Influence of environmental factors on zoospores of Saprolegnia diclina

[ 4 13 ] Tram. Br . mycol. Soc. 8:2 (3), 413-421 ( 1984)

Printed in Great Britain

INFLUENCE OF ENVIRONMENTAL FACTORS ON ZOOSPORES OF SAPROLEGNIA DICLINA By S. N. SMITH, R. A. ARMSTRONG AND J. J. RIMMER Department of Biological Sciences, University of Aston, Birmingham B4 7ET The numbers of zoospores produced by a pathogenic strain of Saprolegnia die/ina and their behaviour are markedly influenced by a variety of environmental variables including temperature, pH, oxygen tension and the presence of biocides. The use of the latter is not recommended, as fish readily succumb to equivalent concentrations of biocides. Analysis of the pattern of the distribution of resulting zoospore cysts demonstrates that zoospores become dispersed by random movement even while in the proximity of the parent colony's nutrient source. However, the presence of amino acids, in particular aspartic and glutamic acid, at concentrations which occur in fish tissue promotes the directed movement of zoospores towards the nutrient source thereby encouraging the colonization of fresh sites. The strains of Saprolegnia spp . pathogenic to fish have received considerable attention. Aspects of their fine structure, life cyle and taxonomy have been extensively investigated by Beakes (1980), Pickering, Willoughby & McGory (1979) and Willoughby (1977, 1978). Although factors controlling the activity of zoospores derived from pathogenic fungi are well documented (Cooke, 1977; Fuller, 1977) little attention has been paid to the environmental factors which may influence the activity and behaviour of zoospores derived from pathogenic Saprolegnia spp. The work presented here investigates a variety of factors which may influence the numbers and activity of zoospores derived from a pathogenic strain of Saprolegnia die/ina Humphrey. Factors include temperature, oxygen tension, pH, metal ions which may become incorporated into aquatic systems as algicides or accidentally discharged through negligence, and a typical herbicide recommended for weed control in aquatic systems. The intense nature of modern fish farming may further promote fungal disease, as such techniques as grading give rise to considerable stress and damage to fish. Disruption of the epidermal layers is commonplace, resulting in loss of a number of metabolites including free amino acids. Royle & Hickman (1963) demonstrated that glutamic acid proved a powerful attractant to zoospores of the plant pathogen Pythium aphanidermatum (Edson) Fitzpatrick. This work also investigates the effects of amino acids on the numbers and directional movement of zoospores of S . die/ina . MATERIALS AND METHODS

Isolation, identification and maintenance A culture of Saprolegnia diclina was isolated from a lesion on a rainbow trout, Salmo gairdneri

Richardson, obtained from the Fish Culture Unit, Aston University. Prior to the isolation of the culture the lesion was thoroughly washed with tap water to remove detritus. Hyphal tips were then removed and placed within a Rapers ring set in glucose-yeast-aureomycin agar. The isolates were incubated at 20° until hyphal tips emerged outside the ring. These were removed with a small disk of agar and transferred to a Petri dish containing enough sterile ' tap water to cover the disk and encourage zoospore production. Single-spore isolates were obtained for the identification of cultures by collecting the discharged zoospores and streaking them on to glucose-yeast agar plates. Individual germinating spores were subsequently isolated on to glucose-peptone agar. The single-spore isolates obtained were classified as Saprolegnia die/ina Type 1 on the following criteria :oogonia sparse, only formed after prolonged incubation at 10° and never at 20°, length/breadth ratio ~ 2 in 43'5 % of oogonia, the walls of which were thin and unpitted. These characteristics match those outlined by Willoughby (1978). Cultures were maintained on glucose-yeast agar at 5° and subcultured every two months. Chambers Position of colon Y-

