Elicitation of Secondary Metabolites in Aquilaria malaccensis Lam. Callus Culture by Crude Mycelial Extract of Fusarium solani and Methyl Jasmonate

Abstract

Agarwood is a resinous wood of great economic value produced by trees from the Thymelaeaceae family in response to stress. The natural formation of agarwood can take decades after exposure to the stressors. Artificial agarwood induction by inoculating the stem with fungi has been successfully demonstrated, but resin accumulation occurs very slowly. Cell suspension and callus cultures may serve as an alternative solution to provide a fast-growing plant material to produce artificial agarwood in a short period. Here, we induced agarwood formation in callus cultures of Aquilaria malaccensis by application of crude mycelial extracts of Fusarium solani strains GSL1 or GSL2, or methyl jasmonate (MeJA). After 20 days of treatment with elicitors, all treated calluses had less dry weight than the control group. The gas chromatography–mass spectrometry analysis identified 33 different secondary metabolites among all samples, four of which were present in all treatments and control, i.e., 1-docosene and 1-octadecene (alkenes), 4-di-tert-buthylphenol (phenolic), and benzenepropanoic acid (fatty acid). The 6-methoxy-2-(4-methoxyphenethyl)-4H-chromene-4-one, a chromone derivative, was only detected in callus elicited with the F. solani strain GSL2 and MeJA. All treated calli produced more fatty acid derivatives than the control group. We conclude that elicitors used in this study can induce the production of agarwood-related chemicals such as chromone and fatty acid in callus culture.

1. Introduction

Agarwood is a resinous wood accumulating in agarwood-producing trees. Resin is a common ingredient in medicines, cosmetics, and perfumes, and is mainly used as an essential oil. Traditionally, agarwood is widely utilized in various religious ceremonies. Nowadays, many modern fragrance industries include agarwood oil as their ingredients. Due to its broad range of users, agarwood is now classified as a high-demand non-timber forest product, and its demand keeps increasing. However, agarwood supply remains limited because it takes decades to produce the resin naturally [1]. Therefore, studies on agarwood formation on resinous wood, both naturally and artificially, are gaining more attention.

In Indonesia, the Aquilaria genus is one of the most abundant agarwood-producing trees. The Aquilaria beccariana Tiegh., Aquilaria cumingiana (Decne.) Ridl., Aquilaria filaria (Oken) Merr., Aquilaria hirta Ridl., Aquilaria malaccensis Lam., and Aquilaria macrocarpa are among the most common species also found in Indonesia [1,2]. These species are generally found in Kalimantan, Sumatra, and occasionally in Papua and Maluku regions. However, the natural formation of agarwood resins from these trees cannot supply the high demand for essential oil production leading to over-harvesting of the agarwood-producing tree. Consequently, Aquilaria species has now been listed in Appendix II by the Convention of International Trade in Endangered Species of Wild Fauna and Flora as potentially threatened with extinction (http://checklist.cites.org, accessed on 2 July 2022).

Various methods have been attempted to induce and simultaneously decrease the duration of agarwood formation artificially. Most studies relied on artificially infecting the tree with fungi inoculation [3,4,5]. Several fungal species have been demonstrated to trigger the formation of agarwood in Aquilaria successfully, i.e., Aspergillus niger [6], Fusarium solani [5,6,7], Lasiodiplodia sp. [8], and Rigidoporus vinctus [9]. Notably, these fungi were also reported to be efficient in inducing agarwood formation in other agarwood-producing genera, namely Gyrinops, indicating its potential use in multiple agarwood-producing species [7]. Induction of agarwood formation by fungi is promising, but resin accumulation still takes years. An alternative induction method is highly desirable given the high demand for agarwood and the need to protect agarwood trees, and in vitro culture may be a promising platform for producing the secondary metabolite relatively faster.

