Chemical Composition of a Supercritical Fluid (Sfe-CO2) Extract from Baeckea frutescens L. Leaves and Its Bioactivity Against Two Pathogenic Fungi Isolated from the Tea Plant (Camellia sinensis (L.) O. Kuntze)

Colletotrichum gloeosporioides and Pseudopestalotiopsis camelliae-sinensis are the two most important tea plant (Camellia sinensis L.) pathogenic fungi. Interest in natural plant extracts as alternatives to synthetic chemical fungicides to control plant pathogens is growing. In this study, the volatile fraction of Baeckea frutescens L. was extracted by supercritical fluid extraction (SFE-CO2), and its chemical composition was analyzed, and investigated for its antifungal activity against C. gloeosporioides and P. camelliae. The major constituents of the volatile fraction were β-caryophyllene (28.05%), α-caryophyllene (24.02%), δ-cadinene (6.29%) and eucalyptol (5.46%) in B. frutescens SFE-CO2 extracts. The terpineol, linalool, terpinen-4-ol and eucalyptol showed strong contact antifungal activity against P. camelliae and C. gloeosporioides with median inhibitory concentration (MIC50) in the range of 0.69 μL/mL to 2.79 μL/mL and 0.62 μL/mL to 2.18 μL/mL, respectively. Additionally, the volatile fraction had high fumigation antifungal activity against P. camelliae and C. gloeosporioides with an inhibition rate between 20.87% and 92.91%. Terpineol presented the highest antifungal activity in the contact and fumigation toxicity assays. Terpineol, linalool, terpinen-4-ol and eucalyptol were associated with the most active chemical compounds in the volatile fraction against the fungi. The results suggest that B. frutescens SFE-CO2 extracts are potential ingredients to develop a natural fungicide for control of tea plant pathogens.


