Volatile Constituents of Endophytic Fungi Isolated from Aquilaria sinensis with Descriptions of Two New Species of Nemania

Algae, bacteria, and fungi, as well as higher plants, produce a wide variety of secondary metabolites known as natural products. Natural products are well known as remarkable sources of many therapeutic agents. The genus Nemania is a wood-decaying fungus that belongs to family Xylariaceae. Nemania is often found as an endophyte in diverse hosts and some species are known to produce useful secondary metabolites. In this study, two Nemania species were isolated as an endophytic fungus from Aquilaria sinensis. Multi-gene phylogenetic studies showed that the newly described strains of Nemania are new to science, and this is the first report of Nemania from the host Aquilaria. One of the fermented species, Nemania aquilariae (KUMCC 20-0268), resulted in five sesquiterpenoids, which were previously reported from agarwood, and their structures were identified by gas chromatography-mass spectrometry (GC-MS). In addition, five different media were investigated in vitro to optimize conditions for growing the fungal biomass of Nemania aquilariae and N. yunnanensis.

Heartwood contains resin-impregnated fragrant wood that is extremely valuable and in high demand throughout the world [14,15]. Healthy Aquilaria trees can only produce agarwood after being subjected to damaging events [3,5,[16][17][18]. In natural forests, agarwood formation occurs slowly and infrequently in old trees, and only 7-10% of Aquilaria trees contain agarwood. When compared with market demand, the supply of agarwood from wild sources is severely inadequate. Unfortunately, indiscriminate felling of trees and overharvesting in hopes of finding the treasured resin have led to the severe depletion of wild trees and other negative impacts on biodiversity [19]. As a result, eight Aquilaria species are now listed on the IUCN red list as endangered species [1,20].
Many artificial induction approaches, viz., chisel nails, burning, trunk breaking, and bark removal, for the development of agarwood via traditional methods have been developed but these methods are slow and produce poor-quality agarwood [3,21,22]. Several techniques inducing agarwood production are described in Tan et al. [23]. So far, more than 300 compounds have been isolated and reported from agarwood and Aquilaria trees [24,25]. Fungal inoculum development in Aquilaria trees first began in 1929 [26]. Later, several researchers isolated fungi from naturally occurring Aquilaria trees in the wild (using healthy or diseased parts) to investigate the role of fungi in agarwood formation, finding that most of the isolated fungi were endophytes [22]. Fungi are some of the organisms involved in inducing agarwood formation, and fungal culture for inoculum can be "pure" or "mixed" [22]. For instance, Fusarium laseritum, Lasiodiplodia theobromae, and Menanotus flavolives are able to promote agarwood formation [25][26][27]. Outcomes may vary between fungal strains and across sites when they are applied.
The genus Nemania is an endophytic fungal genus that has been reported on several hosts. Endophytic fungi of Nemania have shown interesting applications associated with its bioactive compounds on many hosts [28][29][30][31][32]. The pattern of Nemania geographic distribution from 1979-2020 is shown in Figure 1 [33]. This genus is distributed mostly in Europe (Denmark, Sweden, and the United Kingdom), Australia, and North America, while only few specimens have been recorded from Asia, Africa, and South America. This clearly shows that Nemania species are more diverse in temperate zones than tropical zones. The aim of this study was to isolate two endophytic fungi from the resin of Aquilaria sinensis collected from Yunnan Province, China. Multi-gene phylogenetic analyses showed two endophytic fungi are new species of Nemania. Besides, one of the species N. aquilariae fungally ferments was investigated for how its volatile organic compounds are formed, which is related to eventually forming agarwood, which was confirmed by GC-MS method. In addition, we optimized the best media for production of fungal biomass yields of the newly described strains' isolates in vitro.

