APPLIED MICROBIOLOGY, Dec. 1971, p. 1012-1016Copyright (© 1971 American Society for Microbiology

Vol. 22, No. 6Prinited in U.S.A.

Stimulation of Mycelial Growth of Endothiaparasitica by Heavy Metals'

CAROL M. ENGLANDER AND MALCOLM E. CORDEN

Botanty and Pla,it Pathology Departmenit, Oregoni State Uniiversity, Corvallis, Oregoni 97331

Received for publication 26 July 1971

Of 16 metal cations tested on agar medium, only copper and iron stimulatedmycelial growth of Endothia parasitica in relatively high concentrations. Similarlyenhanced growth was produced in high (32%) glucose concentrations and also whenthe fungus was grown on cellophane placed over the agar surface. E. parasiticasecreted large amounts of oxalate that precipitated primarily as calcium oxalateat the periphery of the fungal colony, causing an opaque halo in the medium. My-celial growth was retarded greatly when calcium oxalate accumulated, but retarda-tion was reversed by copper and iron salts that prevented accumulation of the cal-cium oxalate crystals. E. parasitica grew well on media containing copper oxalateand copper-calcium oxalate but grew poorly with calcium oxalate as the carbonsource and was inhibited by sodium oxalate in the medium. The specificity by whichonly copper and iron salts stimulated mycelial growth suggested that the metal andoxalate ions interact to form specific oxalate complexes that reverse the inhibitionof simple oxalate salts. This probably accounts for enhanced growth in the presenceof otherwise toxic levels of metals and oxalate. The stimulation did not occur inliquid cultures.

During an investigation of the toxicity of coppersalts to various fungal species, we noted thatmycelial growth of Endothia parasitica (Murr.)And. was stimulated by high concentrations ofCuSO4 that inhibited Aspergillus niger, a copper-tolerating fungus. Stimulation at high copperconcentrations precludes a micronutrient functionfor copper. Although fungi can develop resistanceto metal toxicants, adaptation generally requirescontinual culture on sublethal levels of the toxi-cant and does not account for growth stimulation.

Stimulation of E. parasitica by high concentra-tions of CuSO4 and the relatively poor growth ofthis fungus on standard culture media suggestthat stimulation may be due to interaction of theCUSO4 with a self-inhibiting fungal metabolite inthe medium, such that both are inactivated. Thepresent study was designed to test this hypothesisand to determine the influence of other metals onthe growth of E. parasitica.

MATERIALS AND METHODSThe influence of metal salts on the growth of E.

parasitica was determined on potato-dextrose-agar

I Journal Paper No. 2809, Oregon Agriculture ExperimentStation, Corvallis. The data presented in this paper were takenfrom a thesis submitted by the first author in partial fulfillmentof the requirements for the M.S. degree, Botany and Plant Pa-thology Department, Oregon State University, Corvallis.

(PDA) prepared from the infusion of 200 g of pota-toes, 20 g of dextrose, and 20 g of agar (Difco) in 1liter of distilled water. In certain experiments, thepotato infusion was filtered through Whatman no. 1paper and then centrifuged at 4,080 X g for 0.5 hr.The potato broth was sterilized by autoclaving withthe dextrose and agar or by filtering through a Milli-pore HA filter (0.45,m) before addition to a steriledextrose-agar solution.

Tubes containing 19 ml of molten PDA were cooledto 45 C, and the desired pH was obtained by adding0.1 N HCl or NaOH. Unless otherwise specified, thepH was adjusted to 5.0. Metal salts were added in 1.0ml of an aqueous solution, and the medium from eachtube was poured into a petri plate. Each plate wasinoculated with a 7-mm disc cut from the peripheryof a colony of E. parasitica on PDA and incubated at23 C.

Mycelial growth of the fungus on solid media wasestimated by linear or dry weight measurements, orby both methods. In each experiment, average colonydiameters were obtained from two perpendicularmeasurements made daily from the time when themost rapidly growing colonies were about 20 mm indiameter until they reached the edge of the petriplates. During this period, the daily growth rate wasrelatively constant.Dry weights of the mycelium from nutrient agar

cultures were obtained by first melting the agar in anautoclave and collecting the mycelial mat on Whatmanno. 4 filter paper. The mats were dried to constantweight at 80 C.

