Comparison of isolates and strains within the brown-rot fungus genus Gloeophyllum using the soil block decay method.
Eleven decay tests, using three isolates of Gloeophyllum trabeum, one isolate of G. separium, plus five pairings (co-cultures) of G. trabeum isolates, were conducted based on standard soil block decay test methods. After 12 weeks, the greatest mean weight loss achieved in southern yellow pine sapwood blocks was 47.5 percent with a pairing of two isolates of G. trabeum (wild type DR 397 X ATCC 11539). The lowest weight loss achieved was 19.1 percent with a wild type isolate (DR 274). A newly obtained (2001) isolate of G. trabeum (MAD 617) achieved significantly better weight loss (43.7%) than a culture of the same isolate that had been used in the current lab since 1991 (24.2%). The results indicate the variability of decay capability of Gloeophyllum isolates due to such factors as age, storage, and origin. Results also demonstrate the difficulty of achieving 50 percent or more weight loss in southern yellow pine sapwood by a selection of Gloeophyllum isolates under optimum conditions using standard test procedures. In addition, it was found that inserting the test block in the soil jar at the time of inoculation resulted in the same amount of weight loss as the standard method of inserting the test block 3 weeks after inoculation; the former method saves time and reduces contamination.
Gloeophyllum trabeum (Pers: Fr.) Murr. (syn. Lenzites trabea) is a brown-rot fungus commonly used in wood preservative and wood products testing. It is the principal brown-rot fungus specified by various testing organizations (WDMA 2001, ASTM 2002, AWPA 2002) for screening in soil block decay testing. Gloeophyllum trabeum is important because it is tolerant to phenolics and arsenic-containing preservatives (AWPA 2002).
In these standard decay tests, a mean of 50 percent weight loss in untreated control sapwood blocks is considered a minimum for valid comparison to treatments; however, this level is not consistently achieved. A brief check of the literature shows that some laboratories using G. trabeum for brown-rot testing report 50 percent mean weight loss or above in southern yellow pine controls (Preston et al. 1983, Chen 1994a, Chen 1995, Worrall et al. 1997, DeGroot et al. 1998). However, when using these methods with G. trabeum over the past 12 years, published papers (e.g., Verhey et al. 2001, Laks et al. 2002) and unpublished proprietary reports from our laboratory have shown that we commonly achieve between 30 and 40 percent weight loss due to decay in untreated controls, although occasionally up to 70 percent weight loss has been achieved in individual blocks. Other labs have also reported less than 50 percent mean weight loss in control southern yellow pine sapwood using these tests with G. trabeum. For example, Amburgey (1979) reported between 21.2 and 34.9 percent mean weight loss in six tests of untreated slash pine when comparing the use of different soils in the test, and Chen (1994b) reported 36.6 percent mean weight loss in untreated loblolly pine.
It is not unusual for an isolate of G. trabeum to weaken in ability to decay wood after continual use and successive transfers. Gramass (1990) concluded that stock cultures of wood decay fungi are subject to progressive senescence even under optimum conditions of strain preservation. Our lab has noted loss in decay capability even after re-isolating cultures directly from decayed wood. Reduced ability to cause decay may be due to genetic changes after growing under laboratory conditions for long periods of time (Smith and Onions 1983, Gramass 1990). For example, older isolates of G. trabeum that have been in use in our lab for some time lose hyphal clamp connections, suggesting reversion to monokaryons and thus putatively less genetically viable (Rayner and Boddy 1988). Choi et al. (2001) have reported that storage and repeated subculturing affect fungal capacity for fruiting and spore production.
The isolate of G. trabeum specified by the three standard soil block test methods mentioned previously is American Type Culture Collection (ATCC) #11539, also designated as Madison (MAD) #617. These are identical isolates given different strain identification numbers; it was originally isolated from fruiting body tissue on a western redcedar pole collected by F.F. Lombard prior to 1977 (exact date is not cited [Jong and Edwards 1991]). We have used this isolate for wood preservative testing in our laboratory for many years; however, occasionally we have had to replace the culture being used with a fresh culture either from ATCC or the Forest Products Laboratory, Madison, WI, due to a weakening in its ability to decay wood.
