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Survival of arthrospores of Geotrichum candidum at sub-zero temperatures
[ 697 ] Trans. Br. mycol. Soc. 82 (4) 697-705 (1984)
Printed in Great Britain
SURVIVAL OF ARTHROSPORES OF GEOTRICHUM CANDIDUM AT SUB-ZERO TEMPERATURES By P. M. ROBINSON AND R. KENNEDY Department of Botany, The Queen's University, Belfast BTl INN Viability declined when arthrospore suspensions of Geotrichum candidum were cooled to - 27°C and there was no difference when viability was determined with or without added nutrients in the recovery medium. Storage at -27° resulted in a further fall in viability and the induction of an exogenous nutrient requirement for germination in part of the arthrospore population. Arthrospores produced on a range of media did not differ significantly in susceptibility to cold storage, but arthrospores from very young (1 day old) cultures were less susceptible to cold treatment than those from older cultures. Viability was constant when assessed in a range of recovery media. There was a further reduction in the viability of cold-treated arthrospores when sucrose, sodium chloride, sodium lauryl sulphate or 2,4dinitrophenol were incorporated into the recovery medium. It is concluded that cold treatment may cause membrane damage which results in leakage of nutrients essential for germination, and that this necessitates, for part of the population, a requirement for an exogenous supply of nutrient for repair and subsequent germination. Several factors influence the survival of fungal spores at low temperatures. The rates at which spores are cooled during freezing and warmed during revival are important factors in relation to viability and have been studied by Mazur (1953), Haskins (1957) and Goos, Davis & Butterfield (1967). In most instances rapid cooling was more detrimental than slow cooling and rapid thawing was less harmful than slow thawing. The temperature to which spores are cooled and the storage period also influence spore viability (Mazur, 1960a).
The conditions of storage influence viability and comparisons have been made between spores suspended in liquid media prior to freezing and spores which were air dried before freezing (Mazur, 1953; Graham, 1956; Mazur, 1960b). The relative humidity during storage influenced survival of dormant sporangiospores of Rhizopus species stored at 0° (Dennis & Hocker, 1981), and Ogunsanya & Madelin (1977) have shown that spores of some species are very sensitive to rapid rehydration after storage for a few hours at chilling temperatures. The damaging effect of rapid rehydration could be offset by exposing spores to a humid atmosphere prior to suspension in the revival medium. This investigation is concerned with the effects of storage of arthrospores of Geotrichum candidum Link at - 27° and 25° and examines some aspects of cold treatment which are relevant to survival. The only report on the tolerance of G. candidum to
low temperatures is by Davis (1951) who recorded that a temperature of - 23° was required to stop growth of G. candidum on butter. MA TERIALS AND METHODS
Routine culture and preparation of arthrospore suspensions
The same strain of G. candidum used for all previous studies with this organism in this laboratory was maintained in the dark at 25° on medical flat slopes of malt agar (MA) which had the following composition (g I-I): Oxoid malt extract, 30; Oxoid agar no. 3, 20. A medical flat slope consisted of a 200 ern" capacity medical flat bottle to which was added 30 em" of medium and after sterilization the slope was laid horizontally until the medium had set. An arthrospore suspension was poured over the surface of the medium and the surplus suspension was decanted. Sufficient arthrospores remained to give a uniform surface growth on the slopes which were incubated for 4 days. Arthrospore suspensions were obtained by washing a slope culture with sterile double distilled water (SDW) and the arthrospore concentration was determined with a haemocytometer. Appropriate dilutions with SDW were made for the various treatments. In one experiment G. candidum was cultured on S agar (SA) which had the following composition (g I-I): glucose, 0'7; MgS0 4 • 7H20, 0'5; KH 2P0 4 , 0-2; NH 4N0 3, 0'1; Oxoid agar no. 3,
Survival of Geotrichum arthrospores 12. In another experiment arthrospores were revived in YMPG liquid of the following composition (g 1-1): Oxoid yeast extract, 3; Oxoid malt extract, 3; Oxoid peptone, S; glucose, 10.
Storage, revival and estimation of viability of arthrospores
The arthrospore suspension was distributed in S ern" lots in S ern diam plastic Petri dishes. Suspensions which were to be frozen contained 104 arthrospores cm:"; suspensions stored at 2So contained S x 105 arthrospores cm", a self-inhibitory concentration to prevent germination during storage. The Petri dish lids were sealed to the bases to reduce evaporation and freeze-drying during storage experiments. The oxygen and carbon dioxide concentrations inside the Petri dishes were monitored during storage of arthrospore suspensions at 2So and found not to differ from the concentrations in the laboratory air. Arthrospore suspensions were cooled to and stored at - 27° in a low-temperature cabinet (Fisons, Loughborough, England). The rates of cooling and warming of the arthrospore suspensions were determined in experiments in which arthrospores were frozen. A copper-constantan thermocouple was immersed in one of the suspensions under test and the time course of temperature was recorded. The simplest revival technique was to incubate the dishes of arthrospores at 30° after cold treatment. This was feasible as low-density arthrospore suspensions of G. candidum germinated in SDW without added nutrients. With this method there was a longer lag prior to germination and a slightly decreased rate of germination than when nutrients were added. Removal of the Petri-dish lid reduced the lag and increased the germination rate of arthrospores in SDW, possibly by reducing the level of a self-produced volatile inhibitor of germination (Robinson, 1980). Revival at 30° without added nutrients and without the Petri-dish lid was selected as one of the two methods used to assess viability. Contamination of the arthrospore suspensions did not prove to be a problem during the relatively short periods of incubation and evaporation was suppressed by the maintenance of a high relative humidity in the incubator. In the other method arthrospores were revived in S liquid which was added in a volume of 1 ern" at an appropriate concentration to compensate for subsequent dilution due to the volume of arthrospore suspension. The Petri-dish lid was not removed and incubation was again at 30°. The two revival methods are compared in Fig. 1 a for suspensions of 10 4 arthrospores cm ? which had not been cold
treated. There was no significant difference between the values for germination determined by both revival techniques after incubation for 7 h. The effect of storage at - 27° for 24 h on viability of arthrospore suspensions is shown in Fig. 1 b. Even though this cold pretreatment reduced viability there was no significant difference between the values obtained for viability by the two revival methods when germination was assessed after 7 h incubation of the arthrospore suspensions. An arthrospore was considered viable if it produced one or more germ-tubes and it was established that such arthrospores were able to form colonies if transferred to a solid medium. Germination of treated and control suspensions was first recorded 7 h after transfer of suspensions to 30°. Germination was then assessed repeatedly at intervals of 1 h from 7 h onwards until no further increase in germination was recorded. Statistical treatment of results
Four replicate suspensions for each treatment were assayed at each sampling time. Viability was determined for a sample of 100 randomly selected arthrospores from each replicate suspension. The data were transformed using the 'angular' or sin " yip transformation and an analysis of variance was carried out for each experiment. A fixed range test, using the least significant range (L.S.R.), enabled comparisons to be made between treatment means (Parker, 1979). In all Figs the values displayed for the viability are the actual mean percentage values recorded; statements about differences between treatment means are based on the analysis of the transformed data.
