A selective medium for the isolation of Saprolegnia ...

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DIRECTORATE OF COLDWATER FISHERIES RES on 10/13/15 For personal use only.

1126

CAN. J. MICROBIOL. VOL. 21, 1975

Our findings reveal that glucose concentration affects the amount of lactose uptake, which concomitantly induces P-D-galactosidase activity in S. rousiimycelium. It is not known yet whether this enzyme plays any role in the formation of sclerotia or in the partial inhibition by lactose of mycelial linear growth in S. rousii. I. CHET,I.. and Y. HENIS.1972. The response of two types of Scleroti~rtn rolfsii to factors affecting sclerotium formation. J. Gen. Microbiol. 73: 483486. 2. HENIS,Y., Y. OKON,and I. CHET.1973. The relationship between early hyphal branching and formation of sclerotia in Sclerolirrm rolfsii. J . Gen. Microbiol. 79: 147-150. 3. K N I . A,. and T . I C H I M I1969. . Production and properties of galactosidases from Corricirrtn ro/fsii. Appl. Microbiol. 18: 1036-1040. A. L. FARR,and 4. LOWRY,0 . H., N. J. ROSEBROUGH, R. J . RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.

5. LYSEK,G., and K . ESSER.1971. Rhythmic mycelial growth in Podospora n~rserinn.11. Evidence for a correlation with carbohydrate metabolism. Arch. Mikrobiol. 75: 360-373. 6. OKON.Y., I. CHET. and Y. HENIS.1972. Lactoseinduced synchronous sclerotium formation in Sclerorirrr?~ rolfsii and its inhibition by ethanol. J . Gen. Microbiol. 71: 465470. 7. OKON,Y.. I. CHET,and Y. HENIS.1973. Effect of lactose, ethanol and cycloheximide on the translocation pattern of radioactive compounds and on sclerotium formation in Sclero~irrtnrolfsii. J . Gen. Microbiol. 74: 251-258. 8. OKON,Y., I. CHET,N. KISLEV.and Y. HENIS.1974. Effect of lactose on soluble-glucan production and on rolfsii Sacc. grown in the ultrastr.ucture of Sclero~i~rm submerged culture. J. Gen. Microbiol. 81: 145-149. 9. PASTAN. I., and R. L. PERLMAN. 1969. Repression of P-galactosidase synthesis by glucose in phosphotlansferase mutantsofEschericl~incoli. J . Biol. Chem. 244: 5836-5842, 10. T R I N C IA. , P., and A. C O L L I N G E 1973. . Influence of L-sorbose on the growth and morphology of Nelrrosporn o o s s n . J . Gen. Microbiol. 78: 179-192.

A selective medium for the isolation of Saprolegnia spp. from freshwater1 H. H. Ho L)epnrltnet~~ qf'Riology, Slole Uni1,ersily College. N P I IPnllz, ~ New York 12561 Accepted February 24. 1975 Ho, H. H. 1975. A selective medium for the isolation ofScrprolegtrirr spp. from freshwater. Can. J . Microbiol. 21: 1 126-1 128. A selective medium was developed for the isolation of Snprolegt~icrspp. from freshwater. Ho. H. H. 1975. A selective medium for the isolation of Snprolegnirr spp. from freshwater. Can. J. Microbiol. 21: 1126-1 128. Un milieu selectif aete developpe pour isoler Snprolegnirr spp. 5 partir d'eau douce. [Traduit par le journal]

The biology of water pollution is of current interest but the role of fungi seems to have been largely neglected (Cooke 1969). Harvey (1952) found that certain species of aquatic Saprolegniaceae, for example Saprolegnia, were always absent from heavily polluted waters. Harvey's observation suggests a correlation between water pollution and population of Saprolegnia spp., a possibility yet to be explored. It is important, therefore, to be able to determine quantitatively Saprolegnia spp. in water, and the best approach 'Received November 29, 1974.

seems to be isolating and enumerating Saprolegnia spp. on agar plates. The method used widely for isolating Saprolegnia spp. is to bait the fungi from water by using boiled, halved hemp seeds (Cannabis satiua L.). It has proved to be an extremely reliable technique but unfortunately it is not satisfactory for quantitative studies. Special media were developed to recover aquatic Phycomycetes (Collins and Willoughby 1962; Fuller and Poyton 1964; Miller 1967) but these were not designed specifically for Saprolegnia spp. The object of this study was t o develop such a selective medium.

1127


NOTES

The basal medium used was half-strength Bacto Emerson yeast-starch agar (YpSs, Difco Laboratories, Detroit, Michigan), which has been used successfully for the culture of many aquatic Phycomycetes (Emerson 1958). It was essential to screen various common antibiotics and fungicides so that the appropriate ones might be incorporated into the medium to suppress bacteria and fungal colonies other than those of Saprolegnia spp. Pentachloronitrobenzene (PCNB, K & K Laboratories, Plainview, New York) was prepared by Farley's method (1972). Pimaricin (Royal Netherlands Fermentation Industries Ltd.) was in the form of "Pimafiicin" suspension (24% pimaricin). All antimicrobial agents were prepared fresh, being dissolved or suspended in sterile distilled water. They were added to the warm melted basal medium just before plating so that the final concentrations were in the order of 1000, 250, and 62.5 mgllitre, or in the case of pimaricin, 10, 2.5, and 0.625 mgllitre. These concentrations were comparable with those used by other workers (Tsao 1970). Saprolegniafernx (Gruith.) Thuret, isolated from a neighboring stream, was used as the test organism. It was grown at 15C on Bacto yeast-starch agar for 3 days, after which discs of mycelium were removed with a sterile No. 1 cork borer and used as the inoculum. Five plates were used per

