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Chloramphenicol resistance in an isolate of Phytophthora infestans
Notes and brief articles HOHL, H . R. (1975). Levels of nutritional complexity in Phytophthora: Lipids, nitrogen sources and growth factors. Phytopathologische Zeitschrift 84, 18-33· LOPATECKI, L. E. & NEWTON, W. (1956). The nutrition of Phytophthora. Canadian Journal of Botany 34, 75 1-757. McMEEKIN, D . (1973). Streptomycin inhibition of Peronospora parasitica and its host reversed by manganese and calcium. Phytopathology 63, 34-36. McMEEKIN, D. (1978). Inhibition and stimulation of growth of Pythium by streptomycin. My cologia 70, 880-883 . OKOROKOV, L. A., SYSUYEV, V. A., TERKHOVA, V. A. & DYAKov, Y. T. ( 1976). [Effects of calcium ions on growing Phytophthora infestans (M ont.) de Bary .) Mikologija Fitopathologija 10,484 (Abstr.). RONCANDORI, R. W. (1965). A nutritional comparison of
373
some species of Phytophthora . Phytopathology 55, 595-599· ROOKE, D . M . & SHATTOCK, R. C. ( 1983). Effect of chloramphenicol and streptomycin on developmental stages of Phytophthora infestans (M ont.) de Bary. J ournal of General Microbiology u9, 1-11. SHATTOCK, R. C. & SHAW, D. S. (1975). Mutants of Phytophthora infestans resistant to, and dependent upon, antibiotics. Transactions of the Br itish M ycological S ociety 64, 2~41. STERNE, R. E. , ZENTMYER, G. A. & BINGHAM, F . T. (1976). The effect of osmotic potential and specific ions on growth of Phytophthora cinnamomi. Phytopathology 66, 1398-1402. VENIS, M . A. ( 1969). Streptomycin inhibition of protein synthesis in peas reversed by divalent cations. Nature, London 221, 1147-1148.
CHLORAMPHENICOL RESISTANCE IN AN ISOLATE OF PHYTOPHTHORAINFESTANS BY DIANA M. ROOKE
* AND
R. C. SHATTOCK
School of Plant Biology, University College of North Wales, Bangor, Gwynedd, LL57 2UW Treatment of wild-type P. infestans with chloramphenicol induced the mitochondria to lose their christae, whereas there was no change with a chloramphenicol-resistant mutant. Resistance appeared to be due to reduced uptake and there was no evidence of antibiotic degradation. Several fungi within the Pythiaceae are inherently sensitive to chloramphenicol (CM) (M archan t & Smith, 1968; Smith & Marchant, 1968; Tsao, 1970; Hendrix, 1974; Shattock & Shaw, 1975; Rawn & Van Etten, 1978), by virtue of unusual permeability characteristics which allow access of antibacterial antibiotics (Voros, 1965; Rawn & Van Etten, 1978). Chloramphenicol, an inhibitor of procaryoticprotein synthesis (Pestka, 1975), impairs mitochondrial function in Pythium ultimum, producing depletion of cytochrome b and cytochrome oxidase, and increase of cytochrome c in the mycelium (Marchant & Smith, 1968; Smith & Marchant, 1968), in similar fashion to other fungi treated with much higher levels of CM (Schatz & Mason, 1974). Loss of normal mitochondrial function in fungi is often accompanied by ultrastructural changes in these organelles (Lloyd, 1974) and in some species alternate electron transport systems may operate (Smith & Marchant, 1968; Lambowitz & Sla yman, 1971). This note describes the effect of CM on the mitochondrial ultrastructure of a wild-type and a resistant mutant of P. infestans. Also considered is
* Present address: Department of Microbiology, The Medical School, The University, Newcastle upon Tyne, NE 1 7RU . Trans . BT, my col. So c. 82 (2), (1984)
