Biodiversity and application prospects of fungal endophytes in the agarwood-producing genera, Aquilaria and Gyrinops (Thymelaeaceae): A review

Faizal A., Hermawaty D., Junita E., Rahmawati A., Azar A.W., Makajanma M.M., Turjaman M.

Gyrinops versteegii is a tropical, agarwood-producing tree. Agarwood is valued for its fragrant resin, which has resulted in over-harvesting of agarwood trees and their protection as endangered species. Agarwood is produced in response to physical, chemical, or biological stress, and considerable research has been devoted to identifying effective methods for artificial induction of agarwood. Among the known agarwood-forming species, most research has been carried out on species in the genus Aquilaria. In the present study, we report results of a preliminary study on the potential of Gyrinops versteegii for agarwood production using seedlings. Agarwood production in the stem of Gyrinops versteegii seedlings was evaluated in response to bark removal (wounding treatment), or inoculation with bacteria (Bacillus sp. or Alcaligenes sp.) or fungi (Fusarium solani or Paecilomyces variotii), or treatment with salicylic acid. Healthy unwounded seedlings were included in the analyses as negative controls. We found that terpene was formed in response to all treatments and mainly accumulated in the interxylary phloem and xylem rays. Analysis for volatile chemicals using gas chromatography-mass spectrometry revealed that sesquiterpenes were only detected in stems treated with the fungus, Fusarium solani, and that chromones were present in stems inoculated with bacteria or fungi. Our results demonstrate that both biological and chemical agents have the potential to induce the biosynthesis of agarwood resin components in Gyrinops versteegii seedlings. Among these, Fusarium solani was the most effective agent. We anticipate that this study will lead to a more elaborate study of agarwood formation in the genus Gyrinops.

Peeraphong L.

Ma S., Fu Y., Li Y., Wei P., Liu Z.

• Fungi extracted from wild agarwood can improve the yield and quality of agarwood. • Four fungi are the species found novel as inoculant for agarwood formation. • The best time to harvest agarwood when the trees inoculated with fungi for one year. • A. sinensis is more suitable for the fungal inducer than A. crassna . Agarwood has high economic value and wide uses, however, the amount of wild agarwood is scarce. Wild agarwood with high oil content may contain strains associated with agarwood production. To improve the formation of agarwood, we produced fungal inducers from a high-oil wild agarwood block containing enrichment fungi. We investigated the composition of strains and inoculated the fungal inducers into Aquilaria trees. We analyzed the content of alcohol-soluble extract, essential oil , agarotetraol, 2-(2-phenylethyl)chromone, 6,7-dimethoxy-2-(2-phenylethyl)chromone and total chromone, and the chemical components of alcohol-soluble extract. We aimed to assess agarwood quality through these evaluations. The results demonstrated that fungi extracted from wild agarwood with high oil content can effectively improve the yield and quality of agarwood, and Gongronella butleri , Saitozyma podzolica , Cladorrhinum bulbillosum , Humicola grisea and Tetracladium marchalianum were the species found novel as inoculant for agarwood formation. After 12 months of inoculation, agarwood produced using A. sinensis had higher quality, the contents of alcohol-soluble extract, essential oil, total chromones, and the total relative contents of sesquiterpenes and chromones were 21.73 % ±2.97 %, 6.07 % ±0.47 %, 12.30 % ±2.21 %, and 97.59 %, respectively. Over 6–18 months of inoculation, agarotetrol content decreased, and 2-(2-phenylethyl) chromone and 6,7-dimethoxy-2-(2-phenylethyl)chromone content first increased and then decreased.

Huang L., Zhu X., Zhou S., Cheng Z., Shi K., Zhang C., Shao H.

