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Socio-economic perspectives of treated wood for the common European market ... suitable for controlling spread of dry rot, particularly to conserve wood and ...
Cost Action E37 Final Conference in Bordeaux 2008 Socio-economic perspectives of treated wood for the common European market

Limiting fungal spread in buildings without the use of toxic biocides Sarah Watkinson1, Monika Tlalka2 1

Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK. e-mail: [email protected] 2

e-mail: [email protected]

Keywords: Serpula lacrymans, remedial treatment, AIB ABSTRACT By preventing amino acid translocation through mycelial networks, the spread of wood decay fungi within building structures can be contained, and sound joinery protected from attack. The amino acid analogue AIB (α-aminoisobutyric acid) competitively excludes utilisable amino acids from the mycelium, and from the nutrient supply network of mycelial cords that enables basidiomycete fungi including S. lacrymans to spread through buildings. AIB inhibits all basidiomycetes tested including Serpula lacrymans, Coniophora puteana, Gloeophyllum trabeum, Lentinus lepideus and Coriolus versicolor. It is effectively non-toxic to non-target organisms and biodegrades in soil. Here we report the application of a radiolabelled 14C-AIB marker to establish the power of locally applied AIB to arrest extension throughout a whole mycelial network, irrespective of the point of application. Real time imaging by PCSI was used to track the velocity and direction of AIB translocation in microcosms designed to mimic building timber elements in a masonry structure. AIB applied at any point in the mycelial network rapidly permeated the whole fungal system, resulting in durable inhibition of extension. Mycelial extension ceased and the development of the foraging network was delayed and disorganised. Experiments on the partitioning of AIB between partially exhausted and fresh wood food sources showed that AIB was preferentially translocated to sites of attack on fresh wood, where it accumulated. AIB is licensed for experimental use on a site by site basis, and is suitable for controlling spread of dry rot, particularly to conserve wood and cellulosic materials in historic buildings infected with Serpula lacrymans. INTRODUCTION AIB inhibits fungal spread by targetting N translocation The mode of action of AIB is not to kill the fungus, but to interfere with the unique physiological and developmental adaptations of wood decay basidiomycetes to conserve nitrogen, a key feature enabling them to use N-poor wood as a sole food source and thus cause decay. AIB (Fig. 1) is a nonmetabolised analogue of the utilisable amino acid alanine, right, with a substituted methyl group. AIB competitively excludes utilisable amino acids from the intracellular pool of free amino acids in the fungal cell. Free amino acids are believed to be the main form in which scarce nitrogen is distributed by mass flow translocation throughout the fungal network.

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Cost Action E37 Final Conference in Bordeaux 2008 Socio-economic perspectives of treated wood for the common European market

Figure 1: Structure of α-aminoisobutyric acid (AIB)

Cellulose decay is nitrogen-limited Wood and other cellulosic materials such as paper provide carbon/energy sources for decay fungi but insufficient nitrogen for maximum rates of growth and enzyme production. Nitrogen import via mycelium strongly enhanced cellulolysis by S. lacrymans (Fig. 2). Figure 2: The effect on cellulolysis of supplying colonising mycelium with a nitrogen source.

S.lacrymans decay S.lacrymans decay + KOH Lentinus lepideus decay L.lepideus decay + KOH

Fig. 2 shows the positive effect of addition of nitrogen to a fungal culture medium on the weight loss of cellulose filter paper supported above the mycelium, and connected to it only via colonising hyphae. Weight loss after incubation is shown for two wood decay fungi, and the effect of 10% KOH wash to remove oligosaccharides resulting from endoglucanase activity is compared for each species. S. lacrymans endoglucanase appeared to show greater enhancement of activity due to N addition than did L. lepideus (C.M. Venables, unpublished data.) Mycelial cords translocate nutrients around the mycelial network Foraging growth by wood decay basidiomycetes requires the development of mycelial cords linking wood food bases across carbon-free areas. Scavenging mycelium can thus extend from wood food bases to collect any available nitrogen present in the environment as nitrate, ammonium, or organic N compounds. Nitrogen in the form of free amino acids is then channelled through the network of cords to supply areas of growth and active wood digestion. The mycelium develops in response to its nutrient environment to form a resource supply network (Bebber et al. 2006). 42

