Effect of mercuric chloride and methylmercuric chloride on spore germination and colony growth rate of Penicillium notatum and Aspergillus niger

Chemoephere No. 3, PP 107 - 114, 1974.

Pergamon Press.

Printed in Great Britain.

Effect of Mercuric Chloride and Methylmercuric Chloride on Spore Germination and Colony Growth Rate of Penicillium notatum and Asper~illus niger.

J. E. Hardcastle and Nara Mavichakana Chemistry and Biology Departments Texas Woman's University Denton, Texas 76204 (Received in USA 27 December 1973; received in UK for publication 19 April 1974) INTRODUCTION

Since fungi are causative agents of many plant diseases, several kinds of mer-

cury compounds have been used as fungicides.

Also, inorganic and organic mercury compounds

have been used as seed and soil disinfectants.

Organic mercury compounds have been found to

be more toxic to some fungi than inorganic mercury compounds 1'2.

However,

little is known a-

bout the effect of sub-lethal amounts of mercury on the germination and growth of common soil fungi.

The objective of this project was to determine the effects of different levels of mer-

curic chloride and methylmercuric chloride on colony growth and spore germination of Asper~illus niger and Penicillium notatum.

These organisms are widely distributed throughout the

biosphere, and are among the most abundant species of soil fungi.

EXPERIMENTAL

The mercuric chloride stock solution was prepared by dissolving 1.354 g. of

HgCI2(ACS reagent grade)

in IOOO ml of distilled water.

The concentration of mercury in this

solution was I000 ppm, and dilutions were made as needed from this stock solution.

The methyl-

mercuric chloride stock solution was prepared by dissolving 0.125 g of methylmercuric chloride (Alfa Inorganics, Ventron, Beverly, Mass.)

in fOOD ml of distilled water.

The concentration

of mercury in this solution was IO0 ppm, and dilutions were prepared as needed from this stock solution. LISHMENT,

Slants of Aspergillus niger and Penicillium notatum (WARDS NATURAL SCIENCE ESTABINC., ROCHESTER, NEW YORK) were washed several

Czapek broth (containing 0.5% agar as a stabilizer). were pooled in sterile 125 ml flasks. Neubauer Counting Chamber.

times with IO ml portions of sterile

The washings for the individual

fungi

Counts of spore suspensions were made by means of a

The final concentration was adjusted to 2 x IO 7 spores per ml.

A slide culture technique 3 was used to show the effect of mercury on the rate of fungal spore germination.

Czapek agar containing different concentrations of mercuric chloride and

methylmercuric chloride was aseptically cut into one cm sqwares using a flamed knife. 107

Bent

108

No. 5

glass tubes which contained sterile water were placed into sterile Petri dishes, and sterile glass slides were placed 6n top of the bent glass tubes. and placed on the slides.

The agar squares were lifted out

One drop of standard spore suspension was inoculated onto the cent-

er of the agar squares and a flamed cover slide was centrally placed on the agar squares. Controls were made by using the Czapek agar containing no mercury.

Each subtreatment was re-

plicated 4 times. The cultures were observed periodically for 48 hours with a compound microscope, 480X magnification.

Germination was considered completed when 80% of the spores in the microscope

field had produced germination tubes.

The time required for this 80% germination to occur

was recorded. To study the effect of mercury on fungal colony growth two ml of HgCl2, or CH3HgCI solution, and 8 ml of Czapek agar were added to sterile Petri dishes.

Such culture plates were

prepared containing O, 0. l, 1.0, 2.0, 5.0, lO,0, 15.0, and 20.0 ppm Hg as mercuric chloride; and O, O.Ol, 0.05, O.10, 0.15, 0.20, 0.30, and 0.50 ppm Hg as methylmercuric chloride.

Each

subtreatment was replicated 4 times. After the media solidified, a drop of standard spore suspension was inoculated onto the center of each agar plate.

The cultures were incubated in the dark at 25 C for 9 days.

The

diameters of the colonies were measured daily with a centimeter ruler.

