AGRICULTURE ROOM
•
COPPER TOLERANCE CIF Whit
WOOD-ROTTING FUNGI
June 19611
No. 2223
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UNITED STATES DEPARTMENT OF AGRIC ITURE
FOREST PRODUCTS LABORATORY
FOREST SERVICE
MADISON 5, WISCONSIN
n Cooperation with the University of Wisconsin
•
COPPER TOLERANCE OF SOME WOOD-ROTTING FUNGI
By
George Y. Young
1
Summary
The generally high tolerance of acid-forming brown-rot fungi and the low
tolerance of white-rot fungi to copper sulfate was confirmed for 16 species,
including Poria cocos
__ Wolf., a fungus associated with the failure of coppernapthenate treated fence posts in Florida, and four other brown-rot species not
previously reported as copper tolerant. The tolerance of the brown-rot species
was strikingly increased by lowering the pH of the substratum from pH 6 to
pH 2 with sulfuric acid. Several mechanisms for fungus tolerance to copper have
been suggested; all require a decrease in the pH of the medium.
Introduction
Samples of pine fence posts containing brown cubical decay at or near the
ground level were received by this office for examination. The posts came
from a farm near Gainesville, Florida, and had been pressure-treated with
copper naphthenate by the solvent-recovery process for a retention of one-half
pound of the preservative per cubic foot of wood.
•
Sections cut from the posts through the decayed wood were placed in a moist
chamber. In a few days mycelium grew over the surface of these sections and
in about two weeks fungus fruiting bodies (sporophores) began to develop. Pure
culture isolations were made from the surface mycelium, the context of the
sporophores, spores from the sporophores, and the brown cubical rotted wood.
In all, 25 isolations from these sources yielded the same fungus, identified by
its cultural characteristics as Poria cocas Wolf., the tuckahoe fungus. This
identification 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 a
polysporous isolate from one of the sporophores.
1
—Pathologist, Forest Disease Laboratory, Forest Service, United States
Department of Agriculture, Beltsville, Maryland.
Report No. 2223
•
The fungus was obviously attacking wood that had been penetrated by copper
naphthenate, indicating that the preservative treatment was ineffective in
preventing decay by it. On that basis it was considered advisable to determine
the copper tolerance of Poria cocos and to include in the test a number of other
decay fungi that have been used in the past in this Laboratory and at the Forest
Products Laboratory, U. S. Forest Service, Madison, Wisconsin.
Materials and Method
2
The toxicant used was the hydrated sulfate of copper, CuS0 . 5H 2 0. Sorted,
2
4
perfectly blue crystals were used. The medium was the standard substratum
formerly used in this Laboratory: 2 percent Fleischmann's diamalt, and 2
percent Difco agar in distilled water. The media and the toxicant solutions
were autoclaved separately for 20 minutes at 15 lbs. pressure, mixed while
hot and, with a pipette, 30 ml. lots were poured into Petri dishes.
The concentrations of copper sulfate used were in geometric progression
with a common ratio of 1.75, i. e., each concentration was 1.75 times that of
the next below and ranged from 0. 1 percent to 2. 87 percent. In terms of
copper, the concentrations are one-fourth of those shown for the copper sulfate.
In sterilizing the copper sulfate solutions by autoclaving, it was found that a
precipitate separated from the solutions. This precipitate was very heavy,
particularly at the higher concentrations. It was dissolved by adding concentrated
sulfuric acid (6613e; sp. Gr. 1. 8355). The heaviest concentration required
12 drops (by eye-dropper) of the acid per liter of agar for clearing. The same
amount of acid was added to each of the other concentrations, including the
controls 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 were
made from fresh Petri dish cultures of each fungus. Uniform discs of inoculum
were cut from an arc equidistant from the center of each culture to insure
that each inoculum was approximately of the same age. The plates were incubated
at room temperature maintained at approximately 25° C.
After 10 days of incubation, 8 of the 17 fungi used in this test had failed to
make 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.
Report No. 2223
-2
•
must have been at the critical level when sulfuric acid was added to clear the
precipitates brought about by autoclaving. The acidity of the medium was pH
2 as shown by the Alkacid Test Ribbon.
