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Production of Diplocarpon rosae conidia on overwintering rose leaves
N otes and brief articles
354
PRODUCTION OF DIPLO CARPO N R OSAE CONIDIA ON OVER WINTERING ROSE LEAVES R . T. A. COOK
R oyal H orticult ural S ociety's Garden, The role of fallen leave s in th e overwintering of Diplo carpon rosae Wolf, the cause of Rose Black Sp ot , has recently been re-examined by Knight & Wheeler (1977). They recovered viabl e conidia from such leaves after the winters of 1972-3, 1973-4 and 1974-5 and also found apothecia (a new record for Britain) after the latter two winters . As they pointed out, several aspects of the perennation of this fungus remain unresolved including the extent to which new acervuli arise on the overwintering mycelium. Information on this and the effect of chemicals on conidial production is reported here. Infected leaves (fioribunda cv, ' I ceberg') were collected on 12 January 1978 and overwintered in nylon-net bags laid on coarse sand in trays on the laboratory roof at Wisley. The infected areas on twelve leaves were carefully mapped to ind icat e the distribution of swollen acervuli. The leaves were placed singly in net bags on the sand, six with the adaxial and six with the abaxial surface upperm ost , and re-examined under a binocular microscope ( x 17) at c. 4-wk intervals. Other leaves were dipped in one of the following chemicals before they were heaped with random orientation in a net bag : 5 % aq. ammonium sulphate, 0'125 % (vjv) aq. Saprol (19 % a.i, (wjv) triforine), 0'2 % maneb (80 % W .P .). Leaves . dipped in water were used as controls. Six replicate samples of each treatment were arranged in a randomized block design. These leaves were not disturbed until 9 March 1978 when the production of conidia and their viability were examined. The remaining leaves were not treated before heaping them in net bags but on these, conidial production and spore viability was determined at intervals of c. 2 wk. Conidia were removed from samples of leaf di sks (1'4 em diam) by shaking in o-oa % "Teepol ', The conidial suspension was decanted, washed by centrifugation and resuspended in 0'5 ml water. Numbers of conidia were assessed using a haemocytometer and viability was determined by streaking suspensions on tap water agar and asses sing germination after 24 h at 20 °C. Similar methods were used to record the production and viability of conidia on leaves remaining on the bushes throughout the same period. Trans. Br. my col. Soc. 73 (2), (1979). 0007-1536/79/2828-5560 $00.35
~\7isley,
Woking, Surrey
In a separate experiment some leaves were overwintered loosely on a tray of sand on the roof, the tray being covered with wire netting as these conditions may be more suitable for apothecial development (Knight & Wheeler, 1977 ). No apothecia of D. rosae were found on any sample, but new acervuli developed within the original lesion s on the mapped leaves . They could be distinguished by their swollen, blisterlike appearance. Acervulus production proved to be very variable and there was no significant difference between leaves overwintered with different surfaces uppermost. Consequently the two sets of data were combined. Fig. 1 illu strates the changes which were observed in on e area. Many new acervuli appeared during the first tw o weeks of the experiment, but subsequently production declined as can be seen in the curve for total numbers (F ig. 2). Once swollen the majority of the acervuli were exposed within 3-5 wk by rupture of the ho st epidermis. Thus by the fifth week c. 50 % of the acervuli were uncovered (F ig. 2). On the other untreated leaves heaped in net bags, both spore release and viability reached a maximum after 5 wk and then fell sharply (Fig. 3). The very rapid decline in viability su ggests that conidia did not live long once the acervuli were exposed. Numbers of conidia recovered from leaves on bushes continued to rise with the passage of winter and as on detached leaves there was a positive relationship between spore release and viability although an initial delay in the increase in spore viability was more noticeable (Fig 3B). Unfortunately, the bushes of cv. 'Iceberg' were pruned back in early March and no leaves remained on the bushes on 23 March. However, an assessment done on nearby plants of fioribunda cv. Chinatown, which were not cut back, indicated that both spore release and viability were greater at this time. There were no clear relationships between weather factors and spore release or viability on either attached or detached leaves. Leaves dipped in triforine were comparatively well preserved in early March, whilst those treated with ammonium sulphate were the most
Printed in Great Britain
© 1979 The British Mycological Society
Notes and briefarticles
355
2 Feb.
19 Jan.
28 Feb.
2mm
L--.-l
28 Mar.
Fig.