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S. N. Smith, R. A. Armstrong and J.J. Rimmer were removed from subcultures and placed in sterile glass Petri dishes containing a small volume of sterile distilled water. Halved hempseeds were individually placed on each disk and incubated for 48 h. The colonized hempseeds were removed from the disks and attached to the end wall of the channels by means of a small amount of silicon grease. Channels were filled with 15 ml of sterile 0'005 M phosphate buffer, the pH of which was modified to assess the effect of pH on zoospore production and activity. Copper sulphate, zinc sulphate and paraquat dichloride (formulated as Gramoxone, ICI) over a range of concentrations were also individually incorporated into phosphate buffer (pH 7'0) to assess their effects on zoospore production and activity. Channels were incubated

Table 1, Concentrations of amino acids incorporated into agar (mM) Alanine Aspartic acid Glutamic acid Glycine Proline

17'87 17'83 19'18 22'52 10'14

Zoospore activity Zoospore activity was assessed by counting the number of encysted zoospores along the channels (4 separate counts each of 2'54 mm" for each individual chamber, Fig. 1) which were set up in the following manner. Disks of mycelium and agar

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46 686'98 866 42 '77 78242'43 593 6'23 55 145'77 (***

3 4 12 2 40 =

p < 0'001)

15561'3 2 21660'69 6520'20 2968'11 137 8'64

11'29*** 15'71 *** 4'73*** 2'15

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SS 639 82'27 147218'45 150249'68 23898 '4° (***

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at 10° , or over a range of temperatures to assess the effects of temperature on zoospore production and activity. The effect of oxygen tension on zoospore production and activity was assessed in channels which were partially sealed after the incorporation of colonized hempseeds and phosphate buffer (pH 7'0). Oxygen tension was controlled through gas mixtures of nitrogen and air fed into the channels through needle valves. The phosphate buffer took approximately two hours to equilibrate full y with the gas mixtures employed. The partial pressures of oxygen generated within the buffer by the gas mixtures employed were ascertained by comparison with a previousl y constructed standard curve relating proportions of gases to the partial pressure of oxygen within the buffer.

F 33'46*** 379'4 2*** 19'64***

Attraction of zoospores The ability of amino acids to att ract zoospores was assessed in the following manner. Samples of tissue from the flank of the carp Cyprinus carpio L., which the strain of S. diclina under investigation was capable of infecting, were gently homogenized and the concentration of free am ino acids within the samples determined with a ' L ocart e ' amino acid anal yser. The five amino acids occurring in the greatest concentrations were diluted in agar (' Oxoid ' NO.3) to give equivalent concentrations of amino acid s to those within carp tissue (T able 1). After sterilization the molten medium was drawn up int o capillary tubes and after sealing one open end of each capillary tube th ey were placed within channels (Fig. 1) which were inoculated and filled with buffer (pH 7 '0) in the manner described above .

4 16

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Fig. 4a, b. Effect of N./air mixtures on zoospore release and distribution of resulting cysts. Analysis of variance tables relating to the above data appear below. Variation SS Df MS F O.

Chambers O. x Chambers Error

18583 '32 166890 '80 49750'66 4796 '26 (***

3 4 12 40 =

6 1 94"44 4 1722'70 4 145 '89 119'23

51'95*** 349 '93*** 34 '77***

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Statistical analysis The experiments testing pH, copper, zinc and paraquat concentration were 4 (or 3) x 5 factorials (4 levels of the factor with 5 distances down the chamber) with each experiment made on three occasions (blocks). The experiments testing temperature, oxygen and amino acids were 6, 5 or 4 x 5 factorials with three replicates in a randomized design. The most appropriate statistical method for analysing these data is z-way analysis of variance (Ridgman, 1975; Snedecor & Cochran, 1967 ). The analysis of variance for each experimental factor tests three null hypotheses. First, that the number of spores averaged over the chambers is the same at different levels of the environmental factor. Second, that the number of spores averaged over the levels of the environmental factor is the same

with distance along the channels. Third, that the distribution of the number of spores is consistent within the different levels of the environmental factor. Hence, for example, a significant main effect of temperature would suggest greater zoospore production at some temperatures while a significant interaction would suggest greater mobility at some temperatures. In addition, an analysis was made of the numbers of encysted zoospores in the control channels (pH 7 '0 and 10°, conditions closely matching those of rivers within the Severn Trent Water Authority, 1979) of each experimental factor to determine whether the distribution of encysted zoospores along the channels was consistent with random diffusion from a point source. Random diffusion would result in a negative exponential depletion of numbers along the channels (Pielou , 1969) while the numbers transformed to logarithms

