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Proceedings
Trans. Brit. mycol. Soc. 47 (I), 143- 145 (1964) Printed in Great Britain
143
PROCEEDINGS MEETINGS 4 October 1963. Meeting held at the School of Pharmacy, University of London, at I 1.0 a.m. with the President, Prof. J. Colhoun, in the Chair. D. PITT (Birmingham). Observations on sharp eyespot disease of cereals. Several isolates of Corticium (Rhizoctonia) solani were obtained from wheat stems bearing sharp eyespot lesions in the summers of 1960 and 1962. Differences in virulence of the isolates, assessed by severity of attack on seedlings, was demonstrated. Isolates taken from different plants in the same crop were shown to differ significantly in respect of virulence. All isolates obtained from wheat lesions attacked both winter and spring varieties of wheat, barley, oats and rye. Several varieties of spring and winter wheats have shown no differences in susceptibility to the disease. Infection of the host plant was shown to take place at any time during the growing period; however, infection was most severe in the earlier stages of growth. Greenhouse experiments demonstrated that the effects of the pathogen on seedlings were serious, resulting in lack of establishment and reduction in height and dry weight. Microscopic examination of disease lesions on mature plants indicated that penetration by the fungus is often deep, and in heavily infected plants may result in partial disintegration of the vascular system. Disease in the field was found to occur on contrasting soil types from light sandy soils to the heavy clays of southern Warwickshire. There was no significant effect of pH over the range 4'7-8'3 as studied in sand culture experiments. The disease on seedlings was favoured by the lowest temperature (9'3° C.) and the lowest soil-moisture content (25"5% W.H.C.) tested. The abnormally high incidence of the disease in the field in 1960 and 1962 has been related to the favourable temperatures and the unusually dry conditions prevailing in the spring of these years and in the autumn of the previous years, times of the year when plants have been found to be most susceptible to attack by the pathogen. Experiments demonstrated the ability of sclerotia to survive under conditions of desiccation in the laboratory and in natural soil. These sclerotia were able to regerminate several times on repeated disturbance, a factor which could compensate, to some extent, for their lack of specificity in response to stimuli. The pathogen rapidly declined to a low level in artificially infested natural soil under either fallow or various cropping regimes. The presence of a ley arrested decline of the pathogen to some extent. The inability of C. solani to colonize buried wheat straw or buckwheat was shown and also its ability for hmited survival in naturally infected straws.
P. D. HEWETT (Official Seed Testing Station, Cambridge). The incidence of Septoria nodorum and Fusarium spp. on wheat. To be published in these Transactions.
R. G. PAWSEY (Alice Holt). The importance of Peridermium pini and Cronartium ribicola on conifers in Britain. Among the many rust fungi that attack timber trees in this country only Peridermium pini Lev. on two-needle pines and Cronartium ribicola]. C. Fischer on five-needle pines are of any considerable economic importance. Recent survey and research work has
144
Transactions British Mycological Society
indicated that the pathogenic potential of C. ribicola, particularly on Pinus strobus, has been greatly overestimated for many years in Britain. It is now thought that if nursery stock can be raised free of infection, P. strobus can be planted in many forest areas which are sufficiently isolated from the most important alternate host, blackcurrant, with low risk of infection in the timber crop. Extensive survey work in the large pine forests in the area south of the Moray Firth has shown that losses in Scots pine caused by Peridermium pini, particularly in crops 25-40 years old, have increased in recent years. Very little is known of the infection biology of this autoecious rust. Although present losses are not serious over the area generally affected, P. pini is regarded as an important threat to production of Scots pine in the future. There is evidence that host susceptibility is related to provenance, or seed origin, and that the intensity of infection during different periods in the life of the crop is related to crop vigour and to the sequence of normal forest management.
I.
(in collaboration with B. Kassanis, Rothamsted Experimental Station). Transmission of tobacco necrosis virus by Olpidium.