Cost Action E37 Final Conference in Bordeaux 2008 Socio-economic perspectives of treated wood for the common European market

Mycelial cords have a specialised multicellular structure (Tlalka et al. 2008), providing an interface across which nutrients absorbed at the hyphal tips, initially into cell vacuoles and cytoplasm, are transported into a wide channel in which mass flow can occur. Cell uptake systems absorb AIB preferentially, displacing utilisable nitrogen from the supply network. Figure 3: Mycelium of Serpula lacrymans growing in a laboratory microcosm.

Fig. 3 shows a controlled microcosm designed to mimic building conditions where wood elements are spatially separated and embedded in damp, nutritionally-inert masonry. Plates were 22cm square dishes filled with a layer of moist black sand. Mycelium was grown from a 1cm3 S. lacrymans infected beech wood block placed in the centre. Further fresh blocks stimulated mycelial cord development in mycelium that grew to form a bridge between inoculum and fresh ‘bait’, in a microcosm designed to induce cord development in saprotrophic cord-forming basidiomycetes (Boddy 1999). Figure 4: Mycelial expansion and cord development inhibited by AIB added at a single point.

Fig. 4: S. lacrymans was grown from a wood block over sand to colonise a second block. Cellulose filter paper was placed on the distal side of the second block, and infiltrated with either 30mg AIB in solution (top), or with water (bottom). Photos at 2, 4 & 6 weeks. (Reproduced from Tlalka et al. 2008). AIB moved against the direction of growth. The entire treated colony was persistently inhibited, and cord development was delayed and disorganised.

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Cost Action E37 Final Conference in Bordeaux 2008 Socio-economic perspectives of treated wood for the common European market

AIB locally applied, acts systemically to stop spread from infected wood through brick The effectiveness of AIB control of mycelial spread of the dry rot fungus Serpula lacrymans was tested in scaled up experiments in a damp cellar. Figure 5: AIB controls spread of dry rot through a brick-built structure

Fig. 5: Six towers of Fletton bricks incorporating a central timber element pre-infected with S. lacrymans were kept under a high relative humidity. 10% AIB solution was infiltrated locally into the infected wood block from the side of the structure when 2 brick courses had been colonised. After seven months the towers were disassembled and each course of bricks inspected for signs of mycelium. In all three treated replicate towers the fungus was restricted to two course of bricks, while in untreated controls, the entire tower had been colonised (Dobson et al. 1993). The aim of the work described here was to test how fast and how extensively locally applied AIB would move through mycelium; to check whether translocation would carry AIB equally towards and away from the direction of growth; and to investigate whether AIB would be preferentially imported into parts of the mycelium network attacking fresh wood. METHODS AND RESULTS Measuring velocity and direction of translocation in real time. Photon counting scintillation imaging (PCSI) was applied to corded networks in realistic microcosms using subtoxic (millimolar) concentrations of radiolabelled AIB as a marker for translocated amino acids (Tlalka et al. 2002, Watkinson et al. 2005). Because AIB is not metabolised, the radioactive label remained attached to the AIB and was not released into metabolism. Figure 6: PCSI image of S. lacrymans translocating 14C-AIB

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Cost Action E37 Final Conference in Bordeaux 2008 Socio-economic perspectives of treated wood for the common European market

PSCI image of mycelium shown in Figure 3, recorded 360h after adding 14C-AIB to the central inoculum, with a fresh wood block added at the advancing margin. White intensity indicates AIB concentration, showing accumulation into margins and the new wood block, top right. (Reproduced from Tlalka et al. 2008) Results showed that AIB was translocated through cords at around 25cm h-1, irrespective of the original direction of growth, and became symmetrically distributed within days, accumulating at the margins and in freshly-colonised wood (Tlalka et al. 2008). Figure 7: Microcosm used to estimate AIB redistribution into new wood. Decayed (central inoculum)