Sample Collection, Fungal Isolation, Preparation of Cultures, and Production of Fungal Biomass
Endophytic fungi species were isolated from dark resinous heartwoods of Aquilaria sinensis collected from Xishuangbanna Dai Autonomous Prefecture (N 21 • 44 38 , E 100 • 21 36 ), Yunnan Province, China. Pieces of agarwood were burned to verify the presence of the agarwood fragrance before being stored in ice boxes and transported to the Kunming Institute Botany laboratory. Samples were cleaned under running tap water to remove dust and then air dried. Samples were cut into 0.5-cm, circular-shaped pieces. The surface of each sample was disinfected by being soaked in 75% ethanol for 1 min, 3% sodium hypochlorite solution for 2 min, and 75% ethanol for 30 s, followed by three rinses in sterile distilled water before finally being dried on sterile tissue papers [34]. All sections were placed in potato dextrose agar plates (PDA, Oxoid, Basingstoke, UK) and incubated at 28 • C for 1-3 days. Hyphal tips of fungal colonies appeared during incubation, so the colonies were transferred to new PDA plates and incubated to obtain pure cultures. New fungal taxa were examined in the pure culture, and photographs, morphological characteristics, and descriptions were completed.
For production of fungal biomass, fresh cultures of Nemania aquilariae and N. yunnanensis were inoculated into the following five liquid broth media (without agar): CzapekDox broth (CDB, oxoid), malt extract broth (MEB, oxoid), potato dextrose broth (PDB, oxoid), Richard broth (RB), and Sabouraud's broth (SB). Broth media (100 mL) were prepared according to the manufacturer's instructions, poured into clean, 150-mL flasks, covered with cotton lids and aluminum foil on the top, and sterilized via autoclaving at 121 • C for 30 minutes. The pure cultures (14 days old) on PDA were cut out near the margin by a 0.5-cm-diameter, sterilized cork borer. Five culture disks were transferred to each media flask (triplicate) under aseptic conditions. The flasks were inoculated at 28 • C on a rotary shaker at a speed of 120 rpm for seven days [35]. After seven days of incubation, mycelial masses were harvested via filtration through Grade 1 Whatman filter paper No. 1 (Madiston, Walton-on-Thames, UK) (initial weight of the filter papers was recorded prior to use), dried at 40-45 • C for 24 h, weighed of biomass, and recorded. Three replicates of biomass in various liquid media were carried out for each treatment, and data are the average of these three assays. Statistical analyses were performed using one-way ANOVA and the Mann-Whitney ranks sum test. Graphs and statistical analyses used Sigmaplot version 12.5 (Systat, San Jose, CA, USA). Analysis of variance of p ≤ 0.05 was used as the threshold for significance. Herbarium specimens were prepared from cultures that were dried in silica gel. The holotypes were deposited in Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China. The ex-type cultures were deposited in the Kunming Institute of Botany culture collection (KMUCC). New taxa were registered in Facesoffungi (FoF) [36] and Index Fungorum [37] 2.2. Genomic DNA Extraction, PCR Amplification, and Sequencing Genomic DNA was isolated from pure fungal cultures using the Biospin Fungus genomic DNA extraction kit-BSC14S1 (Bioflux, Kunming, China). Polymerase chain reaction (PCR) was used to amplify partial gene regions of Internal Transcribed Spacers (ITS), 28S ribosomal RNA (LSU), RNA polymerase II second largest subunit (RPB2), beta-tubulin (BT), and actin (ACT), using primers shown in Table 1. Total volume of PCR mixtures for amplifications was 25 µL [38]. Purification and sequencing of PCR products were performed by TsingKe Biotech, Kunming, Yunnan, China.

Phylogenetic Analyses
Sequence data generated in this study were checked for the quality of chromotagrams, and raw forward and reverse sequences were assembled using Geneious Pro.v4.8.5. Subjected to Basic Local Alignment Search Tool (BLAST) searches in the nucleotide database of GenBank (http://blast.ncbi.nlm.nih.gov, accessed on 16 April 2021) to determine their most probable closely related taxa. Sequence data were retrieved from GenBank based on BLAST searches and recent publications [44]. Sequence alignments were carried out through MAFFT v.6.864b [45] and the alignments were manually improved where necessary. Sequence data sets were combined using BioEdit [46].
Dissyanake et al. [47] were followed for construction of the combined phylogenetic trees using maximum likelihood (ML) and Bayesian Inference posterior probabilities (BYPP). The GTR+I+G model of nucleotide substitution and searches for model selected for ML were applied. Bootstrap supports were obtained by running 1000 pseudo-replicates. Bayesian Inference analysis was conducted when two parallel runs were performed using the default settings in addition to the following adjustment: Six Markov chains were run simultaneously for 5,000,000 generations, trees were sampled every 100th generation, and 20% of trees representing the burn-in phase were discarded. The remaining 80% of trees were used to calculate probability proportional to size (PPs). Bootstrap support values for ML and BYPP were given next to each node in the phylogenetic trees (  (Table 2). Table 2. Names, isolate numbers, and GenBank accession numbers of the fungal taxa used for the phylogenetic analyses of this study.

