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Secondary homothallism in Bremia lactucae
Vol. 78, Part
February 1982
1
Trans, Br , mycol. S oc. 78 (1) 1-9 (1982)
Printed in Great Britain
SECONDARY HOMOTHALLISM IN BREMIA LACTUCAE By R. W. MICHELMORE AN D D. S. INGRAM Th e Botany S chool, University of Cambridge, D owning S treet, Cambridge CB23EA, UK M any isolates of B remia lactucae are heterothallic ; some, howe ver, seemed to be self-fertile. Single conidial lin es were derived from one self-fertile isolate. The majority of the se lines were self-fert ile, but some were self-ste rile. The self-fertile lines were stable over man y asexual generations when subcultured using large numbers of conidia. Attempts to restore self-fertility by cult uri ng self-sterile lines together were not successful. Both normal and abnormal patt ern s of gametangial developm ent were observed by scann ing electron mi croscopy of a self-fertile line. The pattern of segregation of self-fertile and self-sterile lines from a self-fertile isolate of B, lactucae is similar to th at described for the hom othallic isolates of Phytophthora drechsleri. It is suggested th at self-fertility in B. lactu cae, as in P. drechsleri, may be due to the chromosomal determinant s of compatibility-typ e being trisomic following numerical nondis junc tion at meiosis. The segregation of self-sterile lines from the self-fertile isolate may have in volved at least tran sitory heterokaryosis. Bre mia lactucae Regel, th e obliga te biotr oph ic pathogen causing downy mildew of lettuce, has recent ly been shown to be heterothallic (M ichelmore & Ingram, 1980). In a survey, th e majori ty of isolates were found to be self-sterile and of two sexual com patibility type s, designated B, and B2 • Some isolates , however, apparently contained th e det erminant s of both compatibility type s as th ey produce d oospores when cultured alone. These self- fertile isolates pr oduced more oospores when cultur ed in combination with an isolate of compatibiliry type B, than when cultur ed alone or with an isolate of comp at ibility type B2 • This paper describes the det ailed investiga tion of one self-fer tile isolate wh ich suggested the existenc e of secondary hornothaliisn an d heterokaryosis in B . lactu cae ; th is is the ErA investigation of such phenomena in th e Pero nospo ra ccac. The stud ies also pro vided furthe r evidence of the extent of the similarity between B. lactucae and the heterothallic Phytop hthora spe cies, M .~ TER I AL :':
A N D M ETHO D S
'1'1:.. pathogen I solate 11\.125 of B. luctucac W 3 S collected from th e lett uce trial grour. d at ! he N ational Institute of Agricult ural HeLm y Ca.n bridge, and was found to p rod uce nn:;r ;', ~e , rC'4ub riy when cultured alone (M ichelmorc ,.'\;- \l~ gr,'r:l , j9\\;)) . Lines were der ived fro m isolate ) ,\ \ 2) as descri bed below. I solates IL4 and IfvLt -+ hav.. bC,~D .icscribcd by Miche1more & Ingram (19ik ) _Tile ,;;" tJt<-:s and lin es were cultured --- - ' - . -- . ... _--- - - - - - - - 1701·77, Part
3 , 1(" J ~
on detached, fully expanded single cotyledons of L actuca sati va L. (lettuce), ev. Briti sh H ilde, laid out in clear plastic boxes (125 x 80 x 20 mm ) and sealed with N escofilm (N ippon Shuji & Kaisha, Osaka, Japan), as already described (M ichelmore & Ingram, 1980). Incubation was at 15 °C, in darkn ess for the first 24 h and then in a growth room irr adiated for 12 h each day by daylight fluorescent tubes giving a light int ensity of 6'31 W m - 2 at the surface of the boxes. When necessary the lines were stored by freezing newly sporulating cotyledons, sealed in culture boxes, to - 20". Pro cedure f or obtaining lines of B. lactucae fro m single conidia
Single cotyledons oflettuce, cv. British Hilde, were detached from seedlings at the stage of first leaf emergence (seedlings approximately 7 days old) and laid abaxial surface uppermost on moist filter paper in Petri dishes (52 mm diam ), one per dish; when possible dishes were set up 24 h prior to inoculation . In early experiments detached cotyledon pair s were used; however, single cotyledons supported more synchrono us sporulation and were used in later experiments. A small droplet of distilled water (approx, 0'02 ml) was placed on the abaxial surface of each cotyledon . A lettuce cotyledon, on which B. lactucae had been sporu lating asexually for 2 or 3 days, was lightly wiped over th e surface of 0'3 % water agar in a small Petri dish to streak the conidi a onto th e agar. This dish was lit from below using a cool fibre-optic light - - - - - - - - - - - - - - - - - -
i -sued 31. December 1981 MY C
78
2
Secondary homothallism in Bremia
source. Single conidia were located using a dissecting microscope (50 x) and picked off using a drawn-out glass microcapillary which was joined to a shortened Pasteur pipette via a catheter tube (the Pasteur pipette was held in the mouth and, by applying positive or negative pressure, the microcapillary could be flushed with water). The conidia were transferred singly from the surface of the agar to the droplet of water on a cotyledon and the microcapillary flushed to create turbulance in the droplet. After each transfer the microcapillary was washed by repeated flushing in two changes of distilled water. The Petri dishes were labelled so that subcultures from the same cotyledon could be grouped. Only a limited number of subcultures were made from anyone cotyledon (usually 10 or 20). Following inoculation the cotyledons in the Petri dishes were transferred to a dark incubator at 15° for 24 h; care was taken not to dislodge the droplets. After 24 h the Petri dishes were sealed with Nescofilm to prevent desiccation and cross-contamination, and then incubated in the growth room. The number of days between inoculation and the onset of asexual sporulation was noted for each cotyledon (usually six to nine days). Infection resulted in up to 45 % of the single conidium inoculations. Checks to ensure that only one spore was transferred were made by picking up single conidia and then replacing them on the agar; only one conidium was transferred in all such transfers. Also, control cotyledons were sham-inoculated after the microcapillary had been washed and without picking up another conidium; when such 'inoculations' were made, they were alternated with the transfer of single conidia. Sporulation was never observed on the sham-inoculated cotyledons. Approximately 2 days after asexual sporulation had commenced the single spore derivatives were subcultured separately on to 30 lettuce cotyledons in each of two culture boxes. The suspensions of conidia were prepared by shaking the cotyledon in 10 ml distilled water; the conidia in the resulting suspensions were washed once by pelleting in a bench centrifuge to remove a water soluble inhibitor of germination and resuspended in 2 ml fresh distilled water. Each cotyledon was inoculated by placing a drop (approx. 0'03 ml) of a suspension (approx. i x 104 conidia ml r") on the abaxial surface using a 2 ml syringe and needle; this procedure often resulted in all the cotyledons becoming infected. All suspensions were prepared and the inoculations made on a laminar-flow airbench to prevent cross-contamination. The culture boxes were sealed and incubated in the growth room as described. After asexual sporulation had
commenced in these subcultures, one culture box of each line was frozen for storage. Determination of sexual compatibility type and virulence phenotype The compatibility type of the derived lines of B. lactucae was determined by culturing each one either alone or in combination with the type isolates IL4 (compatibility type B I ) or IM44 (compatibility type B2 ) (Michelmore & Ingram, 1980). Using a syringe and needle as described above, 20 single cotyledons in each of three culture boxes were inoculated with a suspension of washed conidia of each line. One day later, one box of each line was further inoculated with isolate IL4 and one box with isolate IM44, by spraying thoroughly with a suspension of conidia. The culture boxes were sealed and incubated in the growth room until decayed and transparent. The cotyledons were then microscopically examined in situ for the presence of oospores. The virulence phenotype of some isolates was determined by inoculating a differential series of 12 resistant lettuce cultivars as has been described in detail elsewhere (Crute & Johnson, 1976; Michelmore & Ingram, 1981 b). Methods of observation The number of cotyledons containing oospores was determined using a dissecting microscope (50 x ). Fresh cotyledons were fixed and cleated in 95 % ethyl alcohol. The material for the observations on the formation of the gametangia was prepared for light microscopy by vacuum infiltration with water and for scanning electron microscopy by chemical fixation and dry-fracture according to the methods of Michelmore & Ingram (1981 a). The intensity of asexual sporulation was determined by shaking the sporulating cotyledons in 10 ml of distilled water and then making counts with a haemocytometer. RESUL TS
Single spore analysis of isolate IM25 One hundred and fourteen lines, each derived from a single conidium of isolate IM25, were bulked up by subculturing on to lettuce cotyledons and the resulting conidia used to determine the sexual compatibility type of each line (Fig. 1). Twenty of the derived lines were of compatibility type B 2, two were of compatibility type B I and 92 (80'7 %) were capable of sexual reproduction when cultured alone and also in combination with at least one of the type isolates (Table 1). The majority of the single spore lines derived from isolate IM25, therefore, were self-fertile.
