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Liberation of spores from mouldy hay
[ 73 ] Trans. Brit. mycol. Soc. 46 (1),73-80 (1963).
LIBERATION OF SPORES FROM MOULDY HAY By P. H. GREGORY AND MAUREEN E. LACEY Rothamsted Experimental Station, Harpenden, Herts. (With 5 Text-figures) Mouldy hay was shaken gently in a small wind-tunnel at various wind speeds and the concentration of the resulting dust-cloud determined by sampling with a cascade impactor. A wind of 1'2 ra.leec. removed 1000 times as many spores from an obviously mouldy hay as from a well-made hay. Sampling at successive intervals during I hr. while comparable samples of hay were being shaken at different wind speeds showed that the number of spores released per minute decreased rapidly from the start; two-thirds were removed in the first 3 min. Subsequent washing removed many more spores: the proportion of all the spores in the hay that could be blown away increased with increasing wind speed, from 2'5 % at 0·6 ta.jscs: to 15'5 % at 4'9 ta.jsec. Although the total number of spores blown away in a given time is roughly proportional to wind speed, the decreases in liberation rate are similar in winds of 0,6, 1'2, 2'3 and 4'9 ui.iscc: Further, the curves for decrease in liberation rate with time for 'total fungi', 'total actinomycetes' and Humicola lanuginosa shaken from a mouldy hay associated with a case of Farmer's lung hay were all similar. Possibly relevant to the liberation of dry-spored fungi in nature is the observation that a sample of hay, which had already liberated 50 million spores/g. dry weight while being blown for 3 I min. at 1'2 m./sec" produced a second typical cumulative liberation curve and liberated another 55 million spores when blown for another 31 min. at 4'9 iu.lsec. This suggests the hypothesis that part of the mechanism of spore liberation by blowing away (' deflation ') is that increasing the wind speed decreases the thickness of the boundary layer of air at the leaf surface, exposing more deeply immersed spores to the pruning action of eddies.
For the study of micro-organisms in hay associated with Farmer's lung and certain animal mycoses the easily removed dust is important as it can enter the respiratory tract during inhalation. Developing a standard method of sampling hay for micro-organisms required preliminary studies of spore-release when a small quantity of hay is shaken in a wind-tunnel and the resulting dust cloud sampled (Gregory & Lacey, 1963). Stepanov (1935) studied the liberation of dry-spored imperfect fungi in air, both in convection currents and in wind in a small wind-tunnel. Although different fungi behaved in different ways, stronger wind currents always removed more spores (until the source of spores was exhausted). Recent work on dry-spored fungi by Zoberi (1961) shows that, with Trichothecium roseum for example, dry air currents remove more spores than damp ones, and faster currents more than slow. Trichoderma viride (a c slime-spored' fungus) did not release any spores in low wind speeds, but at higher speeds released many, often in clumps; with this species the humidity of the air was less important than for dry-spored fungi. With most of the fungi studied by Zoberi, the rate of decrease of spore liberation with time was as pronounced as spore release from hay studied here.
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Transactions British Mycological Society
Spores of Mucor ramannianus were not removed at any air speeds tested up to 16·6 m. /sec., with relative humidities from zo-qo %. Our method entails revolving 20-30 g. of hay in a perforated drum in a wind-tunn el at controlled wind speeds. The cascade impactor (May, 1945) sampling at 20 I.jrnin. (a suitable rate for the range of particle sizes encountered in hay) is used as an efficient trap to catch spores for microscopical examination. The number of spores released from the sample of hay during shaking is estimated from one count across each band of spore 1000
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Fig. I. Cumulative totals of spores released from samples of hay blown at a wind speed of 1'2 iu. lsec., plotted on a log. log. scale. H I, associated with acut e pneumonia of calves ('before cleaning', as received ; and 'after cleaning', after shaking vigorously in a wind of 2'7 m./sec.); H 2 , associated with acute Thresher's lung ; H 3 and H 4, good barn-dried hays; H 44, hay associated with Farmer's lung.
deposit (trace) on each of the four slides of the cascade impactor, and from this the total number liberated per gram dry weight of hay is calculated (Gregory & Lacey, 1963). Spores trapped on the slides were classified for convenience into visual categories including: actinomycetes (mainly Streptomycetaceae), Aspergillus- and Penicillium-like forms, Cladosporium, Humicola lanuginosa, Mucoraceae, and Trichothecium roseum.