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55

Df 2 4 8 2 30

5493 '17 12444 '49 17866, 83 18'00 33 6 '3 2 (***

=

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of pH, temperature and oxygen tension on the average number of encysted zoospores (per unit area per da y) and the distribution of these enc ysted zoospores. The numbers of encysted zoospores (F ig, 2a ) are sign ifican tly affect ed by pH, with the greatest numbers of enc ysted zoospores being found at pH 7'0. Although growth was observed, R ESUL TS no enc ysted zoospore was detected at pH 4'0 or 9'0. Anal ysis of the distribution pattern of enc ysted In addition to affecting the number of enc ysted zoospores demonstrates (F = 103 , P < 0 '01 ) that zoospores pH also affects their distribution in control treatments the distribution of encysted (F ig, 2b ), particularly at pH 5'0, where few cysts zoospores within the channels is the result of are found far from the parent colony, random diffusion. F igs 2-4 demonstrate the effects The effect of temperature on average numbers of would result in a linear depletion, A test of linearity of the logarithmic values was made by anal ysis of variance with the total sums of the sq uares partitioned into linear effect and deviations from regression (Ridgman, 1975).

14

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SS 1193'9 1 90655'39 1276'90 69'99 1106'97 (***

MS 397'97 22663. 85 16"41 34'99 27'67

Df 3 4 12 2 40 =

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14'3 8*** 819'07*** 3'84*** 1'26

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zoospores is shown by Fig. 3 a, the greatest numbers ofcysts being found in channels incubated at 20°. The effect oftemperature on the distribution of encysted zoospores is shown by Fig. 3b. At a lower temperature the pattern of distribution does not appear to be markedly affected; however, there is a significant interaction between treatment and mobility as evidenced by the great numbers of cysts found in channels incubated at 25°, the great majority of which are found in close proximity to the parent colony. The effect of oxygen tension on average numbers of encysted zoospores is shown by Fig. 4a; partial pressures of oxygen between 103 and 154 mmHg do not greatly reduce the average number of cysts. However, a reduction in partial pressure of oxygen to 51 rnmHg dramatically reduced the average number of encysted zoospores. A further reduction of partial pressure to 10 rnmHg halts growth and zoospore production. The effect of oxygen tension on the distribution of encysted zoospores is shown

by Fig. 4b. The distribution pattern of cysts is in the main not markedly affected, being primarily governed by numbers of zoospores released. A partial pressure of 124 rnmHg does, however, appear mildly stimulatory to zoospore activity. The effect of copper and zinc ions on average numbers of encysted zoospores is shown by Figs 5 a and 6a. Copper ions drastically curtail zoospore production at a concentration of only o·5 mg/l ; concentrations in excess of 1 '0 mg/I halt zoospore production. Zinc ions are far less' toxic, with only a gradual decrease in the average number of encysted zoospores with increasing concentrations of the ion. The zinc ion, however, does significantly reduce the activity of zoospores, with few cysts being found far from those parent colonies exposed to zinc ions. The paraquat ion like the metal ions is also toxic, markedly reducing the average numbers of encysted zoospores with increasing concentrations (Fig. 7a). In similar fashion to the copper ion the paraquat ion does not appear to alter

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SS 9678-50 2395 6-65 30191-32 37-78 3416-67 (***

=

MS Df 3226-16 3 59 89-16 4 12 25 15-94 18-89 2 85-42 4° P < 0-001)

the distribution pattern of cysts, which appears to be mainly governed by the numbers of zoospores released. Fig. 8a, b demonstrates the interaction of amino acids and zoospore production and mobility; while three of the amino acids appear somewhat neutral in their effect, glutamic acid and proline appear somewhat stimulatory to zoospore production, However, the majority of the amino acids tested here appear attractive in varying degrees to zoospores. Of the amino acids tested, glutamic acid and aspartic acid were particularly attractive and to a lesser extent glycine and proline. Alanine exerts little effect, proving neither attractive not stimulatory to zoospore production.