R. W. Michelmore and D. S. Ingram Isolate IM25 144 lines (produced oospores - - derived from ~-when cultured alone) single conidia
Derived lines bulked-up
~
3
I nocu lated alone
} Determination of the Orwith B,-type isolate compatibility type of each line Orwith B,-type isolate
Isolate IM25, L2 69 lines ~Inoculated alone ~ Determination of the (produced oospores ~derived from Orwith B,-type isolate compatibili~y type of when cultured alone) single conidia Orwith B,-type isolate each line Fig.
Table
1.
1.
Single spore analysis of a self-fertile isolate of B. lactucae.
Sexual compatibility type composition of lines of Bremia lactucae derived from single conidia of self-fertile isolates Compatibility type composition
Parental isolate 1M25 IM25,L2 B, only r B 2 only j
n, >
B2
_ _ _ _ _ _ _ _ _ _- - - - - - A -
_
rr:
B, only
B, > B 2
2 1
B, < B2 88
B 2 only
o
B, = B 2 4
o
0
30
38
20
Total 114 69 183
Self-sterile, heterothallic lines Produced oospores more frequently with the B 2 type-isolate than} with the B, type-isolate Produced oospores as frequently with that B 2 type-isolate as with Self-fertile the B, type-isolate lines Produced oospores less frequently with the B 2 type-isolate than with the Br type-isolate
One self-fertile line, IM25,L2, was chosen for further study. This line continued to produce oospores with similar frequency when subcultured alone over ten asexual generations. A further 69 lines were derived, each from a single conidium of line IM25,L2; the resulting conidia were used to determine the compatibility type of each line without an asexual generation for bulking up (Fig. 1). This was done as it was thought that fluctuations in compatibility type composition might occur during subculturing. Thirty-eight of the lines derived from line IM25,L2 seemed to be B 2 , one to be B, and 30 to be self-fertile (Table 1). Nearly all the self-fertile lines produced large numbers of oospores when cultured with the B, type-isolate but formed oospores infrequently and in small groups when cultured alone or with the B 2 type-isolate; they were therefore considered to be predominantly B 2 • The frequency of oospore production was similar when a self-fertile line was cultured alone and when it was cultured with the B 2 type-isolate. No self-fertile lines were found which produced more oospores with the B 2 typeisolate than with the B, type-isolate. The self-fertile lines derived from isolate IM25,
L2 produced oospores in only 1 or 2 cotyledons out of the 20 inoculated, when cultured alone or with the B 2 type-isolate. Several of the lines scored as self-sterile B 2 lines might, therefore, have been self-fertile, and this would account for the lower proportion of self-fertile lines recorded from line 1M25,L2 as compared to the progeny of IM25. Because the bulking-up stage had been omitted (Fig. 1), only small numbers of conidia could be used to inoculate each cotyledon, resulting in the low frequencies of oospore production (Michelmore & Ingram, 1980). All cotyledons which became infected with B. lactucae following inoculation with single conidia were cleared in 95 % ethyl alcohol and examined microscopically for the presence of oospores. Only two cotyledons were observed to contain oospores, even though the determinants of both compatibility types must have been present in over 80% of infections as shown by the analysis of compatibility type. The time between inoculation with single conidia and the onset of asexual sporulation was not correlated with compatibility type. Sporulation occurred 6-10 days after inoculation and self1-2
4
Secondary homothallism in Bremia
fertile lines sporulated simultaneously with the self-sterile lines. Two self-sterile lines derived from isolate IMz5 (IMz5,R7, compatibility type B 1 ; 1Mz5,P11, compatibility type B2) , line IMz5,Lz and the B 1 line derived from line IMz5,Lz were each inoculated on to a differential series of resistant lettuce cultivars and shown to have the same virulence phenotype as isolate 1Mz5. They were, therefore, not contaminants and virulence did not appear to be segregating in isolate IMz5. Sporulation of self-sterile lines derived from isolate 1Mz5 The self-sterile lines 1Mz5,R7 (compatibility type B1) and IMz5,P11 (compatibility type B 2) were each derived from single conidia of isolate IMz5. Cotyledons were inoculated with each line alone or with the two lines together (1: 1 ratio) at two different inoculum concentrations (Michelmore, 1979). These self-sterile lines sporulated in a manner which was indistinguishable from heterothallic isolates studied previously: no oospores were produced when the lines were cultured singly but large numbers of oospores and few conidia were produced when they were cultured together; sexual and asexual reproduction commenced simultaneously; the time interval between inoculation and sporulation was dependent upon the concentration of conidia in the inoculum; and high levels of sexual reproduction accelerated decay of the lettuce tissue. Also, the gametangia developed in a manner similar to that observed between other heterothallic isolates. Although both lines, when cultured alone, started to sporulate simultaneously, line 1Mz5,R7 sporulated more heavily than line IMz5,P11, producing over twice as many conidia as line IMz5,Pl1 at one inoculum level. Also, cotyledons decayed more rapidly when inoculated with line IMz5,P11. These phenomena were observed in several experiments with these two lines and suggest that during sporulation line IMz5,P11 had a less-balanced relationship with lettuce cv. British Hilde than line 1Mz5,R7. Genetic factors in addition to compatibility type may, therefore, have been segregating in isolate 1Mz5. Whether this segregation is related to the segregation of compatibility type in B. laetucae requires further investigation, but these observations emphasize the capacity of B. laetucae for somatic variation. Subculture of mixtures of heterothallic derivatives of isolate 1Mz5 Experiments described above suggested that selfsterile components might be segregating from a heterokaryotic mycelium. The formation and
100
90
80 OJ '" 0c.
70 0 '" 0 Cl
c:
c ~
60
c: 0 o
c '" 50 0
""0
OJ
e-
+-'
0
o ""0
40
so
.E c:
15
30
d'2.
