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Phylloplane mycoflora of bracken, Pteridium aquilinum
[ 3°5 ] Trans. Br, mycol. Soc. 62 (I), 305-3II (1974) Printed in Great Britain
PHYLLOPLANE MYCOFLORA OF BRACKEN, PTERIDIUM AQUILINUM By B. E. S. GODFREY Department of Biological Sciences, Sir John Cass School of Science and Technology, City of London Polytechnic, 31 Jewry Street, London, EC3N 2EY The ph ylloplane mycoflora on th e fronds of bracken, Pteridiumaquilinum, was followed during two seasons and was shown to be similar to that on angiosperm leaves. The succession involved Aureobasidiumpullulans, Botrytis cinerea and Cladosporium herbarum followed by Alternaria alternata and Epicoccum purpurascens, A leachate prepared from living fronds enhanced germ hyphal length and stimulated the production of extra germ-tubes from conidia of Botrytis cinerea, although it did not affect percentage germination.
During the past century a significant amount of information has been accumulated concerning the non-parasitic mycoflora of healthy leaf surfaces, which was reviewed by Last & Deighton (1965); since then there have been further reports of investigations on a variety of plants both from this country and abroad (see Preece & Dickinson, 1971). Most of the leaves studied have been from angiosperms; among the limited observations on plants of other groups are those of Kendrick & Burges (1962) and Rishbeth & Meredith (1957) on Pinus, and Kilbertus (1968) on a moss, Pseudoscleropodium purum. In spite of many records of fungi found associated with living bracken, Pteridium aquilinum L. (Kuhn), there appears to hav e been no systematic study of the phylloplane mycoflora of this plant, or indeed of any other pt eridophyte. This paper presents the results of an investigation of the surface mycoflora of green bracken fronds carried out during the summers of 1967 and 1968. Ruinen (1956) and Last & Deighton (1965) suggested that phylloplane fungi may subsist on materials leached from plant surfaces by water, and a number of other authors have reported that plant leachates can affect the germination and growth of fungal conidia. Brown (1922) correlated an increased electrical conductivity of water drops on petals of Coleus with increased germination of conidia of Botrytis cinerea in the same water drops . Kovacs & Szeoke (1956), on the other hand, demonstrated that high concentrations of leachates inhibited germination while low concentrations enhanced it. Both Hafiz (1952) and Kovacs (1955) showed an inhibition of conidial germination with aqueous leachate preparations, while Topps & Wain (1957) demonstrated the same effect using ether extracts of leachates from a number of different plants. The report presented here shows the effect of leachate prepared from living bracken fronds on B. cinerea. 20
MY C 62
306
Transactions British Mycological Society MATERIALS AND METHODS
Thephylloplane flora Collections of Pteridium aquilinum were made at a site on Hayes Common, Kent, from plants growing amongst a typical mixed heathland community. Sampling began as soon as the first fronds had expanded in early May, and it continued at monthly intervals until November, when the fronds were turning brown. Pinnae were cut from five different plants on each occasion, put into clean, unused polythene bags and taken to the laboratory within 2 h, where they were immediately washed using the technique outlined by Hogg & Hudson (1966). Ten whole pinna segments (average size 16 mm") were cut at random from each of the five pinnae, giving a total of 50 pinna segments, and each one was washed separately in 5-10 ml sterile distilled water for 10 min on a shaking machine. This was repeated seven times. Preliminary experiments showed that by the seventh washing only a few bacteria remained in the water. The washed pinna segments were placed separately in the centre of fifty 9 ern Petri dishes of PDA and incubated in the dark at 25°C. The cultures were examined after 6 days and thereafter at 6-day intervals over a period of 6 weeks. The number of records for each fungal species obtained each month (maximum possible 50), was determined and expressed as a percentage. Bracken frond leachate An aqueous leachate of bracken frond was prepared by immersing five freshly collected pinnae in 200 ml deionized water. Care was taken to ensure that no cut surfaces came into contact with the water, and the pinnae were