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Efficacy of rapamycin in modulating autophagic activity of Botrytis cinerea for controlling gray mold
Postharvest Biology and Technology 150 (2019) 158–165
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Efficacy of rapamycin in modulating autophagic activity of Botrytis cinerea for controlling gray mold
T
Danying Maa,b, Dongchao Jia,b, Zhanquan Zhanga, Boqiang Lia, Guozheng Qina, Yong Xua, ⁎ Tong Chena, , Shiping Tiana,b,c a
Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China University of Chinese Academy of Sciences, Beijing, 100049, China c Key Laboratory of Post-Harvest Handling of Fruits, Ministry of Agriculture, Beijing, 100093, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Autophagy Botrytis cinerea Membrane integrity Rapamycin
Postharvest diseases severely deteriorate the intrinsic quality and commercial value of fruits and vegetables, leading to significant economic losses for farmers and increased costs for consumers. In the present study, rapamycin was effective in suppressing mycelial growth of Botrytis cinerea and disease severity of gray mold on harvested apple, pear and grape fruits. The inhibitory mechanism was attributed to a modulation in autophagic activity and membrane permeability rather than the induction of reactive oxygen species. Increases in the degree of membrane peroxidation and the leakage of cytosolic contents were also detected, supporting the data on reduced cell viability. The results indicated that rapamycin may be a promising alternative for controlling gray mold caused by B. cinerea on harvested produce.
1. Introduction Botrytis cinerea is the causal agent of gray mold on more than two hundred species worldwide, leading to great losses in fruits and vegetable production (Dean et al., 2012). Up to now, numerous approaches have been attempted to control gray mold, including biocontrol agents (Droby et al., 2009; Liu et al., 2013), natural antimicrobial polymers (Ji et al., 2018) and substances generally regarded as safe (GRAS) (Elad et al., 2016; Qin et al., 2010; Sanzani et al., 2014). Among them, synthetic fungicides are still widely used, particularly in developing countries. However, abuse of commercial fungicides has resulted in increasing risks in environmental pollution and food contamination (Elad et al., 2016; Tian et al., 2016). Therefore, it is urgent to further explore new targeting sites and underlying mechanisms for alternative antifungal agents. Originally isolated from Streptomyces hygroscopicus, rapamycin is a macrolide compound showing inhibitory effects on Candida albicans (Vézina et al., 1975). During the past several decades, it was further demonstrated that rapamycin can induce physiological variations similar to those under nitrogen or carbon deficiency in Podospora anserine (Dementhon et al., 2003), and Fusarium graminiarum hyphae showed morphological abnormalities following rapamycin treatment (Yu et al., 2014). As rapamycin has been widely used as an interfering agent in the
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studies on autophagy, a macro-molecule degradation process in which cells recycle cytoplasmic contents under specific conditions (Marobbio et al., 2012; Martínez-Cisuelo et al., 2016; Paghdal and Schwartz, 2007), it is envisioned that the inhibitory mechanisms of rapamycin may be also attributed to induced autophagic activities. However, the results are scarce for its application in phytopathogens for postharvest produces. Recently, several lines of evidence suggest that it is also safe to be used as a health-promoting drug, since rapamycin in nano-molar doses can efficiently bind to newly synthesized mTOR before it binds to Rictor, and thus prevent formation of mTORC2 (Mukhopadhyay et al., 2016). Moreover, rapamycin can postpone aging and extend lifespan at low doses without detectable side effects, and thus it has been approved by FDA as drugs for treating aging-related diseases (Ehninger et al., 2014). Utilizing in vitro and in vivo antifungal activity assay, the present study aimed to evaluate the efficacy of rapamycin in controlling gray mold on harvested fruits. It was found that rapamycin exhibited antifungal eff ;ects by modulating autophagic activities and membrane permeability rather than inducing reactive oxygen species. Moreover, the degree of membrane peroxidation and the leakage of cytosolic contents also increased. The results may further broaden our insights towards antifungal mechanisms of rapamycin and provide guidance for handling of postharvest fresh produce.
Corresponding author at: Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Xiangshan, Haidian District, Beijing, 100093, China. E-mail address: [email protected] (T. Chen).
https://doi.org/10.1016/j.postharvbio.2019.01.005 Received 3 August 2018; Received in revised form 8 January 2019; Accepted 11 January 2019 Available online 16 January 2019 0925-5214/ © 2019 Elsevier B.V. All rights reserved.
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2. Materials and methods
510, LT515), PI detection (BP 546/12, FT 580, LP 590), MDC detection (G365, FT395, LP420).
2.1. Chemicals 2.7. Total RNA extraction and RT-qPCR Rapamycin was purchased from Sigma-Aldrich (San Louis, MO, USA).
Total RNAs were extracted using Trizol reagent (Tiangen, Beijing, China) as reported by Ji et al. (2018). After first strand cDNA was synthesized using PrimeScript RT reagent kit with gDNA eraser (TaKaRa, Dalian, China), RT-qPCR was performed on Step One Plus RealTime PCR system (Thermo Fisher Scientific) using SYBR premix ex Taq (TaKaRa, Dalian, China). Primer Premier 5.0 (PREMIER Biosoft, USA) was used to design the primers, as listed in Supplementary Table 1. The RT-qPCR procedures were as follows: pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, 40 cycles. BcTubulin was used as the reference gene for normalization, and relative expression levels were calculated using the 2−△△Ct method (Livak and Schmittgen, 2001).
2.2. Pathogens Botrytis cinerea B05.10 was cultivated on potato dextrose agar (PDA) at 23 ± 2 °C for 7–10 d (Ji et al., 2018). Afterwards, spores were harvested, filtrated against sterile gauze and counted as previously described (Qin et al., 2010). 2.3. Fruits Apple (Malus domestica Borkh.), pear (Pyrus bretschneideri Rehd.) and grape (Vitis labrusca L.) fruits were harvested at commercial maturity. Fruits without injuries and lesions were sterilized with 2% (v/v) sodium hypochlorite, rinsed with water and air-dried before use. Total soluble solids (TSS, %) were measured according to the method described by Yun et al. (2013), and titratable acid (TA, %) was determined using the digital acidity meters (GMK-835, G-WON HITECH CO., LTD, Korea) following the manufacturer’s instructions (Obenland et al., 2011).
2.8. Detection of membrane lipid peroxidation degree and electrical conductivity Membrane lipid peroxidation degree was examined by determining malondialdehyde (MDA) content using thiobarbituric acid (TBA) method (Pereira et al., 2003). After the spores were incubated in PDB on a shaker at 180 rpm and 25 °C for 3 d, rapamycin was added at various concentrations and the cells were further incubated for 0, 4, 8 and 12 h before measurement. Electrical conductivity of the incubation medium was measured using a conductivity meter (EC215, HANNA Instrument; Lee et al., 1998). Spores were incubated in 200 mL PDB (1 × 106 spores mL−1) at 180 rpm for 3 day at 25 °C. After washing with water, the mycelia (3 g wet weight) were suspended in 40 mL sterile water containing rapamycin at various concentrations, and the measurements were performed at 0, 4, 8 and 12 h after further incubation. To rule out possible influences from background conductivity of rapamycin, the results were expressed as changes relative to the initial values of measurement.
