Antifungal effects of lycorine on Botrytis cinerea and possible mechanisms

Botrytis cinerea cause postharvest diseases on fruit and lead economic losses. Application of environment-friendly natural compounds is an alternative for synthetic fungicides to control postharvest disease. Lycorine is an indolizidine alkaloid which is widely used for human drug design, however, application of lycorine in controlling postharvest disease and the underlying mechanisms have not been reported. In this study, the effects of lycorine on mycelium growth, spore germination, disease development in apple fruit, cell viability, cell membrane integrity, cell wall deposition, and expression of mitogen-activated protein kinase (MAPK) and GTPase of B. cinerea were investigated. Our results showed that lycorine was effective in controlling postharvest gray mold caused by B. cinerea on apple fruit. In the in vitro tests, lycorine strongly inhibited spore germination and mycelium spreading in culture medium. Investigation via fluorescein diacetate and propidium iodide staining suggested that lycorine could damage the membrane integrity and impair cell viability of B. cinerea. Furthermore, the expression levels of several MAPK and GTPase coding genes were reduced upon the lycorine treatment. Taken together, lycorine is an effective and promising way to control postharvest disease caused by B. cinerea.


Introduction
Fungal pathogens cause severe postharvest diseases on fruit and lead to considerable economic losses during transportation and storage . Botrytis cinerea, the causal agent of gray mold, could infect over two hundred plant species, including a number of common fruits (Dean et al. 2012). Synthetic fungicides have been widely used to control both pre-and postharvest diseases of fruit. However, indiscriminate use of synthetic fungicides has caused widespread concerns over food security and environmental contamination (Droby et al. 2009), which encourages researchers to identify and develop environmentfriendly antifungal compounds for controlling postharvest diseases. A great number of natural chemicals have been found to be effective in controlling fungal disease in postharvest fruit till now. Treatment with phytohormones such salicylic acid and methyl jasmonate could induce the plant immunity and decrease postharvest decay in fruit Cao et al. 2013;Wang et al. 2014;Yao et al. 2005). Plant essential oils and some isolated constituents could directly inhibit the growth of fungal pathogen in postharvest fruit (Ji et al. 2018;Mendel et al. 2002). Natural compounds such as chitosan and boron showed strong antimicrobial effect on fungal pathogens (Liu et al. 2007;Meng et al. 2010;Qin et al. 2010). Lycorine, an indolizidine alkaloid, is identified in Amaryllidaceae plants. In recent years, researchers discovered that lycorine and their derivatives have excellent properties including anti-tumor (Yui et al. 1998(Yui et al. ), anti-inflammatory (Ç itoglu et al. 2012, antimalarial (Kogure et al. 2008), anti-virus (Liu et al. 2011, and other functions. In addition, lycorine is safe for most people when taken by mouth in low amounts. Thus, lycorine and their derivatives have been widely used for drug design. However, to our knowledge, application of lycorine in controlling postharvest disease and the underlying mechanisms have not been reported. The objectives of this research were to (i) study the inhibitory activity of lycorine on B. cinerea both in vitro and in vivo; (ii) evaluate mechanisms by which lycorine inhibit growth of B. cinerea via analyzing cell viability, cell wall structure, and cell membrane integrity.

Chemicals
Lycorine  was purchased from Solarbio Science & Technology (Beijing). Lycorine was resolved in DMSO to make the stock solution at 0.696 mol L -1 before use.

Pathogens
Botrytis cinerea was isolated from infected tomato fruit and maintained on potato dextrose agar (PDA). For germination and inoculation tests, spores were obtained from 10-day old PDA cultures and suspended to 10 5 CFU mL -1 with ddH 2 O containing 0.05% tween 80.

Fruit
Healthy apple fruit (Malus pumila Mill cv. Fuji) at commercial maturity were purchased from market. Fruit without physical injuries and infections were selected based on size uniformity. Before treatments, fruit surfaces were disinfected with 2% sodium hypochlorite for 3 min, then rinsed with tap water, and air-dried.