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To determine whether growth stimulation occurs inliquid culture, E. parasitica was grown on potatobroth medium (PDA without agar). Erlenmeyer flasks(250 ml) containing 30 ml of the broth medium wereautoclaved; the medium was adjusted to pH 5.0 andinoculated with a 7-mm plug of E. parasitica. Thecultures were incubated at about 23 C for 7 days.The mycelial mats were then collected on glass filterpaper and dried to constant weight at 60 C.The copper content of culture media was determined

by atomic absorption analysis and was 2.5 X 10-,3.2 X 10-6, and 5.7 X 10-6 M for PDA, PDA with acellophane cover, and PDA with 320 g of dextrose/liter, respectively. In all cases, the copper content ofthe media from impurities in the components wasinsufficient to stimulate growth of E. parasitica.

RESULTS

Influence of metal salts on growth ofE. parasitica.The growth of E. parasitica on PDA was sig-nificantly stimulated by CuSO4 over a wide rangeof concentrations, as estimated by either mycelialgrowth rate or dry weight accumulation after 7days of growth (Fig. 1A). The mycelial growthrate was enhanced by CuSO4 as the reaction in-creased from pH 4.0 to 8.0, but when copper wasabsent growth was relatively poor over this pH

;05 104 ¶53 162 idmolar conc. of CuSO4

FIG. 1. Growth ofE. parasitica on media with variousamounts of CuS04 added. (A) Mycelial growth rateand dry weight accumulation on PDA; (B) dry weightaccumulation in potato-dextrose broth cultures; (C) my-celial growth rate with and without cellophane overPDA.

TABLE 1. Effect of pH on the growth rate of E.parasitica on PDA containing CuS04

Mycelial growth rate (mm/24 hr) at variouspH of the concn of CUS04 added to the medium

culture mediumoa 5 X 10-5 Ms5X 10-4m 5 X 10-3lM

3.0 1.5 2.2 1.4 0.74.0 2.6 4.4 5.9 4.95.0 2.4 9.4 9.2 7.16.0 1.1 9.4 9.3 5.17.0 1.4 8.9 9.7 6.18.0 2.0 5.9 8.1 7.2

a Copper content of PDA was 2.5 X 10-6 M.

TABLE 2. Effect of metal salts on the growth rate ofE. parasitica

Mycelial growth rate (mm/24 hr) at two metal salt concna

Metal salt

5 X 10-4M 5 X 10-3 M

CuSO4*5H20 ........... 9.3 6.0CuCl................. 9.3 8.6FeC12*4H20 ............ 9.9 3.7FeC13*6H20 ............ 10.2 4.1ZnS04-7H2O ........... 2.8 1.5NiCl2*6H20 ............ 0.9 0.1MgSO4- 7H20.1.1 1.3CoC12*6H20 ............ 2.1 1.5MnSO4*H20. 1.2 1.8CaCl2*2H20 ............ 1.5 2.2KCl................. 1.1 1.1BaC12*2H20 ............ 1.7 1.7AgNO3 ................. 1.4 0PbCl2 .................. 0.5 1.0CrCl3- 6H20.0.7 0CdC12 .................. 1.8 0HgCl2.0 0

On media without metal salts added, E.parasitica had an average growth rate of 1.5mm/24 hr.

range (Table 1). The growth on PDA with nocopper added was generaUy arrested when thecolony attained a diameter of about 40 mm and,thus, never reached the edge of the petri plate.Of 16 other metal compounds tested in PDA,

only salts of Cu+, Fe2+, and Fe'+ enhancedmycelial growth to a similar degree at relativelyhigh concentrations (Table 2). Zinc sulfateslightly increased the growth rate at a concen-tration of 5 X 10-4 M, but the other metal saltsfailed to enhance growth significantly at thisconcentration.

Relative to the corresponding controls to whichno copper was added, growth in liquid mediumwas slightly stimulated at the lower copper con-

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centrations but was inhibited at high concentra-tions that were stimulatory on solid medium(Fig. iB). Thus, enhanced growth at relativelyhigh copper concentrations appears to depend oncomponents in the agar or is characteristic ofgrowth on solid media.

E. parasitica was grown on several natural andsynthetic solid media to determine whether stimu-lation is unique on PDA. Copper sulfate wasadded at a final concentration of 1.6 X 10-4Mto 2% water-agar; filter-sterilized PDA; auto-claved PDA; Campbell's medium (3) containingagar, salts, biotin, thiamine, dextrose, andasparagine; and Leonian's medium (11) con-taining agar, salts, peptone, maltose, and maltextract.When no copper was added, the fungus grew

poorly on all of the above media, but when cop-per was present growth was stimulated aboutninefold. Thus, stimulation by copper is notunique on PDA and is unaffected by the methodof medium sterilization.