RESULTS

The effects of HgCl 2 on the germination time of spores of Aspergillusniger

Penicillium notatum are presented in Table {A.

and

When no mercury was present, the time re-

quired 80% germination of spores of Asper~illus ~

and Penicillium notatum was 8 hours.

No increase in germination time over control values was observed with a mercury concentration of 0.1 ppm.

The germination time increased in a nearly linear manner as the concentration of

mercury increased above the 0.1 ppm level.

Spores of ~. niger did not germinate at mercury

concentrations of 5 ppm and higher within the 48 hour observation period. rum germinated in the presence of mercury concentrations up to 15 ppm.

Spores of P. nota-

No. 5

109

Table I.

Effect of (A) mercuric chloride and (B) methylmercuric chloride on the rate of spore germination of Asper~illus niger and Penicillium notatum.

Hg in nutrient agar, ppm A.

Mercuric chloride

0

O.l

1.0

2.0

Asper~illus niger Penicillium notatum

5.0

lO.O

15.0

8 hr*

8

13

16

8

8

12

15

20

24

24

O.Ol

0.05

O. lO

0.15

12

16

.

8

12

15

Organism B.

Methylmercuric chloride

0

Asper~illus niger

8 hr*

Penicillium notatum

8

.

. 18

0.20

0.30

. 20

24

*Time required for 80% of the spores to produce germ tubes. These times represent the average of 4 replicates, and the average times were rounded to the nearest hour. Table IB shows the effect of methylmercuric chloride on the germination of spores of A. niger and P. notatum.

No increase in the germination time over control values of P. notatum

was observed with a mercury concentration of O.Ol ppm. then increased as the mercury concentration

increased.

The germination time of P. notatum At 0.5 ppm Hg 80% germination of P.

notatum spores did not occur within the 48 hour observation period.

However, 0.Ol ppm of Hg

did delay the germination of ~. niger for four hours and 0.05 ppm Hg doubled the germination time.

At higher mercury concentrations 80% germination of A. niger spores was not observed

within the 48 hour incubation period. Mercury, both inorganic and organic, delayed the other stages of development to the same extent that germination was delayed. The effects of HgCl Ill.

on the growth rate of the test fungi are presented in Tables II and

The growth rate of P. notatum decreased gradually as the mercury concentration

ed up to 15 ppm, and no growth occurred at 20 ppm.

increas-

Up to 2 ppm Hg did not affect the growth

rate of ~. [email protected] greatly, however, 5 ppm Hg markedly reduced the growth rate, and above 5 ppm no growth occurred.

Ii0

No,

Table II.

Effect of mercuric chloride on colony size and growth rate of Asper~illus niger

Incubation time

Mercury Concentration in Agar (ppm)

(days) 0

O.l .

.

l.O

2.0

5.0

I

.

2

0.78*

0.78

0.78

0.64

3

1.76

1.76

1.76

1.54

4

7.07

7.07

7.07

6.60

-

5

15.21

15.21

15.21

14.86

-

6

23.76

22.90

22.90

22.06

0.78

7

33.18

32.67

33.18

32.67

2.14

8

38.48

38.48

38.48

37.39

6.16

9

50.27

50.27

50.27

47.17

]2.25

lO.0

15.0

20.0

.

*Area (cm2), average of 4 replicates.

Average deviations of above values were ~ 5%.

Table Ill. Effect of mercuric chloride on colony size and growth rate of Penicillium notatum

Incubation time

(days)

Mercury Concentration in A~ar (ppm) 0

0. I

l.O

2.0

5.0

10.O

15.0

I

-

-

2

0.20*

0.16

0.20

0.16

0.13

0.16

0.13

3

0.71

0.66

0.71

0.59

0.57

0.50

0.50

4

2.99

3.05

2.90

2.75

2.69

2.55

2.27

5

4.71

4.91

4.53

4.53

4.41

4.34

4.16

6

6.38

6.61

6.16

5.73

5.73

5.73

5.52

7

8.30

8.55

8.04

7.55

9.62

7.07

7.07

8

12.88

12.88

12.57

12.57

12.38

II.95

II.64

9

15.90

15.90

15.55

15.90

15.21

15.21

14.86

~Area (cm2), average of 4 replicates. Average deviations of above values were + 5%.

