OXIDATIVE SYSTEMS OF WOOD DECAY FUNGI

• Sponsoring Institution: Forest Service/USDA
• Project Status: COMPLETE
• Funding Source: USDA INHOUSE
• Reporting Frequency: Annual
• Accession No.: 0212931
• Project Start Date: Jul 23, 2007
• Project End Date: Jul 22, 2012
• Program Code: [(N/A)]- (N/A)

Recipient Organization
FOREST PRODUCTS LABORATORY
ONE GIFFORD PINCHOT DRIVE
MADISON,WI 53726

Performing Department
FOREST PRODUCTS LAB, MADISON LAB HQ - MADISON, WI

Non Technical Summary
This research seeks to identify and understand the oxidative systems of wood decay fungi so that we can increase the efficacy of these naturally-occurring organisms in bioconversion technologies, devise new environmentally-sound ways to protect wood in use, and accelerate decomposition of forest litter. The principal research needs in this area are to identify, at the genetic and biochemical level, (1) the systems that selective white-rot fungi use to modify wood during bioprocessing and (2) the systems brown rot fungi use to penetrate the lignin in wood and depolymerize the cellulose.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
123	0650	1000	20%
123	4020	1000	20%
123	4020	1040	20%
511	0650	1000	20%
511	4020	1000	10%
511	4020	1040	10%

Knowledge Area
511 - New and Improved Non-Food Products and Processes; 123 - Management and Sustainability of Forest Resources;

Subject Of Investigation
0650 - Wood and wood products; 4020 - Fungi;

Field Of Science
1040 - Molecular biology; 1000 - Biochemistry and biophysics;

Keywords

bioconversion

thinnings

biotechnology

oxidative fungi

mechanisms

wood decay

Goals / Objectives
The objectives of this research are to understand which aspects of fungal metabolism need to be optimized and which need to be deselected to obtain high-performance organisms and enzymes for technological application. The principal research needs in this area are to identify, at the genetic and biochemical level, (1) the systems that selective white-rot fungi use to modify wood during bioprocessing and (2) the systems brown rot fungi use to penetrate the lignin in wood and depolymerize the cellulose.

Project Methods
This research involves basic investigations into the mechanisms of lignin and cellulose degradation by selected white rot and brown rot fungi. Emphasis will be on elucidating the chemistry of extracellular oxidative systems that these fungi produce, and identifying the genes and enzymes that are responsible for producing the oxidants. The underlying hypothesis for the work is that, because enzymes such as cellulases are too large to penetrate sound wood, decay fungi produce low-molecular-weight metabolites that can penetrate the lignin matrix in wood and generate oxidants in situ to initiate depolymerization of lignin or cellulose. Subsequent reactions between ferrous iron and reduced oxygen species then produce hydroxyl radicals, which are very strong oxidants that cleave both cellulose and lignin. Research also indicates that the fungi probably employ quinone reductase enzymes to maintain their hydroquinones in the reduced state and thus drive the reduction of iron and oxygen. It is not yet known how the fungi synthesize the hydroquinones, or how the hydroquinone-driven oxidation system is coordinated with production of enzymes such as cellulases, or how widespread this biodegradative mechanism is among wood decay fungi. We will address these problems in the following ways: 1. Begin to elucidate the biochemical pathways these fungi use to biosynthesize hydroquinones. These pathways could be key targets for inhibitors of wood decay, which might then find use as new wood preservatives. 2. Identify the quinone reductases, and the genes that encode them, from wood decay fungi. These enzymes are also potential targets for inhibition. Alternatively, it may be possible to up-regulate these reductases, with the result that the fungi are better biodegraders of lignocellulosic wastes or organopollutants. 3. Determine how hydroquinone production and quinone reductase expression is coordinated with the production of extracellular cellulases. This question is important because the selective delignification of wood by fungi is a potentially useful process that relies on the early production of small agents. 4. Determine, by screening a variety of white, brown, and soft rot fungi, how widespread the hydroquinone-dependent biodegradative mechanism is. Identify new biodegradative mediators that these fungi may use instead of hydroquinones. 5. Identify and develop new nano-protection systems for wood 6. Identify and characterize new metabolic pathways involved in wood decay that might serve as targets for inhibition of decay or provide new opportunities for bioconversion technologies. Specific emphasis will be on extracellular carbohydrate metabolism related to production of peroxide, reductants, and organic acids important during incipient decay of lignocellulosics. 7. Communicate the findings in high-quality scientific papers in refereed journals and in lectures before a variety of audiences.

