Accelerted detection of brown-rot decy: Comprison of soil block test, chemicl nlysis, mechnicl properties, nd immunodetection C. A. Clusen S. N. Krtl Abstrct Erly detection of wood decy is criticl becuse decy fungi cn cuse rpid structurl filure. The objective of this study ws to compre the sensitivity of different methods purported to detect brown-rot decy in the erly stges of development. The immunodignostic wood decy (IWD) test, soil block test/cke pn test, mechnicl property tests, nd chemicl nlysis were evluted in southern yellow pine blocks nd stkes exposed to Posti plcent for 5 weeks. The IWD test ws percent positive in blocks fter 5 dys of incubtion; similrly, IWD ws percent positive in stkes fter 3 dys. Weight loss ws not ccurte for mesuring decy in stkes using the cke pn method, but significnt weight loss (1 8%) occurred in blocks fter4 weeks ofexposure to the fungus in soil block tests. Mximum compressive strength (MCS) nd modulus of elsticity (MOE) were reduced 2 1 nd 13 percent, respectively, in blocks exposed to the fungus for 2 weeks. In stkes, MOE nd modulus ofrupture were reduced 9 percent nd 19 percent, respectively, fter 4 weeks of exposure. Arbinn, xyln, nd rhmnn decresed rpidly in blocks, exceeding 30 percent fter 4 weeks ofexposure. In stkes, only glctn ws decresed beyond 30 percent fter4 weeks. Smple size nd shpe ws significnt fctor in successfully detecting erly stges of brown-rot decy in lbortory tests. Blocks fostered rpid coloniztion in soil block tests, nd most methods tested were ble to detect the fungus sooner in blocks thn in stkes. The IWD test ws the most rpid method of detecting P. plcent (from 3 to 5 dys), followed by reduction in MCS in blocks. D ecy cused by brown-rot fungi is the most prevlent nd destructive type of wood deteriortion becuse it cn cuse rpid structurl filure. Annul losses of over $1 billion in the United Sttes result from fungl deteriortion of untreted or indequtely treted wood (Scheffer 1973). Losses re difficult to quntify. but it is estimted tht 10 percent of the nnul timber hrvested in the United Sttes is used to replce wood tht hs deteriorted in service (Zbel nd Morrell 1992). Although incipient brown-rot decy occurs shortly fter brown-rot fungi initite coloniztion nd relese enzymes, there is no visible evidence of dmge to the wood. However, chemicl chnges during initil coloniztion result in mesurble reductions in strength before mesurble weight loss (Schmidt et l. 1978, Wilcox 1978, Immur 1993, Kim et l. 1996). During erly decy, slight chnges in color or texture occur, but decy is not yet obvious (Zbel nd Morrell 1992, Clusen et l. 2001). By the time 1 percent weight loss is relized, lbortory tests show tht losses in toughness rnge from 6 percent to greter thn 50 percent. Strength losses my exceed 50 percent by the time 10 percent weight loss is incurred (Highley 1999). The uthors re, respectively, Reserch Microbiologist, USDA Forest Service, Forest Products Lb., One Gifford Pinchot Drive, Mdison, WI 53705-2398; nd Assistnt Professor, Deprtment of Forest Biology nd Wood Protection Technology, Forestry Fculty, Istnbul Univ., Istnbul, Turkey. The uthors would like to cknowledge the technicl ssistnce of Mrshll Begel nd Lori Schroeder of the Engineering Mechnics Lbortory, Forest Products Lb., Mdison, WI. This pper ws received for publiction in June 2002. Article No. 9509. Forest Products Society 2003. Forest Prod. J. 53(11/12):90-94. 90 NOVEMBER/DECEMBER 2003