1.
28 Apr.
Appearance of acervuli of Diplocarpon rosae on a mapped area of rose leaf at intervals during overwintering (1978). Covered acervulus (.), exposed acervulus (0).
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2.
/
----
2
4
10 8 6 Weeks from 12 Jan. 1978
12
14
16
Populations of acervuli of Diplocarpon rosae on detached rose leaves. Total acervuli (-0-), exposed acervuli (......).
Trans. Br. mycol, Soc. 73 (2), (1979). 0007-1536/79/2828-5560 $00.35
Printed in Great Britain
© 1979 The British Mycological Society
Notes and brief articles
356
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Fig. 3. The productionof conidia(-0-) of Diplocarponrosae and their viability(- -e- -)on detached(A) and attached (B) rose leaves. (All data from rose cv. 'Iceberg' except wk 10 recording for B where 'Chinatown' used.) decayed. Conidiophores of Botrytis cinerea were present on many leaves and ammonium sulphate appeared to encourage colonisation by this fungus. Triforine significantly suppressed (P ~ 0'01) conidial production (as measured in March); so did ammonium sulphate, but maneb had no effect (Table 1). None of the treatments was shown to Trans. Br. mycol. Soc. 73 (2), (1979). 0007-1536/79/2828-5560 $00.35
have a significant effect on spore viability at this time probably because of the variability of germination and the long period (8 wk) elapsing between treatment and assessment of results. As with these experiments, Green (1931) was also unable to detect apothecial D. rosae at Wisley. It is not clear whether this is due to
Printed in Great Britain
© 1979 The British Mycological Society
Notes and brief articles Table. 1 Effects of chemical treatment of rose leaves on overwintering of Diplocarpon rosae Treatment" Control Ammonium sulphate Triforine Maneb S.E.
Conidial Conidial production (conidiajcm - x 103) germination ( %) 16 4'8 10 23 15 N.S.
1'5 0'5 4"9 ± 1'2
* For details, see text .
adverse environmental conditions or whether the local strains of the fungus are sexually infertile. This investigation does indicate, however, that new acervuli can develop and produce viable spores throughout the winter within existing blackspot lesions. In 1978 many viable conidia were recovered from leaves at the end of March. These could act as primary inoculum; indeed there was evidence of infection on newly emerged rose leaves in the Garden at this time. It seems likely also that infected leaves remaining on bushes are a more important source of such inoculum. In this respect roses, such as ramblers or climbers, which are not cut back early in the year would be favourable sites for the overwintering of the fungus and it would be interesting to see whether or not
357
triforine or ammonium sulphate applied to the bushes in mid-January would suppress spore release in the spring as effectively as when applied to fallen leaves. There was an indication that ammonium sulphate promoted more rapid decomposition of the leaves which were then less favourable for the development of D. rosae. It might act in a similar way to urea, which when applied to apple orchards in autumn hastens leaf fall and leaf decomposition and subsequently suppresses overwintering Venturia inaequalis (Burchill & Cook, 1971). Since D. rosae can also cause lesions on green stems the relative importance of this as a means of overwintering needs investigation.
REFERENCES
BURCHILL, R. T. & COOK, R. T. A. (1971). The interaction of urea and micro-organisms in suppressing the development of perithecia of Venturia inaequalis (Cke) Wint. In Ecology of Leaf Surface Microorganisms (ed. T. F . Preece and C. H. Dickinson), pp. 471-483. London and New York : Academic Press. GREEN, D . E. (1931). Experiments and observations on the incidence and control of the blackspot disease of roses. Journal of the Royal Horticultu ral Society 56, 18-30. KNIGHT, C. & WHEELER, B. E. J. (1977). Perennation of Diplocarpon rosae on rose leaves. Transactions of the British Mycological Society 69, 385-389.
