molecules

Article Antifungal Effect of and from officinalis on Alternaria alternata Causing Tobacco Brown Spot

1,2, 1,3, 1 1 2 Ya-Han Chen †, Mei-Huan Lu †, Dong-Sheng Guo , Ying-Yan Zhai , Dan Miao , Jian-Ying Yue 2, Chen-Hong Yuan 1, Ming-Min Zhao 2,* and De-Rong An 1,*

1 College of Protection and State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling 712100, China; [email protected] (Y.-H.C.); [email protected] (M.-H.L.); [email protected] (D.-S.G.); [email protected] (Y.-Y.Z.); [email protected] (C.-H.Y.) 2 College of agriculture, Inner Mongolia Agricultural University, Huhhot 010018, China; [email protected] (D.M.); [email protected] (J.-Y.Y.) 3 Microbial Resources of Research Center, Microbiology Institute of Shaanxi, Xian 710043, China * Correspondence: [email protected] (M.-M.Z.); [email protected] (D.-R.A.); Tel.: +86-157-7136-0659 (M.-M.Z.); +86-158-2909-7529 (D.-R.A.); Fax: +86-0471-430-1169 (M.-M.Z.); +86-029-8708-2710 (D.-R.A.) These authors contributed equally. †  Received: 5 May 2019; Accepted: 30 May 2019; Published: 6 June 2019 

Abstract: In this study, two compounds, magnolol and honokiol, were extracted from Magnolia officinalis and identified by LC-MS, 1H- and 13C-NMR. The magnolol and honokiol were shown to be effective against seven pathogenic fungi, including Alternaria alternata (Fr.) Keissl, Penicillium expansum (Link) Thom, Alternaria dauci f.sp. solani, Fusarium moniliforme J. Sheld, Fusarium oxysporum Schltdl., Valsa mali Miyabe & G. Yamada, and Rhizoctonia solani J.G. Kühn, with growth inhibition of more than 57%. We also investigated the mechanisms underlying the potential antifungal activity of magnolol and honokiol. The results showed that they inhibited the growth of A. alternata in a dose-dependent manner. Moreover, magnolol and honokiol treatment resulted in distorted mycelia and increased the cell membrane permeability of A. alternata, as determined by conductivity measurements. These results suggest that magnolol and honokiol are potential antifungal agents for application against plant fungal diseases.

Keywords: Magnolia officinalis; magnolol; honokiol; antifungal activity; Alternaria alternata

1. Introduction Magnolia officinalis is an important herb in traditional Chinese and Japanese medicine for treating conditions such as gastrointestinal disorders, thrombotic stroke, allergic disease, typhoid fever, anxiety, and nervous disturbance [1]. M. officinalis contains a diverse group of biologically active compounds, including magnolol, honokiol, 4-O-methylhonokiol, , and other neolignan compounds [2,3]. Magnolol and honokiol are the two major secondary metabolites found in the ethanolic extracts of Chinese M. officinalis at concentrations of 1.25% and 1.81%, respectively [4,5]. Several reports showed that magnolol and honokiol possess antibacterial, anti-inflammatory, antifungal, anti-cancer, proapoptotic, and anti-osteoclastogenetic activities [3,6–11]. For example, they exhibit potent antifungal activity against human fungal pathogens, such as Trichophyton mentagrophytes, Candida albicans, and Cryptococcus neoformans [4–6,12–14]. Tobacco is an important economic plant worldwide for cigarette production. Alternaria alternata (Fr.) Keissl is the causal agent of tobacco brown spot, which is one of the most harmful diseases of