agitated from time to time during the soaking. After 1 h the first batch of pinnae was replaced by a second, similar batch. The leachate was evaporated to dryness under vacuum in a rotary evaporator at 45°C, and the residue redissolved in 0'5 ml deionized water, giving a 14 K solution (Kovacs & Szeoke, 1956). Two further concentrations of 7 and 3'5 K were then prepared. A suspension of Botrytis cinerea conidia was prepared from a culture grown on PDA in the dark for fourteen days at 22° by adding deionized water and shaking. Mycelial fragments were removed by straining through two layers of surgical gauze and the spore concentration was adjusted to between 3 x 104 and 6 x 10 4 conidia per ml. Glass Petri dishes lined with moistened filter paper were used as incubation chambers, each containing a clean 76 x 25 mm glass microscope slide supported on a bent glass rod. One 0'02 ml drop of conidial suspension was placed at each end of the eight slides. Four replicate 0'02 ml drops from each of the leachate solutions were added to separate drops of conidial suspension and mixed thoroughly. As a control 0'02 ml aliquots of deionized water were used instead of leachate. The cultures were incubated in the dark at 22° and after 20 h fixed by adding one drop oflactophenol tinged with cotton blue. The experiment was assessed in three ways: germinated and ungerminated
Bracken mycoflora. B. E. S. Godfrey
307
conidia in each of ten, randomly selected, non-overlapping high-power fields were counted; the number of germ-tubes emerging from germinated conidia was recorded and the length of ten randomly selected germ-tubes was measured for each of the replicated treatments.
Direct microscopic examination oj pinna segments Pinna segments from samples collected in May and September were stained for direct microscopic study using the method described by Preece (1959)· RESULTS
The phylloplane flora The results are presented in Table I. During the two seasons, eighteen microfungi were recorded from washed pinna segments plated on to PDA. Samples collected in May showed that only three species were present at very low frequency on the young newly emerged fronds. ByJune Aureobasidium pullulans, Cladosporium herbarum and Botrytis cinerea were regularly present on the frond surface and once established, these three species remained at a high level of frequency until October. A. pullulans remained abundant in the November samples whereas C. herbarum and B. cinerea showed a marked decline. Alternaria alternata (= A. tenuis) and Epicoccum purpurascens were also frequent members of the phylloplane flora, although they did not make any significant appearance until later in the season than the other three species. Both were relatively common on the frond surface until October after which they showed a fall in frequency. Thirteen other species occurred only sporadically, although Penicillium sp. was abundant in June and July 1968, and Mucor hiemalis and Cephalosporium sp. were common on the fronds in August 1967. The colonization patterns observed in both years were similar. The total number of fungal species recorded each month is also shown in Table I. It can be seen that there was a general increase in the number of fungal species recorded as the fronds matured. Bracken frond leachate The results obtained with bracken frond leachate on the germination of conidia and early growth of germ-tubes of Botrytis cinerea are shown in Table 2. These results were analysed statistically by analysis of variance; the figures for percentage germination were transformed to arcsin values before computation. Analysis of the germination results showed that there was no statistically significant difference between the control and any of the treatments. Germ hyphal length results, on the other hand, gave a variance ratio of 31'3 which is significant at the 0'1 % level. The least significant difference, calculated from the residual mean square, was 145'2 at the I % level of significance, and on the basis of that figure all the germ-tubes were significantly longer than the control. Comparison of the number of emergent germ hyphae using a series of 2 x 2 contingency tests, showed that the 14 K solution stimulated 20-2
VJ
o
00
~ ~
Table
I.
Percentage frequency occurrence
~:::
of microfungi in the phylloplane of Pteridium aquilinum
o· ~
May
June
July
August
September
October
November
~
,---A-----.
,---A-----.
~
,---A-----.
