The manner by which lignin is degraded in-situ in natural substrates by white rot fungi still remains a controversial issue particularly the distribution and role(s) played by lignin degrading enzymes (i.e. manganese II peroxidase and lignin peroxidase). In the present study, use was made of anti-manganese II peroxidase and immunolabelling techniques in conjunction with transmission electron microscopy (TEM) to study the spatial distribution of manganese II peroxidase during degradation of wood and woody fragments by Phanerochaete chrysosporium and Lentinus edodes. Intracellularly, manganese II peroxidase was found localized in the peripheral regions of the fungal cell cytoplasm in association with both the outer cell membrane and membranes within characteristic vesicular bodies. In addition the enzyme was frequently found localized at the interfacial regions of the cell membrane and inner fungal cell wall. Using double immunolabelling procedures and in addition anti-lignin peroxidase, the cytoplasmic distribution of the two lignin degrading enzymes was compared. Both enzymes showed a fairly similar peripheral cytoplasmic localization although manganese II peroxidase tended to be more concentrated compared to lignin peroxidase in peripheral vesicular bodies. Extracellularly, and in solid wood samples manganese II peroxidase was found localized in all wood cell wall regions of either Betula verrucosa, Populus sp. or Fagus sylvatica decayed by either Phanerochaete chrysosporium or Lentinus edodes at both early and late stages of degradation. In particular, manganese II peroxidase was localized in characteristic zones of degradation produced within the secondary wood cell wall regions. These regions displayed a more open structure compared to unattacked wood cell walls and were easily penetrated by lignin degrading enzymes as judged by infiltration and double immunolabelling studies with highly purified and partially purified manganese II and lignin peroxidases. With Lentinus edodes a very characteristic pattern of lignin degradation was noted in which the middle lamella regions between wood cells was selectively degraded. In these regions manganese II peroxidase was found concentrated and associated with its degrading matrix. An extracellular distribution of manganese II peroxidase associated with wood fragments was also observed in liquid cultures of Phanerochaete chrysosporium grown under conditions optimal for peroxidase production. Despite the immersed conditions, similarities between the patterns of attack and extracellular distribution of the enzyme as for solid wood were noted. With both solid wood and wood fragments, manganese II peroxidase penetration was restricted to regions showing structurally modification, and penetration into undecayed cell walls was not observed. The present work suggests a close substrate-enzyme association during wood cell wall and lignin degradation under natural conditions, and in addition, a close correlation between changes in the micromorphology of decay and manganese II peroxidase distribution. Possible reasons for the failure of previous and similar immunolabelling studies to show such a correlation with lignin degrading enzymes are briefly discussed.

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