Phthalic acid esters (PAEs) are a class of lipophilic chemicals widely used as plasticizers and additives to improve various products’ mechanical extensibility and flexibility. At present, synthesized PAEs, which are considered to cause potential hazards to ecosystem functioning and public health, have been easily detected in the atmosphere, water, soil, and sediments; PAEs are also frequently discovered in plant and microorganism sources, suggesting the possibility that they might be biosynthesized in nature. In this review, we summarize that PAEs have not only been identified in the organic solvent extracts, root exudates, and essential oils of a large number of different plant species, but also isolated and purified from various algae, bacteria, and fungi. Dominant PAEs identified from natural sources generally include di-n-butyl phthalate, diethyl phthalate, dimethyl phthalate, di(2-ethylhexyl) phthalate, diisobutyl phthalate, diisooctyl phthalate, etc. Further studies reveal that PAEs can be biosynthesized by at least several algae. PAEs are reported to possess allelopathic, antimicrobial, insecticidal, and other biological activities, which might enhance the competitiveness of plants, algae, and microorganisms to better accommodate biotic and abiotic stress. These findings suggest that PAEs should not be treated solely as a “human-made pollutant” simply because they have been extensively synthesized and utilized; on the other hand, synthesized PAEs entering the ecosystem might disrupt the metabolic process of certain plant, algal, and microbial communities. Therefore, further studies are required to elucidate the relevant mechanisms and ecological consequences.

Auri A., FARIDAH E., SUMARDI S., HARDIWINOTO S.

Abstract. Auri A, Faridah E, Sumardi, Hardiwinoto S. 2021. The effect of crown pruning and induction of Acremonium sp. on agarwood formation in Gyrinops caudata in West Papua, Indonesia. Biodiversitas 22: 2604-2611. Agarwood-producing trees have been planted by the community, but have currently not produced agarwood resin. This research examines the crown pruning effect and fungi inoculation effectivity on the formation of agarwood resin in Gyrinops caudata. The environmental modification was also ascertained based on the real environmental situation in natural forests. Furthermore, it was assumed that environmental conditions significantly influenced the growth, G. caudata tree association, and fungi infection. This method involved the tree crown cover modification. Tree crown was categorized into three different classes, i.e. dense crown (no pruning); moderate (pruning 25%), and sparse (pruning 50%). The results showed that inoculating fungi of Acremonium sp. to infection coverage area after three months produced the best results, due to Fcount 79.671. However, crown prunings obtained through heavy, moderate and non-pruning did not show any effect on stem infection spread. In the sixth month, it was observed that the fungi inoculation factor of Acremonium sp. had a significant effect on infection spread formation by Fcount of 168.894. The change in wood internal tissue also had a highly significant effect as depicted by Fcount 461.516. The fragrance level treatment also showed a considerable effect with Fcount 290.385.

Faizal A., Esyanti R.R., Adn'ain N., Rahmani S., Azar A.W., Iriawati, Turjaman M.

Abstract Agarwood forms in the heartwood of trees in the family Thymelaeaceae in response to wounding, infection, or other stresses. Its formation is random and takes decades in natural populations, which are harvested for their aromatic compounds. This harvest has led to declining population, and many agarwood producing trees are considered endangered. Therefore, an alternative source would be desirable. We established an in vitro shoot culture method for one agarwood species, Aquillaria malaccensis. Agarwood production was elicited by introducing methyl jasmonate (MeJA) and crude extracts of Fusarium solani into the liquid culture medium. A high concentration of MeJA resulted in necrotic shoot tissue, while application of the crude extracts had no effect on growth of the shoots. Interestingly, gas chromatography-mass spectrometry (GC-MS) analysis of MeJA-treated shoots revealed the presence of several agarwood compounds, including sesquiterpenes and chromone derivative. In addition, GC-MS analysis of shoot-treated with the extracts revealed the presence of alkanes, aromatic compounds, and fatty acid derivatives. It may be that different elicitors induce the production of different compounds in A. malaccensis in vitro shoot cultures and could be used to manipulate the accumulation of different products in culture.

Oshima T., Ito M.