Molecules 2019, 24, 2140; doi:10.3390/molecules24112140 www.mdpi.com/journal/molecules Molecules 2019, 24, x FOR PEER REVIEW 2 of 12 Molecules 2019, 24, 2140 2 of 11 Tobacco is an important economic plant worldwide for cigarette production. Alternaria alternata (Fr.) Keissl is the causal agent of tobacco brown spot, which is one of the most harmful diseases of tobacco,tobacco, causing causing huge huge crop crop losses losses around around the the world world [15–17]. [15–17 ].In InChina, China, the the annual annual incidence incidence area area of 2 A.of alternataA. alternata is aboutis about 100,000 100,000 hm hm2, leading, leading to economic to economic losses losses [8]. [Infection8]. Infection of A. of alternataA. alternata leadsleads to the to appearancethe appearance of small of small yellowish-brown yellowish-brown round round spots spots on on the the tobacco tobacco . leaves. Initially, Initially, the the spots spots are blade-shaped and and then then change change into into a a larger larger circle circle [18], [18], which which could could significantly significantly affect affect the quantity and quality quality of of tobacco tobacco [19–21]. [19–21]. To To control control this this disease, disease, chemical chemical reagents, reagents, for for example, example, dimetachlone, dimetachlone, have been widely used in the fieldfield [[20].20]. However, However, chemical chemical agents agents have have the the potential potential to to develop fungicidefungicide resistance resistance in in plant pathogens and caus causee environmental pollution, resulting in serious health risks risks to to animals animals and and human human beings. beings. Indeed, Indeed, many many countries countries have have restricted restricted the the application application of chemicalof chemical fungicides fungicides to tocontrol control plant plant diseases diseases [2 [222].]. Therefore, Therefore, the the plant-derived plant-derived fungicides fungicides are promising alternatives, alternatives, since since they they are are mostly mostly non-phytotoxic, non-phytotoxic, systematic, systematic, readily readily biodegradable, biodegradable, and environmentallyand environmentally safe. safe. InIn recent years, many studies have reported the antifungal activity of plant-derived fungicides against A.A. alternataalternata. Plumbagin,. Plumbagin, a typea type of naphthoquinone, of naphthoquinone, isolated isolated from leaves from ofleavesNepenthes of Nepenthes ventricosa, ventricosainhibited, theinhibited growth the of growthA. alternata of A. [alternata23]. Jing [23]. et al. Jing have et al. found have found that eugenol, that eugenol, an essential an essential oil from oil fromthe buds the ofbudsSyringa of Syringa oblata Lindl,oblata exhibitedLindl, exhibited the complete the complete inhibition inhibition of mycelial of mycelial growth ofgrowthA. alternata of A., alternatasuggesting, suggesting its potential its potential application application as a fungicide as a fungicide [21]. [21]. The extracts of M. officinalis officinalis showed potent antifungal activity against many plant pathogenic fungi.fungi. However, itsits antifungalantifungal activity activity against againstA. A. alternata alternatahas has not not been been reported. reported. Here, Here, we identifywe identify two twocompounds, compounds, magnolol magnolol and honokiol, and honokiol, from M. from officinalis M. officinalis. We found. We that found these twothat activethese compoundstwo active compoundshave the potential have the to inhibitpotential mycelium to inhibit growth mycelium effectively growth and effectively to increase and cell to membraneincrease cell permeability membrane permeabilityof A. alternata of. A. Their alternata antifungal. Their eantifungalffects on Penicillium effects on Penicillium expansum (Link) expansum Thom, (Link)Alternaria Thom, Alternaria dauci f.sp. daucisolani, f.sp.Fusarium solani, moniliforme Fusarium moniliformeJ. Sheld, Fusarium J. Sheld, oxysporum FusariumSchltdl., oxysporumValsa Schltdl., mali Miyabe Valsa &mali G. Miyabe Yamada, & and G. Yamada,Rhizoctonia and solani RhizoctoniaJ.G. Kühn solani were J.G. also Kühn investigated were also in investigated vitro. in vitro. 2. Results and Discussion 2. Results and Discussion 2.1. Identification of Magnolol and Honokiol from M. officinalis 2.1. Identification of Magnolol and Honokiol from M. officinalis The purities of compounds magnolol and honokiol were identified using high-performance liquid The purities of compounds magnolol and honokiol were identified using high-performance chromatography (HPLC). As shown in Figure1, their purities were higher than 98%. The structures liquid chromatography (HPLC). As shown in Figure 1, their purities were higher than 98%. The of magnolol and honokiol were then confirmed by 1H-NMR, 13C-NMR (Figure2), and HP-MS structures of magnolol and honokiol were then confirmed by 1H-NMR, 13C-NMR (Figure 2), and spectra (Figure3), respectively. Their spectral data were identical to that of the previously reported HP-MS spectra (Figure 3), respectively. Their spectral data were identical to that of the previously literature [24–26]. reported literature [24–26].

(a) (b)

FigureFigure 1. 1. TheThe HPLC chromatogram of magnolol and honokiol. ( a) Magnolol; ( b) honokiol.

MoleculesMolecules2019 2019, 24,, 2140 24, x FOR PEER REVIEW 3 of3 11 of 12

Molecules 2019, 24, x FOR PEER REVIEW 3 of 12

HO HO

OH OH magnolol magnolol

(a) (a) HO HO HO HO

honokiol honokiol (b(b) )

FigureFigureFigure 2. Sample 2. 2. Sample Sample and andand structure structurestructure of the of thethe compound compoundcompound identified identified identified as as magnolol as magnolol magnolol and and and honokiol. honokiol. honokiol. ((aa)) Magnolol;(Magnolol;a) Magnolol; (b) honokiol.(b(b) )honokiol. honokiol.

+TOF +TOF MS: 0.0596 MS: 0.0596 min from min Sample from Sample 1 (ADRGDS20181123APCIP-2) 1 (ADRGDS20181123APCIP-2) of DRGDS20181123APCIP-2.wiff of DRGDS20181123APCIP-2.wiff Max. 6368.0 cps.Max. 6368.0 cps. TIC of TIC+TOF of +TOFMS: from MS: Samplefrom Sample 1 (ADRGDS20181123APCIP-2) 1 (ADRGDS20181123APCIP-2) of DRGDS2 of DRGDS20181123APCIP-2.wiff0181123APCIP-2.wiff (DuoSpray (DuoSpray ()) ()) Max. Max. 5.0e5 5.0e5 cps. cps. a=7.02829347220602400e-004,a=7.02829347220602400e-004, t0=2.76363532840233130e-001 t0=2.76363532840233130e-001 (DuoSpr ay(DuoSpr ()) ay ())

