EFFECT of MIXTURES of CARBON DISULFIDE and METHYLISOTHIOCYANATE on SURVIVAL of WOOD-COLONIZING FUNGI Edwin F

Home , Carbon disulfide

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 decomposition 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 fungitoxic, 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. The aerated sporulation that is a common feature of this block was placed into a 36-ml stainless steel species (Bessey 1959). canister containing a stainless steel ball and 5 CFUs in some non-fumigant-exposed con- ml of sterile distilled water. The canister was trol blocks tended to decline slightly over the shaken for 5 seconds on a KlecoB 4100 Pul- 10-day test period. This effect was more no- verizer (Kleco Kinetic Manufacturing Co., Vi- ticeable in I. lacteus than in any other species. salia, CA). After maceration, the steel ball was Efforts were made to humidify the atmosphere removed with a magnetic stir bar wand, and by bubbling the air in the test apparatus through the macerated wood suspension was diluted to distilled water before it passed over the blocks. 30 ml with additional distilled water. A 1.5- Condensation along the walls of the tubing in- ml aliquot of the suspension was added to 10 dicated that moist air was reaching the blocks, ml of molten (45 C) 1.8% malt extract agar, but signs of dryness appeared after 7 to 8 days. and the mixture was poured into petri dishes The CFU declines in controls may reflect loss and allowed to solidify. Three plates were pre- of viability due to drying during the exposure pared from each block. The plates were in- period, although declines did not always occur. cubated at room temperature, and the colonies For example, CFUs of A. carbonica growing that developed in each plate were counted. on pine increased with time. Despite declines These results were compared with colony in some species, differences between chemi- counts from control blocks exposed to air flow cally exposed samples and controls were gen- without fumigant through a separate line in erally of a magnitude that permitted compar- isons between the treatments. the fumigation apparatus (Fig. 1). The data were expressed as number of colony-forming Carbon disulfide units (CFUs) in order to reflect the possibility Fumigation with CS, appeared to stimulate that colonies could arise from both hyphal the number of CFUs in most of the treatments fragments and spores. The data were used to between the first and fourth days of exposure construct concentration x time (CT) curves (Table 2). For P. placenta on Douglas-fir, in- for assessing the amount of chemical necessary creases in CFUs up to 263% (for 500 ppm) and to kill each fungus at selected exposure times. 420% (8,000-9,000 ppm) were obtained. For The CT values necessary to kill 90% of pro- the same species on pine, increases ranged from pagules (CT,,) were calculated from the re- 182% (500 ppm) to 370% (8,000-9,000 pprn). gression lines on these curves. Many fungi produce thick-walled chlamydo- Canessa and MorreN-SURVIVAL OF WOOD-COLONIZING FUNGI 21 1 spores that are able to withstand long expo- gesting that the fumigant might be effective sures to toxic substances or to other adverse upon even longer exposures. Drying of wood environmental conditions (Zabel and Morrell in the fumigation system at these longer ex- 1992). Antrodia carbonica produced large posures, however, would cause a correspond- amounts of thick-walled chlamydospores, but ing decrease in CFUs, obscuring potential fu- I. lacteus, which does not produce this type of migation effects. Several fungi, including A. structure, was less tolerant of the treatments. carbonica on Douglas-fir, G. saepiarium on The lowest level of CS, (500 ppm) had little ponderosa pine, T. versicolor on pine, and H. effect on CFUs for any of the species tested. resinae on Douglas-fir and ponderosa pine, CFUs for P. placenta, G. saepiarium, T. ver- were relatively unaffected by this exposure. sicolor, and H. resinae exceeded 100% after 10 Antrodia carbonica is an important colonizer days of fumigation. Some fungi growing under of Douglas-fir heartwood, and the survival of sulfur-deficient regimes have been shown to this species in the presence of CS, would be a be more resistant to sulfur-containing fumi- major drawback if this fumigant were the only gants and even stimulated when fumigated for decomposition product of metham sodium 2 h with MITC (Cobb 1972). Sulfur is neces- (Eslyn 1970; Graham and Corden 1980). Sim- sary for fungal growth and reproduction (Lilly ilarly, G. saepiarium is an important colonizer and Horace 195 1). The ability of fungi to ox- of untreated pine, although studies suggest that idize sulfur in vitro has long been recognized it is not an important colonizer of preserva- (Waksman 19 18; Armstrong 192 1). Sulfur tive-treated southern pine (Zabel et al. 1980). content in wood is generally less than 0.1% for The survival of H. resinae at the highest CS, species such as Douglas-fir and pine (Mingle level is not surprising in light of the well-known and Boubel 1968). It is possible that some fun- tolerance of this species to a variety of bio- gi metabolize CS, or MITC to satisfy sulfur cides. This fungus occasionally has been iso- needs. These effects may help explain the ini- lated from Douglas-fir poles (Zabel et al. 1980), tial increase in CFUs observed for most of the but in creosote-treated southern pine utility species fumigated with CS,, although more re- poles, it was the most frequently isolated fun- fined studies of sulfur balance in fumigated gus (Zabel et al. 1985). The possible effects of fungi would be required to confirm this hy- this fungus on residual fumigant levels in wood pothesis. are unclear. Hormoconis resinae is capable of Increasing CS, levels to 3,000-4,000 ppm utilizing creosote as a sole carbon source, but produced declines in CFUs for virtually all of its ability to utilize CS, is unknown (Marsden the fungi tested, but this level was only effec- 1954; Kerner-Gang 1976). tive against I. lacteus, which produced no vi- These results demonstrate that exposure of able CFUs at the end of the treatment. wood colonized by various decay and non- Exposure to 8,000-9,000 ppm of CS, re- decay fungi to low levels of CS, reduces the sulted in decreased CFUs for all of the fungi number of CFUs in the wood, but fumigants tested, although many species survived a 10- at these levels generally were not lethal. Meth- day exposure. Initial stimulation of CFUs was am sodium is a relatively short-lived treat- again noted for A. carbonica, P. placenta, and ment, with reinvasion of poles by decay fungi G. saepiarium on both Douglas-fir and pine, characteristically starting only 3 to 7 years after and for I. lacteus and T. versicolor on pine. treatment (Helsing et al. 1984; Zabel et al. 1985; Prolonged exposure resulted in very low levels Zabel and Wang 1988). Metham sodium's lim- of survival for A. carbonica on pine, P. pla- ited residual time in the wood may permit centa, I. lacteus, and G. trabeum on both survival of fungal propagules in zones where Douglas-fir and pine, G. saepiarium on Doug- diffusion of decomposition products is limited. las-fir, and T. versicolor on Douglas-fir, sug- These zones might include wet pockets, knots, 212 WOOD AND FIBER SCIENCE, JULY 1995, V. 27(3)