whether resistance is due to chemical modification of CM or reduced permeability of the fungus, as described for resistant bacteria (Pestka, 1975), Mycelial cultures of P. infestans isolate B .25 (sensitive to CM) and Cr (resistant to CM) (Shattock & Shaw, 1975; Rooke & Sharrock, 1983 ) were grown for electron microscopy as wefts on cellophane squares (5 mm x 5 mm) (Rooke & Shattock, 1983) in Petri dishes containing liquid rye B medium (10 ml ) with 0, zoo or 500 /lg mr' CM for three days at 18 DC. For electron microscopy entire wefts were fixed in 1 % (w Iv) aq. osmium tetroxide (z hat 40 ) , dehydrated in acetone and embedded in resin (Taab Labs, U .K.) polymerised at 60 0 for 48 h. Thin silver-gold sections (silver 600-90 nm; gold 90-150 nm, Peachey, 1958) were mounted on nitrocellulose-coated copper grids, stained with uranyl acetate (z %, w I v) and post-stained in Reynolds lead citrate. Specimens were examined with a Corinth z75 AEI electron microscope at an operating voltage of 80 kV and' observations were restricted to young areas of the prepared material. The hyphal ultrastructure of isolates B. 25 and Cr in the absence of CM was similar and showed cytoplasmic organization typical of P . infestans (E rlich & Erlich, 1966; Fehrmann, 1971) and other
Printed in Great Britain
374
Notes and brief articles
Table 1. Chloramphenicol content of mycelium of isolates of P . infestans grown in liquid rye B medium with or without chloramphenicol at 18°C
Isolate B .25
Chloramphenicol in medium (pg mr") o
Chloramphenicol in mycelium (pg ml" mg? dry weight)* Duration antibiotic exposure (h) 6
180
Cr
4 2 '9±4'S
o 180
* Means ±standard errors; 6 replicates. - no detectable antibiotic. Oornycetes (G rove, Bracker & Morre, 1970), including Golgi dictyosomes, cytoplasmic vesicles and an extensive endomembrane system. The mitochondria in longitudinal sections of hyphae (Fig. 1) were predominantly long (up to 3 Jlm), flexuous and irregularly shaped; their profiles were clearly differentiated with darkly stained, doublelayered membranes containing a moderately electron dense matrix and numerous pleomorphic christae which were roughly spherical in transverse section (F ig. 1). The abundance of mitochondrial sections within the wild-type isolate B.25 grown with CM (200 Jlg ml " ) was unchanged but the mitochondria themselves appeared swollen. The inner and outer mitochondrial membranes retained their osmiophilic appearance but, in contrast to mitochondria of Pythium ultimum treated with CM (M archant & Smith, 1968) which lacked only ATPase particles, enclosed an almost electrontransparent matrix, almost devoid of christae (F igs 2, 3). Mitochondria of the resistant isolate Cr treated similarly showed no obvious differences from mitochondria of the control material (F igs 4, 5), suggesting that the mitochondria are a site of acti on of CM in Phytophthora at antibacterial concentrations. For antibiotic assay, standard weft cultures (Rooke & Shattock, 1984) in liquid medium were incubated for 5 days before adding CM ( 1 8 0 ug ml ", producing about 90 % inhibition of growth of the sensitive isolate) or sterile distilled water (contro ls). Incubation was continued for various periods between 6 hand 5 days before mycelial harvest. Two or three replicates were included for analytical work and five for dry weight estimation . The nature of resistance to CM was inve stigated by comparing uptake of antibiotic into the hyphae of B .25 and Cr. Mycelium harvested directly from liquid cultures without prior washing (washing rapidly removed CM from hyphae) was blended in 5 ml 0'1 M KH 2P0 4 /K 2HP0 4 buffer (pH 7'0) and Trans. Br. my col. So c. 82 (2) , ( 1984)
assayed for CM content by the method of Hughes & Diamond (1964).