Mouldy hay. P. H. Gregory and Maureen E. Lacey
75
DIFFERENT BATCHES OF HAY
One of the objects of the present work is to estimate quantitatively and qualitatively the spore content of any batch of hay. To test the method, hays obviously differing widely in mould content were shaken in a wind of 1'2 m.jsec, Hays used were H 1 (associated with acute pneumonia of calves); H 2 and H 44 (associated with cases of Thresher's lung and Farmer's lung, respectively); and H 3 and H 4 (good hays, partly artificially dried, from the National Institute of Agricultural Engineering, Silsoe, Beds.), Samples of each were shaken in the wind-tunnel for I, 2,4, 8, 16 t-
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2. Release of spores by blowing H I (broken lines) in winds of 0,6, 1'2, 2'3, and m.fsec. and H 44 (solid lines) in winds of 1'2, 2'2 and 4'2 m./sec. a, Cumulative totals of spores plotted on log. log. scale; b, log. number of spores liberated per minute plotted against linear time scale.
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and 32 min. in succession (a total of 63 min.). Fresh slides were inserted in the cascade impactor after each interval, so that a cumulative curve of total liberated against time could be plotted. Fig. 1 shows over a r ooo-fold difference between the best and mouldiest hays in cumulative totals of spores removed after blowing for successive short periods. Later work suggests that had higher wind speeds been used the differences would have been even greater. In attempts to clean a large quantity of mouldy hay (as a possible way
76
Transactions British Mycological Society
of making it safe for feeding to stock) H r was also tested after it had been shaken vigorously by hand in a wind of 2'7 m.leec, This treatment decreased the total spore liberation to about one-twentieth of the original value (Fig. I). EFFECT OF WIND SPEED
Different wind speeds on comparable samples of hay Samples ofH r were blown successively for r, 2,4,8, r6 and 32 min. as before, using a different wind speed for each sample: 0,6, r '2, 2'3 and 4'9 m.Isec. It was at once evident that the higher the wind speed the more 1000 , . . . . - - - - - - - - - - - - - - - - - - - - . . ,
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spores were released in a given time, the number being roughly proportional to wind speed over the range tested. When the cumulative totals of numbers of spores liberated are plotted against time on log.-log. paper the lines for the different wind speeds are more or less parallel (Fig. 2 a). Plotting the results as log. number of spores released per minute against
Mouldy hay. P. H. Gregory and Maureen E. Lacey
77
time (Fig. 2 b) shows the fall-off to be extremely rapid. Similar tests were done with H 44 at three wind speeds-r '2,2'2 and 4'2 m./sec. in succession for r, r, 2, 4, 8, r6, and 32 min. (a total of 64 min. )-and although H 44 contained many more spores than H r, both samples liberated in the same way (Fig. 2 a, b). The curves for decreasing rate of liberation for the 110 100 c-,
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Transactums British Mycological Society various constituents of the total spore load were also very similar, as shown in Fig. 3, where total fungi, total actinomycetes and Humicola lanuginosa, blown from H 44 at 1'2 and 4'2 m./sec. are plotted separately. As a further stage in developing the method, total spore fractions blown out of the samples during 63 min. at four wind speeds were compared with the total removed later by shaking in four changes of water with detergent (the perforated drum was also washed and its spores added to the total) and counted on a haemocytometer slide. The proportion of the total spore content of the hay that could be recovered by the different wind speeds was: 0·6 m./sec., 2'5 %; 1'2 m./sec" 4'2 %; 2'3 m./sec., 3'9 %; and 4'9 m./sec., 15'5 %.
