Maintaining cultures of ectomycorrhizal and plant pathogenic fungi in sterile water cold storage1
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DALHOUSIE UNIVER on 12/09/12 For personal use only.
DONALD H. MARXA N D WILLIAM J . DANIEL USDA Fori~stSei.~.ii,r, So~it/~rri.ster~tr Forrst E.rperirnerrt Stotiorr, Fore.st,lv Scierrce.~Lrrboi.otory, Corltorr Street, Atherrs, Georgirr 30602 Accepted September 19, 1975 MARX,D. H . , i ~ n dW. J . D A N I E L 1976. . Maintaining c~rlturesof ectomycorrhizal and plant pathogenic fi~ngiin sterile water cold storage. Can. J . Microbiol. 22: 338-341. Mycelial cultures of 64 isolates of 14 species of ectomycorrhizal firngi and 27 isolates of IS species of plant pathogenic fungi were grown on agar medium in Petri dishes. Mycelial discs, 8 rnm in diameter, were removed from the c ~ ~ l t ~and ~ r estored s in sterile distilled water in test tubes at 5°C. Sixty-four, 61, and 41 isolates ofthe symbiotic fungi were viable after 1.2, and 3years storage respectively. Only 19, 10, and 8 isolates of the pathogenic fungi were viable after 1, 2, and 3 years storage, respectively. Time in pure culture before water storage did not affect viability of any fungal species following water storage. After 3 years storage, fo~rrfirngi (three syrnbiontsand one pathogen) were tested and f o ~ ~ ntod have retained their original growth Ixtes and rootinfecting abilities on pine seedlings. The same four isolates, however, maintained on agar slants at 5 %and s ~ ~ b c ~ r l t ~every l r e d 4 to 6 months, grew slower and did not infect a s many feeder roots of pine as the water-stored isolates. MARX,D. H.. et W. J . D A N I E L .1976. Maintaining cultures of ectomycorrhizal and plant pathogenic fi~ngiin sterile water cold storage. Can. J. Microbiol. 22: 338-341. Soixante-quatre isolats de 14 especes de champignons ectornycorrhizate~~rs et 27 isolats d e 15 especes de charnpignons phytopathogenes furent rnis en culture S L I agar ~ dans des plats d e Petri. Des disques de mycelium de 8 mrn de diametre furent prelevees et places a 5 "C dans des tubes i essai contenant de I'eau distillee sterile. Des charnpignons syrnbiotiques, 64, 61 et 41 isolats sont derneures viables apres 1, 2 et 3 annees d'entreposage, respectivernent. Des pathogenes fongiques, seulement 19, 10 et 8 isolats sont demeures viables apres 1, 2 et 3 annees d'entroposage, respectivernent. La duree de culture pure qui precede I'entreposage dans I'eau n'affecte pas la viabilite des diverses especes fongiques apres I'entreposage dans I'eau. Apres 3 annees d'entreposage, quatre charnpignons (trois symbiontes et un pathogene) furent testes: ils avaient conserve leurs taux originels de croissance et leur aptitude infecter les racines des plantules de pin. Toutefois, lorsque ces rnerne quatre isolats furent maintenus sur agar en pente B 5 "C et repiques a tous les 4 ou 6 mois, leurs taux d e croissance furent inferieurs e t ils ne purent infecter autant de racines de pin que le pouvaient les isolats conserves dans I'eau. [Traduit par le journal]
Introduction A major problem frequently encountered in research on root disease and ectomycorrhizae is the maintenance of specific fungi and their physiological characteristics over extended periods. After several years of repeated subculturing on agar media and storage at 5 "C, these fungi either die or lose their virulence or symbiotic potential. Various techniques have been developed to preserve fungal cultures. Lyophilization (1 I), covering cultures with mineral oil (2), freeze-drying (5), and other cryogenic methods (1, 10) have been used on a variety of fungi and other microorganisms t o preserve viability and physiological traits over long 'Received August 11, 1975.
periods of time. Most techniques, however, are time consuming and some require specialized equipment. Lyophilization techniques are not satisfactory on habitually nonsporulating cultures of ectomycorrhizal fungi. In 1956, Kelman (3) reported a simple method for storing cultures of Pseu(loronzor7ns solnrznce~tr~inz in sterile distilled water, tap water, and pH 7.0 phosphate buffer at 5 "C or 21 "C. The bacterium retained virulence for 220 days at 21 "C storage. Later, Kelman and Person (4) reported other isolates of this bacterium retained virulence after 18-24 months storage at 22 "C in sterile distilled water. Person (9) adapted this technique to store A s c o c h y t n isolates. He found that mycelial agar discs of this fungus stored for 20-25 months at 10 "C or 10-12 "C in sterile distilled water
MARX AND DANIEL: PLANT PATHOGENIC FUNGI
TABLE1. Viability of isolates of ectoniycorrhizal fungi after storage at 5
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DALHOUSIE UNIVER on 12/09/12 For personal use only.