Introduction
As an alternative to using chemical fungicides, plant extracts have attracted the attention of chemical companies as they may be used as botanical fungicides. Baeckea frutescens L. is an important medicinal plant belonging to the Myrtaceae family [1] and is found in Peninsular Malaysia and Sumatra and distributed along coastal areas of southeastern China and Australia [2]. B. frutescens has various bioactive properties, such as antibacterial [1,3], antioxidant [2], anticancer [4], anti-inflammatory [5] and insecticidal activities [1]. The leaves of B. frutescens are rich in various volatile constituents such as terpenoids, sesquiterpenes and phenylpropanoids, which are secondary metabolites associated with numerous bioactivities [1,5,6].
The tea plant (Camellia sinensis L.) is a crucial commercial crop in world [7]. Tea, made from fresh leaves of C. sinensis, is a prominent beverage worldwide and is the second most consumed nonalcoholic beverage after water [8]. The tea plant suffers from biotic stresses of some pathogenic fungi, which is a serious concern for the tea industry as this condition impacts on reduced tea yields, decreased quality and damaged human health [9,10]. Several Pestalotiopsis-like species are common phytopathogens that cause tea grey blight disease and result in severe losses (10 to 20%) in tea production [11,12]. Pseudopestalotiopsis camelliae-sinensis was shown to cause grey blight disease on tea plants in China [13]. Some research has showed that planting disease-resistant varieties of C. sinensis has a certain effect on controlling the tea plant disease infected by Pestalotiopsis species [14]. In addition, the Colletotrichum species is also a common group of plant pathogens that are responsible for anthracnose diseases [15,16]. Colletotrichum gloeosporioides could lead to tea anthracnose disease, which a is serious foliar disease of the tea plant and causes severe damage accompanied by high yield losses [17]. Meanwhile, C. gloeosporioides-contaminated tea beverage may be harmful to human health [10]. It was recently reported that the leaves of tea plants infected by C. gloeosporioides were reduced in 30-60% of the tea products [18,19]. Altogether, P. camelliae and C. gloeosporioides are two of most destructive pathogens of foliar disease on tea plants and strategies to inhibit their growth are highly necessary.
At present, synthetic chemical fungicides are still the primary control approach to prevent fungal-related diseases in tea leaves. However, the indiscriminate use of these chemical fungicides not only reduces the quality of tea products and causes tea security crises, but also represents a harmful condition for human health and the natural environment [20][21][22]. Nowadays, effective and environmentally safe plant protection products which protect against phytopathogenic fungi are urgently needed in modern agriculture [23,24]. Therefore, replacing synthetic chemical fungicides by botanical alternatives could be a suitable choice to avoid toxicological and environmental-related issues. For instance, the botanical secondary metabolites, such as alkaloids [25], flavonoids [26] and volatile organic compounds [27], may act as antibacterial active substances to inhibit plant diseases. Therefore, botanical fungicides have been considered as environmentally friendly alternatives to synthetic chemical fungicides.
Traditionally, the volatile fraction of plants is extracted by steam distillation and organic solvent extraction. Supercritical fluid extraction (SFE) utilizes supercritical fluids, which exhibit liquid-like as well as gas-like properties, and has become an effective method for separating and extracting more apolar compounds, as well as the extraction of polyphenols, lipids, and essential oils [28][29][30]. Furthermore, conventional extraction with organic solvents has some disadvantages, such as high energy costs, and the possible loss of volatile compounds during the removal of the solvent [31]. Therefore, supercritical fluids are attractive for extracting flavors present in natural materials [28]. The supercritical fluid extraction with carbon dioxide (SFE-CO 2 ), a green extraction method, provides the final product without organic solvent residues [32]. Due to the properties of CO 2 , such as being odorless, colorless, safe, nontoxic and recyclable [28], SFE-CO 2 has been widely used for the extraction of volatile compounds [28]. To the best of our knowledge, there are still no reports on the chemical composition of the supercritical fluid (SFE-CO 2 ) extract of B. frutescens leaves and its bioactivity against P. camelliae and C. gloeosporioides. Therefore, the aims of this study were to investigate the chemical Plants 2020, 9, 1119 3 of 16 composition of the SFE-CO 2 extract from B. frutescens using GC-MS. In addition, the antifungal effects of SFE-CO 2 extract from B. frutescens against P. camelliae and C. gloeosporioides were also evaluated. The main research outline applied in this study is shown in Figure 1.

Plant Materials
The leaves of Baeckea frutescens were collected at the Guangxi Forestry Research Institute, Nanning, Guangxi Province, China in September of 2018. The B. frutescens trees were cultivated in the resource garden of Guangxi Forestry Research Institute. B. frutescens were authenticated by Prof. Wen-Hui Liang (Guangxi Forestry Research Institute). A voucher specimen (No. GX201816) has been deposited in the State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University. A total of 2160 g B. frutescens leaves were collected from nine randomly selected trees. The leaves were freeze-dried in a freeze dryer (model Martin Christ ALPHA 1-4 LD, Osterode am Harz, Germany). The dried leaves (10 mm × 1 mm, 4% humidity) were ground into powder (filtered through a 200-mesh sieve) and stored at −20 ℃.

Isolation and Identification of Fungal Pathogens
Diseased tea leaves were collected from the tea plantation located in Cuona County, Shannan city, Tibet Autonomous Region in China. The pathogenic fungi were isolated from diseased leaves that presented visible grey blight disease and anthracnose symptoms using traditional method as previously described [12,33] and with slight modification. Briefly, the symptomatic leaves were surface-sterilized in 1% NaClO for 2 min, then in 70% ethanol for 1 min, rinsed three times in sterile water, and then margins of lesions were cut into small pieces and transferred into potato dextrose agar medium (PDA, Difco Company). The culture was incubated at 25 °C for 3 to 5 days until fungal