2.4. In vitro inhibitory efficacy on B. cinerea growth Rapamycin was supplemented to PDA to generate a concentration gradient (0, 0.1, 1, 10, 50 and 100 nM). Afterwards, 5 μL spore suspension (1 × 105 spores mL−1) was dropped in the center of each dish and cultured at 23 ± 2 °C, the colony diameter was determined every 24 h, and the experiments were performed in triplicates. Furthermore, spores were incubated in potato dextrose broth (PDB) containing rapamycin at various concentrations at 25 °C to examine spore germination rate and germ tube length. In the comparison to commercially available fungicides, toxicity regression equations and median effect concentrations (EC50) were calculated according to Taylor et al. (2002).
2.9. Cytoplasmic content leakage assay Leakage of cytoplasmic contents from mycelia was determined according to Lewis and Papavizas (1987) with minor modifications. Spores were grown in PDB at 25 °C and 180 rpm, and the mycelia were harvested after incubation for 3 d. After pooled and rinsing, the mycelia were re-suspended in 100 mL sterile water containing rapamycin at various concentrations, then incubated on a shaker at 25 °C for another 2, 4, 6, and 8 h. Afterwards, the filtrates were used to determine nucleic acids and soluble carbohydrate contents after removing the mycelia. Leakage of nucleic acids was measured by determining the optical density at 260 nm (OD260) (Barbas et al., 2007; Cai et al., 2015), anthrone reagent with glucose was used as the standard to quantify the release of soluble carbohydrates (Morris, 1948), and Bradford (1976) was employed to quantitate the release of proteins. The experiments were performed in triplicates.
2.5. In vivo antifungal activity assay In vivo antifungal activity was determined according to Ji et al. (2018). Fruits were punched at the equator (2 mm wide and 5 mm deep) and 5 μL spore suspension (2 × 105 spores mL−1) was inoculated in each wound. After drying, 10 μL rapamycin solutions at various concentrations were added to the wounds. Finally, the fruits were kept at 23 ± 2 °C in 95% humidity for 4 d, and the disease severity was determined by measuring the lesion diameter. Each treatment was composed of three replicates, 25 fruit per replicate; sterile water was used as the control. 2.6. Fluorescence microscopy Dual-fluorescence staining was performed using fluorescein diacetate (FDA, 50 mg L−1; Sigma-Aldrich, San Louis, MO, USA) and propidium iodide (PI, 20 mg L−1; Molecular Probes, Eugene, OR, USA) as previously described by Xing et al. (2018). Intracellular ROS and mitochondria were detected using 2,7-dichlorodihydrofluorescein diacetate (DCHF-DA) (10 μM, Molecular Probes, Eugene, OR, USA) and MitoTracker Red CMXRos (abbreviated as MitoTracker, 1 μM, Molecular Probes, Eugene, OR, USA) according to Qin et al. (2011). Autophagic vacuoles were stained using monodansylcadaverine (MDC; Sigma-Aldrich, San Louis, MO, USA) as previously described by VeneaultFourrey et al. (2006). After staining, B. cinerea spores were examined under a fluorescence microscope (Zeiss Axioskop 40, Gottingen, Germany) as follows: FDA and DCHF-DA detection (EX BP 450–490, FT
2.10. Transmission electron microscopy (TEM) The procedures were performed according to the method according to Waterham et al. (1994) with minor modifications: the samples were successively fixed in 1.5% (w/v) KMnO4 at room temperature for 20 min and 6% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at 0 °C for 60 min, followed by postfixation in 0.5% (w/v) OsO4 and 2.5% (w/v) K2Cr2O7 in cacodylate buffer at 0 °C for 90 min. After dehydration in ethanol series, the samples were embedded in LR white; finally, ultrathin sections were prepared with a diamond knife and examined under a JEM-1230 electron microscope (JEOL, Tokyo, Japan). 159
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Fig. 1. Rapamycin suppressed mycelial growth of B. cinerea in a dose-dependent manner (A) Inhibitory eff ;ects of rapamycin on colony diameter. (B) Statistical analysis for colony diameters. The experiments were performed in triplicates and the data were represented by means ± standard deviations. Columns marked with diff ;erent letters indicate significant diff ;erence at p < 0.05 (Duncan’s multiple range test). (C) The hyphae at the edge of the colonies after incubation with rapamycin were shorter and denser with more branches compared to the control hyphae. Bar represented 20 μm.
Fig. 2. Effects of rapamycin on spore germination and germ tube elongation of B. cinerea. (A) Morphology of B. cinerea cells in the presence of rapamycin; (B) Rapamycin supplement led to vacuolation in the cytoplasm, as suggested by arrows in the inlets; (C–D) Statistical analysis on spore germination and germ tube length was performed in triplicates and approximately 200 spores were counted for each replicate. Data were represented by means ± standard deviations. Bar represented 20 μm. Columns marked with diff ;erent letters indicate significant diff ;erence at p < 0.05 (Duncan’s multiple range test).
statistically significantly (p < 0.05), Duncan's multiple range test was performed.
2.11. Statistical analysis Statistical data were analyzed using SPSS software (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was performed to determine the significance of differences. F-values of treatment effects were listed in Supplementary Table 2. When the differences were 160
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Fig. 3. In vivo efficacy of rapamycin on disease severity (lesion diameter) of gray mold on harvested apple and pear fruits. The lesion diameters on harvested apple fruits (A) and pear fruits (B) were shown as histograms. The experiments were performed in triplicates and the data were represented by means ± standard deviations. Columns marked with diff ;erent letters indicate significant diff ;erence at p < 0.05 (Duncan’s multiple range test).
Fig. 4. Autophagic activities were significantly induced by rapamycin. (A) Detection of autophagic activation with monodansylcadaverine (MDC) in B. cinerea cells. Arrows indicated autophagic vacuoles stained by MDC; Bar represented 20 μm. (B–E) Ultrastructural variations in wild-type and rapamycin-treated cells. (B, D) Control cells; (C, E) Rapamycin-treated cells. AV, autophagic vacuoles; M, mitochondria; N, nuclei, O, oil droplets, bars represented 5 μm (B, C) and 2 μm (D, E); (F) RT-qPCR analysis for the genes related to autophagic activities. The experiments were performed in triplicates and the data were represented by means ± standard deviations. Statistical significance was determined using Student’s t-test in different samples: * p < 0.05; ** p < 0.01.