Various essential oils from plants and fragrance components such as monoterpenes have been discovered to reduce spontaneous movements in mice; thus, it has been made clear that the odor itself has the sedative activity. In the present study, we examined the sedative activity of the odors of fragrance components added to eye drops; l-menthol, d-camphor, phenylethyl alcohol, and geraniol, which are often used as refreshers or preservatives. Each fragrance component was administered by the inhalation route to mice, and the sedative effects were evaluated using an open field test. The results showed that four components administered via inhalation to mice significantly decreased the amount of spontaneous motor activity in a dose-dependent manner, indicating that all four components have a sedative effect. The optimal concentrations at which l-menthol, d-camphor, phenylethyl alcohol, and geraniol showed the highest sedative activity were 4 × 10–2 mg per cage, 4 × 10–4 mg per cage, 4 × 10–2 mg per cage, and both 4 × 10–4 and 4 × 10–2 mg per cage, respectively. The AUC graph of geraniol was represented as a W-shaped curve, suggesting that the sedative action of geraniol was biphasic. The present finding demonstrates a new perspective on a possible pharmacological property of eye drop additives used with no expected pharmacological functions.

Li J., Thangaiyan R., Govindasamy K., Wei J.

This study aimed to investigate the antihyperlipidemic and anti-inflammatory effect of zingiberene (ZBN) on isoproterenol-(ISO) induced myocardial infarction in rats. ZBN (10 mg/kg b.wt.) was orally administered to rats for 21 days and ISO (85 mg/kg b.wt.) was subcutaneously injected into the rats at 24 h intervals for the last 2 consecutive days. We observed increased serum creatine kinase, creatine kinase-MB, cardiac troponin T, and I levels in ISO-treated MI rats. Conversely, ZBN oral administration significantly prevented in cardiac marker enzyme activities in ISO-mediated rats. We also noticed that ZBN oral administration prevented ISO-induced expression of lipid peroxidative markers, total cholesterol, triglycerides, phospholipids, free fatty acids, very-low-density lipoprotein cholesterol (VLDL-C), low-density lipoprotein cholesterol (LDL-C) to the normal basal level. Furthermore, ZBN restored ISO-mediated antioxidant status, increased level of high-density lipoprotein cholesterol (HDL-C), and tissue phospholipids to the near-normal levels. Besides, ZBN pre-treatment significantly reduced the level of inflammatory markers (TNF-α, IL-6, NF-κB, and IL-1β) in ISO-induced MI in rats. We noticed that ZBN pretreatment inhibited the pro-apoptotic proteins Bax and cytochrome c and increased the Bcl-2 expression in ISO induced rats. The gene expression profiling by qRT-PCR array illustrates that ZBN treatment prevents the ISO mediated activation of cardiac markers, inflammatory, and fibrosis-related genes in the heart tissue. Taken together, pre-treatment with ZBN attenuated ISO-induced MI resolved exhibits the anti-inflammatory and antiapoptotic effect.

Kinjo Y., Wada K., Oe M., Hou D., Takahashi M.

Altinoz M.A., Ozpinar A., Seyfried T.N.

Abstract High grade glial tumors (HGGs) including anaplastic astrocytoma (WHO Grade-III) and glioblastoma multiforme (GBM, WHO Grade-IV) are among the most malignant cancers known to man. Due to their defective mitochondria, HGG cells consume glucose via glycolysis even in the presence of oxygen. Overall survival is worse in HGG patients that are hyperglycemic. Unlike normal neural cells, HGG cells cannot efficiently metabolize ketone bodies for energy. Thus, a metabolic treatment based on therapeutic ketosis (reduced glucose with elevated ketone bodies) was proposed to treat GBM and was supoported from preclinical studies. Caprylic (octanoic) acid, a monocarboxylated saturated fatty acid, is among the best producers of ketone bodies and induces necrosis of experimental tumors at high dose. Caprylic acid is enriched in coconut and in goat's milk. It is also a posttranslational modifier of the ghrelin hormone and is produced in trace amounts in human tissues. Caprylic acid is a straight-chain isomer of the antiepileptic valproic acid, which is used in treatment of HGG-associated seizures and which may increase survival in GBM patients according to epidemiological observations. Among the valproic acids analogs tested, caprylic acid is the most potent molecule to block C6 astrocytoma cell growth in vitro and accumulates selectively within glial cells as shown by Positron Emission Tomography in vivo. Caprylic acid blocks glycolysis both in healthy liver and in malignant liver cells, which is more prominent in the latter and also lowers blood glucose. Noteworthy, caprylic acid exerts neuroprotective- and mitochondria-protective effects in several models of neurodegenerative diseases. Boost injections of caprylic acid at non-toxic levels during classical ketogenic metabolic therapy may fortify antitumor actions and reduce systemic toxicity by differential programming of mitochondrial and other metabolic pathways.