DONALD H. MARXA N D WILLIAM J . DANIEL USDA Fori~stSei.~.ii,r, So~it/~rri.ster~tr Forrst E.rperirnerrt Stotiorr, Fore.st,lv Scierrce.~Lrrboi.otory, Corltorr Street, Atherrs, Georgirr 30602 Accepted September 19, 1975 MARX,D. H . , i ~ n dW. J . D A N I E L 1976. . Maintaining c~rlturesof ectomycorrhizal and plant pathogenic fi~ngiin sterile water cold storage. Can. J . Microbiol. 22: 338-341. Mycelial cultures of 64 isolates of 14 species of ectomycorrhizal firngi and 27 isolates of IS species of plant pathogenic fungi were grown on agar medium in Petri dishes. Mycelial discs, 8 rnm in diameter, were removed from the c ~ ~ l t ~and ~ r estored s in sterile distilled water in test tubes at 5°C. Sixty-four, 61, and 41 isolates ofthe symbiotic fungi were viable after 1.2, and 3years storage respectively. Only 19, 10, and 8 isolates of the pathogenic fungi were viable after 1, 2, and 3 years storage, respectively. Time in pure culture before water storage did not affect viability of any fungal species following water storage. After 3 years storage, fo~rrfirngi (three syrnbiontsand one pathogen) were tested and f o ~ ~ ntod have retained their original growth Ixtes and rootinfecting abilities on pine seedlings. The same four isolates, however, maintained on agar slants at 5 %and s ~ ~ b c ~ r l t ~every l r e d 4 to 6 months, grew slower and did not infect a s many feeder roots of pine as the water-stored isolates. MARX,D. H.. et W. J . D A N I E L .1976. Maintaining cultures of ectomycorrhizal and plant pathogenic fi~ngiin sterile water cold storage. Can. J. Microbiol. 22: 338-341. Soixante-quatre isolats de 14 especes de champignons ectornycorrhizate~~rs et 27 isolats d e 15 especes de charnpignons phytopathogenes furent rnis en culture S L I agar ~ dans des plats d e Petri. Des disques de mycelium de 8 mrn de diametre furent prelevees et places a 5 "C dans des tubes i essai contenant de I'eau distillee sterile. Des charnpignons syrnbiotiques, 64, 61 et 41 isolats sont derneures viables apres 1, 2 et 3 annees d'entreposage, respectivernent. Des pathogenes fongiques, seulement 19, 10 et 8 isolats sont demeures viables apres 1, 2 et 3 annees d'entroposage, respectivernent. La duree de culture pure qui precede I'entreposage dans I'eau n'affecte pas la viabilite des diverses especes fongiques apres I'entreposage dans I'eau. Apres 3 annees d'entreposage, quatre charnpignons (trois symbiontes et un pathogene) furent testes: ils avaient conserve leurs taux originels de croissance et leur aptitude infecter les racines des plantules de pin. Toutefois, lorsque ces rnerne quatre isolats furent maintenus sur agar en pente B 5 "C et repiques a tous les 4 ou 6 mois, leurs taux d e croissance furent inferieurs e t ils ne purent infecter autant de racines de pin que le pouvaient les isolats conserves dans I'eau. [Traduit par le journal]
Introduction A major problem frequently encountered in research on root disease and ectomycorrhizae is the maintenance of specific fungi and their physiological characteristics over extended periods. After several years of repeated subculturing on agar media and storage at 5 "C, these fungi either die or lose their virulence or symbiotic potential. Various techniques have been developed to preserve fungal cultures. Lyophilization (1 I), covering cultures with mineral oil (2), freeze-drying (5), and other cryogenic methods (1, 10) have been used on a variety of fungi and other microorganisms t o preserve viability and physiological traits over long 'Received August 11, 1975.
periods of time. Most techniques, however, are time consuming and some require specialized equipment. Lyophilization techniques are not satisfactory on habitually nonsporulating cultures of ectomycorrhizal fungi. In 1956, Kelman (3) reported a simple method for storing cultures of Pseu(loronzor7ns solnrznce~tr~inz in sterile distilled water, tap water, and pH 7.0 phosphate buffer at 5 "C or 21 "C. The bacterium retained virulence for 220 days at 21 "C storage. Later, Kelman and Person (4) reported other isolates of this bacterium retained virulence after 18-24 months storage at 22 "C in sterile distilled water. Person (9) adapted this technique to store A s c o c h y t n isolates. He found that mycelial agar discs of this fungus stored for 20-25 months at 10 "C or 10-12 "C in sterile distilled water
MARX AND DANIEL: PLANT PATHOGENIC FUNGI
TABLE1. Viability of isolates of ectoniycorrhizal fungi after storage at 5