1967
1967
1968
1967
1968
1967
1968
~
1968
1968
~
1967 96 66 22
80 52 64 4
100 38 60 4 12 38
100 60 50 74 76 62
90 78 66
100 78 60 54 64 28
100 74 58 52 60 2
100
36
94 96 52 22 14 14
3 52 42
98 24 76 2 42 12
7
9
9
10
9
6
14
9
1967 1968•
Aureobasidium pullulans (de Bary) Arnaud
Cladosporium herbarum (Pers.) Link ex S. F. Gray Botrytis cinerea Pers. ex Pers. Alternaria altemata (Fr.) Keissler Epicoccum purpurascens Ehrenb. ex Schlecht Other fungi Total number of species recorded on washed pinna segments
-
-
-
2 2
-
-
2
-
3
-
-
16 6
-
• No records were available for May 1968.
~3
18
II
~2
1967
1968
100 36 32 28 26 34
90 26 54 16 2 22
14
II
'" tJj ~.
g; ~ g C"
O!i.
B
~
~.
~
Bracken mycoflora. B. E. S. Godfrey Table
309
The effect of bracken frond leachate on the germination ofconidia and hyphal growth ofBotrytis cinerea
2.
Number of conidia germinating with Effective leachate concen tration
Mean per centage germination (as arcsin values)
Mean germ hyphal length (pm)
} x 14K l X7 K l X3'5 K Control
63'0 65'8 66'2 63'0
633.6 360.8 2 85'5 97'3
,------"----,
One Two hypha hyphae 345 545 587 473
192 72 19 72
significantly more conidia to produce two germ hyphae than either the other treatments or the control. Also the number of conidia with two germ hyphae was depressed in the 3'5 K solution compared with the 7 K solution and the control.
Direct microscopic observation ofpinna segments Microscopic examination of cleared pinna segments from living fronds showed that in both May and September, conidia, microsclerotia and fungal hyphae were present on the frond surface. In May some of the conidia had apparently germinated, although by September germinated conidia were much more frequent and hyphal growth was quite extensive. Conidia and microsclerotia of Aureobasidium pullulans and Cladosporium herbarum were often found in th e grooves on the pinna surface above the veins, and hyphae too, tend ed to grow along these grooves. DISCUSSION
Under normal conditions the surfaces of healthy bracken fronds bear a mycoflora similar to that found on other plants from temperate regions (Dickinson, 1965, 1967; Hogg & Hudson, 1966; Diem, 1967; Holloman, 1967; Pugh & Williams, 1968; Hislop & Cox, 1969; Pugh & Mulder, 1971; Pugh & Buckley, 1971). Very young fronds were apparently free from microfungi, suggesting that the tight rolling of the frond during development prevents contamination as it grows through the soil. After unrolling, the fronds quickly acquired a phylloplane flora consisting of Aureobasidium pullulans, Cladosporium herbarum, Botrytis cinerea, Alternaria alternata and Epicoccum purpurascens, all of which persisted until the fronds had died. Hudson (1968), and others, have drawn attention to similar patterns of succession on a variety of leaves. Microscopic examination of cleared pinna segments showed that some microfungi were actively growing on the surface; hyphae, mainly growing along the veins, were occasionally seen on young fronds collected in May, although they were much more common later in the year. As well as hyphae, conidia and microsclerotia were also very common, again occurring more frequently in the region of the veins than elsewhere. The epidermal cells above the veins tend to be slightly lower than the surrounding
310
Transactions British Mycological Society
