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A simple, reliable method of producing perithecia of Gaeumannomyces gramnis var. tritici and its application to isolates of Phialophora spp.
Notes and brief articles
350
MENNA, M. E. & MANTLE, P. G. (1978). The role of penicillia in ryegrass staggers. Research in Veterinary Science 24, 347-351. PATTERSON, D. S. P., ROBERTS, B. A., SHREEVE, B. J., MACDONALD, S. M. & HAYES, A. W. (1979). Tremorgenic toxins produced by soil fungi. Applied and Environmental Microbiology 37, 172-173. Dr
PITT, J. 1. (1979a). Penicillium crustosum and P. simplicissimum, the correct names for two common species producing tremorgenic mycotoxins. Mycologia 71, 1166- 1177. PITT, J. I. (1979b). The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic Press.
A SIMPLE, RELIABLE METHOD OF PRODUCING PERI THEClA OF GAEUMANNOMYCES GRAMNIS VAR. TRITICI AND ITS APPLICATION TO ISOLATES OF PHIALOPHORA SPP. BY J. B. SPEAKMAN* School of Biological Sciences, University of East Anglia, Norwich NR4 7 TJ, England
The exact requirements for producing perithecia in isolates of the take-all fungus, Gaeumannomyces graminis (Sacc.) Arx & Olivier var. tritici J. Walker (Ggt) are not known, although perithecia have been grown successfully on a variety of agar media, on sterilized natural media and on dead or living hosts. A review of the literature has been published recently by Holden & Hornby (1981). The opinion generally held is that there is no consistent method of producing fertile perithecia (Walker, 1975). Holden & Hornby (1981) tested 57 isolates offungi of the Gaeumannomyces-Phialophora complex for their ability to produce perithecia in vivo and in vitro by a range of methods. Their results with isolates of Ggt indicated that no one method was the best, and that in vivo methods were more effective than in vitro methods. Application of the methods to isolates of Phialophora radicicola Cain var. graminicola Deacon (Prg, re-named P. graminicola (Deacon), Walker, 1980) and P. radicicola Cain var. radicicola sensu Deacon (Prr, re-named Phialophora sp. (lobed hyphopodia), Walker, 1980) gave no positive result, although perithecia of G. cylindrosporus Hornby, Slope, Gutteridge & Sivanesan (anamorph Prg) have been obtained on infected seedling roots in other studies (Hornby, 1980; Hornby, Slope, Gutteridge & Sivanesan, 1977). The method proposed here used sterile wheat seedlings growing on water agar in plastic Petri dishes, thus providing a medium which both maintained the pathogenicity of all the isolates and which supported the production of fertile perithecia in all isolates of Ggt tested. Isolates of Ggt, Prg and Prr used in the study are listed in Table 1. Stock cultures were maintained on slants of full-strength potato dextrose agar
* Present address: Biologische Bundesanstalt,Schlosskoppelweg 8, D-2305 Heikendorf-Kitzeberg, West Germany. Trans. Br. Mycol. Soc. 79 (2), (1982).
(PDA), a medium which contained (per I), 4g Difco potato extract, 20 g dextrose and 15 g Oxoid NO.3 agar, adjusted with 0'1 M-NaOH to pH 6'5-7'0, under liquid paraffin in McCartney bottles at 4 0c. Full-strength PDA has been successfully used to grow fungi for storage under paraffin elsewhere (Speakman & Lewis, 1980; Holden & Hornby, 1981). Immediately prior to the seedling infection tests, Petri dish cultures of the isolates on full-strength PDA were prepared from the stock cultures. Seeds of Triticum aestivum cv. Cappelle-Desprez were initially soaked for approx. 20 h in tap water. The water was poured away and a drop of wetting agent (Tween 80) added to the seeds, which were then covered with sodium hypochlorite solution (minimum available chlorine 8 %) for 30 min. The hypochlorite solution was poured away and the seeds were rinsed a minimum of four times with fresh changes of sterile distilled water. Alternatively, a 0'1 '10 silver nitrate solution was used. After 10 min immersion in this solution, the seeds were initially rinsed with a o·5 % sodium chloride solution, which precipitates the remaining silver ions and halts the sterilizing effect, followed by three changes of sterile distilled water. The seeds were then allowed to germinate on Petri dishes of PDA maintained at room temperature (20-22 0 ) for 48 h. The seeds retained their viability and the majority were found to be surface-sterile following this treatment. Surface-sterile seedlings were subsequently transferred individually to plastic Petri dishes containing 20 ml distilled-water agar and these were then inoculated (close to the seminal roots) with an agar disk taken from a culture of the isolate under test. Adhesive tape was used to prevent the developing seedling raising the lid of the Petri dish. The prepared Petri dishes were then placed in a northfacing window and incubated under natural
Printed in Great Britain
Table Identity in paper Ggt 1* 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Prg 1:1: 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 13 Prr 1 2 3 4 5 6 7 8 9 10 II
1.