Different wind speeds on the same sample ofhay Because many more spores were released from different samples of a given hay at high than at low wind speeds, single samples were tested successively at different wind speeds to make sure that the effect was not from sampling error. A sample of H I was blown for a succession of intervals for a total of g r min. at 1'2 ui.lsec., and then for another 31 min. at 4'9 m./sec. Although over 50 million spores were released in the first 3 I min. blowing, a further 55 million spores were released during a second period of 31 min. at the higher wind speed (Fig. 4). In a similar experiment two samples of hay were blown for four separate minutes, one with increasing wind speeds at each successive minute, and one with decreasing wind speed. Many more spores were removed when the wind speed was increased at each minute (Fig. 5a); in fact the number of spores liberated per minute stayed about constant for the first three minutes, and even increased during the fourth. The numbers of spores liberated at each wind speed were: 14'7 million/g. at 0·6 m./sec., 12'5 at 1'2 ta.leec., 11'4 at 2'3 m.lsec. and 39'9 million at 4'9 m./sec. In the series with decreasing wind speed at each change 38 million spores were released during the first minute at 4'9 m./sec. (Fig. 5b), but fewer than 3 million during the next 3 min. at lower wind speeds.
DISCUSSION
As a result of these experiments it was decided, for routine testing of hay, to use a total of 3 min. blowing in a wind speed of 4'2 m./sec. This wind speed is isokinetic when the cascade impactor samples air at 20 l./min. and it will release about 15 % of the total washable spore load from the sample. The period of 3 min. was considered adequate as it released about 65 % of the total blowable at that wind speed. Most of the fungi present in dust from mouldy hay are imperfect fungi producing 'dry-spores' (Mason, 1937) on conidiophores rising above the substratum. The spores are liberated passively by mechanical shock or wind; they are not actively discharged by energy produced by the fungus by any of the methods described by Ingold (1953). The Streptomycetaceae are also dry-spored, though little is known about their liberation, and
Mouldy hay. P. H. Gregory and Maureen E. Lacey
79
Hesseltine (1960) suggests that wetting may be an important factor in their dispersal. Bacteria are also removed from hay by air. Hirst (1959) points out that it is not always easy to define when a spore is liberated; spores may be separated from the structure that bore them but still not have moved perceptibly from it. This occurs commonly in hay where spore chains of such Fungi Imperfecti as Aspergillus fumigatus break off leaving masses of dry loose spores in the hay, which will be liberated from the hay only when it is shaken in air. Thus there are two stages in spore liberation from hay; many spores will be separated from the structure which bore them, and then actually liberated from the hay later; other spores may be released from the sporophore and hay mass simultaneously. Some of the Mucorales present in hay are 'dried-spore shedders' (Dobbs, 1942). At maturity the sporangium will burst, forming a drop containing spores (= primary dispersal) ; this drop will dry and when the hay is later shaken in air spores will be liberated mainly by mechanical shock (= secondary dispersal). Although spores of bacteria and actinomycetes cannot be distinguished on the cascade impactor slides under the microscope, cultural techniques show that many bacteria are liberated from hay. As bacteria are not usually liberated into dry air except on 'rafts', it may be that they behave like the slime-spored Mucorales, being first dried and then liberated by mechanical shock. Liberation of dry spores from hay is now seen to follow a regular curve of decreasing numbers with increasing time of blowing which is remarkably uniform: (I) at different wind speeds; (2) for different batches of hay with widely differing mould content; and (3) for the many types of spores released, including the dry chains of Aspergillus, Penicillium, Trichothecium, Cladosporium and the minute actinomycete spores; the single dry spores of Humicola lanuginosa; the 'slime heads' of Trichoderma viride and Cephalosporium; the 'dried-spore shedders' of the Mucorales; and for bacteria. The cumulative liberation curve is quite unlike the radio-isotope decay curve (plot of log. concentration against time being non-linear in Figs. 2 b and 3) and does not appear to resemble a second- or third-order reaction. This research, which is concerned only with the gross effect ofliberating large numbers of spores from hay, may also give information which is typical of spore release from dry substrata, and so be relevant to the epidemiology of