Chloramphenicol was not detected by chemical assay in the mycelial homogenates of either isolate after 6 h incubation whereas the longer exposures resulted in increasing amounts of detectable antibiotic (T able 1). After 48 h approximately three times as much antibiotic was recorded from the mycelium of B .25 grown with CM compared with isolate Cr . Thin layer chromatography revealed that resistance was not due to chemical modification of CM. Culture media, homogenates and aqueous CM standard (180 Jlg mr") were extracted twice with cold ethyl acetate and the extracts chromatographed on silica gel F254 plates with chloroform-methanol (95 : 5 v Iv) solvent, after the method of Shaw (1967). Chromatograms were viewed with long wavelength UV light to locate the spots.
Fig. 1. Mitochondria (m) in a hypha of a sensitive isolate of P. infestans grown without chloramphenicol, showing typical appearance. Christae (c) arrowed. Length of bar marker 1'0 lIm.
Pr imed in G reat Britain
Notes and brief articles
375 3
Figs 2, 3. Mitochondria in transverse sections of hyphae of P. infestans (B.25) grown with chloramphenicol (zoo flg ml "). Reduced number of christae and electron density of matrix (mx) with intact inner and outer mitochondrial membranes. Figs 4, 5. Mitochondria in transverse sections of hyphae of P. infestans (Cr) grown with chloramphenicol (200 flg ml "), Well-defined osmiophilic mitochondrial membranes and numerous christae. Length of bar marker 0'1 flm in Figs. 2-5.
Table 2 shows that extracts of isolates B.25 and Cr grown with CM showed spots in positions of the chloramphenicol standard. In the absence of enzyme degradation of CM by the isolates it seems likely that reduced uptake of the antibiotic by Cr is the most plausible explanation of resistance. Rawn & Van Etten (1978) considered the cell surface as one site of action of CM since short exposures (60 min) inhibited amino acid transport into hyphae of sensitive isolates of Pythium ultimum and impaired intracellular protein Trans. Br. mycol. Soc. 82 (2), (1984)
synthesis. However, this study does not preclude other mechanisms of CM resistance, such as ribosomal resistance, as reported in P. palmivora (Carnes & Leary, 1977). The authors thank Mrs A. Griffiths, Mr G. Stuart and Mr A. Davies for assistance with electron microscopy. This work was carried out under the tenure of an S.E.R.C. Research Studentship by D.M.R.
Printed in Great Britain
Notes and brief articles Table
2.
Thin layer chromatography of extracts of mycelial homogenates or medium samples from isolates of
P. infestans (T he fungus was grown in liquid rye B medium or in medium with chloramphenicol (180 Jig ml:") for 5 days at 18 "C . Silica gel , chloroform/methanol solvent (95/5, v/v), 20 0 • Data from a single representative experiment.) Rf values of spots viewed under UV
Sample Standard Isolate B.25 Isolate Cr
Medium
0'22
0'24
180 Jig ml"! CM Control 180 p,g ml " CM Control 180 p,g rnl " CM
-
0'21 0'22
no detectable spots.
