Efficacy and mechanism of Mentha haplocalyx and Schizonepeta tenuifolia essential oils on the inhibition of Panax notoginseng pathogens

Industrial Crops & Products 145 (2020) 112073

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Efficacy and mechanism of Mentha haplocalyx and Schizonepeta tenuifolia essential oils on the inhibition of Panax notoginseng pathogens

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Chuan-Jiao Chena, Qing-Qing Lia, Zi-Ying Zenga,b, Su-Su Duana,b, Wei Wangc, Fu-Rong Xua, Yong-Xian Chenga,b, Xian Donga,* a

College of Pharmaceutical Sciences, Yunnan University of Chinese Medicine, Kunming, 650500, People’s Republic of China Guangdong Key Laboratory for Genome Stability & Disease Prevention, School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen, 518060, People’s Republic of China c Feixian Agriculture Bureau, Feixian, 273400, People’s Republic of China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Panax notoginseng Pathogenic fungi Mentha haplocalyx Essential oils Action mechanisms Antifungal activity

In order to control the occurrence of Panax notoginseng (Burk.) F.H. Chen diseases, the antifungal effects and action mechanism of essential oils (EOs) against the pathogenic fungi were investigated. The antifungal effect of Mentha haplocalyx Briq. and Schizonepeta tenuifolia Briq. essential oils (EOs) were determined in vivo and in vitro to develop green fungicides to eradicate fungi pathogens affecting Panax notoginseng. The inhibition rates of M. haplocalyx and S. tenuifolia EOs (50 mg/mL) on Fusarium oxysporum, Fusarium solani, Cylindrocarpon destructans, Pythium aphanidermatum, Botrytis cinerea, Colletotrichum gloeosporioides and Rhizoctonia solani reached 100.00 % and were assessed by the Oxford cup method. The chemical constituents of M. haplocalyx and S. tenuifolia EOs were analyzed by GC–MS. Low MIC values of M. haplocalyx EO, S. tenuifolia EO and its main components against six fungal strains from P. notoginseng were found, which indicated all have substantial antifungal activity. Furthermore, synergistic effects between the EOs and the agrochemical hymexazol against P. notoginseng pathogens were observed. F. oxysporum was chosen as dominant fungi pathogen to investigate the mechanism of action of M. haplocalyx EO. The antifungal effect of M. haplocalyx EO on F. oxysporum in vitro was investigated. The high M. haplocalyx EO concentrations inhibited the spore germination rates, mycelia dry weights and spore yields and disturbed the mycelia soluble protein and reducing sugar contents; it also increased the cell membrane permeability. M. haplocalyx EO also induced decreased SDH and NADH oxidase activities and caused extreme alterations in ultrastructures compared with the control. Altogether, the antifungal mechanism of M. haplocalyx EO may seemed to consist in penetrating and dissolving the mitochondrial membranes. Overall, M. haplocalyx EO and its major compounds show potential to be used as natural alternatives to commercial fungicides for controlling F. oxysporum infection of P. notoginseng.

1. Introduction Plant disease is a major negative factor in agricultural management, and fungi-infected plants result in economic losses in farm production. Continuous cropping obstacles and fungal infestation are also crucial threats to the farming of Panax notoginseng (Burk.) F.H. Chen, which belongs to the Ginseng genus of the Araliaceae family. This plant has the functions of activating blood circulation, alleviating pain and is a traditional Chinese medicinal material with a large medicinal and economic value (Dong et al., 2003). Panax notoginseng is a perennial herbaceous plant that grows in warm and humid environments; it is vulnerable to various adverse environmental considerations and

harmful organisms in its particular growth environment (Guo et al., 2006). The number of pathogenic species against this plant has been increasing (Fuad Mondal et al., 2013). For example, Rhizoctonia solani, Botrytis cinerea, Colletotrichum gloeosporioides, Alternaria panax primarily cause aboveground diseases, while underground disease is primarily cause by Pythium aphanidermatum, Fusarium oxysporum, Fusarium solani, and Cylindrocarpon destructans (Miao et al., 2006; Jiang et al., 2011). Fungal diseases can decrease the yield and quality of P. notoginseng, thus affecting its effective components and medicinal efficacy (Zhang and Wang, 2010). The control of P. notoginseng fungal infection frequently relies solely on the use of agrochemicals, such as metalaxyl, dimethomorph and

The English of the manuscript has been checked by the AJE. Please see www.aje.com. ⁎ Corresponding author. E-mail addresses: [email protected] (Y.-X. Cheng), [email protected] (X. Dong). https://doi.org/10.1016/j.indcrop.2019.112073 Received 30 July 2019; Received in revised form 18 December 2019; Accepted 27 December 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. Colony morphology (1), mycelia structure (2) and spore morphology (3) of the seven fungi. (a) F. oxysporum, (b) F. solani, (c) C. destructans, (d) P. aphanidermatum, (e) B. cinerea, (f) C. gloeosporioides and (g) R. solani. The R. solani belongs to Agonomycetaceae, of which do not produce spores.

activities of F. oxysporum. There are no previous reports on controlling the pathogens of P. notoginseng through using M. haplocalyx and S. tenuifolia EOs. Thus, the aim of this study was to evaluate the efficacy of M. haplocalyx and S. tenuifolia EOs regarding its antifungal activity against the seven pathogens of P. notoginseng diseases in vivo and in vitro. The dominant pathogen (F. oxysporum) was then selected to further explore the antifungal mechanism of M. haplocalyx EO to clarify the true action targets. This study provides a theoretical basis for the research and development of green and effective antibacterial agents for controlling P. notoginseng diseases.

hymexazol, which can cause environmental pollution, pesticide residues and put human and animal health at risk (Han et al., 2019). With growing concerns about environmental protection, much more attention has been given to the use of natural antimicrobial agents for fighting plant diseases. As a result, natural antimicrobial agents such as essential oils (EOs) have been extensively used. EOs are considered to be vital for plant defense, as often possess antifungal properties (Tajkarimi et al., 2010). What’s more, there are several advantages include not contributing to pesticide resistance and degrading easily in the environment; therefore, it can be used as novel botanical antifungal agents to defeat plant diseases (Isman, 2015). Mentha haplocalyx Briq. and Schizonepeta tenuifolia Briq. belong to the Labiatae family and its EOs demonstrated broad-spectrum antifungal activities, being widely use in the pharmaceutical and food industries (Cao et al., 2011; Fung and Lau, 2013). Several studies have found that the EOs of Labiatae have a significant antifungal effect against some agricultural fungi, including Phytophthora infestans (Olanya and Larkin, 2006), Macrophomina phaseolina (Moghaddam et al., 2013), Aspergillus niger (Marotti et al., 1994) and F. oxysporum (Tyagi and Malik, 2011). Notably, the EO of M. haplocalyx had showed strong antifungal properties (Kang et al., 2013), whereas S. tenuifolia EO possesses antipyretic, antiviral and antifungal activities (Fung and Lau, 2013). Previous studies have proposed that the antifungal activity of EOs is especially related with the concentration in phenol compounds such as eugenol (Ma et al., 2019). The cell membrane contributed to maintaining life activities and stabilizing intracellular constituents, such as soluble protein and reducing sugars. Some research demonstrated the cell membrane is one of action targets when invaded by fungal agents (Wei et al., 2008). The antimicrobial mechanism of EOs may cause cell membrane breakage due to the lipophilic components of EOs passing easily through the cell membrane (Shukla et al., 2012). The intracellular compounds can flow when the cell membrane is damaged, which could disturb cell membrane permeability, finally changing the relative electrical conductivity (Liu et al., 2010; Kim et al., 2009). The morphological alterations of fungi treated with EOs can be assessed by scanning electron microscope (SEM) and transmission electron microscope (TEM). Succinate dehydrogenase (SDH) activity and NADH oxidize activity play vital roles in the respiratory chain. The respiratory chain of fungi was found to be inhibited by blocking electron transfer, which is a primary mechanism of antifungal action (Wong et al., 1971). Therefore, we further investigated the effect of M. haplocalyx on the SDH and NADH oxidase

