluded that there can be significant variations in the fertilities of auxotrophs derived from the same monoconidial wild-type culture, and that this intra-strain variation of fertility may be due to pleiotropic effects of the auxotrophic mutations.
Morphology of interspecific hybrids Subcultures of all the interspecific hybrids listed in Tables 2 and 3 were made both by taking mass hyphal-tip transfers from the growing edge of the original diploid sectors, and also by making monoconidial cultures. Both types of subculture were grown on PE. All hybrid diploids grew at about the same linear rate as intra-specific diploids and haploid strains. Hybrid diploids differed from other strains in two principal morphological features; conidial production and the morphology of dark resting structures. They generally formed few conidiophores with verticillate branching, and most conidia were borne in slimy masses resemblingpoinnotes. The individual conidia of hybrid diploids were uninucleate and about the same length as those from intra-specific diploids (Hastie, 1964). Hybrid diploids grown on PE formed less black mycelium than other strains, and their black structures were unlike those of either V. alboatrum or V. dahliae (PI. 47). The black torulose hyphae of V. albo-atrum do not proliferate laterally and the blackening is often continuous along a hypha. The microsclerotia of V. dahliae develop from single hyphae by cell divisions in all planes (Isaac, 1949), and blackening is restricted to themicrosclerotia.
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Table 5. Classification of random conidia from 3 week old diploid cultures derivedfrom crosses in the first block (Table 2) , Cross
108x 16/1/4 108 x 17/8/6 176 x 16/1/4 I76x 108
Types of conidia
,
,
Haploid
Diploid
Aneuploid
Total
0 0 0 237
113 84 123
27 16 28 22
140 100 15 1 270
II
The black structures on hybrid diploids were intermediate in that they showed very limited lateral proliferation, and generally the blackened regions of the mycelium were discrete. Random conidial analysis
Diploid sectors from all fertile crosses of the first block (Table 2) were subcultured on PE by mass hyphal tip transfers. Three independent diploid sectors of each cross were subcultured in this way, and four replicate cultures were made from each of these diploid sectors. Conidial suspensions were prepared from each of these cultures after 3 weeks growth, and incubated on PE plates. It was assumed that each colony had grown from a single conidium, and on inspection of each colony it was decided whether the original conidium had contained a diploid, haploid or aneuploid nucleus by applying the criteria given earlier. The results given in Table 5 are the averages of the progenies from the replicate cultures. The results from cross 176 x roS are obviously different from those of the other three crosses. The diploids from this cross readily yielded conidia containing haploid nuclei as has previously been reported for crosses between strains of V. albo-atrum (Hastie, 1964, 1970, 1973). Crosses between strains of V. dahliae gave diploids which equally readily form haploid segregants (A. C. Hastie, unpublished), and it is therefore concluded that an absence of haploid conidia from samples taken under the above conditions is peculiar to V. albo-atrum x V. dahliae crosses. This conclusion was supported by similar observations made on progenies from the crosses in blocks 2 and 3. The euploid (both haploid and diploid) isolates recorded in Table 5, together with further samples of conidial progeny from these crosses, were replicated on appropriately supplemented media to determine their nutritional requirements. More than rooo random monoconidial isolates from the V. albo-atrum x V. dahliae hybrids were examined, and only one was auxotrophic. It was from cross roS x 17/S/6 and required inositol andp-aminobenzoic acid; the nutritional requirements ofstrain roS. Also, only one of these isolates was haploid. Both the haploid and diploid isolates from cross 176 x roS, the intraspecific cross, showed segregation and recombination for all genetic markers as expected from earlier investigations (Hastie, 1964) and unpublished work with crosses between strains of V. dahliae. The rare occurrence of diploid auxotrophic segregants from
Verticillium hybrids. A. C. Hastie Table 6. Phenotypes of random monoconidial progeny from sectors on colonies grownfrom selected aneuploid conidia
Parental diploid
w8x 16/1/4
No. of aneuploids with diploid sectors
No. of aneuploids with haploid sectors
Phenotypes of random progeny. from haploid sectors
o
12 12
o
II
I
II
I
12
o
II
I
II
I
II II
I
12 12
o o
10
2
26 haploid DM inos paba (not sampled) 26 haploid MS nic 7 haploid DM inos paba, { 19 diploid H prototrophs 26 haploid DM inos paba (not sampled) 26 haploid MS prototrophs 14 haploid MS nic meth, { 12 diploid H prototrophsf
DM = dark mycelial; MS = microsc1erotial; H = hyaline. * Twenty-six conidia were taken randomly from each haploid sector. t These 26 progeny had much smaller microsc1erotia than all other microsc1erotial strains.
crosses of V. albo-atrum x V. dahliae indicates that mitotic recombination is much less frequent in them than in intraspecific diploids (Hastie, 1968).