cells. Pugh & Buckley (1971) suggested a number of reasons for veinal distribution of fungi, including the washing of conidia into the grooves by rain water. There may also be differences in the nature of leachates from the veinal regions which encourage spores to germinate and grow. Clearly some of the microfungi (including Aureobasidium pullulans and Cladosporium herbarum) observed growing from plated pinna segments, represented forms which were actively growing on the frond surface. Dickinson (1965) suggested that C. herbarum was capable of growing on living leaves of Halimione portulacoides, particularly during the summer months. Ruscoe (1971) found A. pullulans colonizing leaf surfaces of Nothofagus truncata when they were quite young, while Pugh & Buckley (1971) also showed fungal growth on Acer leaves from July onwards. Ruinen (1956) and Last & Deighton (1965) suggested that phylloplane fungi might well subsist on substances leached from leaves into free water on the surface. Tukey (1971) reviewed the evidence which showed that appreciable quantities of a variety of different substances are leached from leaves by water; it has been shown that such leachates can affect germination and growth of certain microfungi (Brown, 1922; Hafiz, 1952; Kovacs, 1955; Kovacs & Szeoke, 1956). The leachate prepared from living bracken fronds had no effect on percentage germination of conidia of Botrytis cinerea, although it did stimulate the production of longer germ hyphae than deionized water. This suggests that it contained nutrients which the hyphae were using as a food source: certainly the longest hyphae were produced in the strongest leachate. Grover (197I) recorded that amino acids and carbohydrates were among the substances identified in leachate from leaves and other parts of Capsicum frutescens. Simple tests on the bracken frond leachate used here also showed that amino acids and carbohydrates were present and could represent the nutrients being used by B. cinerea in this experiment. A higher proportion of conidia germinated with two germ-tubes in the leachate than in the deionized water control. This was particularly marked in the most concentrated leachate. This may be simple nutritional effect, although studies using ether extracts of a bracken frond leachate (Godfrey, 1972) showed a similar result. The ether extract gave a strong positive reaction when tested with a reagent for phenols but showed little evidence of the presence of amino acids and carbohydrates. It is possible, therefore, that the augmentation of number of germ hyphae may be associated with phenolic substances in the leachate. Further work is needed to clarify this point. The material in this paper forms part of a thesis accepted by the University of London for the Ph.D. degree. I am grateful to Dr J. K. Spearing for encouragement and advice and Dr C. H. Dickinson for reading and commenting on this manuscript.
Bracken mycoflora. B. E. S. Godfrey REFERENCES
BROWN, W. (1922) . Studies in the physiology of parasitism. Annals of Botany36,101-119. DICKINSON, C. H. (1965). The mycoflora associated with Halimione portulacoides. III. Fungi on green and moribund leaves. Transactions of the British Mycological Society 48, 603-6 10. DICKINSON, C. H . (1967)' Fungal colonisation of Pisum leaves. Canadian Journal of Botany 45, 9 15-927. DIEM, H. G. (1967). Microorganismes de la surface des feuilles. I. Obs ervations preliminaires de la population microbienne de l'Orge, Bulletin de l'Ecole Na tionale superieure agronome de Nancy 9, 102-108. GODFREY, B. E. S. (1972). A study of the microfungal flora ofleaves of bracken, Pteridium aquilinum L. (K uhn), with reference to their role in the phyllosphere. Ph.D. thesis, University of London. GROVER, R. K. (1971). Participation of host exudate chemicals in appressorium formation by Colletotrichum piperatum. In Ecology of leaf-surface micro-organisms (ed. T. F. Preece and C. H. Dickinson). London and New York: Academic Press. HAFIZ, A. (1952). Basis of resistance to Mycosphaerella blight. Phytopathology 42,422-424. HISLOP, E. C. & Cox, T. W. (1969). The effect of captan on the non-parasitic microflora of apple leaves. Transactions of the British Mycological Society 52, 223-235. HOGG, B. M. & HUDSON, H.]