20.0

No. 3

iii

As is shown in Table IV much lower concentrations of methylmercuric chloride affected the growth rates of the test fungi.

The growth rate of ~. niBer was significantly retarded by

0.Ol ppm Hg, and no growth occurred at higher mercury concentrations.

The growth rate of

P. notatum was not significantly affected by O.Ol ppm Hg, but 0.05 ppm Hg reduced the growth rate, and higher mercury concentrations completely inhibited growth.

Table IV.

Effect of methylmercuric chloride on colony size and growth rate of Asper~illus niger and Penicillium notatum

Incubation time (days) 0

Mercury Concentration 0.01 0.05 0 ..Aspergillus n i g e r .

.

in Agar (ppm) 0.01 0.05 Penicillium notatum

I

.

.

2

1.13*

0.78

0.28

0.20

0.28

3

3.14

2.01

1.04

0.78

0.64

4

8.55

4.52

3.30

3.14

1.43

5

16.62

9.62

5.I0

5. I0

3.14

6

25.52

13.20

6.83

6.83

4.52

7

35.78

19.64

8.55

8.55

6.16

8

39.59

26.42

13.52

14.19

9.08

9

52.17

33.18

16.62

17.35

12.57

~

*Area (cm2), average of 4 replicates. Average deviations of above values were + 5%. There are four steps in asexual

cycle of ~. niger and P. notatum:

germination, mycelium formation, and sporangium formation.

spore swelling, spore

In the control plates, micro-

scopic observation showed spore swelling occurred after 4 hour incubation;

after 8 hours in-

cubation 80% of the spores had germinated; from the 9th to the 20th hour the mycelia began growing; and after about 24 hours of incubation the sporangium appeared. growth studies,

the colonies could not be measured on the first day of incubation.

The growth characteristics of the control ferent from one another. the

In the colony

colonies of ~. niq~r and L. notatum were dif-

A. niger tended to grow in a more loosely dispersed colony, and in

9 day incubation period grew to a larger diameter (and total area) than P. notatum.

112

No. 3

60

50

0 • o O •

NO MERCURY, A. NIGER 5.0 ppm Hg(HgCI2), A. NIGER 0.01 ppm Hg(CH3HgCI), A. NIGER NO MERCURY, P. NOTATUM 0.05 ppm Hg(CH3HgCI), P. NOTATUM

40 =E

0

(~ v

30 ILl n" <{

0 20

I0

I 2

:5

4

5

6

INCUBATION TIME (DAYS)

7

B

9

No. 3

113

P. notatum grew in compact colony, and in the 9 day incubation period grew to only half the diameter of ~. niger.

No abnormalities

sence of mercury were observed

DISCUSSION

in fungal pigmentation

and morphology due to the pre-

in these experiments.

Results reported here indicate that the spores and mycelia of Penicillium notat-

u m are more resistant to mercury,

both inorganic and organic,

Fungal spore walls contain chitin, cellulose,

than those of Aspergillus

protein lipids, and nucleic acid derivatives,

and thus, a wide variety of ligands are available to complex with mercury cations itial uptake process 4 with the sulfhydryl

niger.

in the in-

Passow, et al 5, reported that mercuric chloride reacted irreversibly

groups of yeast cell membrane.

perhaps major site of reaction

These data suggest that the initial and

is at the spore wall or fungal membrane.

Ashworth and Am in 6

suggest that the mechanism of resistance may be that intracellular pools of sulfhydryl poun0s protect enzyme systems by forming complexes with the mercury. mounts of these ligand materials present

A difference

com-

in the a-

in the fungi may account for the differences

in

tolerance to mercury. Methylmercuric

chloride was found to be more effective than mercuric chloride

ing the germination of both ~. niger and P. notatum. was required to delay the germination

Whereas

in delay-

l.O ppm Hg as mercuric chloride

of P. notatum by 4 hours, only 0.05 ppm Hg as methyl-

mercuric chloride was required to produce the same effect.