Plotnikov M.B., Plotnikova T.M.

The use of neuroprotective agents for stroke is pathogenetically justified, but the translation of the results of preclinical studies of neuroprotectors into clinical practice has been a noticeable failure. One of the leading reasons for these failures is the one-target mechanism of their activity. p-Tyrosol (Tyr), a biophenol, is present in a variety of natural sources, mainly in foods, such as olive oil and wine. Tyr has a wide spectrum of biological activity: antioxidant, stress-protective, anti-inflammatory, anticancer, cardioprotective, neuroprotective and many others. This review analyzes data on the neuroprotective, antioxidant, anti-inflammatory, anti-apoptotic and other kinds of Tyr activity as well as data on the pharmacokinetics of the substance. The data presented in the review substantiate the acceptability of tyr as the basis for the development of a new neuroprotective drug with multitarget activity for the treatment of ischemic stroke. Tyr is a promising molecule for the development of an effective neuroprotective agent for use in ischemic stroke.

Tanapichatsakul C., Pansanit A., Monggoot S., Brooks S., Prachya S., Kittakoop P., Panuwet P., Pripdeevech P.

Thirty-four endophytic fungal isolates were obtained from the leaves of the medicinal plant Polyscias fruticosa, and their antagonistic activities against the growth of the common tomatoes plant pathogenic fungus Athelia rolfsii were initially screened using a dual culture assay. The endophytic fungus MFLUCC 17-0313, which was later molecularly identified as Diatrype palmicola, displayed the highest inhibition percentage (49.98%) in comparison to the others. This fungus was then chosen for further evaluation. Its culture broth and mycelia from a 10 L scale were separated and extracted using ethyl acetate, methanol, and hexane. Each extract was tested for antifungal activity against the same pathogen using a disc diffusion assay. Only the crude hexane extract of fungal mycelium showed antifungal activity. The hexane extract was fractioned using sephadex gel filtration chromatography and each fraction was tested for antifungal activity until the one with the highest inhibition percentage was obtained. The bioactive compound was identified as 8-methoxynaphthalen-1-ol using nuclear magnetic resonance spectroscopy and mass spectrometry. The minimum inhibition concentration of 8-methoxynaphthalen-1-ol was demonstrated at 250 µg/mL against the selected pathogen. Using the leaf assay, the solution of 8-methoxynapthalen-1-ol was tested for phytotoxic activity against A. rolfsii and was found to have no phytotoxic effects. These results showed that 8-methoxynaphthalen-1-ol has the potential for controlling the growth of A. rolfsii, the cause of Southern blight disease on tomatoes. This study may provide the foundation for future use of this compound as a biofungicide.

Xie Y., Li L., Chen Y., Yang Y., Xu H., Wang Z., Yang L.