20
10
II
III
IV
V
Number of asexual generations
Fig. 2. Frequency of oospore production by B. lactucae in cotyledons of lettuce cv. British Hilde, over five asexual generations. Lettuce cotyledons were initially inoculated with a mixture of self-sterile isolates IM25,R7 (B 1) and 1M25,P11 (B2) in: (i) a 10:1 ratio et 5 x 10 4 conidia ml- 1 (A) or 1 x 103 conidia ml " (6); (ii) a 1: 10 ratio at 5 x to' conidia ml" (.) or 1 x 103 conidia ml " (D). Each treatment line was subcultured every 7 or 8 days at these concentrations of conidia, onto 120 fresh cotyledons for the treatments inoculated with 5 x 10' conidia ml- 1 and on to 150 fresh cotyledons for the treatments inoculated with 1 x 103 conidia ml- 1• Each value represents the percentage of infected cotyledons in which oospores were produced.
segregation of heterokaryons has been demonstrated in Phytophthora megasperma var. sojae using auxotrophic and drug-resistant mutants (Long & Keen, 1977). To investigate the basis of selffertility, attempts were made to reform self-fertile heterokaryons from self-sterile lines derived from isolate IMz5. Suspensions of washed conidia of lines IM z5, R7 and IMz5,P11 were sprayed on to cotyledons
R. W. Michelmore and D. S. Ingram in the manner already described (M ichelmore & Ingram, 1980) at two concentrations, 5 x 10~ and 1 x 103 ml- I , each in the ratios 10: 1 and 1: 10, IM25, R7: IM25,PU. The treatments inoculated with 5 x 10~ conidia ml'< were cultured on 120 single detached cotyledons and the treatments inoculated with 1 x 103 conidia ml- I were cultured on 150 cotyledons. As checks, each line was also cultured alone at 5 x 10~ conidia ml" on 60 single cotyledons. The six treatments were subcultured every 7 or 8 days on to fresh cotyledons for five asexual generations. The incidence of oospore production was determined after each round of culture. After five asexual generations, spores from each experimental treatment were cultured in combination with each of the type isolates and examined for oospore production, to determine the compatibility type composition. The frequency of oospore production declined during subculturing and after five asexual generations, none of the treatments produced oospores (Fig. 2). The decline was most rapid in the treatments inoculated with 5 x 10~ conidia ml- I • Oospores were never observed in the check treatments of each isolate cultured alone. The compatibility type that was the minor component at the beginning of the experiment was eliminated during five asexual generations. As no stable self-fertile isolates were obtained in this experiment, nor in similar experiments repeated with other heterothallic isolates, it was concluded that heterokaryons with respect to sexual compatibility type were not regularly formed between isolates of opposite compatibility type under the conditions of these experiments. The failure to produce self-fertile isolates by culturing isolates of opposite compatibility types together does not demonstrate that hyphal fusion resulting in heterokaryosis never occurs; the experimental procedures may not have favoured heterokaryon formation or may not have detected heterokaryons if they occurred at low frequencies. Continual formation and segregation of heterokaryons, however, do not appear to be the basis for the self-fertility observed in B. lactucae, Formation of gam etangia by line 1M25,L2
Studies of sexual reproduction by heterothallic isolates of B. lactu cae have shown that the formation of the gametangia is preceded by a tight and complex interaction between hyphae presumed to be of opposite compatibility type; oogonia were never observed to occur without an associated antheridium (M ichelmore & Ingram, 1981 a). Light-microscopical observation s of sexual reproduction in cotyledons inoculated with line IM25, L2 indicated that the gametangia did not develop
5
in the manner observed with heterothallic isolates. Cotyledons in which oogonia had been observed by light microscopy were therefore prepared for scanning electron microscopy and the development of the gametangia was examined more closely. In the self-fertile line IM25,L2 close interactions between two or more vegetative hyphae preceded the development of sexual hyphae and gametangia (Fig. 3) as had been observed with heterothallic isolates. However, a different pattern of sexual development was also observed. In zones where sexual reproduction occurred, many thin flexuous hyphae which had often collapsed during preparation were seen (Figs 3-6); they had arisen either as branches from vegetative hyphae or, apparently, from the apex of a vegetative hypha (Fig. 5). These hyphae had often grown for considerable distances through the cotyledons, branching irregularly with inconsistent dimensions. Contact between the thin hyphae and other vegetative hyphae seemed to have often resulted in the further production of thin hyphae; where interactions between thin hyphae had occurred, there were usually no tight associations, but instead the branching pattern was irregular and loose (F ig. 6). It was often impossible to observe the antheridiurn developing beside an oogonium; in one half-cotyledon many oogonia were observed but only one antheridium was seen (F ig. 3). There was, however, always contact between the oogonium and developing thin hyphae or occasionally a vegetative hypha (Fig. 7). On one occasion, a hypha1 swelling, very similar in appearance to an antheridium, was observed without an associated oogonium (Fig. 8). Oogonia and antheridia therefore seemed to be able to develop independently of each other in this self-fertile isolate, although contact with other hyphae was still necessary. Light microscopy confirmed the SEM observations; often antheridia were small or not visible, but in some cotyledon s the oogonia and antheridia regularly developed together. The thin hyphae gave the impression of a much increased hyphal density in zones of the cotyledon where sexual reproduction occurred. Sexual reproduction tended to occur in limited patches rather than evenly throughout the cotyledon and was less synchronous than when heterothallic isolates of opposite compatibility type were cultured together. When infected cotyledons were vacuum infiltrated with water and then immediately observed with the light microscope, vigorous cytoplasmic streaming up and down the vegetative hyphae, in and out of the haustoria and to a lesser extent in and out of the developing sexual hyphae, could be seen. In a heterokaryotic mycelium therefore the nuclei which determine the event s initiating sexual repro-
6
Secondary homothallism in Bremia
Figs. 3-8. Scanning electron micrographs showing the development of gametangia in a self-fertile line of B. lactucae in cotyledons of lettuce, cv. British Hilde, following inoculation with line IMz5, Lz at 5 x 10' conidia ml ". Seven days after inoculation the cotyledons were chemically fixed, dehydrated, critical point dried, dry fractured and sputter-coated with gold (Michelmore & Ingram, 1981 a). The figures show the following: Fig. 3. the tight associations which were sometimes observed (double arrow); in the background are the only oogonium and antheridium observed to be developing together in this half-cotyledon; Fig. 4. the irregular morphology of the thin hyphae and their tendency of collapse during preparation; Fig. 5. the extensive growth of the thin hyphae; Fig. 6. the loose branching pattern of the sexual hyphae;
R. W. Michelmore and D. S. Ingram
7
8 Fig. 7. an oogonium developing apparently without an attached antheridium ; Fig. 8. the thin sexual hyphae and what appears to be an antheridium which has developed without an oogonium. a = antheridium s = sexual hyphae h = haustorium u = underside of the lower epidermis 0= oogonium v = vegetative hyphae p = plant mesophyll cell Scale bar = 5,um
duction may not be the same as those determining subsequent sexual development. This may at least partly explain the loose and apparently disorganized nature of sexual development in self-fertile isolates. DISCUSSION
These studies demonstrate that the self-fertile isolate of B. lactucae, IM25, exhibited a form of homothallism and was not a mixture of heterothallic isolates of opposite compatibility types. Selffertile lines have been reported for other heterothallic members of the Peronosporales: Phytophthora spp. (Mortimer, Shaw & Sansome, 1977), Pythium syluaticum (Pratt & Green, 1973) and Peronospora parasitica (De Bruyn, 1937). The most detailed studies have been made with Phytophthora drechsleri where genetic and cytological experiments have indicated that the self-fertile lines are trisomic for the determinants of compatibility type (Mortimer et al., 1977; Sansome, 1980; Sansome, Mortimer & Shaw, unpub!.). Cytological studies have demonstrated that the cytology of B. lactucae is similar to that of P. drechsleri, and isolate IM25 appears to have an extra chromosome associated at meiosis with a reciprocal translocation (Sansome & Michelmore, unpub!.). The pattern
of asexual segregation of heterothallic components described in this paper for B. lactucae is similar to that observed in some lines of P. drechsleri. The self-fertility of isolate IM25 and derived lines of B.lactucae may therefore be a similar form of secondary homothallism. Isolate IM25 was repeatedly subcultured asexually for most of a 3-year period and showed no decrease in the tendency to produce oospores; the self-fertile line IM25,Lz seemed to have a similar sexual stability. When isolates of opposite compatibility type were subcultured together, however, one compatibility type was eliminated from the population very quickly, often within three asexual generations and always within five, depending on the concentration of conidia in the inoculum. This suggests that the phenotypes of lines IM25,R7 and IM25,P11 were the result of segregation from isolate IM25 rather than that isolate IM25 was a mixture of unrelated lines. The self-fertile isolates detected in the survey reported previously (Michelmore & Ingram, 1980) were also probably homothallic as they had been subcultured over several or many asexual generations. The isolates W1, W3 and W5 used in previous studies of sexual reproduction of B. lactucae (Tommerup, Ingram & Sargent, 1974; Ingram, Tommerup &
