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Effects of cryopreservation methods in liquid nitrogen on viability of Puccinia abrupta var. partheniicola urediniospores
Mycol. Res. 96 (6): 473-476 (1992)
473
Printed in Great Britain
Effects of cryopreservation methods in liquid nitrogen on viability of Puccinia abrupta var. partheniicola urediniospores
A. N. G. HOLDEN International Institute of Biological Control, Silwood Park, Buckhurst Road, Ascot, Berkshire SL5 7TA, UK
D.SMITH International Mycological Institute, Ferry Lane, Kew, Surrey TW9 3AF, UK
Viability and infectivity of dry harvested spores of Puccinia abrupta var. partheniicola were successfully preserved by cooling to and storage at ca - 190°C. Spores remained viable for 32 d following thawing and were able to cause normal infections. The fungus as infected tissue supporting spores was less readily preserved. When the cryoprotectants glycerol, dimethyl sulphoxide, trehalose and polyvinyl pyrrolidone were added prior to freezing, both viability and infectivity were reduced. The ability to cryopreserve this fungus and to retain infectivity for long periods after thawing enhance its use as a biocontrol agent of Parthenium hysterophorus, parthenium weed, for transit to recipient areas.
The effects of cooling rate and addition of various recognized cryoprotectants were studied for the cryopreservation of urediniospores of the rust Puccinia abrupta Diet. & Holw. var. partheniicola Oackson) Parmelee. Cryopreservation in liquid nitrogen was preferred to storage by freeze-drying. Preservation by the latter proved to be inadequate, as spore germination on recovery was less than 10 %. The host specificity of this rust has recently been screened at JIBC prior to its release in Queensland as part of the biological control programme for Parthenium hysterophorus L. (parthenium weed). This study was instigated to establish a method for the preservation of the spores prior to their shipment to Australia, requiring the viability and infectivity of the spores to be maintained for 8-10 d after thawing from storage.
METHODS AND MATERIALS Collection of spores Urediniospores of Puccinia abrupta var. partheniicola 1M! 348242 (JIBC isolate 3), were collected into 1'5 ml (12'5 x 44 mm) sterile polypropylene cryogenic vials from leaves of artificially inoculated, glasshouse-grown Parthenium hysterophorus. Collections were made 21 d after inoculation either as spores free of leaf material, by their suction directly from the leaf, or as spores on leaf material, by cutting four to five 1 mm 2 leaf sections bearing pustules.
Trial 1 Prior to freezing, cryoprotectant was added to vials containing the fungus in O· 5 ml aliquots, agitated and allowed to
equilibrate at 20°C for 1 h. An equal number of vials to which glycerol was not added were frozen at the same time.
Cooling to - 196°C The vials were cooled at one of five different rates. For cooling rates of 0'5° min-I, 1° min-I, 10° min- 1 and 30° min- 1 the vials were placed in the chamber of a Kryo 16 Series JI controlled rate cooler. For a cooling rate of ca 200° min- 1 vials were plunged directly into liquid nitrogen. All tubes were cooled rapidly to -196° with the rate of cooling determined from the time required to cool between ambient and - 40°. Vials were frozen to allow three replicates of each treatment to be sampled at each cooling rate at each sampling time after thawing. Frozen vials were stored in a 350 1 LNR storage vessel in a metal drawer-rack inventory-control system partially immersed in liquid nitrogen. The vials were kept in the vapour phase throughout the storage period, although contact of the drawer racks with the liquid phase ensured a storage temperature ca -190°. The cultures were thawed after three months by rapid agitation of the vials in a water bath at 35° until the last crystals of ice had melted.
Trial 2 Spores on or free of leaf tissue were collected into cryogenic vials as described for trial 1. Equal numbers of vials were treated with one of PVP (polyvinyl pyrrolidone), DMSO (dimethylsulphoxide), or trehalose as cryoprotectants added as solutions (10 % v Iv distilled water). An equal number of vials were frozen without the addition of cryoprotectant.
474
Cryopreservation of Puccinia abrupta The procedure for the subsequent cooling of vials at one of three rates (0'5° min-I, 10° min-lor 200° min-I) were as described for trial 1.
