Fungal flora of soil polluted with copper

Soil Viol. Bioehem. Vol. 17, No. 6. pp. 785-790, 1985 Printed in Great Britain. All rights reserved

FUNGAL

0038-0717/85 $3.00 + 0.00 Copyright 0 1985 Pergamon PressLtd

FLORA OF SOIL POLLUTED WITH COPPER

HIROKI YAMAMOTO, KADZUNORI TATSUYAMA Faculty

of Agriculture,

Shimane

University,

(Areepred

and TOSIHARU UCHIWA

Matsue,

Shimane

690, Japan

15 April 1985)

Summary-The effects of copper pollution on the soil fungal flora was investigated. Soils treated with 100, 200, 400. 800 or 1600 pg Cu gg’ were used for experiments to study changes in fungal populations,

especially the development and dominance of copper-tolerant fungi. Fungi were sampled 1, 3 and 5 months after copper treatment. All the correlation coefficients between the copper contents and the number of fungal colonies plated were positive. The higher the copper concentration in soil. the more 1OOO~gCu ml-’ tolerant fungi were isolated. The relative number of 1000 pg Cu ml-’ tolerant fungi from the soil treated with 1600 t.cgCu gg’ was about 305; of those of the control 14 days after treatment. Within the limits of this experiment, the increase in fungal populations was directly correlated with the increase of dominant Cu-toierant fungi. From control soils, containing low quantities of copper, lOOO/lgCu ml-’ tolerant fungi were also isolated: whereas. from soils containing high amounts of copper, some Cu-sensitive fungi were isolated. Most of the IOOOhgCuml-' tolerant fungi were Penicillium spp. It was concluded that the genus PeniciNium may be dominant in soils polluted with copper.

The rapid increase in industrial activities has been responsible for a serious problem of soil contamination with heavy metals. Accordingly, there have been a number of investigations reporting the chemical form, behavior and plant response to toxic levels of heavy metals in soil. Moreover, the effects of heavy metals on microorganisms have been investigated on aspects of the N-cycle, e.g. ammonification, nitrification, (Quraishi and Cornfield, 1973; Tyler, 1975; Wilson, 1977; Mathur and Preston, 1981; Rother ef al., 1982; Yamamoto et al., 1983) as well as the C-cycle, e.g. soil respiration, cellulose degradation, (Bhuiya and Cornfield, 1972; Chaney et af., 1978; Doelman and Haanstra, 1979a; Tatsuyama et al., 1981). Many studies on the effects of heavy metals, particularly copper and mercury, on fungal cells have been reported as a result of their value as fungicides (Parry and Wood, 1958; Ashida, 1965; Ross, 1975). Less attention has been given to the effects of heavy metals on microbial ecology. Doelman and Haanstra (1979b) and Olson and Thornton (1982) investigated the effects of heavy metals on the bacterial microflora. Kendrick (1962), Lawrey (1977) and Zibilske and Wagner (1982) studied the fungal distribution in soil polluted with heavy metais. Tatsuyama et al. (1974, 1975, 1977) and Yamamoto et al. (1981) reported the effects of cadmium and copper on soil fungal flora. In the latter studies, the authors found that the fungal flora was simplified and that Penicillium spp were dominant. In spite of these reports, the effect of heavy metals on the soil fungal flora is not well known. We investigated the effect of copper on the soil fungal flora, especially the development and dominance of Cu-tolerant fungi.

used were as follows: pH(KC1) 5.0; organic matter 2.8%; total N 0.45%; cation-exchange capacity 16.5 m-equiv 100 g-‘; texture: coarse sand 20x, fine sand 29.0x, silt lS.l%, clay 27.6%; maximum moisture-holding capacity 44.0%. The soil was collected from a paddy field (Shimane University Central Farm), sieved (~2 mm) and placed in plastic containers (15 x 6 x IO cm). These were then kept under greenhouse conditions (non-heated, frost free) and watered to maintain the moisture content at approximately 20%. After 6 months, the soil was adjusted to 100,200,400, 800 or 1600 pg Cu gg’ dry soil by uniform spraying with CuS04 solution. These treatment made the final moisture contents about 25%. Control soil was also adjusted to a similar moisture content with deionized water. All soils were then maintained under the previous conditions for the duration of the experiment. After 1, 3 and 5 months from the time of copper treatment about 60 g of soil was taken from 3 containers per treatment. These soil samples were used for copper analysis and microbial experiments.