A second series of tests was made, similar in every respect to the above
except that the copper sulfate solutions were sterilized by steaming in the
Arnold steamer to avoid the formation of the precipitate and the necessity to
add sulfuric acid for clearing. The initial acidity of the medium of this second
series was pH 6.
Results
The fungi that failed to grow at the pH 2 level included all four of the white-rot
species 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 extremely
sensitive to copper, the lowest concentration of 0. 1 percent copper sulfate
being sufficient to check their growth entirely.
Of the fungi that did make growth at the pH 2 level, all made substantial growth
and 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 to
stop its growth on the pH 6 media, at the pH 2 level only the highest concentration
of the series (2. 87 percent) checked its growth. Merulius americanus Burt
was 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 growth
at 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 as
well as the fungistatic concentration at the pH 6 level was 2. 87 percent. At
the pH 2 level it made growth, although very slow, even in the highest concen
tration of the series. Measurable growth in this highest concentration did not
begin until the 22nd day of incubation and on the 38th day the diameter of the
colony was 35 mm. The two isolates of Poria cocos Wolf. also were very
tolerant to copper, especially in the low pH range. In this test P. monticola
which has heretofore been regarded as perhaps the most tolerant (1, 5).,_-5
•
The "lag period" to which Horsfall (8) calls attention and frequently observed
in 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 more
acid medium and in all concentrations of copper, even in the controls, attesting
to its acidophilous character as well.
-Underlined numbers in parentheses refer to the literature cited at the end of
this report.
Report No. 2223
-3-
For species that failed to grow in the more acid agar the killing point concentrations were usually higher than the inhibiting concentrations. For the acidtolerant 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 is
conditioned by the acidity of the medium. Worthy of note in this respect is
that the four white-rot species used in this test are all in the lowest tolerance
group. Also in the lowest tolerance group are the brown-rot fungi, Lenzites
saepiaria (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 with
those of other tests involving copper sulfate in agar (10,13); copper naphthenate
in agar (7); and copper napthenate in wood (1, 3, 4, 5, 14). In addition to the 10
brown-rot fungi that were moderately to highly tolerant of copper in the present
study, the following 7 have been previously reported tolerant to one or more
copper compounds in agar or wood: Fomes subroseus (Weir. Overh.; Lentinus
lepideus Fr. (1); Merulius lacrymans Fr. (1); Polyporus sulfureus Fr. (10);
Poria vaillantii (Fr.)) Gke.; P. vaporaria (Fr.)) Cke. (10); and a Ptychogaster
sp. (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. adustus
Fr. (1); Poly. hirsutus Fr. (1); Schizophylum commune Fr. (10); and Stereum
purpureum (Fr. ) Fr. (10). In all of these reports, copper tolerance is generally
associated with the brown-rot fungi and intolerance with the white. Only in
Cowling's (1) results is there any material departure from this.
Rabanus (10) and Shimazono and Takubo (12) have suggested that the high
tolerance 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 oxalate
thus rendering the copper metabolite inert. However, if precipitation of the
metal 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 with
respect to zinc tolerance. Zinc is a more potent poison than copper and zinc
oxalate 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 brownrot fungi in their metabolism, irrespective of the acid produced, may be more
important as a factor in their copper tolerance. In this study sulfuric acid was
used and, in the case of Coniophora puteana (Fr. ) Karst., its tolerance to
copper was increased more than nine-fold at the pH 2 level. Lin (9) found that
hydrochloric acid antidoted copper sulfate for spores of Sclerotinia fructicola
Wint. in the pH range of 2 to 6 covered by this study. Horsfall (8) attributes
the greater tolerance in acid substrata as due to the protection of amitio acids
against replacement of their hydrogen by chelating the copper. The copper
tolerance of the oxalic-acid-producing brown-rot species, he continues, may
thus be due not to the low solubility of copper oxalate but mainly to the lowering
of the pH of the substratum.
•
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 pentachlorophenol. 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 the
effectiveness 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 of
a coal tar creosote, a petroleum containing pentachlorophenol
or copper-naphthenate and mixtures of them. Amer. Wood
Preservers' 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.
•
Report No. 2223
-6-
•
•
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. Wood
Preservers' 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 by
the wood-destroying fungi. Bul. of the (Japan) Government
Forest 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.
•
Report No. 2223
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