|
Project summary:
Establishing a reference collection of sequences: A reliable and extensive source of morphological taxonomic information is required to develop molecular detection tools for wood decay fungi in Australia (only 5% of the probable number of species of fungi in Australia have been classified and described in detail. This information does not exist and because of the lack of morphological mycologists in Australia who have the capacity to describe new species will probably take many years to develop. The strategy used here for establishing reference sequences has several limitations as shown by the work in this thesis. Out of 111 sporocarps and 70 isolates obtained from them, sequencing definitively coupled only 10 pairs of sporocarps and cultures. Only four of these 10 matching pairs were identified to species level by DNA sequence analysis. Only 30 sequences out of 57 obtained directly from sporocarp tissue supported at some level (seven to species level) the original morphological identifications attributed to the sporocarps analysed. Eleven sporocarp sequences and 12 isolate sequences, obtained from 19 sporocarps identified to species or genus level did not support the morphological identifications.
There are several compounding reasons for this low rate of success in obtaining
identified sequences for a reference collection. i) The success rate of isolations obtained from sporocarps is approximately 70%. Isolates cultured may be a contaminant of the sporocarp – approximately 41% of 96 DNA sequences for the 56 fungal isolates supported the morphological identification of the sporocarps from which the fungal isolates had been cultured. ii) There is little expertise available to morphologically identify sporocarps (only 84 out of 111 sporocarps were morphologically identified to species or genus level before DNA analysis). Even experts find the morphological identification of wood decay fungal sporocarps difficult. iii) DNA is not successfully extracted from the sporocarps and/or cultures, either not at all or with high concentrations of compounds that inhibit molecular analyses. iv) The primers that are used may not amplify all fungal species. v) Contaminant DNA is obtained from sporocarps meaning that there are multiple PCR products which interfere with the readability of sequencing results. vi) In addition to all the above limitations there are no matching sequences available in public databases as Australian wood decay fungi have not been widely studied, sequences may not be reliable when present as many sequences submitted to Genbank do not include isolate of herbarium accession details. For example, two sporocarps and two isolates do not have corresponding sequences of Ryvardenia, Fomes hemitephrus, Xylobulus illudens and Postia caesia respectively available on both Genbank and private databases for comparison.
Despite the above limitations, molecular analyses of this type will eventually allow the construction of a sequence reference library and the importance of this study is to highlight the problems and pitfalls involved. There was also the benefit of molecular identifications in the absence of morphological identifications. Sequences were derived from 15 of 27 unidentified sporocarps and there were seven identifications to species or genus level which could possibly have been correct in respect to the type of
sporocarp collected.
Identification of cultures obtained from ecological studies of rotten wood types found in coarse woody debris: In ecological research where a large number of wood decay isolates are obtained this study showed that molecular analyses are useful (although many of the above listed limitations apply) for: confirming, refining or reallocating the identity of isolates that have characteristic morphology in rotting wood or in culture – either by direct comparison to known sequences and/or phylogenetic analysis e.g. 11 isolates that had been identified as Polyporus mylittae (since renamed Laccocephalum mylittae). The closest BLAST match results for those isolates were Diplomitoporus lindbladii (currently accepted as Poria lindbladii) and Postia subcaesia (currently accepted as Tyromyces subcaesius), though no sequences of L. mylittae or other Laccocephalum species were available for comparison. A herbarium specimen of L. mylittae was obtained and the ITS sequenced, but this sequence did not match that of any of the 11 isolates which were therefore accepted as belonging to a different species in the Polyporaceae. One of the caveats very relevant to the identification of wood decay fungi isolated directly from rotting wood is the lack of sequences available for comparison. The sporocarps associated with these fungi might be rare in nature. The only method for ascertaining whether the fungal flora on coarse woody debris (CWD) is the same as that inside a log is to survey, identify and analyse the sporocarps that appear on CWD over an extended period. Then destructively sample the CWD to analyse, using cultural and molecular techniques, the fungi associated with the rotting wood. Such a project is in progress at the Warra Long Term Ecological Research site but will take another 3-4 years to complete. Another problem highlighted by this study is the high level of contamination of cultures that has probably taken place during storage and routine subculturing, as the molecular identification bears no relation to the original morphological grouping e.g. isolates identified as Basidiomycetes were identified by sequencing as Xylariaceae. This is always going to be a problem with large-scale studies resulting in over 1000 cultures. This may be overcome in the future by grouping isolates through enzyme tests and a quick morphological examination, then keeping only one or two representative isolates but DNA of other isolates in the same group or DNA analysis before the cultures are destroyed.