Several reviews have compiled the agarwood chemical constituents identified from various Aquilaria species revealing the complexity of the chemical constituent of the resin [10,11,12]. All authors agreed that sesquiterpenes and 2-(2-phenylethyl)-4H-chromen-4-one (chromones) derivatives are agarwood’s predominant constituents. With more than 60 sesquiterpenes and 50 chromones compounds, the most notable derivatives are agarofuran (α-agarofuran, β-agarofuran), eudesmanes (selina-3,11-dien-9-ol, epi-γ-eudesmol), agarol, agarospirol, α-guaiene, chromones (2-(2-phenylethyl)-chromones, and the highly oxidized 5,6,7,8-tetrahydro-2-(2-phenylethyl)-chromones). Some fatty acids (e.g., dodecanoic, tetradecanoic, pentadecanoic, and hexadecenoic) and phenol are detected depending on extraction method [10]. Because agarwood is a product of plant response to environmental stress, the composition of chemical constituents often varies among species or geographical origin. Quality assessment of the resin has been evolving from traditional qualitative of physical appearance and aroma sensation to modern quantitative assessment using electronic nose (E-nose) measurement and GC-MS analysis [13]. Nevertheless, a standard grading system recognized across the agarwood sourcing regions is still unavailable [14]. Consequently, there are a few terms to describe the oil quality used by the sourcing countries; for example, Japan qualified high quality agarwood (Kanankoh) by the presence of α-guaiene in the resin. Meanwhile, other countries such as Malaysia, Vietnam, and Papua New Guinea use the ABC system, where grading is determined by resin coverage and colour, wood density (sinking in water), and burn aroma. Scientific approaches using GC-MS, HPLC, electronic nose, and x-ray is gaining more attention but is still underutilized [14].

Some secondary metabolites commonly found in agarwood resin were reported to be produced in the calli and cell suspension following elicitation with biological or chemical agents [15,16,17]. In an experiment using Aquilaria, the sesquiterpenes α-guaiene, α-humulene and δ-guaiene, and chromone derivatives phenylethylchromones were detected in native calli culture [15]. Treatment with salicylic acid (SA) and methyl jasmonate (MeJA) induced sesquiterpenes α-guaiene, α-humulene, and δ-guaiene as early as 2 weeks post treatment. Meanwhile, the production of chromones derivative that starts quite late coincides with increased cell death. The author suggests that cell death may contribute to the production of phenylethylchromones via oxydoagarochromones (OACs). In Gyrinops walla, both calli and cell suspensions were subjected to agarwood production using chemical and biological elicitors [16]. They produced four sequiterpenoids, i.e., α-cadinol, β-caryophyllene, α-guaiene, and γ-selinene. Adding salicylic acid and methyl jasmonate to calli and cell suspension medium yielded higher percentages of α-cadinol and β-caryophyllene. Meanwhile, calli treatment with sterilized fungal homogenate (carbohydrate equivalents) induced α-cadinol, β-caryophyllene, and α-guaiene. These studies showed the potential of establishing a robust system for sustainable agarwood production via cell culture.

Methyl jasmonate at a concentration of 100 µM has been reported to induce α-guaiene, α-humulene, and δ-guaiene in calli culture of A. crasna and A. sinensis [15]. Crude extract of Trichoderma as a biological elicitor at a concentration of 8 mg/L has been reported in studies using A. malaccensis calli to successfully induce 8-epi-γ-eudesmol, α-guaiene, and alloaromadendrene oxide-1 [18]. Our previous work using the same concentration demonstrates induction of sesquiterpene (3.5-di-tert-butyl-4-hydroxybenzaldehyde) and chromone (4H-1-benzopyran-4-one) when elicited with MeJA and predominantly induced fatty acid derivatives, aromatic compounds, and alkanes when elicited with fungal crude extract [17]. In the present study, A. malaccensis calli were used to investigate eliciting effect of 100 µM MeJA and 8 mg/L F. solani crude mycelial extract on agarwood constituents’ production. This study expects to provide preliminary information for agarwood production using plant tissue culture system.

2. Materials and Methods

2.1. Generation of Callus

Callus of A. malaccensis was produced following induction method described by Faizal et al. [17] and Estiyanti et al. [19] with some modifications. Leaf explants of A. malaccensis were incubated on Murashige and Skoog (MS) basal salt medium [20] supplemented with 2 mg/L of 2,4-dichlorophenoxyacetic acid and 0.1 mg/L of 6-benzylaminopurine. Successfully initiated callus was maintained by regular subculturing every 4 weeks into a new callus-inducing media.