In vitro antifungal activity assay
To evaluate the effects of lycorine on mycelium growth of B. cinerea, aliquots of lycorine stock solution were added to potato dextrose agar (PDA) medium to generate final concentrations of 0, 1, 2, and 5 mmol L -1 respectively; in addition, extra DMSO were added into the medium to make DMSO at same concentration. The PDA media were poured into sterilized Petri dishes (90 mm in diameter). 2 mm of mycelium together with agar were cut form the growing edge of 3 days-old cultures of B. cinerea, and transferred into the center of each Petri dish. Then the plates were cultured at 22°C and the diameter of colony was determined. Each treatment contained three replicates. To evaluate the effects of lycorine on spore germination and germ tube elongation of B. cinerea, spores were incubated in potato dextrose broth (PDB) containing lycorine at various concentrations. At least 100 spores were counted to calculate the germination rate. Measurement of germ tube length were performed on triplicates of 50 spores using ImageJ.
In vivo antifungal activity assay The in vivo assay was carried out according to the method described previously (Ji et al. 2018). Apple fruit were wounded (3 mm wide and 4 mm deep) at the equator using a sterilized scalpel. Droplets of 5 lL of spore suspension at 10 5 spore mL -1 were inoculated into the wounds and air-dried for 1 h. Then 5 lL of lycorine solution diluted in ddH 2 O at 0, 5, 10, and 30 mmol L -1 were added into the wounds. Fruit treated with ddH 2 O was used as the control. Then the treated fruit were kept in moisture environment and stored at room temperature, and lesion diameter were recorded at 2, 3, and 4 day. Each treatment contained three replicates with 10 fruit per replicate, and the experiment was performed twice.

Fluorescence microscopy
Botrytis cinerea spore were incubated in PDB containing lycorine as mentioned above. Fluorescent dyes fluorescein diacetate (FDA; Sigma) was used to determine cell viability; propidium iodide (PI; Sigma) was used to determine membrane integrity; Calcofluor white M2R (Sigma) was used to stain cell wall. After staining, B. cinerea spores were examined using a fluorescence microscope (LEICA DM4000M). At least 100 spores were counted to calculate the percentage of staining.

RNA extraction and qRT-PCR
Botrytis cinerea were grown on cellophane paper plated on the PDA containing different concentration of lycorine. Then mycelium was collected and disrupted in liquid nitrogen by grinding in a mortar with a pestle, and RNA was extracted using TRIzol Reagent (Invitrogen). Reverse transcription was conducted with Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher) according to the manufacturer's instructions. The Quantitative real-time PCR was then performed using FastStart Universal SYBR Green Master (Roche, Switzerland) with a LightCycler 96 System (Roche, Switzerland) instrument. Relative expression levels of three mitogen-activated protein kinase (MAPK), including BcMPS1 (BCIN_02g 08170), pathogenicity MAP kinase 1 (BcPmk1, BCIN_09g02390), and protein kinase A (BcPKA, BCIN_16g01130), and three GTPase, including Bccd-c42 (BcBCIN_13g00090), BcRac (BCIN_01g02000), BcRas1 (BcIN_12g05760), were estimated using the 2 -DDCt method. The B. cinerea actinA coding gene (BCIN_16g02020) was used as an endogenous control for normalization. The primers used for quantitative RT-PCR are listed in Table S1.

Statistical analysis
Statistical analysis was performed with SPSS version 10.0 (SPSS Inc., Chicago, USA). Data analyzed by one-way ANOVA, and mean separations were determined using Duncan's multiple range test. Differences at P \ 0.05 were considered significant.

Lycorine inhibited mycelial growth and spore germination in vitro
The in vitro assay showed that lycorine markedly inhibited mycelial growth of B. cinerea with a dosedependent manner (Fig. 1). The colony diameter of the control sample was about 50 mm after inoculation for 3 day; whereas that of the sample treated with lycorine at 1 mmol L -1 was smaller than half of the control. Furthermore, the mycelial growth was completely inhibited when the lycorine concentration increased to 5 mmol L -1 (Fig. 1b). Germination is the first step for B. cinerea spore to infect plant host. Thus, the effect of lycorine on spore germination was examined. As shown in Fig. 2, the inhibitory effect of lycorine on germination also showed a dose-dependent manner. After incubation for 10 h, about 95% of spore in the control sample was germinated; in comparison, the germination rate was only 17% in the sample treated with 5 mmol L -1 lycorine (Fig. 2a,  b). In addition, the germ tube elongation was completely inhibited by 5 mmol L -1 lycorine (Fig. 2c).