8
Secondary homothallism in Bremia
Dixon, 1975) are also likely to have been homothallic for the same reason. The infrequent and patchy production of oospores by isolates W1, W3 and W5 was similar to that observed in the selffertile derivatives of isolate IM25. Studies are now needed to determine whether self-fertile isolates of B. lactucae exist which do not segregate self-sterile components and are therefore similar to some stable self-fertile forms of P. drechsleri. The data for the sexual compatibility of the lines derived from single conidia suggested that selfsterile components with opposite compatibility types could segregate from one mycelium and reproduce sexually; the majority of the mycelia, however, apparently contained the determinants of both compatibility types (B1+ 2 mycelia), and it is less evident what sexual phenotypes were exhibited by such B1+ 2 mycelia. Large numbers of oospores were produced only when self-fertile lines were cultured with the Bj-type isolate; when self-fertile lines were cultured with the Bctype isolate, no more oospores were produced than when they were cultured alone. When heavy oospore production occurred following inoculation with a self-fertile line in combination with a B, isolate there was very little asexual sporulation, indicating that the majority of the mycelia were involved in sexual reproduction (M ichelmore & Ingram, 1980). The B1+ 2 mycelia therefore seemed to respond as compatibility type B 2 when in contact with B 1 mycelia but responded rarely, if at all, as compatibility type B 1 when in contact with B2 mycelia. The observations sugge st that oospore production in self-fertile lines cultured alone resulted from the interaction between the hyphae of B 1 mycelia and the hyphae of B1+ 2 or B 2 mycelia, rather than that sexual reproduction involved only one mycelium which the term homothallism strictly implies. Therefore, the sexual reproduction of all isolates of B. lactucae so far studied, whether self-fertile or self-sterile, may be considered to be essentially heterothallic. Oospores were produced only infrequently in the cotyledons in which the single spore isolations were made (2 out of 183), even though approximately 80 % of the lines were subsequently found to be self-fertile. This is probably due to the threedimensional pattern of the hyphal growth within the host tissue. Mycelia tend to radiate out from the point of establishment; when there was only one infection in a cotyledon, the contacts between hyphae of opposite compatibility type which are necessary for sexual reproduction would have been infrequent. However, oospores were observed occasionally to develop from single infections, and therefore, sexual reproduction must have occurred between hyphae of the same mycelium, but the
above discussion suggests that vegetative segregation of the compatibility types would have occurred first. As lines of B. lactucae with defined marker genes do not exist, the possibility cannot be excluded that the data reflect the segregation of nonchromosomal determinants. Evidence is accumulating, however, that sexual compatibility type in heterothallic Phytophthora spp. is controlled by chromosomal determinants and it is useful to consider the consequences of a similar interpretation for B. lactucae. Self-fertile (homothallic) isolates of predominantly heterothallic Phytophthora spp, have frequently been recovered in the progeny after sexual reproduction and are thought to have been the result of numerical non-disjunction at meiosis producing a gametic nucleus which was disornic for the determinants of compatibility type and which gave rise to a trisomic zygote (Mortimer et al., 1977; Sansome, Mortimer & Shaw, unpub!.). It is possible that the self-fertile isolates of B. lactucae similarly resulted from non-disjunction at meiosis (Sansome & Michelmore, unpub!.). This would imply that the BI+2 mycelia of a self-fertile isolate of B. lactucae are heterogeneous populations of nuclei of at least three types: unstable trisomic nuclei (B, nuclei) and nuclei with phenotypes of only one compatibility type (B, or B 2 nuclei) which arose either as the result of chromosome loss or due to somatic recombination from B, nuclei. Depending on the relative rates of formation and segregation of the heterothallic nuclei, heterokaryosis may only be transitory, and heterokaryotic mycelium may be uncommon. Many more of the derived single spore lines were compatibility type B2 than compatibility type B 1 • The excess of B 2 lines over B, lines may not necessarily indicate a differential segregation of the two nuclear types from B, nuclei, but rather the differential elimination of B, nuclei. When heterothallic isolates were subcultured as mixtures, the rarer compatibility type was quickly lost from the mixture during successive asexual subculturing. Therefore, as the B" nuclei appear to determine a self-sterile B2 phenotype, then even if B1 and . B2 nuclei formed at equal rates, B, segregants would only be rarely detected among single conidia. B2 segregants would tend not to be eliminated. Many self-fertile isolates of P. drechsleri derived from oospores were not stable and lost their selffertility when subcultured vegetatively (Mortimer et al., 1977). For the asexual subculture of B. lactucae, inoculations were made with large numbers of conidia (which are multinucleate and germinate direct) which were produced by many mycelia. This could have resulted in the apparent
R. W. Michelmore and D. S. Ingram stability of self-fertility over many asexual generations in a continually segregating population. The stability of such isolates in nature may be less as the levels of infection are low. Self-fertility appears to be common, however, as 7 out of 39 isolates of Bi lactucae surveyed produced oospores when cultured alone (Michelmore & Ingram, 1980). Secondary homothallism may be merely a consequence of a heterothallic mating system that is stabilized by a reciprocal translocation. Alternatively, as many downy mildews predominantly form restricted angular lesions, each probably resulting from one or a few conidia, there may have been a selective advantage for some members of the population in a heterothallic species to be potentially inbreeding. The observations reported in this paper provide further evidence of the similarities between sexual systems of B. lactucae and the heterothallic Phytophthora spp. There are, however, small differences: B, nuclei of B. laetucae appear to determine selfsterility rather than self-fertility; no differences in growth rate were detected between the self-fertile and the self-sterile lines derived from single conidia of B. lactucae whereas self-sterile sectors of P. drechsleri grew faster than the self-fertile sectors (Mortimer et al., 1977); some self-sterile A 2 isolates of P. drechsleri have been observed to revert to self-fertility and occasionally to selfsterile A l sectors, but this was not observed with lines IM25,R7 or IM25,P11. It is likely that heterothallism has evolved independently several times in the Peronosporales and, therefore, although in several species the stability of the sexual system may depend upon reciprocal translocation heterozygosity, small differences are to be expected. We wish to thank the Agricultural Research Council for a Research Support Grant. R. W. M. also wishes to thank the Dr Lucy Ernst Scholarship Fund for additional financial support. We thank Dr E. Sansome for helpful discussions and Mr B.
9
Goddard and Mrs R. Chapman for technical assistance. REFERENCES
CRUTE, 1. R. & JOHNSON, A. G. (1976). The genetic relationship between races of Bremia lactucae and cultivars of Lactuca sativa. Annals of Applied Biology 83, 125- 137. DE BRUYN, H. L. G. (1937). Heterothallism in Peronospora parasitica. Genetica 19, 553-558. INGRAM, D. S., TOMMERUP, 1. C. & DIXON, G. R. (1975). The occurrence of oospores in lettucecultivars infected with Bremia lactucae Regel. Transactions of the British Mycological Society 64, 149-154. LONG, M. & KEEN, N. T. (1977). Evidence for heterokaryosis in Phytophthora megasperma var, sojae. Phytopathology 67, 670-674. MICHELMORE, R. W. (1979). A study of sexual reproduction by Bremia lactucae Regel. Ph.D. Thesis, University of Cambridge. MICHELMORE, R. W. & INGRAM, D. S. (1980). Heterothallism in Bremia lactucae Regel. Transactions of the British Mycclogical Society 75, 47-56. MICHELMORE, R. W. & INGRAM, D. S. (1981 a). The origin of gametangia in heterothallic isolates of Bremia lactucae Regel. Transactions of the British Mycological Society 76, 425-432. MICHELMORE, R. W. & INGRAM, D. S. (1981b). The recovery of sexual progeny following sexual reproduction of Bremia laetucae Regel. Transactions of the British Mycological Society. 77, 131-137. MORTIMER, A. M., SHAW, D. S. & SANSOME, E. R. (1977). Genetical studies of secondary homothallism in Phytophthora drechsleri. Archives of Microbiology 111, 255-259. PRATT, R. G. & GREEN, R. J. (1973). The sexuality and population structure of Pythium syloaticum, Canadian Journal of Botany 51, 429-436. SANSOME, E. R. (1980). Reciprocal translocation heterozygosity in heterothallic species of Phytophthora and its significance. Transactions of the British Mycological Society 74, 175-185. TOMMERUP, 1. C., INGRAM, D. S. & SARGENT, J. A. (1974). Oospores of Bremia lactucae. Transactions of the British Mycological Society 62, 145-150.