Progress 07/23/07 to 07/22/12

Outputs
OUTPUTS: Decay fungi that cause white rot and brown rot of wood or plant litter have key roles in global carbon cycling and forest health. The agents they use to deconstruct lignin and other wood polymers include a poorly understood group of enzymatic and nonenzymatic agents that not only have an important ecological role, but also have potential industrial applications in woody biomass utilization. In FY2012, research focused on the oxidative and hydrolytic enzymes some of these fungi use to deconstruct lignocellulose. The brown rot fungus Postia placenta was shown to secrete a complex of cellulose-hydrolyzing enzymes, whose properties were extensively characterized. In addition, a newly discovered ligninolytic peroxidase was discovered and characterized from Ceriporiopsis subvermispora, and was shown to have properties that may contribute to selective lignin degradation by this white rot fungus. Finally, a peroxygenase enzyme from the litter decay fungus Agrocybe aegerita was shown to oxidize many pharmaceutical compounds, which suggests peroxygenases may have utility in fine chemicals synthesis. These investigations open new avenues for research and development involving lignocellulose-degrading fungi. Results of these studies were disseminated in influential peer-reviewed journals. Termination Report: In FY2008, a new spectroscopic approach showed for the first time that a brown rot fungus degrades lignin. In FY2009, a fungal peroxygenase was shown to cleave diverse ethers, including some environmentally significant pollutants. In FY2010, a brown rot fungus was found to produce a laccase, and this enzyme was shown to have a biodegradative role. In FY201l, degraded lignin structures in brown-rotted wood were identified for the first time, and the results pointed to a nonselective free radical mechanism for fungal attack on the wood. In FY2012, a lignin peroxidase was characterized for the first time from a selective white rot fungus that was previously thought to lack this type of enzyme. This problem area will be continued in the new Research Work Unit Description. PARTICIPANTS: Significant participants during FY2010 included the Consejo Superior de Investigaciones Científicas in Madrid, Spain and the International Graduate School of Zittau, Germany.

Impacts
This research resulted in a change in knowledge regarding the ability of brown rot fungi to cleave cellulose, discovered a new type of ligninolytic peroxidase with potential bioprocessing applications, and also pointed to a biotechnological use for fungal peroxygenases.

Publications

• Langan, Paul; Gnanakaran, S.; Rector, Kirk D.; Pawley, Norma; Fox, David T.; Cho, Dae Won; Hammel, Kenneth E. 2011. Exploring new strategies for cellulosic biofuels production. Energy and Environmental Science. 4: 3820-3833.
• Poraj-Kobielska, Marzena; Kinne, Matthias; Ullrich, René; Scheibner, Katrin; Kayser, Gernot; Hammel, Kenneth E.; Hofrichter, Martin. 2011. Preparation of human drug metabolites using fungal peroxygenases. Biochemical Pharmacology. 82: 789-796. DOE 10.1016/j.bcp.2011.06.020.
• Ryu, Jae San; Shary, Semarjit; Houtman, Carl J.; Panisko, Ellen A.; Korripally, Premsagar; St. John, Franz J.; Crooks, Casey; Siika-aho, Matti; Magnuson, Jon K.; Hammel, Kenneth E. 2011. Proteomic and functional analysis of the cellulase system expressed by Postia placenta during brown rot of solid wood. Applied and Environmental Microbiology. 77(22): 7933-7941.
• Fernandez-Fueyo, Elena; Ruiz-Duenas, Francisco J.; Miki, Yuta; Martinez, Marta Jesus; Hammel, Kenneth E.; Martinez, Angel T. 2012. Lignin-degrading peroxidases from genome of selective ligninolytic fungus Ceriporiopsis subverispora. The Journal of Biological Chemistry. 287(20): 16903⿿16916.

