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The effect of saprophytes on infection of leaves of Brassica spp. By Alternaria brassicicola
Notes and Brief Articles
193
REFERENCES
BRACKER, C. E. & WILLIAMS, C. M. (1966). Comparative ultrastructure of developing sporangia and asci in fungi, pp. 307-308. In Sixth International Congress for Electron Microscopy (ed. R. Uyeda). Tokyo: Maruzen Co. CARROLL, G. C. (1966). A study of the fine structure of ascosporogenesis in Saccobolus kerverni and Ascodesmis sphaerospora. Ph.D. Thesis. University of Texas. CARROLL, G. C. (1967), The fine structure of ascospore delimitation in Saccobolus keruerni. Journal ofCell Biology 33,218-224. COLSON, B. (1938). The cytology and development of Phyllactinia corylea. Annalsof Botany 52,38 1-402. DODGE, B. O. (1928). Spore formation in asci with fewer than eight spores. Mycologia 110,18-21. DODGE, B. O. (1929). Nature of spores and segregation of sex factors in Neurospora. Mycologia Ill, 222-231. FAULL, J. H. (1912). The cytology of Laboulbenia chaetophora and L. gyridinarum. Annals of Botany 116, 325-335. GRAFF, P. W. (1932). The morphological and cytological development of Meliola circinance. Bulletin of Torrey Botanical Club 59, 241-266. HAYMAN, D. S. (1964)' Rosellinia limoniispora: nuclear changes in the developing ascus. Canadian Journal of Botany 4ll, 13-2 I • HElM, P. (1932). Observations sur Ie noyau des Ascomycetes. Revue de Mycologic, Paris 17,3-38. JENKINS, W. A. (1932). The development of Cordyceps agariciformis. Mycologia 116, 220-243. JONES, S. G. (1925). Life history and cytology of Rhytisma acerinum (Pers) Fries. Annals of Botany 39,41-45. JONES, S. G. (1926). The development of the perithecium of Ophiobolus graminis Sacco Annals of Botany 40, 607-629. REEVES, D. B. (1967). The fine structure of ascospore formation in Pyronema domestica, Mycologia 59, 1018-1033. RAYMOND,J. R. (1934). Contribution a la connaissance cytologique des Ascomycetes. Botaniste 116, 371-533. THITE, A. N. (1972). Cytology and development of ascus and ascospores in Meliola asyridicola Hans. Proceedings 59th Indian Science Congress, Calcutta, 1972. WARD, H. M. (1882). Researches on morphology and life history of a tropical pyrenomycete fungus. Quarterly Journal of Microscopical Science 1111, 347-357. WARD, H. M. (1883). Morphology and development of perithecium of Meliola a genus of epiphyllous fungi from the Philippines. Transactions of Royal Scoiety, London 174, 583-599.
THE EFFECT OF SAPROPHYTES ON INFECTION OF LEAVES OF BRASSICA spp. BY ALTERNARIA BRASSICICOLA MARGARET A. PACE AND R. CAMPBELL
Department of Botany, The University, Bristol, BS8 1 UG The leaf surface may support the growth of many different microorganisms (Last & Deighton, 1965). Several authors have described antagonism by saprophytic phylloplane organisms towards leaf pathogens (Leben, 1965; van den Heuvel, 1971; McBride, 1971; Last & Warren, 1972) and it has been suggested (Last & Deighton, 1965; Last & Warren, 1972) that a leaf which is able to support a microflora antagonistic to a pathogen may have greater resistance to disease than one which cannot. Trans. Br. mycol. Soc. 63 (I), (1974). Printed in Great Britain 13
M YC
63
194
Transactions British Mycological Society
During a study of the dark leaf spot disease of Brassica spp. caused by Alternaria brassicicola (Schwein.) Wiltshire it was found that the microflora of mature cabbage leaves was similar to that reported for other temperate plants (Last & Deighton, 1965; Dickinson, 1973). Of the thirty-eight fungal species isolated by a washing technique, eleven were antagonistic to A. brassicicola in culture. Of these, Aureobasidium pullulans (de Bary) Arn. and Epicoccum nigrum Link ex Fr. were found to be leaf surface residents sensu Leben (1965). The growing colonies of these two fungi inhibited mycelial growth of A. brassicicola and their germinating spores inhibited its germination. Au. pullulans and E. nigrum were therefore tested to see if their presence on leaves reduced infection by A. brassicicola. Six-week-old seedlings of cabbage (Brassica oleracea var. capitata cultivar January King) and Brussels sprout (Brassica oleracea var. gemmifera cultivar Cambridge Number One) were grown at approximately 20°C in Levington's compost under artificial lighting in a greenhouse. Preliminary experiments indicated that 85-100 % infection by A. brassicicola could be consistently obtained by wounding leaves with