(Received for publication 12 November 1980)
February 1982
1
Trans, Br , mycol. S oc. 78 (1) 1-9 (1982)
Printed in Great Britain
SECONDARY HOMOTHALLISM IN BREMIA LACTUCAE By R. W. MICHELMORE AN D D. S. INGRAM Th e Botany S chool, University of Cambridge, D owning S treet, Cambridge CB23EA, UK M any isolates of B remia lactucae are heterothallic ; some, howe ver, seemed to be self-fertile. Single conidial lin es were derived from one self-fertile isolate. The majority of the se lines were self-fert ile, but some were self-ste rile. The self-fertile lines were stable over man y asexual generations when subcultured using large numbers of conidia. Attempts to restore self-fertility by cult uri ng self-sterile lines together were not successful. Both normal and abnormal patt ern s of gametangial developm ent were observed by scann ing electron mi croscopy of a self-fertile line. The pattern of segregation of self-fertile and self-sterile lines from a self-fertile isolate of B, lactucae is similar to th at described for the hom othallic isolates of Phytophthora drechsleri. It is suggested th at self-fertility in B. lactu cae, as in P. drechsleri, may be due to the chromosomal determinant s of compatibility-typ e being trisomic following numerical nondis junc tion at meiosis. The segregation of self-sterile lines from the self-fertile isolate may have in volved at least tran sitory heterokaryosis. Bre mia lactucae Regel, th e obliga te biotr oph ic pathogen causing downy mildew of lettuce, has recent ly been shown to be heterothallic (M ichelmore & Ingram, 1980). In a survey, th e majori ty of isolates were found to be self-sterile and of two sexual com patibility type s, designated B, and B2 • Some isolates , however, apparently contained th e det erminant s of both compatibility type s as th ey produce d oospores when cultured alone. These self- fertile isolates pr oduced more oospores when cultur ed in combination with an isolate of compatibiliry type B, than when cultur ed alone or with an isolate of comp at ibility type B2 • This paper describes the det ailed investiga tion of one self-fer tile isolate wh ich suggested the existenc e of secondary hornothaliisn an d heterokaryosis in B . lactu cae ; th is is the ErA investigation of such phenomena in th e Pero nospo ra ccac. The stud ies also pro vided furthe r evidence of the extent of the similarity between B. lactucae and the heterothallic Phytop hthora spe cies, M .~ TER I AL :':
A N D M ETHO D S
'1'1:.. pathogen I solate 11\.125 of B. luctucac W 3 S collected from th e lett uce trial grour. d at ! he N ational Institute of Agricult ural HeLm y Ca.n bridge, and was found to p rod uce nn:;r ;', ~e , rC'4ub riy when cultured alone (M ichelmorc ,.'\;- \l~ gr,'r:l , j9\\;)) . Lines were der ived fro m isolate ) ,\ \ 2) as descri bed below. I solates IL4 and IfvLt -+ hav.. bC,~D .icscribcd by Miche1more & Ingram (19ik ) _Tile ,;;" tJt<-:s and lin es were cultured --- - ' - . -- . ... _--- - - - - - - - 1701·77, Part
3 , 1(" J ~
on detached, fully expanded single cotyledons of L actuca sati va L. (lettuce), ev. Briti sh H ilde, laid out in clear plastic boxes (125 x 80 x 20 mm ) and sealed with N escofilm (N ippon Shuji & Kaisha, Osaka, Japan), as already described (M ichelmore & Ingram, 1980). Incubation was at 15 °C, in darkn ess for the first 24 h and then in a growth room irr adiated for 12 h each day by daylight fluorescent tubes giving a light int ensity of 6'31 W m - 2 at the surface of the boxes. When necessary the lines were stored by freezing newly sporulating cotyledons, sealed in culture boxes, to - 20". Pro cedure f or obtaining lines of B. lactucae fro m single conidia
Single cotyledons oflettuce, cv. British Hilde, were detached from seedlings at the stage of first leaf emergence (seedlings approximately 7 days old) and laid abaxial surface uppermost on moist filter paper in Petri dishes (52 mm diam ), one per dish; when possible dishes were set up 24 h prior to inoculation . In early experiments detached cotyledon pair s were used; however, single cotyledons supported more synchrono us sporulation and were used in later experiments. A small droplet of distilled water (approx, 0'02 ml) was placed on the abaxial surface of each cotyledon . A lettuce cotyledon, on which B. lactucae had been sporu lating asexually for 2 or 3 days, was lightly wiped over th e surface of 0'3 % water agar in a small Petri dish to streak the conidi a onto th e agar. This dish was lit from below using a cool fibre-optic light - - - - - - - - - - - - - - - - - -
i -sued 31. December 1981 MY C
78
2
Secondary homothallism in Bremia
source. Single conidia were located using a dissecting microscope (50 x) and picked off using a drawn-out glass microcapillary which was joined to a shortened Pasteur pipette via a catheter tube (the Pasteur pipette was held in the mouth and, by applying positive or negative pressure, the microcapillary could be flushed with water). The conidia were transferred singly from the surface of the agar to the droplet of water on a cotyledon and the microcapillary flushed to create turbulance in the droplet. After each transfer the microcapillary was washed by repeated flushing in two changes of distilled water. The Petri dishes were labelled so that subcultures from the same cotyledon could be grouped. Only a limited number of subcultures were made from anyone cotyledon (usually 10 or 20). Following inoculation the cotyledons in the Petri dishes were transferred to a dark incubator at 15° for 24 h; care was taken not to dislodge the droplets. After 24 h the Petri dishes were sealed with Nescofilm to prevent desiccation and cross-contamination, and then incubated in the growth room. The number of days between inoculation and the onset of asexual sporulation was noted for each cotyledon (usually six to nine days). Infection resulted in up to 45 % of the single conidium inoculations. Checks to ensure that only one spore was transferred were made by picking up single conidia and then replacing them on the agar; only one conidium was transferred in all such transfers. Also, control cotyledons were sham-inoculated after the microcapillary had been washed and without picking up another conidium; when such 'inoculations' were made, they were alternated with the transfer of single conidia. Sporulation was never observed on the sham-inoculated cotyledons. Approximately 2 days after asexual sporulation had commenced the single spore derivatives were subcultured separately on to 30 lettuce cotyledons in each of two culture boxes. The suspensions of conidia were prepared by shaking the cotyledon in 10 ml distilled water; the conidia in the resulting suspensions were washed once by pelleting in a bench centrifuge to remove a water soluble inhibitor of germination and resuspended in 2 ml fresh distilled water. Each cotyledon was inoculated by placing a drop (approx. 0'03 ml) of a suspension (approx. i x 104 conidia ml r") on the abaxial surface using a 2 ml syringe and needle; this procedure often resulted in all the cotyledons becoming infected. All suspensions were prepared and the inoculations made on a laminar-flow airbench to prevent cross-contamination. The culture boxes were sealed and incubated in the growth room as described. After asexual sporulation had
commenced in these subcultures, one culture box of each line was frozen for storage. Determination of sexual compatibility type and virulence phenotype The compatibility type of the derived lines of B. lactucae was determined by culturing each one either alone or in combination with the type isolates IL4 (compatibility type B I ) or IM44 (compatibility type B2 ) (Michelmore & Ingram, 1980). Using a syringe and needle as described above, 20 single cotyledons in each of three culture boxes were inoculated with a suspension of washed conidia of each line. One day later, one box of each line was further inoculated with isolate IL4 and one box with isolate IM44, by spraying thoroughly with a suspension of conidia. The culture boxes were sealed and incubated in the growth room until decayed and transparent. The cotyledons were then microscopically examined in situ for the presence of oospores. The virulence phenotype of some isolates was determined by inoculating a differential series of 12 resistant lettuce cultivars as has been described in detail elsewhere (Crute & Johnson, 1976; Michelmore & Ingram, 1981 b). Methods of observation The number of cotyledons containing oospores was determined using a dissecting microscope (50 x ). Fresh cotyledons were fixed and cleated in 95 % ethyl alcohol. The material for the observations on the formation of the gametangia was prepared for light microscopy by vacuum infiltration with water and for scanning electron microscopy by chemical fixation and dry-fracture according to the methods of Michelmore & Ingram (1981 a). The intensity of asexual sporulation was determined by shaking the sporulating cotyledons in 10 ml of distilled water and then making counts with a haemocytometer. RESUL TS
Single spore analysis of isolate IM25 One hundred and fourteen lines, each derived from a single conidium of isolate IM25, were bulked up by subculturing on to lettuce cotyledons and the resulting conidia used to determine the sexual compatibility type of each line (Fig. 1). Twenty of the derived lines were of compatibility type B 2, two were of compatibility type B I and 92 (80'7 %) were capable of sexual reproduction when cultured alone and also in combination with at least one of the type isolates (Table 1). The majority of the single spore lines derived from isolate IM25, therefore, were self-fertile.
