Characterization of Composition and Antifungal Properties of Leaf Secondary Metabolites from Thirteen Cultivars of Chrysanthemum morifolium Ramat

Chrysanthemum morifolium Ramat is an ornamental plant of worldwide cultivation. Like many other species in the family Asteraceae, C. morifolium is a rich producer of secondary metabolites. There are two objectives in this study: (I) to determine and compare the diversity of apolar secondary metabolites among different cultivars of C. morifolium and (II) to compare their properties as antifungal agents. To attain these objectives, we selected 13 cultivars of C. morifolium that are commonly used for making chrysanthemum tea as experimental materials. Leaves at the same developmental stage were collected from respective mature plants and subjected to organic extraction. The extracts were analyzed using gas chromatography–mass spectrometry. A total of 37 apolar secondary metabolites including 26 terpenoids were detected from the 13 cultivars. These 13 cultivars can be largely divided into three chemotypes based on chemical principal components analysis. Next, the extracts from the 13 cultivars were examined in in vitro assays for their antifungal properties against three species of pathogenic fungi: Fusarium oxysporum, Magnaporthe oryzae, and Verticillium dahliae. Significant variability in antifungal activity of the leaf extracts among different cultivars was observed. The 13 cultivars can be divided into four groups based on their antifungal activities, which could be partly correlated to the contents of terpenoids. In short, this study reveals large variations in chemical composition, particularly of terpenoids, of leaf secondary metabolites among different cultivars of C. morifolium and their different abilities in functioning as antifungal agents.


Introduction
Plants make diverse phytochemicals with most of them defined as secondary, or specialized, metabolites [1]. Among the best-known classes of plant secondary metabolites are terpenoids, phenylpropanoids, and alkaloids [1,2]. Plant secondary metabolites have many biological and ecological functions, such as for defense against insect herbivores and pathogenic microorganisms and for establishment of mutualistic interactions [3][4][5][6][7]. In addition to their significance in fundamental plant biology and evolution, plant secondary metabolites have played an important role in human civilization. They have been widely used as medicines (e.g., artemisinin), spices (e.g., capsaicin), and agrochemicals (e.g., pyrethrin) [8][9][10]. Despite the wide applications of plant secondary metabolites, the vast diversity, biological functions, and potential applications of secondary metabolites from many plants remain poorly characterized. Chrysanthemum morifolium Ramat is one of them.
Chrysanthemum morifolium, also known as florist's daisy, belongs to the Asteraceae family, which is arguably the largest family in eudicots [11]. Originated in China, several thousands of cultivars of C. morifolium have been developed. Most of the cultivars are used as garden plants or cut flowers. Nevertheless, some cultivars of C. morifolium have been developed for culinary and medical uses due to antioxidant and antimicrobial activities [12,13], especially as herbal teas made from flower heads [14]. In fact, the medicinal use of C. morifolium was noted in the first major work dedicated to Chinese Materia Medica known as Shennong Bencao Jing. Secondary metabolites from some C. morifolium cultivars have been analyzed. Pertaining to this study, essential oils extracted from some C. morifolium cultivars were shown to be dominated by terpenoids [15][16][17][18][19]. Nevertheless, systematic comparisons of composition of secondary metabolites and their biological roles from comparable cultivars are generally lacking.
We have started to systematically characterize the chemical composition, biosynthesis, and biological functions of apolar secondary metabolites in C. morifolium. The aim of the present study was to determine the chemical composition of apolar secondary metabolites from leaves of different cultivars of C. morifolium and to determine whether these compounds have any inhibitory effect on fungal pathogens. Such knowledge may facilitate the breeding of novel cultivars of C. morifolium with enhanced resistance to fungus pathogens and lay the foundation for developing new antifungal agents.

Chemical Composition of Apolar Secondary Metabolites of C. morifolium Leaves
A total of 13 cultivars of C. morifolium (Table 1) commonly used for making chrysanthemum tea were selected for this study. To simplify cultivar references between the main text and figures/tables, these 13 cultivars were coded as Cm1 to Cm13 (Table 1). To avoid variations due to different developmental stages, the third leaf from the top of individual mature plants for each of the cultivars was collected and subjected to organic extraction. All extracts were then analyzed using gas chromatography-mass spectrometry (GC-MS). The number of apolar secondary metabolites detected from the 13 cultivars ranged from 15 to 26 (Table S1). In total, 37 apolar secondary metabolites were detected in three chemical classes: terpenoids, fatty acid derivatives, and benzenoids. Leaves of the cultivar 'Xiao xiang ju' (Cm 10) exhibited the highest concentration of total apolar secondary metabolites (982.35 ± 89.08 µg g −1 fresh weight), followed by the cultivars 'Wan gong ju' (Cm 9) (947.08 ± 90.28 µg g −1 fresh weight) and 'Xiao huang ju' (Cm 10) (770.31 ± 211.56 µg g −1 fresh weight) ( Figure 1). Table 1. Cultivars of C. morifolium used in this study.