MATERIALS AND METHODS

Soils

The physical and chemical properties of the soil 785

Copper analysis

Copper in the soils was analyzed by atomic absorption spectrophotometry (Hitachi 170-40) after the one of six extraction procedures described below. (1) Water extraction: Five gram aliquots of each soil sample were placed in 100ml Erlenmeyer flasks and 50 ml of deionized water was added. Then the mixture was shaken for I h, and centrifuged at 1000 g for 50 min. The copper in the supernatant was determined. (2) 1 M MgC& extraction: The water extraction method was used, but 1 M MgCl, solution was substituted for deionized water. (3) 0.1 M EDTA extraction: Again the water extraction method was used, but 0.1 M EDTA solution was substituted for deionized water. (4) 0.1 M HCl or 1 M HCl extraction: Five gram aliquots were shaken for 1 h with 50 ml of 0.1 M or

1%

HIR~KI

YAMAM~T~

1 M HCl and the soil-acid mixture was filtered (Toyo Roshi, No. 2). The copper in the filtrate was determined. (5) Wet digestion: One gram aliquots were transfered to 100 ml Kjeldahl flasks to which 1 ml of H,SO,, 5 ml of HNO, and 20ml of HCIO, were added. The soil-acid mixture was heated and thickened by evaporation, after cooling, the mixture was then filled to 10 ml with 0.1 M HCI. The copper in the solution was directly determined. Microbial

Copper extracted with 1 M HCI or HClO, digestion were the highest, whereas the lowest was with H,O extraction. The values obtained after 1 month were greater than expected values with HClO,, 1 M HCl and 0.1 M HCI. These results may be attributed to the accumulation of treated copper in the soil surface layer by capillary action and other factors. Although copper contents estimated in the soil decreased considerably for a few months after the treatment, copper levels became comparatively stable after 3 months. To evaluate the availability of copper to the microorganisms, correlation coefficients between the copper contents of the soils and the number of fungal colonies are shown in Table 2. At all sampling times with all of the extraction methods, positive correlations were found and the coefficients for 1000 pg Cu ml- ’ tolerant fungi were highest and significant at P = 0.01. The coefficients for copper content in 0.1 M EDTA extract were higher than those in other extracts, it was suggested that the relation between copper content in 0.1 M EDTA extract and the number of fungal colonies was close in the soil polluted with copper.

experiments

Fungal propagules were enumerated by the agar plate method. It is well known that the number of fungal colonies on an agar plate may not reflect their activity in soil in a quantitative way because most colonies appearing on the plate may be derived from spores. However. fungal sporulation originates from mycehal activity, so under certain situations such as in this study, the relative number of colonies indicates the degree of dominance of a species in the soil sample. PSA (potato sucrose agar), containing 50 pg streptomycin ml-’ and 50 pg rose Bengal ml-‘, was used for plate counts. Copper (CuSOJ was added to provide 10, 100 or 1000 pg Cu ml-’ medium. In all cases 10 g aliquots of the each soil sample was diluted to 1 :lOOO with sterile water, and 1 ml of the soil-water mixture was pipetted onto five independent plates. After 5 days at 28°C the colonies were counted. In this study, all colonies appearing on copper-containing medium were considered to be Cu-tolerant strains. Fungi growing on control and copper treatment plates incubated for 5-10 days were isolated. Hyphal tips of individual colonies on every plates were picked up and transferred to fresh PSA slants. Tube cultures were preserved at room temperature and identified according to Gilman (1957), Barnett and Hunter (1972) and Udagawa (1978).