RESUL TS
Effect of storage at - 27° on arthrospore viability
Arthrospore suspensions stored at 0° for 3 weeks did not decline in viability when assessed with added nutrients. When stored at - SO there was a slight fall in viability to 90 % during this time. At a storage temperature of - 27° there was a fall in viability (assessed with and without added nutrients) to approximately 70 % after only 24 h storage (Figs 1 b, 2). When arthrospores were revived with nutrients, viability fell to 60 % after storage for 2 days at - 27° and remained approximately at this level for the next 12 days of storage (Fig. 2). Between 14 and 28 days of storage there was a further fall in viability to 30 %. The trend in arthrospore viability with storage at - 27° as determined without added nutrients is also shown in Fig. 2. After storage for 3 days a significant
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difference in viability was obtained by the two revival techniques, the lower viability being recorded for revival without added nutrients. In the absence of nutrients no further decrease in viability occurred during storage for the following 25 days. After storage for 28 days there was no significant
Arthrospores injured by cold treatment (but still able to germinate with added nutrients) may have deteriorated during further storage to the point where revival by added nutrients was no longer possible. To determine if damage also occurred during storage at 25°, and whether or not this could be rectified by addition of nutrients, arthrospore suspensions were stored at a self-inhibitory concentration for 10 weeks. During the first 4 week period of storage, the length of the previous experiment, there was no significant fall in viability as determined by either revival method. After 6 weeks of storage there was no significant drop in viability assessed by addition of nutrients but without added nutrients there was a significant drop in viability to 73 %. After storage for 10 weeks both revival techniques gave viability estimates between 60 and 70% (Fig. 3a). Effect of cold pretreatment on viability of arthrospores stored at 25°
Arthrospore suspensions were stored at - 5° for 3 h prior to long-term storage at 25°. Such pretreatment might render an arthrospore population more sensitive to storage damage at 25°. There was an
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Cb) Pretreated at -5° for 3 h prior to storage at 25°. Viability was assessed with Ce) and without CO) added nutrients.
initial 5 % drop in viability (estimated by both revival techniques) which was maintained for 5 weeks (Fig. 3b). After 7 weeks storage there was a decline in viability (without added nutrients) to 35%, whereas viability (plus nutrients) was 80%. From 7 to 14 weeks viability without added nutrients remained constant but with nutrients it fell to 30 %, a value not significantly different from that given by revival in the absence of added nutrients.
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Decline in arthrospore viability during cooling In order to determine the effect of the initial cooling component of cold storage on viability, viability was determined at various times during the period in which arthrospore suspensions were cooled to - 27°. The results are shown in Fig. 4 and viability was assessed with added nutrients. Cooling to 0° did not result in a significant fall in viability but as the temperature fell from - 1 to - 7° there was a sharp fall in viability to 89 %. Thereafter viability declined steadily to 72 % as the temperature fell to - 24°. Further cooling to - 27° did not result in any further decrease in viability. This time course of cooling was typical for the initial cooling treatment applied to arthrospore suspensions used in the present investigation. The cooling rate never exceeded 1° min-I. It took approximately 27 min for the temperature of a suspension at - 27° to reach 30°, the incubation temperature used for revival.
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Effect of culture age and culture medium on viability of cold-treated arthrospores To determine any effect that culture age might have on the susceptibility of arthrospores to cold treatment a series of medical flat slope cultures of G. candidum was harvested at selected times from inoculation. The resultant arthrospore suspensions,
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from cultures ranging in age from 1 day to 8 weeks , were pretreated at - 27° for 24 h and viability was assessed with and without added nutrients. The results for revival with added nutrients are shown in Fig. 5 and the youngest arthrospore population (from the 1 day old culture) was the most resistant to the cold treatment. The results for revival without added nutrients are not shown as they did not differ significantly from those for revival with added nutrients. Experiments were carried out to investigate the effect of the culture medium on the viability of cold-treated arthrospores. Synchronised sporulation was induced in cultures growing on MA, SA and distilled water agar as described by Park & Robinson (1969) and Robinson (1978). Arthrospores were harvested as soon as possible after formation and assayed for susceptibility to storage at - 27° during a period of 4 weeks. No marked differences were obtained at the various assay times between the viability of populations of arthrospores produced on these three culture media. Each population gave results, for revival with and without added nutrients, similar to those obtained in Fig . 2 but there was a tendency for a higher viability to be obtained at each assay time. The h igher viabilities in the present experiment may have reflected the usc of young arthrospores (1 to 2 h old ) in contrast to the older population (from a 4 day old culture) in Fig . 2 . This conclusion is supported by the results for the effect of culture age on the viability of cold-treated arthrospores.