Code
Cultivar Collection Locality

PCA and HCA Analysis Based on the Terpenoid Contents
Since terpenoids represent the dominant class of apolar secondary metabolites in most cultivars examined, principal component analysis (PCA) and hierarchical clusters analysis (HCA) based on the terpenoid contents in the leaf extracts were conducted to define the relationship of these 13 cultivars. The chemical PCA horizontal axis explained 26.10% of the total variance, while the vertical axis explained a further 18.14% ( Figure 3A). The HCA based on the Euclidean distance between groups placed the 13 cultivars into 3 groups (I, II, and III) ( Figure 3B).

PCA and HCA Analysis Based on the Terpenoid Contents
Since terpenoids represent the dominant class of apolar secondary metabolites in most cultivars examined, principal component analysis (PCA) and hierarchical clusters analysis (HCA) based on the terpenoid contents in the leaf extracts were conducted to define the relationship of these 13 cultivars. The chemical PCA horizontal axis explained 26.10% of the total variance, while the vertical axis explained a further 18.14% ( Figure 3A). The HCA based on the Euclidean distance between groups placed the 13 cultivars into 3 groups (I, II, and III) ( Figure 3B).
Group II also contained three cultivars ('Da yang ju' (Cm 5), 'She yang hong xin ju' (Cm 12), and 'Su ju 9' (Cm 11)), whose extracts were specialized in high concentrations of α-curcumene (148.68 ±  Table 1. Control refers to the treatment with organic solvent ethyl acetate, which served as a negative control. Data were presented as means ± standard deviations based on three replicates. * indicates significance at the statistical level (p < 0.05) compared to the control. (D) Representative growth of Fusarium oxysporum, Magnaporthe oryzae, and Verticillium dahlia treated with a control (ethyl acetate) or with an extract made from 'Huang ju' (Cm 8) leaves.

Antifungal Activity of Apolar Secondary Metabolites from C. morifolium Leaves
Many apolar secondary metabolites detected from C. morifolium, especially terpenoids, are known to have antimicrobial activities [21][22][23][24][25]. To evaluate the antimicrobial properties of the apolar secondary metabolites from C. morifolium leaves, we tested three species of pathogenic fungi, Fusarium oxysporum, Magnaporthe oryzae, and Verticillium dahliae. F. oxysporum exhibits variations in infecting C. morifolium and other species of Chrysanthemum [26,27]. Identifying metabolites with antifungal activities may provide a chemical interpretation for the varied resistance or tolerance. M. oryzae and V. dahliae are both important fungal pathogens causing enormous economic losses and model fungal pathogens [28][29][30][31]. M. oryzae is best known as the causal agent of the rice blast disease. V. dahliae, a soil-borne pathogen found in temperate and subtropical zones, infects both herbaceous and woody host plants, especially the Solanaceae: tobacco, potatoes, peppers, and eggplant. Screening effective antagonistic chemicals would be of great economic significance for managing diseases caused by these and other fungal pathogens.
Apolar secondary metabolites extracted from the leaves of the 13 cultivars of C. morifolium exhibited differences in their antifungal properties against the three species of fungi (  Figure 4C). Among these cultivars, 'Huangju' (Cm 8) showed the strongest inhibitory activity against all three species of fungi ( Figure 4D). Our results point to a possible application of Chrysanthemum leaves as a source of fungicide for the control of certain fungal pathogens such as F. oxysporum, M. oryzae, and V. dahliae. To this end, it will be important to compare the antifungal activities of Chrysanthemum leaf extracts with those of comparable commercial products.