2. Materials and methods 2.1. Materials and chemicals In order to compare to the chemical composition of EOs of fresh and dry M. haplocalyx, the study used dry M. haplocalyx and fresh M. haplocalyx and S. tenuifolia from the Labiatae family that were grown in Yunnan Province. The materials were obtained as a commercial product from the Yunnan Jinfa Pharmaceutical Co., Ltd. (Kunming, Yunnan). The plant materials were identified by one of the authors (Yong-Xian Cheng). The aerial parts of dry M. haplocalyx and fresh M. haplocalyx and S. tenuifolia (300 g) were cut off into pieces respectively and then subjected to hydro-distillation for 8 h using 2400 mL of deionized water. The EO yields were estimated on a dry-weight basis (w/w). Once obtained, the EOs were dried using anhydrous sodium sulfate (Na2SO4) and stored in darkness at -20℃ until use. A panel of fungi including F. oxysporum, F. solani, C. destructans, P. aphanidermatum, B. cinerea, C. gloeosporioides and R. solani were tested. All fungi were cultured and identified by Sangon Co., Ltd. (Shanghai, China). The mycelia structure and spore morphology of the fungi were detected using a microscope (Ci-S, Nikon,Co. Ltd., Tokyo, Japan) at 100x magnification (Fig. 1). D-Carvone (≥ 99 %) and hymexazol (≥ 97 %) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; p-menthan-3-one (≥ 99 %) was purchased from Shanghai Macklin Blochemical Co., Ltd.; D-limonene (≥ 95 %) was obtained from Shanghai Nine-Dinn Chemistry Co., Ltd.; and (-)-menthone (≥ 98 %) was procured from Shanghai Ai-Lan Chemical Technology Co., Ltd..

2

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where FIC (EO) = MIC (EO) in combination/MIC (EO) alone; FIC (hymexazol) = MIC (hymexazol) in combination/MIC (hymexazol) alone. The ∑ FICi values were interpreted as follows: synergy relationship (FICi ≤ 0.5); addition relationship (0.5 < FICi ≤ 1); irrelevant relationship (1.0 < FICi ≤ 4.0); antagonistic relationship (FICi > 4.0).

2.2. Measurement of antifungal activity of EOs against seven pathogens in vitro The antifungal effect in vitro was determined according to the Oxford cup method (Chen et al., 2019). The M. haplocalyx and S. tenuifolia EOs and hymexazol were dissolved in 10/1000 DMSO and 1/ 1000 Tween-80 (1-DMSO-T) solution, and the final EO concentration was 50 mg/mL. 1-DMSO-T was used as negative control, and 5 mg/mL of hymexazol suspension was used as positive control. Under aseptic conditions, all suspensions were filtered with a 0.22 μm organic filter to obtain the aseptic filtrate. A 5 mm mycelium disk was obtained from the fungal colony and was placed in the center of the culture dish. And four Oxford cups were then placed in the dish and 200 μL of filtered suspension was added. Each treatment was repeated five times and cultured in a microorganism incubator at 28℃. The inhibitory rates of the fungi was calculated as previously reported (Chen et al., 2019).

2.6. The effect of M. haplocalyx EO on F. oxysporum infecting P. notoginseng The effect of M. haplocalyx EO on the ability of F. oxysporum to infect P. notoginseng in vivo were determined by Chen et al. (2019). A 2:1 ratio of vermiculite and sterilized quartz sand were used as the P. notoginseng culture medium. M. haplocalyx EO at a concentration of 1/8 MIC (0.12 mg/mL) was thoroughly mixed into the culture medium and allowed to incubate for 5 d. The F. oxysporum colonies were washed three times with sterile water and then adjusted to a concentration of 1 × 107 spores/mL. Healthy P. notoginseng were inoculated with the spore suspension for 3 h. To elucidate the prevention and control effect of M. haplocalyx EO on F. oxysporum, the biomass, disease incidence and disease index of P. notoginseng were measured 20 days after inoculation. The plant disease incidence and disease index were calculated based on a previous report (Chen et al., 2019). The fresh weight of the roots, stems and leaves were obtained through accurate weighing; the dry weight was then measured after incubation in a drying chamber at 40℃ for 7 days. Three experimental groups were set up in the experiment. Ten seedlings were used per group, and each treatment was repeated five times. CK: The healthy P. notoginseng seedlings were transplanted into culture medium directly. Fo: The P. notoginseng seedlings inoculated with F. oxysporum for 3 h, which were then transplanted into culture medium. FMO: The P. notoginseng seedlings inoculated with F. oxysporum for 3 h, which were then transplanted into the culture medium containing 1/8 MIC M. haplocalyx EO (0.12 mg/mL).

2.3. GC–MS analyses The chemical compositions of the dry M. haplocalyx and the fresh M. haplocalyx and S. tenuifolia EOs were analyzed by GC–MS. The GC apparatus was an Agilent Technologies 7890B-5977B with an apolar Agilent HP-5MS capillary column (30 m ×0.25 mm, film thickness of 0.25 μm). The flow rate of carrier gas helium was 1.5 mL/min. The ionization potential, electron ionization detector and injector temperatures were set at 70 eV, 250℃ and 230℃, respectively. The scanning range was 30−550 m/z. The compositions were identified in the NIST 14 Libraries database. 2.4. Minimum inhibitory concentration (MIC) assay Minimum inhibitory concentration values were determined on the basis of the methods described by Ma et al. (2019). Briefly, the EOs, main components and hymexazol were prepared in 20/1000 DMSO with 1/1000 Tween-80 (2-DMSO-T) as a suspension, and filtered through 0.22 μm Millipore filters. The EOs concentrations ranged from 75.00-0.15 mg/mL, and the main compositions and hymexazol were also obtained in eight concentrations, ranging from 2.50-0.0048 mg/mL by two-fold dilution. The fungi (except R. solani) were added to 20 mL of 1/4 liquid Potato Dextrose Agar (PDA); the concentration of the fungal suspensions was then adjusted to 1 × 104 spores/mL. The fungal suspensions (150 μL) and filter-sterilized EOs (50 μL) were mixed in the wells of a 96-well microtiter plate. The positive control was 150 μL of fungal suspension with 50 μL of 2-DMSO-T. Eight replicates were carried out for each sample. All treatments were maintained at 28℃. The absorbance of the suspensions was measured at 595 nm with a microplate reader (1510, Thermo, Thermo Fisher Scientific Co. Ltd., Waltham, USA). The antifungal activity was ascertained based on the absorbance.

