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Effect of heavy metals (Pb, Zn, Cu and Cd) on germination of conidia of Cylindrocarpon destructans (Zinssm. ) Scholten
Zentralbl. Mikrobiol. 147 (1992),261-269 Gustav Fischer Yerlag lena [Laboratory of Microbiology, Institute of Biology, Nicolaus Copernicus University, Toruli, Poland]
Effect of Heavy Metals (Pb, Zn, Cu and Cd) on Germination of Conidia of Cylindrocarpon destructans (ZINSSM.) SCHOLTEN H. R6zYCKI With 2 Figures
Key words: Cylindrocarpon destructans, germination of conidia, inhibitory action of lead, zinc, copper and cadmium
Summary Studies were carried out on the effect of heavy metals (Pb, Zn, Cu and Cd) on germination of conidia (both % germination and germ tube length) in three isolates of Cylindrocarpon destructans (one - non-pathogenic and two - pathogenic to fir and pine seedlings). The inhibitory effect of heavy metals upon % spore germination and germ tube length increased in the following order: Zn < Pb < Cd -so Cu. Germ tube length was a more sensitive indicator of heavy metals toxicity (except Pb) than % spore germination. The relationship between germ tube length and percentage of germination was evaluated by cubic regression equations. Analysis of variance (ANOYA) has shown that metal concentrations affected both parameters of spore germination more strongly than the isolates. There were differences in reactions of different isolates to the presence of heavy metals tested.
Zusammenfassung Der Effekt von Schwermetallen (Pb, Zn, Cu, Cd) auf die Konidien-Keimung von Cylindrocarpon destructans wurde an 3 Isolaten (I nicht pathogen, 2 pathogen fiir Tannen- und Kiefern-Samlinge) untersucht. Der Hemmeffekt der Schwermetalle auf die Sporenkeimung (%) und auf die Lange des Keimschlauches stieg in der Reihenfolgen Zn < Pb < Cd -so Cu. Die Keimschlauchlange war ein empfindlicheres MaB fiir die Schwermetall-Toxizitat (auBer bei Pb) als die Sporenkeimung (% ). Die Korrelation zwischen Keimschlauchlange und prozentualer Sporenkeimung wurde mittels kubischer Regression bestimmt. Yarianzanalysen (ANOYA) zeigten, daB die beiden Parameter der Sporenkeimung starker durch Metalle beeinfluBt wurden als die Isolate. Die verschienen Isolate reagierten unterschiedlich auf die getesteten Metalle.
Cylindrocarpon destructans (ZINSSM.) SCHOLTEN (c. radicicola Wr.) (BOOTH 1966) has been isolated from the roots of both healthy and diseased plants (GERLACH 1964, Matturi and STENTON 1964, TAYLOR and PARKINSON 1964). Although according to MATT URI and STENTON (1964) this organism is not specialized in the direction of parasitism its pathogenicity for many different plants has been well documented (GERLACH 1964, MANKA et al. 1968, MANKA and GIERCZAK 1971, KOWALSKI 1980, 1982, DAHM and STRZELCZYK 1987, UNESTAM et al. 1990). Particularly C. destructans seems to be responsible for poor natural regeneration in Abies alba in Southern Poland (KOWALSKI 1980, 1982). At least part of these regions may be affected by heavy metal-smelting industry in Upper Silesia. Accumulation of heavy metals (the source - industrial emmisions) may cause the decrease of the rate of decomposition of organic matter - as a result of diminished soil microbial activity (JORDAN and LECHEVALIER 1975, BADURA et al. 1984a). There is a need of complex studies on the ecological impact of heavy metal pollution on soil microorganisms. Actually the effects of
262
H. R6ZYCKI
heavy metal pollution of microbial communities in soil, soil enzymatic activity, as well as on pure cultures of soil microbes have been extensively studied (JORDAN and LECHEV ALlER 1975 , BABICH and STOTZKY 1977, BADURA et al. 1984a, b, 1986a, b, PACHA 1988). As yet nothing is known about the effects of heavy metals on C. destructans . If this fungus would be sensitive to given heavy metal(s) it could serve as a bioindicator of the pollution with this metal. Therefore the present studies on spore germination C. 4estructans have been undertaken.
Materials and Methods Three isolates of Cylindrocmpon destructans (ZINSSM.) SCHOLTEN (two - No.6 and No.8 - pathogenic, and one - No. I - nonpathogenic to fir and pine seedlings) were used in this study . The fungsl isolates were kindly provided by Prof. Dr. Stefan Kowalski of the Agricultural University, Department of Forestry (Forest Pathology Laboratory), Cracow, Poland. The heavy metals tested were added to the modified liquid Czapek-Dox medium of the following composition (g. 1-1): 30 sucrose, 2 NaN0 3, 0.4 MgCIz· 6 H20, 0.5 KCl, 0.01 FeS04' 7HzO, 1000 ml H20 dest.; pH == 5.0 (phosphate was ommited and MgS04 was replaced by MgCl 2 to avoid precipitation of insoluble lead salts). Metals were tested in a variety of concentrations within the following ranges (as established in preliminary experiments): Pb 2 + (acetate) from 5 to 50 ppm, Zn 2 + (sulphate) from 50 to 2000 ppm, Cu2 + (sulphate) from 0.2 to 10 ppm, and Cd 2+ (acetate) from 0.5 tp 10 ppm. IO-fold concentrated solutions of the appropriate salts were autoclaved separately and then were mixed with the sterile medium at the ratio I : 9. For studies on the effect of heavy metals on spore germination the spore suspension prepared from 2 - 3 weeks old agar cultures [Potato Dextrose Agar (Difco), 20-22°C, at the light of fluorescent tubes (day light) - to induce sporulation] were used. After centrifugation the suspension (3000 r.p.m. during IS min.) was washed with sterile distilled water and repeatedly centrifuged. 30 !J.l portions of the media were pippeted on microscopic slides and inoculated with 3 !J.l of heavy conidial suspension (\ -5 . 106 spores per ml). The slides were placed in Petri dishes and kept in a moist chamber and incubated at 26°C for 16 h. Then the microcultures were fixed with lactophenol and covered with cover slips. During examination of germination 300 spores were considered in each experimental combination (3 replications - 100 spores each). The germ tube length (50 in each combination) was also considered. The results were expressed as percentage inhibition of spore germination (germ tube length) as compared to the control according to the following formula: Inhibition of spore germination (germ tube length) in percent = [(A-B)/A] . 100. A - germination of spores (germ tube length) in control (water instead of heavy metal salt solution), B - germination of spores (germ tube length) in presence of heavy metal. The results were presented as ED (effective dose) curves from which the EDso values (doses of heavy metal inhibiting 50% spore germination/germ tube length) were assigned. The relationship between germ tube length (y) and % spore germination (x) was evaluated by calculating polynominal regression equations and appropriate correlation coefficients. To evaluate the differences between isolates in reactions to the heavy metals tested 2 factor (isolates· metal concentrations) analysis of variance (ANOVA) was applied. A statistical package "Stats plus" (StatSoft, Tulsa, Oklahoma, U.S .A.) was used.
