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Cytological studies on Phytophthora nicotianae var. parasitica in relation to mating type
[ 87 ] Trans
s-. mycol. Soc. 84
(1), 87--93 (1985)
Printed in Great Britain
CYTOLOGICAL STUDIES ON PHYTOPHTHORA NICOTIANAE VAR. PARASITICA IN RELATION TO MATING TYPE By E. SAN SOME Stonecroft; Post Office Lane, Lighthorne, Warwick CV35 oAP Three out offour isolates of P. nicotianae var. parasitica from Papua New Guinea, were found to have an extra chromosome belonging to the mating type complex. It is suggested therefore that the occurrence of homothallic isolates in this species may be due to trisomy of the mating type locus. In one culture a 'Type II' trisomic appeared to have arisen from a 'Type I' trisomic, presumably by somatic crossing-over. Also an isolate of Ai mating type changed to A2 mating type after repeated sub-culturing. Associated with this change in mating type was the tendency to produce abnormal oogonia in combinations with antheridia of other species. The hypothesis is put forward that in this species, the A locus shows a 'position effect' being active on the AB chromosome but inactivated if transferred to AD by crossing-over between the Aa locus and the translocation point. The' position effect' hypothesis could also account for the production of oospores reported for single spore cultures of some heterothallic species under certain conditions such as ageing. It has recently been shown (Sansome, 1980) that heterothallism in Phytophthora de Bary is associated with a reciprocal translocation involving two pairs of chromosomes called the' mating type complex'. Self-fertile isolates of P. drechsleri Tucker differed in stability; some gave Al and A2 as well as AIA2 types when subjected to single zoospore analysis. The segregated A2 isolates were also unstable and give rise to both AIA2 and A; types (Mortimer, Shaw & Sansome, 1977). Cytological examination of the self-fertile isolates showed that one of the chromosomes of the mating type complex was present in duplicate (Sansome, unpubl.). Selffertility is therefore assumed to be due to the trisomic condition of the mating type locus, self-fertile isolates being Aaa. In this study, P. nicotianae van Breda de Haan var. parasitica (Dastur) Waterhouse was examined cytologically. This is a normally heterothallic species in which self-fertile isolates have been obtained but in which oospore production in heterothallic individuals has also been observed in old cultures (Tsao et al., 1980) or cultivars treated with chloroneb (Ko, 1981; Noon & Hickman, 1974)·
mating type from the same isolation 7007(1) of A2 mating type and 7007(2) of Ai mating type. The original isolate (7007) was of A; mating type and was kept as two sub-cultures. Repeated sub-cultures from the edge of the colony were made from only one ofthese. When mating type was re-tested after several years, this culture (7007(1)) was found to be A2. A sub-culture from the second original sub-culture (7°°7(2)) was found to be of Al mating type as originally determined. A change in mating type from Ai to A2 had therefore occurred during the successive sub-culturing of 7007(1). Phytophthora megakarya Brasier & Griffin, isolates P. 132 (Ar) and P. t84 (A2), and P. palmivora (Butl.) Butl., isolates P.13t (A2) and P. 80 (Al) (Sansome et al., 1979; Sansome, 1980) were used as markers in matings with the P. nicotianae var. parasitica isolates. The paired cultures were grown in the dark on carrot agar and aceto-orcein squashes of gametangia were made as previously described (Sansome 1976).
MATERIALS AND METHODS
In the case of the mating between 7164 and P. 132, a small proportion of 7164 oogonia were distorted. The distortion took the form of elongation of the oogonium or production of a small branch (Fig. 3). These abnormalities could be the result of a defect in oogonial wall formation. The mating between 7016 and P. 184 failed to
The cultures of P. nicotianae var. parasitica were obtained from Dr F. Arentz of the Forest Research Station, Bulolo, Papua New Guinea and the numbers given to them are those of the Bulolo collection. They comprise an At isolate (7016), an A2 isolate (7164) and two cultures of opposite
RESUL TS AND PRELIMINARY DISCUSSION
Abnormal oogonial development of P. nicotianae var. parasitica in interspecific matings
88
Cytology of Phytophthora
2
3
Figs 1-3. Abnormal oogonial development in P. nicorianae var . para sitica isolates (x 1375). Fig. 1. 7007(1) oogonium x P132 (P . megakarya). Fig. 2. 7007(1) oogonium x P80 (P . palmivora). Fig. 3. 7164 oogonium x P132 (P. megakarya).