Identification of fungi directly from rotten wood types found in coarse woody debris: One of the problems experienced was the non-amplification of DNA. Twenty-six samples of rotten wood taken from 19 decayed logs at six sites gave rise to a total of 80 DNA samples, 17 PCR products, 10 readable sequences. The by-products of wood decay most likely prohibited amplification of the DNA template. Diluting out the inhibitors could also mean diluting out the DNA past the threshold of detection. Amplification of two samples was successful after purification of the fungal DNA using Polyvinylpolypyrrolidone (PVPP). A cloning step to separate the different fungal templates before sequencing was necessary in the majority of samples. Three wood samples originating from one rotting wood sample yielded very different fungi including the groups of Basidiomycota, Ascomycota and even Zygomycota. As a sample of rotting wood is a microcosm of fungi, not just one or several basidiomycete wood-decay fungi, and the primers used were fungal specific not basidiomycete specific, this result is expected. Depending on the target fungi required, primers specific to a smaller sub-set of fungi could be used. If only an idea of the different groups of fungi present is required then an analytical technique such as TRFLP should be employed with relevant fungal specific primers. Eight different fungi were identified, with very low levels of confidence, from the rotten wood samples, six were Basidiomycetes. However, the fungi identified did not match the original fungi cultured from these samples. The only basidiomycete identified with any level of confidence was a Ganoderma species that in phylogenetic analysis was close to a species from oil palm, indicating possible contamination of DNA samples. We believe that this low rate of success in identifying fungi directly from rotten wood samples is attributable to the fact that these samples were not processed immediately they were harvested or frozen at -80 °C and then processed. In this study, the samples were left at 4 °C before analysis and it is likely that, even at this low temperature, the basidiomycete fungi present were out competed by other fungi. Further analyses to detect and identify fungi direct from wood will be carried out. This pilot study to directly extract fungi from rotted wood material was a useful learning experience.
Increase in our knowledge of the molecular profiles of wood decay fungi: This project has been valuable as a pilot study for understanding the problems involved in the detection of wood decay fungi direct from rotting wood samples. It has also added a number of new sequences of fungi associated with coarse woody debris and attached to named or tagged sporocarps and/or cultures that can be included in both Genbank and the Hobart Forest Health sequence databases. This type of information is being applied to ecological studies of the fungal diversity in the wet sclerophyll forest of southern Tasmania and ultimately will inform industry on how best to manage the forest matrix for conservation values. By sequence comparison, we have identified cultures and associated sporocarps of several taxa of commonly recognised wood decay fungi. Cosmopolitan species of Hypholoma fasciculare (P0916, LF495) and Trametes versicolor were found in logs of all decay stages. The taxa Postia, Xylaria and Stereum have been shown to be present in both the living veteran E. obliqua trees and E. obliqua logs in the wet sclerophyll forest. Some other species from the wet sclerophyll ecosystem were putatively identified as Athelia and Phlebia, which are known decay species and have been previously recorded on Eucalyptus logs. Several cultures of resupinate fungi were identified by their sequences in this study. Resupinate fungi are very common on CWD in the wet sclerophyll forest and will be the focus of future taxonomic and biodiversity studies.
Methodology: (1) Establishing a reference collection of sequences; (2) Identification of cultures obtained from ecological studies of rotten wood types found in coarse woody debris; (3) Identification of fungi directly from rotten wood types found in coarse woody debris
Datasets: None available.
Publications: Trang, T.T. (2007). Resistance screening to fungal diseases for plantation eucalypts in Vietnam: molecular tools to assist fungal detection and identification. Masters Thesis, University of Tasmania, Hobart.
|