R. W. Michelmore and D. S. Ingram Isolate IM25 144 lines (produced oospores - - derived from ~-when cultured alone) single conidia
Derived lines bulked-up
~
3
I nocu lated alone
} Determination of the Orwith B,-type isolate compatibility type of each line Orwith B,-type isolate
Isolate IM25, L2 69 lines ~Inoculated alone ~ Determination of the (produced oospores ~derived from Orwith B,-type isolate compatibili~y type of when cultured alone) single conidia Orwith B,-type isolate each line Fig.
Table
1.
1.
Single spore analysis of a self-fertile isolate of B. lactucae.
Sexual compatibility type composition of lines of Bremia lactucae derived from single conidia of self-fertile isolates Compatibility type composition
Parental isolate 1M25 IM25,L2 B, only r B 2 only j
n, >
B2
_ _ _ _ _ _ _ _ _ _- - - - - - A -
_
rr:
B, only
B, > B 2
2 1
B, < B2 88
B 2 only
o
B, = B 2 4
o
0
30
38
20
Total 114 69 183
Self-sterile, heterothallic lines Produced oospores more frequently with the B 2 type-isolate than} with the B, type-isolate Produced oospores as frequently with that B 2 type-isolate as with Self-fertile the B, type-isolate lines Produced oospores less frequently with the B 2 type-isolate than with the Br type-isolate
One self-fertile line, IM25,L2, was chosen for further study. This line continued to produce oospores with similar frequency when subcultured alone over ten asexual generations. A further 69 lines were derived, each from a single conidium of line IM25,L2; the resulting conidia were used to determine the compatibility type of each line without an asexual generation for bulking up (Fig. 1). This was done as it was thought that fluctuations in compatibility type composition might occur during subculturing. Thirty-eight of the lines derived from line IM25,L2 seemed to be B 2 , one to be B, and 30 to be self-fertile (Table 1). Nearly all the self-fertile lines produced large numbers of oospores when cultured with the B, type-isolate but formed oospores infrequently and in small groups when cultured alone or with the B 2 type-isolate; they were therefore considered to be predominantly B 2 • The frequency of oospore production was similar when a self-fertile line was cultured alone and when it was cultured with the B 2 type-isolate. No self-fertile lines were found which produced more oospores with the B 2 typeisolate than with the B, type-isolate. The self-fertile lines derived from isolate IM25,
L2 produced oospores in only 1 or 2 cotyledons out of the 20 inoculated, when cultured alone or with the B 2 type-isolate. Several of the lines scored as self-sterile B 2 lines might, therefore, have been self-fertile, and this would account for the lower proportion of self-fertile lines recorded from line 1M25,L2 as compared to the progeny of IM25. Because the bulking-up stage had been omitted (Fig. 1), only small numbers of conidia could be used to inoculate each cotyledon, resulting in the low frequencies of oospore production (Michelmore & Ingram, 1980). All cotyledons which became infected with B. lactucae following inoculation with single conidia were cleared in 95 % ethyl alcohol and examined microscopically for the presence of oospores. Only two cotyledons were observed to contain oospores, even though the determinants of both compatibility types must have been present in over 80% of infections as shown by the analysis of compatibility type. The time between inoculation with single conidia and the onset of asexual sporulation was not correlated with compatibility type. Sporulation occurred 6-10 days after inoculation and self1-2
4
Secondary homothallism in Bremia
fertile lines sporulated simultaneously with the self-sterile lines. Two self-sterile lines derived from isolate IMz5 (IMz5,R7, compatibility type B 1 ; 1Mz5,P11, compatibility type B2) , line IMz5,Lz and the B 1 line derived from line IMz5,Lz were each inoculated on to a differential series of resistant lettuce cultivars and shown to have the same virulence phenotype as isolate 1Mz5. They were, therefore, not contaminants and virulence did not appear to be segregating in isolate IMz5. Sporulation of self-sterile lines derived from isolate 1Mz5 The self-sterile lines 1Mz5,R7 (compatibility type B1) and IMz5,P11 (compatibility type B 2) were each derived from single conidia of isolate IMz5. Cotyledons were inoculated with each line alone or with the two lines together (1: 1 ratio) at two different inoculum concentrations (Michelmore, 1979). These self-sterile lines sporulated in a manner which was indistinguishable from heterothallic isolates studied previously: no oospores were produced when the lines were cultured singly but large numbers of oospores and few conidia were produced when they were cultured together; sexual and asexual reproduction commenced simultaneously; the time interval between inoculation and sporulation was dependent upon the concentration of conidia in the inoculum; and high levels of sexual reproduction accelerated decay of the lettuce tissue. Also, the gametangia developed in a manner similar to that observed between other heterothallic isolates. Although both lines, when cultured alone, started to sporulate simultaneously, line 1Mz5,R7 sporulated more heavily than line IMz5,P11, producing over twice as many conidia as line IMz5,Pl1 at one inoculum level. Also, cotyledons decayed more rapidly when inoculated with line IMz5,P11. These phenomena were observed in several experiments with these two lines and suggest that during sporulation line IMz5,P11 had a less-balanced relationship with lettuce cv. British Hilde than line 1Mz5,R7. Genetic factors in addition to compatibility type may, therefore, have been segregating in isolate 1Mz5. Whether this segregation is related to the segregation of compatibility type in B. laetucae requires further investigation, but these observations emphasize the capacity of B. laetucae for somatic variation. Subculture of mixtures of heterothallic derivatives of isolate 1Mz5 Experiments described above suggested that selfsterile components might be segregating from a heterokaryotic mycelium. The formation and
100
90
80 OJ '" 0c.
70 0 '" 0 Cl
c:
c ~
60
c: 0 o
c '" 50 0
""0
OJ
e-
+-'
0
o ""0
40
so
.E c:
15
30
d'2.