produce any P. nicotianae var. paras itica oogonia so 7016 was mated with P . 131. In th is case the P. nicotianae var. parasitica oogonia were mostly round, occasionally oval, and distorted oogonia were not observed. When 7007(2) was mated with P . 184 it also produced distorted oogonia but less frequently. However, when the derived A2 (7007 (1» was mated with either P. 132 or P.80, it produced many
distorted oogonia (F igs 1, 2). Moreover, the distortion of the individual oogonia was more pronounced in 7007(1) than in 7164 and 7007(2). It appears that the change in mating type is accompanied by an increase in the dosage of whatever is responsible for the production of distorted oogonia . No distorted oogonia were observed in the mating between 7007(1) and 7007(2) so the
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Figs 4-11. Configurations observed in isolates of P. nicotianae var. parasitica (x 4500) together with diagrammatic interpretations. Fig. 4. (a) 7007(2) (x P184 P. megakarya), (b) interpretation. Fig. 5. (a) 7007(2) (x P131 P. palmivora), (b) interpretation. Fig. 6. (a) 7007(2) (x P184 P. megakarya), (b) interpretation. Fig. 7. (a) 7007(2) (x P184 P. megakarya), (b) interpretation. Fig. 8. (a) 7007(1) (x P80 P. palmivora), (b) interpretation. Fig. 9. (a) 7007(1) (x P132 P. megakarya), (b) interpretation. Fig. 10. (a) 7007(1) (x P132 P. megakarya), (b) interpretation. Fig. 11. (a) 7016 (x P131 P. palmivora), (b) interpretation.
phenomenon may only occur in interspecific matings. It is possible however that in the 7007(1) X 7007(2) mating, all the oogonia were formed by the 7007(2) parent. Cytological observations and interpretation
All the isolates examined had the reciprocal translocation complex characteristic of heterothallic species but three of the isolates examined
appeared to be trisomic, having an extra chromosome belonging to the mating type complex. The A2 isolate 7164 had an association of four chromosomes. The A1 isolate 7016 had the configuration illustrated in Fig. 11, which would appear to be a ring of four plus one duplicate. This isolate is therefore interpreted as being a 'Type I' trisomic (sensu Sansome, 1980). The mating type locus might or might not be on the extra chromosome.
9°
Cytology of Phytophthora
Culture 7007(2) which had been kept in stock without repeated sub-culturing was also Ar in mating type since it reacted with A2 isolates of both P. megakarya and P. palmivora. A ring of four chromosomes was readily observed. The ring often appeared to have a loop believed to be due to the presence of an extra chromosome as illustrated in Figs 4, 6, 7. Figure 5 shows a chain with a loop. Culture 7007(2) is therefore interpreted as being a 'Type I' trisomic, closely resembling 7016. 7°°7(1), the sister culture to 7007(2) had been subjected to vegetative sub-culturing and had changed from A1 to A2 mating type in the process. This could have been due to the original isolate being mixed or heterokaryotic but it is thought more probable that the change was the result of somatic crossing-over. A ring of chromosomes was not observed in 7001(1) but complex chains (Figs 8, 10) were seen and an unpaired chromosome was seen in some cases. Culture 7007(1) is therefore interpreted as being a 'Type II' trisomic (sensu Sansome, 1980; Michelmore & Sansome, 1982); that is, a type having two pairs of chromosomes linked by an extra chromosome partly homologous with each pair. This extra chromosome is believed to be the univalent observed in some nuclei. A 'Type II' trisomic can be derived from a 'Type I' by somatic crossing-over as follows (Fig. 12). The chromosome complex can berepresentedAB. BC. CD . DA. AB. The A and C segments containing the centromeres will be referred to as the proximal segments, the B and D segments as the distal segments. As shown in Fig. 12a, crossing-over anywhere in the proximal C segment followed by adjacent disjunction gives CB, CB and CD, CD as a result of a non-cross-over chromatid of one chromosome segregating with a cross-over chromatid of the other chromosome. With AB, AB, AD from the other chromosomes of the complex this will give the combinations AB, AB, AD, CB, CB and AB, AB, AD, CD, CD. The first combination is monosomic for D and tetrasomic for B and being more unbalanced than the original trisomic would be unlikely to survive. The second combination would be a 'Type II' trisomic and in the absence of lethal recombinants should be as viable as the original 'Type I'. It is suggested therefore that 7007(1) is a 'Type II' trisomic which is believed to have arisen from a 'Type I' (7007(2») trisomic by somatic crossingover. Accompanying this change in the mating type complex were two other changes: a change in mating type from A1 to A2 and an intensification of oogonial distortion when mated with P. megakarya and P. palmivora.