sub jected to various types of stress during recovery from cold storage at - 27° for 24 h. For these experiments arthrospore suspensions were prepared from 1 day old cultures. The recovery medium, with and without added nutrients, was amended by the addition of sucrose in some cases and of sodium chloride in others. Addition of up to 60 % (w I v) sucrose, with or without added nutrients, had a negligible effect on the viability of arthrospores which had not been cold treated. There was a marked effect of sucrose addition on the viability of cold-treated arthrospores which fell to 13 % when revival was with added nutrients plus 60 % (w I v) sucrose (F ig. 6 a). Although at the highest sucrose concentrations tested there was evidence of lower viabilities in suspensions revived without added nutrients (these results are not included), there was no significant difference between the viability of arthrospores revived in the presence of up to 20 % (w / v) sucrose with added nutrients (F ig. 6a ) or without added nutrients (F ig. 6b ). When cold-treated arthrospores were revived with added nutrients containing from 0 to 6 % (w Iv ) sodium chloride (F ig. 7) the viability fell to 73 % at a sodium chloride concentration of 6 % (w/ v). Data for recovery in the absence of added nutrients are not shown but under these conditions arthrospores were more susceptible to stress due to sodium chloride. In fact, 0'1 % (w/v ) sodium chloride reduced viability to 72 %. Added nutrients are obviously essential for the arthrospores to withstand the osmotic and/or ionic stress imposed by quite low concentrations of sodi um ch loride. The enhanced susceptibility of cold-treated arthrospores to the presence of sucrose or sodium chloride during recovery could have been partly due to an effect on the permeability of the cells as a result of membrane damage during cold treatment . With thi s in mind cold-treated arthrospores were allowed to recover in media containing the surface active agent sodium lauryl sulphate. In the presence of added nutrients, arthrospore viability fell markedly at concentrations of sodium lauryl sulphate which had no significant effect on arthrospore suspensions which had not been cold treated (F ig. 8).
Survival of Geotrichum arthrospores
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Fig. 7. Effect of addition of sodium chloride during revival of arthrospores of G. candidum pretreated at - 27° for 24 h. e, control (without cold pretreatment); 0, with cold pretreatment. Viability assessed with added nutrients.
Arthrospores were revived in solutions of z.a-dinirrophenol, an agent which uncouples oxidative phosphorylation, shifting ATP synthesis to substrate level phosphorylation. The solutions were buffered at pH 5"4. Preliminary results established that low concentrations of 2,4-dinitrophenol inhibited germination of arthrospore sus-
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pensions which had not been cold-treated and which were revived without added nutrients. When arthrospores were revived with added nutrients 2,4-dinitrophenol at 60 p.p.m. caused a marked reduction in the viability of cold-treated arthro-
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spores when compared with a control series which had not been cold treated (F ig. 9). This suggests an increase in energy requirement for germination of cold-treated arthrospores. Arthrospores were revived in buffered solutions of kanamycin monosulphate which inhibits protein synthesis. Again it was nece ssary to add nutrients during revival and there was no significant effect of up to 800 p.p.m. of kanamycin monosulphate in the revi val medium, irrespective of whether or not the arthrospores had been cold treated. Although these results seemed to indicate that protein synthesis was not a prerequisite for germination after cold treatment more work would be required to substantiate this view. Storage at - 27° was only for 24 h and a longer period may have induced a requirement for protein synthesis prior to germination. It is known that in some organisms storage damage may result from long-term effects of low temperature on proteins and changes in the structure of membranes and enz ymes have been described (L evitt , 1972).
DISCUSSION
In an y discussion of the effects of low-temperature storage on fungal spores it is important to consider the two main sources of potential injury, namely, loss of viability due to the cooling and warming component of storage and to storage itself. Rapid cooling rates are associated with a greater loss in
viability of fungal spores than slow cooling rates and in the pre sent study the cooling rate never exceeded 1° min" in an attempt to minimize freezing damage. This rate is very slow compared with the rates of 50 to 300° min-I used with Sac charomy ces cerev isiae and which proved more harmful than a rate of 1° rnin " (M azur, 1961, 1967). Ninety per cent of the water in yeast cells is able to freeze (W ood & Rosenberg, 1957 ; Souzu, Nei & Bito, 1961 ; Mazur, 1963). Cells cooled rapidly retained more of their or igin al water (M azur, 1967) and would be subject to more damage by intracellular ice formation than cells cooled slowl y. In the present work a cooling/warming cycle took approximately 4 h and resulted cons istently in a loss of viability (assessed with or without added nutrients) in about 30 % of the arthrospore suspension. Separation of the cooling and warming components of the cycle by 3 day s of storage at - 27° resulted in a further loss in viability which at 3 days became lower when assessed without added nutrients than when assessed with added nutrients. The simplest hypothesis would be that cooling (in conjunction with the later warming component of the freeze/thaw cycle) kills part of the arthrospore population and damages another portion such that some arthrospores of this portion become nonviable during this initial storage period whereas others become nutrient-dependent for germination. Further storage results in exacerbation of this damage such that the formerly nutrient-dependent fraction fails to germinate in the presence of added nutrients and viability of the population as a whole falls to approximately 40 % after 4 weeks . When arthrospores were stored at 25° a loss in viability was onl y apparent after storage for 5 weeks . It cannot be assumed that this loss in viability was due to the same type of damage induced by low-temperature storage but it is of interest that the damage could be rectified by addition of nutrients after storage for 6 weeks but not after 10 weeks. A 24 h pretreatment at - 5°, apart from inducing an initial slight fall in viability, did not result in a further decline in viability during storage for 5 weeks at 25°. At 7 week s there was a dramatic fall in viability, particularly when assessed without added nutrients . This could have been due to a delayed action effect of th e cold pretreatment augmenting the slight decline in viability which occurred at approximately thi s time in an arthrospore su spension not subjected to a cold pretreatment. What is the mechanism of damage during cold treatment? There was no significant fall in viability during cooling to 0 ° but below zero the viability fell quite markedly. Arthrospores could have been