Chemical Composition of the B. Frutescens Leaf SFE-CO2 Extract
The yield of the extract obtained by SFE-CO 2 was 2.2% (w/w relative to dry material weight). The major compounds of the SFE-CO 2 extract of were analyzed using GC-MS, and the peaks with matching similarly of more than 80% were accepted as candidate compounds. These compounds were further confirmed and identified by comparing the mass spectra with those from National Institute of Standards and Technology (NIST)mass spectral (http://webbook.nist.gov/chemistry/) and National Institutes of Health (NIH) databases, as well as by calculating the retention index (RI) and comparing RI from some of the previous literature records. Some major compounds were identified by co-injection of available standard compounds, such as α-pinene, β-pinene, eucalyptol, γ-terpinene, linalool, terpinen-4-ol, terpineol, β-caryophyllene and α-caryophyllene. The GC-MS separation of SFE-CO 2 extract is shown in supplementary material Figure S1.The chemical composition of the SFE-CO 2 extract is listed in Table 1, along with their retention indices, relative contents and identification.

Contact Antifungal Activity of B. frutescens SFE-CO 2 Extract
The SFE-CO 2 extract of B. frutescens leaves exhibited an inhibitory effect on mycelial growth of two tea plant pathogenic fungi as shown in Figure 2. The mycelial growth of P. camelliae and C. gloeosporioides was strongly inhibited and this effect was dose-dependent as shown in Figure 2 and Table 2.
This is the first report in which the chemical composition of a supercritical fluid (SFE-CO2) extract of B. frutescens leaves is provided. β-Caryophyllene, α-caryophyllene, δ-cadinene and eucalyptol were the major components.

Contact Antifungal Activity of B. frutescens SFE-CO2 Extract
The SFE-CO2 extract of B. frutescens leaves exhibited an inhibitory effect on mycelial growth of two tea plant pathogenic fungi as shown in Figure 2. The mycelial growth of P. camelliae and C. gloeosporioides was strongly inhibited and this effect was dose-dependent as shown in Figure 2 and Table 2. As seen in Figure 2, from concentrations of 6 μg/mL to 50 μg/mL, the SFE-CO2 extract of B. frutescens leaves showed a significantly limited mycelial growth of both fungi. In addition, the extract presented stronger inhibited activity against C. gloeosporioides than against P. camelliae ( Figure 2) as also observed by the lower MIC50 value for C. gloeosporioides. The median inhibitory concentration (MIC50) of the B. frutescens leaf SFE-CO2 extract against two phytopathogenics was also evaluated ( Table 2). The inhibitory activity of B. frutescens SFE-CO2 extract against C. gloeosporioides exhibited the lowest MIC50 value of 4.79 μg/mL, while the MIC50 against P. camelliae was 5.11 μg/mL. However, compared with other previously published papers, our research exhibited that the B. frutescens SFE-CO2 extracts have stronger antifungal activity. For example, three plants (oregano, thyme and ajwain) SFE-CO2 extracts showed broad-spectrum antifungal activity against four Aspergillus species fungi with MIC values in the range of 128-1024 μg/mL [39]. In addition, the SFE-CO2 extract of Prunus persica leaves presented MIC50 of 62.50 μg/mL against Candida As seen in Figure 2, from concentrations of 6 µg/mL to 50 µg/mL, the SFE-CO 2 extract of B. frutescens leaves showed a significantly limited mycelial growth of both fungi. In addition, the extract presented stronger inhibited activity against C. gloeosporioides than against P. camelliae ( Figure 2) as also observed by the lower MIC 50 value for C. gloeosporioides. The median inhibitory concentration (MIC 50 ) of the B. frutescens leaf SFE-CO 2 extract against two phytopathogenics was also evaluated ( Table 2). The inhibitory activity of B. frutescens SFE-CO 2 extract against C. gloeosporioides exhibited the lowest MIC 50 value of 4.79 µg/mL, while the MIC 50 against P. camelliae was 5.11 µg/mL. However, compared with other previously published papers, our research exhibited that the B. frutescens SFE-CO2 extracts have stronger antifungal activity. For example, three plants (oregano, thyme and ajwain) SFE-CO 2 extracts showed broad-spectrum antifungal activity against four Aspergillus species fungi with MIC values in the range of 128-1024 µg/mL [37]. In addition, the SFE-CO 2 extract of Prunus persica leaves presented MIC 50 of 62.50 µg/mL against Candida albicans [38]. In our study, the B. frutescens SFE-CO 2 extract revealed a stronger antifungal activity against two fungi (MIC 50 values of 4.79 µg/mL and 5.11 µg/mL, respectively). Overall, results showed that C. gloeosporioides may be the most sensitive micro-organism for B. frutescens SFE-CO 2 extract.