Position effect hypothesis to explain the change in mating type from A 1 to A2 The position of the mating type locus on the translocation complex affects how readily A1 will segregate from the heterozygote A2 asa consequence of somatic crossing-over during vegetative growth (Sansome, 1980). If the Aa locus is on a proximal segment, segregation would only occur as the result of a double cross-over resulting in the transposition of A and a. If the Aa locus is on a distal segment, segregation could occur if crossing-over occurred between the Aa locus and the translocation point. When the extra chromosome is present the situation is changed. In a 'Type I' trisomic, if A is on the proximal segment of the AB chromosome and a on the proximal segment of AD (Fig. 12b), an ABA, ABA, ADa type (A2) can become ABA, ADa, ADa (probably self-fertile) by crossing-over in the A segment. Similarly, the converse can also happen. If however a is on the distal B segment of chromosome AB, (Fig. 12C) a disomic A2: ABa, BCA, AD, AD, CD, with crossing-over between AB and AD can also give trisomic homothallic (Aaa) progeny, i.e. ABa ABa, BCA, CD, DA. Segregation of the A2 mating type from an A1 culture cannot be explained simply by crossing-over if A is dominant to a. It would have to be assumed either that a mutated to A or that an inhibitor of A had been lost. One further explanation which would account for this change however is that A is only functional on one chromosome. Thus ABA, ADa would be Az for mating type but if the A locus is transposed to AD it becomes non-functional and can be represented as (A). Fig. 13 illustrates how A can be transferred from AB to AD by somatic crossing-over. Crossing-over in the A segment between the proximal end of the chromosome and the mating type locus gives AaD and AAB in which the A locus remains on the AB chromosome. However, if crossing-over occurs in the region between the mating-type locus and the translocation point, the result is AAD and AaB. If A is only functional when on the AB chromosome, this will result in the conversion of the active A to inactive (A). If for instance the original 'Type I' trisomic 7007(2) is ABa, AD(A), ADa it would be of A1 mating type as observed. Crossing-over between the mating type locus and the translocation point (between ABa and AD (A) would give ABA and ADa and the resultant trisomic nucleus would be Aaa homothallic (ABA, ADa plus AD(A) or ADa). Such a homothallic type could give rise to an A2 type by further crossing-over between ABA and ADa resulting in ABA being duplicated rather than ABa. Such an Az isolate would be unstable since
91
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Fig. 12. Diagrammatic representation illustrating how new combinations may result from crossing-over in a 'Type I' trisomic. A and mark a the possible position of the mating type locus. (a) Crossing-over in the proximal C segment followed by segregation of a non-cross-over chromatid of one chromosome with a cross-over chromatid of the other thus giving a 'Type II' trisomic (non-reversible), (b) and (c) Crossing-over in the proximal A segment followed by segregation of a non-cross-over chromatid of one chromosome with
Cytology of Phytophthora
92 (a)
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Fig. 13. Diagrammatic representation of crossing-over between ABA and ADa. (a) Cross-over between the A locus and the proximal end of the chromosome; (b) Cross-over between the A locus and the translocation point.
crossing-over in the proximal A segment would lead to a reversion from A2 to homothallic. The A2 culture 7°°7(1) is thought to be a 'Type II' trisomic, assumed to have arisen from' Type I' by crossing-over in the proximal C segment. Such crossing-over could give rise to a 'Type II' trisomic with a on the linking chromosome and Aa or AA on one of the homologous pairs. Ifthe paired chromosomes started out heterozygous for Aa, they could readily become homozygous by somatic crossing-over, resulting in an AAa type, presumably A2 for mating type. Crossing-over in the A segment involving the linking chromosome AD and one of the paired AB chromosomes could lead to two new combinations of chromosomes, namely AD, AD, AB, CD, CD (tetrasomic for D but monosomic for B, therefore probably inviable) and AB, AB, AB, CD, CD. If the maintenance of the structurally heterozygous condition is dependent on a balanced lethal system it is possible that the latter type would also be eliminated, in which case the 'Type II' trisomic would be stable after the paired chromosomes had become homozygous for the A locus. It is assumed that culture 7007(1) must have passed through a homothallic stage before becoming Az. This may have been before or after crossing-over in the C segments led to the change from a 'Type I' to a 'Type II' trisomic. Since transfers were taken from the colony edge it is likely that the A2 type was favoured as a result of a faster growth rate (Sansome, 1980).