REFERENCES
CARNES, D. W. & LEARY,]. V. (1977). The sensitivity of Phytophthora palmioora to chloramphenicol. Proceedings of the American Phytopathological Society 4, 94-95 . ERLICH, M. A. & ERLICH, H . G . ( 1966). Ultrastructure of the hyphae and haustoria of Phytophthora infestans and hyphae of Phytophthora parasitica . Canadian Journal of Botany 44, 1495-1503 . FEHRMANN, H . (1971). Vergleich der Ultrastruktur in H yphen von Phytophthora-Arten. Archives of Microbiology 77, 47-58. GROVE, S. N., BRACKER, C. E . & MORRE, D .]. ( 1970). An ultrastructural basis for hyphal tip growth in Pythium ultimum. American Journal of Botany 57, 245-266. HENDRIX, ]. W. (1974). Physiology and biochemistry of growth and reproduction in Pythium. Proceedings of the American Phytopathological Society 1, 207-210. HUGHES, D. W . O . & DIAMOND, L. K . (1964) . Chloramphenicol in blood. Simple chemical estimations in patients receiving multiple antibiotics. S cience 144, 296-297· LAMBOWITZ, A. M. & SLAYMAN, C . W. (1971). Cyanideresistant respiration in Neurospora crassa. Journal of Bacteriology 108, 1087-1096. LLOYD, D . (1974) . The M itochondria of M icroorganisms, London, New York : Academic Press. MARCHANT, R. & SMITH, D. G . (1968). Effect of chloramphenicol on growth and mitochondrial structure of Pythium ultimum.Journal of General Microbiology 50, 39 1-397 . PEACHEY, L. D. (1958). Thin sections. I. A study of sect ion thickness and physical distortion during
Trans . Br. my col. Soc. 8% (2), (1984)
Mycelial homogenate
microtomy. Journal of Biophysical and Biochemical Cytology 4, 233-242. PESTKA, S. (1975 ). Chloramphenicol. In Antibiotics. Vol. III. Mechanism of Action of Antimicrobial and Antitumour Agents (ed . ] . W. Corcoran & F. E. Hahn), pp . 310-395. Berlin, New York: Springer Verlag. ROOKE, D. M. & SHATTOCK, R. C . (1983). Effect of chloramphenicol and streptomycin on isolates of Phytophthora inf estans (M on t .) de Bary. J ournal of General Microbiology U9, 1-11 . RAWN, C. D . & VAN ETTEN,] . L. (1978). Mechanisms of antibacterial sens itivity in Pythium ultimum. Journal of General Microbiology 108, 133-139. SCHATZ, G . & MASON, T. L. (1974). The biosynthesis of mitochondrial proteins. Annual Review of Biochemistry 43,5 1-87. SHATTOCK, R. C . & SHAW, D . S. (1975). Mutants of Phytophthora infestans resistant to , and dependent upon antibiotics. Transa ctions of the British Mycological Society 64, 29-41. SHAW, W . V. (1967). The enz ymatic acetylation of chloramphenicol by extracts of R-factor res istant Escherichia coli. Journal of Biological Chemistry %4%, 68 7-693. SMITH,D . G. & MARCHANT, R. ( 1968). Chloramphenicol inhibition of Pythium ultimum and Rh odotorula glutinis. Archiv fur M ikrobiologie 60, 262-274. TSAO, P. H . (1970) . Selective media for the isolation of pathogenic fungi. Annual Review of Phytopathology 8, 157-186. VOROS,]. (1965 ). Streptomycin sensitivity of Oomycetes due to increased absorption of streptomycin by their mycelia . Phy topathologische Zeits chrift 54, 249-257.
Printed in Great Br itain
373
some species of Phytophthora . Phytopathology 55, 595-599· ROOKE, D . M . & SHATTOCK, R. C. ( 1983). Effect of chloramphenicol and streptomycin on developmental stages of Phytophthora infestans (M ont.) de Bary. J ournal of General Microbiology u9, 1-11. SHATTOCK, R. C. & SHAW, D. S. (1975). Mutants of Phytophthora infestans resistant to, and dependent upon, antibiotics. Transactions of the Br itish M ycological S ociety 64, 2~41. STERNE, R. E. , ZENTMYER, G. A. & BINGHAM, F . T. (1976). The effect of osmotic potential and specific ions on growth of Phytophthora cinnamomi. Phytopathology 66, 1398-1402. VENIS, M . A. ( 1969). Streptomycin inhibition of protein synthesis in peas reversed by divalent cations. Nature, London 221, 1147-1148.