0.05 0.05 267.1382267.1382 5.0e5 5.0e5 63686368 4.8e5 4.8e5 6000 4.6e5 6000 4.6e5 4.4e5 4.4e5 5500 4.2e5 5500 4.2e5 4.0e5 4.0e5 5000 3.8e5 5000 3.8e5 3.6e5 3.6e5 4500 3.4e5 4500 3.4e5 3.2e5 3.2e5 4000 3.0e5 4000 3.0e5 2.8e5 2.8e5 3500 2.6e5 3500 2.6e5 2.4e5 3000 2.4e5Intensity, cps 2.2e5 Intensity, cps 3000 Intensity, cps 2.2e5 2.0e5 Intensity, cps 2500 2.0e5 1.8e5 2500 1.8e5 1.6e5 2000 1.6e5 1.4e5 2000 1.4e5 1.2e5 1500 1.2e5 1.0e5 1500 1.0e5 8.0e4 1.99 1000 6.0e4 8.0e4 0.47 1.25 1.99 261.0909 531.2543 4.0e4 1000 6.0e4 0.47 1.25 500 261.0909 531.2543 2.0e4 4.0e4 0.0 500 2.0e4 0.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.92.0 0 Time, min 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 0.0 m/z, Da 0.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.92.0 0 Time, min 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z, Da (a) (b) TIC of +TOF MS: from Sample 2 (ADRGDS20181123APCIP-1) of DRGDS20181123APCIP-1.wiff (DuoSpray ()) Max. 1.2e6 cps. +TOF MS: 0.0656 min from Sample 2 (ADRGDS20181123APCIP-1) of DRGDS20181123APCIP-1.wiff Max. 4.5e4 cps. (a) a=7.02829341773204870e-004, t0=2.62826036975815080e-001 (DuoSpray ()) (b) 0.06 1.25e6 TIC of +TOF MS: from Sample 2 (ADRGDS20181123APCIP-1) of DRGDS20181123APCIP-1.wiff (DuoSpray ()) Max. 1.2e6 cps. +TOF MS: 0.0656 min from Sample 2 (ADRGDS20181123APCIP-1)531.2523 of DRGDS20181123APCIP-1.wiff Max. 4.5e4 cps. 4.5e4 1.20e6 a=7.02829341773204870e-004, t0=2.62826036975815080e-001 (DuoSpray ()) 4.4e4 0.06 1.25e61.15e6 531.2523 4.2e44.5e4 1.20e61.10e6 4.0e44.4e4 1.15e61.05e6 3.8e44.2e4 1.10e61.00e6 3.6e44.0e4 1.05e69.50e5 3.4e43.8e4 1.00e69.00e5 8.50e5 3.2e4 9.50e5 3.6e4 8.00e5 9.00e5 3.0e43.4e4 7.50e5 8.50e5 2.8e43.2e4 7.00e5 8.00e5 2.6e43.0e4 6.50e5 2.4e4 7.50e5 2.8e4 6.00e5 7.00e5 2.2e4 Intensity, cps 5.50e5 2.6e4 6.50e5 Intensity, cps 2.0e4 5.00e5 2.4e4 6.00e5 1.8e4 4.50e5 2.2e4 Intensity, cps 5.50e5 1.6e4

4.00e5 Intensity, cps 2.0e4 5.00e5 3.50e5 1.4e4 1.8e4 4.50e53.00e5 1.2e4 1.6e4 4.00e52.50e5 1.0e4 267.1373 1.4e4 3.50e52.00e5 8000.0 548.2793 3.00e5 1.2e4 1.50e5 6000.0 2.50e51.00e5 1.0e4 4000.0 267.1373 2.00e55.00e4 8000.0 2000.0 548.2793 1.50e5 0.00 0.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.92.0 0.06000.0 1.00e5 Time, min 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 4000.0 m/z, Da 5.00e4 2000.0 0.00 0.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.92.0 0.0 (Time,c )min 100 200 300 400 500 600(d 700) 800 900 1000 1100 1200 1300 1400 1500 m/z, Da FigureFigure 3. The 3. identification The( c)identification of magnolol of andmagnolol honokiol and by high-performancehonokiol by high-performance liquid(d) chromatography liquid/ chromatography/mass spectrometry (HPLC/MS) chromatogram. (a,b) Magnolol; (c,d) honokiol. massFigure spectrometry 3. The (HPLC identification/MS) chromatogram. of magnolol (a,b) Magnolol;and ho (nokiolc,d) honokiol. by high-performance liquid chromatography/mass spectrometry (HPLC/MS) chromatogram. (a,b) Magnolol; (c,d) honokiol.

Molecules 2019, 24, 2140 4 of 11

1 Magnolol (5,50-diallyl-2,20-dihydroxybiphenyl): colorless solid; H-NMR (500 MHz, CD3OD): δ (ppm) 3.35 (d, J = 6.7 Hz, 4H), 4.86 (s, 2H), 5.02–5.10 (m, 4H), 5.95–6.03 (m, 2H), 6.88 (d, J = 8.2 Hz, 2H), 13 7.04–7.06 (m, 4H); C-NMR (125 MHz, CD3OD): δ (ppm) 40.3, 115.5, 117.4, 127.6, 129.7, 132.6, 133.1, + 139.3, 153.2; HR-MS (ESI): m/z calcd for C18H18O2 ([M + H] ) 267.1378, found 267.1382. 1 Honokiol (5,50-diallyl-2,40-dihydroxybiphenyl): colorless solid; H-NMR (500 MHz, CD3OD): δ (ppm) 3.30 (d, J = 6.7 Hz, 2H), 3.41 (d, J = 6.5 Hz, 2H), 4.86 (s, 2H), 5.00–5.11 (m, 4H), 5.93–6.09 (m, 2H), 13 6.79–7.26 (m, 6H); C-NMR (125 MHz, CD3OD): δ (ppm) 35.3, 40.4, 115.3, 115.5, 116.8, 127.3, 128.7, 129.1, 129.9, 131.4, 131.5, 131.9, 132.5, 138.4, 139.5, 153.2, 155.5; HR-MS (ESI): m/z calcd for C18H18O2 ([M + H]+) 267.1378, found 267.1373. Given the fact that usage of synthetic and mechanical fungicides have associated adverse effects, it is important to develop environmentally friendly and biodegradable botanical fungicides to produce green and safe food products for animal and human consumption [22].