2.7. Analysis of the antifungal activity of M. haplocalyx EO against spore germination rates, dry mycelial weight and spore yields of F. oxysporum The spore germination rates of F. oxysporum were determined with the surface germination method in 2 % water agar medium (Chen et al., 2012). Under aseptic conditions, F. oxysporum was added to 20 mL of sterile water and was then adjusted to 1 × 104 spores/mL. Next, 99 mL of water agar and 1 mL of M. haplocalyx EO or hymexazol were diluted with 2-DMSO-T in an Erlenmeyer flask to obtain the final EO concentrations of 2 MIC, MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC, and 1/16 MIC. Each culture dish then received 20 mL of water agar medium. Hymexazol and 2-DMSO-T were used as positive and negative control, respectively. After the culture medium was cooled, 25 μL of spore suspension was added to each culture dish. The spore suspensions were evenly coated in a clockwise direction with a sterile coating rod on the medium. After uniform coating, the petri dishes were placed in a microbial incubator for 72 h at 28℃ in the dark. Each treatment was repeated five times, and the spore germination rates were calculated. Germination rate (%) = germination spores / total spores × 100 The dry mycelial weight and spore yields of F. oxysporum were previously described by Zhang et al. (2018). M. haplocalyx EO and hymexazol were diluted with 2-DMSO-T and added to Erlenmeyer flasks containing 99 mL of PDA medium and 1 mL of F. oxysporum to obtain the final concentrations of 2 MIC, MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC and 1/16 MIC. Hymexazol and 2-DMSO-T were used as the positive control and negative control, respectively. The flasks were shaken at 180 rpm and 28℃ for 5 days in a constant temperature oscillator. Hyphae were obtained by vacuum suction filtration after culturing, and the residue on the filter paper was dried at 80℃; the dry mycelial

2.5. Synergistic effects of M. haplocalyx and S. tenuifolia EOs and chemical fungicide The chessboard method was used to determine the synergistic effects between a chemical fungicide (hymexazol) and plant-based EOs, with minor modifications (Lv et al., 2011). The MIC values of M. haplocalyx EO, S. tenuifolia EO and hymexazol were obtained from the above MIC assay. The respective concentrations ranging from 8 MIC-1/ 16 MIC were obtained using two-fold dilution. The filter-sterilized EOs, hymexazol and fungal suspensions were added to a 96-well microtiter plate. The subsequent steps were the same as in section 2.4. The synergistic effects between EOs and hymexazol were evaluated by the fractional inhibitory concentration index (FICi) values (Gutierrez et al., 2008): ∑ FICi = FIC (EO) + FIC (hymexazol) 3

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following formula: Relative electrical conductivity (%) =(V1-V0) / V2 × 100

weight was then measured. In the above experiments, the spores were determined by a biological microscope (xs-212-202, JEOEC, Co. Ltd., Tokyo, Japan), and the spores yields were finally obtained. Each treatment was performed five times with two replicates each.

2.12. Determination of SDH and NADH oxidase activities

2.8. SEM and TEM

The SDH and NADH activities were measured in 0.1 g of fresh mycelia grounded with liquid nitrogen, following the kit instructions (Beijing Splarbio Science and Technology Co., Ltd., Beijing, China).

Fusarium oxysporum fungal suspensions treated with the M. haplocalyx EO at different concentrations (control, MIC, 1/4 MIC) were observed by SEM (S-3000 N, Hitachi, Co. Ltd., Tokyo, Japan) and TEM (JEM-1011, JEOL, Co. Ltd., Tokyo, Japan) according to a previous study, with minor modifications (Devi et al., 2010; Li et al., 2016). In a sterile environment, 99 mL of PDA liquid medium and 200 μL F. oxysporum fungal suspensions were added to a conical flask, and M. haplocalyx EO were diluted with 2-DMSO-T for final concentrations of MIC and 1/4 MIC. All treatments were shaken at 180 rpm and 28℃ for 2 days. The mycelia suspension was centrifuged at 8500 g for 8 min to obtain the mycelia and spores. The mycelia and spores were sampled and prepared in ultrathin sections and observed under SEM and TEM.

2.13. Statistical analysis Statistical analysis was performed with SPSS Statistics 19.00 using one-way ANOVA and Duncan's multiple comparisons test. 3. Results and discussion 3.1. Fungi morphology The morphological characteristics of the seven fungi are shown in Fig. 1. The colonies of F. oxysporum were examined during the 7 days of incubation on PDA at 28℃. In the early growing stage, white aerial mycelia were more frequently formed, which then became dark purple in the later growing stage (Fig. 11a). Hyphae were septate (Fig. 1, 2a). Fusarium oxysporum produced abundant oval and ellipsoid microconidiophores with 1–2 septa measuring 3.16–8.53 × 4.39–11.21 μm (Fig. 1, 3a). Macroconidia were inequilaterally spindle-shaped with three septa measuring 5.70–20.34 × 8.92–23.33 μm in size. These mycological characteristics were in accordance with previous descriptions of F. oxysporum (Booth, 1970). The colony morphology, mycelia structure and spore morphology of F. solani are shown in Fig. 1, 1b, 2b, and 3b. The aerial mycelia were white, flourishing and nonseptate (Fig. 1, 1b and 2b) and had many microconidia (Fig. 1, 3b). The microconidia were long-oval, oval, or kidney-shaped, measuring 6.10–13.56 × 2.08–3.94 μm with usually one septum, and were similar to those in a previous report (Booth, 1971). The mycelia of C. destructans was initially white and then became rust-colored. The terminal branches were usually more appressed, whereas the basal branches were often divergent; more importantly, it had no septa (Fig. 1, 1c and 2c). Conidiophores were abundant and reached a golden-to-brown color (Fig. 1, 3c). The conidia usually contained 1–3 septa measuring 2.10–6.52 × 4.71–9.12 μm each. The mycelia and conidiophores were the same as found by Mao et al. (2014). A previous study reported that P. aphanidermatum was one of the most important plant pathogenic fungi that could cause crop decay and root rot (Chen et al., 2009). The colony of P. aphanidermatum was circular on PDA after 2 days of incubation; the texture was cotton-like, and the aerial mycelia were flourishing (Fig. 1, 1d). The mycelia were not separated and had no septa (Fig. 1, 2d), and the oospores were globular, smooth and 4.50–8.12 μm in size (Fig. 1, 3d). As a soil-borne underground disease, the fungus colony morphology, mycelia structure and spore morphology were similar to a previous report (Chen et al., 2009). The conidiophores of B. cinerea were clustered and branched; and were pale white in the early stage and became gray-white in the later stage (Fig. 1, 1e). There was multiple septum of the mycelia, whose top was spherical (Fig. 1, 2e). Conidia (4.05–9.23 μm) were spherical subspherical and dark gray; and were clustered in the top of the branch and formed a grape spike, which could easily fall off (Fig. 1, 3e). The appearance of hypha and conidia were the same as described in a previous report (Zhang et al., 2017). The colonies of C. gloeosporioides were soft and villous. The edge was neat, and the septum hypha was white (Fig. 1, 2f) in the early growth stage, subsequently changing to light brown (Fig. 1, 1f). The conidia were single-celled, colorless and round and 3–9 μm in size (Fig. 1, 3f). There are few reports on the morphology of C. gloeosporioides from P. notoginseng, and the results of the current study could provide useful