704
Survival of Geotrichum arthrospores
damaged by the formation of intracellular ice humidity (Barer & Joseph, 1958). A reduced water crystals although the slow cooling rate would have content in spores could result in more solute keptthis to a minimum (Mazur, 1968). Intracellular precipitation and more mechanical stress during ice formation would have resulted in disruption of slow freezing. These factors could result in a membranes and cell organelles and in solute reduction in viability. In the present work it was precipitation. Damage to the cytoplasmic mem- significant that arthrospores harvested from 8 week brane could have resulted in a subsequent leakage old cultures had a viability of approximately 5 % of metabolites during revival. whereas arthrospores harvested from 4 day old It is more difficult to explain the fall in viability cultures and stored at 25° for 8 weeks had a viability which occurred during storage at - 27° but this of 65-80 %, depending on whether or not nutrients could have been due to a long exposure to were added during revival. Obviously continued concentrated solutes (Mazur, 1968) and mechanical contact with the culture has a detrimental effect stress imposed by extracellular ice formation which could be partly due to a greater water loss (Sutcliffe, 1977). In the present investigation it from arthrospores in contact with the culture might be expected that the slow cooling rate would compared to arthrospores stored in SDW. This have resulted in a greater water loss from the aspect of the work requires further investigation, arthrospores than if much faster rates had been possibly using refractometric techniques to employed. The resultant arthrospore contraction establish the water content of arthrospores would have increased when extracellular ice (Barer & Joseph, 1958). formation occurred so placing the protoplasm under even greater mechanical stress. Dehydrated R. K. wishes to acknowledge the award of a protoplasm would be stiffer and more susceptible research scholarship from the Department of to mechanical stress. A sustained stress of this Education for Northern Ireland. nature may have been a relevant factor in the decline in viability during cold storage and would have augmented any damage caused by the initial REFERENCES cooling operation. BARER, R. & JOSEPH, S. (1958). Concentration and mass The suggestion that damage to the cytoplasmic measurements in microbiology. Journal of Applied membrane was partly responsible for failure of Bacteriology 21, 146-159. some cold-treated arthrospores to germinate, and DAVIS,]. G. (1951). The effectofcoldon micro-organisms in relation to dairying. Proceedings of the Society for for nutrient dependence in others, is supported by Applied Bacteriology 14,216--242. the increased sensitivity of cold-treated arthrospores to osmotic and/or ionic stress during DENNIS, C. & HOCKER, J. (1981). Effect of relative humidity on the chillingsensitivityof sporangiospores recovery. The increased sensitivity of cold-treated of Rhizopus species. Transactions of the British Mycoarthrospores to sodium lauryl sulphate is also conlogical Society 77,179-183. sistent with this type of damage which would affect Goos, R. D., DAVIS, E. E. & BUTTERFIELD, W. (1967). cell permeability. Treatment with 2,4-dinitroEffect of warming rates on the viability of frozen phenol enhanced inhibition of cold-treated arthrofungous spores. Mycologia 59, 58-66. spores and indicated an increased energy demand GRAHAM, S. O. (1956). Germination responsesof Ustilago tritici (Pers.) Rostr. teliospores in relation to lyophiliduring resuscitation. This increase in energy zation. I. Some factors affectingmortality before and requirement could have resulted from the necessity after sublimation. Research studies of State University of to repair cold damage and from the leakage of Washington 24, 3°7-317. metabolites critical for germination during revival HASKINS, R. H. (1957). Factors affecting survival of after cold treatment. lyophilizedfungalsporesand cells. Canadian Journal of There are no reports on the effect of spore age in Microbiology 3, 477-485. relation to tolerance of cold storage. In the present LEVITT, J. (1972). Responses of Plants to Environmental investigation the terms 'culture age' and 'arthroStresses. London: Academic Press. spore age' are almost synonymous since the MAZUR, P. (1953). Studies of the effects of low temperature and dehydration on the viability of technique for seeding medical fiat slopes resulted fungous spores. PhD Thesis, Harvard University. in arthrospore formation within 24 h and the maximum number of arthrospores was present MAzUR, P. (1960a). The effects of subzero temperatures on microorganisms. In Recent Research in Freezing and within 24-48 h from inoculation. The arthrospores Drying (ed. A. S. Parkes & A. U. Smith), pp. 65-77. became more susceptible to cold treatment as they Oxford: Blackwell. aged on a culture. The mechanism of this effect is MAZUR, P. (1960b). Physical factors implicated in the not known but spores of some fungal species lose death of microorganisms at subzero temperatures. water as they age on a culture, even though the Annals of the New York Academy of Science 85, atmosphere is maintained at 100 % relative 610-6 29.
P. M. Robinson and R . Kennedy MAZUR, P. (1961). Manifestation of injury in yeast cells exposed to subzero temperatures. I. Morphological changes in freeze-substituted and in 'frozen-thawed' cells. Journal of Bacteriology 8z , 662-672. MAzUR, P . ( 1963). Studies on rapidly frozen suspensions of yeast cells by differential thermal analysis and conductometry, B iophysical Journal 3, 3 23-353 . MAzUR, P . (1967). Physical chemical basis of injury from intracellular freezing in yeast. In Cellular Injury and Resistance in Freezing Organisms (ed. E. Asahina), pp . 171-189. Japan : Institute of Low T emperature Science, Hokkaido University. MAzUR, P. (1968). Survival of fungi after freezing and desiccation . In The Fungi, vol. III (ed. G . C. Ainsworth & A. S. Sus sman ), pp . 325-394. London : Academic Pre ss. OGUNSANYA, O. C. & MADELlN, M . F . (1977). Sensitivity of Botryodiplodia ricinicola conidia to mild chilling. Transactions of the British Myc ological Society 69, 191-195· PARK, D. & ROBINSON, P. M. (1969). Sporulation in
7°5
Georrichum candidum. Transactions of the British My cological S ociety 52, 213-222. PARKER, R. E . ( 1979). Introductory Statisticsfor B iology . Studies in Biology, no. 43. London : Edward Arnold. ROBINSON , P. M . (1978). Phase transformations. In Practical Fungal Physiology, pp. 53-55 . New York : Wile y & Sons. ROBINSON, P . M. (1980). Autotropisrn in germinating arthrospores of Geotrichum candidum. Transactions of the British Mycological Society 75,151-153 . SOUZU, H ., NEI, T. & BITo, M . ( 1961). Wat er of microorganisms and its freezing with special reference to the relation between water content and viability of yeast and coli cells. Low Temperature S cience Bt9, 4~57 ·
SUTCLIFFE, J. ( 1977). Effects of mechanical stress. In Plants and Temperature. Studies in Biology, no. 86, pp . 45-46. London : Edward Arnold. WOOD, T. H. & ROSENBERG, A. M . (1957). Freezing in yeast cells. Biochimica et Biophysi ca Acta z5 , 78-87.