Progress 07/23/07 to 07/22/12

Outputs
OUTPUTS: This project is complete. PARTICIPANTS: This project is complete. TARGET AUDIENCES: This project is complete. PROJECT MODIFICATIONS: This project is complete.

Impacts
This project is complete.

Publications

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Decay fungi that cause white rot and brown rot of wood or plant litter have key roles in global carbon cycling, humus formation, and pollutant detoxification in forest soils. The agents they use to deconstruct wood polymers and other recalcitrant chemicals include a poorly understood group of enzymatic and nonenzymatic oxidants that not only have an important ecological role, but also have potential industrial applications in woody biomass utilization. In FY2010, research focused on the biotransformations accomplished by two types of secreted fungal oxidative enzyme, a white rot peroxygenase and a brown rot laccase. The peroxygenase was shown to catalyze several environmentally important oxidations, including the cleavage of hazardous ethers. The laccase was shown to oxidize a secreted fungal metabolite-a hydroquinone-to produce reactive oxygen species that have a key role in the biodegradation of lignocellulose. These investigations open new avenues for research and development of processes for lignocellulose bioconversion, organopollutant remediation, and fine chemicals production. Results of these studies were disseminated in influential peer-reviewed journals. Events: Co-Organizer and Chair of two symposia at the International AAAS Annual Meeting, San Diego, CA, including the symposium Science of the Small: NanoBioTechnology under the Biological Microscope. Served on the Organizing Committee of the International AAAS 2010 Annual Meeting, Biological Sciences Section, San Diego, CA, proposed and sponsored nine symposia. Services: The research carried over from our previous RWU-4502 on invasive species lead to co-authoring a Forest Service Research Strategy, external consulting for the Kansas state DNR regarding quarantine issues with a fungal disease of black walnut, provided recommendations that assisted the decisions for new quarantine requirements, and consulting for USDA-APHIS and IFQRG-UN-FAO-IPPC. PARTICIPANTS: Significant participants during FY2010 included the International Graduate School of Zittau, Germany. TARGET AUDIENCES: Academic, governmental and industrial scientists engaged in research for the protection of wood in service and/or conversion of lignocellulose to value-added products.

Impacts
This research resulted in a change in knowledge regarding the role of fungal oxidants in bioconversions. Specifically, the roles of two enzymes in these processes were shown to be more extensive than previously realized.