Results The results shown in Fig. 1 present the inhibition of spore germination and germ tube length by heavy metals. ED50 of heavy metals for three isolates of C. destructans are presented in Table l. It appears from Fig. 1 and Table 1 that in three strains studied the inhibitory effect of heavy metals upon both spore germination and germ tube length increased in the following order: Zn < Pb < Cd :$ Cu (values of ED50 for Pb and Zn were by 1- 3 and 2-4 orders of magnitude higher than those for Cu and/or Cd, respectively). % spore germination was affected similarly by lead and zinc in all isolates studied (Fig. 1 and Tab. 1). Lead caused very strong inhibition of this process within very narrow range of concentration (10-17 ppm). Zinc inhibited spore germination gradually within broad range of concentration (100-2000 ppm) (Fig. 1). Nonpathogenic strain No. 1 was severalfold less sensitive (% spore germination) to copper and cadmium than the remaining two isolates (Nos. 6 and 8) (Fig. 1, Tab. 1).
263
Effect of Heavy Metals on Germination
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log of metal concentration (ppm) Fig. I. Inhibition of germination of conidia (% spore germination and germ tube length) of Cyiilldrocarpoll destruetalls by heavy metals (Pb, Zn, Cu and Cd).
Table 1. Values of E0 50 (in ppm) of Pb2+ . Zn 2 + , Cu2+ and Cd 2 + for % spore germination and germ tube length in three isolates of Cylilldroearpoll destruetaJl~. Metal
% germination E0 50
Germ tube length E050
Pb Zn
16.380 820.950
11.04 67.39
Cu
1.640
O.IIS
Cd
1.780
0.631
6
Pb Zn Cu Cd
16.380 719.730 0.247 0.572
16.380 45.410 0.247 0.187
8
Pb Zn Cu Cd
12.1S0 848.400 0.235 0.572
11.41 89.13 0.0035 0.0178
Isolate
No.
264
H.
R 6 ZYCKI
In our studies germ tube length in C. destructans was affected more strongly be heavy metals (except lead ) than % spore germination (Fig. 1, Tab . 1) . In the all strains studied the values of EDso for Znl germ tube length were by one order of magnitude lower than those for % spore germination (Tab. 1) . EDso for Cu and Cd either did not reveal any difference (strain No.6 , Cu), or were also lower (3 - 800 times, depending on metal and strain tested) for germ
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% Germ inat io n Fig. 2. The relationship between % spore germination (x) and germ tube length (y) in three isolates of Cylindrocarpon destructalls as affected by Pb, Zn, Cu and Cd. T he "total" regression curves (for all the metals tested in each isolate) are drawn as solid li nes. The appropriate regression equations and correlation coefficients are given in Tab. 2.
tube length than for percentage germination of conidia (Tab . 1). It points out that the germ tube length is a more sensitive indicator of Zn, Cu and Cd toxicity than % spore germination in C. destructans (Fig. 1, Tab . 1) . Fig . 2 and Tab . 2 present the relationship between average germ tube length (y) and % spore germination (x) in 3 fungal isolates studied . It is obvious that regression is non-linear. In
Effect of Heavy Metals on Germination
265
Table 2. Regression equations and correlation coefficients (% spore germination - x, germ tube length - y) the supplement for Fig. 2. Isolate No
6
8
Metal
Regression equation
Correlation coefficient (R)
n (number of observations)
Pb Zn Cu Cd Total
Y = -10.837 + 4.722x - 0.1455x 2 + 0.001274x 3 y = 20.775 - 4.072x + 0.06592x2 Y = -8.814 + 5.219x - 0.1636x2 + 0.001383x 3 Y = -14.937 + 5.918x - 0.1788x2 + 0.001440x 3 y = -33.306 + 5.945x -0.1672x 2 + 0.001462x 3
0.9524 0.9844 0.9899 0.9238 0.9894
9 7 6 7 29
Pb Zn Cu Cd Total
y y y y
Y = -5.316 + 2.320x - 0.03778x2 + 0.000309x 3 = -6.246 + 5.716x - 0.1375x2 + 0.000978 x3 = 1.184 - 0.5391 x + 0.07541 x2 - 0.000584x 3 = -0.2184 + 0.07812x + 0.04016x l - 0.000212x 3 = -18.363 + 4.226x - 0.1186x2 + 0.000949x 3
0.8873 0.9778 0.9993 0.9985 0.8141
11 8 8 38
Pb Zn Cu Cd total
Y = -4.698 + 2.458x - 0.07744x2 + 0.000730x 3 y = 2.585 + 0.9448x - 0.04636x2 + 0.000599x 3 Y = -1.193 + 1.432x - 0.04340x 2 + 0.000493x 3 Y = 2.156 - 0.00928x + 0.01873x 2 y = -2.742 + 2.837x - 0.08445x 2 + 0.000717x 3
0.9323 0.9943 0.9998 0.999 0.8516
7 9 9 8 33
II
almost all cases (except strain No.8, cadmium) cubic regression equations were successfully fitted (regression and correlation significant at P :s 0.05). For the following metals tested strain No. 1 (non-pathogenic) revealed very similar regression germ tube length - spore germination. Isolate No.8 showed higher variation (than No.1) in this respect and isolate No. 6 - the highest variation (both pathogenic). In controls and in low metal concentration treatments germ tube length was in the following order: isolate No.1> No.6> No.8 (up to 300 !im in No. 1 and up to 200 !im in No.8) (Fig. 2, Tab.2). Analysis of variance (ANOV A) has shown that metal concentrations affected both % spore germination and germ tube length more strongly than the isolates (the effect of isolates was stronger in the presence of Pb and Cd than in the presence of Cu and Zn). Newman-Keuls multiple range test has shown that pathogenic isolate No. 6 was more tolerant to Pb and Zn than two remaining ones (No.1 - non-pathogenic and No.8 - pathogenic). Non-pathogenic isolate No.1 was more tolerant to Cu and Cd than two remaining ones (Tab. 3 and 4).