Cu-tolerant

Changes in copper contents in the soil Table 1 shows changes in copper contents in the treated soil estimated by various extraction methods. Table

I. Changes

in Cu contents Treatment (PgCug~~‘)

I

3

5

The values are average

Control 100 200 400 800 1600 Control 100 200 400 800 1600 Control 100 200 400 800 I600 of triplicates.

in the treated HCIO, 22 ND 301 481 986 1870 ?I 146 242 472 955 I700 21 II6 I85 360 833 1550

fungi in the Cu-treated

soil

Figure 1 gives the relative number of fungal colonies plated on medium containing copper. The results show that the numbers of 10 and 100 pg Cu ml-’ tolerant fungi are almost the same as that of fungi plated on the control medium. On the other hand, 1000pg Cuml-’ tolerant fungi were seldom isolated from the soil treated with 0, 100 or 2OO/*g Cu gg’. But, at higher soil copper concentrations, a greater number of 1000 pg Cu ml-’ tolerant fungi were plated. Within 1 month of treatment, the relative number of 1000 pg Cu ml-’ tolerant fungi was greater than 30”/, of the control in soil treated with 8OOpgCug-’ or 16OOpgCug-‘; and 15:; in soil treated with 400 pg Cu gg’. To determine the time more precisely when 1OOOpgCu ml-’ tolerant fungi had been selected, Cu-treated soils were plated after 1, 3, 7, 14 and 30 days (Fig. 2). The relative number of 1000 pg Cu ml-’ tolerant fungi from the soil treated with 1600 pg Cu gg’, was about 8:/i of the control at

RESULTS

Months after treatment

et al.

soil estimated

Cu contents IMHCI 15 ND 290 415 918 1810 IS 133 225 448 935 1850 12 92 I51 310 675 I490

ND = Not determined

by various

(pg Cu g-‘) 0. I MHCI 13 ND 275 378 848 1670 IO 115 193 353 730 I400 8.0 x4 II7 227 605 I270

extraction

estimated EDTA II ND I50 288 800 I700 I3 II0 I95 325 778 1630 IO 65 118 271 640 I290

procedures

by M&I, 0.4 ND 30 80 205 460 0.3 6.8 20 49 140 590 0.4 2.0 5.5 27 134 415

Hz0 0.5 ND X.0 33 63 200 0.3 0.7

1.1 2.8 21 I65 0.7 0.9 I.1 2.3 15 140

Cu and fungal flora

787

Table 2. Correlation coefficients between the Cu contents estimated by various extraction procedures and the number of IO. 100 and 1000 uaCu ml-’ tolerant fungal colonies Months after treatment

Tolerant HClOd 0

I

10

loo IO00

3

0 10 100

i 000 5

0 IO 100 1000

0.149 0.403 0.030 0.976** 0.738 0.809 0X67*

0.951** 0.862* 0.88S 0.918*” n 974**

I M ncI 0. I79 0.431 0.025 0.980** 0.767 0.816* 0.849’ 0.972** 0.878* 0.898% 0.929** 0.984**

Cu contents estimated by 0.1 M EDTA

1MM&I,

0.1MHCI 0.184 0.436 0.027 0.980+* 0.741 0.802 0X42* 0.966** 0.872, 0.893’ 0.925’* 0.981”

0.185 0.444 0.043 0.991** 0.749 0.812 0.863+ 0.962** o.s94* 0.912* 0.940’. 0.990**

0.173 0.429 0.046 0.990** 0.748 0.777 0.787 0.9W’ 0.908* 0.923** 0.935** 0.990**

H,O 0.210 0.441 0.032 0.97s** 0.779 0.744 0.680 0.9X9** 0.904s 0.915* 0.921** 0.971**

*Significant at P = 0.05: **significant at P = 0.01

7 days and nearly 3O”i,at 14 days. In soils treated with 800 and 400 pg Cu g- ‘, the relative number increased sharply between 14 and 30 days after the treatment. Five species of 1000 pg Cu ml-’ tolerant fungi were isofated from these soils after 30 days. Four of these species, frequently isolated, were identified as Peniciilium spp or Paecilomyces lilaciniis. Fungal ,jlora in the G-treated

soil

Table 3 shows the fungal flora in the soil after 5 months. The fungi listed in Table 3 were limited to those which were isolated more than five times in all treatments and the &-containing plates. Asprrgiilus niger (l), Curaularia trifalii (2), Fus-

“,

(a f

I month

(C)