PCA and HCA Analysis Based on the Antifungal Effect
PCA coupled with HCA based on the values of fungal colony diameters was conducted to evaluate the correlation between the apolar secondary metabolites and the antifungal activities of the extracts made from the 13 cultivars. The PCA horizontal axis explained 70.16% of the total variance, while the vertical axis explained a further 19.03% ( Figure 5A). The HCA based on the Euclidean distances between groups indicated that all the cultivars can be classified into four groups ("a", "b", "c", and "d") according to their colony diameter. Both the PCA ( Figure 5A) and the HCA ( Figure 5B) analysis showed that Group a was composed of 'Huang ju' (Cm 8) and 'Huang xiang li' (Cm 6), with smallest colony growth diameter, indicating the strongest antifungal activity of these two cultivars. Group b was represented by 'Hang bai ju' (Cm 3), 'Qi yue bai' (Cm 7), 'Da yang ju' (Cm 5), 'She yang hong xin ju' (Cm 12), and the control. Being opposite to Group a, Group b was considered as the most inactive against the studied fungus species. None of the cultivars showed significant inhibitory activity on F. oxysporum and M. oryzae. However, among these cultivars, 'Hang bai ju' (Cm 3), 'Qi yue bai' (Cm 7), and 'She yang hong xin ju' (Cm 12) had significant inhibition activity on V. dahliae. Group c was composed of three cultivars, 'Chu ju' (Cm 1), 'Xiao xiang ju' (Cm 4), and 'Jin si huang ju' (Cm 2), whose extracts displayed significant antifungal activities against M. oryzae and V. dahliae. Group d was represented by the remaining four cultivars ('Bai xiang li' (Cm 13), 'Wan gong ju' (Cm 9), 'Su ju 9' Cm 11), and 'Xiao huang ju' (Cm 4)), whose extracts showed strong inhibitory activities against M. oryzae. Among these four cultivars, the extract of 'Bai xiang li' (Cm 13) exhibited an inhibitive effect on the growth of all three species of fungi, while 'Wan gong ju' (Cm 9), 'Su ju 9' (Cm 11), and 'Xiao huang ju' (Cm 10) showed the inhibitive effect only to M. oryzae. Many terpenoid compounds identified in C. morifolium leaves have been reported to have an antifungal effect on various species of fungi [21][22][23][24][25]. By comparing the PCA/HCA results based on terpenoid contents and antifungal activities, the antifungal effects of several cultivars (including 'Da yang ju' (Cm 5), 'Huang xiang li' (Cm 6), 'Huang ju' (Cm 8), 'She yang hong xin ju' (Cm 12)) could be associated with several terpenoids, including zingiberene, β-sesquiphellandrene, α-curcumene, (E)-β-farnesene, and α-farnesene. To establish causal correlation between these individual terpenes and antifungal activities will be interesting future research.  Table 1.

Conclusions
In this study, we showed both similarities and differences in chemical composition of apolar secondary metabolites in leaves among 13 cultivars of C. morifolium. With terpenoids being the dominant class of secondary metabolites, some terpenoids occur in all these cultivars (Table S1), suggesting conserved biosynthesis and biological functions. Nonetheless, there are also large variations in the qualities and quantities of terpenoids among these cultivars (Table S1). Because these plants were cultivated under the same optimal conditions, the variations are most likely genetically  Table 1.

Conclusions
In this study, we showed both similarities and differences in chemical composition of apolar secondary metabolites in leaves among 13 cultivars of C. morifolium. With terpenoids being the dominant class of secondary metabolites, some terpenoids occur in all these cultivars (Table S1), suggesting conserved biosynthesis and biological functions. Nonetheless, there are also large variations in the qualities and quantities of terpenoids among these cultivars (Table S1). Because these plants were cultivated under the same optimal conditions, the variations are most likely genetically determined, probably reflecting both the rapid evolution of the terpene biosynthetic pathway and the complex domestication/breeding history of C. morifolium [20,32]. Through the glimpse of this study, enormous variations in terpene chemistry can be expected to be discovered among the thousands of cultivars of C. morifolium. Such vast diversity of terpenoid chemistry could be explored for various applications. As revealed in this study, terpenoids of C. morifolium may be used as antifungal agents (Figures 4 and 5). PCA and HCA analyses collectively showed that antifungal properties of apolar secondary metabolites of C. morifolium leaves could be partly attributed to the contents of some terpenoids, including zingiberene, β-sesquiphellandrene, α-curcumene, (E)-β-farnesene, α-farnesene, and 1,8-cineole. This information could be used to facilitate the breeding of new cultivars of C. morifolium with enhanced resistance to fungal pathogens and lay a foundation for developing new terpenoid-based antifungal agents.

Plant Material
All 13 cultivars of C. morifolium were cultivated in the Chrysanthemum Germplasm Resource and Preservation Center, Nanjing Agricultural University, China (118 • 98 N, 32 • 07 E). All plants were grown in the greenhouse under the same conditions (at 27-32 • C; with light intensity at 700-1100 lux; humidity at 75%-87%). The third leaf from the top for individual plants at the reproductive stage (full blooming) for each cultivar was collected and subjected to organic extraction.