20
10
II
III
IV
V
Number of asexual generations
Fig. 2. Frequency of oospore production by B. lactucae in cotyledons of lettuce cv. British Hilde, over five asexual generations. Lettuce cotyledons were initially inoculated with a mixture of self-sterile isolates IM25,R7 (B 1) and 1M25,P11 (B2) in: (i) a 10:1 ratio et 5 x 10 4 conidia ml- 1 (A) or 1 x 103 conidia ml " (6); (ii) a 1: 10 ratio at 5 x to' conidia ml" (.) or 1 x 103 conidia ml " (D). Each treatment line was subcultured every 7 or 8 days at these concentrations of conidia, onto 120 fresh cotyledons for the treatments inoculated with 5 x 10' conidia ml- 1 and on to 150 fresh cotyledons for the treatments inoculated with 1 x 103 conidia ml- 1• Each value represents the percentage of infected cotyledons in which oospores were produced.
segregation of heterokaryons has been demonstrated in Phytophthora megasperma var. sojae using auxotrophic and drug-resistant mutants (Long & Keen, 1977). To investigate the basis of selffertility, attempts were made to reform self-fertile heterokaryons from self-sterile lines derived from isolate IMz5. Suspensions of washed conidia of lines IM z5, R7 and IMz5,P11 were sprayed on to cotyledons
R. W. Michelmore and D. S. Ingram in the manner already described (M ichelmore & Ingram, 1980) at two concentrations, 5 x 10~ and 1 x 103 ml- I , each in the ratios 10: 1 and 1: 10, IM25, R7: IM25,PU. The treatments inoculated with 5 x 10~ conidia ml'< were cultured on 120 single detached cotyledons and the treatments inoculated with 1 x 103 conidia ml- I were cultured on 150 cotyledons. As checks, each line was also cultured alone at 5 x 10~ conidia ml" on 60 single cotyledons. The six treatments were subcultured every 7 or 8 days on to fresh cotyledons for five asexual generations. The incidence of oospore production was determined after each round of culture. After five asexual generations, spores from each experimental treatment were cultured in combination with each of the type isolates and examined for oospore production, to determine the compatibility type composition. The frequency of oospore production declined during subculturing and after five asexual generations, none of the treatments produced oospores (Fig. 2). The decline was most rapid in the treatments inoculated with 5 x 10~ conidia ml- I • Oospores were never observed in the check treatments of each isolate cultured alone. The compatibility type that was the minor component at the beginning of the experiment was eliminated during five asexual generations. As no stable self-fertile isolates were obtained in this experiment, nor in similar experiments repeated with other heterothallic isolates, it was concluded that heterokaryons with respect to sexual compatibility type were not regularly formed between isolates of opposite compatibility type under the conditions of these experiments. The failure to produce self-fertile isolates by culturing isolates of opposite compatibility types together does not demonstrate that hyphal fusion resulting in heterokaryosis never occurs; the experimental procedures may not have favoured heterokaryon formation or may not have detected heterokaryons if they occurred at low frequencies. Continual formation and segregation of heterokaryons, however, do not appear to be the basis for the self-fertility observed in B. lactucae, Formation of gam etangia by line 1M25,L2
Studies of sexual reproduction by heterothallic isolates of B. lactu cae have shown that the formation of the gametangia is preceded by a tight and complex interaction between hyphae presumed to be of opposite compatibility type; oogonia were never observed to occur without an associated antheridium (M ichelmore & Ingram, 1981 a). Light-microscopical observation s of sexual reproduction in cotyledons inoculated with line IM25, L2 indicated that the gametangia did not develop
5
in the manner observed with heterothallic isolates. Cotyledons in which oogonia had been observed by light microscopy were therefore prepared for scanning electron microscopy and the development of the gametangia was examined more closely. In the self-fertile line IM25,L2 close interactions between two or more vegetative hyphae preceded the development of sexual hyphae and gametangia (Fig. 3) as had been observed with heterothallic isolates. However, a different pattern of sexual development was also observed. In zones where sexual reproduction occurred, many thin flexuous hyphae which had often collapsed during preparation were seen (Figs 3-6); they had arisen either as branches from vegetative hyphae or, apparently, from the apex of a vegetative hypha (Fig. 5). These hyphae had often grown for considerable distances through the cotyledons, branching irregularly with inconsistent dimensions. Contact between the thin hyphae and other vegetative hyphae seemed to have often resulted in the further production of thin hyphae; where interactions between thin hyphae had occurred, there were usually no tight associations, but instead the branching pattern was irregular and loose (F ig. 6). It was often impossible to observe the antheridiurn developing beside an oogonium; in one half-cotyledon many oogonia were observed but only one antheridium was seen (F ig. 3). There was, however, always contact between the oogonium and developing thin hyphae or occasionally a vegetative hypha (Fig. 7). On one occasion, a hypha1 swelling, very similar in appearance to an antheridium, was observed without an associated oogonium (Fig. 8). Oogonia and antheridia therefore seemed to be able to develop independently of each other in this self-fertile isolate, although contact with other hyphae was still necessary. Light microscopy confirmed the SEM observations; often antheridia were small or not visible, but in some cotyledon s the oogonia and antheridia regularly developed together. The thin hyphae gave the impression of a much increased hyphal density in zones of the cotyledon where sexual reproduction occurred. Sexual reproduction tended to occur in limited patches rather than evenly throughout the cotyledon and was less synchronous than when heterothallic isolates of opposite compatibility type were cultured together. When infected cotyledons were vacuum infiltrated with water and then immediately observed with the light microscope, vigorous cytoplasmic streaming up and down the vegetative hyphae, in and out of the haustoria and to a lesser extent in and out of the developing sexual hyphae, could be seen. In a heterokaryotic mycelium therefore the nuclei which determine the event s initiating sexual repro-
6
Secondary homothallism in Bremia
Figs. 3-8. Scanning electron micrographs showing the development of gametangia in a self-fertile line of B. lactucae in cotyledons of lettuce, cv. British Hilde, following inoculation with line IMz5, Lz at 5 x 10' conidia ml ". Seven days after inoculation the cotyledons were chemically fixed, dehydrated, critical point dried, dry fractured and sputter-coated with gold (Michelmore & Ingram, 1981 a). The figures show the following: Fig. 3. the tight associations which were sometimes observed (double arrow); in the background are the only oogonium and antheridium observed to be developing together in this half-cotyledon; Fig. 4. the irregular morphology of the thin hyphae and their tendency of collapse during preparation; Fig. 5. the extensive growth of the thin hyphae; Fig. 6. the loose branching pattern of the sexual hyphae;
R. W. Michelmore and D. S. Ingram
7
8 Fig. 7. an oogonium developing apparently without an attached antheridium ; Fig. 8. the thin sexual hyphae and what appears to be an antheridium which has developed without an oogonium. a = antheridium s = sexual hyphae h = haustorium u = underside of the lower epidermis 0= oogonium v = vegetative hyphae p = plant mesophyll cell Scale bar = 5,um
duction may not be the same as those determining subsequent sexual development. This may at least partly explain the loose and apparently disorganized nature of sexual development in self-fertile isolates. DISCUSSION
These studies demonstrate that the self-fertile isolate of B. lactucae, IM25, exhibited a form of homothallism and was not a mixture of heterothallic isolates of opposite compatibility types. Selffertile lines have been reported for other heterothallic members of the Peronosporales: Phytophthora spp. (Mortimer, Shaw & Sansome, 1977), Pythium syluaticum (Pratt & Green, 1973) and Peronospora parasitica (De Bruyn, 1937). The most detailed studies have been made with Phytophthora drechsleri where genetic and cytological experiments have indicated that the self-fertile lines are trisomic for the determinants of compatibility type (Mortimer et al., 1977; Sansome, 1980; Sansome, Mortimer & Shaw, unpub!.). Cytological studies have demonstrated that the cytology of B. lactucae is similar to that of P. drechsleri, and isolate IM25 appears to have an extra chromosome associated at meiosis with a reciprocal translocation (Sansome & Michelmore, unpub!.). The pattern