Introduction Agarwood is a highly valuable fragrant resinous wood which is widely used as traditional Chinese medicines, perfumes, incense and decorations. Due to its high economic value and excessive demand, this leads to a rising price and proliferation of fake commodities. Thus, strict authenticity identification and quality evaluation of agarwood are of great significance. Objective To establish a simple, rapid and non-destructive technique for identifying the authenticity of agarwood. Methods Liquid extraction surface analysis mass spectrometry (LESA-MS) was firstly proposed to identify the authenticity of 62 agarwood samples without sample preparation. In addition, multivariate statistical models and thin-layer chromatography (TLC) method were used to analyse and verify the results of LESA-MS. Results Representative compounds of agarwood were detected by LESA-MS. A characteristic 2-(2-phenylethyl)chromone compound (m/z 319.1) was treated as a key chemical marker to identify agarwood and its counterfeits rapidly. Several other chromones ions were identified and used as additional evidence for authentic samples. A total of 62 samples were visually discriminated as two groups by principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA), and the specific characteristic marker was highlighted. Moreover, the qualitative results of the conventional TLC method were in agreement with the LESA-MS approach. Conclusion The proposed LESA-MS method was successfully applied in the direct qualitative analysis of agarwood from different sources. This study indicated great feasibility and practicality of LESA-MS in the rapid identification of agarwood, and provided a non-destructive and meaningful preliminary screening tool for the agarwood industry.

Faizal A., Azar A.W., Turjaman M., Esyanti R.R.

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.

Sun Y., Zhang H., Li Z., Yu W., Zhao Z., Wang K., Zhang M., Wang J.

Agarwood, a species of resinous heartwood, is a precious medicinal plant and a type of rare natural spice, which is widely used in medicine, cosmetics, religious activities, and other fields. In this study, agarwood samples from eight different regions across four countries were analyzed by comprehensive two-dimensional gas chromatography-quadrupole time-of-flight mass spectrometry. A total of 232 species were identified (the match factors of these compounds were above 750). The main compounds of agarwood are oxygenated sesquiterpenes and chromones. The compositions of India1 and Malaysia2 were significantly different from those of other samples, which might be attributed to the different production processes of agarwood. For further investigation, factor analysis was conducted for six agarwood samples. The results showed that the data classification possessed a regional characteristic; according to the retention time and relative content, characteristic compositions were determined by factor scores. Finally, the differences of characteristic compositions were simply analyzed, and the reasons were speculated.

Chen X., Yang Y., Liu Y., Sui C., Wei J.

Yan H., Zhang J., Tan Z., Chen Y., Li C., Li S., Liu H., Zhang W.

AbstractTwo undescribed letendrones A−B (1–2), along with three known compounds, ZL‐6 (3), dankasterone B (4), and minimoidione B (5) were isolated from the Aquilaria‐derived fungus Letendraea helminthicola A820. The structures of 1 and 2 were established by analysis of spectroscopes including 1D/2D NMR, IR, UV, and HRESIMS. Among them, the configuration of 1 was further confirmed by single‐crystal X‐ray diffraction. Letendrones A and B were the new phenalenyl derivatives with radical form that were firstly found in nature. In addition, bioactivity of these compounds was evaluated and compounds 3–5 exhibited inhibitory activity against LPS‐induced NO production in macrophages with IC50 values of 4.64, 13.90, and 34.07 μM. Furthermore, potential targets of the new compounds were analyzed by molecular docking in silico. As a result, compound 1 showed high binding with predicted 5‐HT2c receptor (▵G=−8.2 kcal/mol) potentially associated with depression disease, and compound 2 showed significant connection with phosphodiesterase 3 A (▵G=−9.4 kcal/mol) probably against cardiovascular disorders. Our findings firstly reported the high symmetry phenalenyl compounds from natural products which would provide a basis for further development and utilization of the secondary metabolites from the endophytic fungus Letendraea helminthicola A820.

Fu C., Huang B., Wang S., Song Y., Metok D., Tan Y., Fan T., Fernie A.R., Zargar M., Wang Y., Chen M., Yu L., Zhu F.