In this paper, we report the testing of a number of different isolates and strains of G. trabeum to compare their ability to decay untreated southern yellow pine sapwood under optimum conditions as defined by standard wood decay tests. Some of the isolates tested were derived from the same source, but separated by space and time due to storage or retrieval conditions; hence, these are referred to as strains of the same isolate. Some of the isolates tested are wild types obtained from fruiting body tissue on wood by the first author. Decay tests were also conducted with two isolates or strains of G. trabeum, which were crossed in petri dishes prior to inoculation of wood blocks (co-inoculated). This was done due to the ability of monocaryotic hyphae of some fungi to fuse (anastomose) and exchange genetic material following contact with another monokaryon or dikaryon of the same species (Rayner and Boddy 1988, Ainsworth and Rayner 1989, Beeching et al. 1989). An isolate of G. sepiarium (Fr.) Karst., a brown-rot fungus commonly found on decaying conifer slash in North America (Boyce 1961, Gilbertson and Ryvarden 1986), and one that was found along with G. trabeum actively colonizing CCA-treated decking (Choi et al. 2003), is also included in the test as a comparison to G. trabeum. In addition, a simple modification is made to the standard method of inoculation that will save time and reduce the level of contamination in the soil block decay test methodology.
Materials and methods
Fungal isolates and strains
Table 1 lists fungus isolates and strains used in the decay tests; all were obtained from fruiting body tissue (dikaryotic). Prior to use in the decay test, cultures were maintained by growing fungi 4 to 6 weeks at room temperature on 2 percent malt agar (2M) in tubes, then refrigerated at 5[degrees]C for up to 1 year, when the process was repeated. Inoculum for wood decay tests was produced by transferring fungi from tubes onto 100-mm petri dishes of 2M and growing fungi at room temperature for 2 to 4 weeks until the plates were covered by mycelium.
Five decay tests were conducted with paired isolates or strains (co-cultures) (Table 2). Pairing was done by placing one small piece (approximately 2 to 5 mm in diameter) of mycelium of one isolate or strain in the center and at five equidistant places in the petri dish, and another five pieces of mycelium of another isolate or strain adjacent to the first piece of mycelium. Cultures were allowed to co-mingle and grow 2 to 4 weeks, as were individual isolates. Rejection zones were not observed, suggesting compatibility of mycelium and the possible exchange of genetic material (Rayner and Boddy 1988).
Soil jar set-up
Southern yellow pine sapwood blocks (19 by 19 by 19 mm) were exposed to fungi based on standard test methods (WDMA 2001, ASTM 2002, AWPA 2002). The methods are the same for these three protocols. A square jar (5 by 5 by 13.5 cm) containing 100[+ or -]1 g of air-dried (50[degrees]C) forest top-soil was wetted with 34[+ or -]1 mL of distilled water to obtain a moisture-holding capacity of approximately 30 percent; pH of the soil was 5.8 (1:1 soil to water); a pine feeder strip (0.5 by 3.2 by 2.0 cm) was placed on top of the soil, and a plastic lid with a 5-mm-diameter hole covered by a strip of adhesive first-aid tape was placed on the jar. Jars were autoclaved for 30 minutes two times, separated by 24 hours. After the jars were cool, a piece of agarmycelium inoculum (approximately 1 by 2 by 0.5 cm) was placed on the feeder strip and an ovendried, pre-weighed pine block, sterilized by autoclaving 15 minutes, was placed firmly on the agar and feeder strip. Lids were wrapped loosely with Parafilm[R] to exclude mites, then jars were incubated at 27[degrees][+ or -] 1[degrees]C, 75[+ or -]4 percent relative humidity for 12 weeks. Five replicate blocks were used per test, except in the feeder strip direct vs. delayed inoculation test where 15 replicate blocks were used (see below).
