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Enhancement of ascospore germination from Aleuria aurantia after cold storage
287 Mycoscience 41 : 287-289, 2000
Note
Enhancement of ascospore germination from Aleuria aurantia after cold storage Shigeru O g a w a , Hideyuki Nagao% A k i k a z u A n d o and Y o s h i h o Nagata ~ D e p a r t m e n t of Bioresources Chemistry, Faculty of Horticulture, Chiba University, M a t s u d o , Chiba 2 7 1 - 8 5 1 0 , Japan A c c e p t e d for publication 12 April 2 0 0 0
Ascospores of Aleuria aurantia did not germinate soon after collection. The treatment with alkali induced germination, but the rate was less than 0 . 0 1 % . After storage of the ascospore at 4 ~ or at - 2 0 ~ for three mo, germination was enhanced, and its rate was approximately 6 0 % . Key Words
A/euria aurantia; alkali t r e a t m e n t ; ascospore g e r m i n a t i o n ; cold storage; Discomycetes.
Aleuria aurantia (Fr.) Fuckel is an a s c o m y c e t e fungus, k n o w n as orange peel m u s h r o o m , t h a t belongs to P y r o n e m a t a c e a e in the class of Discomycetes. This m u s h r o o m is o f t e n observed in a u t u m n on freshly broken grounds or edges of forest roads in Japan, and k n o w n to be distributed all over the w o r l d . In the course of studies on this fungus, w e found a p h e n o m e n o n t h a t a germination rate of the ascospore from A. aurantia w a s enhanced d r a m a t i c a l l y after cold storage. Fruit bodies o f A . aurantia w e r e collected in October, 1 9 9 8 near Mt. Akagi, Gunma Pref., Japan. As s h o w n in Figs. 1 A - C , a fruit body of A. aurantia w a s thin and fragile, and c u p - t o saucer-shaped w i t h a d i a m e t e r of 2 0 100mm. H y m e n i u m w a s s m o o t h and bright redorange. Eight ascospores of 1 4 - 1 6 x 10 m m w e r e observed in an ascus. Paraphyses w e r e slight clavate w i t h thick-endings at their tips. A s c o s p o r e s w e r e hyaline and elliptical w i t h distinct coarsely reticulate o r n a m e n t a t i o n . Present address: Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba, 305-8572, Japan. ~ Corresponding author.
Table 1. Expt. No. 1 2 3 4
T w o droplets w e r e observed inside of an ascospore. For induction of ascospore g e r m i n a t i o n in some d i s c o m y c e t e fungi, a t r e a t m e n t w i t h a certain chemical reagent or w i t h heat w a s reported to be effective. In A s c o b o l u s species, the t r e a t m e n t w i t h dilute alkali f o l l o w e d by incubation at 3 7 ~ induced germination at the rate of 8 0 - 9 0 % (Yu, 1954). In A. aurantia the treatm e n t w i t h nonyl-alcohol w a s reported to induce germination, but the rate w a s only less than 1 ~ (Paden, 1972). In this study ascospores of A. aurantia w e r e collected f r o m m a t u r e apothecia puffing on a sheet of paper. A piece of this paper w a s w a s h e d in distilled w a t e r , and the suspension w a s stored at 4 ~ or at - - 2 0 ~ for 3 mo. A l i q u o t s of the suspension w e r e transferred onto p o t a t o d e x t r o s e agar (PDA)-plates (Nissui) containing 100/~g/ml of c h l o r a m p h e n i c o l (Sigma). The plates were incubated at 2 0 ~ for 4 - 7 d in the dark. A n u m b e r of germ tubes appearred w a s c o u n t e d microscopically and the rate of g e r m i n a t i o n w a s calculated. For alkaline t r e a t m e n t , the spore suspension w a s brought to 0 . 2 N of KOH and a l l o w e d to stand for 2 0 rain, after w h i c h aliquots of the suspension w e r e transferred onto PDA-plates.
Germination of ascospores of A. aurantia. ~
Treatment no 0.2 N KOH for 20 min 4~ for 3too - 2 0 ~ for 3 m o
No. of ascospores No. of ascospores Germination rate observed/plate germinated/plate (%) 3.3• 10 s 3.3• 3.0• 3.0•
0 19_+5 177_+57 183_+32
0 <0.01 59_+19 61_+11
* Spore suspension with 3.3 x 105 ascospores was spread on each plate. In Expt. 1 and 2, all the ascospores in a plate were observed, and in Expt. 3 and 4, randomly 300 ascospores/plate were observed microscopically. Plates were incubated at 20~ for 7 d (Expt. 1 and 2), and for 4 d (Expt. 3 and 4), respectively. No. of ascospores germinated/plate was the average of 5 plates.