2.2. Preparation of MeJA Solution and Crude Mycelial Extract of F. solani

Methyl jasmonate solution was used as a chemical elicitor and prepared in 96% ethanol, followed by sterilization with a 0.2 µm syringe filter. Two F. solani strains used in this study, Gorontalo (GSL1) and Jambi (GSL2), were generously provided by the National Institute for Research and Innovation. Prior to the experiment, F. solani was activated in Potato Dextrose Agar (PDA) medium and propagated in Potato Dextrose Broth (PDB) medium for 3 weeks [7]. Crude mycelial extracts of F. solani were prepared as previously described by Faizal et al. [17]. Mycelia were harvested and filtered through Whatman filter paper (No. 1). The filtrate was washed twice with sterile distilled water, freeze-dried, and ground to a powder using mortar. Mycelia extract powder was dissolved in sterile distilled water as an elicitor.

2.3. Callus Treatment

Approximately 3 g of callus were transferred into MS basal salt medium containing 100 µM MeJA or 8 mg/L crude extracts of F. solani. The callus was incubated in the media containing elicitor for 20 days at room temperature in the dark [21].

2.4. Estimation of Growth Rate and Doubling Time of Callus

Every 5 days, callus was harvested and dried at 50 °C for 24 h before being weighed. The growth rate [22] was then estimated using the following formula:

$$Specificgrowthrate=\frac{\ln x-\ln x_0}{t}$$

in which x₀ is the initial dry weight, and x is the dry weight after a particular time (t).

The specific growth rate value was then used to calculate the doubling time [22] using the following equation:

$$Doublingtime:\frac{\ln 2}{growthrate}$$

2.5. Measurement of Cell Viability

Callus was immersed in 0.04% (w/v) of 2,3,5-triphenyl tetrazolium chloride (TTC) for 18–22 h at room temperature in the dark, followed by rinsing with distilled water and sonication in 3 mL 95% ethanol for 1 h. The relative cell viability was determined by measuring the absorbance of cell-free ethanol solution at 480 nm with a spectrophotometer [21].

2.6. Sample Extraction and Gas Chromatography–Mass Spectrometry Analysis

A 0.1 g callus from each treatment was freeze dried, ground into a powder using mortar, and transferred into an Erlenmeyer flask filled with ethyl acetate. The extraction process was done by leaving the tissue homogenate on a rotary shaker for 3 days [17]. The extracts were then centrifuged at 100× g for 10 min at room temperature. Approximately 10 mL of supernatant was transferred to a new Erlenmeyer flask, allowed to air dry for 7 days, and then redissolved in 1 mL ethyl acetate. The GC-MS analysis was done using an Agilent 6890 GC, model number Agilent 19091S-433 with an HP-5MS column and a 0.25 mm × 30 m × 0.25 um capillary. Identification of the chemical components was based on the comparison of the calculation of their retention time and authentic mass spectral data with the existing mass spectral libraries (NIST 20 and Wiley libraries). The detected compounds were then subjected to metabolomics data analysis using the R program and displayed as a heatmap.

3. Results

For simplification, the term GSL1-treated and GSL2-treated refer to calluses treated with elicitors derived from mycelial of F. solani strain GSL1 and GSL2, respectively.

3.1. The Growth Rate of Callus Treated with Elicitors or MeJA Was Less Than That of Untreated Callus

At the end of the experiment (day 20), all calluses had a similar brownish appearance (Figure 1). Furthermore, no significant differences (p-value > 0.05) were found in either callus dry weight, average growth rate, or doubling time (Figure 2 and

Table 1. Average relative growth rate and doubling time of the A. malaccensis callus treated with F. solani strain GSL 1 or GSL2 mycelium, or MeJA, or control untreated callus. No significant differences were observed between treatments (p-value > 0.05, n = 3).

