AGRICULTURE ROOM

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COPPER TOLERANCE CIF WhitWOOD-ROTTING FUNGIJune 19611

No. 2223

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FOREST PRODUCTS LABORATORY

MADISON 5, WISCONSIN

UNITED STATES DEPARTMENT OF AGRIC ITURE

FOREST SERVICE

n Cooperation with the University of Wisconsin

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COPPER TOLERANCE OF SOME WOOD-ROTTING FUNGI

By

1George Y. Young

Summary

The generally high tolerance of acid-forming brown-rot fungi and the lowtolerance of white-rot fungi to copper sulfate was confirmed for 16 species,including Poria cocos Wolf., a fungus associated with the failure of copper-__napthenate treated fence posts in Florida, and four other brown-rot species notpreviously reported as copper tolerant. The tolerance of the brown-rot specieswas strikingly increased by lowering the pH of the substratum from pH 6 topH 2 with sulfuric acid. Several mechanisms for fungus tolerance to copper havebeen suggested; all require a decrease in the pH of the medium.

Introduction

Samples of pine fence posts containing brown cubical decay at or near theground level were received by this office for examination. The posts camefrom a farm near Gainesville, Florida, and had been pressure-treated withcopper naphthenate by the solvent-recovery process for a retention of one-halfpound of the preservative per cubic foot of wood.

Sections cut from the posts through the decayed wood were placed in a moistchamber. In a few days mycelium grew over the surface of these sections andin about two weeks fungus fruiting bodies (sporophores) began to develop. Pureculture isolations were made from the surface mycelium, the context of thesporophores, spores from the sporophores, and the brown cubical rotted wood.In all, 25 isolations from these sources yielded the same fungus, identified byits cultural characteristics as Poria cocas Wolf., the tuckahoe fungus. Thisidentification was confirmed by Dr. J. L. Lowe after examination of the sporo,-phore and the spore prints. Culture No. 104264-Sp, listed in Table 1, is apolysporous isolate from one of the sporophores.

1—Pathologist, Forest Disease Laboratory, Forest Service, United States

Department of Agriculture, Beltsville, Maryland.

Report No. 2223

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The fungus was obviously attacking wood that had been penetrated by coppernaphthenate, indicating that the preservative treatment was ineffective inpreventing decay by it. On that basis it was considered advisable to determinethe copper tolerance of Poria cocos and to include in the test a number of otherdecay fungi that have been used in the past in this Laboratory and at the ForestProducts Laboratory, U. S. Forest Service, Madison, Wisconsin.

Materials and Method

2The toxicant used was the hydrated sulfate of copper, CuS0

4. 5H2 0. Sorted,

2perfectly blue crystals were used. The medium was the standard substratumformerly used in this Laboratory: 2 percent Fleischmann's diamalt, and 2percent Difco agar in distilled water. The media and the toxicant solutionswere autoclaved separately for 20 minutes at 15 lbs. pressure, mixed whilehot and, with a pipette, 30 ml. lots were poured into Petri dishes.

The concentrations of copper sulfate used were in geometric progressionwith a common ratio of 1.75, i. e., each concentration was 1.75 times that ofthe next below and ranged from 0. 1 percent to 2. 87 percent. In terms ofcopper, the concentrations are one-fourth of those shown for the copper sulfate.

In sterilizing the copper sulfate solutions by autoclaving, it was found that aprecipitate separated from the solutions. This precipitate was very heavy,particularly at the higher concentrations. It was dissolved by adding concentratedsulfuric acid (6613e; sp. Gr. 1. 8355). The heaviest concentration required12 drops (by eye-dropper) of the acid per liter of agar for clearing. The sameamount of acid was added to each of the other concentrations, including thecontrols which contained no added copper, in order to establish uniform.conditions of growth.

The species of fungi used in this test are listed in Table 1. Inoculations weremade from fresh Petri dish cultures of each fungus. Uniform discs of inoculumwere cut from an arc equidistant from the center of each culture to insurethat each inoculum was approximately of the same age. The plates were incubatedat room temperature maintained at approximately 25° C.

After 10 days of incubation, 8 of the 17 fungi used in this test had failed tomake growth even in the control plates. The pH of the medium, it was realized,

2—Copper sulfate was used because this compound is most generally used in tests

of copper toxicity, and to avoid any delay in procuring the naphthenate.

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Report No. 2223 -2

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must have been at the critical level when sulfuric acid was added to clear theprecipitates brought about by autoclaving. The acidity of the medium was pH2 as shown by the Alkacid Test Ribbon.

A second series of tests was made, similar in every respect to the aboveexcept that the copper sulfate solutions were sterilized by steaming in theArnold steamer to avoid the formation of the precipitate and the necessity toadd sulfuric acid for clearing. The initial acidity of the medium of this secondseries was pH 6.

Results

The fungi that failed to grow at the pH 2 level included all four of the white-rotspecies and four of the brown rots (table 1). All of these, except Poria

oleracea Da y . and Lomb. and P. ca.rbonica Overh. , were also extremelysensitive to copper, the lowest concentration of 0. 1 percent copper sulfatebeing sufficient to check their growth entirely.