3. Results
control, whereas 100 nM rapamycin completely abolished mycelial growth of B. cinerea. Moreover, in the presence of rapamycin, the mycelia at the edges of colonies were shorter and developed more branches (Fig. 1C). After the comparison to commercially available fungicides, it can be found from the toxicity equation and EC50 that rapamycin can effectively suppress mycelial spreading of B. cinerea (Supplementary Table 3).
3.1. In vitro efficacy of rapamycin on mycelial growth As revealed by the decreased colony diameters with increasing rapamycin doses (Fig. 1A-B), mycelial growth of B. cinerea on PDA was significantly suppressed by rapamycin at room temperature and the inhibitory eff ;ects were dose-dependent. When rapamycin dose was 1 nM, the colony diameter was approximately 50% for that of the 161
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Fig. 5. Rapamycin impaired membrane permeability and attenuated cell viability. (A) Dual-fluorescence staining of B. cinerea cells with FDA and PI. Except a low percentage of the cells were co-stained by FDA and PI, most of the cells were either stained by FDA or stained by PI; (B) B. cinerea cells stained by FDA significantly decreased with increasing rapamycin dose; (C) Rapamycin impaired membrane permeability in a dose-dependent manner, as revealed by PI staining. Bars represented 20 μm. Columns marked with diff ;erent letters indicate significant diff ;erence at p < 0.05 (Duncan’s multiple range test).
3.2. Efficacy of rapamycin in controlling gray mold on harvested fruits
3.4. Changes in membrane integrity and cell viability of B. cinerea
Spore germination and germ tube elongation of B. cinerea were also significantly suppressed in the presence of rapamycin, and the inhibitory efficacies were positively correlated with the rapamycin dose (Fig. 2). Notably, compared to the control, it was found that B. cinerea cells underwent vacuolation after incubation with rapamycin, as indicated by arrows in Fig. 2B. Moreover, rapamycin also significantly alleviate disease severity of gray mold in harvested apple, pear and grape fruits, and the lesion diameters demonstrated dose-dependent decreases with increasing rapamycin dose (Fig. 3 and Supplementary Fig. 1). Taking total soluble solids (TSS) and titratable acid (TA) as examples, rapamycin did not show significant influence on fruit quality (Supplementary Fig. 2).
Dual fluorescence staining with fluorescein diacetate (FDA) and propidium iodide (PI) were performed to check cell viability and membrane permeability of B. cinerea cells. As a result, B. cinerea cells were readily stained by FDA in the control group, whereas the cells stained by FDA significantly decreased after incubation with rapamycin (81.73% for 50 nM rapamycin and 66.77% for 100 nM rapamycin, Fig. 5). Compared with the control group, more cells were stained by PI in the presence of rapamycin (18.40% for 50 nM rapamycin and 32.51% for 100 nM rapamycin, Fig. 5), indicating that rapamycin impaired membrane integrity and attenuated cell viability in B. cinerea. Moreover, rapamycin elevated MDA contents and electrical conductivity over the entire experimental process for 12 h, whereas MDA contents and electrical conductivity of the control cells were stable at basal levels (Fig. 6A-B). Similarly, leakages of cytosolic contents occurred at 1 h after rapamycin application, the releasing rates increased over 8 h, and the released quantities of nucleic acids, soluble proteins and carbohydrates were positively correlated with rapamycin doses (Fig. 6C-E).
3.3. Variations in autophagic activities and expression of autophagy-related genes (ATGs) Given the phenomenon of vacuolation following rapamycin application, MDC staining and transmission electron microscopy (TEM) were further performed to examine the cytological and ultrastructural variations. Upon incubation with rapamycin, brilliant punctate structures associated with MDC fluorescence were detected in B. cinerea cells (Fig. 4A). As shown in the TEM images in Fig. 4B-E, the cytoplasmic content was dense and homogeneous in control cells, and numerous mitochondria in various shapes were distributed adjacent to the nuclei, whereas autophagic structures were not detected; in contrast, vacuolar structures encompassing degraded cytoplasmic contents appeared after incubation with rapamycin, which occupied large areas in the cytosol, and some oil droplets were also found in autophagic vacuoles. Furthermore, previously reported marker genes for autophagic activities were also examined using RT-qPCR analysis (Ren et al., 2017, 2018). The results showed that BcTOR, BcATG1, BcATG8, BcATG14 were significantly up-regulated after rapamycin treatment (Fig. 4F).
3.5. Rapamycin did not induce accumulation of reactive oxygen species (ROS) In the dual-fluorescence examination of ROS accumulation and mitochondrial membrane potential, almost all of the cells were stained by MitoTracker, whereas only 30.22% of the rapamycin-treated cells were stained after incubation with 50 nM rapamycin, and 100 nM rapamycin completely abolished the association of red fluorescence with mitochondria in B. cinerea cells (Fig. 7). Notably, rapamycin did not induce obvious ROS bursts and no further accumulation of intracellular ROS was detected in the cytosol of B. cinerea spores (Fig. 7), as determined by the fluorescence signal of DCHF-DA. 4. Discussion Harvested fruits and vegetables are metabolically active organisms, 162
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Fig. 6. Detection for membrane lipid peroxidation degree and releases of cytosolic macromolecules. Malondialdehyde (MDA) content (A), electrical conductivity (B), leakages of nucleic acids (C), soluble proteins (D) and soluble carbohydrates (E) were determined. Mycelia were transferred into distilled water in the presence of rapamycin at various concentrations (0 [control], 10, 50 and 100 nM) and incubated at 25 °C. The experiments were performed in triplicates and the data were represented by means ± standard deviations. Statistical significance was determined using Student’s t-test in different samples: * p < 0.05; ** p < 0.01.