of asexual segregation of heterothallic components described in this paper for B. lactucae is similar to that observed in some lines of P. drechsleri. The self-fertility of isolate IM25 and derived lines of B.lactucae may therefore be a similar form of secondary homothallism. Isolate IM25 was repeatedly subcultured asexually for most of a 3-year period and showed no decrease in the tendency to produce oospores; the self-fertile line IM25,Lz seemed to have a similar sexual stability. When isolates of opposite compatibility type were subcultured together, however, one compatibility type was eliminated from the population very quickly, often within three asexual generations and always within five, depending on the concentration of conidia in the inoculum. This suggests that the phenotypes of lines IM25,R7 and IM25,P11 were the result of segregation from isolate IM25 rather than that isolate IM25 was a mixture of unrelated lines. The self-fertile isolates detected in the survey reported previously (Michelmore & Ingram, 1980) were also probably homothallic as they had been subcultured over several or many asexual generations. The isolates W1, W3 and W5 used in previous studies of sexual reproduction of B. lactucae (Tommerup, Ingram & Sargent, 1974; Ingram, Tommerup &
8
Secondary homothallism in Bremia
Dixon, 1975) are also likely to have been homothallic for the same reason. The infrequent and patchy production of oospores by isolates W1, W3 and W5 was similar to that observed in the selffertile derivatives of isolate IM25. Studies are now needed to determine whether self-fertile isolates of B. lactucae exist which do not segregate self-sterile components and are therefore similar to some stable self-fertile forms of P. drechsleri. The data for the sexual compatibility of the lines derived from single conidia suggested that selfsterile components with opposite compatibility types could segregate from one mycelium and reproduce sexually; the majority of the mycelia, however, apparently contained the determinants of both compatibility types (B1+ 2 mycelia), and it is less evident what sexual phenotypes were exhibited by such B1+ 2 mycelia. Large numbers of oospores were produced only when self-fertile lines were cultured with the Bj-type isolate; when self-fertile lines were cultured with the Bctype isolate, no more oospores were produced than when they were cultured alone. When heavy oospore production occurred following inoculation with a self-fertile line in combination with a B, isolate there was very little asexual sporulation, indicating that the majority of the mycelia were involved in sexual reproduction (M ichelmore & Ingram, 1980). The B1+ 2 mycelia therefore seemed to respond as compatibility type B 2 when in contact with B 1 mycelia but responded rarely, if at all, as compatibility type B 1 when in contact with B2 mycelia. The observations sugge st that oospore production in self-fertile lines cultured alone resulted from the interaction between the hyphae of B 1 mycelia and the hyphae of B1+ 2 or B 2 mycelia, rather than that sexual reproduction involved only one mycelium which the term homothallism strictly implies. Therefore, the sexual reproduction of all isolates of B. lactucae so far studied, whether self-fertile or self-sterile, may be considered to be essentially heterothallic. Oospores were produced only infrequently in the cotyledons in which the single spore isolations were made (2 out of 183), even though approximately 80 % of the lines were subsequently found to be self-fertile. This is probably due to the threedimensional pattern of the hyphal growth within the host tissue. Mycelia tend to radiate out from the point of establishment; when there was only one infection in a cotyledon, the contacts between hyphae of opposite compatibility type which are necessary for sexual reproduction would have been infrequent. However, oospores were observed occasionally to develop from single infections, and therefore, sexual reproduction must have occurred between hyphae of the same mycelium, but the
above discussion suggests that vegetative segregation of the compatibility types would have occurred first. As lines of B. lactucae with defined marker genes do not exist, the possibility cannot be excluded that the data reflect the segregation of nonchromosomal determinants. Evidence is accumulating, however, that sexual compatibility type in heterothallic Phytophthora spp. is controlled by chromosomal determinants and it is useful to consider the consequences of a similar interpretation for B. lactucae. Self-fertile (homothallic) isolates of predominantly heterothallic Phytophthora spp, have frequently been recovered in the progeny after sexual reproduction and are thought to have been the result of numerical non-disjunction at meiosis producing a gametic nucleus which was disornic for the determinants of compatibility type and which gave rise to a trisomic zygote (Mortimer et al., 1977; Sansome, Mortimer & Shaw, unpub!.). It is possible that the self-fertile isolates of B. lactucae similarly resulted from non-disjunction at meiosis (Sansome & Michelmore, unpub!.). This would imply that the BI+2 mycelia of a self-fertile isolate of B. lactucae are heterogeneous populations of nuclei of at least three types: unstable trisomic nuclei (B, nuclei) and nuclei with phenotypes of only one compatibility type (B, or B 2 nuclei) which arose either as the result of chromosome loss or due to somatic recombination from B, nuclei. Depending on the relative rates of formation and segregation of the heterothallic nuclei, heterokaryosis may only be transitory, and heterokaryotic mycelium may be uncommon. Many more of the derived single spore lines were compatibility type B2 than compatibility type B 1 • The excess of B 2 lines over B, lines may not necessarily indicate a differential segregation of the two nuclear types from B, nuclei, but rather the differential elimination of B, nuclei. When heterothallic isolates were subcultured as mixtures, the rarer compatibility type was quickly lost from the mixture during successive asexual subculturing. Therefore, as the B" nuclei appear to determine a self-sterile B2 phenotype, then even if B1 and . B2 nuclei formed at equal rates, B, segregants would only be rarely detected among single conidia. B2 segregants would tend not to be eliminated. Many self-fertile isolates of P. drechsleri derived from oospores were not stable and lost their selffertility when subcultured vegetatively (Mortimer et al., 1977). For the asexual subculture of B. lactucae, inoculations were made with large numbers of conidia (which are multinucleate and germinate direct) which were produced by many mycelia. This could have resulted in the apparent
R. W. Michelmore and D. S. Ingram stability of self-fertility over many asexual generations in a continually segregating population. The stability of such isolates in nature may be less as the levels of infection are low. Self-fertility appears to be common, however, as 7 out of 39 isolates of Bi lactucae surveyed produced oospores when cultured alone (Michelmore & Ingram, 1980). Secondary homothallism may be merely a consequence of a heterothallic mating system that is stabilized by a reciprocal translocation. Alternatively, as many downy mildews predominantly form restricted angular lesions, each probably resulting from one or a few conidia, there may have been a selective advantage for some members of the population in a heterothallic species to be potentially inbreeding. The observations reported in this paper provide further evidence of the similarities between sexual systems of B. lactucae and the heterothallic Phytophthora spp. There are, however, small differences: B, nuclei of B. laetucae appear to determine selfsterility rather than self-fertility; no differences in growth rate were detected between the self-fertile and the self-sterile lines derived from single conidia of B. lactucae whereas self-sterile sectors of P. drechsleri grew faster than the self-fertile sectors (Mortimer et al., 1977); some self-sterile A 2 isolates of P. drechsleri have been observed to revert to self-fertility and occasionally to selfsterile A l sectors, but this was not observed with lines IM25,R7 or IM25,P11. It is likely that heterothallism has evolved independently several times in the Peronosporales and, therefore, although in several species the stability of the sexual system may depend upon reciprocal translocation heterozygosity, small differences are to be expected. We wish to thank the Agricultural Research Council for a Research Support Grant. R. W. M. also wishes to thank the Dr Lucy Ernst Scholarship Fund for additional financial support. We thank Dr E. Sansome for helpful discussions and Mr B.
9
Goddard and Mrs R. Chapman for technical assistance. REFERENCES
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(Received for publication 12 November 1980)