2.9. Preparation of F. oxysporum mycelia Under aseptic conditions, 1 mL of F. oxysporum fungal suspension and 99 mL of PDA medium was added to an Erlenmeyer flask and incubated in an orbital shaker at 180 rpm and 28℃. After 4 days, mycelia were collected and washed three times with sterile distilled water and prepared for the following tests. 2.10. The effect of M. haplocalyx EO on soluble protein content and reducing sugar content of F. oxysporum mycelia Essential oil of M. haplocalyx was diluted with 2-DMSO-T and then added to Erlenmeyer flasks containing 40 mL of sterile distilled water and 2 g of fresh mycelia to attain the final concentrations of 2 MIC, MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC and 1/16 MIC. All samples were centrifuged at 9500 rpm for 5 min at 4℃. The mycelia soluble protein content was determined by Coomassie brilliant blue G-250 solution (Hatada et al., 2006). The clear upper layer liquid (0.2 mL) was added to 1 mL of G-250 solution, and the absorbance was determined at 595 nm after 2 min of mixing. Each treatment was repeated four times. The soluble protein content (mg) was calculated using a protein standard curve. The reducing sugar content of the mycelia was determined by the 3,5-dinitrosalicylic acid (DNS) method (Hatada et al., 2006). The clear upper layer liquid (0.5 mL) was mixed with 0.5 mL of DNS and then boiled for 5 min. The mixture was cooled to room temperature and added to 4 mL of sterile distilled water. The absorbance was determined at 540 nm. The reducing sugar content (μg) was calculated according to the reducing sugar standard curve. Each treatment was repeated four times, and two experiments were performed. 2.11. The effect of M. haplocalyx EO on the relative electrical conductivity of F. oxysporum The relative electrical conductivity was studied according to the method of Yan et al. (2010). Two grams of fresh mycelia was blended with 40 mL of sterile distilled water and M. haplocalyx EO to attain the final concentrations of 2 MIC, MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC and 1/ 16 MIC. Hymexazol and 2-DMSO-T were used as positive and negative control, respectively. After mixing, the electrical conductivity was measured as the initial conductivity value (V0). The electrical conductivity values of the solutions were determined every 30 min by an electrical conductivity meter; being denoted as V1. After 8 h, the sterile distilled water containing the F. oxysporum mycelia was boiled for 5 min and cooled at room temperature to determine the final conductivity (V2). The relative electrical conductivity was calculated using the 4

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Fig. 2. (A) Inhibition of M. haplocalyx and S. tenuifolia essential oils (EOs) on seven fungi. Pathogenic fungi were (a) F. oxysporum, (b) F. solani, (c) C. destructans, (d) P. aphanidermatum, (e) B. cinerea, (f) C. gloeosporioides and (g) R. solani. The different treatments were (1) 1-DMSO-Tween was as negative control, (2) M. haplocalyx EO, (3) S. tenuifolia EO, (4) Hymexazol was as positive control. (B) Inhibition rates of M. haplocalyx and S. tenuifolia EOs on seven fungi. Each data point represents the mean ± SD of five replicates. Different letters on the same treatment represent significant differences (P < 0.05) among different fungus.

extensively used in the pharmaceutical industry; therefore, issues regarding its diseases receive substantial attention. The seven fungal strains selected in this study could cause P. notoginseng diseases, including underground diseases (F. oxysporum, F. solani, C. destructans and P. aphanidermatum) and aboveground diseases (B. cinerea, C. gloeosporioides and R. solani), which seriously reduce the yield and quality of P. notoginseng (Miao et al., 2016; Ling et al., 2017). From the experimental results, we found that M. haplocalyx and S. tenuifolia EOs had strong inhibitory effects on the above fungi, all of which were 100.00 %. These pathogens infest the plant mainly through spores that first attack the host cells; the fungus then produces a large number of spores, and finally invades into the host cells, killing them (Lengeler et al., 2000). M. haplocalyx and S. tenuifolia EOs could fundamentally suppress the mycelia growth via diffusion.

and reliable information for future research. Rhizoctonia solani belongs to Agonomycetaceae, which do not produce spores (Deng et al., 2005). Therefore, the spore morphology could not be observed for this fungus, and the MIC assay could not be performed. The mycelia of R. solani were cotton-flocculent and spider-filamentous, initially colorless or white, gradually becoming varying degrees brown, and producing brown pigments (Fig. 1, g). The sclerotium were nearly spherical or irregular in shape, had a rough surface, were brown or black-brown in color, and were produced on the surface of the culture medium or closely attached to the cover and wall of the culture dish (Fig. 1, 2g). The microscopic morphology of R. solani has not yet been reported; thus, this is the first report of the microscopic observations of R. solani from P. notoginseng. 3.2. In vitro antifungal activity of EOs

3.3. Chemical compositions The M. haplocalyx and S. tenuifolia EOs reached inhibition rates of 100.00 % against the seven pathogenic fungi of P. notoginseng (Fig. 2B), indicating that these EOs had excellent inhibitory effects. The highest inhibition rate of hymexazol was reached on C. gloeosporioides (100.00 %), followed by B. cinerea (88.81 %), P. aphanidermatum (88.00 %), F. oxysporum (87.48 %), R. solani (86.00 %) and C. destructans (54.66 %), whereas a low inhibition was observed against F. solani (11.06 %). Panax notoginseng is a well-known medicinal plant that is

The dry M. haplocalyx and fresh M. haplocalyx and S. tenuifolia EOs were obtained by steam distillation extraction, with yields of 0.09 %, 0.15 % and 0.39 %, respectively. GC–MS analysis was carried out to describe the chemical compositions of these EOs (Table S1). A total of 49 compounds were identified in the EO from dry M. haplocalyx, being D-carvone (29.71 %) and D-limonene (7.86 %) the main components. In contrast, 40 components were identified in the EO from fresh M. 5

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3.5. Synergistic interactions

haplocalyx, 27 in common with the EO from dry samples. D-Carvone (37.87 %) and D-limonene (9.98 %) were also the main components, being higher its amounts than in the EO from dry samples probably due to the effect of storage time and treatment. These results are in accordance with the GC–MS analysis performed by Cao et al. (2011), which also detected high amounts of D-carvone and D-limonene in M. haplocalyx EO. Meanwhile, 27 compounds were described in the EO from dry S. tenuifolia, 12 in common with EO from dry M. haplocalyx and 11 with EO from fresh M. haplocalyx. The main compounds were (-)-menthone (39.23 %), p-menthan-3-one (30.28 %) and D-limonene (6.95 %), being in concordance with results obtained by Du et al. (2014). Variances in the content of EO chemical constituents for an specific plant species can be due to the cultivation conditions, climate conditions, extraction methods, soil conditions, latitude, altitude, sunshine intensity, collection season and other factors (Singh and Pandey, 2018).