GENERAL DISCUSSION
Phytophthora nicotianae var. parasitica is normally a heterothallic species in which homothallic isolates have been repeatedly observed. Three of the four isolates from Papua New Guinea investigated in this study had an extra chromosome belonging to the mating type complex. This suggests that the presence of an extra chromosome is relatively common in this species and supports the view that homothallic isolates are trisomic and Aaa for mating type. Such homothallic types could be relatively stable if they are' Type II' trisomies with A in the chromosome linking the two homologous pairs. Such types will be less stable if they are 'Type I' trisomies, Another phenomenon reported for P. nicotianae var. parasitica and other species, e.g. P. capsid Leonian (W. Kamjaipai, pers, comm.), is the production of oospores by normally heterothallic isolates in aged cultures (Tsao et al., 1980) or after treatment with chloroneb (Ko, 1981). W. Kamjaipai (pers. comrn.) found that after making single zoospore isolations from fertile regions of an otherwise heterothallic A2 culture, some were A2 like the parent and some were At. The production of oospores in such cultures therefore seemed to be due to the transformation of some nuclei to A1. Such nuclei could then react with the original A2 nuclei and result in sexual reproduction. In species where a position effect is postulated to account for the segregation ofA2 isolates from an A 1
E. Sansome type, it is to be expected that A2 cultures would also segregate Ai types as the result of crossing-over between the translocation point and the Aa locus. Thus if the A2 is ABA, BC, CD, DAa with A only active when on the chromosome AB, then crossingover between the translocation point and the Aa locus would give cross-over chromatids ABa and DA(A). Segregation could result in a nucleus carrying the two cross-over chromatids. Such a nucleus would be balanced and of Ai mating type (a(A)). Since such an Ai isolate carries A in the suppressed (A) form it could segregate an A2 type in the same way as it arose itself, i.e. by crossing-over between the Aa locus and the translocation point. Thus, species in which the A locus is only active on one chromosome could have two genotypes conditioning Ai mating type. One genotype (aa) would be unable to give an A2 type by somatic crossing-over; the other ((A)a) would be able to segregate A2 isolates under the same conditions and at the same rate as Ai isolates arise from A2 cultures. It should be possible to test this hypothesis experimentally. I am indebted to Dr F. Arentz for supplying the cultures of P. nicotianae var. parasitica and for information concerning their origins. The assistance of Mr R. Sampson in producing the plates and of Dr Ian Crute in preparing the manuscript is also gratefully acknowledged. Thanks are also due to Professor J. K. A. Bleasdale, Director, National Vegetable Research Station, for the provision of
93
photographic materials and facilities; this work was completed while the author held the status of visiting worker at this institute.
REFERENCES
Ko, W. H. (1981). Reversible change of mating type in Phytophthora parasitica.fournalofGeneral Microbiology 125,451-454· MICHELMORE, R. W. & SANSOME, E. R. (1982). Cytological studies of heterothallism and secondary homothallism in Bremia lactucae. Transactions of the British Mycological Society 79, 291-297. MORTIMER,A. M.,SHAW,D. S.& SANSOME,E. R. (1977). Genetical studies of secondary homothallism in Phytophthora drechsleri. Archives of Microbiology 111, 255-259. NOON,J. P.&HICKMAN,C. J.(1974).Oosporeproduction in a single isolate of Phytophthora capsici in the presence of chloroneb. Canadian Journal of Botany 52, 591-595. SANSOME, E. (1976). Gametangial meiosis in Phytophthora capsici. Canadian Journal of Botany 54,1535-1545. SANSOME, E. (1980). Reciprocal translocation heterozygosity in heterothallic species of Phytophthora and its significance. Transactions of the British Mycological Society 74, 175-185. SANSOME, E., BRASIER, C. M. & SANSOME, F. W. (1979). Further cytological studies on the' L' and'S' types of Phytophthora from cocoa. Transactions of the British Mycological Society 73, 193-302. TSAO, P. H., UGALE, R., HOBBS, S. & FARIH, A. (1980). Control of homothallic oospore formation in Phytophthora parasitica by culture manipulation. Transactions of the British Mycological Society 75, 153-156.