Segregation in selected spontaneous aneuploids The very rare recovery of haploid segregants in random samples of conidia from interspecific diploid cultures prompted a search for a selective device which would faciliate the recovery of spontaneous haploid segregants from these strains. It is known that haploid nuclei are formed from diploid nuclei through intermediate aneuploid stages in parasexual systems. An attempt was therefore made to find aneuploid coni dia from interspecific diploids which would grow to form colonies with haploid sectors. Random samples of conidia from eight interspecific diploid cultures of cross I76 x 16/ I /4 and eight diploids of cross I76 x 16/ I /4 were spread on PE. Slow-growing colonies which were assumed to be aneuploids were selected after 2 days incubation. Inocula of the selected slow growing colonies (aneuploids) were transferred to further dishes of PE and incubated for up to 5 weeks. All these aneuploids formed fast-growing sectors, and conidia from these sectors were examined to decide whether they were haploid or diploid. The results are given in Table 6. Most aneuploids formed sectors with large diploid conidia and the mycelium had the morphology of an interspecific diploid. Other aneuploids formed sectors with small conidia, and these sectors generally had the mycelial characters of either V. albo-atrum or V. dahliae. Most haploid sectors were progeny tested, and it was shown that conidia from most of them had parental genotypes. One haploid sector was exceptional in that it was prototrophic, and also formed very small microsclerotia.
Transactions British Mycological Society Table 7. Phenotypes of random monoconidial progeny taken from cultures of the interspecific diploid 108 x 16/ I /4 grown in presence and absence if benlate Progeny from benlate containing medium Parental diploid 16/1 16/IA 16/2 16/2A
16/3
16/4
,
Haploids 1 prototroph 23 nic meth 2 prototrophs 1 nic meth 24 inospaba 43 inospaba 1 inospaba nic 3 nic meth 1 methpaba 42 nic meth 143
Progeny from benlate free medium
Diploids
Haploids
51 prototrophs 52 prototrophs 28 prototrophs 24 prototrophs 1 inos
1 prototroph
1 prototroph 2 nic meth 8 prototrophs 16 7
Diploids 51 52 52 52
---.
prototrophs prototrophs prototrophs prototrophs
52 prototrophs
52 prototrophs 3 11
These results show that haploid segregants can be recovered from some aneuploids. Presumably these are aneuploids with near haploid genomes, whereas the aneuploids which formed diploid sectors had near diploid genomes. Effectofbenlate ongrowthandsegregation ofhybrid diploids Benlate is known to cause haploidization in diploid strains of Aspergillus nidulans when incorporated in the culture medium (Hastie, 1970a). Attempts to demonstrate benlate-induced haploidization in diploid strains of V. albo-atrum have been unsuccessful apparently because the frequency of spontaneous haploidization is already high in these diploids (A. C. Hastie, unpublished). The effect of benlate on the growth and segregation of hybrid diploids was investigated after inoculating small pieces of hybrid cultures (about I mms) on PE containing various concentrations of the fungicide. A concentration of 0·5 mg/l benlate in PE greatly restricted growth of the hybrid mycelium, but after 3 weeks incubation on this medium some growth, sectoring and conidiation occurred. Random samples of about 50 conidia were taken from six cultures of the hybrid diploid 108 x 16/1/4 which had been grown on PE for 3 weeks, and also from six parallel cultures which had been grown on PE containing 0·5 mgjl benlate. Cultures from these conidia were examined to decide their ploidy and nutritional requirements. The results are given in Table 7. Nearly half the conidia from cultures grown on PE containing benlate were haploid, but in the absence of benlate only one out of 3 I 2 conidia was haploid. These are the proportions of haploids recovered, and they may not show the true frequencies with which haploids are formed in the presence and absence of benlate. Haploids with the same genotypes recovered from the same culture may be members of a clone arising from one haploidization event. The minimum number of haploidization events detected in the six cultures grown on benlate was ten, and only one