MACFARLANE
Olpidium was maintained on lettuce grown in sand culture and zoospore suspensions were obtained by placing infected roots in dilute (1/20) Hoagland's solution for 10 min. Transmission experiments were made by immersing roots of small lettuce or Mung bean (Phaseolus aureus) seedlings in zoospore suspensions to which purified tobacco necrosis virus (TNV) was added. Three days later the lettuce roots were crushed and inoculated to leaves of French bean (P. vulgaris) on which TNV produces necrotic local lesions, the number of lesions giving a measure of the virus content of the assayed roots. On Mung bean roots necrotic lesions formed one day after exposure to zoospores and virus. Virus infection occurred only when roots were exposed to virus and Olpidium zoospores. This confirms American reports of the apparent transmission of TNV by Olpidium (Teakle, 1962). The identity of the transmitting agent with Olpidium, will, of course, remain in some doubt until Olpidium can be obtained free from other micro-organisms. It is assumed here that Olpidium does, in fact, transmit the virus. Transmission was favoured by low salt concentration (dilution 1/20) and neutral or slightly alkaline reaction of the medium and depended also on the concentrations of virus and zoospores. With abundant zoospores, transmission to lettuce was obtained with as little as 0'05 p.g virus/I. When there was plenty of virus (say 5 p.g/I.) 50-100 zoospores/ml. were effective. There was not always a strict correlation between fungus infection, as judged by the presence and number of zoosporangia in the root, and virus infection. Perhaps penetration of zoospores which fail to develop is sufficient to transmit virus. For example, virus is transmitted when the fungus is killed by heating the inoculated root after sufficient time is allowed for penetration, Some Olpidium isolates were naturally contaminated with TNV, but, from these, some virus-free cultures were obtained by inoculating plants with very low numbers of zoospores. One of these isolates was contaminated by another virus transmitted by Olpidium, lettuce big vein virus (Campbell & Grogan, 1963) but, unlike TNV, it was not lost in this way. Zoospores which had been mixed with TNV were partially washed free from virus by centrifugation. Further evidence that the relationship between TNV and Olpidium is superficial came from experiments with antiserum to TNV. Roots were treated with zoospores that had been exposed to virus before or after antiserum was added to the virus. Antiserum prevented transmission in both instances, but a much greater concentration of antiserum was needed if zoospores were mixed with virus before antiserum was added. This suggests that the virus is principally on the zoospore surface, where it is presumably protected from the aggregating effects which allow fewer antibodies to render virus in suspension unavailable for transmission. REFERENCES
CAMPBELL, R. N. & GROGAN, R. G. (1963). Big-vein virus of lettuce and its transmission by Olpidium brassicae. P~ytopathology, 53, 2.')2-259. TEAKLE, D. S. (1962). Transmission of tobacco necrosis virus by a fungus, Olpidium brassicae. Virology, 18, 224-231.
Proceedings
145
L. G. WILLOUGHBY (Ambleside). Observations on some aquatic fungi in Australia. To be published in the Proceedings of the Royal Society of Victoria and in the Journal of Ultrastructure Research.
M. W.
DICK
(Reading). Some recent work on filamentous water moulds.
A study of the Leptomitaceae has now been started, based at present on four species (Leptomitus lacteus (Roth) Ag., Apodachlya brachynema (Hildeb.) Prings., A. minima Coker and Apodachlyella completa (Humph.) Indoh), all of which have been isolated from the environs of Blelham Tarn in the English Lake District, and are being maintained in pure culture. Preliminary investigations of cultural requirements, using a glucose-peptone broth as a basic medium, hyphal tips in agar as a source of inoculum and dry weight as a criterion of growth, have revealed different responses to the pH of the medium. L. lacteus and Apodachlya brachynema were similar, differing only in the pH optima (6-6·6 and 5,6-6'2, respectively); both grew at pH 5'3-6'9, but gave little or no growth in neutral or alkaline media. On the other hand, A. minima and Apodachlyella complete grew well at all pH values tested (5'3-8), although growth of both was slightly better in acid media. In addition there were indications that Apodachlya minima might have a second optimum below pH 5'3. Temperature has proved surprisingly critical for Apodachlyella completa. Above 20° C. there was no appreciable growth, the hyphal tips becoming inflated and filled with clumped, granular protoplasm. The phenomenon was reversible: removal to an incubator at 19°, even after several days at 23°, resulted in normal growth being resumed after less than 48 hr. Attempts to find a completely defined medium for A. complete have not yet succeeded. Yeast extract is not essential when peptone is provided as the nitrogen source, but it is required when mixed amino acids (including methionine), nitrate or ammonium ions (including ammonium thiosulphate) are supplied. No growth occurred in the presence of nitrite ions. An illustrated account of the morphology of A. completa was given.