a needle point, placing a standard sized drop (approximately 0'025 ml) of spore suspension (16 X lOG spores/ml) over the wound and keeping the plants in a humidity of approximately 100 %. Plants were inoculated with the standard drops of spore suspensions as follows (final spore concentrations in parentheses): (I) A. brassicicola (16 x IOG/ml); (2) E. nigrum (16x IOG/ml); (3) Au. pullulans (36 x IOG/ml); (4) A. brassicicola (16 X IOG/ml) + E. nigrum (16 x IOG/ml); (5) A. brassicicola (16 x IOG/ml) +Au. pullulans (36 x IOG/ml). The plants were incubated for 4 days. At the end of this period successfully infected wounds had given rise to lesions greater than 2 mm diameter, which were brown with a chlorotic halo and which eventually spread and killed the leaf. Necrotic areas smaller than 2 mm diameter often formed around the wound. These were more darkly pigmented than the spreading lesions, had no chlorotic halo and never progressed. They were thought to be a hypersensitive reaction and were not counted as successful infections. The data for the proportion of successful infections are presented in Fig. I. Inoculation with E. nigrum and Au. pullulans alone produced no lesions. An analysis of variance showed no significant differences between the two hosts for the same treatments, but the differences between treatments on the same host were significant at the I % level. Student's t tests showed that the levels of infection were significantly lower, at the I % level, with the antagonists present than with the pathogen alone. The effect of pre-incubation was investigated by placing spore suspensions of the antagonists on the wounds 14 h before inoculating with A. brassicicola. This time interval allowed more than 90 % germination of E. nigrum and the commencement of active budding by Au. pullulans, Final spore concentrations were the same as in the previous experiment. Trans. Br. mycol. Soc. 63 (I), (1974)' Printed in Great Britain
195
Notes and Brief Articles
The reduction in successful infection by A. brassicicola was greater (Fig. 2) than it was with simultaneous inoculation (Fig. I). This suggests that active growth of the saprophytes on the leaf is important in their antagonism. In support of this possibility a Penicillium sp. isolated as a nonresident from mature cabbage leaves, showed antagonism towards A. brassicicola in culture but had no effect when inoculated onto leaves where its spores failed to germinate. Cabbage
100
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5
3
4
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Treatments
Fig. 1. Percentage (mean of three values) of successful infection obtained on cabbage and Brussels sprout leaves. Treatments: I = A. brassicicola alone; 2 = E. nigrum alone; 3 = Au. pullulans alone; 4 = A. brassicicola-s-E, nigrum; 5 = A. brassicicola-s- Au. pullulans. The vertical bars are the standard deviations from the mean. Cabbage 100
Sprout
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Fig. 2. Percentage of successfulinfections ootained on cabbage and Brusselssprout leaves after pre-incubating the antagonist for 14 h. Treatment abbreviations as in Fig. 1. Trans. Br. mycol. Soc. 63 (I), (1974). Printed in Great Britain
196
Transactions British Mycological Society
Resident antagonistic organisms capable of active growth on the leaf may compete for nutrients or may produce inhibitory substances. Some evidence for the latter hypothesis was the 50 % reduction in successful infection when A. brassicicola spores for inoculation were suspended in a culture filtrate of Au. pullulans, instead of in water. The Au. pullulans had been grown in Czapek-Dox liquid medium which itself enhanced infection compared with controls in water. E. nigrum filtrates had no consistent effect on the leaf infection rate. In culture, the filtrates of both antagonists inhibited spore germination and mycelial growth of A. brassicicola. This study has shown that it is possible to reduce successful infection by A. brassicicola through the use of antagonistic fungi. The inter-relations between host, parasite and saprophytes are complex and delicately balanced (McBride, 1971; Last & Warren, 1972) and there are many difficulties in extrapolating from results obtained under greenhouse conditions to biological control in the field. The use of fungicides, including systemic ones such as benomyl, might seem an easier way than biological control to combat plant diseases, but there are