TABLE2. Effect offumigation with CS2 on number of colony-forming units (CFUs) produced by seven species offungi on Douglas-jr or ponderosa pine, expressed as a % of control^.^

A. carbonica P. placenla I. lacteus G. trabeum G. saeprarium T. versicolor H. resinae Time (davsl D-fir Pine D-fir Pine D-fir Pine D-fir Pine D-fir Pine D-fir Pine D-fir Pine 500 ppm CS2 100 100 100 105 94 93 114 106 136 101 93 101 127 107 103 90 107 126 101 93 140 117 88 107 133 95 88 115 100 77 107 107 70 3,0004,000 ppm CS2 100 100 100 39 203 291 51 173 330 7 200 300 6 142 53 2 118 31 2 95 37 0 106 22 0 134 19 0 126 14 0 103 14 8,000-9,000 ppm CS2 0 100 100 100 100 100 100 100 1 122 150 420 370 109 90 32 2 117 148 363 191 150 76 81 3 87 130 333 152 75 76 107 4 70 84 180 92 34 98 121 5 83 67 152 42 3 122 49 6 64 25 90 16 1 90 30 7 71 8 62 6 0 61 13 8 47 3 42 3 0 46 16 9 32 1 5 1 0 4 3 10 30 0 10 0 1 0

