Molecular phylogeny of wood decay fungi of hardwood and their ability to produce laccase that correlates with triphenylmethane dye decolorization

Though Sri Lanka belongs to one of the 34 biodiversity hotspots of the world, its microfolora specially fungi are not well studied and underrepresented in the global literature. Here we report the fungal species diversity of decaying hardwood of a Sri Lankan dry zone forest for the first time. Decaying hardwoods were collected from historically important Dimbulagala forest reserve, Sri Lanka and fungi associated with these woods were isolated. Out of 35 fungal species identified using morphological and molecular methods, 11 species were first records in Sri Lanka. All the tested isolates were able to utilize wood as the sole carbon source and produced varying degrees of laccase. Isolates of Perenniporia tephropora, Coriolopsis caperata, Gymnopilus dilepis, Fusarium solani and Vanderbylia fraxinea were among the top six laccase producers. Except Fusarium solani, the rest of the isolates showed more than 70% decolorization of the of triphenylmethane dye and there was a significant positive correlation between laccase production and dye decolorization. To the best of our knowledge laccase production and dye decolorization ability of Vanderbylia fraxinea and Gymnopilus dilepis have never been reported in the fungal kingdom before. Perenniporia tephropora was isolated from one of the strongest decay resistant hardwood species, Ebony (Diospyros ebenum) also known as dark wood and V. fraxinea was isolated from another medicinally important hardwood Neem (Azadirachta indica). Findings of this study confirms that decaying hardwood of Sri Lanka provide unexplode a unique niche for discovering fungal species with biotechnological applications such as high laccase producers and dye decolorizers.


Introduction
Fungi are considered to be miniature metabolic factories having versatile secondary metabolites 45 and enzymes with the potential to be used as work horses in diverse array of biotechnological 46 applications [1]. Based on the Hawksworth's [2] estimation, there are abut of 1.5 million fungal species present on earth, which is considered as the baseline estimation. However, recent 48 metagenomics studies suggested that actual numbers might be closer to 3.5 to 5.1 million species 49 or much higher than this [3]. This uncertainty in the numbers is partially due to lack of advanced 50 molecular based thorough studies in the tropics where incredibly rich diversity has been reported 51 [4]. Hawksworth [5] also suggested that much of the undescribed fungal species could be present 52 in the tropics and it is reviewed in Aime and Brearley [4]. 53 Sri Lanka, a tropical island in the Indian ocean along with the Western Ghats, belongs to one of 54 the 34 biodiversity hotspots in the world. Though its plant and animal diversity is well studied 55 [6,7], microbial studies, especially fungal studies remain in its infancy. Furthermore, biodiversity 56 hotspot concept of Sri Lanka should not be an exception for microbial diversity including fungi. 57 However, most of the fungal studies in Sri Lanka have mainly focused on macro-fungi using 58 morphological characters [8]. Moreover, these studies have mainly concentrated on the wet zone 59 forests. On the other hand, dry zone forest ecosystems in the country spread over 22 % of area in 60 Sri Lanka, whereas the total forest cover is about 26.6%. Dimbulagala (7°51'40.5"N 61 81°07'05.5"E) is an isolated hill covered with dry zone forest and it is rich with strong and 62 economically important hardwood bearing plant species. This region is contacted with minimum 63 anthropological activities for many years mainly due to 30-year long civil war in this region and 64 was the study site of the current study. An unexplored niche, decaying hardwood, of Dimbulagala forest reserve was selected with the aim of describing the species diversity of 66 hardwood decay fungi for the first time in Sri Lanka. 67 For a healthy forest ecosystem, it is critical to recycle carbon stored in these hardwood and litter. 68 Among many organisms such as beetles, flies, slime molds, bacteria, slugs and snails, primary 69 decomposers that recycle carbon are fungi. It is known that wood, specially hardwood is a 70 challenging substrate to degrade as it has very low nitrogen content (commonly C: N is about 71 500:1) and the presence of various fungal toxic compounds. Therefore, fungi specially Berrya cordifolia [11][12][13]. These plant species produce hardwood with high economic value and 81 are resistant to degradation. However, mycelial mats and mushrooms were often observed in 82 decaying hardwoods in these areas indicating the involvement of fungal for the decay process. If 83 that is the case, fungal species found in association with these decaying hardwood will be able to 84 utilize wood as the sole carbon source. Therefore, the second objective was to assess the ability of 85 the fungal isolates to grow on wood dust media. It was hypothesized that fungal species capable 86 of growing on such hardwood have the ability to produce high amounts of laccases.
Fungal laccases have been identified not only as cell wall degraders, but also as strong oxidizers 88 with the ability to oxidize di-and polyphenols, aromatic amines, and detoxify environmental 89 effluents of food, paper, pulp and textile dyes [14,15]. Among these abilities of laccase, industrial 90 dye decolorization has been the subject of many studies [16,17] and laccases are often considered as 91 a "Green Tool" in biotechnology [18]. Basidiomycetes have often found to be high laccase 92 producers as well as dye decolorizers. It was also intended to determine whether the isolates of the 93 current study are capable of decolorizing an industrial dye, triphenylmethane with the hope of 94 identifying novel dye decolorizers.

