Resistance of certain Basidiomycetes to freezing

Notes and brief articles

554 REFEREN CE S

COGGINS, C. R . ( 1980). Decay of Timb er in B ui/dings. Dry R oc, We e R oc and Ocher Fungi. East G rins tea d , U .K. : Rentokil, SEGMULLER, J. & WALCHLI, O. (1975). M on ograph ic info rmat ion on S erpula (M eru/ius) lacrymans (Sch um . ex Fr.) S. F . Gray accord ing to the ' M odel Questionnai re for Preparation of M onographic Cards for

Woo d-Des troy ing F ungi ' . The Interna tional Research Group on Wood Preserva tio n: Work ing G roup 1, Biological Pr oblems. D ocum ent N o. IRG/ WP /133, IRG Secre tariat , Stockh olm . WALTERS, N . E . M . (1972). Ca se histories of th e dry rot fun gus Se rpula lacrymans Gray. CSI R O ForeseP roduces Ne uisletter, no. 387,4- 7. WALTERS, N . E. M . (1973). Au stralian hou se fungi. CS IRO Forese Pr oduces Tech nical Notes, no. 13, 1- 25.

A SIMPLE TECHNIQUE FOR STUDYING I N S I T U DE VELOPMENT OF ZYGOSPORES B Y PEN ELOPE J. ANSE LL Department of B iological S ciences, Chelsea College, University of London, Horten sia Road, London SWlO oQR

Although many techniques have been described for observing fungal structures (Booth, 1971; Cole & Kendrick, 1968; Cole, Nag Raj & Kendrick, 1969; Fletcher, 1976), the following was found to be particularly useful in observing zygospore development ofthe heterothallic fungus , M ortierella indohii Ch ien . The apparatus may be set up in 5-10 min , the fungu s receives adequate aeration and the agar does not dr y out rapidly. Molten agar, Czapek-Dox, was poured into a Petri dish to a depth of 0'5-1 ern, and allowed to set. A channel approximately 8' S x 1 ern was cut in the agar. Us ing sterile forceps a NO.1 coverslip , 2 x ~ in (Baird & Tatlock, London Ltd) was sterilized in ethanol and either allowed to dr y in air or flamed, th en dipped into a beaker containing sterile molten agar. The coverslip was rem oved and the agar allowed to set in air for about 1 min then placed over the channel with the long edge parallel to th e channel. An inoculum plug of each stra in was placed either on the agar in the plate ad jacent to the coverslip or on the coverslip itself, and the lid placed on the plate . When hypha e had extended to th e agar on the coverslip, the coverslip was gently lifted and inverted, so that fungal growth was on the lower surface of the coverslip. Agar on the top surfac e was then scraped off and the exposed glass was wiped clean in preparation for observation and photograph y. M. indohii zygospores were pr oduced after 4-5 day s. When continuous microscopi c

observations were made over several days, with the lid off, a dr op of 20 % glycerol was pipetted into the channel to prevent desiccation. Covers lips longer than 2 in were impractical as they often cracked or shattered when sterilized or when coated with agar. High-power observation s could be made easily as the lower surface of the agar was well with in the focal length of both x 40 and x 100 objective lenses. This rap id and simple technique could probably be used to obser ve th e submerged, surface and low growing aerial structures of other fungi. I would like to thank Dr T . W. K. Young for his helpful advice.

R E F ERE NCES

BOOTH, C. ( 1971). Introduct ion to general meth ods. In Methods in M icrobiology , vol. rvr ed. C. Booth), pp . 1-47. Lon don , U .K . : Academic Press. COLE, G . T ., & KENDRICK, W. B. (1968). A th in culture chamber for time-lapse photomicrography of fung i at high magnification s. Mycologia 60, 340-344. COLE, G . T ., N AG RAJ, T . R. & KENDRICK, W. B. (1969). A simple techn iq ue for time-lapse photograph y of microfungi in plate culture. Mycologic 61, 72fr-730. FLETCHER, J. (1976). Construction and use of a windowed Petri-dish for continuous observation and ph otography of subme rged fun gal structures. Transactions of the Brit ish M y cological Society 66, 367-369.

RESISTANCE OF CERTAIN BASIDIOMYCETES TO FREEZING B Y C. T . INGOLD I I

Buckner's Close, B enson, Oxf ord OX9 6LR

Onl y a few of the larger Basidiomycetes have sporophores that are active in th e depth of wint er. N otable among these is the agari c Fla mmulina Tr ans .

s-. mycol . So c.