a Values represent the average of 3 replicates. or other wood defects. If decomposition con- often produced an initial stimulus of CFUs ditions shift heavily towards production of CS,, during the first 2 days of fumigation, especially some fungi may survive as chlamydospores in the 5 and 10 ppm MITC treatments. and later be able to germinate when conditions The lowest concentration (5 ppm) produced again become suitable for microbial growth. declines in CFUs for all species except T. versicolor, which showed significant increases in Fungitoxicity of MITC CFUs after the fifth or sixth day on both Doug- As expected, MITC had a more dramatic las-fir and pine (Table 3). This effect may re- effect than CS, on CFU numbers for all fungal flect delayed stimulation under low fumigant species in the study (Table 3). Like CS,, MITC concentrations, but reasons for the lag are un- Canessa and MorreN-SURVIVAL OF WOOD-COLONIZING FUNGI 2 13

TABLE3. Eflect of fumrgation with MITC on number of colony-forming units (CFUs) produced by seven species of fungi on Douglas-jir or ponderosa pine, expressed as % of control^.^

A. P. placenta I. lacteus G. trabeum G. saepiarium T. versicolor H. resinae Time carbonica (days) D-firb D-fir Pine D-fir Pine D-fir Pine D-fir Pine D-fir Pine D-fir Pine 5 ppm MITC 100 100 124 14 93 27 39 28 26 25 25 24 14 12 6 6 1 3 0 1 0 1 10 ppm MITC 100 100 84 126 119 330 93 19 19 22 6 11 3 5 0 1 0 0 0 0 0 0 18 ppm MITC 100 100 216 103 122 68 3 17 1 11 0 0 0 0 0 0 0 0 0 0 0 0

a Values represent the average of 3 replicates. Data for A. carbonica on ponderosa pine was lost because of contamination

clear. Relatively high CFU levels were found to react to environmental changes, especially for A. carbonica on Douglas-fir and P. placenta with regard to numbers and types of resistant on pine. (The data for A. carbonica on pine structures. For example, G. trabeum and G. were lost during MITC fumigations because of saepiarium were characterized by high CFU contamination.) Little or no fungal survival on counts at the beginning of fumigation, but these either wood was found for I. lacteus, G. tra- levels declined rapidly with time, suggesting beum, G. saepiarium, and H. resinae. Differ- that these species were very susceptible to fu- ences in the number of CFUs among species migation under the conditions of this experi- (Table 3) reflect the different fungi's abilities ment. Both species produce large numbers of 214 WOOD AND FIBER SCIENCE, JULY 1995, V. 27(3)

TABLE4. Effect offumigation with 3.000-4.000 pprn CS2 and 5ppm MITC on number of colony-forming units (CFUs) produced by seven species offungi on Douglas-jir or ponderosa pine, expressed as % of control^.^

A P. placenta I. lacteus G. trabeum G. saepiarium T versicolor H. resinae Time carbonica (days) f fir^ D-fir Pine D-fir Pine D-fir Pine D-fir Pine D-fir Pine D-fir Pine

