1/- Agarwood is studied as the resinous secondary metabolites produced by the natural microbial infection. 

The current study investigated the range of microbial infection in agarwood trees collected from various parts of India. A total of 17 isolates were collected and identified based on the morphological and molecular studies. The study revealed that the agarwood was naturally infected with Aspergillus, Lasiodiploidia, Chaetomium, Fusarium and Penicillium species. Further studies on enzyme activities involved in the pathogenesis process showed the higher cellulase, ligninolytic and laccase activities in Aspergillus isolate AR13 when compared to other isolates. The current study has offered a potential opportunity to further strengthen the research on possible development of microbial strains for artificial inoculation in agar trees to induce agarwood formation.

Agarwood is a resinous substance occurring in Aquilaria trees of Thymelaeceae family that is found in the foothills of Himalayas to the rain forests of Papua New Guinea. The tree has the unique feature of producing resin- impregnated heartwood which is fragrant and economically highly valued depending on the oleoresin content of the wood. The wood is used to manufacture some of the medicines, incense and perfumes across Asia and Europe. The agarwood is reputed to be the most expensive wood in the world and in the consumer countries it ranged from a few dollars per kg for low quality material to more than US$30,000 per kg of top quality wood [1] . The formation of agarwood is mainly attributed to the defense reaction of trees either physically or chemically when they are exposed to biotic and abiotic stresses [2] - [4] . The agarwood causal agents so far studied as physical [5] , chemical and biological agents [6] . The form of mechanical injury is considered as physical inducer of agarwood formation while the induction of chemicals, viz. oil, sugar and methyl jasmonate are reserved as chemical inducers. However, the quality and quantity of agarwood formation were not found to be greater in case of the physical and chemical inducers. Later, the studies on the biological agents in rainforest areas on the infection site of the agarwood trees revealed that the fungal microbes could be the potential agents to induce the formation of agarwood [7] [8] . As a response to the fungal infection, the tree produces a high resin in volatile organic compounds that aids in suppressing or retarding the growth of the fungus. As the fungi caused injury to the tree trunk, the tree underwent several biochemical reactions and produced a white, milky substance called oleoresin. Once the production of aromatic trunk or agarwood is complete, the tree slowly starts drying up, signaling its readiness to be harvested. From this, it is understood that resin wood or agarwood is the result of oleoresin accumulation in response to fungal infection. However, little information is available on the potential fungi and its enzymatic activities that are associated with the formation of agarwood. In this circumstance, it is believed that the study on isolation and characterization of fungi associated with agarwood formation could be the first step in proceeding further research work on the standardization and development of artificial inoculation agents so as to produce the high quality agarwood. With this background information, the current study was carried out with the objectives: 1) identification of fungi from naturally infected agarwood; 2) study of the enzymatic activities of agar wood fungi.

2/- Morphological Identification of Agarwood Fungi

A total of 17 fungal cultures were isolated from agarwood infected samples collected from Dibrugarh, Hojai and Rain Forest Research Institute, Jorhat of Assam, Sarkkapathi, Aliyar Nagar, Tamil Nadu and Kumily, Idukki District of Kerala, India. Of these, 3 isolates were from Dibrugarh (DIB1, DIB2, DIB3) and 4 isolates from Jorhat (RFRI4, RFRI5, RFRI6, RFRI7) of Assam, 6 isolates from Tamil Nadu (AR10, AR11, AR12, AR13, AR14, AR15) and 4 isolates from Kerala (K1, K2, K3 and K4). The colony characters such as colour of the mycelia mat, texture, mycelia growth of individual fungal isolates were observed on PDA medium. The mycelium and conidial characters of the individual fungal isolates were examined under microscope. The observations on colony characters and conidial characters were aided in tentative identification of the fungal cultures (Table 1). The fungi were identified as Aspergillus, Penicillium, Fusarium, Lasiodiploidia and Chaetomium.

3/- Enzymatic Activities of Agarwood Fungi

A significantly higher activity of cellulase was measured in Aspergillus isolate AR13 (1181.952 μmole∙L−1∙min) isolated from Aliyar Nagar of Tamil Nadu followed by Aspergillus isolate K1 (1140.608 μmole∙L−1∙min) whereas the lesser activity was observed in Chaetomium isolate K4 (449.92 μmole∙L−1∙min) (Table 2).