Of the fungi that did make growth at the pH 2 level, all made substantial growthand in much higher concentrations of copper than they did at the pH 6 level(fig. 1). Coniophora puteana (Fr. ) Karst. was outstanding in this respect.Whereas a concentration of only O. 18 percent of the toxicant was sufficient tostop its growth on the pH 6 media, at the pH 2 level only the highest concentrationof the series (2. 87 percent) checked its growth. Merulius americanus Burtwas only moderately tolerant to copper on the standard medium (0.31 percent),but a three-fold concentration (0.94 percent) was required to check its growthat pH 2. This three-fold advantage at the pH 2 level is also exhibited by Poria

monticola Murr. and Daedalea quercina Fr. Poria radiculosa (Peck.) Sacc.showed the highest tolerance to copper of the species tested, the fungicidal aswell as the fungistatic concentration at the pH 6 level was 2. 87 percent. Atthe pH 2 level it made growth, although very slow, even in the highest concentration of the series. Measurable growth in this highest concentration did notbegin until the 22nd day of incubation and on the 38th day the diameter of thecolony was 35 mm. The two isolates of Poria cocos Wolf. also were verytolerant to copper, especially in the low pH range. In this test P. monticolawhich has heretofore been regarded as perhaps the most tolerant (1, 5).,_-5

The "lag period" to which Horsfall (8) calls attention and frequently observedin fungi grown on toxic substrata, is well shown in this work (fig. 1). P.monticola is the only exception. This species grew as well or better in the moreacid medium and in all concentrations of copper, even in the controls, attestingto its acidophilous character as well.

-Underlined numbers in parentheses refer to the literature cited at the end ofthis report.

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For species that failed to grow in the more acid agar the killing point concentra-tions were usually higher than the inhibiting concentrations. For the acid-tolerant species, however, the inhibiting and killing concentrations appeared equal.

Discus sion

The present work at two pH levels and with copper sulfate as the toxicant,indicates that for a number of brown-rot fungi, tolerance to copper sulfate isconditioned by the acidity of the medium. Worthy of note in this respect isthat the four white-rot species used in this test are all in the lowest tolerancegroup. Also in the lowest tolerance group are the brown-rot fungi, Lenzitessaepiaria (Fr.) Fr. and L. trabea (Fr. ) Fr. It should be mentioned here,however, that while these last two species cause brown-cubical decay in wood,on gallic and tannic acid agar media they sometimes give weak oxidase reactions(2), and thus in this respect tend to react like white rotters.

The difference between the fungi in this test are in general agreement withthose of other tests involving copper sulfate in agar (10,13); copper naphthenatein agar (7); and copper napthenate in wood (1, 3, 4, 5, 14). In addition to the 10brown-rot fungi that were moderately to highly tolerant of copper in the presentstudy, the following 7 have been previously reported tolerant to one or morecopper compounds in agar or wood: Fomes subroseus (Weir. Overh.; Lentinuslepideus Fr. (1); Merulius lacrymans Fr. (1); Polyporus sulfureus Fr. (10);Poria vaillantii (Fr.)) Gke.; P. vaporaria (Fr.)) Cke. (10); and a Ptychogastersp. (10). For the white-rot species, in addition to the four in the present study,intolerance to copper compounds has been reported for Fomes pini (Fr.) Karst.(I); Pleurotus ostreatus Fr. (10); Polyporus abietinum Fr. (1); Poly. adustusFr. (1); Poly. hirsutus Fr. (1); Schizophylum commune Fr. (10); and Stereumpurpureum (Fr. ) Fr. (10). In all of these reports, copper tolerance is generallyassociated with the brown-rot fungi and intolerance with the white. Only inCowling's (1) results is there any material departure from this.

Rabanus (10) and Shimazono and Takubo (12) have suggested that the hightolerance of the brown rots is due to their habit of producing oxalic acid which,it was supposed, precipitates the copper into the insoluble form of the oxalatethus rendering the copper metabolite inert. However, if precipitation of themetal into the inert oxalate form were the principal factor in copper tolerance,the brown rots should show an even greater superiority over the white rots withrespect to zinc tolerance. Zinc is a more potent poison than copper and zincoxalate more insoluble and more inert than copper oxalate. In Richards' (11)study, however, the white rots actually outranked the brown rots in zinc tolerance

Report No. 2223 -4-

•This study indicates that the low pH reaction generally produced by the brown-rot fungi in their metabolism, irrespective of the acid produced, may be moreimportant as a factor in their copper tolerance. In this study sulfuric acid wasused and, in the case of Coniophora puteana (Fr. ) Karst., its tolerance tocopper was increased more than nine-fold at the pH 2 level. Lin (9) found thathydrochloric acid antidoted copper sulfate for spores of Sclerotinia fructicola Wint. in the pH range of 2 to 6 covered by this study. Horsfall (8) attributesthe greater tolerance in acid substrata as due to the protection of amitio acidsagainst replacement of their hydrogen by chelating the copper. The coppertolerance of the oxalic-acid-producing brown-rot species, he continues, maythus be due not to the low solubility of copper oxalate but mainly to the loweringof the pH of the substratum.