List of Gaeumannomyces and Phialophora isolates Original code 01 PO 73/58 PO 75/78 G 73/1 G 73/ 2 G 73/3 G 74/1 G 74/2 G 74/4 G 74/ 5 M 320 M 356 M 379 W 85 W 87 W 88 W 89 W 90 W 91 W 125 W 126 W 156 80 bi 80 b3 80 Cl 80 c3 PPR 71/30 PR 73/1 PR 73/2 PR 73/3 PR 73/4 PR 73/5 PR 73/6 PR 73/7 PR 73/8 PR 73/10 PR 74/1 PR 74/2 PR 75/1 PR 75/2 PR 75/3 PR 75/4 W 122 P 1/81 PR 74/3 PR 74/4 B2 B3 B7 B 11 Br 1 Br 5 Br 8 Sb 1 Sb 8
Provenance (host, place, date , isolated by) Gaeumannomyces graminis var. tritici (N) , Norw ich , 1973t, I. Geeson W, Cambridge, 1973t, PRS W, Cambridge, 1973t, PRS W, Haughley, 1973, IBS W, Haughley, 1973, IBS W, Haughley, 1973, IBS W, Haughley, 1974, JBS W , Haughley, 1974, JBS W, Haughley, 1974, IBS W, Norwich, 1974, IBS M , Braunschweig, 1977, WK M . Saarbriicken, 1978, WK M , Saarbriicken, 1979, WK W, Braunschweig, 1978, WK W, Berlin, 1978, WK W, Saarbrucken, 1978, WK W, Saarbriicken, 1978, WK W, Muxall, 1978, WK W, Muxall, 1978, WK W, Hohenschulen, 1980, IBS W, Hohenschulen, 1980, IBS W, Braunschweig, 1981, IBS W, Kitzeberg, 1980, HM W, Kitzeberg, 1980, HM W, Kitzeberg, 1980, HM W, Kitzeberg, 1980, HM Phialophora radicicola var , graminicola Grass, Burle y, 1973t, PRS Grass, Norwich, 1973, JBS Grass , Norwich, 1973, IBS Grass , Norwich, 1973, IBS Grass, Norwich, 1973, IBS Grass, Norwich, 1973, JBS Grass, Norwich, 1973, JBS Grass, Norwich, 1973, IBS Grass, Norwich, 1973, IBS Lolium perenne, Haughley, 1973, IBS Gra ss, Norwich, 1974, IBS Gra ss, Norwich, 197th IBS L . p4renne, Reckenhdlz, 1975,,]BS L. perenne, Reckenholz, 1975, IBS L. perenne, Reckenholz, 1975, IBS L. multiflorum, Unterengstringen, 1975, IBS L. perenne, Kitzeberg, 1980, JBS L. multiflorum, Hornsee, 1981, IBS Phialophora radicicola var . radicicola M, Norwich, 1974, JBS M, NorwicI\ 1974, JBS BM, Berlin, 198o'IJBS BM , Berlin, 1980, JBS BR, Berlin, 1980, JBS BW, Berlin, 1980, JBS BM, Braunschweig, 1980, JBS BW, Braunschweig, 1980, JBS BM , Braunschweig, 1980, IBS BM, Saarbriicken, 1980, IBS BM , Saarbriicken, 1980, IBS
M = Maize (Zea may s), W = Wheat ( T riticum aestivum ), (N ) = no information . BM = Bioassay of ma ize soil, BR = Bioassay of winter rape soil, BW = Bioassay of winter wheat soil. WK = W. Kriiger, HM = H . Mielke, PRS = P. P.. Scott, JBS = J . B. Speakman. * Originally identified as Ophiobolus graminis. t Date recei ved. :\: Originally identified as Phialophora radicicola. Trans . Br. Mycol. Soc. 79 (2), (1982).