autophagic machinery is possibly a target that has been largely unexplored. As a major pathway for bulk turnover of organelles and other cytoplasmic constituents, autophagy is likely to be incompatible with rapid fungal growth, and thus it is a promising drug target to selectively induce autophagy via pharmacological interference (Liu et al., 2012; Ren et al., 2017; Veneault-Fourrey et al., 2006). In the present study, rapamycin induced obvious vacuolation in the cytosol of cells, therefore, we were prompted to examine the variations in autophagic activity, in order to further dissect the mechanisms by which rapamycin exhibited the antifungal activity. As a result, significant autophagic vacuoles with cytoplasmic contents and oil droplets were observed in B. cinerea cells following rapamycin treatment. Moreover, B. cinerea cells treated with rapamycin demonstrated punctate structures associated with MDC fluorescence, whereas the control cells only exhibited weak and diffused fluorescence. These findings were similar to the results reported previously that pre-autophagosomal structures accumulated in the lumens of vacuoles after rapamycin induction (Ren et al., 2017, 2018). Notably, different from most previously reported antifungal agents that provoked ROS accumulation (Cai et al., 2015; Heller and
and appropriate postharvest handling is essential for prolonging their shelf life and maintaining the quality of fresh produce (Chen et al., 2015; Droby et al., 2009; Ji et al., 2018). It is estimated that more than 30% of harvested fruits and vegetables could not reach the customers mainly due to postharvest decay (Tian et al., 2016), and infection by fungal pathogens is above all the most destructive for fresh produce (An et al., 2016; Dean et al., 2012). In the present study, it was found rapamycin displayed dose-dependent inhibitory effects on mycelial growth of B. cinerea and disease severity in harvested fruits. It was found that rapamycin displayed higher efficacy in suppressing mycelial spreading of B. cinerea in the comparative analysis with some commercial fungicides. Nevertheless, we also noticed the apparent difference in antimicrobial activity between in vitro and in vivo assay, which was also previously reported indeed (Fang et al., 2011; Gong et al., 2016; Sanzani et al., 2009; Xu et al., 2018). The difference may be resulted from higher adaptability to host environment compared to that in in vitro tests, and complex intrinsic properties of hosts could not be ruled out also, such as availability of carbon and sulphur sources and other nutrients, moisture content, pH as well as other factors. In terms of studies on inhibitory mechanisms of antifungal agents, 163
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5. Conclusion In summary, rapamycin is efficient in suppressing mycelial growth of B. cinerea and disease severity of gray mold on harvested fruits. It exhibits inhibitory effects by modulating autophagic activity and membrane permeability rather than inducing ROS burst, which is promising as an alternative choice for postharvest handling of fresh produce. Acknowledgements The authors thank Prof. Paul Tudzynski from Westfälische Wilhelms-Universität Münster of Germany for providing B. cinerea B05.10 strain and Mrs. Fengqin Dong for her help in TEM analysis. The work was supported by National Key Research and Development Program of China (2017YFD0401301, 2016YFD0400903) and NSFC (31530057, 30672210). 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.postharvbio.2019.01. 005. References An, B., Li, B.Q., Li, H., Zhang, Z.Q., Qin, G.Z., Tian, S.P., 2016. Aquaporin8 regulates cellular development and ROS production, a critical component of virulence in Botrytis cinerea. New Phytol. 209, 1668–1680. Barbas 3rd., C.F., Burton, D.R., Scott, J.K., Silverman, G.J., 2007. Quantitation of DNA and RNA. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.ip47. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Biochem. Anal. Biochem. 72, 248–254. Cai, J.H., Chen, J., Lu, G.B., Zhao, Y.M., Tian, S.P., Qin, G.Z., 2015. Control of brown rot on jujube and peach fruit by trisodium phosphate. Postharvest Biol. Technol. 99, 93–98. Chen, J., Li, B.Q., Qin, G.Z., Tian, S.P., 2015. Mechanism of H2O2-induced oxidative stress regulating viability and biocontrol ability of Rhodotorula glutinis. Int. J. Food Microbiol. 193, 152–158. Dean, R., van Kan, J.A., Pretorius, Z.A., Hammond-Kosack, K.E., Di Pietro, A., Spanu, P.D., Rudd, J.J., Dickman, M., Kahmann, R., Ellis, J., Foster, G.D., 2012. The top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 13, 414–430. Dementhon, K., Paoletti, M., Pinan-Lucarré, B., Loubradou-Bourges, N., Sabourin, M., Saupe, S.J., Clavé, C., 2003. Rapamycin mimics the incompatibility reaction in the fungus Podospora anserina. Eukaryot. Cell 2, 238–246. Droby, S., Wisniewski, M., Macarisin, D., Wilson, C., 2009. Twenty years of postharvest biocontrol research: is it time for a new paradigm? Postharvest Biol. Technol. 52, 137–145. Ehninger, D., Neff, F., Xie, K., 2014. Longevity, aging and rapamycin. Cell. Mol. Life Sci. 71, 4325–4346. Elad, Y., Pertot, I., Cores-Prado, A.M., Stewart, A., 2016. Plant hosts of Botrytis spp. In: Fillinger, S., Elad, Y. (Eds.), Botrytis—the Fungus, the Pathogen and Its Management in Agricultural Systems. Springer, Berlin, pp. 413–486. Fang, X.L., Li, Z.Z., Wang, Y.H., Zhang, X., 2011. In vitro and in vivo antimicrobial activity of Xenorhabdus bovienii YL002 against Phytophthora capsici and Botrytis cinerea. J. Appl. Microbiol. 111, 145–154. Gong, L., Tan, H.B., Chen, F., Li, T.T., Zhu, J.Y., Jian, Q.J., Yuan, D.B., Xu, L.X., Hu, W.Z., Jiang, Y.M., Duan, X.W., 2016. Novel synthesized 2, 4-DAPG analogues: antifungal activity, mechanism and toxicology. Sci. Rep. 6, 32266. Heller, J., Tudzynski, P., 2011. Reactive oxygen species in phytopathogenic fungi: signaling, development, and disease. Annu. Rev. Phytopathol. 49, 369–390. Ji, D.C., Chen, T., Ma, D.Y., Liu, J.L., Xu, Y., Tian, S.P., 2018. Inhibitory eff ;ects of methyl thujate on mycelial growth of Botrytis cinerea and possible mechanisms. Postharvest Biol. Technol. 142, 46–54. Lee, H.J., Choi, G.J., Cho, K.Y., 1998. Correlation of lipid peroxidation in Botrytis cinerea caused by dicarboximide fungicides with their fungicidal activity. J. Agr. Food Chem. 46, 737–741. Lewis, J.A., Papavizas, G.C., 1987. Permeability changes in hyphae of Rhizoctonia solani induced by germling preparations of Trichoderma and Gliocladium. Phytopathology 77, 699–703. Liu, X.H., Gao, H.M., Xu, F., Lu, J.P., Devenish, R.J., Lin, F.C., 2012. Autophagy vitalizes the pathogenicity of pathogenic fungi. Autophagy 8, 1415–1425. Liu, J., Sui, Y., Wisniewski, M., Droby, S., Liu, Y., 2013. Utilization of antagonistic yeasts to manage postharvest fungal diseases of fruit. Int. J. Food Microbiol. 167, 153–160 (Review). Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408.
Fig. 7. Microscopic detection on reactive oxygen species (ROS) and mitochondrial potential abolishment. (A) Rapamycin abolished mitochondrial membrane potential, whereas cytoplasmic accumulation of ROS was not detected; (B) The percentage of B. cinerea cells associated with DCHF-DA fluorescence were not detected, whereas the percentage of the cells stained by MitoTracker significantly decreased after co-incubation with increasing rapamycin concentration. Bar represented 20 μm. Columns marked with diff ;erent letters indicate significant diff ;erence at p < 0.05 (Duncan’s multiple range test).