Discussion Up to date most of the studies on the effects of heavy metals on the soil microflora was dealing with the action of salts of these metals on microbial popUlations in soil as well as on pure cultures of saprophytic soil microbes (A VAKY AN 1967, BADURA et al. 1984a, b, 1986a, b). Less attention has received the action of heavy metals on the growth and development of plant pathogenic fungi - except testing of these metals as potential fungicides (HASALL 1977, KEAST et al. 1985). In the present work considerable differences in the action of different heavy metals on spore germination of Cylindrocarpon destructans were stated (the toxicity order: Zn < Pb < Cd:s Cu). Also AVAKYAN (1967) observed differences in toxicity of heavy metals towards saprophytic soil bacteria and yeasts. Cadmium was more toxic than zinc to Macrophomina phaseolina - both in pure cultures and in soil (DUBEY and DWIVEDI 1988). 19 Zentralbl. Mikrobiol., Bd. 147, 3-4
266
H. R6zYCKI
Table 3. 2-factor analysis of variance (ANOYA) comparing the effects of isolates and heavy metal concentrations on % spore germination of Cylindrocarpon destructans (% germination in 3 replicate sets of 100 conidia; per cent values were transformed: y = arc sin ]IX). Metal
Effect of:
Sum of squares
df*
Mean square
F parameter
P (Significance)
Ph
Isolates Concentrations Isol. . Conc. Error
4342.54 30955.05 4115.37 905.49
2 4 8 30
2171.27 7738.76 514.42 30.18
71.9 256.4 17.0
0.00000 0.00000 0.00000
Zn
Isolates Concentrations Iso!. . Cone. Error
193.68 25001.83 775.11 760.29
2 4 8 30
96.84 6250.46 96.89 25.34
3.8 246.6 3.8
0.03235 0.00000 0.00360
Cu
Isolates Concentrations Isol. . Conc. Error
3410.02 21754.10 1600.73 716.48
2 4 8 30
1705.01 5438.53 200.09 23.88
71.4 227.7 8.4
0.00000 0.00000 0.00004
Cd
Isolates Concentrations Isol. . Conc. Error
2452.60 20548.10 566.12 303.33
2 4 8 30
1226.30 5137.02 70.76 10.11
121.3 508.1 7.0
0.00000 0.00000 0.00001
Newman-Keuls multiple range test**
Pb: Zn: Cu: Cd:
Isolate No.1
No. 6
46.9787 48.2629 47.9013 45.8234
60.4386 52.6303 29.1188 34.3319
b ab b c
No.8 c b a b
36.4351 48.1967 29.7685 27.9856
a a a a
Explanations: four metal concentrations and no metal control were considered * - degrees of freedom ** - average values indicated in a given row by the same letter do not differ significantly (P
~
0.05).
It is difficult to compare the results obtained in the present studies with any data from the literature, because little work on the effects of heavy metals on fungal spore germination has been done (STRZELCZYK 1968, KOJIMA and URITANI 1978). We found that Cd2+ and particularly Cu 2 + were very toxic to spores of C. destructans (EDso' s: 0.255 to 1. 78 ppm for % spore germination and 0.0035 to 0.63 for germ tube length). According to HISLOP and PARK (1962, 1963 - c. f. HALSALL 1977) Hg2+ ion (contained in phenyl mercuric nitrate) was equally inhibitory for germination of zoospores of Phytophthora palmivora (EDso: 0,02 ppm). Also STRZELCZYK (1968) observed very strong inhibitory activity of phenyl mercuric acetate and mercuric chloride to spore germination of fungi deteriorating old books. Cu 2 + has been shown to be toxic to zoospores of Phytophthora at the level of 10- 7 M (0.00635 ppm) (HALSALL 1977, KEAST et al. 1985) revealing equal- or higher toxicity than we stated for conidia of C. destructans. In our studies Pb2 + exerted moderately toxic action on spore germination of C. destructans. In contrast with other metals tested it was inhibitory (both for % spore germination and germ tube length) within very narrow range of concentrations (10-17 ppm). It can be
Effect of Heavy Metals on Germination
267
Table 4. 2-factor ANOV A comparing the effects of isolates and heavy metal concentrations on germ tube length of Cylindrocarpon destructans [50 germ tubes were measured in each combination; values of germ tube length were transformed: y = log(x + 1)]. Metal
Effect of:
Sum of squares
df*
Mean squares
F parameter
P (Significance)
Pb
Isolates Concentrations Iso!. . Conc. Error
89.54 265.56 104.08 107.22
2 4 8 735
44.77 66.39 13.01 0.14
306.9 455.1 89.2
0.00000 0.00000 0.00000
Zn
Isolates Concentrations Iso!. . Cone. Error
3.70 227.68 8.31 61.24
2 4 8 735
1.85 56.92 1.04 0.08
22.2 683.1 12.5
0.00000 0.00000 0.00000
Cu
Isolates Concentrations Iso!. . Conc. Error
11.90 242.50 6.70 53.29
2 4 8 735
5.95 60.63 0.84 0.07
82.1 836.2 11.6
0.00000 0.00000 0.00000
Cd
Isolates Concentrations Iso!. . Conc. Error
37.44 244.76 9.97 55.78
2 4 8 735
18.72 61.19 1.25 0.08
246.7 806.3 16.4
0.00000 0.00000 0.00000
Newman-Keuls multiple range test**
Pb: Zn: Cu: Cd:
Isolate No. 1
No.6
1.47383 b 1.3697 a 1.37742 b 1.49039 c
1.92569 1.54061 1.08941 1.21845
No.8 c c a b
1.07998 1.43762 1.13746 0.94312
a b a a
Explanations: as in Tab. 3.
