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Long-term storage and viability of aquatic oomycetes
Notes and Brief Articles
611
LONG-TERM STORAGE AND VIABILITY OF AQUATIC OOMYCETES GILLIAN CLARK AND M. W. DICK
Department of Botany, Plant Science Laboratories, University of Reading, Whiteknights, Reading Dick (1965) described a method for maintaining stocks of Saprolegniaceae which had been in use since 1957 and which is now used for cultures at the Commonwealth Mycological Institute (Dr Onions, personal communication). Refrigerated cultures were stated to remain viable for periods of up to 2 years and in one case 3 years. When the Aquatic Phycomycete Culture Collection was established at Reading in 1967 it was decided to test the longevity of duplicate stock cultures up to the point at which cultures were in danger of drying up. Between 1965 and 1968 a total of 375 stock culture flasks of more than 70 species of Oomycetes (mostly Saprolegniaceae) had been put into cold storage. Each flask initially contained about 150 ml of glass-distilled water, a small cube of tellurite agar with the inoculum and a single hemp seed. The solid substrates were bound together by mycelial growth within 24 h (Dick, 1965). The flasks were firmly plugged with muslin-bound cotton wool and protected from dust with strong brown paper tightly secured by an elastic band. These flasks were kept in a glass-fronted cupboard in a large cold store held at 4 "C. In 1972 this store was needed for other purposes and the flasks were transferred to a domestic refrigerator at the same temperature. An automatic defrosting unit eliminated the need to open the door, except to empty the drip tray occasionally. No flask was opened either for the addition of water or fresh nutrient while in storage. When removed from the refrigerator for testing, hemp seeds were added to the flasks together with fresh glass distilled water if necessary. Good vegetative growth was usually present after 48 h. Microscopic examination was carried out on these cultures after 2 weeks at room temperature. Twenty cultures had dried up and were not viable by December 1973. By that date a considerable number of flasks contained less than 30 ml of water and could be expected to dry up in a few months. Table 1 shows the record of viability for the remaining 355 cultures. Of the 104 dead cultures, only six contained no viable organism of any kind, while almost all the other flasks contained fungi imperfecti (mostly Cladosporium or Penicillium spp.). Very few flasks contained microscopically detectable bacterial contamination. Less than 2 % of the viable cultures were contaminated. No pattern could be seen in relation to death in axenic culture. Thus it would appear that the major disadvantage of this method of long-term storage is mould contamination due to growth Trans. Br. mycol. Soc. 63 (3), (1974), Printed in Great Britain
Transactions British Mycological Society
612
Table Age of culture 1-2 years 2-3 years 3-4 years 4-5 years 5--6 years 6-7 years 7-8 years Totals
1
Viable
Dead
Total
110 7 4
8 5
127
84
3 25 1
5
8
104
355
I I
118 12 5 1 211
through the plug in the humid atmosphere of the cold store. A second long-term experiment is now underway to test survival using other wrapping membranes to overcome this problem. The three species alive after 8 years were Saprolegnia furcata Maurizio, Achlya mucronata Ziegler and Phytophthora gonapodyides (Petersen) Buisman. Six of the seven species of Pythiaceae tested were viable at 6 years. Some species (notably certain sections of the genus Achlya) rapidly lose the ability to form oogonia in culture, often by the first or second transfer, but this lack of resting spore formation does not appear to affect the ability to survive long-term storage. The use of solid submerged substrates has prevented further analysis of the source of the viable inoculum, though the majority of the visible hyphae of all cultures appear to be autolysed. In order to minimize the number of transfers through which each stock culture has been processed, the viable cultures from long-term storage have been substituted for the existing stock cultures. REFERENCE
DICK, M. W. (1965). The maintenance of stock cultures of Saprolegniaceae. Mycologia 57,828-83 1 •
EVIDENCE FOR THE GENOTYPIC CONTROL OF SPORE SIZE IN CLAVICEPS PURPUREA A. R. LOVELESS AND JANET M. PEACH
Department ofBiological Sciences, Portsmouth Polytechnic Sclerotia of Claviceps purpurea (Fr.) Tul. vary widely in size and shape according to the host on which they occur. For example, sclerotia on Nardus stricta range from 3-10 mm long by 0'7-0'9 mm thick, whereas those on Secale cereale, the type host, may be as long as 50 mm and as wide as 8 mm. If, as is commonly assumed, wild grasses provide a source of inoculum for cereals, it would seem that the form assumed by a sclerotium of C. purpurea is a host-dependent character. The results of appropriate Trans. Br. mycol. Soc.