Delayed vs. direct inoculation test
The above soil block decay tests were set up by inoculating feeder strips and immediately placing blocks in jars (direct inoculation). However, the standard soil block decay methods for brown-rot fungi call for inoculating feeder strips, incubating for 3 weeks, then opening jars for placement of decay blocks (delayed inoculation). The following additional test was conducted to compare simultaneous inoculation of feeder strips and placement of blocks in jars with placement of blocks following 3 weeks incubation of the feeder strips.
A co-culture of G. trabeum ATCC 11539 and MAD 617R (new) was used in the comparison test. Fifteen replicate jars were used for each test. Jars were prepared as described previously. In one set, the feeder strips were inoculated and allowed to colonize for 3 weeks before placement of the soil block. In the other set of jars, the feeder strip, inoculum, and test block were added simultaneously. Jars were incubated identically to jars in the strain test described previously.
Results are shown in Table 3. The highest weight loss (47.5%) was produced by the G. trabeum isolate pairing, ATCC 11539 X DR 397; however, this was not significantly different from weight loss produced by ATCC 11539 alone (38.1%), although it was significantly different from weight loss caused by DR 397 alone (30.3%). The other isolate of G. trabeum that caused weight loss not significantly different than the highest weight loss was the new isolate of MAD 617 (43.7%). The only isolate of G. separium which was used. DR 275, also caused weight loss (39.6%) not significantly different from the highest weight loss caused by G. trabeum.
The older isolate of MAD 617 produced significantly less weight loss (24.2%) compared to the newly obtained isolate of MAD 617 (43.7%). This suggests a change in the ability to decay wood when fungi are maintained under laboratory conditions for extensive time periods. When the new isolate of MAD 617 was crossed with ATCC 11539, as was done in the feeder strip test, weight loss was 33.7 and 35.8 percent as compared to 27.6 percent obtained when the old isolate of MAD 617 was crossed with ATCC 11539; this suggests that the new isolate improved or imparted virulence to the ATCC strain.
The wild type isolate of G. trabeum, DR 274, caused significantly lower weight loss (19.1%) than all other isolates or pairings. When this isolate was paired with the more virulent ATCC 11539 isolate, which by itself had caused among the highest weight losses, the crossing significantly improved weight loss (26.0%) compared to the wild type alone. This is in contrast to pairing ATCC 11539 with the wild type isolate DR 397, which resulted in the highest weight loss (47.5%), white the isolate DR 397 alone resulted in significantly lower weight loss (30.3%).
Crossing of isolates appears to have improved decay virulence in some cases. For example, the wild type isolate of G. trabeum DR 274 produced only 19.1 percent weight loss, but when it was crossed with the old isolate of MAD 617 the co-culture produced better weight loss (30.2%) than either of the strains themselves (only significantly with DR 274). Conversely, when G. trabeum ATCC 11539 was crossed with MAD 617 (old), the combination produced significantly less weight loss (27.6%) than did ATCC 11539 alone (38.1%). Also, when the ATCC 11539 strain was crossed with the new isolate of MAD 617, as was done in the feeder strip test, weight losses were improved (33.7% and 35.8%) compared to the crossing with the old isolate of MAD 617 (27.6%).
Delayed vs. direct inoculation test
Using the same fungus co-culture (G. trabeum ATCC 11539 X MAD 617 [new]), no significant difference (p = 0.05) was seen in mean percent weight loss between blocks directly inoculated (33.7%, SD 9.8) or blocks that were placed on feeder strips that were pre-colonized by G. trabeum for 3 weeks (delayed inoculation) (35.8%, SD 8.9) (Table 3).
1. In these 11 tests with 3 isolates of various ages and histories of G. trabeum, the optimum 50 percent weight loss, as described by AWPA (2002), was not achieved; the highest weight loss was 47.5 percent. The results illustrate the difficulty of obtaining 50 percent weight loss in solid southern yellow pine sapwood in brown-rot decay soil block tests when using G. trabeum under the conditions of the test.
2. Crossing of two different isolates by co-culturing fungi in petri dishes appears to impart characteristics of either of the fungi, and may, in some cases, reinvigorate an isolate of a culture of G. trabeum to decay wood.