explained by: low mobility of Pb 2 + ions, adsorption of lead on spore cell walls and precipitation of lead phosphate salts (inside the cells) at pH > 6 (A VAKY AN 1967, BADURA et al. 1986b). In the present work very low toxicity of zinc to spore germination of C. destructans was stated (very weak stimulatory activity of Zn2+ at the lowest concentration (50 ppm) was observed). Low toxicity of Zn 2 + to the population of fungi in soil was also found by BADURA et al. (1979). Zn 2 + (at the concentration 10- 4 M = 6.54 ppm) is known to stimulate germination of conidia on C eratocystis Jimbriata (KOJIMA and URIT ANI 1978). We found stronger inhibitory activity of zinc, copper and cadmium to germ tube length than to % spore germination in C. destructans. It points out that germ tube length is a better indicator of their toxicity than % spore germination. Germ tube length usually has not been estimated in routine studies on different factors affecting fungal spore germination (STRZELCZYK 1968, STRZELCZYK et al. 1984, 1986). Amino acids and
268
H. ROZYCKI
Regression and correlation analysis of germ tube length versus % spore germination has shown the differences in this relationship under the effect of different heavy metals but only in two studied here pathogenic isolates of C. destructans - not in the non-pathogenic one. It may indicate some physiological differences between these isolates. But it is difficult to relate these differences to the potential pathogenicity of these fungal strains - this needs further studies. The applied in our work ANOV A has shown stronger effects of metal concentrations than those of isolates on parameters of spore germination. It may point out that sensitivity of isolates to heavy metals was of the same order of magnitude (impossible to distinguish: sensitive-versus resistant isolates). Differences in reactions of different fungal strains to the presence of heavy metals tested (shown by ANOVA and Newman-Keuls test) corresponded well with the values of EDso's - except those for Zn (% spore germination and germ tube length) and for eu (germ tube length only). This disagreement between the results of the two methods of evaluation of heavy metal toxicity to fungal strains suggests the need of further studies on improvement of these methods. In present work only the effects of heavy metals on spore germination of C. destructans were studied. Their action on mycelial growth may be different, it is known that concentrations of heavy metals inhibitory to mycelial growth my be by serveral orders of magnitude higher than those inhibitory to spore germination (HALSALL 1977, KEAST et al. 1985). Therefore further studies on the effects of heavy metals on mycelial growth of C. destructans are needed (they are in progress in our laboratory).
Acknowledgements I express my best thanks to Prof. Dr. Edmund Strzelczyk for his help and critical appraisal of the manuscript. The financial support from the Ministry of National Education is gratefully acknowledged.
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Effect of Heavy Metals on Germination
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HALSALL, D. M.: Effect of certain cations on the formation and infectivity of Phytophthora zoospores. 2. Effects of copper, boron, cobalt, manganese, molybdenum, and zinc ions. Can. J. Microbial. 23 (1977),
1002-1010. JORDAN, M. J., LECHEVALlER, M. P.: Effect of zinc-smelter on forest soil microflora. Can. J. Microbial. 21
(1975), 1855-1865. KEAST, D., TONKIN C., SANFELlEU, L.: Effects of cooper salts Qn growth and survival of Phytophthora cinnamomi in vitro and on the antifungal activity of actinomycete populations from the roots of Eucalyptus marginata and Banksia grandis. Aus!. J. Bot. 33 (1985), 115-129. KOJIMA, M., URITANI, 1.: Studies on factors in sweet potato root which induce spore germination. Plant Cell Physiol. 19 (1978), 91-97. KOWALSKI, S.: Cylindrocarpon destructans (ZINS.) SCHOLT. sprawca zamierania samosiewu jodly (Abies alba MILL.) w niekt6rych drzewostanach g6rskich poludniowej Polski. Acta Agr. et Silvestria, Ser. Silvestris 19
(1980),57-73. KOWALSKI, S.: Role of mycorrhiza and soil fungi in natural regeneration of fir (Abies alba MILL.) in Polish Carpathians and Sudetes. Eur. J. For. Path. 12 (1982), 107-1l2. MANKA, K., GIERCZAK, M.: 0 czynnikach sprawczych zgorzeli siewek sosny zwyczajnej w woj. poznanskim. Zesz. Probl. Pos!. Nauk Rain. 127 (1971), 87-95. MANKA, K., GIERCZAK, M., PRUSINKIEWICZ, Z.: Zamieranie siewek cisa (Taxus baccata L.) w Wierzchlesie na tie zepol6w saprofitycznych grzyb6w srodowiska glebowego, Prace Kom. Nauk Rain. i Kom. Nauk Lesn. PTPN 25 (1968),177-195. MATTURI, S. T., STENTON, H.: Distribution and status in the soil of Cylindrocarpon species. Trans. Brit. Mycol. Soc. 47 (1964), 577-587. PACHA, J.: Wp!yw zwi,!zk6w tr6j- i szesciowartosciowego chromu na aktywnosc wybranych enzym6w w glebie kwasnej i zasadowej (English summary). Acta BioI. Silesiana 9 (1988), 50-862. R6zYCKI, H., STRZELCZYK, E.: Effect of some amino- and organic acids on growth and spore germination of Cylindrocarpon destructans (ZINS,/SCHOLT.). Zbl. Mikrobiol. 143 (1988), 405-412. STRZELCZYK, A. B.: Influence of antifungal vapors on spore germination of fungi isolated from deteriorated old books. Can. J. Microbial. 14 (1968), 901-906. STRZELCZYK, E., R6zYCKI, H., MlcHNIEwlcz, M.: Effect of bacteria and actinomycetes on germination of conidia of Cylindrocarpon destructans isolates pathogenic to Abies alba and Pinus sylvestris. Eur. 1. For. Path. 14 (1984),265-270. STRZELCZYK, E., R6zYCKI, H., MICHNIEWICZ, M.: Growth and spore germination of Cylindrocarpon destructans in associative cultures with bacteria and actinomycetes. Eur. J. For. Path. 16 (1986), 11-15. TAYLOR, G. S., PARKINSON, D.: Studies on fungi in the root region. II. The effect of certain environmental conditions on the development of root surface mycofloras of dwarf bean seedlings. Plant, Soil 20 (1964),
34-42. UNESTAM, T., BEYER-ERICSON, L., STRAND, M.: Involvement of Cylindrocarpon destructans in root death of Scots pine seedlings: pathogenic behaviour and predisposing factors. Scand. J. For. Res. 4 (1990) (in print). Author's address: Dr. HENRYK R6zYCKI, Laboratory of Microbiology, Institute of Biology, Nicolaus Copernicus University, ul. Gagarina 9,87-100 Torun, Poland.