79 (3), (1982)

uelutipes (Fr .) Karst. (I ngold, 1981). Further, fruit-bodies of the gelatinous fungi (Auriculariales, Tremellales and Dac rymycetales ) and some Aph yl-

P rim ed in Great B ritain

Notes and brief articles Table

Species Auricularia mesenterica A. auricula-judae

Exidia glandulosa Stereum hirsutum Coriolus versicolor

1.

555

Rate of spore discharge in certain fungi before and after freezing

Mean hourly rate of discharge at room temperature for the 12 h period before freezing ( x 10- 3 )

733 39 8 239 10500 6260

Mean hourly rate of discharge at room temperature for successive 12 h periods following the period of freezing (x 10- 3 )

Days at -18±2°

o-12h

12-24 h

24-36 h

5 3 3

26 32 10 6 0

550 43 42 26 0

890 99 113 100 0

5 3

370

o

1100

o

-, Not determined.

lophorales (e.g. Stereum spp., Phlebia merismoides Fr., Coriolus versicolor (Fr.) Quel.) can also winter successfully. Generally these fungi can be dried, and on re-wetting soon liberate spores again. No doubt any species that can endure drying can also withstand temperatures considerably below zero in the dry condition. However, a successful winter fungus must be able to survive freezing in the fully hydrated state. In connexion with a winter fungus, a distinction should be made between one in which the sporophores persist during the cold period having been formed under milder conditions earlier in the year (e.g. Auricularia spp., Calocera spp. and Stereum spp.), and one, especially F. uelutipes, in which new sporophores continue to be formed throughout the winter. There are also some agarics which may be found at any time of year. A common example is Tubariafurfuracea (Pers. ex Fr.) Gillet which, during a brief mild spell in mid-winter, can rapidly develop a crop of sporophores. This note reports some observations on the ability of certain fungi to liberate spores after having been frozen. In the active state the bracket sporophore of Auricularia mesenterica (S. F. Gray) Pers. is a greatly hydrated jelly. On drying it becomes shrivelled and horny, and spore discharge ceases. On re-wetting it quickly takes up water and discharge soon starts again (Buller, 1922). The cycle of drying and wetting can be repeated many times without impairing spore liberation in the wet periods (Rockett & Kramer, 1974). In an experiment with A. mesenterica, a fully hydrated specimen collected from an elm stump in January 1981 was wired, through two minute holes in the plastic, to the lid of a Petri-dish which was then placed over a crystallizing dish (8 em diam x 4 ern). Any discharged spores fell into the dish. When appropriate these were suspended Trans. Br. mycol. Soc. 79 (3), (1982)

in water and their number estimated using a haemocytometer. The sporophore used weighed 9'7 g and had an hymenial surface of 18 em", For the first 12 h period the fungus was at room temperature (14-18°C). Then, still wired to its plastic lid, it was placed in a 'deep freeze' at -18±2° for 5 days. It was then removed, when it was found to be frozen hard, and again placed at room temperature. For the next i : 5 days the mean hourly rate of spore discharge was determined over each 12 h period (Table 1). It is clear that the fungus survived freezing, and within 2 days had returned to about the original level of spore liberation. Finally the specimen was allowed to become air-dry. It then weighed 0'72 g. During the drying process spore discharge was followed qualitatively. When the weight had fallen to 0'90 g, spores were still being discharged, but at a very low rate. Similar experiments were carried out using Auricularia auricula-judae (Bull. ex St-Am.) Wetts., Stereum hirsutum (Willd. ex Fr.) S. F. Gray and Corio Iusversicolor collected in south Oxfordshire in January 1981, and also Exidia glandulosa (St-Am.) Fr. kindly supplied by Prof J. Webster from Exeter. The results are also shown in Table 1. It will be seen that, with the exception of C. versicolor, all the species tested were able to survive being frozen, although recovery in the rate of spore discharge was slow in S. hirsutum. The severe conditions of January 1982 provided an opportunity to determine if certain sporophores could survive very low temperature in the field. At Benson R.A.F. airfield the minima on 13, 14 and 15 January were respectively -13'3°, -18'7° and -16'6°. The value of -18'7° was the lowest since records began in 1939. On 18 January sporophores of Flammulina velutipes, Auricularia mesenterica and A. auricula-judae were collected within a mile of the airfield. Without further treatment they were

Printed in Great Britain

Notes and brief articles placed over glass slides at room temperature. Two hours later each had produced a den se deposit of discharged basidiospores. The capacity of gelatinous Basidiomycetes to survive freezing has already been noted. Gaurnann (1928) remarked in reference to Tremellales: 'They are very resistant to temperature changes and endure - 20°C without injury. '