a Values represent the average of 3 replicates. Data for A. carbonica on ponderosa pine was lost because of contamination thin-walled arthrospores, which may be less The highest rates of CFU decline were resistant to MITC fumigation than chlamyd- achieved with the 18 pprn MITC treatment. ospores. However, the presence of even lim- Only H. resinae on Douglas-fir produced vi- ited surviving CFUs could facilitate fungal able colonies after 10 days of fumigation, and "reinvasion" far from the fumigant applica- CFU levels there were only 1% of those found tion point, where MITC levels might be ex- for the untreated controls. pected to be low. The possibility of a surviving microflora in fumigated poles merits further Fungitoxicity of the CS,/MITC mixture study. Fungal recolonization of some wood Because both CS, and MITC are produced species is often quite slow, and the species during metham sodium decomposition, these present appear to differ from those found in products may act synergistically to enhance non-fumigant-treated wood (Zabel et al. 1985; fungal control. The initial results suggested that Giron and Morrell 1989). Survival structures 3,000-4,000 pprn CS, and 5 pprn MITC each of some fungi may play a major role in this produced CFU declines that were usually no- process. ticeable but not lethal. These levels were sub- Exposures to 10 pprn MITC produced a fast- jected to further study in combination. er decline of CFUs for all species (Table 3). The initial CFU stimulus noted with CS, or On both Douglas-fir and pine, I. lacteus, G. MITC alone was absent in the mixture; as be- trabeum, and G. saepiarium showed rapid de- fore, however, sampling was performed every clines in CFUs until the seventh to ninth days, 24 h, and a stimulus earlier in the fumigation when these species succumbed. As in the CS, might have been missed by our procedures. and 5 pprn MITC fumigations, initial increases Mixtures of sublethal dosages of CS, (3,000- in CFU levels were evident in most species 4,000 ppm) and MITC (5 ppm) were generally during the first days of treatment, but the levels more fungitoxic than CS, alone at 8,000-9,000 were much lower. Only A. carbonica on pine, pprn or MITC alone at 10 pprn for A. carbon- P. placenta and T. versicolor on Douglas-fir, ica, P. placenta, I. lacteus, and G. trabeum and H. resinae on both woods survived a 10- (Table 4). T. versicolor had shown high CFUs day exposure to 10 pprn MITC, and even these for the 5 pprn MITC fumigation, but this effect species experienced marked CFU declines was less pronounced with the mixture. Fu- compared with results in the CS, and 5 pprn migant mixtures did not noticeably reduce the MITC treatments. CFUs of G. saepiarium or H. resinae compared Canessa and Morrell-SURVIVAL OF WOOD-COLONIZING FUNGI

Cumulative Concentration (CT) CS, O.OEOO 1.4E+8 2.8E+8 4.2E+8 5.6E+8 7.OE+8 8-4E+8 2.5 -1 -. CS, 2.2------AMITC -.- h lCSdMlTC a . 0 . -= 1.9- ----0-* .-9 l a=-- 1.6- W--. --. . a a -- a 1.3- a

Cumulative Concentration (CT) MITC FIG.2. Effect of sublethal dosages of CS2 (3,0004,000 ppm) and MITC (5 ppm) and a CS2/M1TC mixture (3,000- 4,000 pprn CS2/5 pprn MITC) on survival of A. carbonica on Douglas-fir. The Y axis represents the logarithm of survival, and the X axes represent the product of concentration by time for CS2 (upper axis) and MITC (lower axis). with the 5 pprn MITC treatment, but the mix- For most fungal species, CT,, values for the ture was more effective than CS, alone at 3,000- fumigant mixture were generally lower than 4,000 ppm. those for either CS, or MITC at sublethal levels (Table 5). For example, CT,, values for the Concentration x time (CT) estimates mixture against A. carbonica on Douglas-fir Although air concentration of a chemical is were less than one third the value for CS, alone, a useful measure of fumigant exposure, fu- or about a fifth the value for MITC (5 ppm) migant studies are more often expressed as alone. The CT,, for the mixture for P. placenta concentration x time or CT values. These val- on Douglas-fir was about a third of the value ues reflect the total dosage to which the fungus for CS, or MITC alone; on pine, effects of the was exposed. The CT required to reduce CFUs mixture were similar to those of CS,. In some to 10% of the control, or CT,,, provides a use- instances, however, CT,, values for the mix- ful measure of chemical effectiveness. ture were higher than those for the individual CT curves for sublethal dosages of each components. For I. lacteus on pine, CT,, val- chemical alone or in mixture show that for ues for the mixture were half again as large as most fungi, the log of survival declined more for CS, alone. For G. saepiarium on Douglas- rapidly with the mixture than with either fu- fir, CT,, values for the mixture were higher migant alone (Figs. 2-8). The effects were more than for MITC. For H. resinae on Douglas-fir variable for T. versicolor (Fig. 7), where the or pine, the CT,, for the mixture was similar relationship between CT and survival changed to that found for sublethal levels of MITC (5 at lower CT values. These effects again reflect PP~). the initial stimulation of CFUs previously dis- CT,, values for the 3,0004,000 pprn CS, cussed. treatment tended to be higher for Douglas-fir WOOD AND FIBER SCIENCE, JULY 1995, V. 27(3)