Effect of Heavy Metals (Pb, Zn, Cu and Cd) on Germination of Conidia of Cylindrocarpon destructans (ZINSSM.) SCHOLTEN H. R6zYCKI With 2 Figures
Key words: Cylindrocarpon destructans, germination of conidia, inhibitory action of lead, zinc, copper and cadmium
Summary Studies were carried out on the effect of heavy metals (Pb, Zn, Cu and Cd) on germination of conidia (both % germination and germ tube length) in three isolates of Cylindrocarpon destructans (one - non-pathogenic and two - pathogenic to fir and pine seedlings). The inhibitory effect of heavy metals upon % spore germination and germ tube length increased in the following order: Zn < Pb < Cd -so Cu. Germ tube length was a more sensitive indicator of heavy metals toxicity (except Pb) than % spore germination. The relationship between germ tube length and percentage of germination was evaluated by cubic regression equations. Analysis of variance (ANOYA) has shown that metal concentrations affected both parameters of spore germination more strongly than the isolates. There were differences in reactions of different isolates to the presence of heavy metals tested.
Zusammenfassung Der Effekt von Schwermetallen (Pb, Zn, Cu, Cd) auf die Konidien-Keimung von Cylindrocarpon destructans wurde an 3 Isolaten (I nicht pathogen, 2 pathogen fiir Tannen- und Kiefern-Samlinge) untersucht. Der Hemmeffekt der Schwermetalle auf die Sporenkeimung (%) und auf die Lange des Keimschlauches stieg in der Reihenfolgen Zn < Pb < Cd -so Cu. Die Keimschlauchlange war ein empfindlicheres MaB fiir die Schwermetall-Toxizitat (auBer bei Pb) als die Sporenkeimung (% ). Die Korrelation zwischen Keimschlauchlange und prozentualer Sporenkeimung wurde mittels kubischer Regression bestimmt. Yarianzanalysen (ANOYA) zeigten, daB die beiden Parameter der Sporenkeimung starker durch Metalle beeinfluBt wurden als die Isolate. Die verschienen Isolate reagierten unterschiedlich auf die getesteten Metalle.
Cylindrocarpon destructans (ZINSSM.) SCHOLTEN (c. radicicola Wr.) (BOOTH 1966) has been isolated from the roots of both healthy and diseased plants (GERLACH 1964, Matturi and STENTON 1964, TAYLOR and PARKINSON 1964). Although according to MATT URI and STENTON (1964) this organism is not specialized in the direction of parasitism its pathogenicity for many different plants has been well documented (GERLACH 1964, MANKA et al. 1968, MANKA and GIERCZAK 1971, KOWALSKI 1980, 1982, DAHM and STRZELCZYK 1987, UNESTAM et al. 1990). Particularly C. destructans seems to be responsible for poor natural regeneration in Abies alba in Southern Poland (KOWALSKI 1980, 1982). At least part of these regions may be affected by heavy metal-smelting industry in Upper Silesia. Accumulation of heavy metals (the source - industrial emmisions) may cause the decrease of the rate of decomposition of organic matter - as a result of diminished soil microbial activity (JORDAN and LECHEVALIER 1975, BADURA et al. 1984a). There is a need of complex studies on the ecological impact of heavy metal pollution on soil microorganisms. Actually the effects of
262
H. R6ZYCKI
heavy metal pollution of microbial communities in soil, soil enzymatic activity, as well as on pure cultures of soil microbes have been extensively studied (JORDAN and LECHEV ALlER 1975 , BABICH and STOTZKY 1977, BADURA et al. 1984a, b, 1986a, b, PACHA 1988). As yet nothing is known about the effects of heavy metals on C. destructans . If this fungus would be sensitive to given heavy metal(s) it could serve as a bioindicator of the pollution with this metal. Therefore the present studies on spore germination C. 4estructans have been undertaken.