There has been a considerable interest in analyze the synergistic effect between EOs to increase the antimicrobial activity. The chessboard assay has commonly been used to evaluate the synergistic effects of EO mixtures in vitro (Gutierrez et al., 2008; Lv et al., 2011). Based on the FICi values, a synergistic effect between M. haplocalyx EO and hymexazol was found against P. aphanidermatum (Table 2). However, no relevant effect was perceived against the other fungi. The combination of S. tenuifolia EO and hymexazol showed a synergistic effect on P. aphanidermatum, and the effect against F. solani was additive. There were no relevant effects on F. oxysporum, C. destructans, B. cinerea, or C. gloeosporioides. As an effective antimicrobial fungicide, hymexazol is widely used in agricultural management. Although its inhibitory effect was better, its use is limited because it easily causes the “3R” problems and does not conform to the concept of modern green scientific development (Szabó, 2008). Actually, most studies have limited to the antifungal effect of a single EO in its reports so far. In the report, in order to improve the effect of fungal inhibition, we studied the antifungal activity between the M. haplocalyx and S. tenuifolia EOs and the hymexazol. The combination of EOs and hymexazol had synergistic, additive and irrelevant effects in the study (Table 2); mostly, the existence of synergistic could sharply reduce the use of chemical agents, which could reduce the pesticide residues (Mutlu et al., 2010; Zhang et al., 2015), and ensure the safety of human being and environment (Wu et al., 2004). Therefore, the synergistic effects is potential to achieve the goals of avoiding fungicide residues and developing green degradable EOs as botanical fungicides. The synergistic effects appeared to be due to various inhibition mechanisms between the EO and agricultural chemical. However, the mechanism of action is still unclear, so that more assays are needed to done.

3.4. Determination of MIC Experiments of MIC were carried out to investigate which principal components of the EOs played an antifungal role. The MIC assay results showed that the dry M. haplocalyx and S. tenuifolia EOs and its major compounds had strong inhibitory effects against the fungi of P. notoginseng (Table 1). The data showed that different EOs and major monomer compounds had different inhibition effects on six strains from P. notoginseng, which indicates that EOs and major compounds had selective inhibition effect. And the antifungal effect of major constituents was higher than the EOs. Other studies have shown that Mentha EO possesses excellent inhibitory activity against Fusarium spp. in vitro (Chessa et al., 2013). In this study, D-carvone and D-limonene were the main compounds of M. haplocalyx EO, whose antifungal activity were stronger than that of M. haplocalyx EO. The MIC values of Dcarvone against F. oxysporum, F. solani, C. destructans, P. aphanidermatum, B. cinerea and C. gloeosporioides ranged from 0.13 mg/mL to 0.62 mg/mL. D-Carvone, D-limonene, and terpenoid compounds have attracted considerable interest owing to its significant antifungal activities (Palá-Paúl et al., 2006). Hymexazol was a commonly used chemical fungicide, which possesses a better antifungal effect (Fard et al., 2016), and it also had lower MIC values, varying from 0.03 mg/ mL to 0.31 mg/mL. It was worth mentioning that inhibition effect of Dcarvone had the same order of magnitude as that of hymexazol. The results revealed that the active ingredients in EOs could significantly inhibit the pathogen spore germination and mycelial growth. The phenomenon may hypothesize that the antifungal properties of the EOs could be attributable to its main constituents. These findings provide a theoretical basis for the application of EOs instead of chemical pesticides.

Table 1 MIC [1] values of EOs

[2]

It could be seen the MIC values of M. haplocalyx and S. tenuifolia EOs and its major compounds from Table 1, the data revealed that the inhibition effect of M. haplocalyx EO against fungi from P. notoginseng were higher than that of S. tenuifolia EO, mostly, the dry M. haplocalyx had strong inhibitory effects against the F. oxysporum than other fungi. In order to explain it more clearly, we chose F. oxysporum and M. haplocalyx as the main research object, to illustrate the antifungal mechanism of action. The disease incidence, disease index and biomass of P. notoginseng were determined, as shown in Table 3. The disease incidence and index of P. notoginseng increased significantly after F. oxysporum inoculation, especially in the disease incidence of 94.00 %, and there was a significant difference between CK and FMO. The disease incidence and index of Fo were higher than those of CK and FMO after F. oxysporum inoculation. It was clear that the growth of P.

from M. haplocalyx and S. tenuifolia on seven fungi (mg/mL). F. oxysporum

M. haplocalyx S. tenuifolia D-carvone D-limonene (-)-menthone p-menthan-3-one Hymexazol

3.6. The curative activity of M. haplocalyx EO against F. oxysporum infection of P. notoginseng

0.95 4.39 0.40 0.42 0.29 1.93 0.12

± ± ± ± ± ± ±

0.11c 0.29a 0.16cd 0.07cd 0.07d 0.37b 0.01d

F. solani 2.61 4.69 0.37 0.84 1.15 0.72 0.16

± ± ± ± ± ± ±

C. destructans 0.26b 0.00a 0.06de 0.16cd 0.10c 0.16c 0.00d

2.35 5.57 0.32 0.78 0.92 0.63 0.18

± ± ± ± ± ± ±

0.00b 0.88a 0.09c 0.10c 0.17c 0.18c 0.02c

[1]

P. aphanidermatum 1.47 1.14 0.13 0.25 0.11 0.46 0.31

± ± ± ± ± ± ±

0.27a 0.28a 0.03b 0.04b 0.02b 0.14b 0.00b

B. cinerea 1.72 1.68 0.26 0.63 1.09 0.86 0.03

± ± ± ± ± ± ±

0.32a 0.24a 0.04cd 0.10bc 0.10b 0.15b 0.01d

C. gloeosporioides 0.72 2.13 0.62 1.77 0.92 2.14 0.15

± ± ± ± ± ± ±

0.28bc 0.22a 0.16bc 0.34a 0.31b 0.23a 0.01c

MIC: minimum inhibition concentration. EOs: essential oils. Note: The data are reported as the means ± SD of eight samples. Different letters on the same column were tested for significance by using ANOVA (analysis of variance) with a significance level of P < 0.05.

[2]

6

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Table 2 The FIC [1] index of EOs

M. haplocalyx FICi Results S. tenuifolia FICi Results

[2]

from M. haplocalyx and S. tenuifolia on six fungi. F. oxysporum

F. solani

C. destructans

P. aphanidermatum

B. cinerea

C. gloeosporioides

2.06 Irrelevant 4.00 Irrelevant

1.50 Irrelevant 0.56 Addition

2.06 Irrelevant 1.06 Irrelevant

0.13 synergy 0.31 synergy

1.06 Irrelevant 1.13 Irrelevant

1.13 Irrelevant 1.06 Irrelevant

[1]

FICi: fractional inhibitory concentration index. EOs: essential oils. Note: ∑ FICi = FIC (EO) + FIC (hymexazol). where FIC (EO) = MIC (EO) in combination/MIC (EO) alone; FIC (hymexazol) = MIC (hymexazol) in combination/MIC (hymexazol) alone. The ∑ FICi values were interpreted as follows: synergy relationship (FICi ≤0.5); addition relationship (0.5 < FICi ≤ 1); irrelevant relationship (1.0 < FICi ≤ 4.0); antagonistic relationship (FICi > 4.0).