2.2. Antifungal Activity of Magnolol and Honokiol against A. alternata Magnolol and honokiol inhibited the mycelial growth of A. alternata in a dose-dependent manner (Table1). The mycelial growth was noticeably reduced at higher concentrations of magnolol and honokiol, indicating that magnolol and honokiol had an inhibitory effect on A. alternata. The growth inhibition of A. alternata was 77% and 91% in the presence of 0.1 mg/mL of magnolol and honokiol, respectively. The antifungal activity of carbendazim was much higher than that of magnolol and honokiol (Table1), when used at concentration with 0.001 mg /mL. At concentrations greater than or equal to 3 mg/mL, magnolol completely inhibited the growth of A. alternata, whereas greater than or equal to 1 mg/mL of honokiol completely inhibited growth. In addition, we found that there was a significant positive correlation between the concentration of magnolol and honokiol, and the inhibitory effects on mycelial growth of A. alternata.

Table 1. Inhibition of magnolol and honokiol at the different concentration on mycelial growth of A. alternata.

Mycelial Growth Inhibition (%) Concentration (mg/mL) Magnolol Honokiol Eugenol Carbendazim 0 0 0 - - 0.001 7 3.18 9 0.81 - 58 4.64 ± ± ± 0.005 16 3.98 20 2.29 - ± ± 0.01 23 2.88 26 1.63 - - ± ± 0.1 77 2.09 91 1.56 11 4.02 - ± ± ± 1 90 3.02 100 * - - ± 3 100 * 100 * - - 5 100 * 100 * - - 7 100 * 100 * - -

Values are presented as mean SD. (-) not tested. * significantly different at P < 0.05 level by Duncan0s new multiple range test. ±

To the best of our knowledge, the antifungal activity of magnolol and honokiol against A. alternata in vitro has not yet been reported. However, several studies have been conducted to evaluate their antifungal and antibacterial activities. For instance, magnolol and honokiol could effectively inhibit the growth of Pyricularia oryzae, Phytophthora infestans, Puccinia recondita, and Colletotrichum capsici in [15]. Carbendazim is a systemic fungicide with both protective and curative activities against a wide range of fungal diseases [27]. Its antifungal activity was much higher than that of magnolol and honokiol (Table1), when used at concentrations of 0.001 mg /mL. However, it has been reported that carbendazim, an endocrine disrupter, has mutagenic and teratogenic effects on mammals at low doses [28]. The mycelial radial growth inhibition rates were 77% and 91% for magnolol and honokiol, respectively, when the concentration of each was 0.1 mg/mL. Moreover, the antifungal activity was Molecules 2019, 24, 2140 5 of 11 much higher than eugenol (0.1 mg/mL), which showed 11% inhibition of mycelial growth. On the basis of the present results, which are also in agreement with the reported by Edris and Farrag (2003), eugenol was completely inactive on Sclerotinia sclerotiorum (Lib), Rhizopus stolonifer (Ehrenb. exFr) Vuill and Mucor sp. (Fisher), but mixing eugenol and linalool in a ratio similar to their concentrations in the original oil was found to enhance the antifungal properties oil indicating a synergistic effect [29]. Concentrations greater than or equal to 3 mg/mL of magnolol completely inhibited the growth of A. alternata, whereas greater than or equal to 1 mg/mL of honokiol completely inhibited the growth. However, no toxicity tests were done on other eukaryotic cells. Concentrations higher than 0.1 mg/mL are too high to show selective toxicity. We evaluated the growth inhibition up to 0.1 mg/mL of both magnolol and honokiol, which caused 77% inhibition in fungal colonies, and this inhibition is sufficient and close to the MIC80 (the minimum inhibitory concentration 80% of microbial growth) value of the antifungal activity of natural products with good results.

2.3. Synergistic Effect of Magnolol and Honokiol In order to examine the synergistic effect of a combination of two compounds on the mycelial growth of A. alternata, we combined two compounds at different ratios and tested their effect with in vitro assays (Table2). The results showed that the mycelial growth inhibition was higher than the other six volume ratios, with a radial growth inhibition of 91%, under the condition of the volume ratio of magnolol and honokiol (0:1). When the volume ratio of magnolol and honokiol was 1:4, a growth inhibition of 89% of mycelial growth was inhibited. When the volume ratio was 1:0, the growth inhibition was only 77%. The results showed that there was not any synergic effect of these two compounds.

Table 2. The synergistic effect of magnolol and honokiol on A. alternata.

The Volume Ration of Magnolol (0.1 mg/mL) and Mycelial Growth Inhibition (%) Honokiol (0.1 mg/mL) 1:0 77 2.09 ± 1:1 84 3.85 ± 1:4 89 2.77 * ± 1:9 88 1.19 ± 9:1 87 1.74 ± 4:1 80 2.11 ± 0:1 91 1.56 ± Values are presented as mean SD. * significantly different at P < 0.05 level by Duncan s new multiple range test. ± 0 2.4. Growth Inhibition and the Effect on Morphology At 7 days post-inoculation, we measured the diameter of the colonies of A. alternata on PDA (potato dextrose agar) plates containing 0.1 mg/mL magnolol or honokiol (Figure4b,c). We observed an apparent decrease in colony size in the PDA plate containing either magnolol (Figure4b) or honokiol (Figure4c) when compared with the control PDA plate (Figure4a). Subsequently, the e ffect of magnolol and honokiol on the inhibition of hyphae development was also evaluated by examining the morphology of A. alternata using scanning electron microphotography (SEM). Figure4d–f show the micrographs of mycelia from SEM analysis. Our results indicate that, in the control plate, fungal hyphae appeared normal and cylindrical with a smooth surface (Figure4d). However, treatment of magnolol and honokiol led to morphological changes in the hyphae of A. alternata. In particular, the mycelia were curly, distorted, aggregated, and partly squashed, when grown in the presence of magnolol (Figure4e). In the case of honokiol treatment, shrunken and distorted mycelia of A. alternata were observed (Figure4f). Molecules 2019, 24, 2140 6 of 11 Molecules 2019, 24, x FOR PEER REVIEW 6 of 12