5monthsafter

0fw

urium oxysporum (43, Penicillium sp. (6), Penicillium sp. (14), Penicillium sp. (17) and an unidentified

fungus (32) were frequently isolated. Of the fungi isolated from the copper treated soil, 40.8% were Penic~~ii~mspp; and 16.2, I&0,8.4,4.4 and 3.1% were species of Aspergi~i~~ Tri~hoderma, Fusarium, Curv&aria and Cladosporium, respectively. Of the fungi plated on 1OOpg Cu ml-’ containing medium (i.e. 100pg Cuml-’ tolerant fungi), 47.9, 17.0, 8.5, 6.4 and 20.2% were Penicillium, Aspergillus, Fusarium, Trichoderma and other genera, respectively. Furthermore, 84.6% of 1000 ,ug Cu ml-’ tolerant fungi were Penicillium spp. With increased copper concentration in the agar medium, the proportion of Penicillium increased. f b I

3 months

offer

(dl

7 months

offer

10

CU concentration

in medium

loo

(pg

tooo

10

loo

1000

10

100

1000

CU ml-‘)

Fig. I. Relative number of Cu-tolerant fungal colonies plated on Cu-containing medium from the soil treated with 100, 200, 400, 800 and ltiOO,~gCu g-’ (the number of colonies on the plates = 100%).

788

HIROKI YAMAMOTO et Treated wifh 4oopq cu q-’

Treated with 0oopq cu q-1

flni-7i-71 I 1

3

lnnr

7 14 30

Days

al.

after

13

714

treatment

Treated 16OOpq

with Cu q-’

I 30

Fig. 2. Changes in relative number of 1000pg Cu ml-’ tolerant fungal colonies for 1, 3, 7, 14 and 30 days after the treatment.

Thirty one species were isolated more than five times from the control soil, and 31, 30,27, 25 and 19 species were isolated from the soils treated with 100, 200, 400, 800 and 1600 pg Cu g-l, respectively. Mucorales (Absidia, Cunninghamella, Mucor) were not isolated from the soils treated with 800 and 16OOpgCug-‘.

DISCUSSION

Although copper is essential for fungi in trace amounts, high concentrations are known to be extremely toxic and are used as fungicides. Many studies have been made on the relationship between pure-culture fungi and copper (Ross, 1975). But, with

Table 3. Fungi isolated from the soils treated with Cu using G-containing after the treatment Control ABCD

Fungi 29 Absidia sp. 114 Cunninghamella 90 23 39 61

I

sp.

Mucor sp. Acremonium sp, Aspergillus candidus Aspergillus fumigatus Aspergillus niger Aspergillus ochraceus Aspergillus sp. Ckxiosporium sp. Cunudaria trifilii ~elmi~th~sp~~iu~l sp. Hum~eo~a sp. Fusarium sp. F~sari~ o.-qvporum Paeciiamwes lilacinus Penicillium sp. Penicillium sp. Penicillium sp. Penicillium sp. Penicillium sp, Peniciliium sp, Penicillium sp. Penicillium sp. PeniciNium sp. Penicillium sp. Penicillium sp. Peniciiiium sp. Pestoloria sp. T~ichod~~~zasp. T~i~hode~m~ sp. T~i~h~derrna sp.

15 26 79 2 103 91 16 45 40 6 8 14 17 21 22 24 25 31 35 37 50 69 10 27 33 32 Unidentified