Tble 1. Results of immunodignostic wood decy (IWD) test on southern yellow pine blocks nd stkes exposed to P. plcent over time. Positive detection of decy Incubtion time Blocks Stkes (dy) - - - - - - - - - - - - - - - - - (%) - - - - - - - - - - - - - - - - - 1 0 67 2 0 83 3 0 4 67 5 6 Ech vlue represents verge of six specimens. Tble 2. Loss in weight nd mechnicl properties for southern yellow pine blocks nd stkes exposed to P. plcent over time. Blocks Stkes Incubtion Weight MOE MCS Weight MOE MOR time loss reduction reduction loss reduction reduction (wk.) - - - - - - - - - - - - - - - - - - - - - - - - - - - - (%) - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 0 3 6 0 5 2 2 3 13 21 0 8 2 3 9 22 37 1 7 8 4 18 34 53 3 9 19 5 26 53 71 7 12 22 Ech vlue represents verge of six specimens. The erly stges of decy hve been the focus of detection reserch for mny yers. If method or combintion of methods cn detect incipient decy in wood nd ccurtely dignose the presence of brown-rot fungi, then remedil steps cn be tken to rrest fungl growth prior to structurl dmge. Methods for detecting the erliest stges of fungl decy include tests for mechnicl strength (i.e., compression test or mesurement of modulus of elsticity [MOE]), electricl conductivity (e.g., moisture meter or shigometer), coustic detection (e.g., coustic emission or stress-wve timer), chemicl nlysis, nd lbortory detection (e.g., culturing, microscopy, or serologicl tests). Culturing nd microscopy re currently considered the only definitive methods. Mesurements of work to mximum lod, toughness, nd impct bending re lso reported to be sensitive mechnicl methods for detecting erly decy (Wilcox 1978). In study on the effect of hemicellulose degrdtion on strength properties of wood, Curling et l. (2001) exposed southern yellow pine stkes to Gloeophyllum trbeum nd nlyzed chemicl composition, mechnicl properties, nd representtive weight loss. Their FOREST PRODUCTS JOURNAL VOL. 53, No. 11/12 results demonstrted rtio of strength to weight loss of pproximtely 40:1. Chemicl nlysis indicted tht erly losses in rbinn nd glctn were ssocited with erly strength loss. Winndy nd Morrell (1993) lso demonstrted reltionship between hemicellulose degrdtion, prticulrly rbinose nd mnnose, nd strength loss. One type of serologicl detection, the immunodignostic wood decy (IWD) test, utilizes nti-xylnse ntibody to detect minute quntities of fungl xylnse (Clusen nd Green III 1996). Becuse hemicelluloses re rpidly removed by brown-rot fungi during the initil stges of coloniztion, the presence of endo-1,4-ß-xylnse, hemicellulse, indictes incipient decy (Clusen et l. 1991). The IWD test hs been shown to detect brown-rot decy fungi prior to loss in wood weight or strength (Clusen et l. 1991, Clusen nd Ferge 1995, Clusen et l. 2001). The objective of this study ws to compre the sensitivity of methods purported to detect brown-rot decy in the erly stges of development. Mterils nd methods Stem-sterilized southern yellow pine blocks (10 by 10 by 10 mm) were exposed to Posti plcent MAD 698 in stndrd soil block test (ASTM 1998). Southern yellow pine spwood (250 mm long by 25 mm tngentil by 10 mm rdil) were exposed to P. plcent MAD 698 in modified cke pn test (Soltis et l. 1992). For the cke pn test, 1 L of 1:1 soil nd vermiculite mixture ws plced in n luminum cke pn. The surfce ws covered with rows of southern pine feeders (42 by 29 by 3 mm). The moisture content of the soil/vermiculite mixture ws djusted to 50 percent of the wter-holding cpcity, nd the test pprtus ws utoclved t 103 kp nd 12 1 C for 45 minutes. When cool, the feeders were inoculted with the fungus by pipetting ml/pn of mcerted 3-week-old liquid culture of P. plcent evenly over the feeders. The test pprtus ws seled in plstic bg to prevent drying nd incubted t 27 C nd 70 percent reltive humidity (RH) for 3 weeks until the feeders were completely covered by fungl growth. Stem-sterilized test specimens were plced on top of the feeders nd incubted t 27 C nd 70 percent RH. Uninoculted stem-sterilized blocks nd stkes served s controls for the block nd stke tests. Six blocks nd six stkes were removed from their respective test pprtus fter 3, 4, 5, nd 6 dys. Six uninoculted control blocks nd stkes were lso tested s follows. Shvings were tken from ech stke using 6.4- mm drill bit. The blocks nd shvings were extrcted in queous 0.1 percent Triton