Materials and Methods Three isolates of Cylindrocmpon destructans (ZINSSM.) SCHOLTEN (two - No.6 and No.8 - pathogenic, and one - No. I - nonpathogenic to fir and pine seedlings) were used in this study . The fungsl isolates were kindly provided by Prof. Dr. Stefan Kowalski of the Agricultural University, Department of Forestry (Forest Pathology Laboratory), Cracow, Poland. The heavy metals tested were added to the modified liquid Czapek-Dox medium of the following composition (g. 1-1): 30 sucrose, 2 NaN0 3, 0.4 MgCIz· 6 H20, 0.5 KCl, 0.01 FeS04' 7HzO, 1000 ml H20 dest.; pH == 5.0 (phosphate was ommited and MgS04 was replaced by MgCl 2 to avoid precipitation of insoluble lead salts). Metals were tested in a variety of concentrations within the following ranges (as established in preliminary experiments): Pb 2 + (acetate) from 5 to 50 ppm, Zn 2 + (sulphate) from 50 to 2000 ppm, Cu2 + (sulphate) from 0.2 to 10 ppm, and Cd 2+ (acetate) from 0.5 tp 10 ppm. IO-fold concentrated solutions of the appropriate salts were autoclaved separately and then were mixed with the sterile medium at the ratio I : 9. For studies on the effect of heavy metals on spore germination the spore suspension prepared from 2 - 3 weeks old agar cultures [Potato Dextrose Agar (Difco), 20-22°C, at the light of fluorescent tubes (day light) - to induce sporulation] were used. After centrifugation the suspension (3000 r.p.m. during IS min.) was washed with sterile distilled water and repeatedly centrifuged. 30 !J.l portions of the media were pippeted on microscopic slides and inoculated with 3 !J.l of heavy conidial suspension (\ -5 . 106 spores per ml). The slides were placed in Petri dishes and kept in a moist chamber and incubated at 26°C for 16 h. Then the microcultures were fixed with lactophenol and covered with cover slips. During examination of germination 300 spores were considered in each experimental combination (3 replications - 100 spores each). The germ tube length (50 in each combination) was also considered. The results were expressed as percentage inhibition of spore germination (germ tube length) as compared to the control according to the following formula: Inhibition of spore germination (germ tube length) in percent = [(A-B)/A] . 100. A - germination of spores (germ tube length) in control (water instead of heavy metal salt solution), B - germination of spores (germ tube length) in presence of heavy metal. The results were presented as ED (effective dose) curves from which the EDso values (doses of heavy metal inhibiting 50% spore germination/germ tube length) were assigned. The relationship between germ tube length (y) and % spore germination (x) was evaluated by calculating polynominal regression equations and appropriate correlation coefficients. To evaluate the differences between isolates in reactions to the heavy metals tested 2 factor (isolates· metal concentrations) analysis of variance (ANOVA) was applied. A statistical package "Stats plus" (StatSoft, Tulsa, Oklahoma, U.S .A.) was used.
Results The results shown in Fig. 1 present the inhibition of spore germination and germ tube length by heavy metals. ED50 of heavy metals for three isolates of C. destructans are presented in Table l. It appears from Fig. 1 and Table 1 that in three strains studied the inhibitory effect of heavy metals upon both spore germination and germ tube length increased in the following order: Zn < Pb < Cd :$ Cu (values of ED50 for Pb and Zn were by 1- 3 and 2-4 orders of magnitude higher than those for Cu and/or Cd, respectively). % spore germination was affected similarly by lead and zinc in all isolates studied (Fig. 1 and Tab. 1). Lead caused very strong inhibition of this process within very narrow range of concentration (10-17 ppm). Zinc inhibited spore germination gradually within broad range of concentration (100-2000 ppm) (Fig. 1). Nonpathogenic strain No. 1 was severalfold less sensitive (% spore germination) to copper and cadmium than the remaining two isolates (Nos. 6 and 8) (Fig. 1, Tab. 1).
263
Effect of Heavy Metals on Germination
!
I
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Isolate 1
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.--.Zn
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3
5
log of metal concentration (ppm) Fig. I. Inhibition of germination of conidia (% spore germination and germ tube length) of Cyiilldrocarpoll destruetalls by heavy metals (Pb, Zn, Cu and Cd).
Table 1. Values of E0 50 (in ppm) of Pb2+ . Zn 2 + , Cu2+ and Cd 2 + for % spore germination and germ tube length in three isolates of Cylilldroearpoll destruetaJl~. Metal
% germination E0 50
Germ tube length E050
Pb Zn
16.380 820.950
11.04 67.39
Cu
1.640
O.IIS
Cd
1.780
0.631
6
Pb Zn Cu Cd
16.380 719.730 0.247 0.572
16.380 45.410 0.247 0.187
8
Pb Zn Cu Cd
12.1S0 848.400 0.235 0.572
11.41 89.13 0.0035 0.0178
Isolate
No.
264
H.
R 6 ZYCKI
In our studies germ tube length in C. destructans was affected more strongly be heavy metals (except lead ) than % spore germination (Fig. 1, Tab . 1) . In the all strains studied the values of EDso for Znl germ tube length were by one order of magnitude lower than those for % spore germination (Tab. 1) . EDso for Cu and Cd either did not reveal any difference (strain No.6 , Cu), or were also lower (3 - 800 times, depending on metal and strain tested) for germ
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160 120 80 40 0
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% Germ inat io n Fig. 2. The relationship between % spore germination (x) and germ tube length (y) in three isolates of Cylindrocarpon destructalls as affected by Pb, Zn, Cu and Cd. T he "total" regression curves (for all the metals tested in each isolate) are drawn as solid li nes. The appropriate regression equations and correlation coefficients are given in Tab. 2.
tube length than for percentage germination of conidia (Tab . 1). It points out that the germ tube length is a more sensitive indicator of Zn, Cu and Cd toxicity than % spore germination in C. destructans (Fig. 1, Tab . 1) . Fig . 2 and Tab . 2 present the relationship between average germ tube length (y) and % spore germination (x) in 3 fungal isolates studied . It is obvious that regression is non-linear. In
Effect of Heavy Metals on Germination
265
Table 2. Regression equations and correlation coefficients (% spore germination - x, germ tube length - y) the supplement for Fig. 2. Isolate No
6
8
Metal
Regression equation
Correlation coefficient (R)
n (number of observations)
Pb Zn Cu Cd Total
Y = -10.837 + 4.722x - 0.1455x 2 + 0.001274x 3 y = 20.775 - 4.072x + 0.06592x2 Y = -8.814 + 5.219x - 0.1636x2 + 0.001383x 3 Y = -14.937 + 5.918x - 0.1788x2 + 0.001440x 3 y = -33.306 + 5.945x -0.1672x 2 + 0.001462x 3
0.9524 0.9844 0.9899 0.9238 0.9894
9 7 6 7 29
Pb Zn Cu Cd Total
y y y y
Y = -5.316 + 2.320x - 0.03778x2 + 0.000309x 3 = -6.246 + 5.716x - 0.1375x2 + 0.000978 x3 = 1.184 - 0.5391 x + 0.07541 x2 - 0.000584x 3 = -0.2184 + 0.07812x + 0.04016x l - 0.000212x 3 = -18.363 + 4.226x - 0.1186x2 + 0.000949x 3
0.8873 0.9778 0.9993 0.9985 0.8141
11 8 8 38
Pb Zn Cu Cd total
Y = -4.698 + 2.458x - 0.07744x2 + 0.000730x 3 y = 2.585 + 0.9448x - 0.04636x2 + 0.000599x 3 Y = -1.193 + 1.432x - 0.04340x 2 + 0.000493x 3 Y = 2.156 - 0.00928x + 0.01873x 2 y = -2.742 + 2.837x - 0.08445x 2 + 0.000717x 3
0.9323 0.9943 0.9998 0.999 0.8516
7 9 9 8 33
II
almost all cases (except strain No.8, cadmium) cubic regression equations were successfully fitted (regression and correlation significant at P :s 0.05). For the following metals tested strain No. 1 (non-pathogenic) revealed very similar regression germ tube length - spore germination. Isolate No.8 showed higher variation (than No.1) in this respect and isolate No. 6 - the highest variation (both pathogenic). In controls and in low metal concentration treatments germ tube length was in the following order: isolate No.1> No.6> No.8 (up to 300 !im in No. 1 and up to 200 !im in No.8) (Fig. 2, Tab.2). Analysis of variance (ANOV A) has shown that metal concentrations affected both % spore germination and germ tube length more strongly than the isolates (the effect of isolates was stronger in the presence of Pb and Cd than in the presence of Cu and Zn). Newman-Keuls multiple range test has shown that pathogenic isolate No. 6 was more tolerant to Pb and Zn than two remaining ones (No.1 - non-pathogenic and No.8 - pathogenic). Non-pathogenic isolate No.1 was more tolerant to Cu and Cd than two remaining ones (Tab. 3 and 4).