AbstractAquilaria sinensis is a significant resin-producing plant worldwide that is crucial for agarwood production. Agarwood has different qualities depending on the method with which it is formed, and the microbial community structures that are present during these methods are also diverse. Furthermore, the microbial communities of plants play crucial roles in determining their health and productivity. While previous studies have investigated the impact of microorganisms on agarwood formation, they lack comprehensiveness, particularly regarding the properties of the microbial community throughout the entire process from seedling to adult to incense formation. We collected roots, stems, leaves, flowers, fruits and other tissues from seedlings, healthy plants and agarwood-producing plants to address this gap and assess the dominant bacterial species in the microbial community structures of A. sinensis at different growth stages and their impacts on growth and agarwood formation. The bacteria and fungi in these tissues were classified and counted from different perspectives. The samples were sequenced using the Illumina sequencing platform, and sequence analyses and species annotations were performed using a range of bioinformatics tools to assess the plant community compositions. An additional comparison of the samples was conducted using diversity analyses to assess their differences. This research revealed that Listeria, Kurtzmanomyces, Ascotaiwania, Acinetobacter, Sphingobium, Fonsecaea, Acrocalymma, Allorhizobium, Bacillus, Pseudomonas, Peethambara, and Debaryomyces are potentially associated with the formation of agarwood. Overall, the data provided in this article help us understand the important roles played by bacteria and fungi in the growth and agarwood formation process of A. sinensis, will support the theoretical basis for the large-scale cultivation of A. sinensis, and provide a basis for further research on microbial community applications in agarwood production and beyond.

Zhang Q., Li R., Lin Y., Zhao W., Lin Q., Ouyang L., Pang S., Zeng H.

Xylem-associated fungus can secrete many secondary metabolites to help Aquilaria trees resist various stresses and play a crucial role in facilitating agarwood formation. However, the dynamics of endophytic fungi in Aquilaria sinensis xylem after artificial induction have not been fully elaborated. Endophytic fungi communities and xylem physio-biochemical properties were examined before and after induction with an inorganic salt solution, including four different times (pre-induction (0M), the third (3M), sixth (6M) and ninth (9M) month after induction treatment). The relationships between fungal diversity and physio-biochemical indices were evaluated. The results showed that superoxide dismutase (SOD) and peroxidase (POD) activities, malondialdehyde (MDA) and soluble sugar content first increased and then decreased with induction time, while starch was heavily consumed after induction treatment. Endophytic fungal diversity was significantly lower after induction treatment than before, but the species richness was promoted. Fungal β-diversity was also clustered into four groups according to different times. Core species shifted from rare to dominant taxa with induction time, and growing species interactions in the network indicate a gradual complication of fungal community structure. Endophytic fungi diversity and potential functions were closely related to physicochemical indices that had less effect on the relative abundance of the dominant species. These findings help assess the regulatory mechanisms of microorganisms that expedite agarwood formation after artificial induction.

WANG H., DING X., ZENG J., ZHU J., DONG W., CHEN H., HUANG S., LI W., MEI W., DAI H.

沉香是瑞香科(Thymelaeaceae)沉香属(Aquilaria)或拟沉香属(Gyrinops)植物在受到自然或人为伤害后产生的含有树脂的木材。作为名贵的香料和传统药材,沉香有抗炎、镇静安神等多种药理活性,其主要化学成分为倍半萜类和2-(2-苯乙基)色酮类化合物。白木香是我国特有的沉香基原植物,随着国内大力发展白木香种植和人工结香技术,人工沉香逐渐取代野生沉香成为国内主要的沉香原材料来源。近年来,"结香早、易结香、品质好"的白木香新品种的选育及应用,使沉香产业发展中的"品种单一、人工结香品质差异大,效益周期长"等制约因素得到极大的改善。2020年,白木香染色体水平基因组精细图谱的发布,为白木香结香机制、活性成分合成基因挖掘等研究奠定了基础。本文对沉香基原植物种类,种质资源鉴定、评价及新品种选育,结香机理和人工结香技术,活性成分的生物合成与调控机制的研究进展进行了总结,并对今后研究趋势进行了展望,以期为深入开展沉香研究、保障产业的可持续发展提供理论参考与技术支撑。

Naziz P.S., Das R., Sen S.