F 37-76*** 70-11*** 29-45*** 0-22

DISCUSSION

The sensitivity of zoospores derived from the strain of Saprolegnia diclina utilized in this study precluded the use of techniques where measured concentrations of zoospores are utilized (Cameron & Carlile, 1977) as any undue manipulation of zoospores encourages rapid encystment. The techniques do however have a number of distinct advantages, in particular zoospores under investigation are produced by colonies exposed to an equivalent treatment, a condition closer to that prevailing in a more natural aquatic environment. In addition an indication of the effects of experimental factors on both numbers of zoospores produced and their activity is manifested by the

S. N. Smith, R. A. Armstrong and J. J. Rimmer

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SS

Df

MS

425 69. 62 273200. 80 98189.22

5 3 15

8513.92 91073. 60 6545·95

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191.33

F 44.5 0 *** 476.00 *** 34. 21***

(*** = P < 0·001)

distribution of cysts. In the absence of suitable attractants, zoospores encyst along the channels in a pattern commensurate with random movement. A modification of this pattern such as the clustering of zoospores around the colony indicates a reduction in activity. Zoospore production and activity may be influenced by a variety of environmental variables. Increasing water temperatures (to an optimum of 20°) increase the numbers of zoospores released with no apparent reduction in activity, thereby increasing the exposure of potential hosts to the pathogen. Any enhanced exposure of healthy fish to the pathogen resulting from increased water temperature will not necessarily result in greater incidence of fungal disease, as poikilotherm immunity may become more vigorous with increasing temperature (Avtalion, Weis & Moalem, 1976). Zoospores are produced and active over a wide range of pH; although lower pH may reduce

the number of spores the great majority are produced at pH 7'0, a value close to that of the majority oflocal river systems (Severn Trent Water Authority, 1979). Zoospore production and activity is also markedly influenced by oxygen tension with a considerable reduction in numbers of zoospores released at low oxygen tensions. Although certain cyprinids and salmonids can survive at oxygen tensions of 4 mmHg and 30 mmHg respectively (Macan, 1963) considerably greater oxygen tensions of 40 mmHg and 130 mmHg respectively are required for unrestrictive movement and feeding, this higher oxygen demand of salmonids also favours fungal zoospores. The production and activity of S. diclina zoospores is sensitive to biocides which may be naturally occurring, such as heavy metals (Whitton & Say, 1975), or introduced, as an algicide or herbicide like paraquat which is recommended for aquatic management programmes. The zoospores

Zoospores of Saprolegnia diclina appear little different from propagules of other fungal species whose physiology is particularly sensitive to disruption by copper and to a lesser extent zinc (Ross, 1975) and paraquat (Smith & Lyon, 1976). However, the use of such chemicals as prophylactic agents is likely to be of little value, as in a similar manner to S. diclina zoospores salmonids and cyprinids are particularly sensitive to copper and to a lesser extent zinc (Alabaster & Lloyd, 1980). S. diclina zoospores like those of many phytopathogenic fungi appear to demonstrate chemotaxis (Fuller, 1977). This response also appears amongst other Saprolegnia spp., with zoospores being attracted to exudates arising from wounds incurred by higher plant roots (Ho, 1975). The agents which attract zoospores may also be common; Royle & Hickman (1963) noted that glutamic acid attracted zoospores of Pythium aphanidermatum in a manner which closely matched that of root materials. In the present study glutamic acid also proved to be one of the strongest attractants to zoospores of S. diclina. Incidence of disease could therefore be reduced by careful handling to minimize damage to fish during stripping and grading, processes which may also encourage corticosteroid changes which may further promote infection by Saprolegnia spp. (Neish, 1977). Many of the studies which have investigated chemotaxis in zoospores have removed the zoospores from the influence of the parent colony and its nutrient source. In the study reported above this was not the case, yet in the absence of attractants other than the parent colony's nutrient source, the zoospores demonstrated a pattern of encystment commensurate with random movement. The addition of suitable attractants encourages a directional response indicating the subtlety of the mechanism that controls chemotaxis. The response is not influenced by the parent nutrient source, thereby allowing the zoospores to be dispersed by passive agents, yet appears influenced by extrinsic nutrient sources, thereby encouraging the colonization of fresh sites. REFERENCES