X- (Sigm, St. Louis, Missouri), nd extrcts were tested by the IWD test (Clusen nd Green III 1996). Another six blocks nd six stkes were removed from their respective test pprtus weekly for 5 weeks, brushed free of mycelium, nd ovendried t 60 C for 48 hours. A mtched number of uninoculted blocks nd stkes were nlyzed weekly. Blocks nd stkes were conditioned t 20 C nd 65 percent RH prior to testing. Blocks were tested for weight loss, rdil compressive strength, nd MOE (ASTM 1998, 1998b). Stkes were tested for weight loss, MOE, nd modulus of rupture (MOR) (ASTM 1998, 1998b). Ech specimen ws nlyzed for crbohydrte content (Dvis 1998). Results Tbles 1 nd 2 show the results of the IWD test nd tests for mechnicl prop- 91
Figure 1. Weight loss, MOE, MCS, nd MOR of southern yellow pine blocks nd stkes exposed to P. plcent for 5 weeks compred to tht of uninoculted controls. (Brs represent stndrd devition; n = 6). Figure 2. Reltionship between weight loss nd strength reduction for loocks () nd stkes (b) exposed to P. plcent for 5 weeks. erties for blocks nd stkes fter vrious periods of exposure to P. plcent. Stkes tested percent positive by IWD fter 3 dys of exposure to the fungus, wheres blocks tested percent positive fter 5 dys. The dt on mechnicl properties show the totl percentge of reduction compred to the sme properties in uninoculted controls. MOE of blocks ws reduced 13 percent fter 2 weeks of incubtion, but MOE of stkes ws reduced only 12 percent fter 5 weeks of incubtion. Of the mechnicl properties tested, mximum compressive strength (MCS) showed the most rpid chnge. In blocks, MCS decresed 21 percent fter 2 weeks. The MOR of stkes decresed 19 percent fter 4 weeks of incubtion. Figure 1 summrizes weight loss, MOE, MCS, nd MOR for blocks nd stkes exposed to P. plcent for 5 weeks. Significnt weight loss (>10%) ws mesured in blocks fter 4 weeks. Weight loss proved to be n inccurte method of mesuring decy in stkes exposed to P. plcent in the modified cke pn method, compred to the stndrd ASTM soil block method (ASTM 1998). The size, shpe, nd greter volume of stkes slowed the progression of fungl coloniztion. Stndrd devition ws lso greter t ech test intervl for stkes compred with blocks. Figure 2 shows the reltionship between strength reduction nd weight loss in blocks nd stkes. In blocks, the reduction in MCS occurred sooner nd more rpidly thn the reduction in MOE (Fig. 2). By the time 9 percent weight loss hd occurred, MCS ws reduced 37 percent. In stkes tested by the cke pn method, MOR ws reduced 19 percent by the time 3 percent weight loss hd occurred. Tble 3 summrizes the results of crbohydrte nlyses for blocks nd stkes. Figure 3 shows the percentge of reduction of ech crbohydrte during the 5-week exposure to P. plcent. For blocks, losses in rbinn, rhmnn, nd xyln exceeded 30 percent fter 4 weeks of exposure when weight loss ws 18 percent, MOE ws reduced 34 percent, nd MCS ws reduced 53 percent. Anlysis of stkes reveled 30 percent loss in glctn fter 4 weeks, when no significnt weight loss hd occurred, MOE ws reduced 9 percent, nd MOR ws reduced 19 percent. 92 NOVEMBER/DECEMBER 2003
Tble 3. Crbohydrte nlysis of blocks nd stkes exposed to P. plcent for 5 weeks. Crbohydrte composition Specimens Time Arbinn Glctn Rhmnn Glucn Xyln Mnnn (wk.) Blocks 0 1 2 3 4 5 Stkes 0 I 2 3 4 1.01 0.87 (0.14) 0.78 (0.08) 0.68 (0.08) 0.55 0.61 (0.06) 1.02 (0.03) 0.93 (0.03) 0.94 0.86 0.82 1.37 (0.12) 1.40 (0.02) 1.10 (0.15) 1.02 (0.16) 1.07 (0.13) 0.86 (0.02) 2.63 2.20 (0.40) 2.13 2.05 1.69 (0.27) 5 0.77 2.04 40.48 5.93 8.98 (0.03) (0.19) (1.79) (0.56) (0.19) Ech vlue represents n verge of six specimens; numbers in prentheses re stndrd devitions. Discussion nd conclusions The IWD test clerly detected P. plcent just 3 dys fter inocultion. This result mirrors observtions from number of studies in which IWD ws ble to detect the presence of decy fungi prior to weight loss (Clusen et l. 1991, Clusen nd Ferge 