Treatment	Specific Growth Rate (g/Day)	Doubling Time (Day)
GSL1	0.15 ± 0.047	4.98 ± 1.625
GSL2	0.16 ± 0.048	4.56 ± 1.426
MeJA	0.16 ± 0.040	4.68 ± 1.308
Control	0.17 ± 0.044	4.31 ± 1.125

). Nevertheless, subtle differences were observed. Figure 2 showed that treated samples had a lower dry weight than the control group 20 days post treatment. Exposure to elicitors appeared to affect both callus average growth rate and doubling time, showing control having the shortest growth rate while GSL1-treated group was the longest.

3.2. The Viability of A. malaccensis Callus Cells in Culture Decreased after Treatment with Elicitors

The fraction of viable callus cells was analyzed every 5 days for 20 days and presented as relative viability compared to callus cells on day 0 (

Table 2. Relative fraction of viable A. malaccensis callus cells treated with elicitors derived from F. solani strain GSL1 or GSL2 mycelium, or MeJA on day 5, 10, 15, and 20 compared to callus cells on day 0. No significant differences were observed between treatments (p-value > 0.05, n = 3).

Days Post Treatment	Treatment
	GSL1	GSL2	MeJA	Control
5	0.59 ± 0.131	0.80 ± 0.069	0.59 ± 0.051	0.81 ± 0.108
10	0.69 ± 0.090	0.63 ± 0.092	0.51 ± 0.038	0.95 ± 0.178
15	0.83 ± 0.059	0.86 ± 0.044	0.72 ± 0.054	1.05 ± 0.185
20	0.92 ± 0.075	0.99 ± 0.298	0.75 ± 0.088	1.16 ± 0.366

). The cell viability varied between treatments, with fewer viable callus cells detected in MeJA-treated group than mycelial extract-treated or control groups. In the control and GSL1-treated group, cell viability decreased on day 5 and then steadily increased until day 20. On the contrary, cell viability of MeJA-treated and GSL2-treated calluses continue to decrease until day 10 before increasing on day 15 and day 20.

3.3. Chromone Derivatives Were Detected in Samples Treated with Methyl Jasmonate, and the Elicitors Derived from F. solani Strain GSL2

GC–MS analysis of ethyl acetate crude extract detected 33 compounds from callus harvested on day 20 post treatment with elicitors (Supplementary Table S1). Figure 3 shows the variety of compounds detected from each treatment group, i.e., alkenes, fatty acids, sterols, amines, terpenes, phenolics, and chromones, and their abundance in each treatment group. Out of the 33 compounds detected, only four were commonly found in all samples. These four compounds are 1-docosene and 1-octadecene (alkenes), 4-di-tert-buthylphenol (phenolic), and benzenepropanoic acid (fatty acid). The remaining 29 compounds were unique to one or two treatment groups or exclusively belonged to the control group. The fatty acid derivatives are the most abundant metabolite compounds detected in all treatment groups, but more were detected in treated calli.

Sesquiterpenes and chromones are the main agarwood chemical constituents [10]. While this study did not detect the former, the latter was the most prevalent agarwood chemical constituent detected in the treated A. mallacensis callus. The chromone derivative, 6-methoxy-2-(4-methoxyphenethyl)-4H-chromene-4-one, was primarily detected in MeJA-treated and GSL2-treated calluses. Another metabolite detected in GSL2-treated callus was squalene, a triterpene, which was also detected in the control group. On the other hand, a high level of (22E)-stigmasta-5,22-dien-3-ol was present only in callus treated with MeJA. Meanwhile, sterol derivative compounds, stigmast-5-en-3-ol and gamma-sitosterol, were exclusively found in the control group.