FigureFigure 4.4. ObservationObservation of of hyphae hyphae morphology morphology of A. of alternata A. alternataby electron by electron microphotography. microphotography. The colony The ofcolonyA. alternata of A. alternataon PDA on plates PDA is shownplates inis (showna–c): ( ain). Control(a–c): (a). PDA Control plate; PDA (b). PDAplate; plate (b). containingPDA plate magnololcontaining (0.1 magnolol mg/mL); (0.1 (c). mg/mL); PDA plate (c). containing PDA plate honokiol containing (0.1 mghono/mL).kiol Hyphae(0.1 mg/mL). morphology Hyphae of A.morphology alternata on of the A. PDAalternata plate on is the shown PDA in plate (d–f is): shown (d). Control in (d– PDAf): (d). plate; Control (e). PDA PDA plate; plate containing(e). PDA plate the magnololcontaining (0.1 the mg magnolol/mL); (f ).(0.1 PDA mg/mL); plate containing(f). PDA plate honokiol containing (0.1mg honokiol/mL). (0.1mg/mL).

TheseThese resultsresults are are consistent consistent with with the previousthe previous study thatstudy many that changes many occurchanges in the occur morphology in the ofmorphology fungal hyphae of fungal after treatment hyphae after with chemicaltreatment fungicides, with chemical chitosan, fungicides, and natural chitosan, botanicals and [natural27–29]. Webotanicals assume [27–29]. that the We mycelium assume changes that the may mycelium generally changes occur may because generally of increased occur because permeability of increased of cells, whichpermeability resulted of incells, the which leakage resulted of small in molecularthe leakage substances, of small molecular ions, lesions, substances, and disturbances ions, lesions, in and cell metabolismdisturbances [ 30in]. cell The metabolism marked leakage [30]. The of cytoplasmicmarked leakage material of cytoplasmic is commonly material used asis commonly an indication used of grossas an indication and irreversible of gross damage and irreversible of the cytoplasmic damage andof the plasma cytoplasmic membranes and plasma [30–33 ].membranes [30–33]. 2.5. Effects of Magnolol and Honokiol on Cell Membrane Permeability of A. alternata 2.5. Effects of Magnolol and Honokiol on Cell Membrane Permeability of A. alternata To address whether both magnolol and honokiol could inhibit the hyphae growth by affecting To address whether both magnolol and honokiol could inhibit the hyphae growth by affecting the cell membrane permeability of A. alternata, we tested the effect of magnolol (0.1 mg/mL) and the cell membrane permeability of A. alternata, we tested the effect of magnolol (0.1 mg/mL) and honokiol (0.1 mg/mL) on the cell membrane permeability of A. alternata for a period of 0–5 h (Figure5). honokiol (0.1 mg/mL) on the cell membrane permeability of A. alternata for a period of 0–5 h (Figure We showed that the cell membrane permeability was significantly increased (P < 0.05) when A. alternata 5). We showed that the cell membrane permeability was significantly increased (P < 0.05) when A. was grown on a PDA plate containing magnolol and honokiol at different time points. The conductivity alternata was grown on a PDA plate containing magnolol and honokiol at different time points. The of the A. alternata suspensions after treatment with magnolol for 1 h was 167.34 S/cm, which is much conductivity of the A. alternata suspensions after treatment with magnolol for 1 h was 167.34 S/cm, higher than that of the control, 102.77 S/cm. The conductivity of the fungal suspension after treatment whichMolecules is 2019 much, 24, x FORhigher PEER than REVIEW that of the control, 102.77 S/cm. The conductivity of the fungal7 of 12 with honokiol was 192.81 S/cm. suspension after treatment with honokiol was 192.81 S/cm. These findings indicate that the antifungal activity of magnolol and honokiol may be associated with the irreversible damage to the cytoplasmic membranes of A. alternata, which results in ion leakage from the cells and an imbalance in osmotic pressure of the intra- and extra-cellular membranes.

FigureFigure 5.5. EEffectsffects ofof magnololmagnolol andand honokiolhonokiol ononthe theextracellular extracellular conductivityconductivity ofofA. A. alternataalternata,, CK:PDBCK:PDB mediummedium containingcontaining thethe samesame volumevolume ofof Tween-20Tween-20 and and distilled distilled water water (1:1000 (1:1000v v/v/v).).

2.6. The Inhibitory Efficiency of Magnolol and Honokiol on Six Phytopathogens

To confirm whether magnolol and honokiol inhibit pathogenic fungi, their antifungal activity was tested on six fungal pathogens, and the results are shown in Table 3. We found that the magnolol (0.1 mg/mL) was effective against P. expansum (Link) Thom, A. dauci f.sp. Solani, F. moniliforme J. Sheld, F. oxysporum Schltdl, V. mali Miyabe & G. Yamada, and R. solani J.G. Kühn, with 80, 70, 79, 76, 100, and 57% growth inhibition, respectively. The honokiol (0.1 mg/mL) treatment had potent inhibitory effect on the above pathogens with the growth inhibition of 81, 80, 82, 89, 100, and 68%, respectively. These findings are in good agreement with the antifungal activity of magnolol and honokiol on Pyricularia oryzae, Phytophthora infestans, Puccinia recondita, and Colletotrichum capsici [15]. Thus, we conclude that magnolol and honokiol are potential antifungal agents against the plant fungal diseases.