Laccase activity of fungal isolates revealed the higher secretion of laccase enzyme by Aspergillus isolates K1 (18.116 Uml−1) and AR13 (17.125 Uml−1). These two isolates showed significantly greater activity of laccase production when compared to the other isolates of agarwood fungi (Table 2).

The results of lignin degradation in LME medium supplemented with tannic acid showed brown layer as indicator of positive reaction for lignin degradation. Of seventeen isolates, Aspergillus isolates AR13 and K1 showed stronger ligninolytic activity. The isolates of Fusarium DIB2, RFRI7 showed the medium activity whereas less activity was observed in Lasiodiploidia DIB3, Chaetomium AR11 and Aspergillus K1 (Table 2).

4/- PCR Amplification of ITS Region of Agarwood Fungi

Out of 17 isolates used in the PCR studies, the isolates RFRI4 (Lasiodiploidia sp.), AR10 (Chaetomium sp.), AR11 (Chaetomium sp.), AR14 (Penicillium sp.) and AR13 and K3 (Aspergillus sp.) have amplified ITS1 and ITS2 primers. The DNA gel electrophoresis showed that the amplified products were around 560 bp. Of this, the PCR product amplified from the isolate AR13 (Aspergillus sp.) was purified and sequenced (Figure 1). The nucleotide sequence data showed 93% homology to Aspergillus genus when blast with NCBI database. The nucleotide sequence of agarwood fungal isolate AR13 was submitted to NCBI and assigned with Accession No. KF263963.

1Note: slope value = 7296. 2Note: Extinction coefficient for Guiacol 6.39. **Enzyme activity is significantly different from the other fungal microbes.

The current study identified the colonization of agarwood by Aspergillus, Penicillium, Fusarium, Lasiodiploidia and Chaetomium fungi. Previous studies have also showed that fungal species such as Aspergillus sp., Botryodiplodia sp. (Lasiodiplodia sp.), Diplodia sp., Fusarium bulbiferum, F. laterium, F. oxysporum, Penicillium sp., Pythium sp. and Trichoderma sp., could infect Aquilaria spp. [8] [16] . The major enzyme activities were observed in Aspergillus isolate AR13 in the present study. This could favour the potential infection of agarwood by Aspergillus fungus. It was also reported in Wiriadinata [17] that agarwood had mainly been attacked by opportunistic fungi from soils of rainforest environment. It is regarded as a weak pathogen and does not cause heavy damage to trees. However, the continuous release of enzymes by this fungus could favour defense mechanism in agarwood. Thus, the enzymes involved in the pathogenesis process were assayed in the current study, of which, Cellulose is the major framework molecule of the plant cell wall existing as microfibrils with matrix molecules (glycoproteins, hemicelluloses, pectins, lignins) filling the spaces between the microfibrils and cellulose chains. Cellulolytic enzymes produced by fungi play a role in softening and disintegrating plant cell walls [11] . Thus all 17 fungal isolates were assessed for cellulase activity by dyediffusion method. The study revealed the higher cellulolytic activity by Aspergillus isolate AR13 when compared to all other isolates. Similarly, lignin is a highly branched heterogeneous polymer found principally in the secondary cell walls of plants, although primary walls can also become lignified. In the current study, all seventeen fungi were showed the lignin degrading activity. The presence of lignin degradation was verified based on the brown oxidation zone present around the colony where in the isolate AR13 (Aspergillus niger) showed higher activity. Similarly, the laccase activity was recorded higher in Aspergillus niger isolate AR 13 in the current study. The pathogenesis process by fungus is determined by the production of laccase activity [18] . Similar results were obtained by Desai et al. [12] where Trichoderma genus produced higher levels of laccase activity under solid state condition.

From the current study, it is believed that the formation of agarwood could be a result of plant defense mechanism towards fungal attacks by producing resinous compounds as secondary metabolite [3] [19] [20] . It is also assumed in the current study that the series of enzymes produced by the pathogenic or saprophytic fungal microorganisms could play a major role in defense mechanism which ultimately becomes responsible for agarwood production. In this context, it is possible to develop Aspergillus niger AR13 as artificial agent to inoculate the agarwood.