4. Discussion

It is well established that secondary metabolites of agarwood resin can be induced by treating agarwood-forming plant tissue culture with elicitors. MeJA or fungal crude extract are the most used, although often at the expense of slower growth rate and reduced cell viability. For example, a study in A. sinensis callus treated with Lasiodiplodia theobromae reported a reduction in growth rate [23], while cell suspension treated with Melanotus flavolivens showed a decrease in biomass [24]. Another study using A. malaccensis callus elicited with mycelial extracts from Lasiodiplodia sp. observed a slower growth rate of treated callus than control [18]. A similar observation was also evidenced in this study; the presence of elicitor in callus media resulted in a slower growth rate than callus, albeit statistically insignificant. MeJA treatment was also reported to inhibit callus and cell suspension growth in Aquilaria [15]. Our experiment showed a decrease in viable cells in callus treated with MeJA compared to other groups. A similar study using A. sinensis callus and A. crasna cell suspension elicited with 100 µM MeJA also showed a reduction of cell viability compared to control, indicating higher susceptibility to cell damage when exposed to elicitor [15]. Further, Okudera & Ito [15] indicate that elicitors may be perceived as stress signals, which in turn trigger the formation of secondary metabolites or cell death as part of plants defense mechanism.

The growth rates shown in Figure 1 depicted that control callus was still in logarithmic phase at the end of the experiment. In logarithmic phase, carbon is allocated mainly for primary metabolisms, such as cell structure formation and respiration. Meanwhile, the production of secondary metabolites is generally observed in cells at stationary phase [25]. The decrease in growth rate and cell viability of treated calluses may indicate a metabolism switch to the production of secondary metabolites or occurrence of cell death. Hypersensitive response (HR) of callus cells in the presence of elicitors may explain the decrease in cell viability [15]. HR is a form of plant defense response against pathogens involving reactive oxygen species (ROS) formation, leading to cell death/ non-viable cells [26]. Dead cells produce signals that are recognized by living cells, known as damage associated molecular patterns (DAMPs), and subsequently activate defense response in living cells [27]. Production of secondary metabolites is considered a form of defense response that DAMPs can trigger, therefore, may explain the concurrence of a decrease in cell viability and an increase in the production of secondary metabolites [28].

When working with actively growing juvenile or tissue culture, it should be considered that the production of secondary metabolites can be varied depending on the growth phase and treatment duration. The ratio of α-guaiene, α-humulene, and δ-guaiene content in 2-week-old A. sinensis suspension cells reportedly varied between 36 h and 7 days post induction with MeJA treatment. The α-humulene was dominant at 36 h, while α-guaiene and δ-guaiene were more pronounced after 7 days of treatment [29]. Another study using 5 days old A. sinensis suspension cells reported increased accumulation of α-guaiene, α-humulene, and δ-guaiene in the first 12 h and subsequently decreasing at 24 h mark [15]. Sesquiterpene derivative, 3.5-di-tert-butyl-4-hydroxybenzaldehyde, was detected 5 weeks post exposure to MeJA in an experiment using a more mature explant, such as shoot culture [17].

Overall, the GC-MS results agree with elicitors role in inducing secondary metabolite formation in agarwood-producing callus cells. Compared to control, more secondary metabolites compounds, such as chromones derivatives, were detected in the treated group. In this study, 6-methoxy-2-(4-methoxyphenethyl)-4H-chromene-4-one was present in calli elicited with MeJA and crude mycelium extract from F. solani strain GSL2. However, they remained undetected in callus elicited with mycelium extract of F. solani strain GSL1. Further, higher detection of chromone derivatives was observed in callus treated with MeJA than the fungal strain GSL2 and was concomitant with low cell viability in MeJA treated calli. A study monitoring the eliciting effects of crude fungal extracts on chromones production in A. sinensis cell culture reported 6-methoxy-2-(4-methoxyphenethyl)-4H-chromene-4-one as one of the major chromones compounds identified in the treated cell suspension [24]. Okudera & Ito also showed a similar relationship between chromone production and cell viability, noting that cell viability decreased coincided with increased chromone production in callus and cell suspensions of Aquilaria [15]. The authors suggested that the production of chromone was the result of cell wall degradation catalyzed by endogenous enzymes. Other studies demonstrating high chromones formation in elicited tissue culture were done in A. sinensis callus when treated with L. theobromae [23] and 100 µM MeJA [21].