Table 3. The inhibitory efficiency of magnolol and honokiol against six phytopathogens.

Mycelial Growth Inhibition Rate（%） Pathogen Magnolol Honokiol (0.1 mg/mL) (0.1 mg/mL) P. expansum (Link) Thom 80 ± 3.95 * 81± 0.23 Alternaria dauci f.sp. solani 70 ± 5.26 80 ± 9.23 * F. moniliforme J. Sheld 79 ± 0.69 82 ±0.69 * F. oxysporum Schltdl. 76 ± 1.54 * 89 ± 1.55 V. mali Miyabe & G. Yamada 100 * 100 * R. solani J.G. Kühn 57 ± 2.74 68 ± 1.89 Values are presented as mean ± SD. * significantly different at P < 0.05 level by Duncan′s new multiple range test.

3. Materials and Methods

3.1. Chemicals and Media

Acetonitrile (CH3CN) and pyridine were purchased from Sigma Chemical Co. (San Francisco, CA, USA). Tween-20 was from Coolaber technology Co. LTD (Beijing, China). Glucose and agar were from Shanghai Bioindustrial Engineering Co. Ltd (Shanghai, China) to prepare the PDA (potato dextrose agar) medium. , petroleum ether, quicklime, and hydrochloric acid were supplied from Sinopharm Group Co. LTD (Beijing, China). was from Sulebao Technology Co. LTD (Beijing, China). Eugenol was purchased from Mckuin Biological Co. LTD (Shanghai, China). All chemicals were of analytical grade. Carbendazim was from Zhongxun Agrochemicals Co. LTD (Jiangxi, China). M. officinalis bark was purchased from the Jihetang Pharmacy (Yangling, China) and has been identified by Professor Xiaoqian Mu at Northwest A & F University, Yangling, Shanxi, China.

Molecules 2019, 24, 2140 7 of 11

These findings indicate that the antifungal activity of magnolol and honokiol may be associated with the irreversible damage to the cytoplasmic membranes of A. alternata, which results in ion leakage from the cells and an imbalance in osmotic pressure of the intra- and extra-cellular membranes.

2.6. The Inhibitory Efficiency of Magnolol and Honokiol on Six Phytopathogens To confirm whether magnolol and honokiol inhibit pathogenic fungi, their antifungal activity was tested on six fungal pathogens, and the results are shown in Table3. We found that the magnolol (0.1 mg/mL) was effective against P. expansum (Link) Thom, A. dauci f.sp. Solani, F. moniliforme J. Sheld, F. oxysporum Schltdl, V. mali Miyabe & G. Yamada, and R. solani J.G. Kühn, with 80, 70, 79, 76, 100, and 57% growth inhibition, respectively. The honokiol (0.1 mg/mL) treatment had potent inhibitory effect on the above pathogens with the growth inhibition of 81, 80, 82, 89, 100, and 68%, respectively. These findings are in good agreement with the antifungal activity of magnolol and honokiol on Pyricularia oryzae, Phytophthora infestans, Puccinia recondita, and Colletotrichum capsici [15]. Thus, we conclude that magnolol and honokiol are potential antifungal agents against the plant fungal diseases.

Table 3. The inhibitory efficiency of magnolol and honokiol against six phytopathogens.

Mycelial Growth Inhibition Rate (%) Pathogen Magnolol Honokiol (0.1 mg/mL) (0.1 mg/mL) P. expansum (Link) Thom 80 3.95 * 81 0.23 ± ± Alternaria dauci f.sp. solani 70 5.26 80 9.23 * ± ± F. moniliforme J. Sheld 79 0.69 82 0.69 * ± ± F. oxysporum Schltdl. 76 1.54 * 89 1.55 ± ± V. mali Miyabe & G. Yamada 100 * 100 * R. solani J.G. Kühn 57 2.74 68 1.89 ± ± Values are presented as mean SD. * significantly different at P < 0.05 level by Duncan s new multiple range test. ± 0 3. Materials and Methods

3.1. Chemicals and Media

Acetonitrile (CH3CN) and pyridine were purchased from Sigma Chemical Co. (San Francisco, CA, USA). Tween-20 was from Coolaber technology Co. LTD (Beijing, China). Glucose and agar were from Shanghai Bioindustrial Engineering Co. Ltd (Shanghai, China) to prepare the PDA (potato dextrose agar) medium. Methanol, petroleum ether, quicklime, and hydrochloric acid were supplied from Sinopharm Group Co. LTD (Beijing, China). Chloroform was from Sulebao Technology Co. LTD (Beijing, China). Eugenol was purchased from Mckuin Biological Co. LTD (Shanghai, China). All chemicals were of analytical grade. Carbendazim was from Zhongxun Agrochemicals Co. LTD (Jiangxi, China). M. officinalis bark was purchased from the Jihetang Pharmacy (Yangling, China) and has been identified by Professor Xiaoqian Mu at Northwest A & F University, Yangling, Shanxi, China.