Organic Extraction of Leaves of C. morifolium
For organic extraction, leaf tissue of each cultivar was ground into powder and ethyl acetate of HPLC grade (Macklin Technology, Shanghai, China) was added as solvent in 5:1 (volume to weight) ratio. After shaking at room temperature for 2 h and a subsequent centrifugation (5000 r/min, 5 min), the organic phase was collected for GC-MS analysis and antifungal bioassays. For preparation of samples for GC-MS analysis, nonyl acetate (CAS:143-13-5, ≥98%, Sigma Aldrich, St Louis, MO, USA) was added to ethyl acetate (0.002%) as an internal standard. For each cultivar, three biological replicates were analyzed.

GC-MS Analysis and Identification of Extract Constituents
The analyses were performed using a GC-MS system (Agilent Intuvo 9000 GC system coupled with an Agilent 7000D Triple Quadrupole mass detector). Separation was performed on an Agilent HP 5 MS capillary column (30 m × 0.25 mm) with helium as carrier gas (1 mL·min −1 of flow rate). The injection volume of each sample was 1 µL. The temperature of the injection port was 260 • C, with a split mode (split ratio = 5:1). The column temperature program of gradient heating was adopted as follows: The temperature was initiated at 40 • C, followed by an increase to 250 • C at a rate of 5 • C/min. The MS conditions included an EI ion source temperature of 230 • C, an ionization energy of 70 eV, and a mass scan range of 40-500 amu. The separated constituents were identified by comparing their mass spectra with the authentic standards or those in the NIST17 MS library (National Institute of Standards and Technology). Retention indices were calculated using a series of C7 to C40 hydrocarbon standard (Sigma-Aldrich, St. Louis, MO, USA). Each constituent was quantified based on the comparison of its peak area with that of the internal standard, and the contents were expressed as µg g −1 fresh weight.
Minor peaks, which were defined as those having a peak area less than 1% of total peak area, were excluded from analysis.

Assessment of Antifungal Activity
Three pathogenic fungi, Magnaporthe oryzae, Verticillium dahliae, and Fusarium oxysporum, were tested in this study. M. oryzae strain Guy11, originally isolated from a rice field, is a model strain for studying rice blast disease [33]. V. dahliae strain V991 was originally isolated from a cotton field [34]. F. xysporum strain CFD-B2 was isolated from the cut flower chrysanthemum 'Shenma' plant in the chrysanthemum experimental station of Nanjing Agricultural University in the summer of 2016. These three species of pathogenic fungi were activated and the agarose plug with mycelium was cut from the edge of the original fungus culture under sterile conditions. Each plug was inverted and the mycelium side placed in contact with the medium in the center of dishes containing PDA, and cultured at 28 • C for five days in an incubator to carry out experiments. Ethyl acetate extracts were screened for antifungal activity in vitro by measurement of inhibitory zone diameter as previously described [21]. In order to investigate the antifungal activity of the extracts, the modified mycelial growth test with malt agar was used [35]. Petri dishes (90 mm in diameter) containing sterilized PDA media were used for the test and a 6 mm plug of mycelial agar, obtained from the edge of three-day-old cultures of fungi, was transferred to the center of each Petri dish. For each Petri dish, based on our preliminary experiment, 200 µL of the extract could cover the entire agar plate and was chosen as the volume of extract in each assay. Also performed were assays with 200 µL of ethyl acetate, the organic solvent used for leaf extraction, which served as a negative control. After the plates were cultured for 5 d at 28 • C, the diameter (mm) of the colony zone was determined with a caliper. All of the experiments were performed in triplicates with each replicate containing 10 plates.

Statistical Analysis
The differences in antifungal activity between extracts from the 13 cultivars and control were analyzed using Student's t-test, and the significance of the differences was determined at p < 0.05. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) using SPSS 22.0 software were conducted to determine relationships among the 13 cultivars in their chemical composition and antifungal activities based on the concentrations of individual terpenoids and the values of fungi growth diameters, respectively. The top two highest principal components (PC1 and PC2) were chosen to construct a loading diagram, according to the factor scores. The tested samples were positioned in the two-dimensional space, with some obvious groupings. HCA based on the squared Euclidean distance and the method of between-group linkages was used to cluster the samples with different relative terpenoid contents and antifungal effect.

Conflicts of Interest:
The authors declare no conflict of interest.