CHLORAMPHENICOL RESISTANCE IN AN ISOLATE OF PHYTOPHTHORAINFESTANS BY DIANA M. ROOKE
* AND
R. C. SHATTOCK
School of Plant Biology, University College of North Wales, Bangor, Gwynedd, LL57 2UW Treatment of wild-type P. infestans with chloramphenicol induced the mitochondria to lose their christae, whereas there was no change with a chloramphenicol-resistant mutant. Resistance appeared to be due to reduced uptake and there was no evidence of antibiotic degradation. Several fungi within the Pythiaceae are inherently sensitive to chloramphenicol (CM) (M archan t & Smith, 1968; Smith & Marchant, 1968; Tsao, 1970; Hendrix, 1974; Shattock & Shaw, 1975; Rawn & Van Etten, 1978), by virtue of unusual permeability characteristics which allow access of antibacterial antibiotics (Voros, 1965; Rawn & Van Etten, 1978). Chloramphenicol, an inhibitor of procaryoticprotein synthesis (Pestka, 1975), impairs mitochondrial function in Pythium ultimum, producing depletion of cytochrome b and cytochrome oxidase, and increase of cytochrome c in the mycelium (Marchant & Smith, 1968; Smith & Marchant, 1968), in similar fashion to other fungi treated with much higher levels of CM (Schatz & Mason, 1974). Loss of normal mitochondrial function in fungi is often accompanied by ultrastructural changes in these organelles (Lloyd, 1974) and in some species alternate electron transport systems may operate (Smith & Marchant, 1968; Lambowitz & Sla yman, 1971). This note describes the effect of CM on the mitochondrial ultrastructure of a wild-type and a resistant mutant of P. infestans. Also considered is
* Present address: Department of Microbiology, The Medical School, The University, Newcastle upon Tyne, NE 1 7RU . Trans . BT, my col. So c. 82 (2), (1984)
whether resistance is due to chemical modification of CM or reduced permeability of the fungus, as described for resistant bacteria (Pestka, 1975), Mycelial cultures of P. infestans isolate B .25 (sensitive to CM) and Cr (resistant to CM) (Shattock & Shaw, 1975; Rooke & Sharrock, 1983 ) were grown for electron microscopy as wefts on cellophane squares (5 mm x 5 mm) (Rooke & Shattock, 1983) in Petri dishes containing liquid rye B medium (10 ml ) with 0, zoo or 500 /lg mr' CM for three days at 18 DC. For electron microscopy entire wefts were fixed in 1 % (w Iv) aq. osmium tetroxide (z hat 40 ) , dehydrated in acetone and embedded in resin (Taab Labs, U .K.) polymerised at 60 0 for 48 h. Thin silver-gold sections (silver 600-90 nm; gold 90-150 nm, Peachey, 1958) were mounted on nitrocellulose-coated copper grids, stained with uranyl acetate (z %, w I v) and post-stained in Reynolds lead citrate. Specimens were examined with a Corinth z75 AEI electron microscope at an operating voltage of 80 kV and' observations were restricted to young areas of the prepared material. The hyphal ultrastructure of isolates B. 25 and Cr in the absence of CM was similar and showed cytoplasmic organization typical of P . infestans (E rlich & Erlich, 1966; Fehrmann, 1971) and other
Printed in Great Britain
374
Notes and brief articles
Table 1. Chloramphenicol content of mycelium of isolates of P . infestans grown in liquid rye B medium with or without chloramphenicol at 18°C
Isolate B .25
Chloramphenicol in medium (pg mr") o
Chloramphenicol in mycelium (pg ml" mg? dry weight)* Duration antibiotic exposure (h) 6
180
Cr
4 2 '9±4'S
o 180
* Means ±standard errors; 6 replicates. - no detectable antibiotic. Oornycetes (G rove, Bracker & Morre, 1970), including Golgi dictyosomes, cytoplasmic vesicles and an extensive endomembrane system. The mitochondria in longitudinal sections of hyphae (Fig. 1) were predominantly long (up to 3 Jlm), flexuous and irregularly shaped; their profiles were clearly differentiated with darkly stained, doublelayered membranes containing a moderately electron dense matrix and numerous pleomorphic christae which were roughly spherical in transverse section (F ig. 1). The abundance of mitochondrial sections within the wild-type isolate B.25 grown with CM (200 Jlg ml " ) was unchanged but the mitochondria themselves appeared swollen. The inner and outer mitochondrial membranes retained their osmiophilic appearance but, in contrast to mitochondria of Pythium ultimum treated