Publications

• Illman, Barbara L.; Yang, Vina W. 2010. Mycoremediation of waste wood treated with CCA. In: Seventh international conference remediation of chlorinated and recalcitrant compounds preliminary program. 2010 March 24-27; Monterey, CA. Battelle 7th International conference program and abstract book, Monterey, CA. 5 p.
• Illman, Barbara. 2010. From landscape to nanoscale level [abstract]. In: Bridging science and society AAAS Annual Meeting. 2010 February 18-22; San Diego, CA. Washington, DC: American Association for the Advancement of Science. A50.
• Kapich, Alexander N.; Korneichik, Tatyana V.; Hatakka, Annele; Hammel, Kenneth E. 2010. Oxidizability of unsaturated fatty acids and of a non-phenolic lignin structure in the manganese peroxidase-dependent lipid peroxidation system. Enzyme and Microbial Technology. 46: 136-140.
• Kinne, Matthias; Poraj-Kobielska, Marzena; Ralph, Sally A.; Ullrich, Rene; Hofrichter, Martin; Hammel, Kenneth E. 2009. Oxidative cleavage of diverse ethers by an extracellular fungal peroxygenase. Journal of Biological Chemistry. 284(43): 29343-29349.
• Kinne, Matthias; Zeisig, Christian; Ullrich, Rene; Kayser, Gernot; Hammel, Kenneth E.; Hofrichter, Martin. 2010. Stepwise oxygenations of toluene and 4-nitrotoluene by a fungal peroxygenase. Biochemical and Biophysical Research Communications. 397: 18-21.
• Wei, Dongsheng; Houtman, Carl J.; Kapich, Alexander N.; Hunt, Christopher G.; Cullen, Daniel; Hammel, Kenneth E. 2010. Laccase and its role in production of extracellular reactive oxygen species during wood decay by the brown rot basidiomycete Postia placenta. Applied and Environmental Microbiology. 76(7): 2091-2097.
• Britton, Kerry; Illman, Barbara; Mann, Gary. 2010. Prevention. In: Dix, Mary Ellen; Britton, Kerry. A dynamic invasive species research vision: opportunities and priorities 2009-29. Gen. Tech. Report WO-79. pp 85-90.
• Hammel, Kenneth E.; Yelle, Daniel J.; Ralph, John; Wei, Dongsheng; Kapich, Alexander N. 2010. Solution-state NMR analysis of dissolved, brown-rotted wood reveals that the responsible fungi are ligninolytic [abstract]. In: 239th American Chemical Society annual meeting. 2010 March 21-25; San Francisco, CA. Washington, DC: American Chemical Society. CELL292.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: Fungal oxidative enzymes have potential applications in the manufacture of value-added products such as specialty chemicals. We have demonstrated that an extracellular peroxygenase from the litter-decomposing fungus Agrocybe aegerita can be used for regioselective hydroxylations that result in the preparation of the herbicide precursor 2-(4-hydroxyphenoxy)propionic acid, as well as the human drug metabolites 5-hydroxypropranolol and 4⿿-hydroxydiclofenac. Additional work this year focused on the cell membrane-bound systems that decomposer fungi use to hydroxylate and transport organic chemicals. The results showed that two potential hydroxylating enzymes (and one likely transporter are up-regulated in the decay fungus Phanerochaete chrysosporium during ligninolytic metabolism. Membrane-associated enzymes involved in ligninolytic oxidant production and in protection of the fungus from these oxidants are also up-regulated under these conditions.

Impacts
Identifying and understanding the redox systems of wood decay fungi is critical to increasing the efficacy of these organisms, and will further the application of their enzymes in novel bioconversion technologies.