THERMOPHILOUS FUNGI ASSOCIATED WITH FRESHLY HARVESTED RICE SEEDS A. J. KUTHUBUTHEEN Department of Botany, University of Malaya, Kuala Lumpur 22-11, Malaysia
Thermophilous fungi have consistently been isolated from a variety of self-heated plant substrates (Cooney & Emerson, 1964 ; Fergus, 1964, 1978; Evans, 1971; Tansey, 1971). These fungi have also been reported from soil, nesting materials of animals and the surface of living green leaves (Apinis & Pugh, 1967; Eggins, von Szilvinyi & Allsopp, 1972; Gauchenaur, 1975; Tansey & Jack, 1976, 1977; Kuthubutheen & Pugh, 1977). The necessary temperature regimes on the natura) substrates may be provided by insolation, geothermal heat, body heat of nesting animals and the metabolic heat of microorganisms. On seeds, thermoTrans . Br . mycol. Soc. 73 (2), (1979).
0007-1536/79/2828-5560 $00.35
philous fungi have mostly been found associated with either heat-damaged or stored seeds (M ulinge & Apinis, 1969; Mulinge & Chesters, 1970; Mills & Bollen, 1976; Flannigan, 1969). In this study, the thermophilous fungi associated with freshly-harvested and dried rice seeds were investigated using six varieties of rice: Mahsuri, Mat Candu, MR7 (Hitam), MR7 (Puteh), Pulut Malaysia and SM 1. The fungi occurring on seeds surface-sterilized in 1% 'Chlorox' for 10 min (SS) and subsequently washed serially in sterile distilled water and on non-surface-sterilized seeds (N SS) were studied using two methods. In the
Printed in Great Britain
© 1979 The British Mycological Society
354
PRODUCTION OF DIPLO CARPO N R OSAE CONIDIA ON OVER WINTERING ROSE LEAVES R . T. A. COOK
R oyal H orticult ural S ociety's Garden, The role of fallen leave s in th e overwintering of Diplo carpon rosae Wolf, the cause of Rose Black Sp ot , has recently been re-examined by Knight & Wheeler (1977). They recovered viabl e conidia from such leaves after the winters of 1972-3, 1973-4 and 1974-5 and also found apothecia (a new record for Britain) after the latter two winters . As they pointed out, several aspects of the perennation of this fungus remain unresolved including the extent to which new acervuli arise on the overwintering mycelium. Information on this and the effect of chemicals on conidial production is reported here. Infected leaves (fioribunda cv, ' I ceberg') were collected on 12 January 1978 and overwintered in nylon-net bags laid on coarse sand in trays on the laboratory roof at Wisley. The infected areas on twelve leaves were carefully mapped to ind icat e the distribution of swollen acervuli. The leaves were placed singly in net bags on the sand, six with the adaxial and six with the abaxial surface upperm ost , and re-examined under a binocular microscope ( x 17) at c. 4-wk intervals. Other leaves were dipped in one of the following chemicals before they were heaped with random orientation in a net bag : 5 % aq. ammonium sulphate, 0'125 % (vjv) aq. Saprol (19 % a.i, (wjv) triforine), 0'2 % maneb (80 % W .P .). Leaves . dipped in water were used as controls. Six replicate samples of each treatment were arranged in a randomized block design. These leaves were not disturbed until 9 March 1978 when the production of conidia and their viability were examined. The remaining leaves were not treated before heaping them in net bags but on these, conidial production and spore viability was determined at intervals of c. 2 wk. Conidia were removed from samples of leaf di sks (1'4 em diam) by shaking in o-oa % "Teepol ', The conidial suspension was decanted, washed by centrifugation and resuspended in 0'5 ml water. Numbers of conidia were assessed using a haemocytometer and viability was determined by streaking suspensions on tap water agar and asses sing germination after 24 h at 20 °C. Similar methods were used to record the production and viability of conidia on leaves remaining on the bushes throughout the same period. Trans. Br. my col. Soc. 73 (2), (1979). 0007-1536/79/2828-5560 $00.35
~\7isley,
Woking, Surrey