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Out of tested 43 fungal isolates that could utilize wood as the sole carbon source, 11 were first 96 records of Sri Lanka. Out of them two species were first time records as laccase producers and possible to clearly identify the plant species that decayed wood pieces belonged to. Samples were 110 air dried for two days and stored in a refrigerator until further use.  Ability of isolated fungi to utilize wood as the sole carbon source 129 Ability of isolated fungi to utilize wood as the sole carbon (C) source was tested according to Swe

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[20]. During this assay most of the isolates those with morphological characters similar to Trichoderma spp. were excluded and therefore, out of 55, 43 isolates were used in further studies.

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[20]. Both wood agar plates with and without glucose were inoculated with 5 mm-diameter 136 mycelial disc obtained from actively growing edge of 7-day old culture from each isolate. Plates 137 were incubated at the room temperature (28 ± 2 ºC) for three days. Colony diameters were 138 measured in two-dimension perpendicular to each other. Experiment was repeated once.  Table 1.  Isolates that showed consistent red coloration were used for quantitative analysis.  where, A initial is the absorbance at the beginning and A final is the final absorbance.

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To determine the correlation between laccase production and the dye decolonization abilities, 229 Pearson's correlation was carried out. Since most of the fungal isolates were sterile (Fig 3) it was intended to use molecular 254 identification method.    as well as coloration patterns while negative control remained without any red color development.

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Quantitative determination of laccase production by fungal spp.

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Isolates were significantly different from each other in their mean absorbance values as measured 298 in 5 th , 8 th and 12 th days indicating significant difference in laccase production (p<0.05) (  However, to the best of our knowledge triphenylmethane dye decolorization ability and laccase 377 production of Vanderbylia fraxinea and Gymnopilus dilepis has never been reported in the fungal 378 kingdom before and we report it for the first time.

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These findings were of high importance, since it is known that synthetic dyes used in the textile, laccase producing ability, they produce relatively low amounts. It has also been reported that 387 number of laccase gene copies could vary among species depending on the life history traits [43].

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Finally, this study reported 11 wood decay fungal species in Sri Lanka as first reports highlighting 390 the importance of a thorough fungal diversity study in Sri Lanka since there is a high potential to 391 report novel biotechnologically important species. It was also proved that most of the decaying wood associated fungi are indeed can utilize wood as the sole C source. To the best of our 393 knowledge, laccase production and triphenylmethane dye decolorization ability of Vanderbylia 394 fraxinea and Gymnopilus dilepis have never been reported in the fungal kingdom before. It was 395 also found that laccase production was positively correlated with the dye decolozination ability.

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Therefore, the selected isolates have very high potential in applying for a greener biotechnology.