Printed in Great Britain
Notes and brief articles
352
'.
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.
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........
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.
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' . ..
Fig. L Fertile per ithecium of Gaeumannomy ces graminis var. trit ici. Fig . 2. Ostiole of the perithecium in Fig . L Fig . 3. Mature ascus being discharged through the ost iole of a perithecium . Fig . 4. The same ascus as in Fig . 3, with visible ascospores.
Trans. Br . My col. Soc. 79 (2), (1982) .
Printed in Grear Britain
Notes and brief articles light conditions at laboratory temperature (20-22 0 ) . Periodic inspections of the dishes were made and the time of initiation of perithecial production was noted. Perithecial initials were observed in all Ggt isolates after approx. 21 days and these developed into fertile perithecia within another 14 days. Fertile perithecia were prepared for examination by stereo-electron microscopy (SEM) by the method described by Lewis & Day (1972), modified as described by Speakman (1977). Figs 1-4 illustrate the results obtained. The necks of the perithecia (Fig. 1) were found to vary greatly in length and the ostiole was often clearly visible (Fig. 2). The ascospores within an ascus as it is being discharged through the ostiole are visible in Figs 3 and 4. Perithecia were not produced by any of the Prg or Prr isolates, even after prolonged (over 6 months) incubation, although Hornby (1980) and Hornby et al. (1977) have reported that perithecia developed on the rotted roots of wheat seedlings infected with isolates of Prg and incubated as described by Hornby (1969). All Prr isolates produced sclerotial bodies. As Prr has been previously isolated mainly from maize (Zea mays L.) (Deacon, 1974; Speakman & Lewis, 1978), infection tests were also prepared using surface-sterilized maize seedlings grown in plastic Petri dishes on distilled-water agar. Fertile perithecia were produced by the isolates of Ggt tested, but none of the Prg or Prr isolates produced perithecia even after prolonged incubation; sclerotial bodies were formed by all Prr isolates. Davis (1925), Willets (1961) and Holden & Hornby (1981) have reported that the production of perithecia by isolates of Ggt tends to become intermittent or cease in cultures maintained for long periods. The oldest isolate used in this investigation was Ggt 1 which, at the time of its being received, had been at least 10 years in the culture collection at the University of East Anglia, Norwich (]. Geeson, pers. comm.). During this period the isolate had been regularly sub-cultured on PDA; it was only weakly pathogenic and perithecial production was low and erratic. Finally, the infection procedure described in this paper may provide a method of maintaining the pathogenicity of isolates. Sections of the seminal roots could be removed 5-7 days after infection, surface-sterilized and plated on sterile Petri dishes of PDA. With this aim in mind, the twenty-six Ggt isolates were re-isolated from wheat seminal roots every 9 months. Apart from isolate Ggt 1,
Trans. Br. Mycol. Soc. 79 (2), (1982). 12
353
pathogenicity was maintained by this procedure and yields of perithecia were high and fairly constant. The author wishes to thank Drs M. C. Cowan and B. G. Lewis for their contributions during this research. REFERENCES
DAVIS, R. J. (1925). Studies on Ophiobolus graminis Sacco and the take-all disease of wheat. Journal ofAgricultural Research 31, 801-825. DEACON, J. W. (1974). Further studies on Phialophora radicicola and Gaeumannomyces graminis on roots and stem bases of grasses and cereals. Transactions of the British Mycological Society 63, 307-327. HOLDEN, M. & HORNBY,D.