Cumulative Concentration (CT) CS, 0.0~00 1 2.8E+8 4.2E+8 5.6E+8 7-OE+8 8.4E+8 3.0 I A -. CS, ------AMITC 2.4-. ' --- . CS,/MITC e-. m-.-r- 1.8-""""---5,0-m, A...... Al ..--- .-. A A e-- 6------.-..-K....k-.-.....-.-.,.-. -=.. -=.. --.. A A 1.2- -*-.\ a\-.. 0.6- --\.0--2.

-lu Cumulative Concentration (CT) MITC .-> L 3 Cumulative Concentration (CT) CS, O.OEO0 1.4E+8 2.8E+8 4.2E+8 5.6E+8 7.OE+8 8.4E+8

O.OEO0 2.OE+5 4.OE+5 6.OE+5 8.OE+5 1.OE+6 1.2E+6 Cumulative Concentration (CT) MITC FIG.3. Effect of sublethal dosages of CS2 (3,0004,000 ppm) and MITC (5 pprn) and a CS2/MITC mixture (3,000- 4,000 pprn CS2/5 ppm MITC) on survival of P. placenta on A) Douglas-fir and B) ponderosa pine. The Y axis represents the logarithm of survival, and the X axes represent the product of concentration by time for CS2 (upper axis) and MITC (lower axis). Canessa and Morrrll-SURVIVAL OF WOOD-COLONIZING FUNGI 2 17

------AMITC CS,/MITC

Cumulative Concentration (CT) CS, O.OEO0 1.4E+8 2.8E+8 4.2E+8 5.6E+8 7.OE+8 8.4E+8 2.5 I I I I I

2.0-

1.5 A 1.o- -.=. . 0.5- .=A B A

0 I I I I I O.OEO0 2.OE+5 4.OE+5 6.OE+5 8.OE+5 1.OE+6 1.2E+6 Cumulative Concentration (CT) MITC FIG.4. Effect of sublethal dosages of CS2 (3,0004,000 ppm) and MITC (5 ppm) and a CS2/MITC mixture (3,000- 4,000 ppm CS2/5 ppm MITC) on survival of I. lacteus on A) Douglas-fir and B) ponderosa pine. The Y axis represents the logarithm of survival, and the X axes represent the product of concentration by time for CS2 (upper axis) and MITC (lower axis). WOOD AND FIBER SCIENCE, JULY 1995, V. 27(3)

Cumulative Concentration (CT) CS, O.OEO0 1.4E+8 2.8E+8 4.2E+8 5.6E+8 7.OE+8 8.4E+8 3.0 I I I I I

2.3-.., , w -A,=*.,.A*. w- w , -=-.. ,-.-.-. -No\.., W 1-6 I..,--A..,, A - CS, ------AMITC '@.. '@.. --a=. A '. -*-.. --- 0.9 a-• **.. CS,/MITC - \-\, .-A.... .* .* --.. -0.K-.. --. -a. 0.2 - a--- @\.\@ -\, @-A~A A .. - -0.5 I I I I I 0 -2 O.OEO0 2.OE+5 4.OE+5 6.OE+5 8.OE+5 1.OE+6 1.2E+6 -a Cumulative Concentration (CT) MlTC .-> L> 3 Cumulative Concentration (CT) CS, '/> O.OEO0 1.4E+8 2.8E+8 4.2E+8 5.6E+8 7.OE+8 8.4E+8 3.0 I I I I I