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Efficacy of rapamycin in modulating autophagic activity of Botrytis cinerea for controlling gray mold
T
Danying Maa,b, Dongchao Jia,b, Zhanquan Zhanga, Boqiang Lia, Guozheng Qina, Yong Xua, ⁎ Tong Chena, , Shiping Tiana,b,c a
Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China University of Chinese Academy of Sciences, Beijing, 100049, China c Key Laboratory of Post-Harvest Handling of Fruits, Ministry of Agriculture, Beijing, 100093, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Autophagy Botrytis cinerea Membrane integrity Rapamycin
Postharvest diseases severely deteriorate the intrinsic quality and commercial value of fruits and vegetables, leading to significant economic losses for farmers and increased costs for consumers. In the present study, rapamycin was effective in suppressing mycelial growth of Botrytis cinerea and disease severity of gray mold on harvested apple, pear and grape fruits. The inhibitory mechanism was attributed to a modulation in autophagic activity and membrane permeability rather than the induction of reactive oxygen species. Increases in the degree of membrane peroxidation and the leakage of cytosolic contents were also detected, supporting the data on reduced cell viability. The results indicated that rapamycin may be a promising alternative for controlling gray mold caused by B. cinerea on harvested produce.
1. Introduction Botrytis cinerea is the causal agent of gray mold on more than two hundred species worldwide, leading to great losses in fruits and vegetable production (Dean et al., 2012). Up to now, numerous approaches have been attempted to control gray mold, including biocontrol agents (Droby et al., 2009; Liu et al., 2013), natural antimicrobial polymers (Ji et al., 2018) and substances generally regarded as safe (GRAS) (Elad et al., 2016; Qin et al., 2010; Sanzani et al., 2014). Among them, synthetic fungicides are still widely used, particularly in developing countries. However, abuse of commercial fungicides has resulted in increasing risks in environmental pollution and food contamination (Elad et al., 2016; Tian et al., 2016). Therefore, it is urgent to further explore new targeting sites and underlying mechanisms for alternative antifungal agents. Originally isolated from Streptomyces hygroscopicus, rapamycin is a macrolide compound showing inhibitory effects on Candida albicans (Vézina et al., 1975). During the past several decades, it was further demonstrated that rapamycin can induce physiological variations similar to those under nitrogen or carbon deficiency in Podospora anserine (Dementhon et al., 2003), and Fusarium graminiarum hyphae showed morphological abnormalities following rapamycin treatment (Yu et al., 2014). As rapamycin has been widely used as an interfering agent in the
⁎
studies on autophagy, a macro-molecule degradation process in which cells recycle cytoplasmic contents under specific conditions (Marobbio et al., 2012; Martínez-Cisuelo et al., 2016; Paghdal and Schwartz, 2007), it is envisioned that the inhibitory mechanisms of rapamycin may be also attributed to induced autophagic activities. However, the results are scarce for its application in phytopathogens for postharvest produces. Recently, several lines of evidence suggest that it is also safe to be used as a health-promoting drug, since rapamycin in nano-molar doses can efficiently bind to newly synthesized mTOR before it binds to Rictor, and thus prevent formation of mTORC2 (Mukhopadhyay et al., 2016). Moreover, rapamycin can postpone aging and extend lifespan at low doses without detectable side effects, and thus it has been approved by FDA as drugs for treating aging-related diseases (Ehninger et al., 2014). Utilizing in vitro and in vivo antifungal activity assay, the present study aimed to evaluate the efficacy of rapamycin in controlling gray mold on harvested fruits. It was found that rapamycin exhibited antifungal eff ;ects by modulating autophagic activities and membrane permeability rather than inducing reactive oxygen species. Moreover, the degree of membrane peroxidation and the leakage of cytosolic contents also increased. The results may further broaden our insights towards antifungal mechanisms of rapamycin and provide guidance for handling of postharvest fresh produce.
Corresponding author at: Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Xiangshan, Haidian District, Beijing, 100093, China. E-mail address: [email protected] (T. Chen).
https://doi.org/10.1016/j.postharvbio.2019.01.005 Received 3 August 2018; Received in revised form 8 January 2019; Accepted 11 January 2019 Available online 16 January 2019 0925-5214/ © 2019 Elsevier B.V. All rights reserved.
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2. Materials and methods
510, LT515), PI detection (BP 546/12, FT 580, LP 590), MDC detection (G365, FT395, LP420).
2.1. Chemicals 2.7. Total RNA extraction and RT-qPCR Rapamycin was purchased from Sigma-Aldrich (San Louis, MO, USA).
Total RNAs were extracted using Trizol reagent (Tiangen, Beijing, China) as reported by Ji et al. (2018). After first strand cDNA was synthesized using PrimeScript RT reagent kit with gDNA eraser (TaKaRa, Dalian, China), RT-qPCR was performed on Step One Plus RealTime PCR system (Thermo Fisher Scientific) using SYBR premix ex Taq (TaKaRa, Dalian, China). Primer Premier 5.0 (PREMIER Biosoft, USA) was used to design the primers, as listed in Supplementary Table 1. The RT-qPCR procedures were as follows: pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, 40 cycles. BcTubulin was used as the reference gene for normalization, and relative expression levels were calculated using the 2−△△Ct method (Livak and Schmittgen, 2001).
2.2. Pathogens Botrytis cinerea B05.10 was cultivated on potato dextrose agar (PDA) at 23 ± 2 °C for 7–10 d (Ji et al., 2018). Afterwards, spores were harvested, filtrated against sterile gauze and counted as previously described (Qin et al., 2010). 2.3. Fruits Apple (Malus domestica Borkh.), pear (Pyrus bretschneideri Rehd.) and grape (Vitis labrusca L.) fruits were harvested at commercial maturity. Fruits without injuries and lesions were sterilized with 2% (v/v) sodium hypochlorite, rinsed with water and air-dried before use. Total soluble solids (TSS, %) were measured according to the method described by Yun et al. (2013), and titratable acid (TA, %) was determined using the digital acidity meters (GMK-835, G-WON HITECH CO., LTD, Korea) following the manufacturer’s instructions (Obenland et al., 2011).
2.8. Detection of membrane lipid peroxidation degree and electrical conductivity Membrane lipid peroxidation degree was examined by determining malondialdehyde (MDA) content using thiobarbituric acid (TBA) method (Pereira et al., 2003). After the spores were incubated in PDB on a shaker at 180 rpm and 25 °C for 3 d, rapamycin was added at various concentrations and the cells were further incubated for 0, 4, 8 and 12 h before measurement. Electrical conductivity of the incubation medium was measured using a conductivity meter (EC215, HANNA Instrument; Lee et al., 1998). Spores were incubated in 200 mL PDB (1 × 106 spores mL−1) at 180 rpm for 3 day at 25 °C. After washing with water, the mycelia (3 g wet weight) were suspended in 40 mL sterile water containing rapamycin at various concentrations, and the measurements were performed at 0, 4, 8 and 12 h after further incubation. To rule out possible influences from background conductivity of rapamycin, the results were expressed as changes relative to the initial values of measurement.
