EFFECT OF MIXTURES OF CARBON DISULFIDE AND METHYLISOTHIOCYANATE ON SURVIVAL OF WOOD-COLONIZING FUNGI Edwin F. Canessa Graduate Student and Jefrey J. Morrell Associate Professor Department of Forest Products Oregon State University Corvallis, OR 9733 1 (Received October 1994) ABSTRACT The fungitoxicity of carbon disulfide (CS,), methylisothiocyanate (MITC), or a mixture of these two gases, to selected wood-degrading fungi was studied by using a fumigation apparatus. Both gases are important decomposition products of metham sodium, the most commonly used fumigant for internal treatment of large wood members. Carbon disulfide (up to 8,000-9,000 ppm) was mildly toxic to most of the test fungi, and MITC (up to 18 ppm) was uniformly toxic. A combination of sublethal levels of both gases (3,000-4,000 ppm CS2/5 ppm MITC) was more toxic than either chemical alone. The results suggest a synergism between various metham sodium decomposition products, and this inter- action may account for the protection afforded by this treatment. Further studies of other decom- position products are suggested. Keywords: Fumigant, metham sodium, carbon disulfide, methylisothiocyanate, fungitoxicity. INTRODUCTION sive study, and its behavior under varying re- Fumigants are widely used in North Amer- gimes is well documented (Zahora and Corden ica to arrest and prevent internal decay of elec- 1985; Zahora and Morrell 1988, 1989a, b). tric utility poles and other large wood mem- Metham sodium decomposition in soil pro- bers (Goodell and Graham 1983). Of these duces significant levels of MITC under a va- fumigants, metham sodium (32.7% sodium riety of conditions, although various other de- n-methyldithiocarbamate) is the most com- composition products are also possible (Thorn monly used (Morrel and Corden 1986). This and Ludwig 1962; Turner and Corden 1963; chemical itself is not highly fungitoxic, but it Elson 1966; Smelt and Leistra 1974). decomposes in the presence of organic com- A number of studies suggest that MITC is a pounds to produce a variety of highly fungi- relatively minor component of metham sodi- toxic compounds including methylisothio- um decomposition in wood, with substantial cyanate (MITC) (Turner and Corden 1963; amounts of carbon disulfide (CS,) and carbon- Elson 1966). The toxicity of MITC to wood- yl sulfide produced (Miller and Morrell 1990; degrading fungi has been the subject of exten- Lebow and Morrell 1993; Morrell 1994). Al- though the latter two compounds can be fun- gitoxic, the levels required far exceed those This is Paper 30 12 of the Forest Research Laboratory, produced by metham sodium decomposition. Oregon State University, Corvallis. Futhermore, because these compounds have Wood and Fiber Scrence. 27(3), 1995, pp. 207-224 O 1995 by the Society of Wood Science and Technology 208 WOOD AND FIBER SCIENCE, JULY 1995, V. 27(3) TABLE1. Fungi evaluated for sensitivity to carbon disuljide and MITC. Name Isolate # Source Antrodia carbonica (Overh.) Ryv & Gilbn. L-8242-sp FRL, Corvallis, OR Postia placenta (Fr.) M. Lars & Lomb. MAD-698 FPL, Madison, WI Irpex lacteus (Fr.:Fr.) Fr. FP-105915-sp FPL, Madison, WI Gloeophyllum trabeum (Fr.) Murr. MAD-6 17 FPL, Madison, WI G. saepiariurn (Fr.) Karst S4UT FRL, Corvallis, OR Trametes versicolor (L.:Fr.)Pilat. A4CD-34 FRL, Corvallis, OR Horrnoconis resinae v. Arx & de Vries PI600 SUNY College of Env. Sci. & For., Svracuse. NY no substantial interactions with the wood, they were incubated at room temperature on a ro- remain in the wood for only short periods after tary shaker (80 rpm) for 7 to 14 days, and then treatment. Despite these characteristics, meth- the mycelium was filtered, resuspended in ster- am sodium provides good protection to Doug- ile distilled water, and blended for 10 sec at las-fir poles over a 7- to 10-year period (Hels- approximately 11,000 rpm. The macerated ing et al. 1984) and to southern pine poles for mycelium mixture from a single flask was 3 to 5 years (Zabel and Wang 1988). This per- transferred to a squeezable bottle and poured formance may reflect synergistic interactions over the blocks in a plastic bag, and the bags between various decomposition products that were heat sealed and incubated 20 to 30 days enhance fungitoxicity of the treatment; how- at 27 C. The ascomycete, H. resinae, was grown ever, there is little information to support this on 1.8% malt extract agar, and the plates were premise. In this report, we describe the effects flooded with sterile distilled water to dislodge of CS, and MITC, alone or in combination, conidia. The conidia were poured over the on survival of seven wood-colonizing fungi. blocks in the same manner as the mycelial sus- pensions. Colonization during the incubation MATERIALS AND METHODS period was periodically assessed by removing Blocks were colonized by six basidiomycetes