3. New tissue isolates of Gloeophyllum fruiting on wood vary widely in their ability to cause decay.
4. Results of the delayed vs. direct inoculation test showed that there is no significant difference in mean percent weight loss in blocks that were inoculated at the same time as the feeder strips compared to blocks that were placed in jars following 3 weeks of colonization of the feeder strips. Time could be saved and contamination reduced in G. trabeum brown-rot soil block decay tests by modifying the standard methods to simultaneously inoculate blocks and feeder strips. This latter method may also be beneficial for other brown-rot fungi.
Table 1. -- Isolates and strains of Gloeophyllum used in the decay tests (all are dikaryotic tissue isolates). Fungus name Isolate designation Date isolated Date obtained by lab G. trabeum ATCC 11539 <1977 1996 G. trabeum MAD 617 (old) <1977 1991 G. trabeum MAD 617 (new) <1977 2001 G. trabeum DR 274 1990 1990 G. trabeum DR 397 1998 1998 G. separium DR 275 1990 1990 Fungus name Habitat/source G. trabeum Fruiting body on western redcedar pole G. trabeum Fruiting body on western redcedar pole G. trabeum Fruiting body on Western redcedar pole G. trabeum Fruiting body on creosote-treated hardwood R.R. tie G. trabeum Fruiting body on aspen log G. separium Fruiting body on jack pine slash Table 2. -- Pairs of isolates and strains of Gloeophyllum trabeum used in the decay tests. ATCC 11539 X MAD 617 (new) ATCC 11539 X MAD 617 (old) ATCC 11539 X DR 274 ATCC 11539 X DR 397 DR 274 X MAD 617 (old) Table 3. -- Mean percent weight loss of southern yellow pine sapwood blocks exposed to test fungi or pairs of fungi. Fungus Isolate and strain designation Mean weight loss (%) G. trabeum DR 397 X ATCC 11539 47.5 (5.5) A G. trabeum MAD 617 (new) 43.7 (6.5) A G. sepiarium DR 275 39.6 (4.4) AB G. trabeum ATCC 11539 38.1 (6.8) ABC G. trabeum ATCC 11539 X MAD 617 (new) delayed inoc. 35.8 (8.9) BCD test G. trabeum ATCC 11539 X MAD 617 (new) direct inoc. 33.7 (9.8) BCDE test G. trabeum DR 397 30.3 (4.6) CDE G. trabeum DR 274 X MAD 617 (old) 30.2 (6.0) CDE G. trabeum ATCC 11539 X MAD 617 (old) 27.6 (2.4) DE G. trabeum DR 274 X ATCC 11539 26.0 (3.7) E G. trabeum MAD 617 (old) 24.2 (3.8) E G. trabeum DR 274 19.1 (1.9) F (a) n=5, except n = 15 for feeder strip delayed vs. direct inoculation test; values in parentheses are standard deviations; means followed by different capital letters are significantly different (p = 0.05).
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Dana L. Richter
Peter E. Laks*
Kimberly M. Larsen
Aimee L. Stephens
The authors are, respectively, Research Scientist, Professor, and Lab Assistant, School of Forest Resources and Environmental Science (former Institute of Wood Research), Michigan Technological Univ., Houghton, MI 49931; and Forest Products Technologist, Paper Testing Laboratory, USDA Forest Products Lab., One Gifford Pinchot Dr., Madison, WI 53705. The authors wish to thank Dr. Jessie A. Micales of the Forest Products Laboratory, Madison, WI, for providing a culture of G. trabeum used in these tests, and the two anonymous reviewers for their careful reading of the manuscript. We also thank numerous industrial sponsors for their funding of decay tests over the years, which made this paper possible. This paper was received for publication in October 2003. Article No. 9766.
*Forest Products Society Member.
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|Author:||Richter, Dana L.; Laks, Peter E.; Larsen, Kimberly M.; Stephens, Aimee L.|
|Publication:||Forest Products Journal|
|Date:||Jan 1, 2005|
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