(Received for publication 18 April 1984)
s-. mycol. Soc. 84
(1), 87--93 (1985)
Printed in Great Britain
CYTOLOGICAL STUDIES ON PHYTOPHTHORA NICOTIANAE VAR. PARASITICA IN RELATION TO MATING TYPE By E. SAN SOME Stonecroft; Post Office Lane, Lighthorne, Warwick CV35 oAP Three out offour isolates of P. nicotianae var. parasitica from Papua New Guinea, were found to have an extra chromosome belonging to the mating type complex. It is suggested therefore that the occurrence of homothallic isolates in this species may be due to trisomy of the mating type locus. In one culture a 'Type II' trisomic appeared to have arisen from a 'Type I' trisomic, presumably by somatic crossing-over. Also an isolate of Ai mating type changed to A2 mating type after repeated sub-culturing. Associated with this change in mating type was the tendency to produce abnormal oogonia in combinations with antheridia of other species. The hypothesis is put forward that in this species, the A locus shows a 'position effect' being active on the AB chromosome but inactivated if transferred to AD by crossing-over between the Aa locus and the translocation point. The' position effect' hypothesis could also account for the production of oospores reported for single spore cultures of some heterothallic species under certain conditions such as ageing. It has recently been shown (Sansome, 1980) that heterothallism in Phytophthora de Bary is associated with a reciprocal translocation involving two pairs of chromosomes called the' mating type complex'. Self-fertile isolates of P. drechsleri Tucker differed in stability; some gave Al and A2 as well as AIA2 types when subjected to single zoospore analysis. The segregated A2 isolates were also unstable and give rise to both AIA2 and A; types (Mortimer, Shaw & Sansome, 1977). Cytological examination of the self-fertile isolates showed that one of the chromosomes of the mating type complex was present in duplicate (Sansome, unpubl.). Selffertility is therefore assumed to be due to the trisomic condition of the mating type locus, self-fertile isolates being Aaa. In this study, P. nicotianae van Breda de Haan var. parasitica (Dastur) Waterhouse was examined cytologically. This is a normally heterothallic species in which self-fertile isolates have been obtained but in which oospore production in heterothallic individuals has also been observed in old cultures (Tsao et al., 1980) or cultivars treated with chloroneb (Ko, 1981; Noon & Hickman, 1974)·
mating type from the same isolation 7007(1) of A2 mating type and 7007(2) of Ai mating type. The original isolate (7007) was of A; mating type and was kept as two sub-cultures. Repeated sub-cultures from the edge of the colony were made from only one ofthese. When mating type was re-tested after several years, this culture (7007(1)) was found to be A2. A sub-culture from the second original sub-culture (7°°7(2)) was found to be of Al mating type as originally determined. A change in mating type from Ai to A2 had therefore occurred during the successive sub-culturing of 7007(1). Phytophthora megakarya Brasier & Griffin, isolates P. 132 (Ar) and P. t84 (A2), and P. palmivora (Butl.) Butl., isolates P.13t (A2) and P. 80 (Al) (Sansome et al., 1979; Sansome, 1980) were used as markers in matings with the P. nicotianae var. parasitica isolates. The paired cultures were grown in the dark on carrot agar and aceto-orcein squashes of gametangia were made as previously described (Sansome 1976).
MATERIALS AND METHODS
In the case of the mating between 7164 and P. 132, a small proportion of 7164 oogonia were distorted. The distortion took the form of elongation of the oogonium or production of a small branch (Fig. 3). These abnormalities could be the result of a defect in oogonial wall formation. The mating between 7016 and P. 184 failed to
The cultures of P. nicotianae var. parasitica were obtained from Dr F. Arentz of the Forest Research Station, Bulolo, Papua New Guinea and the numbers given to them are those of the Bulolo collection. They comprise an At isolate (7016), an A2 isolate (7164) and two cultures of opposite
RESUL TS AND PRELIMINARY DISCUSSION
Abnormal oogonial development of P. nicotianae var. parasitica in interspecific matings
88
Cytology of Phytophthora
2
3
Figs 1-3. Abnormal oogonial development in P. nicorianae var . para sitica isolates (x 1375). Fig. 1. 7007(1) oogonium x P132 (P . megakarya). Fig. 2. 7007(1) oogonium x P80 (P . palmivora). Fig. 3. 7164 oogonium x P132 (P. megakarya).