(R eceived for publication 15 September 1983)
Printed in Great Britain
SURVIVAL OF ARTHROSPORES OF GEOTRICHUM CANDIDUM AT SUB-ZERO TEMPERATURES By P. M. ROBINSON AND R. KENNEDY Department of Botany, The Queen's University, Belfast BTl INN Viability declined when arthrospore suspensions of Geotrichum candidum were cooled to - 27°C and there was no difference when viability was determined with or without added nutrients in the recovery medium. Storage at -27° resulted in a further fall in viability and the induction of an exogenous nutrient requirement for germination in part of the arthrospore population. Arthrospores produced on a range of media did not differ significantly in susceptibility to cold storage, but arthrospores from very young (1 day old) cultures were less susceptible to cold treatment than those from older cultures. Viability was constant when assessed in a range of recovery media. There was a further reduction in the viability of cold-treated arthrospores when sucrose, sodium chloride, sodium lauryl sulphate or 2,4dinitrophenol were incorporated into the recovery medium. It is concluded that cold treatment may cause membrane damage which results in leakage of nutrients essential for germination, and that this necessitates, for part of the population, a requirement for an exogenous supply of nutrient for repair and subsequent germination. Several factors influence the survival of fungal spores at low temperatures. The rates at which spores are cooled during freezing and warmed during revival are important factors in relation to viability and have been studied by Mazur (1953), Haskins (1957) and Goos, Davis & Butterfield (1967). In most instances rapid cooling was more detrimental than slow cooling and rapid thawing was less harmful than slow thawing. The temperature to which spores are cooled and the storage period also influence spore viability (Mazur, 1960a).
The conditions of storage influence viability and comparisons have been made between spores suspended in liquid media prior to freezing and spores which were air dried before freezing (Mazur, 1953; Graham, 1956; Mazur, 1960b). The relative humidity during storage influenced survival of dormant sporangiospores of Rhizopus species stored at 0° (Dennis & Hocker, 1981), and Ogunsanya & Madelin (1977) have shown that spores of some species are very sensitive to rapid rehydration after storage for a few hours at chilling temperatures. The damaging effect of rapid rehydration could be offset by exposing spores to a humid atmosphere prior to suspension in the revival medium. This investigation is concerned with the effects of storage of arthrospores of Geotrichum candidum Link at - 27° and 25° and examines some aspects of cold treatment which are relevant to survival. The only report on the tolerance of G. candidum to
low temperatures is by Davis (1951) who recorded that a temperature of - 23° was required to stop growth of G. candidum on butter. MA TERIALS AND METHODS
Routine culture and preparation of arthrospore suspensions
The same strain of G. candidum used for all previous studies with this organism in this laboratory was maintained in the dark at 25° on medical flat slopes of malt agar (MA) which had the following composition (g I-I): Oxoid malt extract, 30; Oxoid agar no. 3, 20. A medical flat slope consisted of a 200 ern" capacity medical flat bottle to which was added 30 em" of medium and after sterilization the slope was laid horizontally until the medium had set. An arthrospore suspension was poured over the surface of the medium and the surplus suspension was decanted. Sufficient arthrospores remained to give a uniform surface growth on the slopes which were incubated for 4 days. Arthrospore suspensions were obtained by washing a slope culture with sterile double distilled water (SDW) and the arthrospore concentration was determined with a haemocytometer. Appropriate dilutions with SDW were made for the various treatments. In one experiment G. candidum was cultured on S agar (SA) which had the following composition (g I-I): glucose, 0'7; MgS0 4 • 7H20, 0'5; KH 2P0 4 , 0-2; NH 4N0 3, 0'1; Oxoid agar no. 3,
Survival of Geotrichum arthrospores 12. In another experiment arthrospores were revived in YMPG liquid of the following composition (g 1-1): Oxoid yeast extract, 3; Oxoid malt extract, 3; Oxoid peptone, S; glucose, 10.
Storage, revival and estimation of viability of arthrospores
The arthrospore suspension was distributed in S ern" lots in S ern diam plastic Petri dishes. Suspensions which were to be frozen contained 104 arthrospores cm:"; suspensions stored at 2So contained S x 105 arthrospores cm", a self-inhibitory concentration to prevent germination during storage. The Petri dish lids were sealed to the bases to reduce evaporation and freeze-drying during storage experiments. The oxygen and carbon dioxide concentrations inside the Petri dishes were monitored during storage of arthrospore suspensions at 2So and found not to differ from the concentrations in the laboratory air. Arthrospore suspensions were cooled to and stored at - 27° in a low-temperature cabinet (Fisons, Loughborough, England). The rates of cooling and warming of the arthrospore suspensions were determined in experiments in which arthrospores were frozen. A copper-constantan thermocouple was immersed in one of the suspensions under test and the time course of temperature was recorded. The simplest revival technique was to incubate the dishes of arthrospores at 30° after cold treatment. This was feasible as low-density arthrospore suspensions of G. candidum germinated in SDW without added nutrients. With this method there was a longer lag prior to germination and a slightly decreased rate of germination than when nutrients were added. Removal of the Petri-dish lid reduced the lag and increased the germination rate of arthrospores in SDW, possibly by reducing the level of a self-produced volatile inhibitor of germination (Robinson, 1980). Revival at 30° without added nutrients and without the Petri-dish lid was selected as one of the two methods used to assess viability. Contamination of the arthrospore suspensions did not prove to be a problem during the relatively short periods of incubation and evaporation was suppressed by the maintenance of a high relative humidity in the incubator. In the other method arthrospores were revived in S liquid which was added in a volume of 1 ern" at an appropriate concentration to compensate for subsequent dilution due to the volume of arthrospore suspension. The Petri-dish lid was not removed and incubation was again at 30°. The two revival methods are compared in Fig. 1 a for suspensions of 10 4 arthrospores cm ? which had not been cold
treated. There was no significant difference between the values for germination determined by both revival techniques after incubation for 7 h. The effect of storage at - 27° for 24 h on viability of arthrospore suspensions is shown in Fig. 1 b. Even though this cold pretreatment reduced viability there was no significant difference between the values obtained for viability by the two revival methods when germination was assessed after 7 h incubation of the arthrospore suspensions. An arthrospore was considered viable if it produced one or more germ-tubes and it was established that such arthrospores were able to form colonies if transferred to a solid medium. Germination of treated and control suspensions was first recorded 7 h after transfer of suspensions to 30°. Germination was then assessed repeatedly at intervals of 1 h from 7 h onwards until no further increase in germination was recorded. Statistical treatment of results
Four replicate suspensions for each treatment were assayed at each sampling time. Viability was determined for a sample of 100 randomly selected arthrospores from each replicate suspension. The data were transformed using the 'angular' or sin " yip transformation and an analysis of variance was carried out for each experiment. A fixed range test, using the least significant range (L.S.R.), enabled comparisons to be made between treatment means (Parker, 1979). In all Figs the values displayed for the viability are the actual mean percentage values recorded; statements about differences between treatment means are based on the analysis of the transformed data.