63
(3), (1974). Printed in Great Britain
611
LONG-TERM STORAGE AND VIABILITY OF AQUATIC OOMYCETES GILLIAN CLARK AND M. W. DICK
Department of Botany, Plant Science Laboratories, University of Reading, Whiteknights, Reading Dick (1965) described a method for maintaining stocks of Saprolegniaceae which had been in use since 1957 and which is now used for cultures at the Commonwealth Mycological Institute (Dr Onions, personal communication). Refrigerated cultures were stated to remain viable for periods of up to 2 years and in one case 3 years. When the Aquatic Phycomycete Culture Collection was established at Reading in 1967 it was decided to test the longevity of duplicate stock cultures up to the point at which cultures were in danger of drying up. Between 1965 and 1968 a total of 375 stock culture flasks of more than 70 species of Oomycetes (mostly Saprolegniaceae) had been put into cold storage. Each flask initially contained about 150 ml of glass-distilled water, a small cube of tellurite agar with the inoculum and a single hemp seed. The solid substrates were bound together by mycelial growth within 24 h (Dick, 1965). The flasks were firmly plugged with muslin-bound cotton wool and protected from dust with strong brown paper tightly secured by an elastic band. These flasks were kept in a glass-fronted cupboard in a large cold store held at 4 "C. In 1972 this store was needed for other purposes and the flasks were transferred to a domestic refrigerator at the same temperature. An automatic defrosting unit eliminated the need to open the door, except to empty the drip tray occasionally. No flask was opened either for the addition of water or fresh nutrient while in storage. When removed from the refrigerator for testing, hemp seeds were added to the flasks together with fresh glass distilled water if necessary. Good vegetative growth was usually present after 48 h. Microscopic examination was carried out on these cultures after 2 weeks at room temperature. Twenty cultures had dried up and were not viable by December 1973. By that date a considerable number of flasks contained less than 30 ml of water and could be expected to dry up in a few months. Table 1 shows the record of viability for the remaining 355 cultures. Of the 104 dead cultures, only six contained no viable organism of any kind, while almost all the other flasks contained fungi imperfecti (mostly Cladosporium or Penicillium spp.). Very few flasks contained microscopically detectable bacterial contamination. Less than 2 % of the viable cultures were contaminated. No pattern could be seen in relation to death in axenic culture. Thus it would appear that the major disadvantage of this method of long-term storage is mould contamination due to growth Trans. Br. mycol. Soc. 63 (3), (1974), Printed in Great Britain
Transactions British Mycological Society
612
Table Age of culture 1-2 years 2-3 years 3-4 years 4-5 years 5--6 years 6-7 years 7-8 years Totals
1
Viable
Dead
Total
110 7 4
8 5
127
84
3 25 1
5
8
104
355
I I
118 12 5 1 211
through the plug in the humid atmosphere of the cold store. A second long-term experiment is now underway to test survival using other wrapping membranes to overcome this problem. The three species alive after 8 years were Saprolegnia furcata Maurizio, Achlya mucronata Ziegler and Phytophthora gonapodyides (Petersen) Buisman. Six of the seven species of Pythiaceae tested were viable at 6 years. Some species (notably certain sections of the genus Achlya) rapidly lose the ability to form oogonia in culture, often by the first or second transfer, but this lack of resting spore formation does not appear to affect the ability to survive long-term storage. The use of solid submerged substrates has prevented further analysis of the source of the viable inoculum, though the majority of the visible hyphae of all cultures appear to be autolysed. In order to minimize the number of transfers through which each stock culture has been processed, the viable cultures from long-term storage have been substituted for the existing stock cultures. REFERENCE
DICK, M. W. (1965). The maintenance of stock cultures of Saprolegniaceae. Mycologia 57,828-83 1 •
EVIDENCE FOR THE GENOTYPIC CONTROL OF SPORE SIZE IN CLAVICEPS PURPUREA A. R. LOVELESS AND JANET M. PEACH
Department ofBiological Sciences, Portsmouth Polytechnic Sclerotia of Claviceps purpurea (Fr.) Tul. vary widely in size and shape according to the host on which they occur. For example, sclerotia on Nardus stricta range from 3-10 mm long by 0'7-0'9 mm thick, whereas those on Secale cereale, the type host, may be as long as 50 mm and as wide as 8 mm. If, as is commonly assumed, wild grasses provide a source of inoculum for cereals, it would seem that the form assumed by a sclerotium of C. purpurea is a host-dependent character. The results of appropriate Trans. Br. mycol. Soc.
63
(3), (1974). Printed in Great Britain