2.3 - C "'-&--..,A A p --=.. A '*', ---...---.--.. A mi! 0.9- '-, ------A--.. C', -0. A-.., -.. '* '* '. A ---0.. A 0.2 - @-@,-@- B *'. -0.5 I I I I 1 O.OEO0 2.OE+5 4.OE+5 6.OE+5 8.OE+5 1 .OE+6 1.2E+6 Cumulative Concentration (CT) MITC FIG. 5. Effect of sublethal dosages of CS2 (3,0004,000 ppm) and MITC (5 ppm) and a CS2/MITC mixture (3,000- 4,000 ppm CS2/5 ppm MITC) on survival of G. trabeum on A) Douglas-fir and B) ponderosa pine. The Y axis represents the logarithm of survival, and the X axes represent the product of concentration by time for CS2 (upper axis) and MITC (lovier axis). Canessa and Morrell-SURVIVAL OF WOOD-COLONIZING FUNGI 219

Cumulative Concentration (CT) CS, O.OEO0 1.4E+8 2.8E+8 4.2E+8 5.6E+8 7.OE+8 8.4E+8 3.0 I I I I I

a .-.-.a 1.8- a H

1.2- "*A,,-... A -0. -0. ------AMITC -a.o. 0.6- -0. A CS,/MITC A--..,, A 0 1 1 I I I I hcn O.OEO0 2.OE+5 4.OE+5 6.OE+5 8.OE+5 1.OE+6 1.2E+6 -0 -V Cumulative Concentration (CT) MITC .-B L Cumulative Concentration (CT) CS, 3 O.OEO0 1.4E+8 2.8E+8 4.2E+8 5.6E+8 7.OE+8 8.4E+8 3.0 I I I I I 2.4- . n . 1.8- •-. [email protected] ¤ ..-a- a ..-.., -0 @a. -a\. 1.2- -..-.. --. -=-. ---. -.a A A="--..oo ---a=-=. 0.6- A ""+.. -*.A-0.. A B

0 I I I I O.OEOO 2.OE+5 4.OE+5 6.OE+5 8.OE+5 1.OE+6 1.2E+6 Cumulative Concentration (CT) MITC FIG.6. Effect of sublethal dosages of CS2 (3,0004,000 ppm) and MITC (5 ppm) and a CS2/MITC mixture (3,000- 4,000 ppm CS2/5 ppm MITC) on survival of G. saepiarium on A) Douglas-fir and B) ponderosa pine. The Y axis represents the logarithm of survival, and the X axes represent the product of concentration by time for CS2 (upper axis) and MITC (lower axis). WOOD AND FIBER SCIENCE, JULY 1995, V. 27(3)

Cumulative Concentration (CT) CS, O.OEO0 1.4E+8 2.8E+8 4.2E+8 5.6E+8 7.OE+8 8.4E+8 3.0 I I I I I

2.5- A .--__-...I,..A .... a-----A-----P-----,. ,@".- ..-.-.--w4 -.-.---.I. 1.5- . ------AMITC 1.o- A

0.5 I 1 I I I hcn 0.OEOO 2.OE+5 4.OE+5 6.OE+5 8.OE+5 1.OE+6 1.2E+6 -0 V Cumulative Concentration (CT) MITC -m .-5 L Cumulative Concentration (CT) CS, 3 u, O.OEO0 1.4E+8 2.8E+8 4.2E+8 5.6E+8 7.OE+8 8.4E+8 3.0 I I I I I I

Cumulative Concentration (CT) MITC FIG.7. Effect of sublethal dosages of CS2 (3,0004,000 ppm) and MITC (5 ppm) and a CS2/MITC mixture (3,000- 4,000 ppm CS2/5 ppm MITC) on survival of T. versicolor on A) Douglas-fir and B) ponderosa pine. The Y axis represents the logarithm of survival, and the X axes represent the product of concentration by time for CS2 (upper axis) and MITC (lower axis). Canessa and Morrell-SURVIVAL OF WOOD-COLONIZING FUNGI 22 1

Cumulative Concentration (CT) CS, O.OEO0 1.4E+8 2.8E+8 4.2E+8 5.6E+8 7.OE+8 8.4E+8 1 I I I I

------AMITC CS,/MITC

I I I I I 4 A 0.4 ! - O.OEO0 2.OE+5 4.OE+5 6.OE+5 8.OE+5 1.OE+6 1.2E+6 -V Cumulative Concentration (CT) MITC .-(d> L Cumulative Concentration (CT) CS, O.OEO0 1.4E+8 2.8E+8 4.2E+8 5.6E+8 7.OE+8 8.4E+8 2.5 1 I I I I