. (1966). Microfungi on the leaves of Fagussy luatica. I. The microfungal succession. Transactions of the British Mycological Society 49, 185-192. HOLLOMAN, D. W. (1967). Observations of the phylloplane flora of potatoes. European PotatoJournal 10, 53-61. HUDSON, H . ]. (1968). The ecology of fungi on plant remains above ground. New Phytologist 67, 837-874. KENDRICK, W. B. & BURGES, A. (1962). Biological aspects of decay of Pinus sylvestris leaf litter. Nova Hedwigia 4, 3 I3-342. KILBERTUS, G. (1968) . Decomposition d 'une mousse: Pseudoscleropodium purum (H edw.) Fleisch. dans la nature. Bulletin de l' Ecole Nationale superieure agronomie de Nancy 10, 20-32. KOVACS, A. (1955) . Uber die Ursachen der unterscheidlichen Resistenz der Zuckerriibensortcn gegen Cercospora beticola Sacco Phytopathologische Zeitschrift 24, 283-298. KOVACS, A. & SZEOKE, E. ( 1956) . Die phytopathologische Bedeutung der kutikularen Exkretion. Phytopathologische Z eitschrift 27, 335-349. LAST, F. T. & DEIGHTON, F. C. (1965). The non-parasitic micro flora on the surfaces of living leaves. Transactions of the British My cological Society 48, 83-99. PREECE, T. F. (1959). A staining method for the study of apple scab infections . Plant pathology 8, 127-129. PREECE, T. F. & DICKINSON, C. H. (1971) Eds. Ecology of leaf-surface micro-organisms. London and New York: Academic Press. PUGH, G.]. F. & BUCKLEY, N. G. (1971). The leaf as a surface for colonisation. In Ecology ofleaf-surface micro-organisms (ed . T. F. Preece and C. H. Dickinson). London and New York: Academic Press. PUGH, G.]. F. & MULDER, J. L. (1971). Mycoflora associated with Typha latifolia. Transactions of the British Mycological Society 57, 273-282. PUGH, G.]. F. & WILLIAMS, G. M. (1968). Fungi associated with Salsola kali. Transactions of the British Mycological Society 51, 389-396. RISHBETH, J. & MEREDITH, D. S. (1957). Surface microflora of pine needles. Nature, London 179, 682-683. R UINEN,]. (1956). Occurrence of Beijerinckia species in the phyllosphere. Nature, London 177, 220-22 I. RUSCOE, Q . W. (1971). Mycoflora of living and dead leaves of Nothofagus truncate, Transactions of the British Mycological Society 56, 463-474. Topps,]. H. & WAIN, R. L. (1957). Fungistatic properties of leaf exudates. Nature, London 179, 652-653. TUKEY, H. B. (1971). Leaching of substances from plants. In Ecology of leaf-surface microorganisms (ed. T. F. Preece and C. H. Dickinson). London and New York: Academic Press.
(Accepted f or publication 5 June 1973)
PHYLLOPLANE MYCOFLORA OF BRACKEN, PTERIDIUM AQUILINUM By B. E. S. GODFREY Department of Biological Sciences, Sir John Cass School of Science and Technology, City of London Polytechnic, 31 Jewry Street, London, EC3N 2EY The ph ylloplane mycoflora on th e fronds of bracken, Pteridiumaquilinum, was followed during two seasons and was shown to be similar to that on angiosperm leaves. The succession involved Aureobasidiumpullulans, Botrytis cinerea and Cladosporium herbarum followed by Alternaria alternata and Epicoccum purpurascens, A leachate prepared from living fronds enhanced germ hyphal length and stimulated the production of extra germ-tubes from conidia of Botrytis cinerea, although it did not affect percentage germination.