still many problems with their use. For example, although benomyl gives good control for particular host-parasite combinations, there are some pathogens, including A. brassicicola (Bollen & Fuchs, 1970), that are resistant to it. The antagonists described in this paper are killed or inhibited by benomyl (unpublished results). It is thus possible that the application ofwide spectrum fungicides for the control of one disease could, through the elimination of natural antagonists resident on the leaf surface, lead to a disease by a previously less important, resistant, pathogen. As no one type of control is likely to be completely satisfactory all possibilities should be studied; it is therefore important that such basic investigations as the present one should be pursued in the hope of eventual practical use, despite the known problems with biological control on a field scale. We thank Mr M. Ames for growing the many plants used in this study. REFERENCES
BOLLEN, G. V. & FUCHS, A. (1970). On the specificity of the in vitro and in vivo antifungal activity ofbenomyl. Netherlands Journal of Plant Pathology ,6,299-312. DICKINSON, C. H. (1973). Effects of ethirimol and zineb on phylloplane microflora of barley. Transactions of the British Mycological Society 60, 423-431. HEUVEL, J. VAN DEN. (1971). Antagonism between pathogenic and saprophytic Alternaria species on bean leaves. Ecology of leaf surface micro-organisms, pp. 537-544. (ed. T. F. Preece and C. H. Dickinson). Academic Press. LAST, F. T. & DEIGHTON, F. C. (1965). The non-parasitic microflora on the surfaces of living leaves. Transactions of the British Mycological Society 48, 83-99. LAST, F. T. & WARREN, R. C. (19Tz). Non-parasitic microbes colonizing green leaves: their form and functions. Endeavour 31, 143-15°. LEBEN, C. (1965). Epiphytic microorganisms in relation to plant disease. Annual Review of Phytopatlwlogy 3, 20g-230' McBRIDE, R. P. (1971). Micro-organism interactions in the phyllosphere of larch. Ecology of leaf surface micro-organisms, pp. 545-555 (ed, T. F. Preece and C. H. Dickinson). Academic Press.
Trans. Br. mycol. Soc. 63 (I), (1974). Printed in Great Britain
193
REFERENCES
BRACKER, C. E. & WILLIAMS, C. M. (1966). Comparative ultrastructure of developing sporangia and asci in fungi, pp. 307-308. In Sixth International Congress for Electron Microscopy (ed. R. Uyeda). Tokyo: Maruzen Co. CARROLL, G. C. (1966). A study of the fine structure of ascosporogenesis in Saccobolus kerverni and Ascodesmis sphaerospora. Ph.D. Thesis. University of Texas. CARROLL, G. C. (1967), The fine structure of ascospore delimitation in Saccobolus keruerni. Journal ofCell Biology 33,218-224. COLSON, B. (1938). The cytology and development of Phyllactinia corylea. Annalsof Botany 52,38 1-402. DODGE, B. O. (1928). Spore formation in asci with fewer than eight spores. Mycologia 110,18-21. DODGE, B. O. (1929). Nature of spores and segregation of sex factors in Neurospora. Mycologia Ill, 222-231. FAULL, J. H. (1912). The cytology of Laboulbenia chaetophora and L. gyridinarum. Annals of Botany 116, 325-335. GRAFF, P. W. (1932). The morphological and cytological development of Meliola circinance. Bulletin of Torrey Botanical Club 59, 241-266. HAYMAN, D. S. (1964)' Rosellinia limoniispora: nuclear changes in the developing ascus. Canadian Journal of Botany 4ll, 13-2 I • HElM, P. (1932). Observations sur Ie noyau des Ascomycetes. Revue de Mycologic, Paris 17,3-38. JENKINS, W. A. (1932). The development of Cordyceps agariciformis. Mycologia 116, 220-243. JONES, S. G. (1925). Life history and cytology of Rhytisma acerinum (Pers) Fries. Annals of Botany 39,41-45. JONES, S. G. (1926). The development of the perithecium of Ophiobolus graminis Sacco Annals of Botany 40, 607-629. REEVES, D. B. (1967). The fine structure of ascospore formation in Pyronema domestica, Mycologia 59, 1018-1033. RAYMOND,J. R. (1934). Contribution a la connaissance cytologique des Ascomycetes. Botaniste 116, 371-533. THITE, A. N. (1972). Cytology and development of ascus and ascospores in Meliola asyridicola Hans. Proceedings 59th Indian Science Congress, Calcutta, 1972. WARD, H. M. (1882). Researches on morphology and life history of a tropical pyrenomycete fungus. Quarterly Journal of Microscopical Science 1111, 347-357. WARD, H. M. (1883). Morphology and development of perithecium of Meliola a genus of epiphyllous fungi from the Philippines. Transactions of Royal Scoiety, London 174, 583-599.