(1981). Methods of producing perithecia of Gaeumannomyces graminis and their application to related fungi. Transactions of the British Mycological Society 77, 107-118. HORNBY, D. (1969). Methods of investigating populations of the take-all fungus (Ophiobolus graminis) in soil. Annals of Applied Biology 64, 503-513. HORNBY, D. (1980). Gaeumannomyces-Phialophora complex. Rothamsted Experimental Station. Reportfor 1979, part 1, p. 171HORNBY, D., SLOPE, D. B., GUTTERIDGE, R. J. & SIVANESAN, A. (1977). Gaeumannomyces cylindrosporus, a new ascomycete from cereal roots. Transactions of the British Mycological Society 69, 21-25. LEWIS, B. G. & DAY, J. R. (1972). Behaviour of uredospore germ tubes of Puccinia graminis tritici in relation to the fine structure of wheat leaf surfaces. Transactions of the British Mycological Society 58, 139-145. SPEAKMAN, J. B. (1977). Interactions between Gaeumannomycesgraminis and Phialophora radicicola in relation to take-all disease of wheat. Ph.D. thesis, University of East Anglia, Norwich. SPEAKMAN, J. B. & LEWIS, B. G. (1978). Limitation of Gaeumannomyces graminis by wheat root responses to Phialophora radicicola. New Phytologist 80, 373-380. SPEAKMAN, J. B. & LEWIS, B. G. (1980). Vitamin requirements of Phialophora radicicola var. graminicola compared with Gaeumannomyces graminis var. tritici. Transactions of the British Mycological Society 74, 4 10-413. WALKER, J. (1975). Take-all disease of Gramineae: a review of recent work. Review of Plant Pathology 54, 113-1 44. WALKER, J. (1980). Gaeumannomyces, Linocarpon, Ophiobolus and several other genera of scolecospored ascomycetes and Phialophora conidial states, with a note on hyphopodia. Mycotaxon 11, 1-129. WILLETS, H. J. (1961). A comparison between Ophiobolus graminis and Ophiobolus graminis var. avenae. Transactions of the British Mycological Society 44, 504-510.
Printed in Great Britain MYC
79
350
MENNA, M. E. & MANTLE, P. G. (1978). The role of penicillia in ryegrass staggers. Research in Veterinary Science 24, 347-351. PATTERSON, D. S. P., ROBERTS, B. A., SHREEVE, B. J., MACDONALD, S. M. & HAYES, A. W. (1979). Tremorgenic toxins produced by soil fungi. Applied and Environmental Microbiology 37, 172-173. Dr
PITT, J. 1. (1979a). Penicillium crustosum and P. simplicissimum, the correct names for two common species producing tremorgenic mycotoxins. Mycologia 71, 1166- 1177. PITT, J. I. (1979b). The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic Press.
A SIMPLE, RELIABLE METHOD OF PRODUCING PERI THEClA OF GAEUMANNOMYCES GRAMNIS VAR. TRITICI AND ITS APPLICATION TO ISOLATES OF PHIALOPHORA SPP. BY J. B. SPEAKMAN* School of Biological Sciences, University of East Anglia, Norwich NR4 7 TJ, England
The exact requirements for producing perithecia in isolates of the take-all fungus, Gaeumannomyces graminis (Sacc.) Arx & Olivier var. tritici J. Walker (Ggt) are not known, although perithecia have been grown successfully on a variety of agar media, on sterilized natural media and on dead or living hosts. A review of the literature has been published recently by Holden & Hornby (1981). The opinion generally held is that there is no consistent method of producing fertile perithecia (Walker, 1975). Holden & Hornby (1981) tested 57 isolates offungi of the Gaeumannomyces-Phialophora complex for their ability to produce perithecia in vivo and in vitro by a range of methods. Their results with isolates of Ggt indicated that no one method was the best, and that in vivo methods were more effective than in vitro methods. Application of the methods to isolates of Phialophora radicicola Cain var. graminicola Deacon (Prg, re-named P. graminicola (Deacon), Walker, 1980) and P. radicicola Cain var. radicicola sensu Deacon (Prr, re-named Phialophora sp. (lobed hyphopodia), Walker, 1980) gave no positive result, although perithecia of G. cylindrosporus Hornby, Slope, Gutteridge & Sivanesan (anamorph Prg) have been obtained on infected seedling roots in other studies (Hornby, 1980; Hornby, Slope, Gutteridge & Sivanesan, 1977). The method proposed here used sterile wheat seedlings growing on water agar in plastic Petri dishes, thus providing a medium which both maintained the pathogenicity of all the isolates and which supported the production of fertile perithecia in all isolates of Ggt tested. Isolates of Ggt, Prg and Prr used in the study are listed in Table 1. Stock cultures were maintained on slants of full-strength potato dextrose agar
* Present address: Biologische Bundesanstalt,Schlosskoppelweg 8, D-2305 Heikendorf-Kitzeberg, West Germany. Trans. Br. Mycol. Soc. 79 (2), (1982).