E. parasitica grows rapidly on cellophaneplaced over PDA (12). Its growth on PDA con-taining various CuSO4 concentrations with orwithout a cellophane cover was therefore meas-ured to determine the possible relationship be-tween enhanced growth over cellophane andstimulation by copper. Cellophane discs (DuPont215 PD) 8.5 cm in diameter were autoclaved,moistened with sterile water, placed wrinkle-freeon PDA in petri plates, and inoculated with 7-mm agar discs of E. parasitica. Mycelial growthwas estimated by linear measurements.

In the absence of copper, the fungus grewrapidly on cellophane placed over PDA. Ratherthan being stimulated by relatively high copperconcentrations that enhance growth on PDAwithout cellophane, its growth was inhibited(Fig. 1C). Cellophane without copper presentappears to stimulate mycelial growth, and, in thepresence of cellophane, the toxicity of CuSO4 wassignificantly increased.

Influence of dextrose on growth of E. parasitica.The concentration of dextrose in PDA was variedto determine whether the mycelial growth ratecould be enhanced by increasing the amount ofcarbon in the medium. At concentrations of160 g/liter and greater, dextrose significantlystimulated mycelial growth (Table 3). Growth ofmost fungi is inhibited by sugar levels above 150g/liter (4), and the remarkable stimulation of E.parasitica at such high dextrose concentrationssuggests that in this case a non-nutrient role of

TABLE 3. Effect of dextrose on the growth rate ofE. parasitica on PDA

Dextrose (g/liter)a Mycelial growth rate(mm/24 hr)

20 1.280 1.6160 4.2240 5.7320 5.2

a The copper content of PDA ranged from 2.5 X10-6 to 5.7 X 10-6 M in media with 20 and 320 gof dextrose/liter, respectively.

FIG. 2. (A) Opaque halo surrounding a colony of E. parasitica growing on PDA; (B) crystals of calciumoxalate trihydrate in the medium that cause the opaque halo (X 1,640).

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in dextrose might result in an action similar to thatof copper on growth.

Accumulation of calcium oxalate in cultures ofE. parasitica. Stimulation of mycelial growth byrelatively high concentrations of copper and therelatively poor growth of E. parasitica on standardculture media suggest that enhanced growth mightresult from interaction of the metal toxicant witha self-inhibiting fungal metabolite such that bothare inactivated in the medium. PDA cultures ofthis fungus contain a distinct opaque halo aroundthe periphery of the colony (Fig. 2A). The halowas found in all cultures on solid media on whichE. parasitica grew poorly but was absent in mediacontaining copper or iron salts where growthwas enhanced. Media from the halo regioncontained many bipyramidal octahedral shapedcrystals that were in close proximity to the hyphae(Fig. 2B) and caused the opaqueness.To obtain crystals for analysis, E. parasitica

was grown on 2% water-agar to avoid the problemof separating the crystals from the starch granulesin PDA. Since the crystals were relatively in-soluble in most solvents, a disc of nylon screen(38 mesh) was placed in a Buchner funnel, andthe media from 10 petri plate cultures were placedon the screen. The stem of the funnel was placedin a centrifuge bottle (250 ml), and the bottle andcentrifuge head were steamed in an autoclave for4 min. The melted agar was centrifuged at 7,970 xg for 2 min at 45 C, the supernatant liquid wasdecanted, and the bottle was rinsed three timeswith distilled water. The pelleted crystals weresuspended in distilled water, poured into anevaporating dish, and dried at 80 C.