Assessment of viability Viability was assessed on thawing and at various times thereafter. Viability was determined by inoculation of a spore suspension (distilled water 0'01 % Tween 20) on to leaves of Parthenium hysterophorus with a fine hair brush. Two leaves were inoculated per replicate sample. Following inoculation, the plants were placed into a dew chamber at 100 % r.h. After 48 h the plants were moved into a plant growth chamber and maintained at 17° for 21 d. During this time the inoculated leaves were monitored for development of symptoms of infection. Viability was recorded as percentage germination and assessed by microscopic examination of leaves sampled, cleared and stained 8 d after inoculation according to the methods of Bruzzese & Hasan (1986). The percentage germination of fresh spores was determined when the cryopreservation vials were prepared, and a base of 100 % germination was established. The actual percentage germination of preserved spores was corrected accordingly. Prior to inoculation spore material was cleared of cryoprotectant by centrifugation, removal of supernatant and resuspension in sterile distilled water, a process that was repeated four times for each sample. Samples without cryoprotectant were treated similarly.
Cryogenic light microscopy Urediniospores were observed during freezing and thawing on a light microscope with a cryogenic stage (CM3; Planer Products Ltd). An Amstrad PC 164050 was used for temperature control of the stage heater. They were cooled from 20 to 5° at a rate of roO min-I, held at 5° for 0'5 min and then cooled at 5, 30 and 100° min- I to -50°.
germination was poorest for spores cooled at the 10° min- I rate, while cooling at a rate of ca 200° min- I gave the highest viability on thawing. In no collection either on or free of leaf material did viability fall in the first two days after thawing. The extent of any recovery in spore viability in the period after thawing, however, appeared to be dependent, not only on the rate at which the material was cooled, but also on the method by which the spores were collected. From day 2 the method of collection markedly affected percentage germination for the spores frozen at 30 min- I and the increase observed in the collection of spores was not apparent in the collection including leaf material, for which viability rapidly declined. Assessment of viability for spores frozen on leaf material was not undertaken beyond 8 d after thawing because of decay of the samples and overgrowth by fungal contaminants. The addition of glycerol further reduced viability (Fig. 1 b), such that for spores frozen on host leaves the highest recovery on thawing, shown by spores frozen at 0'5° min-I, was only 18 %. Viability on thawing was lowest for spores cooled at 30° min-I. The addition of glycerol to spores collected separately from the host material similarly did not improve preservation (Fig. 1 b). The addition of glycerol improved preservation at the two slower cooling rates in relation to the faster rates, recovery rising to over 40 % by day 4 for spores cooled at a rate of 0'5° min-I. In contrast to the results from collections to which no cryoprotectant was added, the faster rates, particularly 200° min-I, significantly impaired preservation. None of the cryoprotectants tested enhanced recovery of viable spores in either the separated collections or those including host material above that of the control samples (Figs 100 ~
~
~--------------------,
(a)
80
RESUL TS AND DISCUSSION Trial 1 Preservation of spores separated from the host material gave high recovery (Fig. 1 a) and retained infectivity, and 2 dafter thawing spore viability in these collections was greater than 75 % irrespective of cooling rate. The highest viability on thawing was observed for spores frozen at the fastest rate (ca 200° min-I). However, the maximum recovery - of 95% germination - was recorded 4 d after thawing (Fig. 1 b). Spores frozen at 10° min- I did not show the same increase; however, high sustained recoveries were observed for spores frozen at each of the three faster cooling rates, such that 72 d after thawing germination of the spores frozen at these rates remained over 80%. Cooling at a rate of 1° min- I was the least effective preparation for storage. Preservation of the spores on the host leaf appeared to be detrimental to spore survival and infectivity (Fig. 1 a). For each of the cooling rates tested, germination on thawing was below 45 % for each of these collections. Throughout the trial
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12345678910111213141516171819 Day Fig. 1. Trial I, Puccinia abrupta var. partheniicola urediniospore viability with time after recovery from cryopreservation. (a) Germination without glycerol. (b) Germination with glycerol. Freezing rate (OC min-I) as spores: - , 0'5; -*-, 1'0; -0-, 10'0; -0-,30; -f:::,,-, 200. Freezing rate on leaves: ---, 0'5; --*--, 1'0; --0--, 10'0; --0--, 30; --f:::,,--, 200.