(PDA), a medium which contained (per I), 4g Difco potato extract, 20 g dextrose and 15 g Oxoid NO.3 agar, adjusted with 0'1 M-NaOH to pH 6'5-7'0, under liquid paraffin in McCartney bottles at 4 0c. Full-strength PDA has been successfully used to grow fungi for storage under paraffin elsewhere (Speakman & Lewis, 1980; Holden & Hornby, 1981). Immediately prior to the seedling infection tests, Petri dish cultures of the isolates on full-strength PDA were prepared from the stock cultures. Seeds of Triticum aestivum cv. Cappelle-Desprez were initially soaked for approx. 20 h in tap water. The water was poured away and a drop of wetting agent (Tween 80) added to the seeds, which were then covered with sodium hypochlorite solution (minimum available chlorine 8 %) for 30 min. The hypochlorite solution was poured away and the seeds were rinsed a minimum of four times with fresh changes of sterile distilled water. Alternatively, a 0'1 '10 silver nitrate solution was used. After 10 min immersion in this solution, the seeds were initially rinsed with a o·5 % sodium chloride solution, which precipitates the remaining silver ions and halts the sterilizing effect, followed by three changes of sterile distilled water. The seeds were then allowed to germinate on Petri dishes of PDA maintained at room temperature (20-22 0 ) for 48 h. The seeds retained their viability and the majority were found to be surface-sterile following this treatment. Surface-sterile seedlings were subsequently transferred individually to plastic Petri dishes containing 20 ml distilled-water agar and these were then inoculated (close to the seminal roots) with an agar disk taken from a culture of the isolate under test. Adhesive tape was used to prevent the developing seedling raising the lid of the Petri dish. The prepared Petri dishes were then placed in a northfacing window and incubated under natural
Printed in Great Britain
Table Identity in paper Ggt 1* 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Prg 1:1: 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 13 Prr 1 2 3 4 5 6 7 8 9 10 II
1.
List of Gaeumannomyces and Phialophora isolates Original code 01 PO 73/58 PO 75/78 G 73/1 G 73/ 2 G 73/3 G 74/1 G 74/2 G 74/4 G 74/ 5 M 320 M 356 M 379 W 85 W 87 W 88 W 89 W 90 W 91 W 125 W 126 W 156 80 bi 80 b3 80 Cl 80 c3 PPR 71/30 PR 73/1 PR 73/2 PR 73/3 PR 73/4 PR 73/5 PR 73/6 PR 73/7 PR 73/8 PR 73/10 PR 74/1 PR 74/2 PR 75/1 PR 75/2 PR 75/3 PR 75/4 W 122 P 1/81 PR 74/3 PR 74/4 B2 B3 B7 B 11 Br 1 Br 5 Br 8 Sb 1 Sb 8