Agarwood oil is one of the costliest essential oils used in perfumery, medicine and aroma. Production of the oil traditionally involves a soaking/fermentation step. Studies have indicated a definite role of the diverse microorganisms growing during the open soaking step, and in the emergent aroma of the essential oil. However, the temporal nature of fermentation and a key functional aspect i.e., the enzymatic properties of the microbes from the fermentation basin have not been studied yet. A total of 20 bacteria and 14 fungi isolated from fermentation basins located in Assam, India, at different soaking periods classified as early (0–20 days), medium (20–40 days) and late (40–60 days) clearly pointed towards an early fungal domination followed by succession of bacteria. The physico-chemical transformations of the wood are controlled by enzymatic properties (cellulase, xylanase, amylase and lipase) of the isolates. The results indicated a strong lignocellulosic substrate modulation potential in the four isolates, viz- Purpureocillium lilacinum (0.354 mg/mL), Mucor circinelloides (0.331 mg/mL), Penicillium citrinum (0.324 mg/mL) and Bacillus megaterium (0.152 mg/mL). The highest culturable abundance (CFU/mL) was found in M. circinelloides (2 × 109) among fungi and B. megaterium (4.5 × 109) among bacteria. The highest cellulase activity was shown by P. lilacinum (0.354 mg/mL) while xylanase and lipase by M. circinelloides (0.873 and 0.128 mg/mL). An interesting revelation was that a substantial proportion of the isolates (70% bacteria and 78% fungi) were positive for lipase activity. This is the first report on the “culturable microbiome” of the agarwood fermentation basin from a temporal and functional bioactivity perspective.

Xie Z., Fan S., Xu J., Xiao H., Yang J., Guo M., Cheng C.

Abstract In the realm of Aquilaria classification and grading, a persistent market uncertainty persists, questioning whether the basis should be geographical distribution or biological origin. In this study, our goal is to shed light on the origin and diversification of the Aquilaria genera. We collected and analyzed a total of 320 specimens belonging to 25 species within the Aquilaria genera. Noteworthy findings include observed leaf similarities among three distinct species—A. yunnanensis, A. sinensis, and A. beccariana. Further results emphasize the effectiveness of matK molecular markers in differentiating Aquilaria species, leveraging 8 stable polymorphic loci. The integration of matK and trnL-trnF not only validates this efficacy but also streamlines the systematic categorization of 34 agarwood products. Delving into the evolutionary status and genetic background of Aquilaria, this study employs molecular clock analyses, revealing four distinct pedigrees: the Chinese pedigree 1 and 2, the Indonesian pedigree, and the Indochina pedigree, aligning with A. sinensis, A. malaccensis, and A. cumingiana, respectively. Notably, we identified A. sinensis and A. malacca as part of the youngest branch of Aquilaria evolution, with A. hirta emerging as the oldest member dating back to 6.78 million years ago. Furthermore, the research challenges previous assumptions by reevaluating G. walla, placing it at 5.75 million years ago, and suggesting its incorporation within the Aquilaria genus rather than considering it as an early divergent species.

Li X., Fang X., Cui Z., Hong Z., Liu X., Li G., Hu H., Xu D.