[2]

3.8. SEM and TEM

notoginseng was poor when inoculated with F. oxysporum (Fig. 3). The invading pathogenic fungus causes vascular blockage and prevents the flow of water, and the aerial part eventually wilts and dies (Pivonia et al., 2002). The plant heights of CK, Fo and FMO were 11.72, 10.67 and 10.58 cm, respectively, we found that there was no significant difference in plant height among Fo, FMO and CK, which may be due to the individual differences of P. notoginseng. Regarding the biomass of P. notoginseng, CK and FMO had significant differences in the fresh and dry weights of the roots, stems and leaves of P. notoginseng compared with Fo. The F. oxysporum is an important pathogen that causes root rot in P. notoginseng. When disease occurs, M. haplocalyx EO combined with hymexazol can be used against the F. oxysporum. With the improvement of living standards and the further enhancement of safety awareness, the antimicrobial activities of plant EOs will have broad developmental prospects. The use of plant EOs in disease control may be a major trend in the future. At present, EO research mainly focuses on finding a wide range of antimicrobial plants and elucidating its antimicrobial activities. However, the antifungal mechanisms of EOs are not yet clear. Here, F. oxysporum was used as a representative strain to study the effects when treated with M. haplocalyx EO.

In vitro studies have shown that M. haplocalyx EO can inhibit the growth of F. oxysporum, so we investigated whether its internal morphology changes. After interpreting the SEM results, we found that the application of M. haplocalyx EO substantially altered the morphology of the mycelia and spores of F. oxysporum. The cell treated with M. haplocalyx EO at 1/4 MIC and MIC, the mycelia and spores appeared distorted with discontinuous areas (Fig. 4B and C). Moreover, when the concentration of M. haplocalyx EO reached the MIC, spores were seldom seen and the mycelia misshapen. The transmission electron microscope could decipher the morphology of the membrane system, mitochondria and vacuoles. The M. haplocalyx EO induced morphological changes in F. oxysporum when incubated with different concentrations for 2 days (Fig. 4). In the control treatment, the cells had a smooth appearance, and the cell wall and cell membrane were uniform, and the mitochondria and vacuoles were observable clearly (Fig. 4a). In contrast, when treated with 1/4 MIC, the fungal cell structures were destroyed, including the cell wall and vacuoles. The major disruption was in the vacuoles, where the volume was increased clearly. Furthermore, the endomembrane system was seriously damaged, including the cell wall and cell membrane, resulting in cells with inhomogeneous thickening (Fig. 4b). When F. oxysporum was treated with the MIC of M. haplocalyx EO, the cell wall was broken, causing the cell contents to flow out, and the mitochondria were indistinct and unidentifiable. Additionally, the internal morphology was markedly disrupted. The F. oxysporum mycelia gradually lost integrity after treatment with M. haplocalyx EO and eventually shrank (Fig. 4c). Many studies have demonstrated the antifungal effects of EOs, but the mechanism has not yet been clarified (Tajkarimi et al., 2010). SEM has also been used to show that EOs can destroy cellular structures, these finding are in accordance with this study (Di Pasqua et al., 2007; Oussalah et al., 2006). Membrane system alterations, such as to the cell wall, cell membrane and cytoplasm (swelling and leakage) were observable by TEM. These results illustrated that M. haplocalyx EO possessed antifungal activity against F. oxysporum causing irreversible changes. The results showed that mitochondria were disrupted, which indicated that M. haplocalyx EO could pass through the cell wall, and then negatively impact organelles. Previous research has shown morphological alterations in Candida albicans via TEM observations, along with irreversible damage to the cell wall, cell membrane and organelles of C. albicans by the EO of Allium sativum L. (garlic oil) (Li et al., 2016). Garlic oil can penetrate the cell membrane due to its lipophilic character (Rasooli and Owlia, 2005; Nogueira et al., 2010). The TEM observations showed that the mitochondria and vacuoles were also damaged in C. albicans cells after treatment with garlic oil (Li et al., 2016). Another study also observed damage in Penicillium funiculosum treated with garlic oil and indicated that EOs could penetrate the mitochondrial membranes (Li et al., 2014). The internal structure of the mitochondria

3.7. Antifungal effect of M. haplocalyx EO against F. oxysporum in vivo The spore germination rates, mycelia dry weights and spore yields of F. oxysporum treated with a series of M. haplocalyx EO concentrations are shown in Table 4. When F. oxysporum was treated with a series of M. haplocalyx EOs (2 MIC, MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC, 1/16 MIC), the spore germination rates were 0 %, 1.84 %, 11.33 %, 27.44 %, 39.00 % and 45.20 %, respectively; however, when treated with hymexazol (MIC), the spore germination rate was 50.13 %. The mycelia dry weights were 0.04 g, 0.04 g, 0.23 g, 0.45 g, 0.57 g and 0.59 g, respectively, when treated with 2 MIC, MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC, 1/16 MIC. The inhibitory effect of hymexazol (MIC) was close to 1/2MIC. A range of M. haplocalyx EO concentrations (2 MIC, MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC, 1/16 MIC) against F. oxysporum showed spore yields (×105 spores/mL) of 0.18, 0.24, 0.33, 2.60, 3.62, and 9.08, respectively. When treated with hymexazol, the spore yield was 0.44 × 105 spores/mL. In the present study, we were selected F. oxysporum as the representative strain and explored the antimicrobial mechanism of M. haplocalyx EO through the following experiments. The infection process by F. oxysporum usually goes through four stages: (1) The spores are first germinated and then the hyphae adhere to the root surface; (2) The spores and mycelia enter the root cortex; (3) The spores grow and reproduce in vascular bundles; (4) The spores and mycelia invade the plant tissues and produce chlamydospore to complete the infection process (Di Pietro et al., 2010). These results indicated that the M. haplocalyx EO had high dose-dependent inhibitory activity against the spore germination rates, mycelia dry weights and spore yields of F. oxysporum, which may be one of the antifungal mechanisms of EO. 7

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5.84 ± 0.84 62.00 ± 3.89a 9.17 ± 0.83b CK Fo FMO

17.50 ± 2.50 94.00 ± 2.45a 25.00 ± 2.89b

b b

Note: CK: The healthy P. notoginseng seedlings were transplanted into culture medium directly. Fo: The P. notoginseng seedlings inoculated with F. oxysporum for 3 h, which were then transplanted into culture medium. FMO: The P. notoginseng seedlings inoculated with F. oxysporum for 3 h, which were then transplanted into the culture medium containing 1/8 MIC (minimum inhibitory concentration) M. haplocalyx EO (0.12 mg/mL). Different letters on the same column were tested for significance by using ANOVA (analysis of variance) with a significance level of P < 0.05.

15.19 ± 0.81ab 14.04 ± 1.36b 17.95 ± 0.58a 12.99 ± 0.01 11.56 ± 0.66a 12.83 ± 0.36a 2.69 ± 0.22 1.76 ± 0.38b 3.15 ± 0.17a 138.44 ± 5.36 80.37 ± 4.37b 120.67 ± 4.43ab 191.88 ± 5.92 131.90 ± 4.56b 188.03 ± 3.56a 11.72 ± 0.25 10.67 ± 0.25b 10.58 ± 0.31b

37.78 ± 2.52 23.55 ± 5.79b 43.26 ± 3.34a

ab

Root

a

Fresh weight (mg) Plant height (cm) Disease index Disease incidence (%) Treatments

Table 3 The curative activity of M. haplocalyx EO against F. oxysporum infection of P. notoginseng.