many diseases including Farmer's lung, animal mycoses such as Aspergillosis, and rust and smut diseases of crop plants. A sample which has already produced a typical cumulative liberation curve while being blown at a low wind speed will proceed to develop another typical cumulative liberation curve, in which as many spores again are liberated, when transferred to a higher wind speed (Fig. 5). Two processes seem likely to produce the typical die-away curve. The amount that the fragments of hay separate from each other depends on the amount of mechanical shock received. In high winds the' tedding' action is more violent, and this is one cause of the larger number ofspores released at successively higher wind speeds. At low wind speeds both spores and sporophores are submerged in a relatively thick boundary layer of air at the plant surface, into which layer
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Transactions British Mycological Society
eddies dip from time to time pruning off the most exposed spores-the effect diminishing with passage of time. The protection afforded by the boundary layer may also explain the small percentage of the total sporeload of the hay sample removed in this method. These processes give rise to characteristic liberation curves, whose slopes (Figs, 2 band 3) are little affected by wind speed over the range 0·6-4'9 m.fsec., or by the nature of the spores liberated whether from moulds, streptomycetes, or from a single species. Whether this die-away curve is generally characteristic of the process of blowing-away of fungus spores, or is merely characteristic of some feature of the wind-tunnel (such as tedding rate or eddy size) has not yet been investigated. REFERENCES
DOBBS, C. G. (1942). On the primary dispersal and isolation of fungal spores. New Phytol. 41, 63-69. GREGORY, P. H. & LACEY, M. E. (1963). Mycological examination of dust from mouldy hay associated with Farmer's lung. ]. gen. Microbiol. 30, 75-88. HESSELTINE, C. W. (1960). Relationships of the Actinomycetes. Mycologia, 52, 460-474. HIRST, J. M. (1959). Spore liberation and dispersal. Plant pathology: problems and progress 1908-1958, pp. 529-538. Madison: University of Wisconsin Press. INGOLD, C. T. (1953). Dispersal infungi, pp. 197. Oxford: Clarendon Press. MASON, E. W. (1937). Annotated account offungi received at the Imperial Mycological Institute. List II, Fasc.g-General Part. Mycol. Pap. 4, 69-99. MAY, K. R. (1945). The cascade impactor: an instrument for sampling coarse aerosols. ]. sci. Instrum. 22, 187-195 .' STEPANOV, K. M.( 1935). (Dissemination of infective diseases of plants by air currents.) (In Russian.) Bull. Plant Prot. Leningr., Ser. 2, Phytopathology, 8, 1-68. ZOBERI, M. H. (1961). Take-off of mould spores in relation to wind speed and humidity. Ann. Bot., Lond., 25, 53-64.
(Accepted for publication 19 April 1962)
LIBERATION OF SPORES FROM MOULDY HAY By P. H. GREGORY AND MAUREEN E. LACEY Rothamsted Experimental Station, Harpenden, Herts. (With 5 Text-figures) Mouldy hay was shaken gently in a small wind-tunnel at various wind speeds and the concentration of the resulting dust-cloud determined by sampling with a cascade impactor. A wind of 1'2 ra.leec. removed 1000 times as many spores from an obviously mouldy hay as from a well-made hay. Sampling at successive intervals during I hr. while comparable samples of hay were being shaken at different wind speeds showed that the number of spores released per minute decreased rapidly from the start; two-thirds were removed in the first 3 min. Subsequent washing removed many more spores: the proportion of all the spores in the hay that could be blown away increased with increasing wind speed, from 2'5 % at 0·6 ta.jscs: to 15'5 % at 4'9 ta.jsec. Although the total number of spores blown away in a given time is roughly proportional to wind speed, the decreases in liberation rate are similar in winds of 0,6, 1'2, 2'3 and 4'9 ui.iscc: Further, the curves for decrease in liberation rate with time for 'total fungi', 'total actinomycetes' and Humicola lanuginosa shaken from a mouldy hay associated with a case of Farmer's lung hay were all similar. Possibly relevant to the liberation of dry-spored fungi in nature is the observation that a sample of hay, which had already liberated 50 million spores/g. dry weight while being blown for 3 I min. at 1'2 m./sec" produced a second typical cumulative liberation curve and liberated another 55 million spores when blown for another 31 min. at 4'9 iu.lsec. This suggests the hypothesis that part of the mechanism of spore liberation by blowing away (' deflation ') is that increasing the wind speed decreases the thickness of the boundary layer of air at the leaf surface, exposing more deeply immersed spores to the pruning action of eddies.