Effect of lycorine in controlling gray mold in apple fruit
Consistent with the results of the in vitro test, lycorine was also effectively in controlling gray mold in apple fruit. As shown in Fig. 3, after inoculation for 2 day, the disease incidence was only 50% in the sample treated with 30 mmol L -1 lycorine, whereas the control sample showed disease symptoms in all wounds (Fig. 3b). The lesion diameters also decreased as the lycorine dose increased. Lycorine at concentration of 30 mmol L -1 significantly alleviated fruit decay in apple fruit with the lesion diameters of 6, 8, 13 mm at 2, 3, 4-day post inoculation; in comparison, the lesion diameters of control fruit were 8, 16, 27 mm, respectively.
Lycorine impaired membrane integrity and decreased cell viability of B. cinerea After incubation for 8 h, B. cinerea spore were collected, stained with FDA and PI respectively, and observed under a fluorescence microscope. The results showed that about 95 % of spores of control were stained with FDA in high fluorescence intensity; whereas only a few spores were stained in the lycorine treated sample (Fig. 4a, c). Meanwhile, less spores were stained with PI in the control sample compared with the lycorine treated sample (Fig. 4b, d). These results suggested lycorine could damage the cell membrane integrity and decrease cell viability of B. cinerea.

Polarized cell wall deposition was impaired by lycorine in B. cinerea
Calcofluor white M2R could be used for staining of microbe cell walls. As shown in Fig. 5, there is an obvious polarized deposition of cell wall compounds in the germ tube of B. cinerea, with intense calcofluor white fluorescence observed in the apical region.
Whereas lycorine treated spore showed uniformly distributed deposition of cell wall and lack polarity.

Lycorine down-regulated MAPK and GTPase in B. cinerea
We also carried out a qRT-PCR assay to assess the effect of lycorine on transcription of MAPK and GTPase coding genes, which are involved in cell growth and virulence of B. cinerea. The results (Fig. 6) suggested that lycorine showed inhibitory effect on expression of GTPase BcMPS1, Bccdc42, BcRac, and BcRas1 with a dose-dependent manner; and lycorine at 2 mmol L -1 significantly depressed expression of the genes compared with the control sample. Meanwhile, treatment with lycorine at 1 mmol L -1 up-regulated the transcription of BcPmk1 and BcPKA; but when the concentration of lycorine come up to 2 mmol L -1 , the transcription of BcPKA was depressed significantly.

