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The water-relations of spore discharge in Epichloe
[ 277 1
THE WATER-RELATIONS OF SPORE DISCHARGE IN EPICHLOE By C. T. INGOLD, Birkbeck College, University (With
2
of London
Text-figures)
To a perithecium from which spores are being actively discharged there must presumably be a steady supply of water making good the loss by evaporation so that the turgidity of the ripening asci may be maintained. Probably most of the lignicolous stromatal Pyrenomycetes (e.g. Necttia spp., Diatrype spp., Endothia spp. and Hypoxylon spp.) depend directly on rain for this water supply and spore discharge is limited to periods during or immediately after rain when the fungal tissue is temporarily turgid. Daldinia concentrica (see Ingold, 1946) is exceptional in being able to discharge spores during prolonged dry periods by virtue of the considerable reserve of water in the stromatal tissue. Epichloe typhina (Fr.) Fr., the choke of grasses, causes a systemic infection of its host and eventually produces its perithecial stroma, during July and August, as a crust around the unexpanded leaves immediately above a node of the stem . The innermost hyphae of the stromatal tissue are in intimate association with the living cells of the host. The experiments reported in this paper were designed to answer the following question: does the fungus during periods of active discharge obtain its water from the host cells and, therefore, indirectly from the transpiration stream of the grass, or is discharge limited to damp periods when the stroma is directly wetted by rain or dew? The specimens used were collected at Sevenoaks, Kent. The form of the fungus on Dactylis glomerata was finally selected for use because in the material available the long thread-like ascospores showed no tendency to break up into part-spores and the units in the spore deposit were 190 x 2JL. On the other hand, from specimens of the fungus on Holcus mollis the units measured 57 x 2JL no doubt due to the breaking up of the ascospores into shorter part-spores. The process appears to be carried still further in the form on Agrostis tenuis since the spore deposit from this strain was composed of units 32 x 2JL. Each measurement oflength is the average of 100 units of the spore deposit chosen at random. It would be interesting to know if these differences obtain in other localities and if the forms on other grasses show still further variations. Each diseased shoot of Dactylis used for an experiment consisted of a length of stem, above this a stroma of Epichloe, and above this again a single green leaf (Fig. I). Each shoot was severed from the plant by a cut made below water to avoid the interruption of the transpiration stream by air blocks in the vessels. With their cut ends in water the shoots were brought indoors for study.
278
Transactions British Mycological Society
It was found that from the fungus on such shoots, freely exposed to the dry air of a room in summer, spore discharge continued for several days so long as the cut ends of the shoots remained in water. Discharge can clearly be seen by the naked eye if, when the sun is shining brightly, the fungus is held more or less between the observer and the sun, but in such a way that the stroma can be viewed against a dark background. Then the individual spores, each about a fifth of a millimetre long, can be seen as needle-like motes in the sunbeam. They first come into view about half a millimetre away from the stroma and then are slowly carried away by gentle air currents. If discharge is very vigorous, there are so many spores in the air around the stroma at any instant that it is impossible to observe discharge from a single ascus, but if spores are not being liberated in too great numbers, the successive discharge of the eight spores from an ascus, which is completed in much less than a second, can be seen. The eight discharged spores, maintaining roughly their formation like a squadron of minute aeroplanes, can often be followed for a few centimetres as they drift away.
x
~
~
!
~-.I'iJ::;.
Fig.
I. Method of measuring rate of spore discharge from stroma of Epichlo«, A, cork support; B, specimen tube; C, glass blocks; D, wooden block ; E, glass slide; F, stem of grass; G, node of grass; H, stroma of Epichlo«; I, leaf of grass.
In studying the rate of spore discharge the arrangement shown in Fig. 1 was used. The cut end of the shoot dipped into water in a specimen tube clamped by a rubber band in a more or less horizontal position to a special cork support (A) consisting of a horizontal and a vertical limb . The stem of the shoot passed through a hole in the vertical limb and through another hole in the cork of the specimen tube (B). In both holes the shoot was wedged firmly with dry cotton-wool. The free leaf of the shoot was held securely in position between two blocks of glass (C). The stroma in this way occupied a horizontal position and below it a wooden block (D) was placed. For observing the rate of spore discharge a microscope slide (E) was used with a square centimetre, sub-divided into square millimetres, etched upon its upper surface. This could quickly be slipped into position under the fungus and it was so arranged that at each observation the etched square occupied exactly the same position relative to the stroma. After a brief exposure (usually fifteen or thirty seconds) the slide was removed and the spores deposited on the square counted under the low power of the microscope. These short exposures were necessary, since it is difficult to count the spores if there are too many, and a single stroma when most active may discharge 2000--10,000 spores a minute. The apparatus was not covered, but was freely exposed to the air of the room, the door and windows
Water-relations of Spore Discharge in Epichloe. C. T. Ingold 279 being closed to avoid draughts. The limitations and errors of the method are obvious, but it can be used to give a reasonably accurate measure of changes in the rate of spore discharge. The effect on discharge of interrupting the transpiration stream was studied by cutting the stem with a razor at the point marked X (Fig. I). Using the method described above, certain facts were clearly demonstrated. In the first place, there tends to be a daily periodicity of discharge with the minimum in the morning and a maximum in the afternoon or evening. This was observed in nearly all the specimens studied. Secondly, considerable fluctuations in th e rate of discharge occur over short intervals ~
.,'./.......!:If.