In a separate experiment some leaves were overwintered loosely on a tray of sand on the roof, the tray being covered with wire netting as these conditions may be more suitable for apothecial development (Knight & Wheeler, 1977 ). No apothecia of D. rosae were found on any sample, but new acervuli developed within the original lesion s on the mapped leaves . They could be distinguished by their swollen, blisterlike appearance. Acervulus production proved to be very variable and there was no significant difference between leaves overwintered with different surfaces uppermost. Consequently the two sets of data were combined. Fig. 1 illu strates the changes which were observed in on e area. Many new acervuli appeared during the first tw o weeks of the experiment, but subsequently production declined as can be seen in the curve for total numbers (F ig. 2). Once swollen the majority of the acervuli were exposed within 3-5 wk by rupture of the ho st epidermis. Thus by the fifth week c. 50 % of the acervuli were uncovered (F ig. 2). On the other untreated leaves heaped in net bags, both spore release and viability reached a maximum after 5 wk and then fell sharply (Fig. 3). The very rapid decline in viability su ggests that conidia did not live long once the acervuli were exposed. Numbers of conidia recovered from leaves on bushes continued to rise with the passage of winter and as on detached leaves there was a positive relationship between spore release and viability although an initial delay in the increase in spore viability was more noticeable (Fig 3B). Unfortunately, the bushes of cv. 'Iceberg' were pruned back in early March and no leaves remained on the bushes on 23 March. However, an assessment done on nearby plants of fioribunda cv. Chinatown, which were not cut back, indicated that both spore release and viability were greater at this time. There were no clear relationships between weather factors and spore release or viability on either attached or detached leaves. Leaves dipped in triforine were comparatively well preserved in early March, whilst those treated with ammonium sulphate were the most
Printed in Great Britain
© 1979 The British Mycological Society
Notes and briefarticles
355
2 Feb.
19 Jan.
28 Feb.
2mm
L--.-l
28 Mar.
Fig.
1.
28 Apr.
Appearance of acervuli of Diplocarpon rosae on a mapped area of rose leaf at intervals during overwintering (1978). Covered acervulus (.), exposed acervulus (0).
20
I0---
16
I
'"0 X
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12
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o -;;:;-
'3 ~
8
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t)
< 4
1
e 0
0
Fig.
2.
/
----
2
4
10 8 6 Weeks from 12 Jan. 1978
12
14
16
Populations of acervuli of Diplocarpon rosae on detached rose leaves. Total acervuli (-0-), exposed acervuli (......).
Trans. Br. mycol, Soc. 73 (2), (1979). 0007-1536/79/2828-5560 $00.35
Printed in Great Britain
© 1979 The British Mycological Society
Notes and brief articles
356
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Fig. 3. The productionof conidia(-0-) of Diplocarponrosae and their viability(- -e- -)on detached(A) and attached (B) rose leaves. (All data from rose cv. 'Iceberg' except wk 10 recording for B where 'Chinatown' used.) decayed. Conidiophores of Botrytis cinerea were present on many leaves and ammonium sulphate appeared to encourage colonisation by this fungus. Triforine significantly suppressed (P ~ 0'01) conidial production (as measured in March); so did ammonium sulphate, but maneb had no effect (Table 1). None of the treatments was shown to Trans. Br. mycol. Soc. 73 (2), (1979). 0007-1536/79/2828-5560 $00.35
have a significant effect on spore viability at this time probably because of the variability of germination and the long period (8 wk) elapsing between treatment and assessment of results. As with these experiments, Green (1931) was also unable to detect apothecial D. rosae at Wisley. It is not clear whether this is due to
Printed in Great Britain
© 1979 The British Mycological Society
Notes and brief articles Table. 1 Effects of chemical treatment of rose leaves on overwintering of Diplocarpon rosae Treatment" Control Ammonium sulphate Triforine Maneb S.E.