Discussion
Lycorine is identified as the most common alkaloid within the Amaryllidaceae family (Zhong et al. 2005). Due to its pharmacological potential, the alkaloid has Fig. 2 Effect of lycorine on spore germination and germ tube elongation of B. cinerea in potato dextrose broth. a Relative photographs of the germinating spore of B. cinerea. b Germination rate of B. cinerea under lycorine treatment; c Germ tube length of B. cinerea after lycorine treatment. A total of 100 spore were counted to calculate the germination rate and germ tube length. The statistical analysis was performed on triplicates of 100 spore and data are means ± standard deviations from three replicates. Columns with different letters represent significant difference at each time point according to Duncan's multiple range test at P \ 0.05. Bar = 50 lm been widely used in medicine design. In the present study, we found that lycorine shows efficient antifungal activity against B. cinerea both in vitro and in vivo. In the in vitro assays, the inhibitory effect of lycorine on B. cinerea was with a dose-dependent manner: the mycelium growth and the spore germ tube elongation were completely inhibited by lycorine at 5 mmol L -1 (Figs. 1, 2). In the in vivo test, lycorine also showed a dose-dependent manner on controlling disease severity in apple fruit (Fig. 3). However, the effect concentration of in vivo tests is relatively higher than that of in vitro tests, which might be explained by the complex environment in the wound of fruit, including carbon sources, pH value, physical barrier of host, Fig. 3 Efficacy of lycorine on disease severity of gray mold caused by B. cinerea on apple fruit. a Disease symptom of representative samples of apple fruit on 2, 3-and 4-day post inoculation. b Statistical analysis of disease incidence. Data are means ± standard deviations from three group of replicates. Columns with different letters represent significant difference at each time point according to Duncan's multiple range test at P \ 0.05. c Statistical analysis of lesion diameter. Data are means ± standard errors from three group of replicates. Columns with different letters represent significant difference at each time point according to Duncan's multiple range test at P \ 0.05 which influence the antifungal effect of lycorine (Ma et al. 2019;Meng et al. 2010).
Cell viability play a vital role in spore germination and expansion of mycelium in fungal pathogen. To investigate whether lycorine showing antifungal activity by impair cell viability of B. cinerea, the FDA and PI staining assays were conducted. FDA is a cell-permeant esterase substrate that can serve as a viability probe. PI is a fluorescent intercalating agent that binds to DNA but cannot passively traverse into cells that possess an intact plasma membrane. The results showed that, compared with the control, fewer spores were stained with FDA (Fig. 4c) and more spore were stained with PI (Fig. 4d) upon the lycorine treatment. These results indicate that lycorine could damage the plasma membrane integrity and decrease the cell viability, which lead to the inhibition of spore germination and decrease in mycelium growth. Similar results were found in the previous reports that lycorine treatment inhibited cellular viability and induced cell death in multiple myeloma cell lines and primary myeloma cells (Jin et al. 2016). Polarized growth of germ tube and fungal hyphae is important for pathogenicity of B. cinerea to plant host , and this kind of polarized growth requires sustained deposition of cell wall components to the apical region (Fischer-Parton et al. 2000). In the present study, treatment of lycorine damage the polarized deposition of cell wall in germinated spore Fig. 4 Effect of lycorine on cell viability and plasma membrane integrity of B. cinerea after incubation for 8 h in potato dextrose broth. a FDA staining assay. b PI staining assay. c FDA staining percentage. d PI staining percentage. The statistical analysis was performed on triplicates of 100 spore and data are means ± standard deviations from three replicates. Columns with Asterisk symbols (*) represent significant difference according to Duncan's multiple range test at P \ 0.05. Bar = 50 lm (Fig. 5); and this kind of impair effect may be attributed to injuries to cell viability.
Mitogen-activated protein kinase (MAPK) signaling pathway regulates various developmental and infection processes in filamentous fungal pathogens (Jiang et al. 2018); and many fungicides kill pathogens by direct targeting at MAPK signaling components. To investigate effect of lycorine on MAPK signaling, the transcription level of three kinases, BcMPS1, BcPKA, and BcPmk1 were measured. The kinase BcMPS1 is conserved in filamentous fungi to regulate cell wall integrity and pathogenesis (Bashi et al. 2016;Li et al. 2012). In the present study, the transcript level of BcMPS1 was significantly down-regulated upon the lycorine treatment (Fig. 6). And the result could also partially explain the impair effect of lycorine on cell wall deposition (Fig. 5). BcPmk1 and BcPKA are important for spore germination, appressorium  Data are means ± standard deviations from three biological replicates. Columns with different letters represent significant difference according to Duncan's multiple range test at P \ 0.05 formation, stress response, and virulence of B. cinerea and other fungal pathogens (Guo et al. 2016;Schumacher et al. 2008;Turra et al. 2014). As showed in Fig. 6, lycorine at concentration of 1 mmol L -1 upregulated the transcription of BcPmk1 and BcPKA, suggesting that the two genes are involved in response to lycorine stress. BcCdc42, BcRas1 and BcRac belong to small GTPases of the Ras superfamily, which participate in various cellular processes; furthermore, the three GTPases are important for polarized growth of B. cinerea (Marschall et al. 2016;Schumacher et al. 2008;). Here we found that lycorine significantly depressed the expression of Bccdc42, BcRas1 and BcRac (Fig. 6), which might lead to inhibition of spore germination and hyphae growth. Taken together, these results suggested that lycorine showing anti-fungal activity by interfering with the MAPK and GTPase signaling.

Conclusions
Lycorine could inhibit pathogenicity of B. cinerea by disrupting cell membrane integrity, decreasing cell viability, impairing polarized growth, and interfering with MAPK and GTPase signaling pathways. lycorine and their derivatives can be used for controlling postharvest gray mold caused by B. cinerea, offering an environmental-friendly alternative for fungicide.