RESUL TS
Effect of storage at - 27° on arthrospore viability
Arthrospore suspensions stored at 0° for 3 weeks did not decline in viability when assessed with added nutrients. When stored at - SO there was a slight fall in viability to 90 % during this time. At a storage temperature of - 27° there was a fall in viability (assessed with and without added nutrients) to approximately 70 % after only 24 h storage (Figs 1 b, 2). When arthrospores were revived with nutrients, viability fell to 60 % after storage for 2 days at - 27° and remained approximately at this level for the next 12 days of storage (Fig. 2). Between 14 and 28 days of storage there was a further fall in viability to 30 %. The trend in arthrospore viability with storage at - 27° as determined without added nutrients is also shown in Fig. 2. After storage for 3 days a significant
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difference in viability was obtained by the two revival techniques, the lower viability being recorded for revival without added nutrients. In the absence of nutrients no further decrease in viability occurred during storage for the following 25 days. After storage for 28 days there was no significant
Arthrospores injured by cold treatment (but still able to germinate with added nutrients) may have deteriorated during further storage to the point where revival by added nutrients was no longer possible. To determine if damage also occurred during storage at 25°, and whether or not this could be rectified by addition of nutrients, arthrospore suspensions were stored at a self-inhibitory concentration for 10 weeks. During the first 4 week period of storage, the length of the previous experiment, there was no significant fall in viability as determined by either revival method. After 6 weeks of storage there was no significant drop in viability assessed by addition of nutrients but without added nutrients there was a significant drop in viability to 73 %. After storage for 10 weeks both revival techniques gave viability estimates between 60 and 70% (Fig. 3a). Effect of cold pretreatment on viability of arthrospores stored at 25°
Arthrospore suspensions were stored at - 5° for 3 h prior to long-term storage at 25°. Such pretreatment might render an arthrospore population more sensitive to storage damage at 25°. There was an
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initial 5 % drop in viability (estimated by both revival techniques) which was maintained for 5 weeks (Fig. 3b). After 7 weeks storage there was a decline in viability (without added nutrients) to 35%, whereas viability (plus nutrients) was 80%. From 7 to 14 weeks viability without added nutrients remained constant but with nutrients it fell to 30 %, a value not significantly different from that given by revival in the absence of added nutrients.
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Decline in arthrospore viability during cooling In order to determine the effect of the initial cooling component of cold storage on viability, viability was determined at various times during the period in which arthrospore suspensions were cooled to - 27°. The results are shown in Fig. 4 and viability was assessed with added nutrients. Cooling to 0° did not result in a significant fall in viability but as the temperature fell from - 1 to - 7° there was a sharp fall in viability to 89 %. Thereafter viability declined steadily to 72 % as the temperature fell to - 24°. Further cooling to - 27° did not result in any further decrease in viability. This time course of cooling was typical for the initial cooling treatment applied to arthrospore suspensions used in the present investigation. The cooling rate never exceeded 1° min-I. It took approximately 27 min for the temperature of a suspension at - 27° to reach 30°, the incubation temperature used for revival.
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Effect of culture age and culture medium on viability of cold-treated arthrospores To determine any effect that culture age might have on the susceptibility of arthrospores to cold treatment a series of medical flat slope cultures of G. candidum was harvested at selected times from inoculation. The resultant arthrospore suspensions,
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from cultures ranging in age from 1 day to 8 weeks , were pretreated at - 27° for 24 h and viability was assessed with and without added nutrients. The results for revival with added nutrients are shown in Fig. 5 and the youngest arthrospore population (from the 1 day old culture) was the most resistant to the cold treatment. The results for revival without added nutrients are not shown as they did not differ significantly from those for revival with added nutrients. Experiments were carried out to investigate the effect of the culture medium on the viability of cold-treated arthrospores. Synchronised sporulation was induced in cultures growing on MA, SA and distilled water agar as described by Park & Robinson (1969) and Robinson (1978). Arthrospores were harvested as soon as possible after formation and assayed for susceptibility to storage at - 27° during a period of 4 weeks. No marked differences were obtained at the various assay times between the viability of populations of arthrospores produced on these three culture media. Each population gave results, for revival with and without added nutrients, similar to those obtained in Fig . 2 but there was a tendency for a higher viability to be obtained at each assay time. The h igher viabilities in the present experiment may have reflected the usc of young arthrospores (1 to 2 h old ) in contrast to the older population (from a 4 day old culture) in Fig . 2 . This conclusion is supported by the results for the effect of culture age on the viability of cold-treated arthrospores.