Alkenes and fatty acids derivatives were two known agarwood compounds that were abundant in this study. Hexadecanoic acid and oleic acid are other metabolites that have a role in the plant’s defensive mechanisms and were detected in this study. Hexadecanoic acid was commonly found in treated samples, while oleic acid was detected only in GSL2-treated callus. The accumulation of hexadecanoic acid (palmitic acid) can signal the formation of fatty acids from carbohydrate precursors [30]. Meanwhile, oleic acid is a fatty acid that is thought to be a precursor to the formation of linoleic acid, a precursor of jasmonic compounds [31,32]. Jayaraman and Mohamed reported the presence of antibacterial activity of hexadecanoid in A. malaccensis cell suspension cultures when treated with Trichoderma sp. [18]. Like hexadecanoic acid, oleic acid was also reported to have antimicrobial activity against bacteria and fungi [33]. Faizal et al. reported the presence of oleic acid in wounded A. malaccensis trees and trees infected with Fusarium, suggesting an association exist between wounding and subsequent infection by bacteria or fungi, which leads to the oleic acid formation [34]. Hexadecanoic acid and oleic acid are fatty acid derivates. More than half of the total secondary metabolites in the analyzed samples were fatty acid derivatives and were found to be more abundant in the treated callus. The presence of elicitors possibly induces the formation and oxidation of fatty acids and may explain the high abundance of fatty acid derivatives found in this study and other studies. For instance, an increase in fatty acid production was concomitant with fungal infections [30], while wounding treatment induced accumulation of octadecane in A. sinensis [35]. Acetyl-CoA is a precursor for both fatty acids and phytosterols biosynthesis [36]. GC–MS results showed a tendency of phytosterol accumulation in control callus while fatty acid was in the treated callus. Meanwhile, MeJA-treated callus accumulated both compounds. Based on this data, it can be speculated that in normal conditions, acetyl-CoA is converted into phytosterol. However, acetyl-CoA may be used to form fatty acids in the presence of pathogen signals such as fungal crude extract. In treatment with MeJA, the oxylipin constituent in MeJA solution may serve as an alternative source to form fatty acid when acetyl-CoA is used to form phytosterol.

Other metabolites with antimicrobial properties observed in this study are the hydrocarbons eicosane, docosane, and octadecane. Detection of alkenes in high abundance was reported in agarwood induction studies and commonly found in healthy and treated explants [17,30]. These studies indicate the role of alkane/alkene in plant protection and response to stress. In addition, previous publication also reported that 1-octadecene compound, the highest in calli treated with GSL1 crude extract, has antimicrobial and antioxidant properties [37]. Lastly, phenolic compounds are one of the secondary metabolites that act as antimicrobial compounds, so these compounds are expected to be found in calli treated with elicitors to facilitate defense response [38,39]. Here, benzenepropanoic acid and 2,4-di-ter-butyl-phenol were also detected at higher levels in the treatment group than in the control group. An earlier study in A. malaccensis found an accumulation of benzene propanoic acid in fungal inoculated trees [40]. The secondary metabolites constituents found in this study strongly indicate that treated callus perceived elicitors as a signal of the presence of pathogen and response accordingly to fight the pathogen off through the formation of chromones and other metabolites with antimicrobial properties.

5. Conclusions

The secondary metabolites produced in A. malaccensis callus elicited by crude mycelial extracts of F. solani strain GSL1 or GSL2 or MeJA were more diverse than those produced in control untreated callus. Elicitation showed a negative correlation between secondary metabolite production and cell viability, showing an increase in secondary metabolite production but, at the same time, decreased cell viability 20 days after treatment. Many secondary metabolites detected in the treated callus had some antimicrobial activities, indicating the callus response to fight pathogens. These results suggest a trade between secondary metabolite production and cell growth where the cell growth rate is reduced when secondary metabolites are needed, such as during plant infection with a pathogen. In the context of chemical constituents produced in calli culture, fatty acids and alkenes were the most predominant chemical constituent; only chromes derivatives were detected in treated calli while sesquiterpenoid compounds remain absent. This indicates that although production of agarwood constituents in calli is still possible, further investigation is needed, such as finding alternative elicitors able to induce a more diverse sesquiterpenoid and chromones compounds in calli culture. Another possible use of the callus culture system is to use it as a platform to produce a more targeted agarwood constituent instead of a near-native mixture.