produce any P. nicotianae var. paras itica oogonia so 7016 was mated with P . 131. In th is case the P. nicotianae var. parasitica oogonia were mostly round, occasionally oval, and distorted oogonia were not observed. When 7007(2) was mated with P . 184 it also produced distorted oogonia but less frequently. However, when the derived A2 (7007 (1» was mated with either P. 132 or P.80, it produced many
distorted oogonia (F igs 1, 2). Moreover, the distortion of the individual oogonia was more pronounced in 7007(1) than in 7164 and 7007(2). It appears that the change in mating type is accompanied by an increase in the dosage of whatever is responsible for the production of distorted oogonia . No distorted oogonia were observed in the mating between 7007(1) and 7007(2) so the
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Figs 4-11. Configurations observed in isolates of P. nicotianae var. parasitica (x 4500) together with diagrammatic interpretations. Fig. 4. (a) 7007(2) (x P184 P. megakarya), (b) interpretation. Fig. 5. (a) 7007(2) (x P131 P. palmivora), (b) interpretation. Fig. 6. (a) 7007(2) (x P184 P. megakarya), (b) interpretation. Fig. 7. (a) 7007(2) (x P184 P. megakarya), (b) interpretation. Fig. 8. (a) 7007(1) (x P80 P. palmivora), (b) interpretation. Fig. 9. (a) 7007(1) (x P132 P. megakarya), (b) interpretation. Fig. 10. (a) 7007(1) (x P132 P. megakarya), (b) interpretation. Fig. 11. (a) 7016 (x P131 P. palmivora), (b) interpretation.
phenomenon may only occur in interspecific matings. It is possible however that in the 7007(1) X 7007(2) mating, all the oogonia were formed by the 7007(2) parent. Cytological observations and interpretation
All the isolates examined had the reciprocal translocation complex characteristic of heterothallic species but three of the isolates examined
appeared to be trisomic, having an extra chromosome belonging to the mating type complex. The A2 isolate 7164 had an association of four chromosomes. The A1 isolate 7016 had the configuration illustrated in Fig. 11, which would appear to be a ring of four plus one duplicate. This isolate is therefore interpreted as being a 'Type I' trisomic (sensu Sansome, 1980). The mating type locus might or might not be on the extra chromosome.
9°
Cytology of Phytophthora
Culture 7007(2) which had been kept in stock without repeated sub-culturing was also Ar in mating type since it reacted with A2 isolates of both P. megakarya and P. palmivora. A ring of four chromosomes was readily observed. The ring often appeared to have a loop believed to be due to the presence of an extra chromosome as illustrated in Figs 4, 6, 7. Figure 5 shows a chain with a loop. Culture 7007(2) is therefore interpreted as being a 'Type I' trisomic, closely resembling 7016. 7°°7(1), the sister culture to 7007(2) had been subjected to vegetative sub-culturing and had changed from A1 to A2 mating type in the process. This could have been due to the original isolate being mixed or heterokaryotic but it is thought more probable that the change was the result of somatic crossing-over. A ring of chromosomes was not observed in 7001(1) but complex chains (Figs 8, 10) were seen and an unpaired chromosome was seen in some cases. Culture 7007(1) is therefore interpreted as being a 'Type II' trisomic (sensu Sansome, 1980; Michelmore & Sansome, 1982); that is, a type having two pairs of chromosomes linked by an extra chromosome partly homologous with each pair. This extra chromosome is believed to be the univalent observed in some nuclei. A 'Type II' trisomic can be derived from a 'Type I' by somatic crossing-over as follows (Fig. 12). The chromosome complex can berepresentedAB. BC. CD . DA. AB. The A and C segments containing the centromeres will be referred to as the proximal segments, the B and D segments as the distal segments. As shown in Fig. 12a, crossing-over anywhere in the proximal C segment followed by adjacent disjunction gives CB, CB and CD, CD as a result of a non-cross-over chromatid of one chromosome segregating with a cross-over chromatid of the other chromosome. With AB, AB, AD from the other chromosomes of the complex this will give the combinations AB, AB, AD, CB, CB and AB, AB, AD, CD, CD. The first combination is monosomic for D and tetrasomic for B and being more unbalanced than the original trisomic would be unlikely to survive. The second combination would be a 'Type II' trisomic and in the absence of lethal recombinants should be as viable as the original 'Type I'. It is suggested therefore that 7007(1) is a 'Type II' trisomic which is believed to have arisen from a 'Type I' (7007(2») trisomic by somatic crossingover. Accompanying this change in the mating type complex were two other changes: a change in mating type from A1 to A2 and an intensification of oogonial distortion when mated with P. megakarya and P. palmivora.