Screening for Fumigation Antifungal Activities of Major Chemical Compounds
In order to quickly screen the antifungal activity of the nine major chemical compounds present in the SFE-CO 2 extract the fumigation method against the mycelial growth of P. camelliae and C. gloeosporioides was used. The results are presented in Figure 3.
Plants 2020, 9, x FOR PEER REVIEW 8 of 16 albicans [40]. In our study, the B. frutescens SFE-CO2 extract revealed a stronger antifungal activity against two fungi (MIC50 values of 4.79 μg/mL and 5.11 μg/mL, respectively). Overall, results showed that C. gloeosporioides may be the most sensitive micro-organism for B. frutescens SFE-CO2 extract.

Screening for Fumigation Antifungal Activities of Major Chemical Compounds
In order to quickly screen the antifungal activity of the nine major chemical compounds present in the SFE-CO2 extract the fumigation method against the mycelial growth of P. camelliae and C. gloeosporioides was used. The results are presented in Figure 3. In Figure 3, it can be seen that the four primary compounds (terpineol, linalool, terpinen-4-ol and eucalyptol) had stronger fumigation activity against P. camelliae and C. gloeosporioides, respectively. By contrast, the other five major compounds (α-pinene, β-pinene, γ-terpinene, βcaryophyllene and α-caryophyllene) only exhibited weak fumigation activity against two tea plant pathogenic fungi, respectively. In the fumigation activity assay, the mycelial growth of P. camelliae and C. gloeosporioides was inhibited by the four relatively higher activity standards (terpineol, linalool, terpinen-4-ol and eucalyptol) as shown in Figure 4.
However, as seen in Figure 3, the other five major compounds (α-pinene, β-pinene, γ-terpinene, βcaryophyllene and α-caryophyllene) showed less than 17% inhibition rate against mycelial growth of P. camelliae and C. gloeosporioides, respectively. These results are in-line with a previous report that showed that α-pinene, β-pinene and β-caryophyllene did not present fumigation inhibitory effects against Aspergillus ochraceus, A. flavus, and A. niger at an air concentration of 56 μg/mL [41]. In Figure 3, it can be seen that the four primary compounds (terpineol, linalool, terpinen-4-ol and eucalyptol) had stronger fumigation activity against P. camelliae and C. gloeosporioides, respectively. By contrast, the other five major compounds (α-pinene, β-pinene, γ-terpinene, β-caryophyllene and α-caryophyllene) only exhibited weak fumigation activity against two tea plant pathogenic fungi, respectively. In the fumigation activity assay, the mycelial growth of P. camelliae and C. gloeosporioides was inhibited by the four relatively higher activity standards (terpineol, linalool, terpinen-4-ol and eucalyptol) as shown in Figure 4.  Conversely, many plant essential oils have been evaluated for fumigation activity against plant pathogens [41,42]. For example, the antifungal activity of cinnamon oil, clove oil, eugenol and geraniol was dose-dependent, and relatively higher doses (8 μL/disc) presented stronger antifungal activities [43]. At a concentration of 10 μL/L air, cinnamon oil, cinnamon bark oil, Litsea cubeba oil, Angelica dahurica oil and thyme oil showed high fumigation inhibitory effects (inhibition rate of 100%) against Villosiclava virens [42]. Trans-cinnamaldehyde, the major component of cinnamon oil and cinnamon bark oil, exhibited strong fumigation activity against Villosiclava virens with an effective medium concentration EC50 of 0.5 μL/L air [42].