A. N. G. Holden and D. Smith
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Fig. 2. Trial 2, Puccinia abrupta var. partheniicola urediniospore
viability with time after recovery from cryopreservation. (a) Germination without cryoprotectant. (b) Germination with DMSO. (c) Germination with trehalose. (d) Germination with PVP. Freezing rate (OC min-I) as spores: -, 0'5; - 0 - , 10'0; -h,-, 200. Freezing rate on leaves: --. 0'5; -0-,10'0; -h,-. 200.
2 a-d). As in trial I, the preservation of spores on leaf material gave lower recoveries than did the collection and freezing of spores separated from the host material. In general viability appeared to be highest between I and 3 d after thawing. Of the cryoprotectants tested, DMSO gave the highest initial recovery of viable, infective spores (Fig. 2 b), but this represented only 40 % of the unfrozen control sample. Although viability increased with time after thawing in samples to which no cryoprotectant was added, in both the DMSO- and trehalose-treated collections the trend was reversed and viability decreased by over 50% in both treatments during the first 4 d after thawing (Fig. 2 b, c). PVP was the least effective of the cryoprotectants tested, with less than 20 % germination irrespective of the method of spore collection (Fig. 2d). It was noticeable that many PVPtreated spores showed signs of leakage of the cell contents through broken cell walls. The few spores that germinated from this treatment characteristically produced deformed germ-tubes, and very few appressoria were observed for these samples during the trials. Significantly, the only inoculations not to produce any symptoms were from PVP-treated spore collections. The extent of pustule development on the host plants was, not surprisingly, very closely related to the viability of the spores as determined by the clearing and staining of the leaves. The monitoring of the plants for symptoms was, however, a valuable verification of the germination data, and did confirm that the ability of the spores to infect the host had not been adversely affected in a way that might not have been detected merely by assessing viability. The spores separated from host material cooled to - 1960 and stored at ca - 1960 gave the highest recovery and infection. Optimum cooling rates appeared to be between 10 and ca 200 0 min-I. The addition of cryoprotectant reduced both viability and infectivity of the spores and leaf infections after freezing and thawing. In many cases a recovery period following thawing was required before optimum recovery and infection were observed. This was particularly apparent following cooling without cryoprotectant. Following this initial recovery period, which probably allowed the repair of damage sustained during freezing and thawing, there was a fall in viability, although this remained at over 80 % in some cases. This recovery phase was less apparent in the presence of cryoprotectant and was absent in the spore collections treated with trehalose. Of the cryoprotectants tested here, DMSO was clearly superior in preserving viability and infectiVity at both slow and fast cooling rates. Glycerol gave better protection at the slower rates of cooling (0'5 0 min-I) than at the faster rates. PVP did not give adequate protection at any of the cooling rates. DMSO is able to penetrate the cell much more rapidly than glycerol and trehalose (Morris, 1976). This contrasts with the mechanism by which protection is conferred by PVP, which is unable to penetrate the cell and normally protects by causing exosmosis of water from the cell, thereby reducing the amount of water available to form ice on freezing (AshwoodSmith & Warby, 1971). The results of these trials clearly demonstrate that the cooling of harvested spores at rates between 10 and ca 200° min- 1 gives optimum survival on
Cryopreservation of Puccinia abrupta thawing, and that infectivity thereafter will remain high for at least 32 d. Although viabilities were checked after only 3 months' storage, Smith (1988) had previously shown that cells once frozen and stored in liquid nitrogen remain viable for extremely long periods. Observations made on the cryogenic light microscope revealed that fast rates of cooling induced intracellular ice formation, but this did not seem to cause rupture of intact urediniospores. However, many did rupture as a result of ice formation. These may have been damaged in some way during harvesting. At slow rates of cooling the spores did not shrink to any great extent. The immature urediniospores shrank more and suffered more observable damage than the mature ones. The addition of a cyroprotectant depressed the freeZing point of the cytoplasm. Cryomicroscopy did show that intracellular ice not resulting in cell rupture was not lethal. Cells frozen in a dry condition would not be exposed to osmotic stress, whereas those frozen in aqueous solutions of cryoprotective agents would. It is this stress rather than ice formation which causes death of the urediniospore. Similar effects on fungal mycelium have been reported (Smith, Coulson & Morris, 1986; Morris, Smith & Coulson, 1988). The increased interest in fungi, particularly the rust