Publications

• Fan, Jun; Hill, Lionel; Crooks, Casey; Doerner, Peter; Lamb, Chris. 2009 Abscisic acid has a key role in modulating diverse plant-pathogen interactions. Plant Physiology. 150: 1750-1761.
• Hammel, K.E.; Yelle, D.J.; Ralph, J.; Wei, D; Kapich, A.N.; Cullen, D.; Shary, S.; Siika-Aho, M.; Crooks, C. 2008. Oxidative and hydrolytic mechanisms for attack on lignocellulose by brown rot basidiomycetes [abstract]. In: Varnai, Aniko; Crestini; Claudia; Viikari, Liisa, eds. The Second Annual Workshop of COST FP0602: Enzymatic fiber modification and hydrolysis. 2008 December 4-5. Biel, Switzerland.
• Hammel, Kenneth E.; Yelle, Daniel J.; Ralph, John; Wei, Dongsheng; Kapich, Alexander N. 2009. A new solution-state NMR approach to elucidate fungal and enzyme/mediator delignification pathways. In: Genomics: GTL Awardee Workshop and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009. February 8-11. Bethesda, MD. 64.
• Hunt, Christopher G.; Hammel, Kenneth E.; Jones, Don Carl. 2009. In vivo mapping of ROS produced by wood decay fungi during early colonization. In: Genomics: GTL Awardee Workshop and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009. 2009 February 8-11. Bethesda, MD. 64.
• Kinne, Matthias; Poraj-Kobielska, Marzena; Aranda, Elisabet; Ullrich, Rene; Hammel, Kenneth E.; Scheibner, Katrin; Hofrichter, Martin. 2009. Regioselective preparation of 5-hydroxypropranolol and 4´-hydroxydiclofenac with a fungal peroxygenase. Bioorganic and Medicinal Chemistry Letters. 19: 3085-3087.
• Kinne, Matthias; Poraj-Kobielska, Marzena; Scheibner, Katrin; Ullrich, Rene; Hammel, Kenneth E.; Hofrichter, Martin. 2009. Regioselective hydroxylation of aromatic compounds by an extracellular fungal peroxygenase from Agrocybe aegerita [abstract]. In: Proceedings of the Annual Conference of the Association for General and Applied Microbiology (VAAM). 2009 March 8-11. Bochum, Germany. 196.
• Kinne, Matthias; Ullrich, Rene; Hammel, Kenneth E.; Scheibner, Katrin; Hofrichter, Martin. 2008. Regioselective preparation of (R)-2-(4-hydroxyphenoxy)propionic acid with a fungal peroxygenase. Tetrahedron Letters. 49: 5950-5953.
• Poraj-Kobielska, Marzena; Kinne, Matthias; Ullrich, Rene; Hammel, Kenneth E.; Kayser, Gernot; Hofrichter, Martin. 2009. Degradation of polycyclic musk fragrances by extracellular fungal oxidoreductases [abstract]. In: Proceedings of the Annual Conference of the Association for General and Applied Microbiology (VAAM). 2009 March 8-11. Bochum, Germany. 196.
• Ralph, John; Lu, Fachuang; Kim, Hoon; Fang, Xiaowen; Norton, Mark; Ellinger, James J.; Markley, John L; Yelle, Daniel; Hammel, Kenneth E.; Ralph, Sally A.; [and others]. 2009. Streamlined method for biomass whole-cell-wall structural profiling. In: Genomics: GTL Awardee Workshop and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009. February 8-11. Bethesda, MD. 18.
• Ralph, John; Lu, Fachuang; Kim, Hoon; Ress, Dino; Yelle, Daniel J.; Hammel, Kenneth E.; Ralph, Sally A.; [and others]. 2009. High-resolution solution-state NMR of unfractionated plant cell walls. In: Proceedings of 15th international symposium on wood, fiber and pulping chemistry. 2009 June 15-18; Oslo, Norway: Oslo, Norway: Congress-Conference AS, www.congrex.no. P-105, 4 p., limited availability.
• Shary, Semarjit; Kapich, Alexander N.; Panisko, Ellen A.; Magnuson, Jon K.; Cullen, Daniel; Hammel, Kenneth E. 2008. Differential expression in Phanerochaete chrysosporium of membrane-associated proteins relevant to lignin degradation. Applied and Environmental Microbiology. 74(23): 7252-7257.

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Recent technical advances in solution state nuclear magnetic resonance (NMR) spectroscopy of lignocellulose have enabled us to make new observations on the strategies wood decay fungi use to access the polymeric sugars in wood. It was previously thought that only white rot fungi degrade the lignin component extensively, but two-dimensional (1)H-(13)C spectra of dissolved wood after extensive attack by brown rot fungi have revealed that the major intermonomer linkages of lignin have been almost completely removed. Similar experiments we have done with soft rot fungi have now established that they also degrade lignin. Our results support the hypothesis that fungal access to the cellulose in wood invariably depends on prior scission of the lignin. We demonstrate that a dehydratase from Phanerochaete chrysosporium produces unstable metabolic intermediates, which undergo competing non-enzymatic reactions, including C-C bond cleavage. Thus, novel acids are produced from glucose by the combined activities of pyranose 2-oxidase and aldose-2-ulose dehydratase. The pyranose 2-oxidase was expressed in E. coli to obtain sufficient enzyme for detailed analysis of reactions with glucose. In this case, glucosone is the main product, as verified by nuclear magnetic resonance (NMR). The glucosone is subsequently oxidized very poorly and incompletely, even at high levels of oxidase. This suggests that the role for pyranose 2-oxidase is to supply glucosone for aldose-2-ulose dehydratase (in contrast to sequential oxidations by the oxidase).