Species Isolates
GenBank Accession Numbers

Species Isolates
GenBank Accession Numbers   Figure 2. Phylogram generated from RAxML analysis based on combined ACT, ITS, LSU, BT, and RPB2 sequence data. Related sequences were obtained from Dayarathne et al. [44]. Bootstrap support values for ML equal to or greater than 60% and BYPP from MCMC analyses equal to or greater than 0.90 are given above/below the nodes. The ex-type strains are indicated in bold. Newly generated sequences are indicated in red bold.

Headspace Solid-Phase Microextraction (HS-SPME) Conditions
Fungal species determined in this study were recultivated on PDA and transferred to 40-mL headspace vials containing 5 mL of PDA solid medium (triplicate). VOCs were extracted by SPME, while uninoculated PDA headspace vial was set as the blank control. The inocula were incubated in a constant temperature incubator at 28 • C for 14 days for solid-phase microextraction. The silicon cap of the vial was closed and conditioned for 250 • C for 30 min by its insertion into the GC injection port under helium atmosphere. The extraction head was inserted into the headspace bottle and adsorbed for 30 min at room temperature (22 • C).

GC-MS Analysis Conditions for the Analyzation of VOC Emissions
The GC-MS conditions were set as follows: The initial temperature was set at 80 • C for 1 min and programmed to increase the temperature at a rate of 20 • C min −1 to 180 • C and then increase by 4 • C min −1 to 230 • C for 2 min. The inlet temperature was 250 • C, the connection port temperature was 290 • C, and desorption was performed for 5 min. Helium with a purity exceeding 99.999% was used as the carrier gas; the flow rate was set at 1.0 mL min −1 (spitless mode). The condition of mass spectrometry sources was set as connection temperature 280 • C. The ionization mode was electron ionization (EI), and the ionization temperature was 230 • C. The MS, four-stage rod temperature was 150 • C. Analyses were performed by setting the electron energy at 70 eV in full-scan mode (m/z 50-600). All identified components were quantified using NIST (National Institute of Standards and Technology) mass spectral database search and GC/MSD ChemStation data analysis software (Agilent Technologies, Santa Clara, CA, USA) and summarized as a percentage of relative peak area, shown in Table 3.

Phylogenetic Analyses
The data set consisted of 58 strains included in the combined sequence analyses, comprising 5193 characters with gaps (349 bp ACT, 1938 bp BT, 841 bp ITS, 848 bp LSU, 1217 bp RPB2). Barrmaelia rhamnicola strains BR1 and CBS 142772 were used as outgroup taxa. Tree topology of the ML analysis was similar to the BYPP. The best scoring ML tree with a final likelihood value of -54284.745986 was presented. The matrix had 2725 distinct alignment patterns, with 50.86% undetermined characters or gaps. Estimated base frequencies were as follows:

Nemania Gray 1821
Nemania was erected for a heterogeneous assemblage of taxa by Gray [49] that belongs to family Xylariaceae. This genus is known to contain saprobes or endophytes from terrestrial or marine environments worldwide [50,51]. There are 55 records of Nemania in Species Fungorum [52]. Notes: Based on BLASTn searches of ACT, ITS, LSU, BT and RPB2 sequence data, Nemania aquilariae showed a high similarity to N. primolutea (ACT=98.62%(EF025592); ITS=99.65%(MG881830); BT=98.52%(EF025607), and RPB2=95.41%(GQ844767) while LSU showed high similarity to N. beaumontii 98.10% (MF161217). In the multi-gene phylogeny, N. aquilariae clustered sister to N. primolutea with 100% in ML and 1.00 in BYPP statistical support (Figure 2). The newly described strain is an endophytic fungus, which does not sporulate in culture so its morphological characteristics cannot be compared with N. primolutea. Sequence comparison results revealed 1.72% (ACT), 3.10% (RPB2), while other genes <1% base pair differences (without gaps) between N. aquilariae and N. primolutea (YMJ 91102001, holotype). Nemania primolutea has also been found in China (Taiwan region) on dead trunk of Artocarpus communis, which differs from N. chrysoconia and N. flavitextura in having carbonaceous tissue between perithecia and absence of perithecial mounds [53]. Based on significant statistical supports in molecular phylogenetic studies, N. aquilariae is introduced herein as a new species on Aquilaria sinensis from Yunnan Province, China. In addition, MEB, PDB, and RB media are most ideal for culturing fungal biomass, while CDB and SB use led to the lowest level for culturing N. quilariae (Figure 4).  Notes: Nemania yunnanensis well separates from other species in Nemania with moderate statistical support in ML analysis ( Figure 2). Based on BLASTn searches of ACT, ITS, LSU, BT and RPB2 sequence data, Nemania yunnanensis showed a high similarity to N. serpens (ACT = 93.13%(KU684031); ITS = 98.70%(MN844431); BT = 90.84%(KU684188) and RPB2 = 92.60%(GQ844773)), while LSU sequence data showed high similarity to N. beaumontii 98.29%(MF161217). As newly described, the strain is an endophytic fungus and does not sporulate in culture. We were not able to compare morphological characteristics with other species in the genus. Based on phylogenetic analyses, N. yunnanensis is introduced herein as a new species on Aquilaria sinensis from Yunnan Province, China. In addition, the best media to support fungal biomass are RB and MEB media, while CDB, PDB, and SB media led to the lowest level for culturing N. aquilariae (Figure 4).

GC-MS Analyses
Five volatile components were found in Nemania aquilariae, accounting for 85.90% of total volatile components (Table 3 and Figure 4). All the five components' structures were confirmed as sesquiterpenoids ( Figure 5). However, no volatile components were detected in Nemania yunnanensis (data not shown). The dominant components of Nemania aquilariae were reported as compound 4 with 43.75%, compound 1 with 20.52%, and compound 5 with 13.38%, respectively ( Figure 6).   Table 3.

Discussion
Several endophytic fungi are known as potentially bioactive metabolite producers in Aquilaria trees, and this is used in agarwood-producing trees [54][55][56]. In this study, two endophytic fungi belonging to Nemania were isolated from the dark resinous wood of Aquilaria sinensis, collected from Xishuangbanna, and this is the first report of Nemania from the host genus Aquilaria. Multi-gene phylogenetic analyses ( Figure 2) showed that new isolates are new species of Nemania. It was also shown that Nemania species separated into three clades when more genes were included ( Figure 2). So, we suggest protein-coding genes are important for resolving the placement of Nemania. In phylogenetic analyses (Figure 2), new isolates grouped in Nemania Clade III, and most of species in this clade were found on decorticated rotten wood and as endophytic fungi but they are not host specific [29,31,57]. Moreover, Euepixylon sphaeriostomum clusters within Nemania which is in consistent with Dayarathne et al. [44].
In this study, N. aquilariae was able to induce the formation of agarwood in Aquilaria sinensis and was capable of producing certain agarwood compounds, such as guaiane-type (2), eudesmane-type (3 and 5), and eremophilane-type (4), and these types of sesquiterpenoids are related to chemical constituents of agarwood [58]. Thus, N. aquilariae can be used in biological fermentation to produce agarwood-related compounds and can also be used to infect other Aquilaria plants in the production of agarwood. Nemania aquilariae is an endophytic fungus from Aquilaria sinensis and can be used as an alternative source for catalyzing the production of agarwood and its key natural ingredients. Nemania aquilariae was shown investigate the relationship between the chemistry and fungal associates of agarwood formed. The species presented that agarwood formation significantly affects the chemical and fungal constituents of agarwood in A. sinensis. In the present study, we indicated that N. aquilariae was able to produce the volatile compounds closely related to a primary determinant of agarwood properties. Thus, only few fungi are being tested for promoting agarwood formation. This species could further influence agarwood formation by injecting the fungi into the trunk, branches, or punch holes and then to subsequently inject fungi into the Aquilaria tree. Those techniques avoid severe damage to Aquilaria trees and also allow for easy agarwood collection. MEB, PDB, and RB nutrient broths are recommended for the cultivation of N. aquilariae for high yield and good quality of biomass.