AN ITS GENE-MEDIATED MOLECULAR DETECTION OF FUNGI ASSOCIATED WITH NATURAL AND ARTIFICIAL AGARWOOD FROM Aquilaria malaccensis

The current study examined fungal diversity in manufactured and natural agarwood samples perceived from A. malecensis trees in a plantation and the East Malaysia rainforest. Isolated fungi were also subjected to molecular analysis. The fungal community composition of healthy and damaged tree samples from both plantation and the wild forest was identified using PCR amplification of the internal transcribed spacer (ITS) region from fungal isolates. Meanwhile, ten groups of fungi isolates are expected to be placed in both natural and inoculated based on 1000 bootstrap values. They validated earlier genomic identification; 55 fungal isolates were discovered in artificial and natural agarwood, including agarwood and healthy wood. Ten fungal groupings were developed based on morphological characterization similarities. Based on NCBI BLAST analysis, molecular identification revealed Fusarium solani, Botryosphaeria theobromae, Polyporales spp, Schizophyllum commune, Aspergillus aculeatus, and Lasiodiplodia theobromae. Fusarium solani excelled in A. aculeatus and Polyporales spp. in agarwood samples and healthy wood. The presence of more fungi species in natural agarwood than in artificial agarwood may be due to the favourable humid and shaded habitat for fungal growth. Nonetheless, synthetic agarwood was able to synthesize agarwood-related chemical compounds. Considering F. solani was often identified in both agarwood, agarwood inoculant may act as a booster for F. solani to begin pathogenicity in artificial agarwood. The current research shows that artificial agarwood may provide quality comparable to natural agarwood and is not influenced by fungus interacting with the tree.

[1]  P. Bhuyar,et al.  Gas chromatography analysis of the microwave-aided extracted agarwood oil from physically induced Aquilaria malaccensis trees in Northern Thailand , 2022, Maejo International Journal of Energy and Environmental Communication.

[2]  A. Ramli,et al.  An Overview of Molecular Basis and Genetic Modification of Floral Organs Genes: Impact of Next-Generation Sequencing , 2022, Molecular Biotechnology.

[3]  S. N. Tajuddin,et al.  Production of Volatile Compounds by a Variety of Fungi in Artificially Inoculated and Naturally Infected Aquilaria malaccensis , 2022, Current Microbiology.

[4]  R. Ramaraj,et al.  Exploration of bioactive compounds and antibacterial activity of marine blue-green microalgae (Oscillatoria sp.) isolated from coastal region of west Malaysia , 2020, SN Applied Sciences.

[5]  P. Pripdeevech,et al.  Incubation of Aquilaria subintegra with Microbial Culture Supernatants Enhances Production of Volatile Compounds and Improves Quality of Agarwood Oil , 2018, Indian Journal of Microbiology.

[6]  N. Kaushik,et al.  Fungal and Bacterial Diversity Isolated from Aquilaria malaccensis Tree and Soil, Induces Agarospirol Formation within 3 Months after Artificial Infection , 2017, Front. Microbiol..

[7]  L. Nahum,et al.  Effectiveness of ITS and sub-regions as DNA barcode markers for the identification of Basidiomycota (Fungi) , 2017, BMC Microbiology.

[8]  Nicholas H Oberlies,et al.  Fungal Identification Using Molecular Tools: A Primer for the Natural Products Research Community , 2017, Journal of natural products.

[9]  A. Faizal,et al.  Formation of agarwood from Aquilaria malaccensis in response to inoculation of local strains of Fusarium solani , 2017, Trees.

[10]  K. T. Parthiban,et al.  Analysis of different grades of Agarwood ( Aquilaria malaccensis Lamk.) oil through GC-MS , 2015 .

[11]  M. Bartlam,et al.  Comparison of ITS and 18S rDNA for estimating fungal diversity using PCR–DGGE , 2015, World Journal of Microbiology and Biotechnology.

[12]  A. Kalra,et al.  Molecular phylogenetic identification of endophytic fungi isolated from resinous and healthy wood of Aquilaria malaccensis, a red listed and highly exploited medicinal tree , 2013 .

