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NGS analysis of fungal OTUs in Aquilaria sp. from French Guyana, Cambodia, Laos and Thailand
2017 - IRG/WP 17-50328
Aquilaria is a tree species belonging to the Thymeleaceae family. When Aquilaria sp. is injured, it produce agarwood. Agarwood is characterized by a darker wood colour than the healthy one and by a strong perfume that is much esteemed by perfumers and some oriental religious communities. The production of agarwood is presumed to depend of environmental factors, among them fungi. The aim of this work is to obtain an overview of fungi present in Aquilaria sp. from different countries. Aquilaria sp. is endemic to South East Asia including notably Cambodia, Laos and Thailand, where it is cultivated to produce agarwood. In French Guiana, native south East Asian farmers would like to locally produce agarwood in their field. That’s why we wonder if fungal communities inducing agarwood are the same between these countries. NGS was used to characterize fungal communities associated with agarwood. 693,961 sequences that cover ITS2 estimated about 250 bp have been obtained. These sequences have been grouped into 535 OTUs, displaying 100% identity. 87 % were Ascomycetes and 10.5 % were Basidiomycetes. These results show differences in fungal communities between aboveground and belowground parts of the tree. Likewise differences between countries within fungal communities were also observed. Notwithstanding our results highlight common OTU between the different countries. Comparisons between aboveground parts and belowground parts and comparisons between different countries showed that several different fungal species may be responsible of the infection, other factors may disturb the community after infection and some samples may be not infected. From this study, some candidates potentially involved in agarwood production were selected. The role of these fungi in the induction of agarwood production will be further studied.
C Zaremski, C Malandain, O Sibourg, C Andary, G Michaloud, M Ducousso, N Amusant, A Zaremski

Biodiversity of wood-decaying fungi in French Guiana sequences of the small subunit (SSU) of ribosomal (r) DNA and the two primer pairs SR6/SR10R and SR7/SR1R
2019 - IRG/WP 19-10936
Fungi from tropical regions are currently under-represented in the classification system. Indeed, difficult access to tropical forests makes it complicated to study fungus species in such environments, unlike in European zones where fungal diversity and taxonomy are much better known. The purposes of this work were: a) to enhance classification by integrating new data that would bring out the importance of certain traits of these fungi, and provide a clearer understanding of how the biodiversity of fungi from the forest ecosystems of French Guiana is organized, particularly those causing wood decay through white rot, brown rot or soft rot; b) to establish a collection of fungal isolates from fruiting bodies collected in French Guiana. This taxonomic study based on sequences of the small subunit (SSU) of ribosomal (r) DNA using the two primer pairs, SR6/SR10R and SR7/SR1R, was carried out on 39 fungus fruiting bodies from Cacao and Régina, in French Guiana. Our protocol enabled extraction, with good repeatability, of fungal DNA from a few mg of pure mycelium. With most of the strains studied we were able to obtain PCR products ranging in size from 376 to 625 base pairs. This study enabled to specify the taxa involved wood decay: 39 species were identified by BlastN. Most of them, in the class of the Basidiomycetes, were the main genera were found, such as Antrodia, Coriolopsis, Fomitopsis, Ganoderma, Poria, Lentinus, Pycnoporus, Auricularia, Gloeophyllum, Trametes, Fomitopsis, Rigidoporus. In addition to the above objectives, the production of pure mycelium from fruiting bodies identified in this study will be used to produce inoculum to test the ability of these fungal species in stimulating oleoresin production in Aquilaria trees from plantation established in French Guyana.
C Zaremski, A Ducousso-Detrez, N Amusant, A Zaremski

Chemical composition of agarwood of Aquilaria crassna Pierre ex. Lecomte induced by Basidiomycetes from French Guiana
2022 - IRG/WP 22-10988
Aquilaria trees subjected to stress induce the formation of a transformed wood named agarwood. The formation of agarwood is a plant defense mechanism that occur in complex interactions with environmental microorganisms. Agarwood chemical compounds are mainly chromones and oxygenated sesquiterpenes such as eudesmol, agarospirol, jinkoh-eremol and valerianol, which are valued in perfumery. Its derivatives, notably the essential oil are therefore expensive. Agarwood essential oil costs 5,000 to 10,000 US $ per kg. Because of this important value and the scarcity of production from the wild, operators are planting Aquilaria and implementing various methods to induce agarwood. Many of these methods are deleterious for trees and even can alter the chemical composition of the essential oil. In this context, a consortium of farmers from French Guiana and scientists and from Centre de cooperation Internationale en Recherches Agronomiques pour le Développement (CIRAD) are developing the Aquil@Guyane project. This project aims at cultivating Aquilaria trees in French Guiana to produce agarwood of controlled chemical composition, respecting the biology and ecology of trees. In this frame we have tested the inoculation of trees with cubic and fibrous rot fungi to induce agarwood development in tree. Fungal strains used in this experiment have been isolated in the vicinity of Guianese Aquilaria plantations. Selected strains have been cultivated on wood test-sticks; infected sticks were then place in holes made with a drill in the trunks of Aquilaria. The yield of agarwood and chemical compositions of essential oils extracted from the so obtain blackened wood have been analyzed enabling to compare the different treatments. First, we evidence that the controlled inoculation of Aquilaria tree with selected fungal strains results in the production of agarwood and thus essential oils. The quality, in terms of composition of essential oils obtained among treatments was distributed between reference white wood and agarwood. The fibrous rot fungi were the most effective in terms of agarwood development and quality and yield of essential oils.
C Zaremski, M Ducousso, C Andary, G Michaloud, C Menut, A Zaremski, N Amusant

Chemotaxonomy of the genus Aquilaria (Thymelaeaceae)
2022 - IRG/WP 22-20687
The genus Aquilaria Lam. (Thymelaeaceae) comprises 21 tree species and is mostly found in Southeast Asia. When the tree is infected (fungi, bacteria, etc.), its wood turns brownish or blackish (called agarwood) due to the secretion of an oleoresin in reaction to the infection. The resin is very fragrant and has been sought after and used for centuries by Buddhists, Hindus and Muslims to make incense for religious ceremonies. This oleoresin is mainly found in species of the genus Aquilaria, but also in a few species of the genera Gyrinops Gaertner and Gonystylus Teijsmann & Binnendijk. It is difficult to distinguish between these species and this lack of taxonomic knowledge has led to over-use of the trees, endangering these endemic species listed in CITES Appendix II. We used chemotaxonomy as a discrimination tool to analyse polyphenolic molecules, secondary metabolites, which are known to act as taxonomic markers in other plants. This work showed the possibility of using some very old herbarium samples, which revealed good conservation of its phenolic profile. It is interesting to note that mangiferin, through its high concentration and constant presence in all the species of the genus Aquilaria analysed, constitutes a chemical marker for this genus. Comparative analysis of these phenolic molecules can therefore be very useful when seeking chemotaxonomic markers.
C Zaremski, N Amusant, C Andary, M Ducousso, S Hul, G Michaloud, A Zaremski