During the past century a significant amount of information has been accumulated concerning the non-parasitic mycoflora of healthy leaf surfaces, which was reviewed by Last & Deighton (1965); since then there have been further reports of investigations on a variety of plants both from this country and abroad (see Preece & Dickinson, 1971). Most of the leaves studied have been from angiosperms; among the limited observations on plants of other groups are those of Kendrick & Burges (1962) and Rishbeth & Meredith (1957) on Pinus, and Kilbertus (1968) on a moss, Pseudoscleropodium purum. In spite of many records of fungi found associated with living bracken, Pteridium aquilinum L. (Kuhn), there appears to hav e been no systematic study of the phylloplane mycoflora of this plant, or indeed of any other pt eridophyte. This paper presents the results of an investigation of the surface mycoflora of green bracken fronds carried out during the summers of 1967 and 1968. Ruinen (1956) and Last & Deighton (1965) suggested that phylloplane fungi may subsist on materials leached from plant surfaces by water, and a number of other authors have reported that plant leachates can affect the germination and growth of fungal conidia. Brown (1922) correlated an increased electrical conductivity of water drops on petals of Coleus with increased germination of conidia of Botrytis cinerea in the same water drops . Kovacs & Szeoke (1956), on the other hand, demonstrated that high concentrations of leachates inhibited germination while low concentrations enhanced it. Both Hafiz (1952) and Kovacs (1955) showed an inhibition of conidial germination with aqueous leachate preparations, while Topps & Wain (1957) demonstrated the same effect using ether extracts of leachates from a number of different plants. The report presented here shows the effect of leachate prepared from living bracken fronds on B. cinerea. 20
MY C 62
306
Transactions British Mycological Society MATERIALS AND METHODS
Thephylloplane flora Collections of Pteridium aquilinum were made at a site on Hayes Common, Kent, from plants growing amongst a typical mixed heathland community. Sampling began as soon as the first fronds had expanded in early May, and it continued at monthly intervals until November, when the fronds were turning brown. Pinnae were cut from five different plants on each occasion, put into clean, unused polythene bags and taken to the laboratory within 2 h, where they were immediately washed using the technique outlined by Hogg & Hudson (1966). Ten whole pinna segments (average size 16 mm") were cut at random from each of the five pinnae, giving a total of 50 pinna segments, and each one was washed separately in 5-10 ml sterile distilled water for 10 min on a shaking machine. This was repeated seven times. Preliminary experiments showed that by the seventh washing only a few bacteria remained in the water. The washed pinna segments were placed separately in the centre of fifty 9 ern Petri dishes of PDA and incubated in the dark at 25°C. The cultures were examined after 6 days and thereafter at 6-day intervals over a period of 6 weeks. The number of records for each fungal species obtained each month (maximum possible 50), was determined and expressed as a percentage. Bracken frond leachate An aqueous leachate of bracken frond was prepared by immersing five freshly collected pinnae in 200 ml deionized water. Care was taken to ensure that no cut surfaces came into contact with the water, and the pinnae were agitated from time to time during the soaking. After 1 h the first batch of pinnae was replaced by a second, similar batch. The leachate was evaporated to dryness under vacuum in a rotary evaporator at 45°C, and the residue redissolved in 0'5 ml deionized water, giving a 14 K solution (Kovacs & Szeoke, 1956). Two further concentrations of 7 and 3'5 K were then prepared. A suspension of Botrytis cinerea conidia was prepared from a culture grown on PDA in the dark for fourteen days at 22° by adding deionized water and shaking. Mycelial fragments were removed by straining through two layers of surgical gauze and the spore concentration was adjusted to between 3 x 104 and 6 x 10 4 conidia per ml. Glass Petri dishes lined with moistened filter paper were used as incubation chambers, each containing a clean 76 x 25 mm glass microscope slide supported on a bent glass rod. One 0'02 ml drop of conidial suspension was placed at each end of the eight slides. Four replicate 0'02 ml drops from each of the leachate solutions were added to separate drops of conidial suspension and mixed thoroughly. As a control 0'02 ml aliquots of deionized water were used instead of leachate. The cultures were incubated in the dark at 22° and after 20 h fixed by adding one drop oflactophenol tinged with cotton blue. The experiment was assessed in three ways: germinated and ungerminated
Bracken mycoflora. B. E. S. Godfrey
307
conidia in each of ten, randomly selected, non-overlapping high-power fields were counted; the number of germ-tubes emerging from germinated conidia was recorded and the length of ten randomly selected germ-tubes was measured for each of the replicated treatments.