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Report No. 2223 -5- 1.5-11

Literature Cited

1. Cowling, Ellis B.1957. The relative preservative tolerance of 18 wood destroying fungi.

For. Prod. Jour. 7: 355-359.

2. Davidson, Ross W., Campbell, W. A., and Blaisdale, Dorothy J.1938. Differentiation of wood-decaying fungi by their reactions on gallic

or tannic acid medium. Jour. Agr. Res. 57: 683-695, illus.

3. Duncan, Catherine G.1953. Soil-block and agar-block-techniques for evaluation of oil-type

preservatives: Creosote, copper-naphthenate and pentachloro-phenol. Forest Pathology Special Release 37, 39 pp., illus.(Processed).

4.1957. Evaluating wood preservatives by soil-block tests. 9. Influence

of different boiling fractions of the petroleum carrier on theeffectiveness of pentachlorophenol and copper-naphthenate.Amer. Wood Preservers' Assoc. Proc., 53: 13-20.

5. and Richards, Audrey C.1950. Evaluating wood preservatives by soil-block tests. 1. Effect of

the carrier on pentachlorophenol solutions. 2. Comparison ofa coal tar creosote, a petroleum containing pentachlorophenolor copper-naphthenate and mixtures of them. Amer. WoodPreservers' Assoc. Proc. 46: 131-145, illus.

6. Findlay, W. P. K.1953. Dry rot and other timber troubles. 267 pp. illus. London

7. Hirt, Ray R.1949. An isolate of Poria xantha on media containing copper.

Phytopathology 39: 31-36.

8. Horsfall, James G.1956. Principles of fungicidal action. Chronica Botanic a. Go. Waltham,

Mass.

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Report No. 2223 -6-

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9. Lin, Ch'Wan Kwang.1940. Germination of the conidia of Sclerotinia fructicola, with special

reference to the toxicity of copper. Cornell Univ. Agr. Exp.Sta. Memoir 223. 33 pp., illus.

10. Rabanus, Ad.1931. Die toximetrische Prufung von Holzkonservierungsmitteln. Angew.

Bot. 13: 352-371 (Partial translation in English, Amer. WoodPreservers' Assoc. Proc. pp. 34-43, 1933).

11. Richards, C. Audrey.1925. Comparative resistance of 18 species of wood-destroying fungi to

zinc chloride. Amer. Wood Preservers' Assoc. Proc. 21:18-22.

12. Shimazono, Hirao, and Takubo, Kenjiro.1952. The biochemistry of wood-destroying fungi: (1st Report) The

Bavendamm's reaction and the accumulation of oxalic acid bythe wood-destroying fungi. Bul. of the (Japan) GovernmentForest Experiment Station 53: 117-125.

13. Tippo, Oswald, Walter, J. M. , Smucker, S. 3. , and Spackman, Wm. , Jr.1947. The effectiveness of certain wood preservatives in preventing the

spread of decay in wooden ships. Lloydia 10: 175-208.

14. Zabel, R.1954. Variations in preservative tolerance of wood-destroying fungi.

Jour. For. Prod. Res. Soc. 4: 166-169.

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SUBJECT LISTS OF PUBLICATIONS ISSUED BY THE

FOREST PRODUCTS LABORATORY

The following are obtainable free on request from the Director, Forest ProductsLaboratory, Madison 5, Wisconsin:

List of publications onBox and Crate Constructionand Packaging Data

List of publications onChemistry of Wood andDerived Products

List of publications onFungus Defects in ForestProducts and Decay in Trees

List of publications onGlue, Glued Productsand Veneer

List of publications onGrowth, Structure, andIdentification of Wood

List of publications onMechanical Properties andStructural Uses of Woodand Wood Products

Partial list of publicationsfor Architects, Builders,Engineers, and RetailLumbermen

List of publications onFire Protection

List of publications onLogging, Milling, andUtilization of TimberProducts

List of publications onPulp and Paper

List of publications onSeasoning of Wood

List of publications onStructural Sandwich, PlasticLaminates, and Wood-BaseAircraft Components

List of publications onWood Finishing

List of publications onWood Preservation

Partial list of publicationsfor Furniture Manufacturers,Woodworkers and Teachers ofWoodshop Practice

Note: Since Forest Products Laboratory publications are so varied in subjectno single list is issued. Instead a list is made up fca . each Laboratorydivision. Twice a year, December 31 and June 30, a list is made upshowing new reports for the previous six months. This is the only itemsent regularly to the Laboratory's mailing list. Anyone who has askedfor and received the proper subject lists and who has had his name placedon the mailing list can keep up to date on Forest Products Laboratorypublications. Each subject list carries descriptions of all other sub-ject lists.

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