with CM (M archant & Smith, 1968) which lacked only ATPase particles, enclosed an almost electrontransparent matrix, almost devoid of christae (F igs 2, 3). Mitochondria of the resistant isolate Cr treated similarly showed no obvious differences from mitochondria of the control material (F igs 4, 5), suggesting that the mitochondria are a site of acti on of CM in Phytophthora at antibacterial concentrations. For antibiotic assay, standard weft cultures (Rooke & Shattock, 1984) in liquid medium were incubated for 5 days before adding CM ( 1 8 0 ug ml ", producing about 90 % inhibition of growth of the sensitive isolate) or sterile distilled water (contro ls). Incubation was continued for various periods between 6 hand 5 days before mycelial harvest. Two or three replicates were included for analytical work and five for dry weight estimation . The nature of resistance to CM was inve stigated by comparing uptake of antibiotic into the hyphae of B .25 and Cr. Mycelium harvested directly from liquid cultures without prior washing (washing rapidly removed CM from hyphae) was blended in 5 ml 0'1 M KH 2P0 4 /K 2HP0 4 buffer (pH 7'0) and Trans. Br. my col. So c. 82 (2) , ( 1984)
assayed for CM content by the method of Hughes & Diamond (1964).
Chloramphenicol was not detected by chemical assay in the mycelial homogenates of either isolate after 6 h incubation whereas the longer exposures resulted in increasing amounts of detectable antibiotic (T able 1). After 48 h approximately three times as much antibiotic was recorded from the mycelium of B .25 grown with CM compared with isolate Cr . Thin layer chromatography revealed that resistance was not due to chemical modification of CM. Culture media, homogenates and aqueous CM standard (180 Jlg mr") were extracted twice with cold ethyl acetate and the extracts chromatographed on silica gel F254 plates with chloroform-methanol (95 : 5 v Iv) solvent, after the method of Shaw (1967). Chromatograms were viewed with long wavelength UV light to locate the spots.
Fig. 1. Mitochondria (m) in a hypha of a sensitive isolate of P. infestans grown without chloramphenicol, showing typical appearance. Christae (c) arrowed. Length of bar marker 1'0 lIm.
Pr imed in G reat Britain
Notes and brief articles
375 3
Figs 2, 3. Mitochondria in transverse sections of hyphae of P. infestans (B.25) grown with chloramphenicol (zoo flg ml "). Reduced number of christae and electron density of matrix (mx) with intact inner and outer mitochondrial membranes. Figs 4, 5. Mitochondria in transverse sections of hyphae of P. infestans (Cr) grown with chloramphenicol (200 flg ml "), Well-defined osmiophilic mitochondrial membranes and numerous christae. Length of bar marker 0'1 flm in Figs. 2-5.
Table 2 shows that extracts of isolates B.25 and Cr grown with CM showed spots in positions of the chloramphenicol standard. In the absence of enzyme degradation of CM by the isolates it seems likely that reduced uptake of the antibiotic by Cr is the most plausible explanation of resistance. Rawn & Van Etten (1978) considered the cell surface as one site of action of CM since short exposures (60 min) inhibited amino acid transport into hyphae of sensitive isolates of Pythium ultimum and impaired intracellular protein Trans. Br. mycol. Soc. 82 (2), (1984)
synthesis. However, this study does not preclude other mechanisms of CM resistance, such as ribosomal resistance, as reported in P. palmivora (Carnes & Leary, 1977). The authors thank Mrs A. Griffiths, Mr G. Stuart and Mr A. Davies for assistance with electron microscopy. This work was carried out under the tenure of an S.E.R.C. Research Studentship by D.M.R.
Printed in Great Britain
Notes and brief articles Table
2.
Thin layer chromatography of extracts of mycelial homogenates or medium samples from isolates of
P. infestans (T he fungus was grown in liquid rye B medium or in medium with chloramphenicol (180 Jig ml:") for 5 days at 18 "C . Silica gel , chloroform/methanol solvent (95/5, v/v), 20 0 • Data from a single representative experiment.) Rf values of spots viewed under UV
Sample Standard Isolate B.25 Isolate Cr
Medium
0'22
0'24
180 Jig ml"! CM Control 180 p,g ml " CM Control 180 p,g rnl " CM
-
0'21 0'22
no detectable spots.