Discussion Up to date most of the studies on the effects of heavy metals on the soil microflora was dealing with the action of salts of these metals on microbial popUlations in soil as well as on pure cultures of saprophytic soil microbes (A VAKY AN 1967, BADURA et al. 1984a, b, 1986a, b). Less attention has received the action of heavy metals on the growth and development of plant pathogenic fungi - except testing of these metals as potential fungicides (HASALL 1977, KEAST et al. 1985). In the present work considerable differences in the action of different heavy metals on spore germination of Cylindrocarpon destructans were stated (the toxicity order: Zn < Pb < Cd:s Cu). Also AVAKYAN (1967) observed differences in toxicity of heavy metals towards saprophytic soil bacteria and yeasts. Cadmium was more toxic than zinc to Macrophomina phaseolina - both in pure cultures and in soil (DUBEY and DWIVEDI 1988). 19 Zentralbl. Mikrobiol., Bd. 147, 3-4
266
H. R6zYCKI
Table 3. 2-factor analysis of variance (ANOYA) comparing the effects of isolates and heavy metal concentrations on % spore germination of Cylindrocarpon destructans (% germination in 3 replicate sets of 100 conidia; per cent values were transformed: y = arc sin ]IX). Metal
Effect of:
Sum of squares
df*
Mean square
F parameter
P (Significance)
Ph
Isolates Concentrations Isol. . Conc. Error
4342.54 30955.05 4115.37 905.49
2 4 8 30
2171.27 7738.76 514.42 30.18
71.9 256.4 17.0
0.00000 0.00000 0.00000
Zn
Isolates Concentrations Iso!. . Cone. Error
193.68 25001.83 775.11 760.29
2 4 8 30
96.84 6250.46 96.89 25.34
3.8 246.6 3.8
0.03235 0.00000 0.00360
Cu
Isolates Concentrations Isol. . Conc. Error
3410.02 21754.10 1600.73 716.48
2 4 8 30
1705.01 5438.53 200.09 23.88
71.4 227.7 8.4
0.00000 0.00000 0.00004
Cd
Isolates Concentrations Isol. . Conc. Error
2452.60 20548.10 566.12 303.33
2 4 8 30
1226.30 5137.02 70.76 10.11
121.3 508.1 7.0
0.00000 0.00000 0.00001
Newman-Keuls multiple range test**
Pb: Zn: Cu: Cd:
Isolate No.1
No. 6
46.9787 48.2629 47.9013 45.8234
60.4386 52.6303 29.1188 34.3319
b ab b c
No.8 c b a b
36.4351 48.1967 29.7685 27.9856
a a a a
Explanations: four metal concentrations and no metal control were considered * - degrees of freedom ** - average values indicated in a given row by the same letter do not differ significantly (P
~
0.05).
It is difficult to compare the results obtained in the present studies with any data from the literature, because little work on the effects of heavy metals on fungal spore germination has been done (STRZELCZYK 1968, KOJIMA and URITANI 1978). We found that Cd2+ and particularly Cu 2 + were very toxic to spores of C. destructans (EDso' s: 0.255 to 1. 78 ppm for % spore germination and 0.0035 to 0.63 for germ tube length). According to HISLOP and PARK (1962, 1963 - c. f. HALSALL 1977) Hg2+ ion (contained in phenyl mercuric nitrate) was equally inhibitory for germination of zoospores of Phytophthora palmivora (EDso: 0,02 ppm). Also STRZELCZYK (1968) observed very strong inhibitory activity of phenyl mercuric acetate and mercuric chloride to spore germination of fungi deteriorating old books. Cu 2 + has been shown to be toxic to zoospores of Phytophthora at the level of 10- 7 M (0.00635 ppm) (HALSALL 1977, KEAST et al. 1985) revealing equal- or higher toxicity than we stated for conidia of C. destructans. In our studies Pb2 + exerted moderately toxic action on spore germination of C. destructans. In contrast with other metals tested it was inhibitory (both for % spore germination and germ tube length) within very narrow range of concentrations (10-17 ppm). It can be
Effect of Heavy Metals on Germination
267
Table 4. 2-factor ANOV A comparing the effects of isolates and heavy metal concentrations on germ tube length of Cylindrocarpon destructans [50 germ tubes were measured in each combination; values of germ tube length were transformed: y = log(x + 1)]. Metal
Effect of:
Sum of squares
df*
Mean squares
F parameter
P (Significance)
Pb
Isolates Concentrations Iso!. . Conc. Error
89.54 265.56 104.08 107.22
2 4 8 735
44.77 66.39 13.01 0.14
306.9 455.1 89.2
0.00000 0.00000 0.00000
Zn
Isolates Concentrations Iso!. . Cone. Error
3.70 227.68 8.31 61.24
2 4 8 735
1.85 56.92 1.04 0.08
22.2 683.1 12.5
0.00000 0.00000 0.00000
Cu
Isolates Concentrations Iso!. . Conc. Error
11.90 242.50 6.70 53.29
2 4 8 735
5.95 60.63 0.84 0.07
82.1 836.2 11.6
0.00000 0.00000 0.00000
Cd
Isolates Concentrations Iso!. . Conc. Error
37.44 244.76 9.97 55.78
2 4 8 735
18.72 61.19 1.25 0.08
246.7 806.3 16.4
0.00000 0.00000 0.00000
Newman-Keuls multiple range test**
Pb: Zn: Cu: Cd:
Isolate No. 1
No.6
1.47383 b 1.3697 a 1.37742 b 1.49039 c
1.92569 1.54061 1.08941 1.21845
No.8 c c a b
1.07998 1.43762 1.13746 0.94312
a b a a
Explanations: as in Tab. 3.