sub jected to various types of stress during recovery from cold storage at - 27° for 24 h. For these experiments arthrospore suspensions were prepared from 1 day old cultures. The recovery medium, with and without added nutrients, was amended by the addition of sucrose in some cases and of sodium chloride in others. Addition of up to 60 % (w I v) sucrose, with or without added nutrients, had a negligible effect on the viability of arthrospores which had not been cold treated. There was a marked effect of sucrose addition on the viability of cold-treated arthrospores which fell to 13 % when revival was with added nutrients plus 60 % (w I v) sucrose (F ig. 6 a). Although at the highest sucrose concentrations tested there was evidence of lower viabilities in suspensions revived without added nutrients (these results are not included), there was no significant difference between the viability of arthrospores revived in the presence of up to 20 % (w / v) sucrose with added nutrients (F ig. 6a ) or without added nutrients (F ig. 6b ). When cold-treated arthrospores were revived with added nutrients containing from 0 to 6 % (w Iv ) sodium chloride (F ig. 7) the viability fell to 73 % at a sodium chloride concentration of 6 % (w/ v). Data for recovery in the absence of added nutrients are not shown but under these conditions arthrospores were more susceptible to stress due to sodium chloride. In fact, 0'1 % (w/v ) sodium chloride reduced viability to 72 %. Added nutrients are obviously essential for the arthrospores to withstand the osmotic and/or ionic stress imposed by quite low concentrations of sodi um ch loride. The enhanced susceptibility of cold-treated arthrospores to the presence of sucrose or sodium chloride during recovery could have been partly due to an effect on the permeability of the cells as a result of membrane damage during cold treatment . With thi s in mind cold-treated arthrospores were allowed to recover in media containing the surface active agent sodium lauryl sulphate. In the presence of added nutrients, arthrospore viability fell markedly at concentrations of sodium lauryl sulphate which had no significant effect on arthrospore suspensions which had not been cold treated (F ig. 8).
Survival of Geotrichum arthrospores
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Arthrospores were revived in solutions of z.a-dinirrophenol, an agent which uncouples oxidative phosphorylation, shifting ATP synthesis to substrate level phosphorylation. The solutions were buffered at pH 5"4. Preliminary results established that low concentrations of 2,4-dinitrophenol inhibited germination of arthrospore sus-
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pensions which had not been cold-treated and which were revived without added nutrients. When arthrospores were revived with added nutrients 2,4-dinitrophenol at 60 p.p.m. caused a marked reduction in the viability of cold-treated arthro-
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spores when compared with a control series which had not been cold treated (F ig. 9). This suggests an increase in energy requirement for germination of cold-treated arthrospores. Arthrospores were revived in buffered solutions of kanamycin monosulphate which inhibits protein synthesis. Again it was nece ssary to add nutrients during revival and there was no significant effect of up to 800 p.p.m. of kanamycin monosulphate in the revi val medium, irrespective of whether or not the arthrospores had been cold treated. Although these results seemed to indicate that protein synthesis was not a prerequisite for germination after cold treatment more work would be required to substantiate this view. Storage at - 27° was only for 24 h and a longer period may have induced a requirement for protein synthesis prior to germination. It is known that in some organisms storage damage may result from long-term effects of low temperature on proteins and changes in the structure of membranes and enz ymes have been described (L evitt , 1972).
DISCUSSION
In an y discussion of the effects of low-temperature storage on fungal spores it is important to consider the two main sources of potential injury, namely, loss of viability due to the cooling and warming component of storage and to storage itself. Rapid cooling rates are associated with a greater loss in
viability of fungal spores than slow cooling rates and in the pre sent study the cooling rate never exceeded 1° min" in an attempt to minimize freezing damage. This rate is very slow compared with the rates of 50 to 300° min-I used with Sac charomy ces cerev isiae and which proved more harmful than a rate of 1° rnin " (M azur, 1961, 1967). Ninety per cent of the water in yeast cells is able to freeze (W ood & Rosenberg, 1957 ; Souzu, Nei & Bito, 1961 ; Mazur, 1963). Cells cooled rapidly retained more of their or igin al water (M azur, 1967) and would be subject to more damage by intracellular ice formation than cells cooled slowl y. In the present work a cooling/warming cycle took approximately 4 h and resulted cons istently in a loss of viability (assessed with or without added nutrients) in about 30 % of the arthrospore suspension. Separation of the cooling and warming components of the cycle by 3 day s of storage at - 27° resulted in a further loss in viability which at 3 days became lower when assessed without added nutrients than when assessed with added nutrients. The simplest hypothesis would be that cooling (in conjunction with the later warming component of the freeze/thaw cycle) kills part of the arthrospore population and damages another portion such that some arthrospores of this portion become nonviable during this initial storage period whereas others become nutrient-dependent for germination. Further storage results in exacerbation of this damage such that the formerly nutrient-dependent fraction fails to germinate in the presence of added nutrients and viability of the population as a whole falls to approximately 40 % after 4 weeks . When arthrospores were stored at 25° a loss in viability was onl y apparent after storage for 5 weeks . It cannot be assumed that this loss in viability was due to the same type of damage induced by low-temperature storage but it is of interest that the damage could be rectified by addition of nutrients after storage for 6 weeks but not after 10 weeks. A 24 h pretreatment at - 5°, apart from inducing an initial slight fall in viability, did not result in a further decline in viability during storage for 5 weeks at 25°. At 7 week s there was a dramatic fall in viability, particularly when assessed without added nutrients . This could have been due to a delayed action effect of th e cold pretreatment augmenting the slight decline in viability which occurred at approximately thi s time in an arthrospore su spension not subjected to a cold pretreatment. What is the mechanism of damage during cold treatment? There was no significant fall in viability during cooling to 0 ° but below zero the viability fell quite markedly. Arthrospores could have been
704
Survival of Geotrichum arthrospores