Direct microscopic examination oj pinna segments Pinna segments from samples collected in May and September were stained for direct microscopic study using the method described by Preece (1959)· RESULTS
The phylloplane flora The results are presented in Table I. During the two seasons, eighteen microfungi were recorded from washed pinna segments plated on to PDA. Samples collected in May showed that only three species were present at very low frequency on the young newly emerged fronds. ByJune Aureobasidium pullulans, Cladosporium herbarum and Botrytis cinerea were regularly present on the frond surface and once established, these three species remained at a high level of frequency until October. A. pullulans remained abundant in the November samples whereas C. herbarum and B. cinerea showed a marked decline. Alternaria alternata (= A. tenuis) and Epicoccum purpurascens were also frequent members of the phylloplane flora, although they did not make any significant appearance until later in the season than the other three species. Both were relatively common on the frond surface until October after which they showed a fall in frequency. Thirteen other species occurred only sporadically, although Penicillium sp. was abundant in June and July 1968, and Mucor hiemalis and Cephalosporium sp. were common on the fronds in August 1967. The colonization patterns observed in both years were similar. The total number of fungal species recorded each month is also shown in Table I. It can be seen that there was a general increase in the number of fungal species recorded as the fronds matured. Bracken frond leachate The results obtained with bracken frond leachate on the germination of conidia and early growth of germ-tubes of Botrytis cinerea are shown in Table 2. These results were analysed statistically by analysis of variance; the figures for percentage germination were transformed to arcsin values before computation. Analysis of the germination results showed that there was no statistically significant difference between the control and any of the treatments. Germ hyphal length results, on the other hand, gave a variance ratio of 31'3 which is significant at the 0'1 % level. The least significant difference, calculated from the residual mean square, was 145'2 at the I % level of significance, and on the basis of that figure all the germ-tubes were significantly longer than the control. Comparison of the number of emergent germ hyphae using a series of 2 x 2 contingency tests, showed that the 14 K solution stimulated 20-2
VJ
o
00
~ ~
Table
I.
Percentage frequency occurrence
~:::
of microfungi in the phylloplane of Pteridium aquilinum
o· ~
May
June
July
August
September
October
November
~
,---A-----.
,---A-----.
~
,---A-----.
1967
1967
1968
1967
1968
1967
1968
~
1968
1968
~
1967 96 66 22
80 52 64 4
100 38 60 4 12 38
100 60 50 74 76 62
90 78 66
100 78 60 54 64 28
100 74 58 52 60 2
100
36
94 96 52 22 14 14
3 52 42
98 24 76 2 42 12
7
9
9
10
9
6
14
9
1967 1968•
Aureobasidium pullulans (de Bary) Arnaud
Cladosporium herbarum (Pers.) Link ex S. F. Gray Botrytis cinerea Pers. ex Pers. Alternaria altemata (Fr.) Keissler Epicoccum purpurascens Ehrenb. ex Schlecht Other fungi Total number of species recorded on washed pinna segments
-
-
-
2 2
-
-
2
-
3
-
-
16 6
-
• No records were available for May 1968.
~3
18
II
~2
1967
1968
100 36 32 28 26 34
90 26 54 16 2 22
14
II
'" tJj ~.
g; ~ g C"
O!i.
B
~
~.
~
Bracken mycoflora. B. E. S. Godfrey Table
309
The effect of bracken frond leachate on the germination ofconidia and hyphal growth ofBotrytis cinerea
2.
Number of conidia germinating with Effective leachate concen tration
Mean per centage germination (as arcsin values)
Mean germ hyphal length (pm)
} x 14K l X7 K l X3'5 K Control
63'0 65'8 66'2 63'0
633.6 360.8 2 85'5 97'3
,------"----,
One Two hypha hyphae 345 545 587 473
192 72 19 72
significantly more conidia to produce two germ hyphae than either the other treatments or the control. Also the number of conidia with two germ hyphae was depressed in the 3'5 K solution compared with the 7 K solution and the control.