fungi, as classical biological control agents necessitates the development of practical and effective methods of storing those organisms. Frequently time or space constraints restrict the amount of fresh inoculum that can be maintained at a given time. A further constraint may arise from a delay between the conclusion of the required host-range screening as part of the quarantine regulations and the granting of permission to import. Whatever the method of storage, viability and infectivity must be sustained not only during storage but also for a period thereafter, to accommodate any delay during the shipment of the organism from the donor to recipient area. Freeze-drying has proved unsatisfactory, and germination was less than 10 % for P. abrupta spores preserved with or without (Accepted 27 December 1991)
476 skimmed milk. The practical implication of the results presented here for the storage of organisms and the subsequent viability are clear. The collection of spores including leaf material, although easier, may severely restrict subsequent storage potential. Similarly the addition of cryoprotectant may be superfluous or indeed detrimental to the viability of the spores after storage. The authors acknowledge the financial assistance of the Queensland Department of Lands in funding the parthenium weed biological control programme, and Mr A. Tomley for supplying parthenium seed. We also acknowledge the help of Mr J. Bayle in growing and maintaining the plants at IIBC and Miss V. Thomas for the study on the cryogenic stage of the light microscope. REFERENCES Ashwood-Smith, M. J. & Warby, C. (1971). Studies on the molecular weight and cryoprotective properties of PVP and Dextran with bacteria and erythrocytes. Cryobiology 8, 453-464. Bruzzese, E. & Hasan, S. (1986). The collection and selection in Europe of isolates of Phragmidium violaceum (Uredinales) pathogenic to species of European blackberry naturalised in Australia. AnlUlls of Applied Biology 108, 527-533.
Morris, G.). (1976). The cryopreservation of Chlorella L Interactions of the cooling rate, protective additive and warming rate. Archives of Microbiology 107,57-62.
Morris, G. J., Smith, D. & Coulson, G. E. (1988). A comparative study of the changes in the morphology of hyphae during freezing and viability upon thawing for twenty species of fungi. Journal of General Microbiology 134, 2897-2906.
Smith, D. (I988). Culture and preservation. In Living Resources for Biotechnology: Filamentous Fungi (ed. D. L. Hawksworth & B. E. Kiesop), pp. 75-99. Cambridge University Press: Cambridge, U.K. Smith, D.. Coulson, G. E. & Morris, G. J. (1986). A comparative study of the morphology and viability of Penicillium expansum and Phytophthora nicotianae during freezing and thawing. Journal of General Microbiology 132, 2013-2021.
473
Printed in Great Britain
Effects of cryopreservation methods in liquid nitrogen on viability of Puccinia abrupta var. partheniicola urediniospores
A. N. G. HOLDEN International Institute of Biological Control, Silwood Park, Buckhurst Road, Ascot, Berkshire SL5 7TA, UK
D.SMITH International Mycological Institute, Ferry Lane, Kew, Surrey TW9 3AF, UK
Viability and infectivity of dry harvested spores of Puccinia abrupta var. partheniicola were successfully preserved by cooling to and storage at ca - 190°C. Spores remained viable for 32 d following thawing and were able to cause normal infections. The fungus as infected tissue supporting spores was less readily preserved. When the cryoprotectants glycerol, dimethyl sulphoxide, trehalose and polyvinyl pyrrolidone were added prior to freezing, both viability and infectivity were reduced. The ability to cryopreserve this fungus and to retain infectivity for long periods after thawing enhance its use as a biocontrol agent of Parthenium hysterophorus, parthenium weed, for transit to recipient areas.
The effects of cooling rate and addition of various recognized cryoprotectants were studied for the cryopreservation of urediniospores of the rust Puccinia abrupta Diet. & Holw. var. partheniicola Oackson) Parmelee. Cryopreservation in liquid nitrogen was preferred to storage by freeze-drying. Preservation by the latter proved to be inadequate, as spore germination on recovery was less than 10 %. The host specificity of this rust has recently been screened at JIBC prior to its release in Queensland as part of the biological control programme for Parthenium hysterophorus L. (parthenium weed). This study was instigated to establish a method for the preservation of the spores prior to their shipment to Australia, requiring the viability and infectivity of the spores to be maintained for 8-10 d after thawing from storage.
METHODS AND MATERIALS Collection of spores Urediniospores of Puccinia abrupta var. partheniicola 1M! 348242 (JIBC isolate 3), were collected into 1'5 ml (12'5 x 44 mm) sterile polypropylene cryogenic vials from leaves of artificially inoculated, glasshouse-grown Parthenium hysterophorus. Collections were made 21 d after inoculation either as spores free of leaf material, by their suction directly from the leaf, or as spores on leaf material, by cutting four to five 1 mm 2 leaf sections bearing pustules.