Stem

a

Leaf

a

Dry weight (mg)

Root

a

Stem

a

Leaf

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suffered severe damage and finally dissolved. According to the Fig. 4, the cell wall may be a target of the M. haplocalyx EO. Thus, the current results showed that M. haplocalyx EO could be used as a botanical fungicide to damage the cell integrity. 3.9. Effect of M. haplocalyx EO on F. oxysporum mycelia content of soluble protein and reducing sugar The mycelia soluble protein content of F. oxysporum is shown in Fig. 5A. There was no difference between seven treatments and the 2DMSO-T within the first 6 h, then the soluble protein content of 2MIC, MIC, 1/2 MIC and 1/4MIC increased sharply until it peaked at 120 h and remained stable, thereafter, the F. oxysporum mycelia soluble protein content reached 99.25, 87.67, 74.45 and 71.25 mg/mL, respectively. When treated with 2 MIC, MIC, 1/2 MIC and 1/4 MIC after 120 h of incubation, the respective soluble protein contents were 82.27 %, 61.01 %, 36.73 % and 30.85 % higher than that of 2-DMSO-T. There was no significant difference in the content change of the 1/8 MIC, 1/ 16 MIC, 2-DMSO-T and hymexazol treatments. As shown in Fig. 5B, the F. oxysporum mycelia reducing sugar content when treated with 2 MIC M. haplocalyx EO continuously increased sharply after incubation for 120 h, finally reaching 0.09 μg/mL. After incubation for 120 h, the reducing sugar content increased to 0.04 μg/mL when treated with the MIC. There was no significant difference among five treatments (1/2 MIC, 1/4 MIC, 1/8 MIC, 1/16 MIC, hymexazol) and the 2-DMSO-T over the120 h incubation. To confirm the targets of M. haplocalyx EO in the membrane system, the soluble protein and reducing sugar contents were determined. Soluble protein and reducing sugars are indispensable cellular components and are fundamental to the function of the fungi. The disturbance of M. haplocalyx EO to the integrity of F. oxysporum was assessed by measuring the soluble protein and reducing sugar contents. A previous report illustrated that EOs attacked the cell wall or cell membrane of the microorganism, damaging the function of the cell membrane and causing the cell contents to leak out; the fungi then eventually died (Denyer et al., 1991). Other studies have shown that the membrane system is the main target of EOs to defeat pathogens (Tian et al., 2012; Yu et al., 2015). The study provides a theory in which the F. oxysporum cell wall may be a primary target of M. haplocalyx EO. 3.10. Effect of M. haplocalyx EO on F. oxysporum cell membrane permeability An additional antifungal mode of action of M. haplocalyx EO against F. oxysporum was confirmed using a cell membrane permeability assay by measuring the relative electrical conductivity. Fig. 5C shows the effect of M. haplocalyx EO on the F. oxysporum cell membrane permeability. Compared with the 2-DMSO-T, the conductivity of M. haplocalyx EO suspension increased significantly in treatments with three concentrations (2 MIC, MIC, 1/2 MIC) after 1.5 h of cultivation. This figure illustrates that this effect occurred in a concentration-dependent manner between the M. haplocalyx EO and F. oxysporum. A slight change to this trend in the other treatments (1/4 MIC, 1/8 MIC, 1/16 MIC and hymexazol) compared with the 2-DMSO-T was observed. Ion homeostasis is important for maintaining the normal metabolic activity of cells (Cox et al., 2001). In fungi, the cell membrane permeability of small ions such as K+, Na+ and H+, which are essential for promoting cell membrane function, helps to maintain a normal metabolism and enzyme activity (Diao et al., 2014). When the fungal cell membrane was destroyed, the cellular contents flowed out; thus, the electrical conductivity sharply increased (Fig. 5C), might be due to fungal cytolysis (Diao et al., 2014; Zhang et al., 2016), finally resulting in cell death (Sharma et al., 2013). The lipophilicity of EOs enables them to preferentially enter the membrane system of the fungi, resulting in membrane leakage and increased membrane permeability and fluidity, thus disturbing the intracellular and extracellular ion 8

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Fig. 3. The curative activity of M. haplocalyx essentail oil (EO) against F. oxysporum infection of P. notoginseng. CK: The healthy P. notoginseng seedlings were transplanted into culture medium directly. Fo: The P. notoginseng seedlings inoculated with F. oxysporum for 3 h, which were then transplanted into culture medium. FMO: The P. notoginseng seedlings inoculated with F. oxysporum for 3 h, which were then transplanted into the culture medium containing 1/8 MIC (minimum inhibitory concentration) M. haplocalyx EO (0.12 mg/mL).

balance. The integrity of the cell membrane is critical for F. oxysporum growth. Therefore, we could investigate the antifungal mechanism by analyzing the leakage of cellular constituents.

Table 4 Inhibitory effects of M. haplocalyx EO on spore germination rates, mycelial dry weights and spore yields of F. oxysporum. Different treatments

Spore germination rates (%)

Mycelial dry weights (g)

2 MIC MIC 1/2 MIC 1/4 MIC 1/8 MIC 1/16 MIC Hymexazol 2-DMSO-T

0.00 ± 0.00f 1.84 ± 0.64f 11.33 ± 1.09e 27.44 ± 2.41d 39.00 ± 1.18c 45.20 ± 1.53b 50.13 ± 3.49b 84.16 ± 2.69a

0.04 0.04 0.23 0.45 0.57 0.59 0.24 0.62

Spore yields (×105 spores/mL)

3.11. Effect of M. haplocalyx EO on the respiratory chain of F. oxysporum ± ± ± ± ± ± ± ±

0.02d 0.00d 0.03c 0.03b 0.03a 0.02a 0.03c 0.03a

0.18 ± 0.02c 0.24 ± 0.00c 0.33 ± 0.04c 2.60 ± 0.13b 3.62 ± 0.32b 9.08 ± 0.50a 0.44 ± 0.06c 10.53 ± 1.41a

From the data, we found that as the concentration of M. haplocalyx EO increased, the activities of SDH and NADH oxidase in F. oxysporum gradually decreased in a clear gradient (Fig. 6). According to research, SDH and NADH exist widely in animals, plants and microorganisms and play a vital role in regulating the metabolism of fungi. And SDH is a mitochondrial marker enzyme. It is a membrane-binding enzyme located on the inner membrane of mitochondria and is one of the key links between respiratory electron transport and oxidative phosphorylation. This enzyme is not only involved in the regeneration of NAD + but is also closely related to the immune response. NADH oxidase is also an oxidoreductase that can oxidize NADH to NAD + directly in the presence of oxygen (Piasecka et al., 2001). In addition, it

MIC: minimum inhibitory concentration. EO: essential oil. Note: The data are reported as the means ± SD of five samples. Different letters on the same column were tested for significance by using ANOVA (analysis of variance) with a significance level of P < 0.05.