Contact Antifungal Activity of Major Activity Components
According to the results of the fumigation activity assay, the four compounds with the highest activity (terpineol, linalool, terpinen-4-ol and eucalyptol) were thoroughly evaluated in relation to their contact antifungal activity against P. camelliae and C. gloeosporioides. The results of contact As shown in Figures 3 and 4, terpineol showed the strongest fumigation inhibitory effects against P. camelliae, presenting an inhibition rate of 92.91%, followed by linalool, terpinen-4-ol and eucalyptol which inhibited 48.68%, 35.20% and 20.87%, respectively. Terpineol also exhibited the highest fumigation inhibitory effect (82.15%) against C. gloeosporioides, followed by linalool, terpinen-4-ol and eucalyptol that inhibited 65.51%, 41.06% and 28.92%, respectively. In addition, α-caryophyllene showed weak fumigation activity (16.93%) against C. gloeosporioides. In a previous report, linalool showed fumigation inhibitory effect (83.7% inhibition) against, Aspergillus ochraceus at an air concentration of 56 µg/mL [39].
Conversely, many plant essential oils have been evaluated for fumigation activity against plant pathogens [39,40]. For example, the antifungal activity of cinnamon oil, clove oil, eugenol and geraniol was dose-dependent, and relatively higher doses (8 µL/disc) presented stronger antifungal activities [41]. At a concentration of 10 µL/L air, cinnamon oil, cinnamon bark oil, Litsea cubeba oil, Angelica dahurica oil and thyme oil showed high fumigation inhibitory effects (inhibition rate of 100%) against Villosiclava virens [40]. Trans-cinnamaldehyde, the major component of cinnamon oil and cinnamon bark oil, exhibited strong fumigation activity against Villosiclava virens with an effective medium concentration EC 50 of 0.5 µL/L air [40].

