Fungal and Bacterial Diversity Isolated from Aquilaria malaccensis Tree and Soil, Induces Agarospirol Formation within 3 Months after Artificial Infection.

Hemraj Chhipa,Nutan Kaushik

doi:10.3389/fmicb.2017.01286

Abstract

Aquilaria malaccensis Lam, commonly known as Agarwood, is a highly valuable species used in production of agar oil from its infected wood, which is utilized in pharmaceutical and perfumery industry. Agar oil formation in agarwood takes years through the natural process which is induced by natural or artificial injury or microbial infection. The role of soil fungi and bacteria in artificial induction is still an unexplored area. In the present study, we isolated the fungal and bacterial community residing inside the stem of A. malaccensis tree and circumventing soil, samples collected from 21 different sites of the north-eastern state Assam of India and explored their potential in induction of Agarospirol (2-(6,10-Dimethylspiro[4,5]dec-6-en-2-yl)-2-propanol) production by artificially infecting the trees with these microorganisms. A total 340 fungi and 131 bacteria were isolated from 50 stem samples, and 188 fungi and 148 bacteria were isolated from 50 soil samples. Highest Shannon (H′ = 2.43) and Fisher (α = 5.57) diversity index was observed in the stem isolates. The dominant fungal genus was Trichoderma in stem with Pi value of 0.18; while in soil, Aspergillus showed dominance with Pi value 0.73. In bacteria, Bacillus genera showed dominance in both stem and soil samples with Pi = 0.62 and 0.51, respectively. Forty fungal and bacterial isolates were used to assess their potential to induce formation of agarwood in A. malaccensis by artificial infection method. Gas chromatography mass spectroscopy (GC-MS) analysis confirmed development of Agarwood by the presence of Agarospirol compound in samples collected after 3 months of the artificial infection. Only 31% of bacterial and 23% of fungal isolates showed their ability in production of Agarospirol by artificial infection method. Bacteria Pantoea dispersa and fungi Penicillium polonicum showed the highest production in comparison to other isolates.

Highlights

Aquilaria malaccensis is an oleoresin-producing tree which occurs in Malaysia, Myanmar, Sumatra, Borneo Island, Philippines, Bangladesh, Vietnam, China, and India (Gibson, 1977; Hai et al, 1999)
In all 340 fungi and 131 bacteria were isolated from 50 stem samples, and 188 fungi and 148 bacteria were isolated from 50 soil samples
These isolates were grouped on the basis of similarity of their external morphology and microscopic examination and representative isolates were used for molecular identification by 16s rRNA region amplification using 518F and 1492 R primers for bacteria and ITS region amplification using ITS1 and ITS4 primers for fungi and sequenced using Merck sequencing services, Bengaluru, India

Summary

Introduction

Aquilaria malaccensis is an oleoresin-producing tree which occurs in Malaysia, Myanmar, Sumatra, Borneo Island, Philippines, Bangladesh, Vietnam, China, and India (Gibson, 1977; Hai et al, 1999). The oleoresin produced by the tree is a highly valuable component of incense, perfumes, drugs, stimulants, cardiac tonics, and carminatives (Mengling et al, 2005). The dark wood of Aquilaria tree having oleoresin is known as agarwood, which emits a pleasant smell on burning and is widely used as incense in Islamic, Buddhist, and Hindu ceremonies. Commercial exploitation and uncontrolled cutting is leading to a decline in number of these trees. Eight species of Aquilaria are listed in Appendix II of the Convention on International Trade in Endangered Species (CITES) of wild fauna and flora (CITES, 1994) and are categorized as vulnerable by the International Union for the Conservation of Nature (IUCN, 2009)

Methods

Results

Discussion

Conclusion