Cu concentration

t* ** I ** * f ** * * **t ** **

I** IL

* ** * *** **

Treatment (~gCu gg’) 100 200 400 800 1600 ABCD ABCD ABCD ABCD ABCD

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plates at 5 months

** *t

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*

I*+

in the isolation medium (gg Cu ml-‘) A: 0, B; 10. C; 100, D. 1000

Cu and fungal

the exception of some investigations of the effects of copper pollution on soil fungi (Kendrick, 1962; Tatsuyama ef al., 1974; L. M. Hartman, unpublished Ph.D. thesis, University of Montana, 1976; Lawrey, 1977; Yamamoto et af. 1981; Zibilske and Wagner, 1982), little attention has been directed to their influence in soil fungal ecology. In earlier field investigation on the effects of copper pollution on soil fungi (Yamamoto et al., 1981), we found positive and significant correlations between the number of fungal colonies and copper contents of soil (P < 0.01). Also the number of fungal species isolated was halved at high copper concentrations, and about 65% of all the isolates were Penicillium. In regard to the latter, we suggested that PenicilIium spp may be dominant in soils polluted with copper. In the present laboratory study, designed especially to study the effects of copper pollution on the development of populations of Cu-tolerant fungi, correlation coefficients between the copper contents of the soils used and the number of fungal colonies were positive as was also found in the results reported previously (Table 3). Kendrick (1962), investigating the soil fungi of a copper swamp, reported that copper content and fungal count fluctuate in the same rather than in opposite directions. Within the limits of the present experiment, we suggest that the increase of Cu-tolerant fungi in proportion to the copper content in the soil resulted in the increase in fungal count. These results suggest that copper may stimulate the growth and subsequent sporulation of some Cutolerant fungi or inhibit the growth of their competitors such as Cu-sensitive microorganisms. However, in some cases no significant correlation was observed between the copper contents of the soils and the numbers of 0, 10 and 100 pg Cu ml-’ tolerant fungi. Also, the differences in correlation coefficient between several Cu-extraction methods were not so clear cut as the one obtained in earlier field investigation. From these results, it seems that a biological equilibrium had not become established as the “climax” microbial community 5 months after the copper treatment. The selection of 1000 pg Cu ml-’ tolerant fungi occurs in a relatively short time in heavily polluted soils (the results of Fig. 2 also support this), whereas, the development of 10 and IOOpg Cu ml-’ tolerant fungi requires more time (more than 5 months). Doelman and Haanstra (1979b), investigating the effect of lead on soil bacterial microflora, reported that the selection of Pbtolerant strains in the soil occurs within a few years, though it may be dangerous to compare directly the adaptation of bacteria toward lead with that of fungi toward copper. Lawrey (1977) and Zibilske and Wagner (1982) reported that heavy metal pollution results in a decrease in soil fungal diversity. In this study, the number of fungal species isolated from the soil treated with 1600 pg Cu g-’ decreased to about twothirds of the one from control soil after 5 months. With increasing copper concentrations both in the agar medium and the soil, the proportion of Penicillium increased. Lawrey (1977) mentioned Penicillium as a dominant fungus in heavily polluted soil, but Zibilske and Wagner (1978), on the contrary, reported that Penicillium and Paecilomyces decrease

flora

with increasing

789

concentrations in soil, while and Aspergillus increase. In the latter study, however, they could identify only three or four dominant colony types and overlooked some other colonies,and it is not possible to compare their results with our investigations. It is quite probable that Penicillium is a dominant genus in Cupolluted soil because most 1000 pg Cu ml-’ tolerant fungi are Penicillium, and the growth or sporulation of some of the Penicilfium isolated from the soil are stimulated in medium containing copper (unpublished data). From the control soil, which contained only low quantities of copper, 1000 pg Cu ml-’ tolerant fungi were also isolated. This fact suggests that tolerance is related to innate properties and that certain strains in soil are endowed with such properties. When the Cu-sensitive strains were eliminated, Cu-tolerant strains were favored and became dominant. This is supported by the results in Fig. 2, where a high proportion of 1OOOpgCuml-’ tolerant fungi appeared soon after the soil had been treated with high quantities of copper. Not all strains isolated from the soil containing high levels of copper were Cu-tolerant, indeed, some were sensitive. The presence of Cu-sensitive strains may be the result of (1) uneven distribution of copper in the soil, where the inside of soil aggregates contain less copper than the outside; (2) tolerance in soil relating to presence of clay minerals, soil organic matter, soil solution, etc.; (3) occupation of habitats where the copper concentation is low, due to the precipitation of copper by other microorganisms. These are probable and interesting possibilities which should be investigated to elucidate fungal ecology in polluted soil. Trichoderma,

copper

Rhizopus

Acknowledgements-The authors express appreciation to Dr David R. Hosford, Department of Biological Science, Central Washington University, for critical reading of this manuscripL This study was supported in part by a Research Grant from the Ministry of Education, Science and Culture of Japan. REFERENCES

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790

HIK~KI YAMAMOTO

in habitats

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