Interestingly, two of our isolates from our collection i.e. Aspergillus and Lasiodiplodia were also reported as endophytic fungi in Mitra and Gogoi [21] . In this context, our cultures should be tested for their endophytic nature and their role in agarwood formation. Although the possibility of inducing agarwood formation by deliberately infecting trees with fungal isolates has been postulated to induce resin, it has to be tested at various agroforestry settings having different environmental factors and weather conditions. Further, the isolate AR13 which is to be tested for agar formation studies should be verified for its host specificity. It is otherwise should be proved as non-pathogenic to other crop plants.

5/- Identification of sesquiterpene synthase genes in the genome of Aquilaria sinensis and characterization of an α-humulene synthase

Phylogenetic relationships of TPSs from Aquilaria sinensis and Arabidopsis thaliana. Fuchsia: AsTPS1-AsTPS32, black: A. thaliana, blue triangles: the six newly discovered TPSs (AsTPS27-AsTPS32). SemBDPS BAL41682.1, a miltiradiene synthase from

Tertiary structural models of AsHS1 from Aquilaria sinensis and comparison with other STSs. a Structural model of AsHS1 with farnesyl diphosphate (FPP) and three Mg 2+ . The C-terminal domain is in bright orange, with three bound Mg 2+ atoms (dark-gray spheres), the N-terminal domain is in light blue, and the hydrophobic structure is the active site. b Detail of the AsHS1 catalytic cleft, showing the

Proposed humulene and guaiene biosynthetic pathways in A. sinensis. ASS1, ASS2 and ASS3 are the δ-guaiene synthases identified by Xu et al. (2013)

6/- Evaluation of incense-resinous wood formation in agarwood ( Aquilaria malaccensis Lam. ) using sonic tomography

Incense-resinous wood of agarwood is a high-value non-timber forest product found in the trunk or branches of Aquliaria and Gyrinops species. Incense-resinous wood of agarwood is formed as a response to tree damage caused by wounding or fungal attack. Detection of such wood in trees has generally been carried out based on natural signs such as dark spots or black marks when peeling back tree bark, but these often yield uncertain results. Sonic tomography can be applied to predict the presence of incense-resinous wood in standing trees. The objective of this study was to evaluate sonic tomography at various trunk heights based on variations in the sound velocity associated with the presence of incense-resinous wood. Ten agarwood trees (Aquilaria malaccensis) were selected for this study; five trees were artificially inoculated with Fusarium solani fungus and the other five were untreated. The results showed that the height of the measurement did not significantly affect the propagation velocity of sound waves or the tomographic results. Sonic tomography revealed that prediction of the deteriorated zone which is indicative of incense-resinous wood formation was 1.1% greater in inoculated trees than in uninoculated trees.

Inoculation process on agarwood trunk: (A) Pattern of fungal inoculation holes in the tree trunk; (B) Wooden peg used to plug the inoculation hole. 

Tree dimensions in inoculated and uninoculated agarwood (A. malaccensis) trees.

Application of sonic tomography on agarwood tree trunk. 

Tree sound wave velocity values based on distance from the ground (height).

Internal condition of the wood as shown by a tomogram. The full spectral color range is shown in grayscale here with solid wood and damaged wood as well as initial attack indicated by the arrows. 

Black marks on an inoculated tree at several heights along the trunk (A); Uninoculated tree with white patches of fungus (B). 

Example of two-dimensional and three-dimensional tomograms at several heights along the agarwood trunk: (A) inoculated tree P1; (B) uninoculated tree P6. (darker areas indicate solid or intact wood, while lighter areas indicate initial attack).

7/ Agarwood Formation Induced by Fermentation Liquid of Lasiodiplodia theobromae, the Dominating Fungus in Wounded Wood of Aquilaria sinensis.