Conidial Conidial production (conidiajcm - x 103) germination ( %) 16 4'8 10 23 15 N.S.
1'5 0'5 4"9 ± 1'2
* For details, see text .
adverse environmental conditions or whether the local strains of the fungus are sexually infertile. This investigation does indicate, however, that new acervuli can develop and produce viable spores throughout the winter within existing blackspot lesions. In 1978 many viable conidia were recovered from leaves at the end of March. These could act as primary inoculum; indeed there was evidence of infection on newly emerged rose leaves in the Garden at this time. It seems likely also that infected leaves remaining on bushes are a more important source of such inoculum. In this respect roses, such as ramblers or climbers, which are not cut back early in the year would be favourable sites for the overwintering of the fungus and it would be interesting to see whether or not
357
triforine or ammonium sulphate applied to the bushes in mid-January would suppress spore release in the spring as effectively as when applied to fallen leaves. There was an indication that ammonium sulphate promoted more rapid decomposition of the leaves which were then less favourable for the development of D. rosae. It might act in a similar way to urea, which when applied to apple orchards in autumn hastens leaf fall and leaf decomposition and subsequently suppresses overwintering Venturia inaequalis (Burchill & Cook, 1971). Since D. rosae can also cause lesions on green stems the relative importance of this as a means of overwintering needs investigation.
REFERENCES
BURCHILL, R. T. & COOK, R. T. A. (1971). The interaction of urea and micro-organisms in suppressing the development of perithecia of Venturia inaequalis (Cke) Wint. In Ecology of Leaf Surface Microorganisms (ed. T. F . Preece and C. H. Dickinson), pp. 471-483. London and New York : Academic Press. GREEN, D . E. (1931). Experiments and observations on the incidence and control of the blackspot disease of roses. Journal of the Royal Horticultu ral Society 56, 18-30. KNIGHT, C. & WHEELER, B. E. J. (1977). Perennation of Diplocarpon rosae on rose leaves. Transactions of the British Mycological Society 69, 385-389.
THERMOPHILOUS FUNGI ASSOCIATED WITH FRESHLY HARVESTED RICE SEEDS A. J. KUTHUBUTHEEN Department of Botany, University of Malaya, Kuala Lumpur 22-11, Malaysia
Thermophilous fungi have consistently been isolated from a variety of self-heated plant substrates (Cooney & Emerson, 1964 ; Fergus, 1964, 1978; Evans, 1971; Tansey, 1971). These fungi have also been reported from soil, nesting materials of animals and the surface of living green leaves (Apinis & Pugh, 1967; Eggins, von Szilvinyi & Allsopp, 1972; Gauchenaur, 1975; Tansey & Jack, 1976, 1977; Kuthubutheen & Pugh, 1977). The necessary temperature regimes on the natura) substrates may be provided by insolation, geothermal heat, body heat of nesting animals and the metabolic heat of microorganisms. On seeds, thermoTrans . Br . mycol. Soc. 73 (2), (1979).
0007-1536/79/2828-5560 $00.35
philous fungi have mostly been found associated with either heat-damaged or stored seeds (M ulinge & Apinis, 1969; Mulinge & Chesters, 1970; Mills & Bollen, 1976; Flannigan, 1969). In this study, the thermophilous fungi associated with freshly-harvested and dried rice seeds were investigated using six varieties of rice: Mahsuri, Mat Candu, MR7 (Hitam), MR7 (Puteh), Pulut Malaysia and SM 1. The fungi occurring on seeds surface-sterilized in 1% 'Chlorox' for 10 min (SS) and subsequently washed serially in sterile distilled water and on non-surface-sterilized seeds (N SS) were studied using two methods. In the
Printed in Great Britain
© 1979 The British Mycological Society