Verticillium hybrids. A. C. Hastie haploidization was detected in the six cultures grown without benlate. This indicates that haploidization is at least ten times more frequent on medium containing benlate. This estimate is of course very approximate. Besides the possible errors arising from clonal multiplication after haploidization, it takes no account of differences in growth rate in the presence and absence of benlate. The recovery of some prototrophic and triauxotrophic haploids shows that some genetic recombination occurred between the V. albo-atrum and V. dahliae genomes, but most of the haploids obtained had parental requirements and morphologies (Table 7). Most haploids either required nicotinic acid and methionine, and formed microsclerotia, or they required inositol and p-aminobenzoic acid, and formed dark mycelia. Although the inositol and p-aminobenozic acid mutants were only regularly recovered in parental combinations of this diploid, and also from other hybrid diploids, these same genes recombined freely in the diploid 176 x 108. The latter is of course derived from two strains of V. albo-atrum. The implications of these variations ofrecombination frequencies in hybrid and selfed diploids will be discussed. DISCUSSION
Heterokaryotic mycelia of Verticillium are very unstable (Hastie, 1962), and if a heterozygous diploid nucleus is formed by nuclear fusion then the heterokaryon is soon overgrown by the diploid mycelium. No attempt was made in this work to estimate the frequencies of hyphal anastomoses and heterokaryon formation. The technique detected only those heterokaryons which formed heterozygous diploids. The formation of these diploids does of course imply the occurrence of hyphal anastomosis, followed by some nuclear migration, and the establishment of at least one heterokaryotic cell. Nuclear fusion (diploidization) was detected by the emergence of a sector ofprototrophic mycelium from an inoculum ofmixed complementary auxotrophs. The diploid nature of these prototrophic sectors was confirmed by examining for relatively large uninucleate prototrophic conidia, and usually by progeny-testing. The results presented indicate that the frequency with which diploids are formed on particular crosses depends upon intrinsic properties of the haploid parent strains involved. Two haploid auxotrophs derived from the same mono-conidial wild-type culture may show significantly different fertilities when each is crossed to a third strain (e.g. 152 or 176 x ro8). This difference may be due to either pleiotropic effects ofthe auxotrophic markers or to differences resulting from the mutagen treatments. These alternatives are now being resolved by a backcross programme. Auxotrophic strains vary in the amounts ofgrowth they make on minimal media, but the effect referred to above is not simply related to the variation in the amount of growth made by the auxotrophs on the selective medium. Diploidization readily occurs between V. albo-atrum auxotrophs derived from the same wild-type strain (Hastie, 1964), and similarly it is shown here and elsewhere (Hastie, 1962) that there is no restriction preventing hyphal anastomsis and diploidization between different wild-type strains of V. albo-atrum. Auxotrophs of V. albo-atrum and V. dahliae equally readily
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formed diploids, and it is assumed that the failure to recover diploids from V I 4 x V I 7 in the first two blocks of crosses was due to the particular auxotrophs involved (Tables 2 and 3). This assumption seems justified by the successful diploidization OfV14 and V17 auxotrophs in the third block of crosses. Relatively few crosses of V. dahliae have been attempted, and the only fertile crosses have involved auxotrophs derived from the same wildtype strain (intra-strain crosses) (A. C. Hastie, unpublished). The failure to recover diploids from the interstrain crosses of V. dahliae (V I 6 x V I 7) requires further investigation. It may be due to properties of the specific auxotrophs used, or to the existence of incompatibility between V. dahliae strains from different countries. Incompatibility could affect hyphal anastomosis, nuclear migration or nuclear fusion. The existence of heterokaryon incompatibility is of course well known in other fungi (Caten & Jinks, 1966). The similarity of the frequencies with which hybrid diploids (V. albaatrum x V. dahliae) and selfed diploids (V. albo-atrum selfed) are recovered is markedly different from the behaviour of hybrid and selfed diploids when subcultured and progeny-tested (Table 5). Both form conidia containing aneuploid nuclei in similar proportions, and this indicates that aneuploids are formed with the same frequency in both. However, hybrid diploids form spontaneous haploid segregants very rarely. It has been inferred that spontaneous haploidization of V. albo-atrum diploids occurs through intermediate aneuploid stages (Hastie, 1973), as in Aspergillus nidulans (Pontecorvo, 1959). Because aneuploid nuclei are readily formed from hybrid diploid nuclei, it follows that the processes required for spontaneous haploidization occur frequently. It is therefore necessary to formulate some hypothesis which will explain why spontaneous haploidization is rarely realized in these diploids. This hypothesis should also explain why most haploids recovered from hybrid