Contact Antifungal Activity of Major Activity Components
According to the results of the fumigation activity assay, the four compounds with the highest activity (terpineol, linalool, terpinen-4-ol and eucalyptol) were thoroughly evaluated in relation to their contact antifungal activity against P. camelliae and C. gloeosporioides. The results of contact antifungal activity of the chemical compounds compared to that of carbendazin are presented in Figure 5. The contact antifungal toxicity of terpineol, linalool, terpinen-4-ol and eucalyptol against P. camelliae and C. gloeosporioides was concentration-dependent.  Terpineol, linalool, terpinen-4-ol and eucalyptol showed inhibitory effects on mycelial growth of P. camelliae at the five different concentrations, Figure 6. As shown in Figures 5 and 6, at 2 µL/mL, terpineol, linalool and terpinen-4-ol exhibited the strongest contact activity with 100% inhibition of P. camelliae. In contrast, at 20 µL/mL, the commercial fungicide carbendazim inhibited 90.64% the growth of P. camelliae ( Figure 5). Linalool, terpineol and terpinen-4-ol exhibited the highest inhibitory effects on the mycelial growth of P. camelliae at 1 µL/mL ( Figure 6). Terpineol presented antifungal activity (26.48% inhibition) at 0.5 µL/mL (Figure 6), whereas eucalyptol showed moderate antifungal activity (37.9% inhibition) against P. camelliae at 2 µL/mL. In addition, compared with the control, other concentrations of the linalool and terpinen-4-ol also showed significantly contact antifungal activity ( Figure 5). Eucalyptol also showed antifungal activity and inhibited mycelial growth of P. camelliae at concentrations higher than 0.25 µL/mL.  However, as expected, carbendazim showed a strong inhibitory effect on the mycelial growth of P. camelliae at concentrations higher than 10 µL/mL (Figure 7). Carbendazim presented the highest antifungal activity (90.64% inhibition) at 20 µL/mL, and it also showed moderate antifungal activity with inhibition rate of 52.17% at 10 µL/mL (Figures 5 and 7). In summary, compared with carbendazim, the tested compounds (terpineol, linalool, terpinen-4-ol and eucalyptol) showed stronger antifungal activity and inhibited mycelial growth of P. camelliae.
The effects of terpineol, linalool, terpinen-4-ol and eucalyptol on the mycelial growth of C. gloeosporioides at the five different concentrations are shown in Figure 8. Compared with the control, all the four chemical compounds had significant difference in the contact antifungal activity and inhibition of mycelial growth of C. gloeosporioides (Figures 5 and 8). The results show that terpineol, linalool and terpinen-4-ol were able to inhibit 100% of mycelial growth ( Figure 8) and at 2 µL/mL it inhibited 100% of C. gloeosporioides growth. On the other hand, eucalyptol inhibited 47.63% of the C. gloeosporioides growth ( Figure 5). Terpineol showed the strongest contact inhibition of C. gloeosporioides mycelial growth, followed by linalool, terpinen-4-ol and eucalyptol ( Figure 8). However, compared with the tested four compounds, carbendazim exhibited the strongest contact antifungal activity and inhibition of mycelial growth of C. gloeosporioides (Figures 5 and 7). Carbendazim exhibited the strongest contact activity with 92.27% inhibition of C. gloeosporioides at 0.4 µL/mL ( Figure 5). In addition, at 0.2 µL/mL and 0.1 µL/mL, carbendazim also inhibited 82.76% and 53.8%of C. gloeosporioides mycelial growth, respectively (Figure 7). However, as expected, carbendazim showed a strong inhibitory effect on the mycelial growth of P. camelliae at concentrations higher than 10 μL/mL (Figure 7). Carbendazim presented the highest antifungal activity (90.64% inhibition) at 20 μL/mL, and it also showed moderate antifungal activity with inhibition rate of 52.17% at 10 μL/mL (Figures 5 and 7). In summary, compared with carbendazim, the tested compounds (terpineol, linalool, terpinen-4-ol and eucalyptol) showed stronger antifungal activity and inhibited mycelial growth of P. camelliae. The effects of terpineol, linalool, terpinen-4-ol and eucalyptol on the mycelial growth of C. gloeosporioides at the five different concentrations are shown in Figure 8. Compared with the control, all the four chemical compounds had significant difference in the contact antifungal activity and inhibition of mycelial growth of C. gloeosporioides (Figures 5 and 8). The results show that terpineol, linalool and terpinen-4-ol were able to inhibit 100% of mycelial growth ( Figure 8) and at 2 μL/mL it inhibited 100% of C. gloeosporioides growth. On the other hand, eucalyptol inhibited 47.63% of the C. gloeosporioides growth ( Figure 5). Terpineol showed the strongest contact inhibition of C. gloeosporioides mycelial growth, followed by linalool, terpinen-4-ol and eucalyptol ( Figure 8). However, compared with the tested four compounds, carbendazim exhibited the strongest contact antifungal activity and inhibition of mycelial growth of C. gloeosporioides (Figures 5 and 7). Carbendazim exhibited the strongest contact activity with 92.27% inhibition of C. gloeosporioides at 0.4 μL/mL ( Figure 5). In addition, at 0.2 μL/mL and 0.1 μL/mL, carbendazim also inhibited 82.76% and 53.8%of C. gloeosporioides mycelial growth, respectively (Figure 7). This result is in-line with other studies that showed that the treatment with an 8 μL/disc of linalool dramatically decreased the conidial germination of C. gloeosporioides to 3.4%, but eugenol showed moderate anti-germination activities with the conidial germination of C. gloeosporioides was 50.4% [43]. At 1 μL/mL, terpineol, linalool, and terpinen-4-ol inhibitedthe mycelial growth by 77.42%, 58.53% and 60.62%, respectively. However, eucalyptol at 1 μL/mL showed relatively weak activity (<20% inhibition) in relation to the mycelial growth of C. gloeosporioides [43]. The median inhibitory concentration (MIC50) values of terpineol, linalool, terpinen-4-ol and eucalyptol compounds were estimated using the logistic analyses model and the results are displayed in Table 3.
In general, the fumigation and contact antifungal activity of plant extracts could not be easily correlated with one specific component. In this study, terpineol, linalool, terpinen-4-ol and eucalyptol showed strong fumigation antifungal activity against P. camelliae and C. gloeosporioides with inhibition rates in the range of 20.87% and 92.91%. Additionally, terpineol, linalool, terpinen-4-ol and eucalyptol also exhibited notable contact antifungal activity against P. camelliae and C. gloeosporioides with MIC 50 values in the range of 0.62 µL/mL to 2.79 µL/mL. The commercial fungicide carbendazim showed significantly contact antifungal activity against P. camelliae and C. gloeosporioides with MIC 50 values of 0.095 µL/mL and 9.70 µL/mL, respectively. Therefore, it seems that terpineol, linalool, terpinen-4-ol and eucalyptol could be considered as the main active ingredients and are responsible for antifungal activities of the SFE-CO 2 extract from B. frutescens leaves.