Analysis of the partially purified crystalsshowed them to be slightly soluble in hot waterand 1 N NaOH, and highly soluble in 1 N HCI,H2SO4, and HNO3 ; they were insoluble in coldwater, 6 M acetic acid, 6 M NH30H, 6 M NH3Cl,ethanol, benzene, anhydrous ether, acetone,hydrogen peroxide (10%), carbon tetrachloride,ethyl acetate, N,N-dimethylformamide, aceto-nitrile, dioxan, and N,N-dimethylacetoacetimide.The crystal structure was altered at 160 to 170 C,and the crystals decomposed at 350 C withoutmelting. Infrared absorption (KBr pellet) showedmajor peaks at 2.95, 3.4, 6.2, 7.25, 7.45, 10.5, and12.8 ,um. The X-ray diffraction pattern (CuK a

radiation and nickel filter) had major peaks at0.619, 0.594, 0.365, 0.296, 0.278, 0.250, 0.241,0.235 ,.0.224,,0.202, and 0.196 nm. On the basis ofthis "analysis, the substance was identified as

calcium oxalate (1, 6). Calcium was the onlymetal found in a sufficient amount (13.5%, w/w)to be theoretically a component of the crystals.The calcium required for formation of these

crystals probably came from the agar, whichcontains about 0.13% calcium (personal com-munication, D. G. Erwin, Difco Laboratories,Detroit, Mich.).Three hydrates of calcium oxalate occur

naturally (9), but only the trihydrate has thebipyramidal octahedral shaped crystals typical ofthose isolated from cultures of E. parasitica. Thegradual addition of oxalic acid to an excess ofcalcium ions approximated the conditions underwhich calcium oxalate is formed in cultures of E.parasitica, and resulted in crystals identical tothose deposited in PDA by the fungus.When a solution of CUSO4 (102 M) was added

to freshly prepared calcium oxalate crystals,they dissolved. Depending on the metal and theconcentration of oxalate and metal ions, simplesalts or metal oxalate complexes with oxalategroups coordinated to the metal ions can form(10). Most metal oxalates are only sparinglysoluble, but their solubility can be increasedthrough complex formation by the addition ofother metal ions (10). For example, the ferric ionis frequently used to form a complex with calciumoxalate, thereby increasing the solubility of theoxalate (13). Thus, the dissolution of calciumoxalate crystals by a CUSO4 solution is probablyattributable to complex formation and may ac-count for the lack of calcium oxalate crystals inE. parasitica cultures containing high levels ofCUSO4 .Growth of E. parasitica on media containing

metal oxalates. E. parasitica was grown on potatoinfusion-agar with various metal oxalates inplace of dextrose to determine whether the formof the oxalate could influence growth of thefungus. Calcium, copper, and copper-calciumoxalates were obtained by precipitation fromsolutions of Ca(N03)2, CUSO4, or both afteraddition of oxalic acid. These oxalates weresterilized dry at 80 C for 24 hr, and were thendissolved or suspended at three concentrations inthe medium. The medium was -adjusted to pH5.0, poured into petri plates (ca. 20 mi/plate),and inoculated. Mycelial growth rate versus

TABLE 4. Growth of E. parasitica on mediacontaining metal oxalates

MycelialMetal oxalate at 10-2 M growth rate

(mm/72 hr)

Copper oxalate..................... 24.0Copper-calcium oxalate .............. 22.2Calcium oxalate ...................... 9.6Sodium oxalate...................... 2.4Control (potato infusion-agar).4.8Control (PDA).9.4

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oxalate concentration was plotted, and the growthrates at 1I0 M were chosen for comparison be-cause they were indicative of results at the otherconcentrations.When copper oxalate and copper-calcium

oxalate were present, fungal growth was sig-nificantly stimulated (Table 4) as on PDA con-taining CuS04 . On the calcium oxalate medium,the growth rate was twice that on potato infusion-agar without oxalate and was about equal togrowth on the medium containing dextrose(PDA). Although calcium oxalate accumulateswhen growth is inhibited, these results suggestthat calcium oxalate is not inhibitory per se; thetoxicity of sodium oxalate, however, suggeststhat high levels of oxalate ions may be inhibitory.

DISCUSSIONBecause of the general toxicity of heavy metal

ions to fungal cells and the relatively high con-centrations of copper that stimulate growth of E.parasitica, it is most probable that the stimulatoryaction of copper is initiated outside the cells in theculture medium.

Limited growth of E. parasitica on natural andsynthetic media and the concomitant accumula-tion of calcium oxalate at the colony edge suggesta possible causal relationship between poorgrowth and oxalate accumulation. Growthstimulation by copper is accompanied by a sub-stantial reduction in the accumulation of calciumoxalate crystals, further indicating a positivecorrelation between poor growth and calciumoxalate accumulation.On the basis of this study, the following

working hypothesis is offered to explain the re-lationship of metal salts and oxalate to the growthof E. parasitica on solid culture media. Oxalateaccumulates and inhibits growth either by directtoxic action or by blocking the uptake of needednutrients (e.g., calcium). When copper or ironsalts are present at stimulatory concentrations,complexes are formed that detoxify the oxalateand the heavy metal.