3.2. Fungal Strains A. alternata (Fr.) Keissl, R. solani J.G. Kühn, F. moniliforme J. Sheld, A. dauci f.sp. Solani, and P. expansum (Link) Thom were obtained from Microbiology Institute of Shaanxi, Shaanxi Province, China. V. mali Miyabe & G. Yamada was provided by the State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A & F University, Yangling, Shanxi, China. F. oxysporum Schltdl. was provided by the Laboratory of Phytopathology, Inner Mongolia Agricultural University, Huhhot Inner Mongolia, China. All fungi were grown on PDA medium at 25 2 C. ± ◦ Molecules 2019, 24, x FOR PEER REVIEW 8 of 12

3.2. Fungal Strains A. alternata (Fr.) Keissl, R. solani J.G. Kühn, F. moniliforme J. Sheld, A. dauci f.sp. Solani, and P. expansum (Link) Thom were obtained from Microbiology Institute of Shaanxi, Shaanxi Province, China. V. mali Miyabe & G. Yamada was provided by the State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A & F University, Yangling, Shanxi, China. F. oxysporum Schltdl. wasMolecules provided2019, 24, 2140by the Laboratory of Phytopathology, Inner Mongolia Agricultural University,8 of 11 Huhhot Inner Mongolia, China. All fungi were grown on PDA medium at 25 ± 2 °C.

3.3. Isolation Isolation and and Purification Purification of Acti Activeve Compounds and St Structureructure Analysis The stem bark of M. officinalis officinalis was rolled up into a column, brown and spicy (Figure6 6).). WithWith thethe method of alkali-extraction and acid-precipitation, 100 g of dried bark was ground into powder by a disintegrator XL600BXL600B (Xiaobao(Xiaobao Electric Electric Co. Co. LTD, LTD, Yongkang, Yongkang, China), China), and and extracted extracted three three times times with with 90% 90%methanol. methanol. The resultingThe resulting crude crude extract extract (1500 mL)(1500 was mL) dried was to dried a paste to and a paste then and incubated then incubated with two timeswith twoquicklime times quicklime for 24 h. After for 24 washing h. After three washing times three using times five timesusing dilutedfive times hydrochloric diluted hydrochloric acid, the extract acid, thepaste extract was concentrated. paste was concentrated. After the concentrating After the co step,ncentrating the extract step, was the dissolved extract inwas chloroform dissolved and in chloroformisolated with and three isolated volumes with of alkalinethree volumes water (0.5% of alkaline NaOH solution).water (0.5% The NaOH crystals solution). were filtered The outcrystals from werethe solution filtered byout adjusting from the thesolution pH value by adjusting to 7. After the drying pH value at a to low 7. temperature,After drying at the a low solution temperature, that was thefiltered solution from that thecrystal was filtered with petroleum from the forcrystal 1 h waswith naturally petroleum cooled for 1 to h room was temperature,naturally cooled and to the room final temperature,product was analyzedand the final by a liquidproduct chromatography-mass was analyzed by a liquid spectrometry chromatography-mass (LC-MS). The structures spectrometry were 1 13 13 (LC-MS).identified The by Hstructures and C-NMR were identified spectra. 1H by and 1H andC-NMR 13C-NMR spectra spectra. were obtained1H and 13 withC-NMR Bruker spectra Avance were III obtained500 MHz with (Bruker Bruker Corporation, Avance III Karlsruhe, 500 MHz Germany).(Bruker Corporation, HPMS were Karlsruhe, recorded Germany). on a Triple HPMS TOF 5600were+ recordedsystem (AB on SCIEX a Triple Co. LTD,TOF Framingham,5600+ system MA, (AB USA). SCIEX The Co. high-performance LTD, Framingham, liquid MA, chromatography USA). The washigh-performance performed on anliquid Agilent 1260chromatography liquid chromatography was performed system (Agilent on Technologiesan Agilent Co.1260 LTD, liquid Santa chromatographyClara, CA, USA) equippedsystem (Agilent with a Zorbax Technologies SB-C18 column,Co. LTD, particle Santa sizeClara, 5 µ CA,m, dimension USA)equipped150 mm withi.d. a 1 × Zorbax9.4 mm, SB-C18 flow rate column,particle 7 mL min− . size 5 μm, dimension 150 mm × i.d. 9.4 mm, flow rate 7 mL min−1.

Figure 6. TheThe sample sample of M.M. officinalis officinalis bark.

3.4. Antifungal Antifungal Activity Activity of Magnol Magnololol and Honokiol on A. alternata Magnolol and honokiol were dissolved in DMSO (1000 mg/mL) mg/mL) and diluted to the required concentration with Tween-20 and distilled water (1:1000 vv/v/v). Antifungal activities of magnolol and honokiol werewere tested tested against againstA. alternataA. alternatain vitro in vitroaccording according to the agarto the dilution agar methoddilution [34method]. Magnolol [34]. Magnololand honokiol and (5 honokiol mL) were (5 added mL) towere sterilized added Petrito sterilized dishes (90 Petri mm diameter)dishes (90 in mm concentrations diameter) in of concentrations0.001, 0.005, 0.01, of 0.001, 0.10, 1.00,0.005, 3.00, 0.01,5.00, 0.10, and1.00, 7.00 3.00, mg 5.00,/mL, and and 7.00 eugenol mg/mL, (0.1 and g /eugenolmL) and (0.1 carbendazim g/mL) and carbendazim(0.001 g/mL),then (0.001 were g/mL),then mixedwith were PDA mixed medium with PDA (15 mL).medium As the(15 control,mL). AsPDA the control, dishes werePDA addeddishes withwere theadded same with volume the same of Tween-20volume of andTween-20 distilled and water distilled (1:1000 waterv/ v(1:1000). A 6 v/v mm). A diameter 6 mm diameter disc of A.disc alternata of A. alternatamycelia mycelia was inoculated was inoculated into theinto centerthe center of the of the plate. plate. After After incubation incubation for for 77 daysdays at 25 2 C, the colony diameter was measured. Each treatment was performed in triplicate. To calculate 25 ±± 2 ◦°C, the colony diameter was measured. Each treatment was performed in triplicate. To calculatethe percentage the percentage of mycelial of growthmycelial inhibition growth in (MGI),hibition the (MGI), following the formulafollowing was formula used: was mycelium used: inhibition (%) = [(Dc Dt)/Dc] 100, where Dc (cm) is the mean colony diameter for the control Petri − × dishes, and Dt (cm) is the mean colony diameter for the treatment Petri dishes.