1995, Clusen et l. 2001) nd strength loss (Clusen et l. 2001). The smpling method my ccount for differences seen in IWD test results from blocks nd stkes. Enzymes were extrcted from intct blocks; for stkes, shvings were used. The in- - - - - - - - - - - - - - - - - - - - - - - - - (%) - - - - - - - - - - - - - - - - - - - - - - - - 0.06 0.05 0.04 0.04 0.08 42.82 (0.87) 42.56 (0.88) 42.46 (1.05) 42.23 (0.94) 41.89 (0.92) 39.13 (0.53) 41.97 (1.12) 42.80 (1.21) 42.07 (0.23) 42.23 (0.50) 41.25 (0.87) FOREST PRODUCTS JOURNAL VOL. 53, NO. 11/12 5.88 (0.81) 5.05 (0.98) 5.60 (0.90) 4.82 (0.93) 3.91 (0.40) 4.36 (0.63) 6.44 (0.19) 6.39 (0.35) 6.61 6.21 6.13 (0.43) 11.32 (0.80) 12.15 (1.08) 10.32 (0.91) 9.65 (0.92) 10.73 (0.16) 7.87 (0.24) 9.84 (0.27) 10.43 (0.57) 10.53 (0.20) 10.62 (0.19) 9.84 (0.81) cresed surfce re of the shvings my hve incresed the mount of enzyme extrcted for detection. The dimensions of the stkes (250 by 25 by 10 mm) used in this study, though necessry for the stndrdized MOR test, hindered erly detection of P. plcent by ll methods except the IWD test. Size, shpe, nd volume of test specimen, s well s configurtion of test pprtus, re importnt fctors in rpid initition of fungl coloniztion. Idelly, test methods for erly brown-rot decy should detect initil chnges in colonized wood or fungl metbolites, nd ccelerted testing therefore relies on rpid fungl coloniztion. MCS showed the erliest nd most rpid decline of mechnicl properties tested in the 19-mm-squre blocks, followed by MOE (Tble 2). Significnt weight loss (>10%) did not occur until blocks hd been incubted for 4 weeks. By 3 weeks, 4: 1 rtio in strength loss to weight loss ws observed. Smith nd Grhm (1983) showed tht compressive strength losses were highly correlted to nd evident before weight loss in Dougls-fir decyed by P. plcent. For stkes, MOR showed the most rpid decline of mechnicl properties tested. Kim et l. (1994) demonstrted tht significnt strength loss could be detected in full-sized lumber fter 5 weeks of exposure to Gloeophyllum trbeum. In our study, stkes showed 6:1 rtio of strength loss to weight loss fter 4 weeks of incubtion. Nevertheless, weight loss ws not greter thn 7 percent, nd stndrd devitions in weight loss vlues were high fter 5 weeks of incubtion. We conclude tht the cke pn method used in this study is not conducive to ccurte or erly weight loss mesurements of stkes. Crbohydrte losses were inconsistent for decyed blocks nd stkes. Crbohydrte nlysis of blocks showed significnt losses (>30%) in rbinn, rhmnn, nd xyln fter 4 weeks of incubtion, while nlysis of stkes showed similr loss for only glctn fter this incubtion period. Coinciding with high losses in rbinn, rhmnn, nd xyln, blocks lso showed significnt weight loss (18%), 34 percent reduction in MOE, nd 53 percent reduction in MCS. On the other hnd, 36 percent reduction in glctn for stkes coincided with no significnt weight loss (3%), 9 percent reduction in MOE, nd 19 percent reduction in MOR. We conclude tht crbohydrte nlysis is not n ccurte or relible method for detecting incipient decy by the decy methods used in this study. This study illustrtes tht the sensitivity of methods for detecting incipient decy vries gretly, nd therefore methods must be used in combintion to evlute in-service wood for decy. A positive IWD does not necessrily indicte potentil strength reduction if conditions for sustined decy do not exist for the prticulr wood member being smpled. Rther, it cn serve s n indictor 93
Figure 3. Totlpercentge of reduction in crbohydrtes in blocks () nd stkes (b) exposed to P. plcent for 5 weeks, compred to crbohydrte reduction in uninoculted controls. for 1) dditionl testing with other methods for erly detection; 2) potentil losses if steps re not tken to protect in-service wood from further exposure to moisture nd decy fungi; or 3) res where remedil tretments to rrest decy re pproprite Literture cited 94 NOVEMBER/DECEMBER 2003