Provenance (host, place, date , isolated by) Gaeumannomyces graminis var. tritici (N) , Norw ich , 1973t, I. Geeson W, Cambridge, 1973t, PRS W, Cambridge, 1973t, PRS W, Haughley, 1973, IBS W, Haughley, 1973, IBS W, Haughley, 1973, IBS W, Haughley, 1974, JBS W , Haughley, 1974, JBS W, Haughley, 1974, IBS W, Norwich, 1974, IBS M , Braunschweig, 1977, WK M . Saarbriicken, 1978, WK M , Saarbriicken, 1979, WK W, Braunschweig, 1978, WK W, Berlin, 1978, WK W, Saarbrucken, 1978, WK W, Saarbriicken, 1978, WK W, Muxall, 1978, WK W, Muxall, 1978, WK W, Hohenschulen, 1980, IBS W, Hohenschulen, 1980, IBS W, Braunschweig, 1981, IBS W, Kitzeberg, 1980, HM W, Kitzeberg, 1980, HM W, Kitzeberg, 1980, HM W, Kitzeberg, 1980, HM Phialophora radicicola var , graminicola Grass, Burle y, 1973t, PRS Grass, Norwich, 1973, JBS Grass , Norwich, 1973, IBS Grass , Norwich, 1973, IBS Grass, Norwich, 1973, IBS Grass, Norwich, 1973, JBS Grass, Norwich, 1973, JBS Grass, Norwich, 1973, IBS Grass, Norwich, 1973, IBS Lolium perenne, Haughley, 1973, IBS Gra ss, Norwich, 1974, IBS Gra ss, Norwich, 197th IBS L . p4renne, Reckenhdlz, 1975,,]BS L. perenne, Reckenholz, 1975, IBS L. perenne, Reckenholz, 1975, IBS L. multiflorum, Unterengstringen, 1975, IBS L. perenne, Kitzeberg, 1980, JBS L. multiflorum, Hornsee, 1981, IBS Phialophora radicicola var . radicicola M, Norwich, 1974, JBS M, NorwicI\ 1974, JBS BM, Berlin, 198o'IJBS BM , Berlin, 1980, JBS BR, Berlin, 1980, JBS BW, Berlin, 1980, JBS BM, Braunschweig, 1980, JBS BW, Braunschweig, 1980, JBS BM , Braunschweig, 1980, IBS BM, Saarbriicken, 1980, IBS BM , Saarbriicken, 1980, IBS
M = Maize (Zea may s), W = Wheat ( T riticum aestivum ), (N ) = no information . BM = Bioassay of ma ize soil, BR = Bioassay of winter rape soil, BW = Bioassay of winter wheat soil. WK = W. Kriiger, HM = H . Mielke, PRS = P. P.. Scott, JBS = J . B. Speakman. * Originally identified as Ophiobolus graminis. t Date recei ved. :\: Originally identified as Phialophora radicicola. Trans . Br. Mycol. Soc. 79 (2), (1982).
Printed in Great Britain
Notes and brief articles
352
'.
-~
- .....
.
J~
........
T
.
'
' . ..
Fig. L Fertile per ithecium of Gaeumannomy ces graminis var. trit ici. Fig . 2. Ostiole of the perithecium in Fig . L Fig . 3. Mature ascus being discharged through the ost iole of a perithecium . Fig . 4. The same ascus as in Fig . 3, with visible ascospores.
Trans. Br . My col. Soc. 79 (2), (1982) .