Agarwood is broadly used in incense and medicine. Traditionally, agarwood formation is induced by wounding the trunks and branches of some species of Aquilaria spp., including A. sinensis. As recently evidenced, some fungi or their fermentation liquid may have the potential of inducing agarwood formation. The present study aimed to analyze the fungi isolated from an agarwood-producing A. sinensis tree and subsequently identify the fungi capable of promoting agarwood formation. We identified a total of 110 fungi isolates based on their morphological characteristics and rDNA ITS sequences. These isolates came from four different layers (namely the decomposing layer, agarwood layer, transition layer, and normal layer) near the agarwood formation site of the trunk. According to the experimental results, most of them belonged to Dothideomycetes (81.82%), while the others to Sordariomycetes (13.64%) or Eurotiomycetes (4.55%). Of note, 88 isolates were shown belonging to the species of Lasiodiplodia theobromae that are most frequently isolated from different layers. In addition, when the fermentation liquid of two isolates of L. theobromae (AF4 and AF12) and one isolate of Fusarium solani (AF21) was inoculated into the A. sinensis wood using the Agar-Wit technique, promoted agarwood formation was observed; however, the effect of AF21 did not keep stable in the later test, while AF4 and AF12 still functioned 1 year later. This study may lay a foundation for exploring the underlying mechanism of agarwood formation as well as fungi application in agarwood production.

Agarwood induced by the branch-cutting method was used for fungi isolation in the present study. Four layers were separately collected. DL decomposing layer, AL agarwood layer, TL transition layer, NL normal layer

The numbers of isolated strains from different layers. The species of Lasiodiplodia theobromae was dominantly spreading in each layer

Agarwood induced by the fermentation liquid of isolated fungi. a Wood inoculated with: 1 the fermentation liquid of NAF6; 2 culture media; 3 the fermentation liquid of AF12; 4 the fermentation liquid of AF4; 5 the patented agarwood inducer; 6 the fermentation liquid of AF21. b Plants that were inoculated with the fermentation liquid of fungi showed yellow leaves; c colony of AF4; d colony of AF12; e colony of NAF6; f colony of AF21; g thin-layer chromatogram of extracts from the wood inoculated with: 1 the fermentation liquid of AF21; 2 the patented agarwood inducer; 3 the fermentation liquid of AF4; 4 the fermentation liquid of AF12; 5 ST; 6 7-dimethoxy-2-(2-phenylethyl) chromone, and six culture media. (Color figure online)

8/- Isolation and identification of Endophytic Fungus Fusarium sp from Agarwood ( Aquilaria sp ) population originated from the forest of Aceh Tamiang district, Indonesia

Aquilaria sp is one of higher plant types that can produce agarwood. Agarwood is commonly stimulated by the biological response of endophytic fungal infection of Fusarium sp . This research aims to isolate and identify endophytic fungus Fusarium sp from Aquilaria sp populations originated from the forest of Aceh Tamiang District. The pure isolates of Fusarium sp derived from Aquilaria sp that reinfected on Aquilaria sp populations to stimulate agarwood formation. The endophytic fungal isolates of Fusarium sp were growth on a selective media of Sabouraud Dextrose Agar (SDA) containing antibiotics Streptomycin 10 µg/dl and Chloramphenicol 25µg/dl. Ten percent of infected Aquilaria sp from the population was taken aseptically. Isolation and identification of endophytic fungus Fusarium sp were conducted from June to August 2016. The results showed that within 10 infected Aquilaria sp , several microbe species were found such as three Fusarium sp , six Aspergillus sp , two Rhizopus sp , two Penicillium sp , one Bacillus sp , one Actinomyces sp and one Streptomyces sp species. The result of identification towards three species of endophytic fungus Fusarium sp that has been purified were srongly suspected to be Fusarium solani, Fusarium oxysporum , and Fusarium monyliformae.

The wood fibre containing agarwood from sampling techniques, a; sample of wood fibre containing agarwood in sterile adhesive plastic, b; endophytic fungus from xylem, c; endophytic fungus from wood fibre of Aquilaria sp.

The endopyhtic fungal isolates of Fusarium solani, Fusarium oxyforum and Fusarium monyliformae grown on SDA media, A; Fusarium oxyforum grown on SDA media, incubation at room temperature, day 5 th incubation, B; Fusarium oxyforum grown on SDA media, incubation at room temperature, on day 7 th of incubation.