800
..........v
/..k··············· :.:
E 700 o
& : 600
.....................-•..•../
5
.
.~ 500
.15.. 400
]
1~ 300
~ 200
8.
rFl
lOO 3
5
79 11 P.M.
Fig.
I
3
5
7 A.M.
9 11
3
'i
7
9
P..\!.
Time (a.M.T.) A and B, curves of rate of spore discharge from two different stromata of Epichloe plotted against time (a .M.T.) . One experiment (A ) performed on 23-24 July, the other (B ) on 18-19 July 1946. Observations of temperature (dotted line ) and relative humidity (interrupted line) are for 23-24 July and should be considered in connexion with A. Arrows, X and T, indicate the time when the stem of the host plant was severed, thu s interrupting the transpiration stream.
2.
of time. Thirdly, and most important, cutting the stem at the point X when the rate of discharge is at a high level rapidly leads to a cessation of spore liberation. The results of two experiments are represented diagrammatically in Fig. 2. Both were conducted on fairly warm july days. In both, cutting the stem at a time when the rate of discharge was high and apparently increasing led to a rapid fall of the rate to zero after an hour or so. These observations indicate that spore discharge can be maintained in fairly dry air without any external supply of water to the stroma, but that the fungus is dependent, like the host tissue with which it is so intimately associated, on the water of the transpiration stream and when this is interrupted by cutting the stem, the rate of spore discharge soon falls to zero.
Transactions British Mycological Society
280
SUMMARY
Spore discharge from Epichloe typhina can be observed with the unaided eye. A method is described for studying the rate of spore liberation. Discharge tends to be periodic with a minimum in the morning and a maximum in the late afternoon or evening. In fairly dry air spore discharge continues for several days provided that the water supply to the host tissue is maintained, but when the transpiration stream is stopped by severing the stem, discharge soon ceases. REFERENCE C. T. (1946). Spore discharge in Daldinia concentrica. Trans. Brit. myc. Soc. 43-5 1•
INGOLD,
(Accepted for publication 3 May 1947)
XXIX,
THE WATER-RELATIONS OF SPORE DISCHARGE IN EPICHLOE By C. T. INGOLD, Birkbeck College, University (With
2
of London
Text-figures)
To a perithecium from which spores are being actively discharged there must presumably be a steady supply of water making good the loss by evaporation so that the turgidity of the ripening asci may be maintained. Probably most of the lignicolous stromatal Pyrenomycetes (e.g. Necttia spp., Diatrype spp., Endothia spp. and Hypoxylon spp.) depend directly on rain for this water supply and spore discharge is limited to periods during or immediately after rain when the fungal tissue is temporarily turgid. Daldinia concentrica (see Ingold, 1946) is exceptional in being able to discharge spores during prolonged dry periods by virtue of the considerable reserve of water in the stromatal tissue. Epichloe typhina (Fr.) Fr., the choke of grasses, causes a systemic infection of its host and eventually produces its perithecial stroma, during July and August, as a crust around the unexpanded leaves immediately above a node of the stem . The innermost hyphae of the stromatal tissue are in intimate association with the living cells of the host. The experiments reported in this paper were designed to answer the following question: does the fungus during periods of active discharge obtain its water from the host cells and, therefore, indirectly from the transpiration stream of the grass, or is discharge limited to damp periods when the stroma is directly wetted by rain or dew? The specimens used were collected at Sevenoaks, Kent. The form of the fungus on Dactylis glomerata was finally selected for use because in the material available the long thread-like ascospores showed no tendency to break up into part-spores and the units in the spore deposit were 190 x 2JL. On the other hand, from specimens of the fungus on Holcus mollis the units measured 57 x 2JL no doubt due to the breaking up of the ascospores into shorter part-spores. The process appears to be carried still further in the form on Agrostis tenuis since the spore deposit from this strain was composed of units 32 x 2JL. Each measurement oflength is the average of 100 units of the spore deposit chosen at random. It would be interesting to know if these differences obtain in other localities and if the forms on other grasses show still further variations. Each diseased shoot of Dactylis used for an experiment consisted of a length of stem, above this a stroma of Epichloe, and above this again a single green leaf (Fig. I). Each shoot was severed from the plant by a cut made below water to avoid the interruption of the transpiration stream by air blocks in the vessels. With their cut ends in water the shoots were brought indoors for study.