2.4. In vitro inhibitory efficacy on B. cinerea growth Rapamycin was supplemented to PDA to generate a concentration gradient (0, 0.1, 1, 10, 50 and 100 nM). Afterwards, 5 μL spore suspension (1 × 105 spores mL−1) was dropped in the center of each dish and cultured at 23 ± 2 °C, the colony diameter was determined every 24 h, and the experiments were performed in triplicates. Furthermore, spores were incubated in potato dextrose broth (PDB) containing rapamycin at various concentrations at 25 °C to examine spore germination rate and germ tube length. In the comparison to commercially available fungicides, toxicity regression equations and median effect concentrations (EC50) were calculated according to Taylor et al. (2002).
2.9. Cytoplasmic content leakage assay Leakage of cytoplasmic contents from mycelia was determined according to Lewis and Papavizas (1987) with minor modifications. Spores were grown in PDB at 25 °C and 180 rpm, and the mycelia were harvested after incubation for 3 d. After pooled and rinsing, the mycelia were re-suspended in 100 mL sterile water containing rapamycin at various concentrations, then incubated on a shaker at 25 °C for another 2, 4, 6, and 8 h. Afterwards, the filtrates were used to determine nucleic acids and soluble carbohydrate contents after removing the mycelia. Leakage of nucleic acids was measured by determining the optical density at 260 nm (OD260) (Barbas et al., 2007; Cai et al., 2015), anthrone reagent with glucose was used as the standard to quantify the release of soluble carbohydrates (Morris, 1948), and Bradford (1976) was employed to quantitate the release of proteins. The experiments were performed in triplicates.
2.5. In vivo antifungal activity assay In vivo antifungal activity was determined according to Ji et al. (2018). Fruits were punched at the equator (2 mm wide and 5 mm deep) and 5 μL spore suspension (2 × 105 spores mL−1) was inoculated in each wound. After drying, 10 μL rapamycin solutions at various concentrations were added to the wounds. Finally, the fruits were kept at 23 ± 2 °C in 95% humidity for 4 d, and the disease severity was determined by measuring the lesion diameter. Each treatment was composed of three replicates, 25 fruit per replicate; sterile water was used as the control. 2.6. Fluorescence microscopy Dual-fluorescence staining was performed using fluorescein diacetate (FDA, 50 mg L−1; Sigma-Aldrich, San Louis, MO, USA) and propidium iodide (PI, 20 mg L−1; Molecular Probes, Eugene, OR, USA) as previously described by Xing et al. (2018). Intracellular ROS and mitochondria were detected using 2,7-dichlorodihydrofluorescein diacetate (DCHF-DA) (10 μM, Molecular Probes, Eugene, OR, USA) and MitoTracker Red CMXRos (abbreviated as MitoTracker, 1 μM, Molecular Probes, Eugene, OR, USA) according to Qin et al. (2011). Autophagic vacuoles were stained using monodansylcadaverine (MDC; Sigma-Aldrich, San Louis, MO, USA) as previously described by VeneaultFourrey et al. (2006). After staining, B. cinerea spores were examined under a fluorescence microscope (Zeiss Axioskop 40, Gottingen, Germany) as follows: FDA and DCHF-DA detection (EX BP 450–490, FT
2.10. Transmission electron microscopy (TEM) The procedures were performed according to the method according to Waterham et al. (1994) with minor modifications: the samples were successively fixed in 1.5% (w/v) KMnO4 at room temperature for 20 min and 6% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at 0 °C for 60 min, followed by postfixation in 0.5% (w/v) OsO4 and 2.5% (w/v) K2Cr2O7 in cacodylate buffer at 0 °C for 90 min. After dehydration in ethanol series, the samples were embedded in LR white; finally, ultrathin sections were prepared with a diamond knife and examined under a JEM-1230 electron microscope (JEOL, Tokyo, Japan). 159
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Fig. 1. Rapamycin suppressed mycelial growth of B. cinerea in a dose-dependent manner (A) Inhibitory eff ;ects of rapamycin on colony diameter. (B) Statistical analysis for colony diameters. The experiments were performed in triplicates and the data were represented by means ± standard deviations. Columns marked with diff ;erent letters indicate significant diff ;erence at p < 0.05 (Duncan’s multiple range test). (C) The hyphae at the edge of the colonies after incubation with rapamycin were shorter and denser with more branches compared to the control hyphae. Bar represented 20 μm.
Fig. 2. Effects of rapamycin on spore germination and germ tube elongation of B. cinerea. (A) Morphology of B. cinerea cells in the presence of rapamycin; (B) Rapamycin supplement led to vacuolation in the cytoplasm, as suggested by arrows in the inlets; (C–D) Statistical analysis on spore germination and germ tube length was performed in triplicates and approximately 200 spores were counted for each replicate. Data were represented by means ± standard deviations. Bar represented 20 μm. Columns marked with diff ;erent letters indicate significant diff ;erence at p < 0.05 (Duncan’s multiple range test).
statistically significantly (p < 0.05), Duncan's multiple range test was performed.
2.11. Statistical analysis Statistical data were analyzed using SPSS software (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was performed to determine the significance of differences. F-values of treatment effects were listed in Supplementary Table 2. When the differences were 160
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Fig. 3. In vivo efficacy of rapamycin on disease severity (lesion diameter) of gray mold on harvested apple and pear fruits. The lesion diameters on harvested apple fruits (A) and pear fruits (B) were shown as histograms. The experiments were performed in triplicates and the data were represented by means ± standard deviations. Columns marked with diff ;erent letters indicate significant diff ;erence at p < 0.05 (Duncan’s multiple range test).
Fig. 4. Autophagic activities were significantly induced by rapamycin. (A) Detection of autophagic activation with monodansylcadaverine (MDC) in B. cinerea cells. Arrows indicated autophagic vacuoles stained by MDC; Bar represented 20 μm. (B–E) Ultrastructural variations in wild-type and rapamycin-treated cells. (B, D) Control cells; (C, E) Rapamycin-treated cells. AV, autophagic vacuoles; M, mitochondria; N, nuclei, O, oil droplets, bars represented 5 μm (B, C) and 2 μm (D, E); (F) RT-qPCR analysis for the genes related to autophagic activities. The experiments were performed in triplicates and the data were represented by means ± standard deviations. Statistical significance was determined using Student’s t-test in different samples: * p < 0.05; ** p < 0.01.