Plant Materials
The leaves of Baeckea frutescens were collected at the Guangxi Forestry Research Institute, Nanning, Guangxi Province, China in September of 2018. The B. frutescens trees were cultivated in the resource garden of Guangxi Forestry Research Institute. B. frutescens were authenticated by Prof. Wen-Hui Liang (Guangxi Forestry Research Institute). A voucher specimen (No. GX201816) has been deposited in the State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University. A total of 2160 g B. frutescens leaves were collected from nine randomly selected trees. The leaves were freeze-dried in a freeze dryer (model Martin Christ ALPHA 1-4 LD, Osterode am Harz, Germany). The dried leaves (10 mm × 1 mm, 4% humidity) were ground into powder (filtered through a 200-mesh sieve) and stored at −20 • C.

Isolation and Identification of Fungal Pathogens
Diseased tea leaves were collected from the tea plantation located in Cuona County, Shannan city, Tibet Autonomous Region in China. The pathogenic fungi were isolated from diseased leaves that presented visible grey blight disease and anthracnose symptoms using traditional method as previously described [12,48] and with slight modification. Briefly, the symptomatic leaves were surface-sterilized in 1% NaClO for 2 min, then in 70% ethanol for 1 min, rinsed three times in sterile water, and then margins of lesions were cut into small pieces and transferred into potato dextrose agar medium (PDA, Difco Company). The culture was incubated at 25 • C for 3 to 5 days until fungal hyphae started to grow from the pieces. Single-hyphal tip was transferred to a new PDA plate to purify isolates and two strains were obtained. To identify the two fungal isolates, DNA was extracted, amplified, and sequenced using universal primers (Internal Transcribed Spacer, ITS1/ITS4). BLAST (Basic Local Alignment Search Tool) analysis of GenBank data showed 100% sequence homology with the ITS sequence of strain for Colletotrichum gloeosporioides (Accession No. KF836743.1) and Pseudopestalotiopsis camelliae-sinensis (Accession No. MK909901.1), respectively. The two pathogenic fungi C. gloeosporioides and P. camelliae cultured using potato dextrose agar medium at 4 • C and stored for future use.

Supercritical Fluid (SFE-CO2) Extract of Baeckea frutescens L.
The extraction was performed with a laboratory scale multi-vessel accelerated supercritical fluid extraction system (model MV-10 ASFE Waters Corporation, Milford, MA, USA). The dried B. frutescens leaf powder (5.0 g) was placed in a 25 mL supercritical fluid extractor vessel and was extracted using supercritical carbon dioxide without any cosolvents. The temperature and pressure were set at 40 • C and 180 bar, respectively. The extract laden Supercritical CO 2 (flow rate 13 min/mL) was sent to extractor vessel through a pressure pump for 70 min, with 20 min dynamic duration, 30 min of static duration and 20 min of dynamic duration, respectively. The extraction efficiency (yield) was 2.2% (110 mg), and the SFE-CO 2 extract was stored for future use.