Partly decayed

Fresh wood (added at bottom left of mycelium)

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5

222

7

11

542

Fig. 7: A three-block microcosm was set up. Mycelium was allowed to establish a corded system linking an infected inoculum to a second fresh block. After the second block had been captured by expanding mycelium, a third fresh block was added. 14C-AIB was then added to the mycelium at the inoculum block. After six weeks incubation the plate was harvested into separate fractions including each of the wood blocks; inoculum, first bait (now partly decayed) and fresh bait. 14CAIB content of each block was assayed by scintillation counting of ethanolic extracts. Left, before 14C-AIB application; right, at harvest, 6 weeks later. Table, right, shows dpi in ethanolic extract of each wood block in 2 replicate experiments (details: Tlalka et al. 2008). Results show preferential import of colonised wood block.

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C-AIB from the whole mycelium into the most recently-

CONCLUSION The results support the view that AIB targets adaptive nitrogen redistribution mechanisms that are unique to wood decay fungi, and which are essential to their ability to spread in buildings and colonise fresh wood sources poor in nitrogen. All basidiomycete wood decay fungi that we have tested (including all species used in preservative test EN 113) are similarly affected (Elliott and Watkinson 1989). No toxic effects of AIB have been recorded for AIB. It has low toxicity and has been used on healthy human volunteers in medical research, as a potential tumour suppressant. It is readily degraded by common soil bacteria (Aaslestad and Larson 1964). AIB thus offers an environmentally sustainable approach to control of decay caused by S. lacrymans and other basidiomycetes in buildings. AIB is particularly applicable to protecting historic cellulosic artefacts from attack in buildings infected by S. lacrymans, while building work is carried out to eliminate damp.

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Cost Action E37 Final Conference in Bordeaux 2008 Socio-economic perspectives of treated wood for the common European market

REFERENCES Aaslestad, H. G. and A. D. Larson (1964) Bacterial Metabolism of 2- Methylalanine. 10 Journal of Bacteriology 88:1296-303. Bebber, D. P., M. Tlalka, J. Hynes, P. R. Darrah, A. E. Ashford, S. C.Watkinson, L. Boddy and M. D. Fricker (2006) Imaging complex nutrient dynamics in mycelial networks, p. 3-21. In: G. M. Gadd, S. C. Watkinson and P. S. Dyer (ed.), Fungi in the Environment. Cambridge University Press, Cambridge. Boddy, L. (1999) Saprotrophic cord-forming fungi: meeting the challenge of heterogeneous environments. Mycologia 91:13-32. Dobson, J., Power, J., Singh, J. and Watkinson, S.C. (1993) The effectiveness of 2aminoisobutyric acid as a translocatable fungistatic agent for the remedial treatment of dry rot caused by Serpula lacrymans in buildings. International Biodeterioration and Biodegradation 3:129-141. Elliot, M. L. and S. C. Watkinson (1989) The effect of α-aminoisobutyric acid on wood decay and wood spoilage fungi. International Biodeteriotation 25:355–371. Tlalka, M., Watkinson, S.C., Darrah, P.R. and Fricker, M. (2002) Continuous imaging of amino acid translocation in intact mycelia of Phanerochaete velutina reveals rapid, pulsatile fluxes. New Phytologist 153: 173-184. Tlalka, M., Fricker, M.D. and S. C. Watkinson (2008) Translocation dynamics in Serpula lacrymans. Applied and Environmental Microbiology 74:2700-2708 Watkinson, S.C., Boddy, L., Burton, K.S., Darrah, P.R.,Eastwood, D., Fricker, M.D. and Tlalka, M. (2005) New approaches to investigating the function of mycelial networks. Mycologist. 19, 11-17. Further information on AIB technology transfer: [email protected] (Isis Innovation, Oxford University)

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