ALABASTER, J. S. & LLOYD, R. (1980). Water Quality Criteria for Freshwater Fish. London: Butterworths, AVTALION, R. R., WEIS, E. & MOALEM, T. (1976). Regulatory effects of temperature upon immunity in

42 1

ectothermic vertebrates. In Comparative Immunology (ed. J. J. Marchalonis),pp. 227-238. Oxford: Blackwell Scientific Publications. BEAKES, G. W. (1980). Electron microscopic study of oospore maturation and germination in an emasculated isolate of Saprolegniaferax. Changes in organelle status and associations. Canadian Journal of Botany 58, 209-227. CAMERON, N. J. & CARLILE, M. J. (1977). Negative geotaxis of zoospores of the fungus Phytophthora. Journal of General Microbiology 98, 599-602. COOKE, R. C. (1977). The Biology of Symbiotic Fungi. London, New York, Sydney, Toronto: Wiley. FULLER, M. S. (1977). The zoospore, hallmark of the aquatic fungi. Mycologia 69, 1-20. Ho, H. H. (1975). Observations on the behaviour of zoospores of a Saprolegnia species. Mycologia 67, 425-428. MACAN, T. T. (1963). Freshwater Ecology. London: Longmans. NEISH, G. A. (1977). Observations on saprolegniasis of adult sockeye salmon, Oncorhynchus nerka Walbaum. Journal of Fish Biology 10, 513-522. PICKERING, A. D., WILLOUGHBY, L. G. & MCGoRY, B. C. (1979). Fine structure of secondary zoospore cyst cases of Saprolegnia isolated from infected fish. Transactions of the British Mycological Society 7:1.,

427-436. PIELOU, E. C. (1969). An Introduction to Mathematical Ecology. London, New York, Sydney, Toronto: Wiley. RIDGMAN, W. J. (1975). Experimentation in Biology. Glasgow, London: Blackie. Ross, I. S. (1975). Some effectsof heavy metals on fungal cells. Transactions of the British Mycological Society 64, 175- 193. ROYLE, D. J. & HICKMAN, C. J. (1963).Analysisoffactors governing in vitro accumulation of zoospores of Pythium aphanidermatum on roots. Canadian Journal of Microbiology 10,201-219. SEVERN TRENT WATER AUTHORITY (1979). Water Quality. Birmingham. SMITH, S. N. & LYON, A. J. E. (1976). The uptake of paraquat by soil fungi. New Phytologist 76, 479-484. SNEDECOR, G. W. & COCHRANE, W. G. (1967). Statistical Methods, 6th ed. Ames: Iowa State University Press. WHITTON, B. A. & SAY, P. J. (1975). Heavy Metals. In River Ecology (ed. B. A. Whitton), pp. 286-311. Oxford, London, Edinburgh, Melbourne: Blackwell Scientific Publications. WILLOUGHBY, L. G. (1977). An abbreviated life cycle in the salmonid fish Saprolegnia. Transactions of the British Mycological Society 69, 133-135. WILLOUGHBY, L. G. (1978). Saprolegniasis of salmonid fish in Windermere: a critical analysis. Journal of Fish Diseases 1,51-67.

(Received for publication 14 October 1982)