[13]  Nicholas A. Bokulich,et al.  Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative, Ultra-High-Throughput Profiling of Fungal Communities , 2013, Applied and Environmental Microbiology.

[14]  Christoph Wittmann,et al.  Characterization and control of fungal morphology for improved production performance in biotechnology. , 2013, Journal of biotechnology.

[15]  L. Raskin,et al.  PCR Biases Distort Bacterial and Archaeal Community Structure in Pyrosequencing Datasets , 2012, PloS one.

[16]  Satoshi Yamamoto,et al.  High-Coverage ITS Primers for the DNA-Based Identification of Ascomycetes and Basidiomycetes in Environmental Samples , 2012, PloS one.

[17]  T. C. Lorenz,et al.  Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies , 2012, Journal of visualized experiments : JoVE.

[18]  N. Nejat,et al.  Morphological and molecular characterization of Fusarium spp. associated with yellowing disease of black pepper (Piper nigrum L.) in Malaysia , 2012, Journal of General Plant Pathology.

[19]  John L. Spouge,et al.  Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi , 2012, Proceedings of the National Academy of Sciences.

[20]  G. B. Golding,et al.  Are similarity- or phylogeny-based methods more appropriate for classifying internal transcribed spacer (ITS) metagenomic amplicons? , 2011, The New phytologist.

[21]  H. Philippe,et al.  Resolving Difficult Phylogenetic Questions: Why More Sequences Are Not Enough , 2011, PLoS biology.

[22]  R. Henrik Nilsson,et al.  Progress in molecular and morphological taxon discovery in Fungi and options for formal classification of environmental sequences , 2011 .

[23]  Pierre Taberlet,et al.  ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases , 2010, BMC Microbiology.

[24]  Rozi Mohamed,et al.  Fungal diversity in wounded stems of Aquilaria malaccensis , 2010, Fungal Diversity.

[25]  G Ronald Jenkins,et al.  Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits , 2010, Analytical and bioanalytical chemistry.

[26]  A. Subramanian,et al.  A fundamental study of the PCR amplification of GC-rich DNA templates , 2008, Comput. Biol. Chem..

[27]  M. Bhuiyan,et al.  Analysis of essential oil of eaglewood tree (Aquilaria agallocha Roxb.) by gas chromatography mass spectrometry , 2008 .

[28]  D. Hickey,et al.  Assessing the effect of varying sequence length on DNA barcoding of fungi , 2007, Molecular ecology notes.

[29]  B. Summerell,et al.  Fusarium laboratory workshops—A recent history , 2006, Mycotoxin Research.

[30]  Ian Humphery-Smith,et al.  Regionalized GC content of template DNA as a predictor of PCR success. , 2003, Nucleic acids research.

[31]  T. Bruns,et al.  ITS primers with enhanced specificity for basidiomycetes ‐ application to the identification of mycorrhizae and rusts , 1993, Molecular ecology.

[32]  S. N. Tajuddin,et al.  Fungi mediated agarwood (A. malaccensis) production and their pharmaceutical applications: A systematic review , 2022, International Journal of Plant Based Pharmaceuticals.

[33]  M. Rahim,et al.  Antioxidant and antibacterial activity of red seaweed Kappaphycus alvarezii against pathogenic bacteria , 2020 .

[34]  Rozi Mohamed,et al.  Understanding Agarwood Formation and Its Challenges , 2016 .

[35]  M. Turjaman,et al.  Development of Agarwood Induction Technology Using Endophytic Fungi , 2016 .

[36]  B. Salleh,et al.  Occurrence and Pathogenicity of Fusarium spp. on the potato tubers in Malaysia , 2011 .

[37]  R. Henry,et al.  DNA extraction from plant tissue , 2008 .

[38]  David L. Hawksworth,et al.  Fungal diversity and its implications for genetic resource collections , 2005 .

[39]  M. Curtis,et al.  in environmental samples , 2002 .

[40]  Chen Hin Keong,et al.  Heart of the matter: Agarwood use and trade and CITES implementation for Aquilaria malaccensis , 2000 .

[41]  T. White.  Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics , 1990 .