Fig. 4. The internal morphological of F. oxysporum were observed by SEM (A, B and C) and TEM (a, b and c). (A, a) control; (B, b) The F. oxysporum were treatment with M. haplocalyx EO at the concentration of 1/4 MIC for 2 days; (C, c) The F. oxysporum were treatment with M. haplocalyx EO at the concentration of MIC for 2 days. SEM: scanning electron microscope; TEM: transmission electron microscope; EO: essential oil; S: spore; M: Mycelia; CW: cell wall; CM: cell membrane; MIT: mitochondria; V: vacuoles. 9

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Fig. 5. The effect of different concentrations of M. haplocalyx essential oil (EO) on soluble protein content (A), reducing sugar content (B) and relative electrical conductivity (C) of F. oxysporum.

treatments.

provides electrons for the respiratory chain in various prokaryotic cells. A previous study revealed that NADH oxidase can be used as a scavenger of intracellular oxygen (Higuchi et al., 2000). The study showed that Curcuma longa could decrease the activity of SDH and NADH oxidase, which might inhibit the TCA cycle and disturb ATP synthesis in the mitochondria, indicating that there are inhibitory effects on the respiratory chain of Fusarium graminearum (Higuchi et al., 2000). The results also found that M. haplocalyx EO suppressed the activity of SDH and NADH oxidase, indicating that the EO could interfere with the respiratory chain in F. oxysporum. The current study showed that the M. haplocalyx EO could disrupt the structure of fungal cells, which may ultimately inhibit the spore germination and mycelia growth. The antifungal effects were related to the disruption of the fungal cell membrane systems and respiratory chain. Future studies should ascertain the EO active ingredients, identify its structures, and modify and synthesize these structures to find targets, specific mechanisms of action, and unique and highly active new fungicides to provide preventive and comprehensive agricultural

4. Conclusion This study demonstrated that M. haplocalyx and S. tenuifolia EOs and its main compounds had excellent antifungal activities against the pathogenic fungi of P. notoginseng in vivo and in vitro. There were good synergistic effects between the EOs and hymexazol against some P. notoginseng pathogens. The spore germination rates, mycelia dry weights and spore yields sharply decreased with increasing M. haplocalyx EO concentration. Changes in the mycelia soluble protein and reducing sugar contents and a decrease in the activities of SDH and NADH oxidase were induced. The cell wall and organelles appear to be the main targets of the M. haplocalyx EO. Altogether, M. haplocalyx EO can penetrate and dissolve the membranes of organelles such as the mitochondria, resulting in fungi growth inhibition. Overall, M. haplocalyx EO and its major compounds show potential to be used natural alternatives to commercial fungicides for controlling the pathogens of

Fig. 6. The effect of different concentrations of M. haplocalyx essential oil (EO) on succinate dehydrogenase activity (A) and NADH oxidases activity (B) of F. oxysporum. 10

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P. notoginseng. Then could provide the basis for developing alternative, pollution-free fungicides.

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Author contributions Yong-Xian Cheng and Xian Dong designed the experiment. Xian Dong and Chuan-Jiao Chen analyzed the data and wrote the paper. Chuan-Jiao Chen, Qing-Qing Li, Zi-Ying Zeng, Su-Su Duan and Wei Wang performed the experiments. Fu-Rong Xu commented the paper. All authors have read and approved the manuscript. Funding This work was financially supported by the National Key Research and Development Program of China “Research and Development of Comprehensive Technologies on Chemical Fertilizer and Pesticide Reduction and Synergism” (2017YFD0201402), the National Natural Science Foundation of China (81660626), Yunnan Provincial Science and Technology Department-Applied Basic Research Joint Special Funds of Yunnan University of Chinese Medicine [2019FF002(-003)], [2017FF116 (-014)], Yunnan Applied basic Research Program-Youth Project (2018FB139), the Scholarship of China Scholarship Council (CSC) and Yunnan Provincial Department of Education Major Project (2019Y0310). Declaration of Competing Interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.112073. References Booth, C., 1970. Fusarium oxysporum. CMI Descriptions of Pathogenic Fungi and Bacteria. Kew, Survey, England. Booth, C., 1971. The Genus Fusarium. Commonwealth Mycological Institute, Kew, Survey, England. Chen, Y., Zhang, A.F., Gao, T.C., Zhang, Y., Wang, W.X., Ding, K.J., Chen, L., Sun, Z., Fang, X.Z., Zhou, M.G., 2012. Integrated use of pyraclostrobin and epoxiconazole for the control of Fusarium head blight of wheat in Anhui province of China. Plant Dis. 96, 1495–1500. https://doi.org/10.1094/pdis-01-12-0099-re. Chen, C.J., Li, Q.Q., Ma, Y.N., Wang, W., Cheng, Y.X., Xu, F.R., Dong, X., 2019. Antifungal effect of essential oils from five kinds of Rutaceae plants-avoiding pesticide residue and resistance. Chem. Biodivers. 16, e1800688. https://doi.org/10.1002/cbdv. 201800688. Chen, Z.M., Wu, H.X., Zeng, H.F., Zhang, S.S., 2009. Identification of Pythium aphanidermatum isolated from tobacco field and the screening of Trichoderma spp. for antagonistic agent. J. Fujian Agric. For. Univ. 38, 11–15. https://doi.org/10.13323/j. cnki.j.fafu(nat.sci.).2009.01.007. Chessa, M., Sias, A., Piana, A., Mangano, G.S., Petretto, G.L., Masia, M.D., Pintore, G., 2013. Chemical composition and antibacterial activity of the essential oil from Mentha requienii Bentham. Nat. Prod. Res. 27, 93–99. https://doi.org/10.1080/ 14786419.2012.658798. Cao, G., Shan, Q., Li, X., Cong, X., Zhang, Y., Cai, H., Cai, B., 2011. Analysis of fresh Mentha haplocalyx volatile components by comprehensive two-dimensional gas chromatography and high-resolution time-of-flight mass spectrometry. Analyst 136https://doi.org/10.1039/c1an15616k. 4653-4561. Cox, S., Mann, C., Markham, J., Gustafson, J., Warmington, J., Wyllie, S., 2001. Determining the antimicrobial actions of tea tree oil. Molecules 6, 87–91. https://doi. org/10.3390/60100087. Deng, Z.S., Zhang, B.C., Sun, Z.H., Xu, W.M., Ren, J.M., 2005. Observation of vegetative mycelial pleomorphism in Rhizoctonia solani. J. Microbiol. 25, 56–58. https://doi. org/10.3969/j.issn.1005-7021.2005.06.015. Denyer, S.P., Hugo, W.B., Hugo, W.B., 1991. Mechanisms of Action of Chemical Biocides:their Study and Exploitation. Blackwell Scientific. Devi, K.P., Nisha, S.A., Sakthivel, R., Pandian, S.K., 2010. Eugenol (an essential oil of clove) acts as an antibacterial agent against Salmonella typhi by disrupting the cellular membrane. J. Ethnopharmacol. 130, 107–115. https://doi.org/10.1016/j.jep.2010. 04.025. Di Pasqua, R., Betts, G., Hoskins, N., Edwards, M., Ercolini, D., Mauriello, G., 2007. Membrane toxicity of antimicrobial compounds from essential oils. J. Agric. Food Chem. 55, 4863–4870. https://doi.org/10.1021/jf0636465.

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