Trial 1 Prior to freezing, cryoprotectant was added to vials containing the fungus in O· 5 ml aliquots, agitated and allowed to
equilibrate at 20°C for 1 h. An equal number of vials to which glycerol was not added were frozen at the same time.
Cooling to - 196°C The vials were cooled at one of five different rates. For cooling rates of 0'5° min-I, 1° min-I, 10° min- 1 and 30° min- 1 the vials were placed in the chamber of a Kryo 16 Series JI controlled rate cooler. For a cooling rate of ca 200° min- 1 vials were plunged directly into liquid nitrogen. All tubes were cooled rapidly to -196° with the rate of cooling determined from the time required to cool between ambient and - 40°. Vials were frozen to allow three replicates of each treatment to be sampled at each cooling rate at each sampling time after thawing. Frozen vials were stored in a 350 1 LNR storage vessel in a metal drawer-rack inventory-control system partially immersed in liquid nitrogen. The vials were kept in the vapour phase throughout the storage period, although contact of the drawer racks with the liquid phase ensured a storage temperature ca -190°. The cultures were thawed after three months by rapid agitation of the vials in a water bath at 35° until the last crystals of ice had melted.
Trial 2 Spores on or free of leaf tissue were collected into cryogenic vials as described for trial 1. Equal numbers of vials were treated with one of PVP (polyvinyl pyrrolidone), DMSO (dimethylsulphoxide), or trehalose as cryoprotectants added as solutions (10 % v Iv distilled water). An equal number of vials were frozen without the addition of cryoprotectant.
474
Cryopreservation of Puccinia abrupta The procedure for the subsequent cooling of vials at one of three rates (0'5° min-I, 10° min-lor 200° min-I) were as described for trial 1.
Assessment of viability Viability was assessed on thawing and at various times thereafter. Viability was determined by inoculation of a spore suspension (distilled water 0'01 % Tween 20) on to leaves of Parthenium hysterophorus with a fine hair brush. Two leaves were inoculated per replicate sample. Following inoculation, the plants were placed into a dew chamber at 100 % r.h. After 48 h the plants were moved into a plant growth chamber and maintained at 17° for 21 d. During this time the inoculated leaves were monitored for development of symptoms of infection. Viability was recorded as percentage germination and assessed by microscopic examination of leaves sampled, cleared and stained 8 d after inoculation according to the methods of Bruzzese & Hasan (1986). The percentage germination of fresh spores was determined when the cryopreservation vials were prepared, and a base of 100 % germination was established. The actual percentage germination of preserved spores was corrected accordingly. Prior to inoculation spore material was cleared of cryoprotectant by centrifugation, removal of supernatant and resuspension in sterile distilled water, a process that was repeated four times for each sample. Samples without cryoprotectant were treated similarly.
Cryogenic light microscopy Urediniospores were observed during freezing and thawing on a light microscope with a cryogenic stage (CM3; Planer Products Ltd). An Amstrad PC 164050 was used for temperature control of the stage heater. They were cooled from 20 to 5° at a rate of roO min-I, held at 5° for 0'5 min and then cooled at 5, 30 and 100° min- I to -50°.