Direct microscopic observation ofpinna segments Microscopic examination of cleared pinna segments from living fronds showed that in both May and September, conidia, microsclerotia and fungal hyphae were present on the frond surface. In May some of the conidia had apparently germinated, although by September germinated conidia were much more frequent and hyphal growth was quite extensive. Conidia and microsclerotia of Aureobasidium pullulans and Cladosporium herbarum were often found in th e grooves on the pinna surface above the veins, and hyphae too, tend ed to grow along these grooves. DISCUSSION
Under normal conditions the surfaces of healthy bracken fronds bear a mycoflora similar to that found on other plants from temperate regions (Dickinson, 1965, 1967; Hogg & Hudson, 1966; Diem, 1967; Holloman, 1967; Pugh & Williams, 1968; Hislop & Cox, 1969; Pugh & Mulder, 1971; Pugh & Buckley, 1971). Very young fronds were apparently free from microfungi, suggesting that the tight rolling of the frond during development prevents contamination as it grows through the soil. After unrolling, the fronds quickly acquired a phylloplane flora consisting of Aureobasidium pullulans, Cladosporium herbarum, Botrytis cinerea, Alternaria alternata and Epicoccum purpurascens, all of which persisted until the fronds had died. Hudson (1968), and others, have drawn attention to similar patterns of succession on a variety of leaves. Microscopic examination of cleared pinna segments showed that some microfungi were actively growing on the surface; hyphae, mainly growing along the veins, were occasionally seen on young fronds collected in May, although they were much more common later in the year. As well as hyphae, conidia and microsclerotia were also very common, again occurring more frequently in the region of the veins than elsewhere. The epidermal cells above the veins tend to be slightly lower than the surrounding
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Transactions British Mycological Society
cells. Pugh & Buckley (1971) suggested a number of reasons for veinal distribution of fungi, including the washing of conidia into the grooves by rain water. There may also be differences in the nature of leachates from the veinal regions which encourage spores to germinate and grow. Clearly some of the microfungi (including Aureobasidium pullulans and Cladosporium herbarum) observed growing from plated pinna segments, represented forms which were actively growing on the frond surface. Dickinson (1965) suggested that C. herbarum was capable of growing on living leaves of Halimione portulacoides, particularly during the summer months. Ruscoe (1971) found A. pullulans colonizing leaf surfaces of Nothofagus truncata when they were quite young, while Pugh & Buckley (1971) also showed fungal growth on Acer leaves from July onwards. Ruinen (1956) and Last & Deighton (1965) suggested that phylloplane fungi might well subsist on substances leached from leaves into free water on the surface. Tukey (1971) reviewed the evidence which showed that appreciable quantities of a variety of different substances are leached from leaves by water; it has been shown that such leachates can affect germination and growth of certain microfungi (Brown, 1922; Hafiz, 1952; Kovacs, 1955; Kovacs & Szeoke, 1956). The leachate prepared from living bracken fronds had no effect on percentage germination of conidia of Botrytis cinerea, although it did stimulate the production of longer germ hyphae than deionized water. This suggests that it contained nutrients which the hyphae were using as a food source: certainly the longest hyphae were produced in the strongest leachate. Grover (197I) recorded that amino acids and carbohydrates were among the substances identified in leachate from leaves and other parts of Capsicum frutescens. Simple tests on the bracken frond leachate used here also showed that amino acids and carbohydrates were present and could represent the nutrients being used by B. cinerea in this experiment. A higher proportion of conidia germinated with two germ-tubes in the leachate than in the deionized water control. This was particularly marked in the most concentrated leachate. This may be simple nutritional effect, although studies using ether extracts of a bracken frond leachate (Godfrey, 1972) showed a similar result. The ether extract gave a strong positive reaction when tested with a reagent for phenols but showed little evidence of the presence of amino acids and carbohydrates. It is possible, therefore, that the augmentation of number of germ hyphae may be associated with phenolic substances in the leachate. Further work is needed to clarify this point. The material in this paper forms part of a thesis accepted by the University of London for the Ph.D. degree. I am grateful to Dr J. K. Spearing for encouragement and advice and Dr C. H. Dickinson for reading and commenting on this manuscript.
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(Accepted f or publication 5 June 1973)