REFERENCES

BULLER, A. H . R. (1922). Researches on Fungi, vol. II . London, U .K . : Longmans, Green. GAUMANN, E . A. (1928). Comparative Morph ology of Fungi (trans. C. W. Dodge). New York: McGraw-HilI. INGOLD, C. T. (1981). Flammulina uelutip es in relation to drying and freezing. Transactions of the British Myc ological S ociety 76, 150-152. ROCKETT, T. R. & KRAMER, C. L. (1974). The biology of sporulation of selected Tremellales. My cologia 66, 9 26-941.

POTENTIAL FOR LONG-TERM STORAGE OF DIPLOCARPON ROSAE CONIDIA IN LIQUID NITROGEN P. CASTLEDINE, B. W. W. GROUT AND A. V. ROBERTS

Department of Biology, North East London Polytechnic, Romford Rd, London E15 4LZ Resistance of roses to different pathogenic races of the blackspot fungus, Diplocarpon rosae (Lib.) Wolf, varies considerably (Bolton & Sveida, 1979; Svejda & Bolton, 198o). Consequently the resistance of a rose to blackspot disease can best be assessed if it is challenged with a range of distinct races of the fungus . Maintenance of these races by in vitro culture is unsuitable for long-term storage, as pathogenicity is lost (L yle, 1938; Frick, 1943; Jenkins, 1954). Storage of infected leaf material in a domestic freezer ( - 15°C) is suitable for short-term storage, but Knight & Wheeler (1978) reponed a marked reduction in germination of conidia after 48 weeks under these conditions. This effect is confirmed by unpublished results of the present authors. In the present investigation the prospect for long-term storage of conidia of D. rosae in liquid nitrogen ( - 196°) was assessed. Table 1. Germinative and infective ability of frozen and unfrozen conidia Germination Treatment Unfrozen control Conidia frozen in suspension with or without DMSO 0 % DMSO 2'5 % DMSO 5'0 % DMSO 10'0 % DMSO Conidia frozen on leaf pieces

( 'X,)

69'2 (S.E. 2'9) 42'9 (S.E. 13'2)

39'4 (S.E. 0'8) 0'0 63'8 (S.E. 1"8 13"3 (S.E. 8'8 ) 16'5 (S.E. 0'6) 6'7 (S.E. 6'5) 53'5 (S.E. 2'4) 6'7 (S.E. 6·5) 69'8 (S.E. 2'9) 92'9 (S.E. 6'9 )

* Estimates based on 5 samples of

t

Leaf disks infected']

on 2 ~/~ water agars ([/0 )

Estimates based on 15 leaf disks .

Trans . Br . my col. S oc. 79 (3), (1982)

100

conidia.

Lesions with acervuli were taken from freshly collected leaves of the rose cultivar "Frensham '. One half of each lesion was placed in a small uncapped polypropylene tube, where it was entrapped by stainless-steel gauze. The tubes were flooded with liquid nitrogen, capped and stored in liquid nitrogen for 24 h . Upon removal, the contained liquid nitrogen was poured off and the tube immediately placed in water at 40° . One minute was allowed for thawing and care was taken to ensure that water did not enter the tube. An aqueous suspension was prepared from thawed leaf pieces and adjusted to 2 X 105 conidia /mi. The ability of conidia in this suspension to germinate and to infect leaves was then tested. The remaining half-lesions were used to prepare suspensions containing 2 x 105 conidia /rnl and, respectively, 0 %, 2'5 %, 5 % and 10 % concentrations of the cryoprotectant dimethyl sulphoxide (Ashwood-Smith & Farrant, 1980). Aliquots of 1 ml were taken from each suspension and transferred to polypropylene tubes which were capped and maintained at room temperature for 30 min to allow time for the dimethyl sulphoxide (DMSO) to penetrate the conidia. The tubes were then immersed in liquid nitrogen for 24 h. To thaw, the tubes were immersed in water at 40° until all the contained ice had melted. Some of the suspension without DMSO was retained and used as unfrozen control material. The ability of conidia from thawed and control suspension to germinate and to infect leaves was then tested. Percentage germination of conidia was assessed on 2 % water agar after 24 h at room temperature (15- 20°). Estimates were based on counts of five samples, each of 100 conidia (T able 1). The ability of conidia to infect leaves was tested by the standard leaf-disk method of Knight &

Printed in Great Britain