REFERENCES
CARNES, D. W. & LEARY,]. V. (1977). The sensitivity of Phytophthora palmioora to chloramphenicol. Proceedings of the American Phytopathological Society 4, 94-95 . ERLICH, M. A. & ERLICH, H . G . ( 1966). Ultrastructure of the hyphae and haustoria of Phytophthora infestans and hyphae of Phytophthora parasitica . Canadian Journal of Botany 44, 1495-1503 . FEHRMANN, H . (1971). Vergleich der Ultrastruktur in H yphen von Phytophthora-Arten. Archives of Microbiology 77, 47-58. GROVE, S. N., BRACKER, C. E . & MORRE, D .]. ( 1970). An ultrastructural basis for hyphal tip growth in Pythium ultimum. American Journal of Botany 57, 245-266. HENDRIX, ]. W. (1974). Physiology and biochemistry of growth and reproduction in Pythium. Proceedings of the American Phytopathological Society 1, 207-210. HUGHES, D. W . O . & DIAMOND, L. K . (1964) . Chloramphenicol in blood. Simple chemical estimations in patients receiving multiple antibiotics. S cience 144, 296-297· LAMBOWITZ, A. M. & SLAYMAN, C . W. (1971). Cyanideresistant respiration in Neurospora crassa. Journal of Bacteriology 108, 1087-1096. LLOYD, D . (1974) . The M itochondria of M icroorganisms, London, New York : Academic Press. MARCHANT, R. & SMITH, D. G . (1968). Effect of chloramphenicol on growth and mitochondrial structure of Pythium ultimum.Journal of General Microbiology 50, 39 1-397 . PEACHEY, L. D. (1958). Thin sections. I. A study of sect ion thickness and physical distortion during
Trans . Br. my col. Soc. 8% (2), (1984)
Mycelial homogenate
microtomy. Journal of Biophysical and Biochemical Cytology 4, 233-242. PESTKA, S. (1975 ). Chloramphenicol. In Antibiotics. Vol. III. Mechanism of Action of Antimicrobial and Antitumour Agents (ed . ] . W. Corcoran & F. E. Hahn), pp . 310-395. Berlin, New York: Springer Verlag. ROOKE, D. M. & SHATTOCK, R. C . (1983). Effect of chloramphenicol and streptomycin on isolates of Phytophthora inf estans (M on t .) de Bary. J ournal of General Microbiology U9, 1-11 . RAWN, C. D . & VAN ETTEN,] . L. (1978). Mechanisms of antibacterial sens itivity in Pythium ultimum. Journal of General Microbiology 108, 133-139. SCHATZ, G . & MASON, T. L. (1974). The biosynthesis of mitochondrial proteins. Annual Review of Biochemistry 43,5 1-87. SHATTOCK, R. C . & SHAW, D . S. (1975). Mutants of Phytophthora infestans resistant to , and dependent upon antibiotics. Transa ctions of the British Mycological Society 64, 29-41. SHAW, W . V. (1967). The enz ymatic acetylation of chloramphenicol by extracts of R-factor res istant Escherichia coli. Journal of Biological Chemistry %4%, 68 7-693. SMITH,D . G. & MARCHANT, R. ( 1968). Chloramphenicol inhibition of Pythium ultimum and Rh odotorula glutinis. Archiv fur M ikrobiologie 60, 262-274. TSAO, P. H . (1970) . Selective media for the isolation of pathogenic fungi. Annual Review of Phytopathology 8, 157-186. VOROS,]. (1965 ). Streptomycin sensitivity of Oomycetes due to increased absorption of streptomycin by their mycelia . Phy topathologische Zeits chrift 54, 249-257.
Printed in Great Br itain