germination was poorest for spores cooled at the 10° min- I rate, while cooling at a rate of ca 200° min- I gave the highest viability on thawing. In no collection either on or free of leaf material did viability fall in the first two days after thawing. The extent of any recovery in spore viability in the period after thawing, however, appeared to be dependent, not only on the rate at which the material was cooled, but also on the method by which the spores were collected. From day 2 the method of collection markedly affected percentage germination for the spores frozen at 30 min- I and the increase observed in the collection of spores was not apparent in the collection including leaf material, for which viability rapidly declined. Assessment of viability for spores frozen on leaf material was not undertaken beyond 8 d after thawing because of decay of the samples and overgrowth by fungal contaminants. The addition of glycerol further reduced viability (Fig. 1 b), such that for spores frozen on host leaves the highest recovery on thawing, shown by spores frozen at 0'5° min-I, was only 18 %. Viability on thawing was lowest for spores cooled at 30° min-I. The addition of glycerol to spores collected separately from the host material similarly did not improve preservation (Fig. 1 b). The addition of glycerol improved preservation at the two slower cooling rates in relation to the faster rates, recovery rising to over 40 % by day 4 for spores cooled at a rate of 0'5° min-I. In contrast to the results from collections to which no cryoprotectant was added, the faster rates, particularly 200° min-I, significantly impaired preservation. None of the cryoprotectants tested enhanced recovery of viable spores in either the separated collections or those including host material above that of the control samples (Figs 100 ~
~
~--------------------,
(a)
80
RESUL TS AND DISCUSSION Trial 1 Preservation of spores separated from the host material gave high recovery (Fig. 1 a) and retained infectivity, and 2 dafter thawing spore viability in these collections was greater than 75 % irrespective of cooling rate. The highest viability on thawing was observed for spores frozen at the fastest rate (ca 200° min-I). However, the maximum recovery - of 95% germination - was recorded 4 d after thawing (Fig. 1 b). Spores frozen at 10° min- I did not show the same increase; however, high sustained recoveries were observed for spores frozen at each of the three faster cooling rates, such that 72 d after thawing germination of the spores frozen at these rates remained over 80%. Cooling at a rate of 1° min- I was the least effective preparation for storage. Preservation of the spores on the host leaf appeared to be detrimental to spore survival and infectivity (Fig. 1 a). For each of the cooling rates tested, germination on thawing was below 45 % for each of these collections. Throughout the trial
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12345678910111213141516171819 Day Fig. 1. Trial I, Puccinia abrupta var. partheniicola urediniospore viability with time after recovery from cryopreservation. (a) Germination without glycerol. (b) Germination with glycerol. Freezing rate (OC min-I) as spores: - , 0'5; -*-, 1'0; -0-, 10'0; -0-,30; -f:::,,-, 200. Freezing rate on leaves: ---, 0'5; --*--, 1'0; --0--, 10'0; --0--, 30; --f:::,,--, 200.
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Fig. 2. Trial 2, Puccinia abrupta var. partheniicola urediniospore
viability with time after recovery from cryopreservation. (a) Germination without cryoprotectant. (b) Germination with DMSO. (c) Germination with trehalose. (d) Germination with PVP. Freezing rate (OC min-I) as spores: -, 0'5; - 0 - , 10'0; -h,-, 200. Freezing rate on leaves: --. 0'5; -0-,10'0; -h,-. 200.
2 a-d). As in trial I, the preservation of spores on leaf material gave lower recoveries than did the collection and freezing of spores separated from the host material. In general viability appeared to be highest between I and 3 d after thawing. Of the cryoprotectants tested, DMSO gave the highest initial recovery of viable, infective spores (Fig. 2 b), but this represented only 40 % of the unfrozen control sample. Although viability increased with time after thawing in samples to which no cryoprotectant was added, in both the DMSO- and trehalose-treated collections the trend was reversed and viability decreased by over 50% in both treatments during the first 4 d after thawing (Fig. 2 b, c). PVP was the least effective of the cryoprotectants tested, with less than 20 % germination irrespective of the method of spore collection (Fig. 2d). It was noticeable that many PVPtreated spores showed signs of leakage of the cell contents through broken cell walls. The few spores that germinated from this treatment characteristically produced deformed germ-tubes, and very few appressoria were observed for these samples during the trials. Significantly, the only inoculations not to produce any symptoms were from PVP-treated spore collections. The extent of pustule development on the host plants was, not surprisingly, very closely related to the viability of the spores as determined by the clearing and staining of the leaves. The monitoring of the plants for symptoms was, however, a valuable verification of the germination data, and did confirm that the ability of the spores to infect the host had not been adversely affected in a way that might not have been detected merely by assessing viability. The spores separated from host material cooled to - 1960 and stored at ca - 1960 gave the highest recovery and infection. Optimum cooling rates appeared to be between 10 and ca 