For the study of micro-organisms in hay associated with Farmer's lung and certain animal mycoses the easily removed dust is important as it can enter the respiratory tract during inhalation. Developing a standard method of sampling hay for micro-organisms required preliminary studies of spore-release when a small quantity of hay is shaken in a wind-tunnel and the resulting dust cloud sampled (Gregory & Lacey, 1963). Stepanov (1935) studied the liberation of dry-spored imperfect fungi in air, both in convection currents and in wind in a small wind-tunnel. Although different fungi behaved in different ways, stronger wind currents always removed more spores (until the source of spores was exhausted). Recent work on dry-spored fungi by Zoberi (1961) shows that, with Trichothecium roseum for example, dry air currents remove more spores than damp ones, and faster currents more than slow. Trichoderma viride (a c slime-spored' fungus) did not release any spores in low wind speeds, but at higher speeds released many, often in clumps; with this species the humidity of the air was less important than for dry-spored fungi. With most of the fungi studied by Zoberi, the rate of decrease of spore liberation with time was as pronounced as spore release from hay studied here.
74
Transactions British Mycological Society
Spores of Mucor ramannianus were not removed at any air speeds tested up to 16·6 m. /sec., with relative humidities from zo-qo %. Our method entails revolving 20-30 g. of hay in a perforated drum in a wind-tunn el at controlled wind speeds. The cascade impactor (May, 1945) sampling at 20 I.jrnin. (a suitable rate for the range of particle sizes encountered in hay) is used as an efficient trap to catch spores for microscopical examination. The number of spores released from the sample of hay during shaking is estimated from one count across each band of spore 1000
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Fig. I. Cumulative totals of spores released from samples of hay blown at a wind speed of 1'2 iu. lsec., plotted on a log. log. scale. H I, associated with acut e pneumonia of calves ('before cleaning', as received ; and 'after cleaning', after shaking vigorously in a wind of 2'7 m./sec.); H 2 , associated with acute Thresher's lung ; H 3 and H 4, good barn-dried hays; H 44, hay associated with Farmer's lung.
deposit (trace) on each of the four slides of the cascade impactor, and from this the total number liberated per gram dry weight of hay is calculated (Gregory & Lacey, 1963). Spores trapped on the slides were classified for convenience into visual categories including: actinomycetes (mainly Streptomycetaceae), Aspergillus- and Penicillium-like forms, Cladosporium, Humicola lanuginosa, Mucoraceae, and Trichothecium roseum.
Mouldy hay. P. H. Gregory and Maureen E. Lacey
75
DIFFERENT BATCHES OF HAY
One of the objects of the present work is to estimate quantitatively and qualitatively the spore content of any batch of hay. To test the method, hays obviously differing widely in mould content were shaken in a wind of 1'2 m.jsec, Hays used were H 1 (associated with acute pneumonia of calves); H 2 and H 44 (associated with cases of Thresher's lung and Farmer's lung, respectively); and H 3 and H 4 (good hays, partly artificially dried, from the National Institute of Agricultural Engineering, Silsoe, Beds.), Samples of each were shaken in the wind-tunnel for I, 2,4, 8, 16 t-
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2. Release of spores by blowing H I (broken lines) in winds of 0,6, 1'2, 2'3, and m.fsec. and H 44 (solid lines) in winds of 1'2, 2'2 and 4'2 m./sec. a, Cumulative totals of spores plotted on log. log. scale; b, log. number of spores liberated per minute plotted against linear time scale.
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and 32 min. in succession (a total of 63 min.). Fresh slides were inserted in the cascade impactor after each interval, so that a cumulative curve of total liberated against time could be plotted. Fig. 1 shows over a r ooo-fold difference between the best and mouldiest hays in cumulative totals of spores removed after blowing for successive short periods. Later work suggests that had higher wind speeds been used the differences would have been even greater. In attempts to clean a large quantity of mouldy hay (as a possible way
76
Transactions British Mycological Society
of making it safe for feeding to stock) H r was also tested after it had been shaken vigorously by hand in a wind of 2'7 m.leec, This treatment decreased the total spore liberation to about one-twentieth of the original value (Fig. I). EFFECT OF WIND SPEED
Different wind speeds on comparable samples of hay Samples ofH r were blown successively for r, 2,4,8, r6 and 32 min. as before, using a different wind speed for each sample: 0,6, r '2, 2'3 and 4'9 m.Isec. It was at once evident that the higher the wind speed the more 1000 , . . . . - - - - - - - - - - - - - - - - - - - - . . ,
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Fig. 3. Number of spores released from H 44, blown at wind speeds of 1'2 and 4'2 tn.] sec., plotted on log. linear scale and classified into: total fungi (f), total actinomycetes + bacteria (a.), and Humicola lanuginosa (H.l.).