THE EFFECT OF SAPROPHYTES ON INFECTION OF LEAVES OF BRASSICA spp. BY ALTERNARIA BRASSICICOLA MARGARET A. PACE AND R. CAMPBELL
Department of Botany, The University, Bristol, BS8 1 UG The leaf surface may support the growth of many different microorganisms (Last & Deighton, 1965). Several authors have described antagonism by saprophytic phylloplane organisms towards leaf pathogens (Leben, 1965; van den Heuvel, 1971; McBride, 1971; Last & Warren, 1972) and it has been suggested (Last & Deighton, 1965; Last & Warren, 1972) that a leaf which is able to support a microflora antagonistic to a pathogen may have greater resistance to disease than one which cannot. Trans. Br. mycol. Soc. 63 (I), (1974). Printed in Great Britain 13
M YC
63
194
Transactions British Mycological Society
During a study of the dark leaf spot disease of Brassica spp. caused by Alternaria brassicicola (Schwein.) Wiltshire it was found that the microflora of mature cabbage leaves was similar to that reported for other temperate plants (Last & Deighton, 1965; Dickinson, 1973). Of the thirty-eight fungal species isolated by a washing technique, eleven were antagonistic to A. brassicicola in culture. Of these, Aureobasidium pullulans (de Bary) Arn. and Epicoccum nigrum Link ex Fr. were found to be leaf surface residents sensu Leben (1965). The growing colonies of these two fungi inhibited mycelial growth of A. brassicicola and their germinating spores inhibited its germination. Au. pullulans and E. nigrum were therefore tested to see if their presence on leaves reduced infection by A. brassicicola. Six-week-old seedlings of cabbage (Brassica oleracea var. capitata cultivar January King) and Brussels sprout (Brassica oleracea var. gemmifera cultivar Cambridge Number One) were grown at approximately 20°C in Levington's compost under artificial lighting in a greenhouse. Preliminary experiments indicated that 85-100 % infection by A. brassicicola could be consistently obtained by wounding leaves with a needle point, placing a standard sized drop (approximately 0'025 ml) of spore suspension (16 X lOG spores/ml) over the wound and keeping the plants in a humidity of approximately 100 %. Plants were inoculated with the standard drops of spore suspensions as follows (final spore concentrations in parentheses): (I) A. brassicicola (16 x IOG/ml); (2) E. nigrum (16x IOG/ml); (3) Au. pullulans (36 x IOG/ml); (4) A. brassicicola (16 X IOG/ml) + E. nigrum (16 x IOG/ml); (5) A. brassicicola (16 x IOG/ml) +Au. pullulans (36 x IOG/ml). The plants were incubated for 4 days. At the end of this period successfully infected wounds had given rise to lesions greater than 2 mm diameter, which were brown with a chlorotic halo and which eventually spread and killed the leaf. Necrotic areas smaller than 2 mm diameter often formed around the wound. These were more darkly pigmented than the spreading lesions, had no chlorotic halo and never progressed. They were thought to be a hypersensitive reaction and were not counted as successful infections. The data for the proportion of successful infections are presented in Fig. I. Inoculation with E. nigrum and Au. pullulans alone produced no lesions. An analysis of variance showed no significant differences between the two hosts for the same treatments, but the differences between treatments on the same host were significant at the I % level. Student's t tests showed that the levels of infection were significantly lower, at the I % level, with the antagonists present than with the pathogen alone. The effect of pre-incubation was investigated by placing spore suspensions of the antagonists on the wounds 14 h before inoculating with A. brassicicola. This time interval allowed more than 90 % germination of E. nigrum and the commencement of active budding by Au. pullulans, Final spore concentrations were the same as in the previous experiment. Trans. Br. mycol. Soc. 63 (I), (1974)' Printed in Great Britain
195
Notes and Brief Articles
The reduction in successful infection by A. brassicicola was greater (Fig. 2) than it was with simultaneous inoculation (Fig. I). This suggests that active growth of the saprophytes on the leaf is important in their antagonism. In support of this possibility a Penicillium sp. isolated as a nonresident from mature cabbage leaves, showed antagonism towards A. brassicicola in culture but had no effect when inoculated onto leaves where its spores failed to germinate. Cabbage