diploids are parental. One hypothesis is that the haploid genomes of V. albo-atrum and V. dahliae are not homologous and differ in both the arrangement and the structure of their genes. Random loss of chromosomes from the corresponding hybrid diploid may only rarely yield balanced and functional haploid nuclei. The haploid chromosome number of V. albo-atrum is probably four (Hastie, 1967; Heale, Gafoor & Rajasingham, 1968). V. albo-atrum diploids would then have four pairs of homologous chromosomes. A haploid segregant derived from such a diploid would contain one set of homologous chromosomes. If we ignore mitotic crossing-over, the number of different haploid genomes which can be derived is 2 4 = 16. These haploid genomes will occur with equal frequency only if the individual chromosomes are lost at random during haploid formation. There would be no restriction on the recombination of genes on non-homologous chromosomes. There would similarly be no restriction on the formation of haploid nuclei and recombination in V. dahliae diploids. However, if the haploid genomes of V. albo-atrum and V. dahliae are not homologous, then hybrid diploids would not necessarily contain pairs of homologous chromosomes. This would impose restrictions on the number of different types of haploids which could be derived from them. The maximum restriction would be that the haploid segregants were only viable if they contained either the V. alba-
Verticillium hybrids. A. C. Hastie
52 1
atrum genome or the V. dahliae genome. In these circumstances there would
be no genetic recombination between the genes introduced into the diploid from the V. albo-atrum and V. dahliae parents. In fact the haploids recovered from such diploids are predominantly parental both regarding morphology and the auxotrophic markers (Tables 6 and 7). The free recombination of the inositol and p-aminobenzoic acid genes (inos-4 paba-7) in V. albo-atrum diploids (108 x 176 and 108 x 152) together with their linked segregation in the hybrid diploids shows that there is restricted recombination between the V. albo-atrum and V. dahliae genomes, although some recombination did occur (Tables 6 and 7). This restricted recombination in diploids synthesized from V. albo-atrum and V. dahliae, together with their rare spontaneous production of haploids, is interpreted as an indication of non-homology between the V. albo-atrum and V. dahliae genomes and justifies maintaining them as separate species. This may seem illogical as hybrid diploids can be synthesized with ease under the highly selective conditions used (Tables 2-4). I find the limited genetic recombination in the hybrid diploids a useful basis on which to erect and maintain the species limits. Given certain specific conditions even man-mouse hybrid cells can be made (Harris & Watkins, 1965). Caution is of course required in drawing conclusions about species limits from the results of a few laboratory crosses. Nevertheless, the results clearly show that the microsclerotial and dark mycelial strains show very limited genetic recombination, unlike selfed diploids, and it seems unwise to follow Fordyce & Green (1964) and rank them as one species. Fordyce & Green (1964) detected genetic recombination between auxotrophic mutants derived from dark mycelial and 'pseudosclerotial' strains of Verticillium. The' pseudosclerotial ' strains were considered identical to V. dahliae Kleb., and they therefore interpreted their results to indicate that both dark mycelial and 'pseudosclerotial' strains should be included in the species V. albo-atrum. However, they reported only the results of crosses between dark mycelial and 'pseudosclerotial' strains, and they required selective techniques to recover segregants and recombinant progeny. I believe V. albo-atrum and V. dahliae should be ranked as distinct species partly because selective techniques are necessary to recover interspecific recombinants. The relationship proposed for V. albo-atrum and V. dahliae is analogous to that known to exist within many genera of flowering plants (Stebbins, 1971). Interspecific hybrids can be formed but they are relatively sterile because of chromosomal non-homology. Extension of this analogy indicates a crucial test for the suggested non-homology in the Verticillium genomes. Sterile interspecific hybrids of flowering plants can be made fertile because, as a result of doubling the chromosome complement, they have two homologous sets of chromosomes. By analogy one may predict that doubling the chromosome complements of V. albo-atrum x V. dahliae hybrids would give allotetraploids. These would readily haploidize but continue to show only limited recombination between the V. albo-atrum and V. dahliae genomes. Attempts to create Verticillium allotetraploids by colchicine treatments have failed.