spores were released in a given time, the number being roughly proportional to wind speed over the range tested. When the cumulative totals of numbers of spores liberated are plotted against time on log.-log. paper the lines for the different wind speeds are more or less parallel (Fig. 2 a). Plotting the results as log. number of spores released per minute against
Mouldy hay. P. H. Gregory and Maureen E. Lacey
77
time (Fig. 2 b) shows the fall-off to be extremely rapid. Similar tests were done with H 44 at three wind speeds-r '2,2'2 and 4'2 m./sec. in succession for r, r, 2, 4, 8, r6, and 32 min. (a total of 64 min. )-and although H 44 contained many more spores than H r, both samples liberated in the same way (Fig. 2 a, b). The curves for decreasing rate of liberation for the 110 100 c-,
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Time (min.)
Fig. 5. Cumulative totals on linear scale. a, A sample blown for four successive 1 min. periods at successively increasing wind speeds; b, another sample blown successively at four decreasing wind speeds.
Transactums British Mycological Society various constituents of the total spore load were also very similar, as shown in Fig. 3, where total fungi, total actinomycetes and Humicola lanuginosa, blown from H 44 at 1'2 and 4'2 m./sec. are plotted separately. As a further stage in developing the method, total spore fractions blown out of the samples during 63 min. at four wind speeds were compared with the total removed later by shaking in four changes of water with detergent (the perforated drum was also washed and its spores added to the total) and counted on a haemocytometer slide. The proportion of the total spore content of the hay that could be recovered by the different wind speeds was: 0·6 m./sec., 2'5 %; 1'2 m./sec" 4'2 %; 2'3 m./sec., 3'9 %; and 4'9 m./sec., 15'5 %.
Different wind speeds on the same sample ofhay Because many more spores were released from different samples of a given hay at high than at low wind speeds, single samples were tested successively at different wind speeds to make sure that the effect was not from sampling error. A sample of H I was blown for a succession of intervals for a total of g r min. at 1'2 ui.lsec., and then for another 31 min. at 4'9 m./sec. Although over 50 million spores were released in the first 3 I min. blowing, a further 55 million spores were released during a second period of 31 min. at the higher wind speed (Fig. 4). In a similar experiment two samples of hay were blown for four separate minutes, one with increasing wind speeds at each successive minute, and one with decreasing wind speed. Many more spores were removed when the wind speed was increased at each minute (Fig. 5a); in fact the number of spores liberated per minute stayed about constant for the first three minutes, and even increased during the fourth. The numbers of spores liberated at each wind speed were: 14'7 million/g. at 0·6 m./sec., 12'5 at 1'2 ta.leec., 11'4 at 2'3 m.lsec. and 39'9 million at 4'9 m./sec. In the series with decreasing wind speed at each change 38 million spores were released during the first minute at 4'9 m./sec. (Fig. 5b), but fewer than 3 million during the next 3 min. at lower wind speeds.
DISCUSSION
As a result of these experiments it was decided, for routine testing of hay, to use a total of 3 min. blowing in a wind speed of 4'2 m./sec. This wind speed is isokinetic when the cascade impactor samples air at 20 l./min. and it will release about 15 % of the total washable spore load from the sample. The period of 3 min. was considered adequate as it released about 65 % of the total blowable at that wind speed. Most of the fungi present in dust from mouldy hay are imperfect fungi producing 'dry-spores' (Mason, 1937) on conidiophores rising above the substratum. The spores are liberated passively by mechanical shock or wind; they are not actively discharged by energy produced by the fungus by any of the methods described by Ingold (1953). The Streptomycetaceae are also dry-spored, though little is known about their liberation, and
Mouldy hay. P. H. Gregory and Maureen E. Lacey