Detoxification mechanisms have been postu-lated to explain the growth of some fungi in thepresence of metal toxicants (2). For example,Poria vaporaria produces oxalic acid in sufficientquantities to detoxify CuS04 in treated wood (8)and other copper salts in an agar medium (7),but only if growth has occurred prior to the ad-dition of the copper salt. No reports have beenfound, however, of enhanced growth by P.vaporaria on media containing copper salts.Calcium oxalate fails to accumulate in stimu-

lated cultures of E. parasitica on PDA containingexcessive dextrose concentrations and when the

APPL. MICROBIOL.

fungus is grown on cellophane over the PDAmedium. High levels of dextrose may stimulategrowth by solubilizing oxalate as it is formed inthe culture medium (5), and cellophane may in-fluence growth either by adsorbing oxalate orby dispersing oxalate over the entire surface of thePDA, thus preventing its concentration aroundthe fungal colony.The precise physiological mode of action of

oxalates and metals on the growth of E. parasticais not known. However, the specificity by whichonly copper and iron salts stimulate mycelialgrowth of E. parasitica suggests the formation ofspecific metal-oxalate complexes that reversetoxicity of the simple oxalate salts.

ACKNOWLEDGMENTS

Acknowledgment is made to M. E. Harward for the X-raydiffraction analyses, D. P. Moore for the cation analyses by atomicabsorption, and J. C. Decius for assistance in infrared spec-troscopy.

The financial assistance of The Mountain Copper Co., Ltd.,and a National Science Foundation Graduate Traineeship aregratefully acknowledged.

LITERATURE CITED

1. American Society for Testing and Materials, Joint Committeeon Powder Diffraction Standards. 1968. Index (inorganic)to the powder diffraction file 1968. ASTM Publ. PDIS-18i.

2. Ashida, J. 1965. Adaptation of fungi to metal toxicants.Annu. Rev. Phytopathol. 3:153-174.

3. Campbell, R. 1967. The interaction of carbon, nitrogen andsterilization of the medium on pycnidial production ofEndothia parasitica. Trans. Brit. Mycol. Soc. 50:413-421.

4. Cochrane, V. W. 1958. Physiology of fungi. John Wiley &Sons, Inc., New York.

5. Hamada, M. 1940. Physiologisch-morphologisch Studienuiber Armillaria mellea (Vahl.) Quel., mit besondererRucksicht auf die Oxalsaiurebildung. Ein Nachtrag zurMykorrhiza von Galeola septentrionalis Reichb. f. Jap. J.Bot. 10:387-463.

6. Hunt, J. M., and M. P. Wishered. 1950. Infrared absorptionspectra of minerals and other organic compounds. Anal.Chem. 22:1478-1497.

7. Keino, K. 1950. Effects of copper salts upon the mycelialgrowth of wood-destroying fungi. I. The formation of theaccumulating zone of copper oxalate. Bull. Govt. ForestExp. Sta. Tokyo 44:71-90.

8. Kind, A. 1945. Die Saure-Produktion von Pilzen und derenEinfluss auf mit Kupfersulfat imprignierte Holzer. Bull.Schweiz. Elektrotech. 35:174-176.

9. Kohlschutter, V., and J. Marti. 1930. Untersuchungen uberPrinzipien der genetischen Stoffbildung. I. Uber Bildungs-formen des Calciumoxalats. Helv. Chim. Acta 13:929-978.

10. Krishnamurtz, K. V., and G. M. Harris. 1961. The chemistryof the metal oxalate complexes. Chem. Rev. 61:213-246.

11. Leonian, L. H. 1924. A study of factors promoting pycnidium-formation in Sphaeropsidales. Amer. J. Bot. 11:19-50.

12. McDowell, L. L., and A. A. DeHertogh. 1968. Metabolismof sporulation in filamentous fungi. I. Glucose and acetateoxidation in sporulating and nonsporulating cultures ofEndothia parasitica. Can. J. Bot. 46:449-451.

13. Osawa, T. 1950. Purity of precipitate and crystal. II. Copre-cipitation of ferric ion and ammonium ion with calciumoxalate. J. Chem. Soc. Jap. Pure Chem. Sect. 71:68-70.

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