explained by: low mobility of Pb 2 + ions, adsorption of lead on spore cell walls and precipitation of lead phosphate salts (inside the cells) at pH > 6 (A VAKY AN 1967, BADURA et al. 1986b). In the present work very low toxicity of zinc to spore germination of C. destructans was stated (very weak stimulatory activity of Zn2+ at the lowest concentration (50 ppm) was observed). Low toxicity of Zn 2 + to the population of fungi in soil was also found by BADURA et al. (1979). Zn 2 + (at the concentration 10- 4 M = 6.54 ppm) is known to stimulate germination of conidia on C eratocystis Jimbriata (KOJIMA and URIT ANI 1978). We found stronger inhibitory activity of zinc, copper and cadmium to germ tube length than to % spore germination in C. destructans. It points out that germ tube length is a better indicator of their toxicity than % spore germination. Germ tube length usually has not been estimated in routine studies on different factors affecting fungal spore germination (STRZELCZYK 1968, STRZELCZYK et al. 1984, 1986). Amino acids and
268
H. ROZYCKI
Regression and correlation analysis of germ tube length versus % spore germination has shown the differences in this relationship under the effect of different heavy metals but only in two studied here pathogenic isolates of C. destructans - not in the non-pathogenic one. It may indicate some physiological differences between these isolates. But it is difficult to relate these differences to the potential pathogenicity of these fungal strains - this needs further studies. The applied in our work ANOV A has shown stronger effects of metal concentrations than those of isolates on parameters of spore germination. It may point out that sensitivity of isolates to heavy metals was of the same order of magnitude (impossible to distinguish: sensitive-versus resistant isolates). Differences in reactions of different fungal strains to the presence of heavy metals tested (shown by ANOVA and Newman-Keuls test) corresponded well with the values of EDso's - except those for Zn (% spore germination and germ tube length) and for eu (germ tube length only). This disagreement between the results of the two methods of evaluation of heavy metal toxicity to fungal strains suggests the need of further studies on improvement of these methods. In present work only the effects of heavy metals on spore germination of C. destructans were studied. Their action on mycelial growth may be different, it is known that concentrations of heavy metals inhibitory to mycelial growth my be by serveral orders of magnitude higher than those inhibitory to spore germination (HALSALL 1977, KEAST et al. 1985). Therefore further studies on the effects of heavy metals on mycelial growth of C. destructans are needed (they are in progress in our laboratory).
Acknowledgements I express my best thanks to Prof. Dr. Edmund Strzelczyk for his help and critical appraisal of the manuscript. The financial support from the Ministry of National Education is gratefully acknowledged.
References AVAKYAN, Z. A.: Comparative toxicity of heavy metals for certain microorganisms. Mikrobiologiya 36 (1967), 446-450. BABICH, H., STOTZKY, G.: Sensitivity of various bacteria including actinomycetes, and fungi to cadmium and the influence of pH on sensitivity. Appl. Environ. Microbiol. 33 (1977), 681-695. BADURA, L., GORSKA, B., ULFIG, K.: Oddzia!ywanie soli cynku i miedzi na drobnoustroje gleby. Cz. I. Reakcje grzyb6w (English summary). Acta BioI. (Katowice) 7 (1979), 89-99. BADURA, L., GALIMSKA-STYPA, R., GORSKA, B., SMYLLA, A.: Wp!yw emisji huty cynku na drobnoustroje glebowe (English summary). Ibid. 15 (1984a), 112-127. BADURA, L., MROZOWSKA, 1., KAJZEREK, B.: Pr6ba adaptacji wybranych szczep6w bakterii glebowych do wzrastaji,lcych st~zen cynku i miedzi (English summary). Ibid. 15 (1984b), 96-1O\. BADURA, L., GORSKA, B., MIKOLAJCZYK, M.: Wp!yw jon6w chromu na mikrogrzyby glebowe (English summary). Acta BioI. Silesiana 3 (1986a), 85-94. BADURA, L., SMYLLA, A., SZUMLAS, 1.: Zbadanie wrailiwosci promieniowc6w najony olowiu oraz pr6ba ich adaptacji (English summary). Ibid. 3 (l986b), 59-67. BOOTH, C.: The genus Cylindrocarpon. Mycol. Pap. 104 (1966), I-57. DAHM, H., STRZELCZYK, E.: Effect of pH, temperature and light on the pathogenicity of Cylindrocarpon destructans to pine seedlings in associative cultures with bacteria and actinomycetes. Eur. 1. For. Path. 17 (1987), 141-148. DUBEY, R. C., DWIVEDI, R. S.: Effect of heavy metals on growth and survival of Macrophomina phaseolina (Toissi) Goid. BioI. Fertil. Soils 6 (1988), 311-314. GERLACH, W.: Beitrage zur Kenntnis der Gattung Cylindrocarpon Wr. IV. Cylindrocarpon radicicola Wr., seine phytopathologische Bedeutung und sein Auftreten als Erreger einer Faule des Usambaraveilchens. Phytopath. Z. 41 (1964), 361-369.
Effect of Heavy Metals on Germination
269
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