2.1 s*e7m-.-a- h...:>*. -0.y. a .-.- '**y a a -.- . 1.7- *:.:> 4:.. 0.: 0.: L..:2~...>. 1.3- *=.>-- A?-.:>. -;=>:Ia --A 0.9- B a

0.5 I I I 1 I O.OEO0 2.OE+5 4.OE+5 6.OE+5 8.OE+5 1.OE+6 1.2E+6 Cumulative Concentration (CT) MITC FIG.8. Effect of sublethal dosages of CS2 (3,000-4,000 ppm) and MITC (5 ppm) and a CS2/MITC mixture (3,000- 4,000 pprn CS2/5 ppm MITC) on survival of H. resinae on A) Douglas-fir and B) ponderosa pine. The Y axis represents the logarithm of survival, and the X axes represent the product of concentration by time for CS2 (upper axis) and MITC (lower axis). 222 WOOD AND FIBER SCIENCE, JULY 1995, V. 27(3)

TABLE5. Concentration x time values necessary to kill 90% of thepropagules (CTya)for seven species offungi exposed to CS2, MITC, or a CSr/MITC mixture. (CTyOvalues are all x 106.) Values in parentheses represent the number of days necessary to reach 90% kill; for the mixture, values in parentheses are for the combined CS2 and MITC treatment.

P. placenta I. lactm A, carbonica D-firb D-fir Pine D-fir Pine

CS2 3,0004,000 ppm 3,172 (42) 1,544 (20) 551 (7) 489 (6.5) 253 (3) 8,000-9,000 ppm 3,497 (1 9) 1,722 (9) 1,193 (6.5) 780 (4) 798 (4) MITC 5 PP~ 6.72 (62) 1.95 (18) 2.2 1 (20.5) 0.83 (7.7) 7.85 (72) 10 PP~ 7.47 (35) 1.99 (9) 1.81 (8.4) 1.10 (5) 1.05 (5) 18 PP~ 1.99 (5) 1.3 (3.3) 1.67 (4.3) 1.16 (3) 1.12 (3) Mixture 3,000-4,000 ppm CS2 942 496 526 208 39 1 5 ppm MITC 1.35 (2.5) 0.7 1 (6.6) 0.75 (7) 0.33 (3) 0.56 (5)

than for ponderosa pine, but this effect was not gest that the fungicidal action of metham so- evident for higher CS, levels (8,000-9,000 dium in wood is far more complex than pre- ppm), for MITC alone, or for the CSJMITC viously thought. While synergistic activity mixtures. Douglas-fir heartwood is less per- among biocides is well documented, the pos- meable than pine sapwood (Panshin and sibility that the various metham sodium de- DeZeeuw 1980); however, Douglas-fir per- composition products can interact to enhance meability to MITC is largely influenced by performance helps explain the higher activity wood moisture content (Zahora 1987). Very of this fumigant compared with that of others. low MITC concentrations, which may not be The results also suggest that interactions may toxic to inactive decay fungi in dry wood, be- occur among the other decompositi~nprod- come fungitoxic in wet wood. Because fungal ucts. Metham sodium decomposes to more growth and active decay will only occur in wood than 14 possible compounds. Of these, only above the fiber saturation point, increased sus- CS, and MITC have been studied in wood. ceptibility ofA. carbonica to MITC in wet wood Further studies using other volatile decom- may be important in determining long-term position products such as methylamine and wood protection. In the present study, mois- hydrogen sulfide may provide important clues ture content in wood was maintained above concerning the factors that maximize the fun- the fiber saturation point, which may help ex- gitoxicity of metham sodium in wood; such plain the excellent fungitoxicity of the fumi- factors could be used to enhance decomposi- gants despite the low MITC concentrations tion to produce the compounds most likely to employed. affect fungal control.

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TABLE5. Extended.

G. trabeum G. saeolanum T. versicolor H. resrnae D-fir P~ne D-fir P~ne D-fir Pine D-fir Pine

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