Position effect hypothesis to explain the change in mating type from A 1 to A2 The position of the mating type locus on the translocation complex affects how readily A1 will segregate from the heterozygote A2 asa consequence of somatic crossing-over during vegetative growth (Sansome, 1980). If the Aa locus is on a proximal segment, segregation would only occur as the result of a double cross-over resulting in the transposition of A and a. If the Aa locus is on a distal segment, segregation could occur if crossing-over occurred between the Aa locus and the translocation point. When the extra chromosome is present the situation is changed. In a 'Type I' trisomic, if A is on the proximal segment of the AB chromosome and a on the proximal segment of AD (Fig. 12b), an ABA, ABA, ADa type (A2) can become ABA, ADa, ADa (probably self-fertile) by crossing-over in the A segment. Similarly, the converse can also happen. If however a is on the distal B segment of chromosome AB, (Fig. 12C) a disomic A2: ABa, BCA, AD, AD, CD, with crossing-over between AB and AD can also give trisomic homothallic (Aaa) progeny, i.e. ABa ABa, BCA, CD, DA. Segregation of the A2 mating type from an A1 culture cannot be explained simply by crossing-over if A is dominant to a. It would have to be assumed either that a mutated to A or that an inhibitor of A had been lost. One further explanation which would account for this change however is that A is only functional on one chromosome. Thus ABA, ADa would be Az for mating type but if the A locus is transposed to AD it becomes non-functional and can be represented as (A). Fig. 13 illustrates how A can be transferred from AB to AD by somatic crossing-over. Crossing-over in the A segment between the proximal end of the chromosome and the mating type locus gives AaD and AAB in which the A locus remains on the AB chromosome. However, if crossing-over occurs in the region between the mating-type locus and the translocation point, the result is AAD and AaB. If A is only functional when on the AB chromosome, this will result in the conversion of the active A to inactive (A). If for instance the original 'Type I' trisomic 7007(2) is ABa, AD(A), ADa it would be of A1 mating type as observed. Crossing-over between the mating type locus and the translocation point (between ABa and AD (A) would give ABA and ADa and the resultant trisomic nucleus would be Aaa homothallic (ABA, ADa plus AD(A) or ADa). Such a homothallic type could give rise to an A2 type by further crossing-over between ABA and ADa resulting in ABA being duplicated rather than ABa. Such an Az isolate would be unstable since
91
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Cytology of Phytophthora
92 (a)
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crossing-over in the proximal A segment would lead to a reversion from A2 to homothallic. The A2 culture 7°°7(1) is thought to be a 'Type II' trisomic, assumed to have arisen from' Type I' by crossing-over in the proximal C segment. Such crossing-over could give rise to a 'Type II' trisomic with a on the linking chromosome and Aa or AA on one of the homologous pairs. Ifthe paired chromosomes started out heterozygous for Aa, they could readily become homozygous by somatic crossing-over, resulting in an AAa type, presumably A2 for mating type. Crossing-over in the A segment involving the linking chromosome AD and one of the paired AB chromosomes could lead to two new combinations of chromosomes, namely AD, AD, AB, CD, CD (tetrasomic for D but monosomic for B, therefore probably inviable) and AB, AB, AB, CD, CD. If the maintenance of the structurally heterozygous condition is dependent on a balanced lethal system it is possible that the latter type would also be eliminated, in which case the 'Type II' trisomic would be stable after the paired chromosomes had become homozygous for the A locus. It is assumed that culture 7007(1) must have passed through a homothallic stage before becoming Az. This may have been before or after crossing-over in the C segments led to the change from a 'Type I' to a 'Type II' trisomic. Since transfers were taken from the colony edge it is likely that the A2 type was favoured as a result of a faster growth rate (Sansome, 1980).