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Hesseltine (1960) suggests that wetting may be an important factor in their dispersal. Bacteria are also removed from hay by air. Hirst (1959) points out that it is not always easy to define when a spore is liberated; spores may be separated from the structure that bore them but still not have moved perceptibly from it. This occurs commonly in hay where spore chains of such Fungi Imperfecti as Aspergillus fumigatus break off leaving masses of dry loose spores in the hay, which will be liberated from the hay only when it is shaken in air. Thus there are two stages in spore liberation from hay; many spores will be separated from the structure which bore them, and then actually liberated from the hay later; other spores may be released from the sporophore and hay mass simultaneously. Some of the Mucorales present in hay are 'dried-spore shedders' (Dobbs, 1942). At maturity the sporangium will burst, forming a drop containing spores (= primary dispersal) ; this drop will dry and when the hay is later shaken in air spores will be liberated mainly by mechanical shock (= secondary dispersal). Although spores of bacteria and actinomycetes cannot be distinguished on the cascade impactor slides under the microscope, cultural techniques show that many bacteria are liberated from hay. As bacteria are not usually liberated into dry air except on 'rafts', it may be that they behave like the slime-spored Mucorales, being first dried and then liberated by mechanical shock. Liberation of dry spores from hay is now seen to follow a regular curve of decreasing numbers with increasing time of blowing which is remarkably uniform: (I) at different wind speeds; (2) for different batches of hay with widely differing mould content; and (3) for the many types of spores released, including the dry chains of Aspergillus, Penicillium, Trichothecium, Cladosporium and the minute actinomycete spores; the single dry spores of Humicola lanuginosa; the 'slime heads' of Trichoderma viride and Cephalosporium; the 'dried-spore shedders' of the Mucorales; and for bacteria. The cumulative liberation curve is quite unlike the radio-isotope decay curve (plot of log. concentration against time being non-linear in Figs. 2 b and 3) and does not appear to resemble a second- or third-order reaction. This research, which is concerned only with the gross effect ofliberating large numbers of spores from hay, may also give information which is typical of spore release from dry substrata, and so be relevant to the epidemiology of many diseases including Farmer's lung, animal mycoses such as Aspergillosis, and rust and smut diseases of crop plants. A sample which has already produced a typical cumulative liberation curve while being blown at a low wind speed will proceed to develop another typical cumulative liberation curve, in which as many spores again are liberated, when transferred to a higher wind speed (Fig. 5). Two processes seem likely to produce the typical die-away curve. The amount that the fragments of hay separate from each other depends on the amount of mechanical shock received. In high winds the' tedding' action is more violent, and this is one cause of the larger number ofspores released at successively higher wind speeds. At low wind speeds both spores and sporophores are submerged in a relatively thick boundary layer of air at the plant surface, into which layer
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eddies dip from time to time pruning off the most exposed spores-the effect diminishing with passage of time. The protection afforded by the boundary layer may also explain the small percentage of the total sporeload of the hay sample removed in this method. These processes give rise to characteristic liberation curves, whose slopes (Figs, 2 band 3) are little affected by wind speed over the range 0·6-4'9 m.fsec., or by the nature of the spores liberated whether from moulds, streptomycetes, or from a single species. Whether this die-away curve is generally characteristic of the process of blowing-away of fungus spores, or is merely characteristic of some feature of the wind-tunnel (such as tedding rate or eddy size) has not yet been investigated. REFERENCES
DOBBS, C. G. (1942). On the primary dispersal and isolation of fungal spores. New Phytol. 41, 63-69. GREGORY, P. H. & LACEY, M. E. (1963). Mycological examination of dust from mouldy hay associated with Farmer's lung. ]. gen. Microbiol. 30, 75-88. HESSELTINE, C. W. (1960). Relationships of the Actinomycetes. Mycologia, 52, 460-474. HIRST, J. M. (1959). Spore liberation and dispersal. Plant pathology: problems and progress 1908-1958, pp. 529-538. Madison: University of Wisconsin Press. INGOLD, C. T. (1953). Dispersal infungi, pp. 197. Oxford: Clarendon Press. MASON, E. W. (1937). Annotated account offungi received at the Imperial Mycological Institute. List II, Fasc.g-General Part. Mycol. Pap. 4, 69-99. MAY, K. R. (1945). The cascade impactor: an instrument for sampling coarse aerosols. ]. sci. Instrum. 22, 187-195 .' STEPANOV, K. M.( 1935). (Dissemination of infective diseases of plants by air currents.) (In Russian.) Bull. Plant Prot. Leningr., Ser. 2, Phytopathology, 8, 1-68. ZOBERI, M. H. (1961). Take-off of mould spores in relation to wind speed and humidity. Ann. Bot., Lond., 25, 53-64.
(Accepted for publication 19 April 1962)