damaged by the formation of intracellular ice humidity (Barer & Joseph, 1958). A reduced water crystals although the slow cooling rate would have content in spores could result in more solute keptthis to a minimum (Mazur, 1968). Intracellular precipitation and more mechanical stress during ice formation would have resulted in disruption of slow freezing. These factors could result in a membranes and cell organelles and in solute reduction in viability. In the present work it was precipitation. Damage to the cytoplasmic mem- significant that arthrospores harvested from 8 week brane could have resulted in a subsequent leakage old cultures had a viability of approximately 5 % of metabolites during revival. whereas arthrospores harvested from 4 day old It is more difficult to explain the fall in viability cultures and stored at 25° for 8 weeks had a viability which occurred during storage at - 27° but this of 65-80 %, depending on whether or not nutrients could have been due to a long exposure to were added during revival. Obviously continued concentrated solutes (Mazur, 1968) and mechanical contact with the culture has a detrimental effect stress imposed by extracellular ice formation which could be partly due to a greater water loss (Sutcliffe, 1977). In the present investigation it from arthrospores in contact with the culture might be expected that the slow cooling rate would compared to arthrospores stored in SDW. This have resulted in a greater water loss from the aspect of the work requires further investigation, arthrospores than if much faster rates had been possibly using refractometric techniques to employed. The resultant arthrospore contraction establish the water content of arthrospores would have increased when extracellular ice (Barer & Joseph, 1958). formation occurred so placing the protoplasm under even greater mechanical stress. Dehydrated R. K. wishes to acknowledge the award of a protoplasm would be stiffer and more susceptible research scholarship from the Department of to mechanical stress. A sustained stress of this Education for Northern Ireland. nature may have been a relevant factor in the decline in viability during cold storage and would have augmented any damage caused by the initial REFERENCES cooling operation. BARER, R. & JOSEPH, S. (1958). Concentration and mass The suggestion that damage to the cytoplasmic measurements in microbiology. Journal of Applied membrane was partly responsible for failure of Bacteriology 21, 146-159. some cold-treated arthrospores to germinate, and DAVIS,]. G. (1951). The effectofcoldon micro-organisms in relation to dairying. Proceedings of the Society for for nutrient dependence in others, is supported by Applied Bacteriology 14,216--242. the increased sensitivity of cold-treated arthrospores to osmotic and/or ionic stress during DENNIS, C. & HOCKER, J. (1981). Effect of relative humidity on the chillingsensitivityof sporangiospores recovery. The increased sensitivity of cold-treated of Rhizopus species. Transactions of the British Mycoarthrospores to sodium lauryl sulphate is also conlogical Society 77,179-183. sistent with this type of damage which would affect Goos, R. D., DAVIS, E. E. & BUTTERFIELD, W. (1967). cell permeability. Treatment with 2,4-dinitroEffect of warming rates on the viability of frozen phenol enhanced inhibition of cold-treated arthrofungous spores. Mycologia 59, 58-66. spores and indicated an increased energy demand GRAHAM, S. O. (1956). Germination responsesof Ustilago tritici (Pers.) Rostr. teliospores in relation to lyophiliduring resuscitation. This increase in energy zation. I. Some factors affectingmortality before and requirement could have resulted from the necessity after sublimation. Research studies of State University of to repair cold damage and from the leakage of Washington 24, 3°7-317. metabolites critical for germination during revival HASKINS, R. H. (1957). Factors affecting survival of after cold treatment. lyophilizedfungalsporesand cells. Canadian Journal of There are no reports on the effect of spore age in Microbiology 3, 477-485. relation to tolerance of cold storage. In the present LEVITT, J. (1972). Responses of Plants to Environmental investigation the terms 'culture age' and 'arthroStresses. London: Academic Press. spore age' are almost synonymous since the MAZUR, P. (1953). Studies of the effects of low temperature and dehydration on the viability of technique for seeding medical fiat slopes resulted fungous spores. PhD Thesis, Harvard University. in arthrospore formation within 24 h and the maximum number of arthrospores was present MAzUR, P. (1960a). The effects of subzero temperatures on microorganisms. In Recent Research in Freezing and within 24-48 h from inoculation. The arthrospores Drying (ed. A. S. Parkes & A. U. Smith), pp. 65-77. became more susceptible to cold treatment as they Oxford: Blackwell. aged on a culture. The mechanism of this effect is MAZUR, P. (1960b). Physical factors implicated in the not known but spores of some fungal species lose death of microorganisms at subzero temperatures. water as they age on a culture, even though the Annals of the New York Academy of Science 85, atmosphere is maintained at 100 % relative 610-6 29.
P. M. Robinson and R . Kennedy MAZUR, P. (1961). Manifestation of injury in yeast cells exposed to subzero temperatures. I. Morphological changes in freeze-substituted and in 'frozen-thawed' cells. Journal of Bacteriology 8z , 662-672. MAzUR, P . ( 1963). Studies on rapidly frozen suspensions of yeast cells by differential thermal analysis and conductometry, B iophysical Journal 3, 3 23-353 . MAzUR, P . (1967). Physical chemical basis of injury from intracellular freezing in yeast. In Cellular Injury and Resistance in Freezing Organisms (ed. E. Asahina), pp . 171-189. Japan : Institute of Low T emperature Science, Hokkaido University. MAzUR, P. (1968). Survival of fungi after freezing and desiccation . In The Fungi, vol. III (ed. G . C. Ainsworth & A. S. Sus sman ), pp . 325-394. London : Academic Pre ss. OGUNSANYA, O. C. & MADELlN, M . F . (1977). Sensitivity of Botryodiplodia ricinicola conidia to mild chilling. Transactions of the British Myc ological Society 69, 191-195· PARK, D. & ROBINSON, P. M. (1969). Sporulation in
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Georrichum candidum. Transactions of the British My cological S ociety 52, 213-222. PARKER, R. E . ( 1979). Introductory Statisticsfor B iology . Studies in Biology, no. 43. London : Edward Arnold. ROBINSON , P. M . (1978). Phase transformations. In Practical Fungal Physiology, pp. 53-55 . New York : Wile y & Sons. ROBINSON, P . M. (1980). Autotropisrn in germinating arthrospores of Geotrichum candidum. Transactions of the British Mycological Society 75,151-153 . SOUZU, H ., NEI, T. & BITo, M . ( 1961). Wat er of microorganisms and its freezing with special reference to the relation between water content and viability of yeast and coli cells. Low Temperature S cience Bt9, 4~57 ·
SUTCLIFFE, J. ( 1977). Effects of mechanical stress. In Plants and Temperature. Studies in Biology, no. 86, pp . 45-46. London : Edward Arnold. WOOD, T. H. & ROSENBERG, A. M . (1957). Freezing in yeast cells. Biochimica et Biophysi ca Acta z5 , 78-87.
(R eceived for publication 15 September 1983)