3. Results
control, whereas 100 nM rapamycin completely abolished mycelial growth of B. cinerea. Moreover, in the presence of rapamycin, the mycelia at the edges of colonies were shorter and developed more branches (Fig. 1C). After the comparison to commercially available fungicides, it can be found from the toxicity equation and EC50 that rapamycin can effectively suppress mycelial spreading of B. cinerea (Supplementary Table 3).
3.1. In vitro efficacy of rapamycin on mycelial growth As revealed by the decreased colony diameters with increasing rapamycin doses (Fig. 1A-B), mycelial growth of B. cinerea on PDA was significantly suppressed by rapamycin at room temperature and the inhibitory eff ;ects were dose-dependent. When rapamycin dose was 1 nM, the colony diameter was approximately 50% for that of the 161
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Fig. 5. Rapamycin impaired membrane permeability and attenuated cell viability. (A) Dual-fluorescence staining of B. cinerea cells with FDA and PI. Except a low percentage of the cells were co-stained by FDA and PI, most of the cells were either stained by FDA or stained by PI; (B) B. cinerea cells stained by FDA significantly decreased with increasing rapamycin dose; (C) Rapamycin impaired membrane permeability in a dose-dependent manner, as revealed by PI staining. Bars represented 20 μm. Columns marked with diff ;erent letters indicate significant diff ;erence at p < 0.05 (Duncan’s multiple range test).
3.2. Efficacy of rapamycin in controlling gray mold on harvested fruits
3.4. Changes in membrane integrity and cell viability of B. cinerea
Spore germination and germ tube elongation of B. cinerea were also significantly suppressed in the presence of rapamycin, and the inhibitory efficacies were positively correlated with the rapamycin dose (Fig. 2). Notably, compared to the control, it was found that B. cinerea cells underwent vacuolation after incubation with rapamycin, as indicated by arrows in Fig. 2B. Moreover, rapamycin also significantly alleviate disease severity of gray mold in harvested apple, pear and grape fruits, and the lesion diameters demonstrated dose-dependent decreases with increasing rapamycin dose (Fig. 3 and Supplementary Fig. 1). Taking total soluble solids (TSS) and titratable acid (TA) as examples, rapamycin did not show significant influence on fruit quality (Supplementary Fig. 2).
Dual fluorescence staining with fluorescein diacetate (FDA) and propidium iodide (PI) were performed to check cell viability and membrane permeability of B. cinerea cells. As a result, B. cinerea cells were readily stained by FDA in the control group, whereas the cells stained by FDA significantly decreased after incubation with rapamycin (81.73% for 50 nM rapamycin and 66.77% for 100 nM rapamycin, Fig. 5). Compared with the control group, more cells were stained by PI in the presence of rapamycin (18.40% for 50 nM rapamycin and 32.51% for 100 nM rapamycin, Fig. 5), indicating that rapamycin impaired membrane integrity and attenuated cell viability in B. cinerea. Moreover, rapamycin elevated MDA contents and electrical conductivity over the entire experimental process for 12 h, whereas MDA contents and electrical conductivity of the control cells were stable at basal levels (Fig. 6A-B). Similarly, leakages of cytosolic contents occurred at 1 h after rapamycin application, the releasing rates increased over 8 h, and the released quantities of nucleic acids, soluble proteins and carbohydrates were positively correlated with rapamycin doses (Fig. 6C-E).
3.3. Variations in autophagic activities and expression of autophagy-related genes (ATGs) Given the phenomenon of vacuolation following rapamycin application, MDC staining and transmission electron microscopy (TEM) were further performed to examine the cytological and ultrastructural variations. Upon incubation with rapamycin, brilliant punctate structures associated with MDC fluorescence were detected in B. cinerea cells (Fig. 4A). As shown in the TEM images in Fig. 4B-E, the cytoplasmic content was dense and homogeneous in control cells, and numerous mitochondria in various shapes were distributed adjacent to the nuclei, whereas autophagic structures were not detected; in contrast, vacuolar structures encompassing degraded cytoplasmic contents appeared after incubation with rapamycin, which occupied large areas in the cytosol, and some oil droplets were also found in autophagic vacuoles. Furthermore, previously reported marker genes for autophagic activities were also examined using RT-qPCR analysis (Ren et al., 2017, 2018). The results showed that BcTOR, BcATG1, BcATG8, BcATG14 were significantly up-regulated after rapamycin treatment (Fig. 4F).
3.5. Rapamycin did not induce accumulation of reactive oxygen species (ROS) In the dual-fluorescence examination of ROS accumulation and mitochondrial membrane potential, almost all of the cells were stained by MitoTracker, whereas only 30.22% of the rapamycin-treated cells were stained after incubation with 50 nM rapamycin, and 100 nM rapamycin completely abolished the association of red fluorescence with mitochondria in B. cinerea cells (Fig. 7). Notably, rapamycin did not induce obvious ROS bursts and no further accumulation of intracellular ROS was detected in the cytosol of B. cinerea spores (Fig. 7), as determined by the fluorescence signal of DCHF-DA. 4. Discussion Harvested fruits and vegetables are metabolically active organisms, 162
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Fig. 6. Detection for membrane lipid peroxidation degree and releases of cytosolic macromolecules. Malondialdehyde (MDA) content (A), electrical conductivity (B), leakages of nucleic acids (C), soluble proteins (D) and soluble carbohydrates (E) were determined. Mycelia were transferred into distilled water in the presence of rapamycin at various concentrations (0 [control], 10, 50 and 100 nM) and incubated at 25 °C. The experiments were performed in triplicates and the data were represented by means ± standard deviations. Statistical significance was determined using Student’s t-test in different samples: * p < 0.05; ** p < 0.01.