143
PROCEEDINGS MEETINGS 4 October 1963. Meeting held at the School of Pharmacy, University of London, at I 1.0 a.m. with the President, Prof. J. Colhoun, in the Chair. D. PITT (Birmingham). Observations on sharp eyespot disease of cereals. Several isolates of Corticium (Rhizoctonia) solani were obtained from wheat stems bearing sharp eyespot lesions in the summers of 1960 and 1962. Differences in virulence of the isolates, assessed by severity of attack on seedlings, was demonstrated. Isolates taken from different plants in the same crop were shown to differ significantly in respect of virulence. All isolates obtained from wheat lesions attacked both winter and spring varieties of wheat, barley, oats and rye. Several varieties of spring and winter wheats have shown no differences in susceptibility to the disease. Infection of the host plant was shown to take place at any time during the growing period; however, infection was most severe in the earlier stages of growth. Greenhouse experiments demonstrated that the effects of the pathogen on seedlings were serious, resulting in lack of establishment and reduction in height and dry weight. Microscopic examination of disease lesions on mature plants indicated that penetration by the fungus is often deep, and in heavily infected plants may result in partial disintegration of the vascular system. Disease in the field was found to occur on contrasting soil types from light sandy soils to the heavy clays of southern Warwickshire. There was no significant effect of pH over the range 4'7-8'3 as studied in sand culture experiments. The disease on seedlings was favoured by the lowest temperature (9'3° C.) and the lowest soil-moisture content (25"5% W.H.C.) tested. The abnormally high incidence of the disease in the field in 1960 and 1962 has been related to the favourable temperatures and the unusually dry conditions prevailing in the spring of these years and in the autumn of the previous years, times of the year when plants have been found to be most susceptible to attack by the pathogen. Experiments demonstrated the ability of sclerotia to survive under conditions of desiccation in the laboratory and in natural soil. These sclerotia were able to regerminate several times on repeated disturbance, a factor which could compensate, to some extent, for their lack of specificity in response to stimuli. The pathogen rapidly declined to a low level in artificially infested natural soil under either fallow or various cropping regimes. The presence of a ley arrested decline of the pathogen to some extent. The inability of C. solani to colonize buried wheat straw or buckwheat was shown and also its ability for hmited survival in naturally infected straws.
P. D. HEWETT (Official Seed Testing Station, Cambridge). The incidence of Septoria nodorum and Fusarium spp. on wheat. To be published in these Transactions.
R. G. PAWSEY (Alice Holt). The importance of Peridermium pini and Cronartium ribicola on conifers in Britain. Among the many rust fungi that attack timber trees in this country only Peridermium pini Lev. on two-needle pines and Cronartium ribicola]. C. Fischer on five-needle pines are of any considerable economic importance. Recent survey and research work has
144
Transactions British Mycological Society
indicated that the pathogenic potential of C. ribicola, particularly on Pinus strobus, has been greatly overestimated for many years in Britain. It is now thought that if nursery stock can be raised free of infection, P. strobus can be planted in many forest areas which are sufficiently isolated from the most important alternate host, blackcurrant, with low risk of infection in the timber crop. Extensive survey work in the large pine forests in the area south of the Moray Firth has shown that losses in Scots pine caused by Peridermium pini, particularly in crops 25-40 years old, have increased in recent years. Very little is known of the infection biology of this autoecious rust. Although present losses are not serious over the area generally affected, P. pini is regarded as an important threat to production of Scots pine in the future. There is evidence that host susceptibility is related to provenance, or seed origin, and that the intensity of infection during different periods in the life of the crop is related to crop vigour and to the sequence of normal forest management.
I.
(in collaboration with B. Kassanis, Rothamsted Experimental Station). Transmission of tobacco necrosis virus by Olpidium.