288
S. Ogawa et al.
Fig. 1. Fruit bodies, asci and ascospores of A. aurantia. A. Fruit bodies (bar: 20mm). B. Asci (bar: 80mm). C. Ascospore (bar: 6 mm). 80 mm). E. Ascospores after cold storage (bar: 80 mm).
D. Ascospores without any treatment (bar:
Germination of ascospore after cold storage As shown in Fig. 1D and Table 1, the ascospore did not germinate soon after collection. When ascospores were treated with 0.2 N KOH for 20 min, a few germinated after 7 d of incubation but the rate of germination was very low (0.01~ On the other hand, ascospores that had been stored at 4 ~ or at - - 2 0 ~ for 3 mo, germinated after 4 d at the rate of approximately 6 0 ~ (Fig. 1E; Table 1 ). As the same rate of germination was observed after storage at 4 ~ and at - 2 0 ~ this phenomenon did not seem to be due to maturation of ascospores during storage, and the cold storage itself should be effective. The results suggest that the ascospore of A, aurantia is necessary to experience cold condition before germination, and the dormancy of the ascospore may be broken by the cold storage. Since the fruit body of this fungus was formed at the end of autumn, and Hwang (1968) noted that mycelia of some discomycete fungus were weak in freezing, our speculation seem to be reasonable
289
for these fungi that survive as ungerminated ascospores during winter. Acknowledgements--We are grateful to Drs. Y. Fujita and M. Arai, Hokuriku National Agricultural Experimental Station, for their technical assistance.
Literature cited Hwang, S. H. 1969. Investigation of ultra-low temperature for fungal cultures I. An evaluation of liquid-nitrogen storage for preservation of selected fungal cultures. Mycologia 60: 613-621. Paden, J.W. 1972. Imperfect stages and the taxonomy of Pezizales. Persoonia6: 405-414. Yu, C.C. 1954. The culture and spore germination of Ascobolus with emphasis on A. magnificus. Amer. J. Bot. 41: 21-30.
Note
Enhancement of ascospore germination from Aleuria aurantia after cold storage Shigeru O g a w a , Hideyuki Nagao% A k i k a z u A n d o and Y o s h i h o Nagata ~ D e p a r t m e n t of Bioresources Chemistry, Faculty of Horticulture, Chiba University, M a t s u d o , Chiba 2 7 1 - 8 5 1 0 , Japan A c c e p t e d for publication 12 April 2 0 0 0
Ascospores of Aleuria aurantia did not germinate soon after collection. The treatment with alkali induced germination, but the rate was less than 0 . 0 1 % . After storage of the ascospore at 4 ~ or at - 2 0 ~ for three mo, germination was enhanced, and its rate was approximately 6 0 % . Key Words
A/euria aurantia; alkali t r e a t m e n t ; ascospore g e r m i n a t i o n ; cold storage; Discomycetes.
Aleuria aurantia (Fr.) Fuckel is an a s c o m y c e t e fungus, k n o w n as orange peel m u s h r o o m , t h a t belongs to P y r o n e m a t a c e a e in the class of Discomycetes. This m u s h r o o m is o f t e n observed in a u t u m n on freshly broken grounds or edges of forest roads in Japan, and k n o w n to be distributed all over the w o r l d . In the course of studies on this fungus, w e found a p h e n o m e n o n t h a t a germination rate of the ascospore from A. aurantia w a s enhanced d r a m a t i c a l l y after cold storage. Fruit bodies o f A . aurantia w e r e collected in October, 1 9 9 8 near Mt. Akagi, Gunma Pref., Japan. As s h o w n in Figs. 1 A - C , a fruit body of A. aurantia w a s thin and fragile, and c u p - t o saucer-shaped w i t h a d i a m e t e r of 2 0 100mm. H y m e n i u m w a s s m o o t h and bright redorange. Eight ascospores of 1 4 - 1 6 x 10 m m w e r e observed in an ascus. Paraphyses w e r e slight clavate w i t h thick-endings at their tips. A s c o s p o r e s w e r e hyaline and elliptical w i t h distinct coarsely reticulate o r n a m e n t a t i o n . Present address: Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba, 305-8572, Japan. ~ Corresponding author.