autophagic machinery is possibly a target that has been largely unexplored. As a major pathway for bulk turnover of organelles and other cytoplasmic constituents, autophagy is likely to be incompatible with rapid fungal growth, and thus it is a promising drug target to selectively induce autophagy via pharmacological interference (Liu et al., 2012; Ren et al., 2017; Veneault-Fourrey et al., 2006). In the present study, rapamycin induced obvious vacuolation in the cytosol of cells, therefore, we were prompted to examine the variations in autophagic activity, in order to further dissect the mechanisms by which rapamycin exhibited the antifungal activity. As a result, significant autophagic vacuoles with cytoplasmic contents and oil droplets were observed in B. cinerea cells following rapamycin treatment. Moreover, B. cinerea cells treated with rapamycin demonstrated punctate structures associated with MDC fluorescence, whereas the control cells only exhibited weak and diffused fluorescence. These findings were similar to the results reported previously that pre-autophagosomal structures accumulated in the lumens of vacuoles after rapamycin induction (Ren et al., 2017, 2018). Notably, different from most previously reported antifungal agents that provoked ROS accumulation (Cai et al., 2015; Heller and
and appropriate postharvest handling is essential for prolonging their shelf life and maintaining the quality of fresh produce (Chen et al., 2015; Droby et al., 2009; Ji et al., 2018). It is estimated that more than 30% of harvested fruits and vegetables could not reach the customers mainly due to postharvest decay (Tian et al., 2016), and infection by fungal pathogens is above all the most destructive for fresh produce (An et al., 2016; Dean et al., 2012). In the present study, it was found rapamycin displayed dose-dependent inhibitory effects on mycelial growth of B. cinerea and disease severity in harvested fruits. It was found that rapamycin displayed higher efficacy in suppressing mycelial spreading of B. cinerea in the comparative analysis with some commercial fungicides. Nevertheless, we also noticed the apparent difference in antimicrobial activity between in vitro and in vivo assay, which was also previously reported indeed (Fang et al., 2011; Gong et al., 2016; Sanzani et al., 2009; Xu et al., 2018). The difference may be resulted from higher adaptability to host environment compared to that in in vitro tests, and complex intrinsic properties of hosts could not be ruled out also, such as availability of carbon and sulphur sources and other nutrients, moisture content, pH as well as other factors. In terms of studies on inhibitory mechanisms of antifungal agents, 163
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5. Conclusion In summary, rapamycin is efficient in suppressing mycelial growth of B. cinerea and disease severity of gray mold on harvested fruits. It exhibits inhibitory effects by modulating autophagic activity and membrane permeability rather than inducing ROS burst, which is promising as an alternative choice for postharvest handling of fresh produce. Acknowledgements The authors thank Prof. Paul Tudzynski from Westfälische Wilhelms-Universität Münster of Germany for providing B. cinerea B05.10 strain and Mrs. Fengqin Dong for her help in TEM analysis. The work was supported by National Key Research and Development Program of China (2017YFD0401301, 2016YFD0400903) and NSFC (31530057, 30672210). 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.postharvbio.2019.01. 005. References An, B., Li, B.Q., Li, H., Zhang, Z.Q., Qin, G.Z., Tian, S.P., 2016. Aquaporin8 regulates cellular development and ROS production, a critical component of virulence in Botrytis cinerea. New Phytol. 209, 1668–1680. Barbas 3rd., C.F., Burton, D.R., Scott, J.K., Silverman, G.J., 2007. Quantitation of DNA and RNA. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.ip47. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Biochem. Anal. Biochem. 72, 248–254. Cai, J.H., Chen, J., Lu, G.B., Zhao, Y.M., Tian, S.P., Qin, G.Z., 2015. Control of brown rot on jujube and peach fruit by trisodium phosphate. Postharvest Biol. Technol. 99, 93–98. Chen, J., Li, B.Q., Qin, G.Z., Tian, S.P., 2015. Mechanism of H2O2-induced oxidative stress regulating viability and biocontrol ability of Rhodotorula glutinis. Int. J. Food Microbiol. 193, 152–158. Dean, R., van Kan, J.A., Pretorius, Z.A., Hammond-Kosack, K.E., Di Pietro, A., Spanu, P.D., Rudd, J.J., Dickman, M., Kahmann, R., Ellis, J., Foster, G.D., 2012. The top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 13, 414–430. Dementhon, K., Paoletti, M., Pinan-Lucarré, B., Loubradou-Bourges, N., Sabourin, M., Saupe, S.J., Clavé, C., 2003. Rapamycin mimics the incompatibility reaction in the fungus Podospora anserina. Eukaryot. Cell 2, 238–246. Droby, S., Wisniewski, M., Macarisin, D., Wilson, C., 2009. Twenty years of postharvest biocontrol research: is it time for a new paradigm? Postharvest Biol. Technol. 52, 137–145. Ehninger, D., Neff, F., Xie, K., 2014. Longevity, aging and rapamycin. Cell. Mol. Life Sci. 71, 4325–4346. Elad, Y., Pertot, I., Cores-Prado, A.M., Stewart, A., 2016. Plant hosts of Botrytis spp. In: Fillinger, S., Elad, Y. (Eds.), Botrytis—the Fungus, the Pathogen and Its Management in Agricultural Systems. Springer, Berlin, pp. 413–486. Fang, X.L., Li, Z.Z., Wang, Y.H., Zhang, X., 2011. In vitro and in vivo antimicrobial activity of Xenorhabdus bovienii YL002 against Phytophthora capsici and Botrytis cinerea. J. Appl. Microbiol. 111, 145–154. Gong, L., Tan, H.B., Chen, F., Li, T.T., Zhu, J.Y., Jian, Q.J., Yuan, D.B., Xu, L.X., Hu, W.Z., Jiang, Y.M., Duan, X.W., 2016. Novel synthesized 2, 4-DAPG analogues: antifungal activity, mechanism and toxicology. Sci. Rep. 6, 32266. Heller, J., Tudzynski, P., 2011. Reactive oxygen species in phytopathogenic fungi: signaling, development, and disease. Annu. Rev. Phytopathol. 49, 369–390. Ji, D.C., Chen, T., Ma, D.Y., Liu, J.L., Xu, Y., Tian, S.P., 2018. Inhibitory eff ;ects of methyl thujate on mycelial growth of Botrytis cinerea and possible mechanisms. Postharvest Biol. Technol. 142, 46–54. Lee, H.J., Choi, G.J., Cho, K.Y., 1998. Correlation of lipid peroxidation in Botrytis cinerea caused by dicarboximide fungicides with their fungicidal activity. J. Agr. Food Chem. 46, 737–741. Lewis, J.A., Papavizas, G.C., 1987. Permeability changes in hyphae of Rhizoctonia solani induced by germling preparations of Trichoderma and Gliocladium. Phytopathology 77, 699–703. Liu, X.H., Gao, H.M., Xu, F., Lu, J.P., Devenish, R.J., Lin, F.C., 2012. Autophagy vitalizes the pathogenicity of pathogenic fungi. Autophagy 8, 1415–1425. Liu, J., Sui, Y., Wisniewski, M., Droby, S., Liu, Y., 2013. Utilization of antagonistic yeasts to manage postharvest fungal diseases of fruit. Int. J. Food Microbiol. 167, 153–160 (Review). Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408.
Fig. 7. Microscopic detection on reactive oxygen species (ROS) and mitochondrial potential abolishment. (A) Rapamycin abolished mitochondrial membrane potential, whereas cytoplasmic accumulation of ROS was not detected; (B) The percentage of B. cinerea cells associated with DCHF-DA fluorescence were not detected, whereas the percentage of the cells stained by MitoTracker significantly decreased after co-incubation with increasing rapamycin concentration. Bar represented 20 μm. Columns marked with diff ;erent letters indicate significant diff ;erence at p < 0.05 (Duncan’s multiple range test).
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