GENERAL DISCUSSION
Phytophthora nicotianae var. parasitica is normally a heterothallic species in which homothallic isolates have been repeatedly observed. Three of the four isolates from Papua New Guinea investigated in this study had an extra chromosome belonging to the mating type complex. This suggests that the presence of an extra chromosome is relatively common in this species and supports the view that homothallic isolates are trisomic and Aaa for mating type. Such homothallic types could be relatively stable if they are' Type II' trisomies with A in the chromosome linking the two homologous pairs. Such types will be less stable if they are 'Type I' trisomies, Another phenomenon reported for P. nicotianae var. parasitica and other species, e.g. P. capsid Leonian (W. Kamjaipai, pers, comm.), is the production of oospores by normally heterothallic isolates in aged cultures (Tsao et al., 1980) or after treatment with chloroneb (Ko, 1981). W. Kamjaipai (pers. comrn.) found that after making single zoospore isolations from fertile regions of an otherwise heterothallic A2 culture, some were A2 like the parent and some were At. The production of oospores in such cultures therefore seemed to be due to the transformation of some nuclei to A1. Such nuclei could then react with the original A2 nuclei and result in sexual reproduction. In species where a position effect is postulated to account for the segregation ofA2 isolates from an A 1
E. Sansome type, it is to be expected that A2 cultures would also segregate Ai types as the result of crossing-over between the translocation point and the Aa locus. Thus if the A2 is ABA, BC, CD, DAa with A only active when on the chromosome AB, then crossingover between the translocation point and the Aa locus would give cross-over chromatids ABa and DA(A). Segregation could result in a nucleus carrying the two cross-over chromatids. Such a nucleus would be balanced and of Ai mating type (a(A)). Since such an Ai isolate carries A in the suppressed (A) form it could segregate an A2 type in the same way as it arose itself, i.e. by crossing-over between the Aa locus and the translocation point. Thus, species in which the A locus is only active on one chromosome could have two genotypes conditioning Ai mating type. One genotype (aa) would be unable to give an A2 type by somatic crossing-over; the other ((A)a) would be able to segregate A2 isolates under the same conditions and at the same rate as Ai isolates arise from A2 cultures. It should be possible to test this hypothesis experimentally. I am indebted to Dr F. Arentz for supplying the cultures of P. nicotianae var. parasitica and for information concerning their origins. The assistance of Mr R. Sampson in producing the plates and of Dr Ian Crute in preparing the manuscript is also gratefully acknowledged. Thanks are also due to Professor J. K. A. Bleasdale, Director, National Vegetable Research Station, for the provision of
93
photographic materials and facilities; this work was completed while the author held the status of visiting worker at this institute.
REFERENCES
Ko, W. H. (1981). Reversible change of mating type in Phytophthora parasitica.fournalofGeneral Microbiology 125,451-454· MICHELMORE, R. W. & SANSOME, E. R. (1982). Cytological studies of heterothallism and secondary homothallism in Bremia lactucae. Transactions of the British Mycological Society 79, 291-297. MORTIMER,A. M.,SHAW,D. S.& SANSOME,E. R. (1977). Genetical studies of secondary homothallism in Phytophthora drechsleri. Archives of Microbiology 111, 255-259. NOON,J. P.&HICKMAN,C. J.(1974).Oosporeproduction in a single isolate of Phytophthora capsici in the presence of chloroneb. Canadian Journal of Botany 52, 591-595. SANSOME, E. (1976). Gametangial meiosis in Phytophthora capsici. Canadian Journal of Botany 54,1535-1545. SANSOME, E. (1980). Reciprocal translocation heterozygosity in heterothallic species of Phytophthora and its significance. Transactions of the British Mycological Society 74, 175-185. SANSOME, E., BRASIER, C. M. & SANSOME, F. W. (1979). Further cytological studies on the' L' and'S' types of Phytophthora from cocoa. Transactions of the British Mycological Society 73, 193-302. TSAO, P. H., UGALE, R., HOBBS, S. & FARIH, A. (1980). Control of homothallic oospore formation in Phytophthora parasitica by culture manipulation. Transactions of the British Mycological Society 75, 153-156.
(Received for publication 18 April 1984)