Table 1. Expt. No. 1 2 3 4
T w o droplets w e r e observed inside of an ascospore. For induction of ascospore g e r m i n a t i o n in some d i s c o m y c e t e fungi, a t r e a t m e n t w i t h a certain chemical reagent or w i t h heat w a s reported to be effective. In A s c o b o l u s species, the t r e a t m e n t w i t h dilute alkali f o l l o w e d by incubation at 3 7 ~ induced germination at the rate of 8 0 - 9 0 % (Yu, 1954). In A. aurantia the treatm e n t w i t h nonyl-alcohol w a s reported to induce germination, but the rate w a s only less than 1 ~ (Paden, 1972). In this study ascospores of A. aurantia w e r e collected f r o m m a t u r e apothecia puffing on a sheet of paper. A piece of this paper w a s w a s h e d in distilled w a t e r , and the suspension w a s stored at 4 ~ or at - - 2 0 ~ for 3 mo. A l i q u o t s of the suspension w e r e transferred onto p o t a t o d e x t r o s e agar (PDA)-plates (Nissui) containing 100/~g/ml of c h l o r a m p h e n i c o l (Sigma). The plates were incubated at 2 0 ~ for 4 - 7 d in the dark. A n u m b e r of germ tubes appearred w a s c o u n t e d microscopically and the rate of g e r m i n a t i o n w a s calculated. For alkaline t r e a t m e n t , the spore suspension w a s brought to 0 . 2 N of KOH and a l l o w e d to stand for 2 0 rain, after w h i c h aliquots of the suspension w e r e transferred onto PDA-plates.
Germination of ascospores of A. aurantia. ~
Treatment no 0.2 N KOH for 20 min 4~ for 3too - 2 0 ~ for 3 m o
No. of ascospores No. of ascospores Germination rate observed/plate germinated/plate (%) 3.3• 10 s 3.3• 3.0• 3.0•
0 19_+5 177_+57 183_+32
0 <0.01 59_+19 61_+11
* Spore suspension with 3.3 x 105 ascospores was spread on each plate. In Expt. 1 and 2, all the ascospores in a plate were observed, and in Expt. 3 and 4, randomly 300 ascospores/plate were observed microscopically. Plates were incubated at 20~ for 7 d (Expt. 1 and 2), and for 4 d (Expt. 3 and 4), respectively. No. of ascospores germinated/plate was the average of 5 plates.
288
S. Ogawa et al.
Fig. 1. Fruit bodies, asci and ascospores of A. aurantia. A. Fruit bodies (bar: 20mm). B. Asci (bar: 80mm). C. Ascospore (bar: 6 mm). 80 mm). E. Ascospores after cold storage (bar: 80 mm).
D. Ascospores without any treatment (bar:
Germination of ascospore after cold storage As shown in Fig. 1D and Table 1, the ascospore did not germinate soon after collection. When ascospores were treated with 0.2 N KOH for 20 min, a few germinated after 7 d of incubation but the rate of germination was very low (0.01~ On the other hand, ascospores that had been stored at 4 ~ or at - - 2 0 ~ for 3 mo, germinated after 4 d at the rate of approximately 6 0 ~ (Fig. 1E; Table 1 ). As the same rate of germination was observed after storage at 4 ~ and at - 2 0 ~ this phenomenon did not seem to be due to maturation of ascospores during storage, and the cold storage itself should be effective. The results suggest that the ascospore of A, aurantia is necessary to experience cold condition before germination, and the dormancy of the ascospore may be broken by the cold storage. Since the fruit body of this fungus was formed at the end of autumn, and Hwang (1968) noted that mycelia of some discomycete fungus were weak in freezing, our speculation seem to be reasonable
289
for these fungi that survive as ungerminated ascospores during winter. Acknowledgements--We are grateful to Drs. Y. Fujita and M. Arai, Hokuriku National Agricultural Experimental Station, for their technical assistance.
Literature cited Hwang, S. H. 1969. Investigation of ultra-low temperature for fungal cultures I. An evaluation of liquid-nitrogen storage for preservation of selected fungal cultures. Mycologia 60: 613-621. Paden, J.W. 1972. Imperfect stages and the taxonomy of Pezizales. Persoonia6: 405-414. Yu, C.C. 1954. The culture and spore germination of Ascobolus with emphasis on A. magnificus. Amer. J. Bot. 41: 21-30.