3.5. Synergistic Effect Test Synergistic effect of magnolol and honokiol was tested against A. alternata. Magnolol and honokiol (0.1 mg/mL each) were mixed at volume ratios of 1:0, 1:1, 1:4, 1:9, 9:1, 4:1, and 0:1 in a total volume of 5 mL and added to sterilized Petri dishes (90 mm diameter) containing PDA medium (15 mL) and Molecules 2019, 24, 2140 9 of 11

Tween-20 (1:1000 v/v). A 6 mm diameter disc of A. alternata mycelia was inoculated into the center of the plate. After incubation for 7 days at 25 2 C, the colony diameter was measured. Each treatment ± ◦ was performed in triplicate.

3.6. Scanning Electron Microscopy (SEM) Analysis A. alternata incubated on PDA containing magnolol (0.1 mg/mL) and honokiol (0.1 mg/mL) for 7 days were used for SEM analysis. Slices of 7 5 3 mm segments were cut from the plates and × × washed three times using normal saline. After keeping the mycelium in 2.5% glutaraldehyde for 2 h at 4 ◦C, samples were washed with 0.1 M phosphate buffer (pH 7.0) four times. The samples were dehydrated in ethanol sequentially with 30, 50, 70, and 90% ethanol for 20 min each wash, and the final wash was absolute ethyl for 30 min. Mycelium was observed under a scanning electron microscope after drying. All mycelium samples were viewed with a Nova NanoSEM 450 (FEI Co. LTD, Hillsboro, OR, USA) at 5.00 kV of magnification.

3.7. Cell Membrane Permeability Test The cell membrane permeability test of A. alternata was performed, as described previously by Lee et al. [35]. A. alternata suspension was incubated in a PDB (potato dextrose broth) medium and rotated with 160 rpm at 25 ◦C for 48 h. Magnolol (0.1 mg/mL) and honokiol (0.1 mg/mL) were then added to the suspension, 5 mL of which were centrifuged at 10,000 rpm for 15 min. The supernatant conductivity was tested by a conductometer at the start of the measurement (0 h) and then every 1 h, up to 6 h. Each sample was repeated three times.

3.8. Antifungal Activity Test Effects of magnolol and honokiol on the mycelial growth of A. alternata (Fr.) Keissl, P. expansum (Link) Thom, F. moniliforme J. Sheld, F. oxysporum Schltdl., V. mali Miyabe & G. Yamada, and R. solani J.G. Kühn were measured. Different ratios of magnolol and honokiol in a total volume of 5 mL were added to sterilized Petri dishes (90 mm diameter) as indicated in Table2 and mixed with PDA medium (15 mL). The same volume of distilled water was added to control PDA dishes instead of magnolol and honokiol. A 6 mm diameter disc of pathogens mycelia was inoculated into the center of the plate. After incubation for 7 days at 25 2 C. The colony diameter was measured as described in Section 3.4 ± ◦ Each treatment was repeated three times.

3.9. Statistical Analysis All data were presented as the mean SD by measuring four independent replicates. The data ± were analyzed using a Data Processing System 15.10 (Hefei, China). The significance of the statistical differences between four means was determined using Duncan0s new complex range method at the 5% level.

4. Conclusions In this study, magnolol and honokiol were purified from M. officinalis and identified by 1H- and 13C-NMR and HP-MS. Magnolol and honokiol showed antifungal activity against A. alternata and other phytopathogenic fungi assayed in this study. The antifungal activity can be associated with the hyphal distortion that resulted from the disruption of the cell membrane integrity. Therefore, magnolol and honokiol could be potentially used as antifungal agents against plant fungal pathogens.

Author Contributions: Y.-H.C., M.-H.L. and D.-S.G. isolated and purified of active compounds and analyzed the structure; D.M. tested synergistic effect; Y.-Y.Z. tested scanning electron microscopy (SEM); J.-Y.Y. and C.-H.Y. analyzed effects of magnolol and honokiol on cell membrane permeability and the inhibitory against 6 fungal pathogens; D.-R.A. and M.-M.Z. designed the experiments and supervised the study. M.-M.Z. and Y.-H.C. wrote the manuscript. Molecules 2019, 24, 2140 10 of 11

Funding: This study was supported by the Demonstration and Application of Control Technology of Plant Pests and Diseases in Shaanxi Province (No. K4030218261), Research and Application of Green Control and Prevention Technology of Cordyceps Chinensis Based on Entomogenous Fungi (No. 110201601023) and Start-up Funding of High-level Talent Researcher in Inner Mongolia Agricultural University (No. NDGCC2016-23). Acknowledgments: We are grateful to Delong Wang for analyses the data of compound structure. We thank the Instrument Shared Platform of Northwest A&F University, the People0s Republic of China, for the LC-MS and NMR. Conflicts of Interest: The authors have no conflict of interest to declare.

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Sample Availability: Samples of the compounds are available from the authors.

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