treatment. The plates were then incubated at 15C in total darkness. At the end of 3 days the colony diameters were measured. Two measurements were made at right angles t o each other through the center of the plate where the inoculum was placed. For the sake of comparison, effect of antimicrobial agents on the linear growth of Saprolegnia ferax was expressed as a percentage determined by the following formula: {(Mean colony diameter of treatment plates (cm)/ Mean colony diameter of control plates (cm)) x 100) and the data presented in Table 1. This study shows that Saprolegniaferax was relatively tolerant to most antimicrobial agents, with the notable exception of tetracycline hydrochloride and rose bengal. The effect of streptomycin and penicillin "G" as antibacterial agents and PCNB and pimaricin as antifungal agents in a medium for the isolation of Saprolegnia spp. was tested in various combinations (Table 2). H o (1975) found that hemp seeds and hemp seed extract attracted zoospores of Saprolegnia sp. which resulted in rapid encystment and germination as well as enhanced hyphal growth. Thus, hemp seed extract was incorporated into the medium to a final concentration of 1% wlv. The hemp seed extract was prepared by grinding 5 g hemp seeds in a mortar and adding 50 ml of distilled water. It was thor-

TABLE 1 Effect of antimicrobial agents on linear growth (expressed as percentage of that obtained on basal medium*) of Saprolegr~i(rfera.u Treatment Antimicrobial agent

1000 mg!l

250 mg/l

62.5 mg/l

Bacterial inhibitors: Vancomycin Tetracyclin hydrochloride Streptomycin sulfate Polymyxin sulfate Penicillin "G" sodium Neomycin sulfate Fungal inhibitors Nystatin Rose bengal Captan Spergon Pentachloronitrobenzene (PCNB) Pimaricin *Basal medium consisted oT20.2 gllitre Dirco Bacto Emerson yeast-starch agar.

10 mg/l

2 . 5 mg/l

0.625 mg/l


1128

CAN. J . MICROBIOL . VOL. 21, 1975

TABLE 2 Effect of antimicrobial agents on isolation of Saprolegt~iaspp. from freshwater

Treatment Controlt Control streptomycin sulfate (400 mg/l) penicillin "G" sodium (400 mg/l) Control streptomycin sulfate (400 mg/l) penicillin "G" sodium (400 mg/l) PCN B (400 rng/l) Control streptomycin sulfate (400 mg/l) + penicillin "G" sodium (400 rng/l) + PCN B (400 mg/l) + pimaricin (0.25 mgil)

+ + + + + +

No. Saprolegnia colonies per plate* 0 5.0 3.1

3.0

*Average o f 10-20 plates. tControl medium consisted of 20.2 gllitre Difco Emerson yeaststarch agar plus 1% wlv hemp seed extract.

oughly mixed and carefully decanted so that most of the seed-coat fragments were left behind in the mortar and discarded. The decanted liquid was then used as 10x hemp seed extract. The medium (about 25 ml) which contained the necessary ingredients was poured into a petri dish and allowed to solidify. A I-ml water sample, freshly collected from a neighboring stream, was pipetted onto the agar surface and spread evenly with a bent glass rod. The plates were incubated at 15C with the dishes slightly open to allow the water sample to dry overnight, and then the cover was replaced. After 3 days of incubation at 15C, small mycelial discs were removed from the fungal colonies that appeared on the plates and were transferred to another petri dish containing distilled water. These were further incubated for several days or longer until sporangia and (or) oospores were produced and the identity of Saprolegnia spp. was confirmed. Nearly all fungal colonies that appeared first on the agar plates were found to be species of Saprolegnia. Media without the antifungal agents seemed to allow the maximum number of Saprolegnia colonies to appear, but they also

supported rapid growth of other fungi, mainly species of Trichoderma, Fusarium, Mucor, and Penicillium, which often overran Saprolegnia colonies. However, these fungi were only found sporadically on plates with PCNB and were almost absent on plates with PCNB and pimaricin. From these results the selective medium for the isolation of Saprolegnia spp. from freshwater consisted of the following ingredients. Bacto Emerson yeast-starch agar (YpSs) 10% hemp seed extract2 Streptomycin sulfate Penicillin "G" s o d i ~ ~ m Pentachloronitrobenzene (PCNB) Pimaricin Distilled water

20.2 100 400 400 400 0.25 900

g

ml rng mg mg mg ml

'Preparation described in text.

Acknowledgment This project was supported in part by an institutional grant from the National Science Foundation. COLLINS, V. G . , and L. G. WILLOUGHBY. 1962. The distribution of bacteria and fungal spores in Blelham Tarn with particular reference to an experimental overturn. Arch. Mikrobiol. 43: 294-307. COOKE,W. B. 1969. Our mouldy earth. A study in the fungi of our environment with emphasis on water. U.S. Dep. Inter., Fed. Water Pollut. Control Adm., Cincinnati, Ohio. EMERSON, R. 1958. Mycological organization. Mycologia, 50: 589-62 1. FARLEY, J . D. 1972. A selective medium for assay of Collerorricltirm coccodes in soil. Phytopathology, 62: 1288-1293. FULLER, M. S., and R. 0 . POYTON.1964. A technique for the isolation of aquatic fungi. BioScience, 14: 45-46. HARVEY, J . V. 1952. Relationship of aquatic fungi to water pollution. J . Water Pollut. Control, 24: 1159-1 164. Ho, H. H. 1975. Observations on the behavior of zoospores of a species of Saprolegnia. Mycologia, 67: 425428. MILLER, C. E. 1967. Isolation and pure culture of aquatic Phycomycetes by membrane filtration. Mycologia, 59: 524-527. TSAO, P. H. 1970. Selective media for isolation of pathogenic fungi. Annu. Rev. Phytopathol. 8: 157-186.