Contact Antifungal Activities Bioassay
The contact antifungal activity of the SFE-CO 2 extract was determined using the toxic medium method. Median inhibitory concentration (MIC 50 ) of the extract was determined using a serial two-fold micro dilution method against the tea plant pathogenic fungi. The stock solution (100 mg/mL) was serially diluted in 30 mL PDA medium at 45-50 • C and mixed to provide different concentrations (50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6 µg/mL, 3 µg/mL) to evaluate inhibitory activities against C. gloeosporioides and P. camelliae. Based on preliminary screening activities, the four individual chemical components with higher antifungal activity were determined, and which were adjusted to a series of concentration gradients medium (2.0 µL/mL, 1.0 µL/mL, 0.5 µL/mL, 0.25 µL/mL and 0.125 µL/mL) to evaluate inhibitory activities against C. gloeosporioides and P. camelliae. Carbendazim, a broad-spectrum fungicide, was used as a positive control and different concentrations were tested. The negative control received the same quantity of acetone mixed with PDA. The 10 mL toxic medium were poured onto aseptic 9 cm plastic Petri dishes. A 5 mm diameter fungal disc of C. gloeosporioides or P. camelliae was immediately inoculated in the center of each PDA plate and plates were incubated in the dark at 25 • C.
After 7 days of incubation at 25 • C in the dark, the colony growth diameter (mm) was measured using a digital caliper. Each test was repeated three times. The growth inhibition was calculated using the formula.
Inhibition rate (%) = (Dc − Dt)/Dc × 100, where: D C , D T -average diameter (mm) of the fungal colony of the control and the treatment, respectively.

Fumigation Antifungal Activities Bioassay
The fumigation activity of the SFE-CO 2 extract was determined as previously described [49] and with slight modifications. A 5 mm diameter disc of C. gloeosporioides or P. camelliae was inoculated in the center of each PDA plate (9 cm, the volume about 60 mL air spaces), and a 6 mm filter paper containing 10 µL of the extract or isolated substance was placed on the center of the inner surface of the Petri dish lid. Plastic Petri dishes (60 mL air spaces) offer 50 mL air spaces after the addition of 10 mL PDA medium, and the final concentration was 200 µL/L air. The negative control was composed of acetone. The plate was immediately sealed with parafilm to prevent any leakage of the standards. Three replicates were used for each concentration, and the PDA plates were placed upside down in the incubator. After 5 days of incubation at 25 • C in the dark, the colony growth diameter (mm) was measured using a digital caliper. The growth inhibition was calculated using the above Formula (1).

Statistical Analysis
Statistical significance was carried out by applying one-way ANOVA followed by Duncan's test with the acceptance level of significance p = 0.05, using SPSS version 22.0 (IBM Corp., Armonk, NY, USA). Logistic analysis was performed using Origin version 2017 software (OriginLab., Northampton, MA, USA) and GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA).

Conclusions
To the best of the authors' knowledge, this is first report that investigates the volatile chemical composition of the SFE-CO 2 extract from B. frutescens leaves. β-Caryophyllene, α-caryophyllene, δ-cadinene, eucalyptol and terpineol are the major compounds of the extract. The extract presented antifungal activity against P. camelliae and C. gloeosporioides. Terpineol, linalool, terpinen-4-ol and eucalyptol are the main active ingredients. Therefore, the SFE-CO 2 extract from B. frutescens leaves could be a potential alternative to traditional synthetic chemical fungicides of tea plant pathogens (P. camelliae and C. gloeosporioides).