Impacts
Identifying and understanding the redox systems of wood decay fungi is critical to increasing the efficacy of these naturally-occurring organisms in bioconversion technologies, devising new environmentally-sound ways to protect wood in use, and accelerating decomposition of forest litter.

Publications

• Shary, Semarjit; Ralph, Sally A.; Hammel, Kenneth E. 2007. New insights into the ligninolytic capability of a wood decay ascomycete. Applied and Environmental Microbiology. 73(20): 6691-6694.
• Hammel, Kenneth E.; Cullen, Dan 2008. Role of fungal peroxidases in biological ligninolysis. Current Opinion in Plant Biology. 11: 349-355
• Yelle, Daniel J.; Ralph, John; Lu, Fachuang; Hammel, Kenneth E. 2008. Evidence for cleavage of lignin by a brown rot basidiomycete. Environmental Microbiology. 10(7): 1844-1849.
• Kersten, Phil; Cullen, Dan. 2007. Extracellular oxidative systems of the lignin-degrading Basidiomycete Phanerochaete chrysosporium. Forest Genetics and Biology. 44: 77-87.
• Ralph, John, Hedenstrom, Mattias; Lu, Fachuang; Kim, Hoon; Yelle, Daniel; Hammel, Kenneth E. 2008. High-resolution solution-state NMR of unfractionated plant cell walls; potential for biomass selection and process optimization (abstract). In: Proceedings PITTCON 2008 Conference. 2008 March 2-7; New Orleans, LA. Pittsburgh, PA: The Pittsburgh Conference. 1730-5.
• Hunt Christopher G.; Hammel, Kenneth. 2008. Development of modular platforms for in vivo mapping of local metabolite concentrations important to cell wall degradation by microorganisms. In: Joint Meeting of Gennomics: GTL awardee workshop VI and Metabolic Engineering Working Group Interagency Conference on Metabolic Engineering 2008. 2008 February 10-13; Bethesda, MD. Oak Ridge, TN: Oak Ridge National Lab. 11: 11-12.
• Hammel, Kenneth E.; Ralph, John; Yelle, Daniel J.; Lu, Fachung; Shary, Semarjit; Ralph, Sally. 2008. A new solution-state NMR approach to elucidate fungal and enzyme/mediator delignification pathways. In: Joint Meeting of Gennomics: GTL awardee workshop VI and Metabolic Engineering Working Group Interagency Conference on Metabolic Engineering 2008. 2008 February 10-13; Bethesda, MD. Oak Ridge, TN: Oak Ridge National Lab. 11: 11.
• Kersten, P.J.; Mozuch, M.D.; de Koker, T.H.; Hirth, K.C.; Yu, S. 2007. Reaction partitioning with aldose-2-ulose dehydratase for production of novel acids (abstract). In: 10th International Congress on Biotechnology in the Pulp and Paper Industry. 2007 June 10-14; Madison, WI. BioC 1.3.
• Mozuch, M.D.; Hirth, K.C.; Kujawa, M.; Haltrich, D.; Cullen, D.; Gaskell, J.; Kersten, P.J. 2007. Sequential oxidations of D-glucose by recombinant pyranose 2-oxidase from Phanerochaete chrysosporium (abstract). In: 10th International Congress on Biotechnology in the Pulp and Paper Industry. 2007 June 10-14; Madison, WI. PS BPBR 2.3.

Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: Characterization of fungal ligninolytic reactions by NMR analysis of completely dissolved lignocellulose. We have data for the brown rot fungus Gloeophyllum trabeum, and the results show that this fungus has cleaved lignin extensively by the time decay is extensive. Collaborators are Dan Yelle and John Ralph. Dan and I have written up the first draft of a manuscript and we will submit to Biomacromolecules later this fall. A parallel project analyzing changes during early decay by brown and white rot fungi is underway by Alex Kapich in my lab. Identification of ligninolytic reactions in xylariaceous ascomycetes. We have shown that Daldinia concentrica degrades the nonphenolic structures of angiosperm lignin, and that C-C cleavage of the lignin side chain is one of the reactions responsible. This work was just published in the second Oct. issue of Appl. Environ. Microbiol. and will be reported as a publication for FY08. Coauthors are Semarjit Shary and Sally Ralph. Diterpene resin acids are major constituents of conifer oleoresin and play important roles in tree defense against insects and microbial pathogens. The abietanes, a subclass of diterpenes, have conjugated double bonds and exhibit characteristic UV spectra. We developed protocol for analysis of abietanes by high performance liquid chromatography (HPLC) using multi-wavelength detection (1). The analytical method has advantages particularly useful in ecological studies where large numbers of samples are required for statistical validation. Furthermore, samples are not derivatized (in contrast to typical GC methods) and therefore biological activities of fractionated components are retained. The method is simple and rapid, employing robust reversed-phase HPLC. Detection at four wavelengths and spectral analysis provides confirmation and/or refinement for quantitative determination of abietic, neoabietic, levopimaric and palustric acids. The utility of the method was demonstrated with methanol extracts of white spruce (Picea glauca) phloem. Developed a high throughput quantitative luminescnce assay that is a technical advance in being able to remotely moniter microbe viability and invasion into plant tissues with broad applicability towards tagging of wood and wood fermenting fungi and the ability to rapidly determine their progress and viability. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
An improved knowledge of fungal mechanisms for attack on wood will aid efforts to use fungi to enhance forest humus quality and to reduce quantities of small diameter woody material on the forest floor. Knowledge of fungal decay mechanisms will also assist in the design of new wood preservatives.

Publications

• Fan, Jun; Crooks, Casey; Lamb Chris. 2007. High-throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens luxCDABE. The Plant Journal. Online publication 7p.: http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-313X.2007.033 03.x
• Kersten, P.J.; Kopper, B.J.; Raffa, K.F.; Illman, B.L. 2006. Rapid analysis of abietanes in conifers. Journal of Chemical Ecology. 32: 2679-2685.
• Hammel, Kenneth E.; Houtman, Carl J.; Hunt, Christopher G.; Yelle, Daniel J.; Ralph, John. 2007. The hydroxyl radical as an agent of lignocellulose decay by fungi (abstract). In: Proceedings of the 10th International Congress on Biotechnology in the Pulp and Paper Industry. 2007 June 10-15: Madison, WI. Madison, WI: Omni Press: 66.
• Ortiz-Bermudez, Patricia; Hirth, Kolby C.; Srebotnik, Ewald; Hammel, Kenneth E. 2007. Chlorination of lignin by ubiquitous fungi has a likely role in global organochlorine production. Proceedings of the National Academy of Sciences. 104(10): 3895-3900.
• Suzuki, Melissa R.; Hunt, Christopher G.; Houtman, Carl J.; Dalebroux, Zachary D.; Hammel, Kenneth E. 2006. Fungal hydroquinones contribute to brown rot of wood. Environmental Microbiology 8(12): 2214-2223.
• Hammel, Kenneth E.; Ralph, John; Yelle, Daniel J. 2007. A new solution-state NMR approach for the characterization of biodegraded lignocellulose (abstract). In: Irbe, Ilze; Biziks, Vladimirs, eds. Proceedings of the International conference: Biodeterioration of wood and wood products. 2007 August 26-29: Riga, Latvia: Perse Ltd.: 36.