200 0 min-I. The addition of cryoprotectant reduced both viability and infectivity of the spores and leaf infections after freezing and thawing. In many cases a recovery period following thawing was required before optimum recovery and infection were observed. This was particularly apparent following cooling without cryoprotectant. Following this initial recovery period, which probably allowed the repair of damage sustained during freezing and thawing, there was a fall in viability, although this remained at over 80 % in some cases. This recovery phase was less apparent in the presence of cryoprotectant and was absent in the spore collections treated with trehalose. Of the cryoprotectants tested here, DMSO was clearly superior in preserving viability and infectiVity at both slow and fast cooling rates. Glycerol gave better protection at the slower rates of cooling (0'5 0 min-I) than at the faster rates. PVP did not give adequate protection at any of the cooling rates. DMSO is able to penetrate the cell much more rapidly than glycerol and trehalose (Morris, 1976). This contrasts with the mechanism by which protection is conferred by PVP, which is unable to penetrate the cell and normally protects by causing exosmosis of water from the cell, thereby reducing the amount of water available to form ice on freezing (AshwoodSmith & Warby, 1971). The results of these trials clearly demonstrate that the cooling of harvested spores at rates between 10 and ca 200° min- 1 gives optimum survival on
Cryopreservation of Puccinia abrupta thawing, and that infectivity thereafter will remain high for at least 32 d. Although viabilities were checked after only 3 months' storage, Smith (1988) had previously shown that cells once frozen and stored in liquid nitrogen remain viable for extremely long periods. Observations made on the cryogenic light microscope revealed that fast rates of cooling induced intracellular ice formation, but this did not seem to cause rupture of intact urediniospores. However, many did rupture as a result of ice formation. These may have been damaged in some way during harvesting. At slow rates of cooling the spores did not shrink to any great extent. The immature urediniospores shrank more and suffered more observable damage than the mature ones. The addition of a cyroprotectant depressed the freeZing point of the cytoplasm. Cryomicroscopy did show that intracellular ice not resulting in cell rupture was not lethal. Cells frozen in a dry condition would not be exposed to osmotic stress, whereas those frozen in aqueous solutions of cryoprotective agents would. It is this stress rather than ice formation which causes death of the urediniospore. Similar effects on fungal mycelium have been reported (Smith, Coulson & Morris, 1986; Morris, Smith & Coulson, 1988). The increased interest in fungi, particularly the rust fungi, as classical biological control agents necessitates the development of practical and effective methods of storing those organisms. Frequently time or space constraints restrict the amount of fresh inoculum that can be maintained at a given time. A further constraint may arise from a delay between the conclusion of the required host-range screening as part of the quarantine regulations and the granting of permission to import. Whatever the method of storage, viability and infectivity must be sustained not only during storage but also for a period thereafter, to accommodate any delay during the shipment of the organism from the donor to recipient area. Freeze-drying has proved unsatisfactory, and germination was less than 10 % for P. abrupta spores preserved with or without (Accepted 27 December 1991)
476 skimmed milk. The practical implication of the results presented here for the storage of organisms and the subsequent viability are clear. The collection of spores including leaf material, although easier, may severely restrict subsequent storage potential. Similarly the addition of cryoprotectant may be superfluous or indeed detrimental to the viability of the spores after storage. The authors acknowledge the financial assistance of the Queensland Department of Lands in funding the parthenium weed biological control programme, and Mr A. Tomley for supplying parthenium seed. We also acknowledge the help of Mr J. Bayle in growing and maintaining the plants at IIBC and Miss V. Thomas for the study on the cryogenic stage of the light microscope. REFERENCES Ashwood-Smith, M. J. & Warby, C. (1971). Studies on the molecular weight and cryoprotective properties of PVP and Dextran with bacteria and erythrocytes. Cryobiology 8, 453-464. Bruzzese, E. & Hasan, S. (1986). The collection and selection in Europe of isolates of Phragmidium violaceum (Uredinales) pathogenic to species of European blackberry naturalised in Australia. AnlUlls of Applied Biology 108, 527-533.
Morris, G.). (1976). The cryopreservation of Chlorella L Interactions of the cooling rate, protective additive and warming rate. Archives of Microbiology 107,57-62.
Morris, G. J., Smith, D. & Coulson, G. E. (1988). A comparative study of the changes in the morphology of hyphae during freezing and viability upon thawing for twenty species of fungi. Journal of General Microbiology 134, 2897-2906.
Smith, D. (I988). Culture and preservation. In Living Resources for Biotechnology: Filamentous Fungi (ed. D. L. Hawksworth & B. E. Kiesop), pp. 75-99. Cambridge University Press: Cambridge, U.K. Smith, D.. Coulson, G. E. & Morris, G. J. (1986). A comparative study of the morphology and viability of Penicillium expansum and Phytophthora nicotianae during freezing and thawing. Journal of General Microbiology 132, 2013-2021.