Printed in Grear Britain
Notes and brief articles light conditions at laboratory temperature (20-22 0 ) . Periodic inspections of the dishes were made and the time of initiation of perithecial production was noted. Perithecial initials were observed in all Ggt isolates after approx. 21 days and these developed into fertile perithecia within another 14 days. Fertile perithecia were prepared for examination by stereo-electron microscopy (SEM) by the method described by Lewis & Day (1972), modified as described by Speakman (1977). Figs 1-4 illustrate the results obtained. The necks of the perithecia (Fig. 1) were found to vary greatly in length and the ostiole was often clearly visible (Fig. 2). The ascospores within an ascus as it is being discharged through the ostiole are visible in Figs 3 and 4. Perithecia were not produced by any of the Prg or Prr isolates, even after prolonged (over 6 months) incubation, although Hornby (1980) and Hornby et al. (1977) have reported that perithecia developed on the rotted roots of wheat seedlings infected with isolates of Prg and incubated as described by Hornby (1969). All Prr isolates produced sclerotial bodies. As Prr has been previously isolated mainly from maize (Zea mays L.) (Deacon, 1974; Speakman & Lewis, 1978), infection tests were also prepared using surface-sterilized maize seedlings grown in plastic Petri dishes on distilled-water agar. Fertile perithecia were produced by the isolates of Ggt tested, but none of the Prg or Prr isolates produced perithecia even after prolonged incubation; sclerotial bodies were formed by all Prr isolates. Davis (1925), Willets (1961) and Holden & Hornby (1981) have reported that the production of perithecia by isolates of Ggt tends to become intermittent or cease in cultures maintained for long periods. The oldest isolate used in this investigation was Ggt 1 which, at the time of its being received, had been at least 10 years in the culture collection at the University of East Anglia, Norwich (]. Geeson, pers. comm.). During this period the isolate had been regularly sub-cultured on PDA; it was only weakly pathogenic and perithecial production was low and erratic. Finally, the infection procedure described in this paper may provide a method of maintaining the pathogenicity of isolates. Sections of the seminal roots could be removed 5-7 days after infection, surface-sterilized and plated on sterile Petri dishes of PDA. With this aim in mind, the twenty-six Ggt isolates were re-isolated from wheat seminal roots every 9 months. Apart from isolate Ggt 1,
Trans. Br. Mycol. Soc. 79 (2), (1982). 12
353
pathogenicity was maintained by this procedure and yields of perithecia were high and fairly constant. The author wishes to thank Drs M. C. Cowan and B. G. Lewis for their contributions during this research. REFERENCES
DAVIS, R. J. (1925). Studies on Ophiobolus graminis Sacco and the take-all disease of wheat. Journal ofAgricultural Research 31, 801-825. DEACON, J. W. (1974). Further studies on Phialophora radicicola and Gaeumannomyces graminis on roots and stem bases of grasses and cereals. Transactions of the British Mycological Society 63, 307-327. HOLDEN, M. & HORNBY,D.(1981). Methods of producing perithecia of Gaeumannomyces graminis and their application to related fungi. Transactions of the British Mycological Society 77, 107-118. HORNBY, D. (1969). Methods of investigating populations of the take-all fungus (Ophiobolus graminis) in soil. Annals of Applied Biology 64, 503-513. HORNBY, D. (1980). Gaeumannomyces-Phialophora complex. Rothamsted Experimental Station. Reportfor 1979, part 1, p. 171HORNBY, D., SLOPE, D. B., GUTTERIDGE, R. J. & SIVANESAN, A. (1977). Gaeumannomyces cylindrosporus, a new ascomycete from cereal roots. Transactions of the British Mycological Society 69, 21-25. LEWIS, B. G. & DAY, J. R. (1972). Behaviour of uredospore germ tubes of Puccinia graminis tritici in relation to the fine structure of wheat leaf surfaces. Transactions of the British Mycological Society 58, 139-145. SPEAKMAN, J. B. (1977). Interactions between Gaeumannomycesgraminis and Phialophora radicicola in relation to take-all disease of wheat. Ph.D. thesis, University of East Anglia, Norwich. SPEAKMAN, J. B. & LEWIS, B. G. (1978). Limitation of Gaeumannomyces graminis by wheat root responses to Phialophora radicicola. New Phytologist 80, 373-380. SPEAKMAN, J. B. & LEWIS, B. G. (1980). Vitamin requirements of Phialophora radicicola var. graminicola compared with Gaeumannomyces graminis var. tritici. Transactions of the British Mycological Society 74, 4 10-413. WALKER, J. (1975). Take-all disease of Gramineae: a review of recent work. Review of Plant Pathology 54, 113-1 44. WALKER, J. (1980). Gaeumannomyces, Linocarpon, Ophiobolus and several other genera of scolecospored ascomycetes and Phialophora conidial states, with a note on hyphopodia. Mycotaxon 11, 1-129. WILLETS, H. J. (1961). A comparison between Ophiobolus graminis and Ophiobolus graminis var. avenae. Transactions of the British Mycological Society 44, 504-510.
Printed in Great Britain MYC
79