Decay Fungi from New Zealand Leaky Buildings: Isolation, Identification and Preservative Resistance

Abstract

Leaky buildings are those that show elevated moisture contents of the framing timber, which can subsequently lead to the establishment of fungal and bacterial decay. Prior to this study, the causative agents of the decay in these leaky buildings were unknown, though it was suspected to be one or more species of decay fungi. Therefore, the overall goal of this multi-disciplinary PhD thesis research was to determine the causative agents of decay in leaky buildings of New Zealand in an effort to develop solutions for both their remediation and future prevention.

Use of molecular biology methodology and classical mycological techniques based on morphology enabled identification of decay fungi from framing timber and air samples of leaky New Zealand buildings and provided insight into relative importance based on isolation frequency. In most cases, fungi colonising Pinus radiata D. Don were isolated to produce pure cultures. Mycelia from these cultures on agar media were collected to extract DNA. To identify the fungi to the species level, polymerase chain reaction (PCR) with fungal specific DNA primer pairs were performed followed by DNA sequencing of the internal transcribed spacer (ITS) region. Identification was by BLAST (Basic Local Alignment Search Tool) search on sequences in known GenBanks. In total, 421 samples from leaky buildings were processed, predominately untreated P. radiata decayed framing timber and also fibre cement boards and building paper. From these, sixty-eight fungal identifications were made. The only taxa that were isolated with significant frequency were identified as 4 basidiomycete species, as follows, along with the number of times they were isolated from the 421 samples:

• Gloeophyllum sepiarium (Wulf.: Fr.) Karst. 13x • Oligoporus placenta (Fries 1865) Gilb. In Ryv.1985 11x • Antrodia sinuosa (Fr.) Karst. 8x • Gloeophyllum trabeum (Fr.) Murr. 4x Although these species were identified repeatedly, in total they represent less than 10% of the total samples and, therefore, it is concluded that the leaky building decay samples represent high fungal biodiversity.

An aerial spore study of internal air, wall cavity air and exterior air of leaky buildings was carried out using a Merck MAS-100 instrument which collects spores directly onto selective media plates. Viable fungal aerial spores were detected at every sampling location tested at the leaky buildings, by the criteria of culturing, with a highest mean of 3714 colony-forming units (CFU) per cubic metre found in the cavities of water-damaged walls. This aerial spore study in conjunction with isolation from decayed wood samples from the same leaky buildings enabled identification of G. sepiarium and A. sinuosa at the same test site. The use of carboxymethylcellulose medium further demonstrated the presence of potential cellulose-degrading fungi within and around the location. Overall, the combination of direct sampling of timber and air sampling proved useful for detection of fungal species variability at a multi-unit building.

Four decay fungi isolated from New Zealand leaky buildings and two standard control decay fungi (Coniophora puteana and Serpula lacrymans) were submitted to laboratory wood block testing to determine the effectiveness of currently used wood framing preservatives under laboratory conditions before and after a standard leaching regime. P. radiata blocks were treated with water based boron copper azole and solvent based IPBC propiconazole plus tebuconazole (1:1) preservatives and exposed to the basidiomycetes for 12 weeks. Mass loss for the fungal decay-infected samples was recorded of up to 55% for preservative-treated samples, up to 62% mass loss for leached samples and up to 58% mass loss for un-preservative treated samples. Additionally, well defined dosage responses and approximate toxic thresholds were obtained for all preservatives tested. Results suggested that the minimum IPBC retention specified by Hazard Class 1.2 of NZS3640:2003 (0.025% m/m) is on the low side, and demonstrated after the 2 week leaching regime complete loss of efficacy of boron at 0.4% m/m boric acid equivalent (BAE).

This PhD research gave a first overview of fungi occurring in New Zealand leaky buildings, and it demonstrated the following key aspects of wood preservation:

1. The isolated test fungus Antrodia sinuosa was more difficult to control with propiconazole plus tebuconazole at retention 0.007% m/m than the known tolerant fungus Oligoporus placenta; 2. Boron at Hazard Class 1.2 retention of 0.4% m/m BAE was not toxic to Oligoporus placenta; 3. Serpula lacrymans exhibited tolerance to the highest retention of 0.06 %m/m tebuconazole plus propiconazole; and 4. Gloeophyllum species appeared susceptible to all wood preservatives.

In order to correlate fungal colonisation and wood decay, colonised wood blocks were studied using light microscopy (LM) and field- emission scanning electron microscopy (FE-SEM). Microscopic observations of P. radiata wood blocks following a standard wood decay test of twelve weeks of fungal colonisation by Serpula lacrymans, Antrodia sinuosa, Oligoporus placenta and Gloeophyllum sepiarium revealed that the two microscopic techniques employed were complementary by allowing features such as pit membranes, chlamydospores or S3/S2 compound middle lamella interface to be photographed in greater detail, allowing for more precise analyses and interpretation of key findings, as follows:

1. Brown rot fungi directly target their apical growth towards degraded pit apetures; 2. Reliance on light microscopy and observed birefringence as a tool to record changes in cell wall crystallinity associated with brown rot decay alone could be misleading; 3. Presence of fine (≤ 1 m) to wide (≥ 3.5 m) bore-hole and hyphal size ranges, and nearly unchanged cell wall thickness of all wood/test fungal combinations, confirmed active decay at moderate to late stages; 4. Some ray parenchyma cells for Antrodia sinuosa, Oligoporus placenta and Gloeophyllum sepiarium colonised blocks were intact throughout late stages of decay, outlining that they were not preferentially degraded early in the brown rot decay process, and 5. Presence of bore-holes, clamp and medallion clamp formation and resting spores (chlamydospores and arthrospores) are fungal specific, can aid in their differentiation and identification, and should be recorded during wood decay studies, as especially resting spores are an important factor when planning remediation strategies.

In summary, this PhD thesis research provided the first comprehensive investigation into the biodiversity of fungi from leaky New Zealand buildings, identified the dominant species and presented details about their micromorphology and their decay patterns. It also demonstrated substantial differences in efficacy of preservative formulations currently (December 2008) approved for framing treatments in New Zealand and possible deficiencies where framing may be subjected to severe leaching. This study also provided the first comparative analyses of viable fungal aerial spores between leaky wall cavities and the surrounding air environment. Subsequently, this research added to the knowledge of the decay fungal species diversity in and around New Zealand leaky buildings, outlined their capabilities to degrade treated and un-treated P. radiata framing timber and illustrated the efficacy of New Zealand approved wood preservatives for their potential as remedial treatment and future prevention.