MACFARLANE
Olpidium was maintained on lettuce grown in sand culture and zoospore suspensions were obtained by placing infected roots in dilute (1/20) Hoagland's solution for 10 min. Transmission experiments were made by immersing roots of small lettuce or Mung bean (Phaseolus aureus) seedlings in zoospore suspensions to which purified tobacco necrosis virus (TNV) was added. Three days later the lettuce roots were crushed and inoculated to leaves of French bean (P. vulgaris) on which TNV produces necrotic local lesions, the number of lesions giving a measure of the virus content of the assayed roots. On Mung bean roots necrotic lesions formed one day after exposure to zoospores and virus. Virus infection occurred only when roots were exposed to virus and Olpidium zoospores. This confirms American reports of the apparent transmission of TNV by Olpidium (Teakle, 1962). The identity of the transmitting agent with Olpidium, will, of course, remain in some doubt until Olpidium can be obtained free from other micro-organisms. It is assumed here that Olpidium does, in fact, transmit the virus. Transmission was favoured by low salt concentration (dilution 1/20) and neutral or slightly alkaline reaction of the medium and depended also on the concentrations of virus and zoospores. With abundant zoospores, transmission to lettuce was obtained with as little as 0'05 p.g virus/I. When there was plenty of virus (say 5 p.g/I.) 50-100 zoospores/ml. were effective. There was not always a strict correlation between fungus infection, as judged by the presence and number of zoosporangia in the root, and virus infection. Perhaps penetration of zoospores which fail to develop is sufficient to transmit virus. For example, virus is transmitted when the fungus is killed by heating the inoculated root after sufficient time is allowed for penetration, Some Olpidium isolates were naturally contaminated with TNV, but, from these, some virus-free cultures were obtained by inoculating plants with very low numbers of zoospores. One of these isolates was contaminated by another virus transmitted by Olpidium, lettuce big vein virus (Campbell & Grogan, 1963) but, unlike TNV, it was not lost in this way. Zoospores which had been mixed with TNV were partially washed free from virus by centrifugation. Further evidence that the relationship between TNV and Olpidium is superficial came from experiments with antiserum to TNV. Roots were treated with zoospores that had been exposed to virus before or after antiserum was added to the virus. Antiserum prevented transmission in both instances, but a much greater concentration of antiserum was needed if zoospores were mixed with virus before antiserum was added. This suggests that the virus is principally on the zoospore surface, where it is presumably protected from the aggregating effects which allow fewer antibodies to render virus in suspension unavailable for transmission. REFERENCES
CAMPBELL, R. N. & GROGAN, R. G. (1963). Big-vein virus of lettuce and its transmission by Olpidium brassicae. P~ytopathology, 53, 2.')2-259. TEAKLE, D. S. (1962). Transmission of tobacco necrosis virus by a fungus, Olpidium brassicae. Virology, 18, 224-231.
Proceedings
145
L. G. WILLOUGHBY (Ambleside). Observations on some aquatic fungi in Australia. To be published in the Proceedings of the Royal Society of Victoria and in the Journal of Ultrastructure Research.
M. W.
DICK
(Reading). Some recent work on filamentous water moulds.
A study of the Leptomitaceae has now been started, based at present on four species (Leptomitus lacteus (Roth) Ag., Apodachlya brachynema (Hildeb.) Prings., A. minima Coker and Apodachlyella completa (Humph.) Indoh), all of which have been isolated from the environs of Blelham Tarn in the English Lake District, and are being maintained in pure culture. Preliminary investigations of cultural requirements, using a glucose-peptone broth as a basic medium, hyphal tips in agar as a source of inoculum and dry weight as a criterion of growth, have revealed different responses to the pH of the medium. L. lacteus and Apodachlya brachynema were similar, differing only in the pH optima (6-6·6 and 5,6-6'2, respectively); both grew at pH 5'3-6'9, but gave little or no growth in neutral or alkaline media. On the other hand, A. minima and Apodachlyella complete grew well at all pH values tested (5'3-8), although growth of both was slightly better in acid media. In addition there were indications that Apodachlya minima might have a second optimum below pH 5'3. Temperature has proved surprisingly critical for Apodachlyella completa. Above 20° C. there was no appreciable growth, the hyphal tips becoming inflated and filled with clumped, granular protoplasm. The phenomenon was reversible: removal to an incubator at 19°, even after several days at 23°, resulted in normal growth being resumed after less than 48 hr. Attempts to find a completely defined medium for A. complete have not yet succeeded. Yeast extract is not essential when peptone is provided as the nitrogen source, but it is required when mixed amino acids (including methionine), nitrate or ammonium ions (including ammonium thiosulphate) are supplied. No growth occurred in the presence of nitrite ions. An illustrated account of the morphology of A. completa was given.