selected blocks from the bags and placing them and one ascomycete by using modifications of on malt extract agar. Growth of the test fungus a previously described procedure (Sexton et al. from the blocks was used as a measure of suc- 1993/94) (Table 1). Blocks (10 by 10 by 3 mm) cessful colonization. of Douglas-fir heartwood (Pseudotsuga men- The colonized blocks were exposed to meth- ziesii (Mirb.) Franco) and ponderosa pine sap- am sodium decomposition products in a fu- wood (Pinus ponderosa Dougl. ex Laws.) were migation apparatus consisting of five 40-ml placed in autoclavable plastic bags equipped wide-mouth glass jars, each capable of holding with a single breathable patch. Approximately a different metham sodium decomposition 100 g of vermiculite (fine grain) and 700 ml product (Fig. 1). The jars were equipped with of distilled water were added, and the bags Teflon @-linedcaps to retard possible fumigant were loosely sealed prior to autoclaving for 20 loss. Teflon tubing (6-mm outer diameter) was min at 120 C. Each bag was then inoculated used to connect the jars so that different ratios with a macerated hyphal/spore mixture of one of the selected fumigants could be introduced of the seven fungi. into the system. The five bottles were in turn Basidiomycete inoculum was prepared by connected to a single mixing vessel connected placing a small agar plug, cut from the edge of to a manifold that distributed the gas mixture an actively growing malt extract agar culture to 12 135-ml glass jars. Each jar contained 10 of the test fungus, into a flask containing 50 blocks colonized by a single fungus. Flow from ml of 1.8% malt extract solution. The flasks the fumigant jars to the mixing chamber was Canessa and Morrt~ll-SURVIVAL OF WOOD-COLONIZING FUNGI Celite Restrictor - - - - - Fumigant "-Controls \ - Manifold Compressed Air FIG. 1. Apparatus employed to fumigate fungus-colonized wood blocks. controlled with Teflon-lined control valves, to operate for 24 h prior to introduction of while flow to individual fumigation chambers fungal-colonized blocks. was controlled through glass restrictor tubes Fumigant concentrations over the course of packed with Celitem (diatomaceous earth) to the trial were assessed by removing air samples produce a flow of 15 ml of fumigant-laden air from a site on the fumigation chamber in which per minute. All gas flows were measured with a rubber serum cap had been inserted. For a bubble flow meter. Fumigant concentrations analysis of CS,, 5 p1 of air was injected into a were varied by increasing the flow of gas from Varian 3700 Gas Chromatograph (GC) a given fumigant component reservoir to the equipped with a Flame photometric detector mixing vessel. with filters specific for sulfur. The GC condi- Air flowing through the apparatus was first tions were as follows: nitrogen flow rate 33.3 humidified by being bubbled through distilled ml/minute; detector temperature 240 C; in- water. Then the air flowed over the jars con- jector temperature 150 C. Column tempera- taining the fumigant, through the mixing jar, ture was 40 C. Separation was achieved with and finally into the jars that would contain the a 3-m by 4-mm (inner diameter) column blocks. Fumigant flow rates were adjusted un- packed with 10°/o Carbowax 20M on 80/100 til the desired concentration of each gas was Supelcoport (Supelco, Bellefonte, PA). For achieved, and then the apparatus was allowed MITC, 200-p1 air samples were injected; GC 210 WOOD AND FIBER SCIENCE, JULY 1995, V. 27(3) conditions were similar except that column RESULTS AND DISCUSSION temperature was increased to 1 10 C. The number of CFUs varied widely among The fumigant chamber was first employed the fungal species, reflecting both the sensitiv- to evaluate the fungitoxicity of CS, at 500, ity of each species to the isolation procedures 3,000-4,000, and 8,000-9,000 pprn (0.5, 3-4, and the presence or absence and relative and 8-9 ng of CS2/p1 of air, respectively) or amounts of conidia or chlamydospores in the MITC at 5, 10, and 18 pprn (5, 10, and 18 ng wood (Tables 2-4). Irpex lacteus, which pro- MITC/ml of air). The results from these trials duced the smallest number of CFUs, showed indicated that 3,000-4,000 pprn CS, and 5 pprn the highest sensitivity to both fumigants. The MITC were sublethal exposure rates, and the absence of asexual spores or other special sur- effects of a mixture of these two gases at the vival structures and the presence of thin to sublethal level were then evaluated. slightly thickened generative hyphae help ex- Fumigations were carried out over 10-day plain the sensitivity of this fungus (Wang and periods. Each day, one colonized block was Zabel1990). Hormoconis resinae produced the removed from each chamber and aerated to largest number of CFUs, reflecting the massive permit the fumigant to dissipate.