100
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+
.s :; <-
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50
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3
4
2
5
3
4
5
Treatments
Fig. 1. Percentage (mean of three values) of successful infection obtained on cabbage and Brussels sprout leaves. Treatments: I = A. brassicicola alone; 2 = E. nigrum alone; 3 = Au. pullulans alone; 4 = A. brassicicola-s-E, nigrum; 5 = A. brassicicola-s- Au. pullulans. The vertical bars are the standard deviations from the mean. Cabbage 100
Sprout
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-
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2
I 4
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I
5
2
3
4
Treatments
Fig. 2. Percentage of successfulinfections ootained on cabbage and Brusselssprout leaves after pre-incubating the antagonist for 14 h. Treatment abbreviations as in Fig. 1. Trans. Br. mycol. Soc. 63 (I), (1974). Printed in Great Britain
196
Transactions British Mycological Society
Resident antagonistic organisms capable of active growth on the leaf may compete for nutrients or may produce inhibitory substances. Some evidence for the latter hypothesis was the 50 % reduction in successful infection when A. brassicicola spores for inoculation were suspended in a culture filtrate of Au. pullulans, instead of in water. The Au. pullulans had been grown in Czapek-Dox liquid medium which itself enhanced infection compared with controls in water. E. nigrum filtrates had no consistent effect on the leaf infection rate. In culture, the filtrates of both antagonists inhibited spore germination and mycelial growth of A. brassicicola. This study has shown that it is possible to reduce successful infection by A. brassicicola through the use of antagonistic fungi. The inter-relations between host, parasite and saprophytes are complex and delicately balanced (McBride, 1971; Last & Warren, 1972) and there are many difficulties in extrapolating from results obtained under greenhouse conditions to biological control in the field. The use of fungicides, including systemic ones such as benomyl, might seem an easier way than biological control to combat plant diseases, but there are still many problems with their use. For example, although benomyl gives good control for particular host-parasite combinations, there are some pathogens, including A. brassicicola (Bollen & Fuchs, 1970), that are resistant to it. The antagonists described in this paper are killed or inhibited by benomyl (unpublished results). It is thus possible that the application ofwide spectrum fungicides for the control of one disease could, through the elimination of natural antagonists resident on the leaf surface, lead to a disease by a previously less important, resistant, pathogen. As no one type of control is likely to be completely satisfactory all possibilities should be studied; it is therefore important that such basic investigations as the present one should be pursued in the hope of eventual practical use, despite the known problems with biological control on a field scale. We thank Mr M. Ames for growing the many plants used in this study. REFERENCES
BOLLEN, G. V. & FUCHS, A. (1970). On the specificity of the in vitro and in vivo antifungal activity ofbenomyl. Netherlands Journal of Plant Pathology ,6,299-312. DICKINSON, C. H. (1973). Effects of ethirimol and zineb on phylloplane microflora of barley. Transactions of the British Mycological Society 60, 423-431. HEUVEL, J. VAN DEN. (1971). Antagonism between pathogenic and saprophytic Alternaria species on bean leaves. Ecology of leaf surface micro-organisms, pp. 537-544. (ed. T. F. Preece and C. H. Dickinson). Academic Press. LAST, F. T. & DEIGHTON, F. C. (1965). The non-parasitic microflora on the surfaces of living leaves. Transactions of the British Mycological Society 48, 83-99. LAST, F. T. & WARREN, R. C. (19Tz). Non-parasitic microbes colonizing green leaves: their form and functions. Endeavour 31, 143-15°. LEBEN, C. (1965). Epiphytic microorganisms in relation to plant disease. Annual Review of Phytopatlwlogy 3, 20g-230' McBRIDE, R. P. (1971). Micro-organism interactions in the phyllosphere of larch. Ecology of leaf surface micro-organisms, pp. 545-555 (ed, T. F. Preece and C. H. Dickinson). Academic Press.
Trans. Br. mycol. Soc. 63 (I), (1974). Printed in Great Britain