Recently, some new Qi-Nan clones of Aquilaria sinensis (Lour.) Spreng which intensively produces high-quality agarwood have been identified and propagated through grafting techniques. Previous studies have primarily focused on ordinary A. sinensis and the differences in composition when compared to Qi-Nan and ordinary A. sinensis. There are few studies on the formation mechanism of Qi-Nan agarwood and the dynamic changes in components and endophytic fungi during the induction process. In this paper, the characteristics, chemical composition, and changes in endophytic fungi of Qi-Nan agarwood induced after 1 year, 2 years, and 3 years were studied, and Qi-Nan white wood was used as the control. The results showed that the yield of Qi-Nan agarwood continued to increase with the induction time over a period of 3 years, while the content of alcohol extract from Qi-Nan agarwood reached its peak at two years. During the formation of agarwood, starch and soluble sugars in xylem rays and interxylary phloem are consumed and reduced. Most of the oily substances in agarwood were filled in xylem ray cells and interxylary phloem, and a small amount was filled in xylem vessels. The main components of Qi-Nan agarwood are also chromones and sesquiterpenes. With an increasing induction time, the content of sesquiterpenes increased, while the content of chromones decreased. The most abundant chromones in Qi-Nan agarwood were 2-(2-Phenethyl) chromone, 2-[2-(3-Methoxy-4-hydroxyphenyl) ethyl] chromone, and2-[2-(4-Methoxyphenyl) ethyl] chromone. Significant differences were observed in the species of the endophytic fungi found in Qi-Nan agarwood at different induction times. A total of 4 phyla, 73 orders, and 448 genera were found in Qi-Nan agarwood dominated by Ascomycota and Basidiomycota. Different induction times had a significant effect on the diversity of the endophytic fungal community in Qi-Nan. After the induction of agarwood formation, the diversity of Qi-Nan endophytic fungi decreased. Correlation analysis showed that there was a significant positive correlation between endophytic fungi and the yield, alcohol extract content, sesquiterpene content, and chromone content of Qi-Nan agarwood, which indicated that endophytic fungi play a role in promoting the formation of Qi-Nan agarwood. Qi-Nan agarwood produced at different induction times exhibited strong antioxidant capacity. DPPH free radical scavenging activity and reactive oxygen species clearance activity were significantly positively correlated with the content of sesquiterpenes and chromones in Qi-Nan agarwood.

Zhang Y., Wu S., Zhang B., Zhou X., Zhou W., Zhang W., Gao X., Chen X.

Fang X., Li X., Zhang Q., Hu H., Hong Z., Liu X., Cui Z., Xu D.

In recent years, some new Qi-Nan clones of Aquilaria sinensis, which have the characteristics of easily induced and high-quality agarwood, have been selected and propagated by grafting seedlings. To date, research on the grafting of Qi-Nan seedlings is limited. In this study, the effects of grafting on physiological characteristics and endophytic fungi of seedlings within a short period after grafting were studied by means of histochemical observations, physiological index determinations and Illumina sequencing. The results showed that the grafting healing time of ordinary A. sinensis was at least 10 days shorter than that of Qi-Nan, according to the degree of lignification of callus, and the tissue basis for the healing was the interxylary phloem. The MDA content of Qi-Nan peaked earlier than that of ordinary A. sinensis. The contents of JA and ABA peaked earlier in ordinary A. sinensis, and the SA content was significantly higher than that of Qi-Nan at 20–40 days; on average, it was 17.33% higher than that of Qi-Nan. A total of 5 phyla, 28 classes, 84 orders, 197 families, 489 genera and 842 species of endophytic fungi were identified before and after grafting. After grafting, the abundance and diversity of endophytic fungi in ordinary A. sinensis and Qi-Nan decreased, and the endophytic fungi were significantly different before and after grafting. In ordinary A. sinensis, before grafting, Dothideomycetes was the dominant class, and after grafting, Sordariomycetes was the dominant class. The dominant class of Qi-Nan before and after grafting was Dothideomycetes. The dominant genus of ordinary A. sinensis before grafting was Devriesia, and after grafting, it was Fusarium; that of Qi-Nan before grafting was Hyweljonesia and after grafting was Arthopyrenia. Functional genes of the endophytic fungi were mostly related to carbohydrate metabolism and energy metabolism, which may be the reason why endophytic fungi promote agarwood formation in Aquilaria trees. The findings suggest that the healing of the grafting interface in A. sinensis seedlings was the result of multiple factors, and endophytic fungi of Qi-Nan scion changed after grafting.