The endophyte fungal sampling location on some Aquilaria sp populations on four different locations in Aceh Tamiang Regency.

Endophytic fungal and bacteria isolates isolated from ten stems of Aquilaria sp from four different forest in AcehTamiang.

(a) dried infected agarwood chips, (b) fresh infected agarwood chips and (c) fresh uninfected agarwood chips

Effect of juvenile Aquilaria malaccensis stem pieces on fungal growth in PDA plates

Antibacterial activity of Aquilaria malaccensis leaves on S. pneumonia, E. coli, S. dysenteriae, K. pneumonia and B. subtillis

9/- Fusarium solani induces the formation of agarwood in Gyrinops versteegii (Gilg.) Domke branches

Agarwood is a resinous wood produced by some members of plant family Thymelaeaceae under certain conditions. Agarwood is highly prized, but its formation requires a long-time process in nature. Therefore, various induction techniques have been explored to hasten the process. In this study, we induced agarwood in Gyrinops versteegii, one of the most abundant agarwood-producing trees in Indonesia. We used 12 trees and wounded four branches on each tree through an injection process. We used two strains of the endophytic fungi Fusarium solani isolated from Gorontalo and Jambi Provinces. After 3 months, the inoculated wood had an extensive resinous zone, when compared to wounded control wood. Gas chromatographic-mass spectrometric analysis of the inoculated samples revealed the presence of several sesquiterpenes characteristic of agarwood. These included alloaromadendrene, β-eudesmol and β-selinene as well as the chromone derivatives 2-(2-phenylethyl) chromen-4-one, 6-methoxy-2-(2-phenylethyl) chromen-4-one, and 6,7-dimethoxy-2-(2-phenylethyl) chromen-4-one. We conclude that this method successfully induced agarwood to form in a matter of months and could be used to enhance the success of agarwood cultivation.

Principal component analysis of the chemical contents of wood samples of G. versteegii. GC-MS results derived from 3-and 6-month samples after treatment were pooled using either methanol extracts (a) or ethyl acetate extracts (b). (Key: UC, healthy, untreated control; GSL1,

Photographs of branches of G. versteegii 4 weeks after the plants had been wounded or wounded and inoculated with F. solani (either strain GSL1 or GSL2). Note the resinous zones that developed around the wounds of the inoculated plants

A branch of G. versteegii 6 months after it was wounded and inoculated with F. solani (a) compared with wounded only (b). Note that the resinous zone has expanded to much of the surface area and deep into the wood after it was inoculated with F. solani as long as 10 sites of injected holes (indicated by black arrows). In contrast with wounded only sample, the resinous was only developed at injection site (indicated by red arrows)

Micrographs of transverse sections of wood tissue from branches of G. versteegii 6 months after treatment and stained with neutral red for sesquiterpenes. a healthy, unwounded tissue; b wounded tissue; c wounded tissue inoculated with F. solani GSL1, and d wounded tissue inoculated with F. solani GSL2. (IP, interxylary phloem; XR, xylem ray parenchyma)

Results from an olfactory test of wood samples of G. versteegii. Samples were taken at 3 and 6 months after treatment. Thirty-four people participants evaluated the level of fragrance intensity of the wood. (Participants rated the fragrance based on the following scale: 1 nearly odorless, 2 little odor, 3 fair odor, 4 strong odor, 5 very strong and pleasant odor)

Heat map showing relative content of likely compounds in extracts of wood samples from branches of G. versteegii. Samples were taken at 3 and 6 months after treatment using either methanol extracts (a) or ethyl acetate extracts (b). Highest percentage area of the compounds is shown as dark-red colour, while lowest percentage area of the compounds is shown as blue colour. (Key: UC, healthy, untreated control; GSL1, samples from wood that had been wounded and inoculated with F. solani GSL1; GSL2, samples from wood that had been wounded and inoculated with F. solani GSL2; W, wood from trees that had been wounded only; 3, samples treated for 3 months; 6, samples treated for 6 months)

10/- Fusarium solani

Nuôi cấy Fusarium solani được trồng trên môi trường dinh dưỡng PDA, 28 ngày sau khi tiêm đĩa thạch từ một nền văn hóa trước đó trên món Ăn Petri 5cm