Also, 5 ppm Hg as mercuric chlor-

ide delayed germination of P. notatum by 12 hours, whereas only 0.O2 ppm Hg as methyl mercuric produced the same effect.

Finally,

the mercury concentrations

required to produce a 16

hour de~ay in germination of P. notatum were 0.3 ppm as methylmercuric mercuric chloride.

This data shows Hg in the form of methylmercuric

ive in about 1/25th the concentration

as Hg in the form of HgCl 2.

nounced with ~. niger in that only O.Ol ppm Hg as methylmercuric lay germination same effect.

chloride and 10 ppm as

chloride to be as effect-

The effect was more prochloride was required to de-

by 5 hours, but l.O ppm Hg as mercuric chloride was required to p~oduce the

This is an effective

Hg as mercuric chloride.

ratio of l part Hg as methylmercuric

These results are in agreement with reports

chloride to lO0 parts in the literature that

show organic mercury to be more toxic than inorganic mercury to Neurospora crassa 7 and other fungi 1,2

114

No. 3

This study has shown that the two fungi, Aspergillus niger and Penicillium notatum, have a certain tolerance for mercuric and methylmercuric chloride, and these organism can grow and reproduce in the presence of relatively high concentrations of mercury (compared to normal environmental

levels).

This is in agreement with previously published reports con-

cerning the effect of mercury compounds on fungi.

Ashworth and Amin 6, Jackson 8, and Mac-

Farlane and Nadeen 9 reported that ~. niger was tolerant to inorganic and organic mercury. Bonaly, et al lO, and Gerardin and Kayser II reported that some strains of Candida utilis were resistant to mercuric ions.

Evidence of these organisms'

(~. ni@er and P. notatum)

ability to absorb large quantities of mercury, and still grow and reproduce with this mercury in their cells, was reported by Hardcastle and Mavichakana 12.

Fungi are abundant in

nature, and their role in the environmental cycling of mercury should be further studied.

ACKNOWLEDGEMENTS This study was supported by The Texas Woman's University, Institutional Grant No. 0961. The authors wish to thank Dr. Mohammed Abou-Ela and Dr. Kenneth Fry, both of TWU Biology Department, for their helpful advice and criticisms concerning the work.

REFERENCES

I.

K. Pirschle, Planta (Abt. E, Z. Wiss. Biol.) 2_~3, 177-224 (1934).

2.

A. Klages, Z. Angrew. Chem. 40, 559-561 (1927).

3.

E. S. Beneke, Medical Laboratory Manual, Burgess Pub. Co., Minneapolis,

4.

J. M. Aronson, In "Fungi" (G. C. Ainsworth and A. S. Sussman, Ed.), Vol. l, pp. 49-76. Academic Press, New York, (1965).

5.

H. Passow, A. Rothstein, and T. W. Clarkson, Pharmac. 13, 185-224 (1961).

6.

L. J. Ashworth and J. T. Amin, Phytopathology 54(2), 1459-1463 (1964).

7.

L. Lander.

8.

C. Jackson.

9.

E. W. MacFarlane and L. V. Nadeen.

(1966).

Nature 230, 452-454 (1971). Plant Dis. Reptr. 4._77, 147-150 (1963).

lO.

R. Bonaly, F. Kayser, and A. Varney.

II.

C. Gerardln, and F. Kayser.

12.

J. E. Hardcastle and N. Mavfchakana.

Soc. Exptl. Biol. Med., Proc. 6_7_7,I18-121 (1948). Bull. Soc. Pharm. Nancy 5_Ll, If-18 (1961).

Soc. Biol. Paris, Compt. Rend. 152, 1780-1782 (1958). Bull. Environ. Contam. Tox.

(in press).