278
Transactions British Mycological Society
It was found that from the fungus on such shoots, freely exposed to the dry air of a room in summer, spore discharge continued for several days so long as the cut ends of the shoots remained in water. Discharge can clearly be seen by the naked eye if, when the sun is shining brightly, the fungus is held more or less between the observer and the sun, but in such a way that the stroma can be viewed against a dark background. Then the individual spores, each about a fifth of a millimetre long, can be seen as needle-like motes in the sunbeam. They first come into view about half a millimetre away from the stroma and then are slowly carried away by gentle air currents. If discharge is very vigorous, there are so many spores in the air around the stroma at any instant that it is impossible to observe discharge from a single ascus, but if spores are not being liberated in too great numbers, the successive discharge of the eight spores from an ascus, which is completed in much less than a second, can be seen. The eight discharged spores, maintaining roughly their formation like a squadron of minute aeroplanes, can often be followed for a few centimetres as they drift away.
x
~
~
!
~-.I'iJ::;.
Fig.
I. Method of measuring rate of spore discharge from stroma of Epichlo«, A, cork support; B, specimen tube; C, glass blocks; D, wooden block ; E, glass slide; F, stem of grass; G, node of grass; H, stroma of Epichlo«; I, leaf of grass.
In studying the rate of spore discharge the arrangement shown in Fig. 1 was used. The cut end of the shoot dipped into water in a specimen tube clamped by a rubber band in a more or less horizontal position to a special cork support (A) consisting of a horizontal and a vertical limb . The stem of the shoot passed through a hole in the vertical limb and through another hole in the cork of the specimen tube (B). In both holes the shoot was wedged firmly with dry cotton-wool. The free leaf of the shoot was held securely in position between two blocks of glass (C). The stroma in this way occupied a horizontal position and below it a wooden block (D) was placed. For observing the rate of spore discharge a microscope slide (E) was used with a square centimetre, sub-divided into square millimetres, etched upon its upper surface. This could quickly be slipped into position under the fungus and it was so arranged that at each observation the etched square occupied exactly the same position relative to the stroma. After a brief exposure (usually fifteen or thirty seconds) the slide was removed and the spores deposited on the square counted under the low power of the microscope. These short exposures were necessary, since it is difficult to count the spores if there are too many, and a single stroma when most active may discharge 2000--10,000 spores a minute. The apparatus was not covered, but was freely exposed to the air of the room, the door and windows
Water-relations of Spore Discharge in Epichloe. C. T. Ingold 279 being closed to avoid draughts. The limitations and errors of the method are obvious, but it can be used to give a reasonably accurate measure of changes in the rate of spore discharge. The effect on discharge of interrupting the transpiration stream was studied by cutting the stem with a razor at the point marked X (Fig. I). Using the method described above, certain facts were clearly demonstrated. In the first place, there tends to be a daily periodicity of discharge with the minimum in the morning and a maximum in the afternoon or evening. This was observed in nearly all the specimens studied. Secondly, considerable fluctuations in th e rate of discharge occur over short intervals ~
.,'./.......!:If.
800
..........v
/..k··············· :.:
E 700 o
& : 600
.....................-•..•../
5
.
.~ 500
.15.. 400
]
1~ 300
~ 200
8.
rFl
lOO 3
5
79 11 P.M.
Fig.
I
3
5
7 A.M.
9 11
3
'i
7
9
P..\!.
Time (a.M.T.) A and B, curves of rate of spore discharge from two different stromata of Epichloe plotted against time (a .M.T.) . One experiment (A ) performed on 23-24 July, the other (B ) on 18-19 July 1946. Observations of temperature (dotted line ) and relative humidity (interrupted line) are for 23-24 July and should be considered in connexion with A. Arrows, X and T, indicate the time when the stem of the host plant was severed, thu s interrupting the transpiration stream.
2.
of time. Thirdly, and most important, cutting the stem at the point X when the rate of discharge is at a high level rapidly leads to a cessation of spore liberation. The results of two experiments are represented diagrammatically in Fig. 2. Both were conducted on fairly warm july days. In both, cutting the stem at a time when the rate of discharge was high and apparently increasing led to a rapid fall of the rate to zero after an hour or so. These observations indicate that spore discharge can be maintained in fairly dry air without any external supply of water to the stroma, but that the fungus is dependent, like the host tissue with which it is so intimately associated, on the water of the transpiration stream and when this is interrupted by cutting the stem, the rate of spore discharge soon falls to zero.
Transactions British Mycological Society
280
SUMMARY
Spore discharge from Epichloe typhina can be observed with the unaided eye. A method is described for studying the rate of spore liberation. Discharge tends to be periodic with a minimum in the morning and a maximum in the late afternoon or evening. In fairly dry air spore discharge continues for several days provided that the water supply to the host tissue is maintained, but when the transpiration stream is stopped by severing the stem, discharge soon ceases. REFERENCE C. T. (1946). Spore discharge in Daldinia concentrica. Trans. Brit. myc. Soc. 43-5 1•
INGOLD,
(Accepted for publication 3 May 1947)
XXIX,