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I thank Mrs 1. Blair for her excellent technical assistance, and I acknowledge the support of the Science Research Council (B/SR/3474). REFERENCES
BUXTON, E. W. & HAsTIE, A. C. (1962). Spontaneous and utra-violet irradiationinduced mutants of Verticillium albo-atrum. Journal of General Microbiology 28, 62563 2. CATEN, C. E. & JINKS, J. L. (1966). Heterokaryosis: its significance in wild homothallic ascomycetes and fungi imperfecti. Transactions of the British Mycological Society 49, 81-93· FORDYCE, C. & GREEN, R • .J. (1964). Mechanisms of variation in Verticillium albo-atrum. Phytopathology 54, 795-798. HARRIS, H. & WATKINS,J. F. (1965). Hybrid cells derived from mouse and man: artificial heterokaryons of mammalian cells from different species. Nature, London 205, 640-646. HASTIE, A. C. (1962). Genetic recombination in the hop-wilt fungus Verticillium alboatrum. Journal of General Microbiology 27, 373-382. HASTIE, A. C. (1964). The parasexual cycle in Verticillium albo-atrum, Genetical Research 5, 3 05-3 15. HAsTIE, A. C. (1967). Mitotic recombination in conidiophores of Verticillium albo-atrum. Nature, London 214, 249-252. HAsTIE, A. C. (1968). Phialide analysis of mitotic recombination in Verticillium. Molecular and General Genetics 102, 232-240. HASTIE, A. C. (1970). The genetics of asexual phytopathogenic fungi with special reference to Verticillium. Root diseases and soil-borne pathogens (ed. T. A. Toussoun, R. V. Bega and P. E. Nelson). Berkeley: University of California Press. HASTIE, A. C. (1970a). Benlate-induced instability of Aspergillus diploids. Nature, London 226, 771. HASTIE, A. C. (1973). Genetic analysis in Verticillium. Pathological wilting of plants (ed. J. S. Sadasivan, C. V. Subramanian, R. Kalyanasundaram and L. SaraswathiDevi), University of Madras. HEALE, J. B., GAFOOR, A. & RAJASINGHAM, K. C. (1968). Nuclear division in conidia and hyphae of Verticillium albo-atrum. Canadian Journal of Genetics and Cytology 10, 3 2 1-340. ISAAC, I. (1949). A comparative study of pathogenic isolates of Verticillium. Transactions of the British Mycological Society 32, 137-157. ISAAC, I. (1967). Speciation in Verticillium. Annual Review of Phytopathology 5,201-221. KLEBAHN, H. (1913). Beitrage zur kenntnis der fungi imperfecti I. Eine VerticilliumKrankheit auf dahlien. Mykologisches Zentralblatt 3, 49-66. MACGARVIE, Q.D. & ISAAC, I. (1966). Structure and behaviour of the nuclei of Verticillium spp. Transactions of the British Mycological Society 49, 687-693. PONTECORVO, G. (1959). Trends in genetic analysis. London: Oxford University Press. PRESLEY, J. T. (1941). Saltants from a monosporic culture of Verticillium albo-atrum. Phytopathology 31, 1135-1139. REINKE, J. & BERTHOLD, G. (1879). Die zeresetzung der kartoffel durch pilse. Untersuchungen aus dem Botanishen Laboratorium der Universitiit Gottingen I, 1-100. ROBINSON, D. B., LARSON, R. H. & WALKER, J. C. (1957). Verticillium wilt of potato in relation to symptoms, epidemiology and variably of the pathogen. Wisconsin Agricultural Experiment Station Bulletin no. 202, 1-49. ROPER, J. A. (1952). Production of heterozygous diploids in filamentous fungi. Experimentia 8, 14-15. SMITH, H. C. (1965). The morphology of Verticillium albo-atrum, V. dahliae and V. tricorpus. New Zealand Journal of Agricultural Research 8, 450-478. STEBBINS, G. L. (1971). Chromosomal evolution in higher plants. London: Edward Arnold Ltd. TALBOYS, P. W. (1960). A culture medium aiding the identification of Verticillium alboatrum and V. dahliae. Plant Pathology 9, 57-58.
Trans. Br, mycol. Soc.
Vol. 60. Plate 47
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Verticillium hybrids. A. C. Hastie EXPLANATION OF PLATE
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All figures , x 400. Fig. I. Dark mycelium of V. albo-atrum, Fig. 2. Microsc1erotia of V. dahliae. Fig. 3. Dark structures of interspecific hyb rids. (V. albo-atrum x V. dahliae).
(Accepted for publication
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MYC 60