The response of growth and patulin production of postharvest pathogen Penicillium expansum to exogenous potassium phosphite treatment

International Journal of Food Microbiology 244 (2017) 1–10

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

The response of growth and patulin production of postharvest pathogen Penicillium expansum to exogenous potassium phosphite treatment Tongfei Lai a,b,1, Ying Wang c,d,1, Yaya Fan b,1, Yingying Zhou b, Ying Bao a, Ting Zhou a,⁎ a

Key Laboratory for Quality and Safety of Agricultural Products of Hangzhou City, College of Life and Environmental Science, Hangzhou Normal University, Hangzhou 310036, China Research Centre for Plant RNA Signaling, College of Life and Environmental Science, Hangzhou Normal University, Hangzhou 310036, China Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China d University of Chinese Academy of Sciences, Beijing 100093, China b c

a r t i c l e

i n f o

Article history: Received 9 August 2016 Received in revised form 9 December 2016 Accepted 26 December 2016 Available online 28 December 2016 Keywords: Penicillium expansum Potassium phosphite Patulin iTRAQ RNA sequencing

a b s t r a c t In this study, the effects of exogenous potassium phosphite (Phi) on growth and patulin production of postharvest pathogen Penicillium expansum were assessed. The results indicated that P. expansum under 5 mmol/L Phi stress presented obvious development retardation, yield reduction of patulin and lower infectivity to apple fruit. Meanwhile, expression analysis of 15 genes related to patulin biosynthesis suggested that Phi mainly affected the early steps of patulin synthetic route at transcriptional level. Furthermore, a global view of proteome and transcriptome alteration of P. expansum spores during 6 h of Phi stress was evaluated by iTRAQ (isobaric tags for relative and absolute quantitation) and RNA-seq (RNA sequencing) approaches. A total of 582 differentially expressed proteins (DEPs) and 177 differentially expressed genes (DEGs) were acquired, most of which participated in carbohydrate metabolism, amino acid metabolism, lipid metabolism, genetic information processing and biosynthesis of secondary metabolites. Finally, 39 overlapped candidates were screened out through correlational analysis between iTRAQ and RNA-seq datasets. These findings will afford more precise and directional clues to explore the inhibitory mechanism of Phi on growth and patulin biosynthesis of P. expansum, and be beneficial to develop effective controlling approaches based on Phi. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Unwanted fungal growth and mycotoxins contamination have become major problems in food processing and preservation. Among numerous destructive postharvest pathogens, Penicillium expansum, the causal agent of blue mold, is by far the most worrisome species of the genus Penicillium (Ballester et al., 2015). It not only causes considerable pome losses during postharvest handling and storage, but also produces patulin, a mycotoxin with mutagenic and carcinogenic effects on human (Li et al., 2015). Traditionally, beside certain prophylactic measures applied to reduce the amount of inoculums in the storage environments, the control of diseases in postharvest fruits is based on the use of synthetic fungicides (Nunes, 2012). However, the progressive losses of their effectiveness due to emergence of resistant mutants and growing concern of consumers over chemical residues have created interest in alternative approaches and developing safer products that have different modes of action from those of the traditional active ingredients (Palou et al., 2016).

⁎ Corresponding author. E-mail address: [email protected] (T. Zhou). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ijfoodmicro.2016.12.017 0168-1605/© 2016 Elsevier B.V. All rights reserved.

Recent reports showed that propolis, salicylic acid, oxalic acid, chitin, boric acid, ultraviolet-C light, allyl isothiocyanate, gamma irradiation, garlic extracts and betel leaf essential oil could directly inhibit the growth of P. expansum and influence the synthesis of patulin (Basak and Guha, 2015; Daniel et al., 2015; Fu et al., 2016; Lai et al., 2016; Manyes et al., 2015; Jeong et al., 2015; Matny et al., 2015; Neto et al., 2016; Silici and Karaman, 2014; Syamaladevi et al., 2015; Zhu et al., 2016). Meanwhile, postharvest application of γ-aminobutyric acid, hot air treatment, methyl jasmonate or algal saccharides could induce defence response and increase resistance to P. expansum in host fruits (Abouraïcha et al., 2015; Wang et al., 2015a; Wang et al., 2015b; Yu et al., 2014). A lot of information and advances concerning the selection of antagonistic microorganisms and different approaches to enhance biological control activities were also achieved (Chen et al., 2015; Fu et al., 2015; Wang et al., 2015c; Yang et al., 2015). Nevertheless, more antifungal substances and the possible inhibitory mechanisms involved are needed to further explore owing to widespread hosts and strong stress tolerance of P. expansum. Phosphite is a general term used to describe phosphorous acid H3PO3 salts and completely different in nature from phosphate. Although phosphite with high solubility and low reactivity with soil components is readily absorbable by plants through phosphate transporters, this group of compounds does not provide plant P nutrition and thus

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cannot complement or substitute phosphate at any rate (Thao and Yamakawa, 2009). Whereas, it can function as either a translocative, non-selective, pre and post emergent herbicide (Manna et al., 2016) or an insecticide against several insect species (Patterson and Alyokhin, 2014). Furthermore, it is widely marketed as a fungicide with a complex mode of action both exhibiting direct toxicity to the pathogen in plants (Bock et al., 2012; Ogoshi et al., 2013; Shearer and Crane, 2012; Silva et al., 2011), as well as indirectly inhibiting pathogen growth through stimulation of host's defence response (Eshraghi et al., 2011; Olivieri et al., 2012; Massoud et al., 2012). Recently, phosphite was also considered as a good fit in integrated postharvest disease management due to its strong antimicrobial activities, less environmental toxicity and lower production costs (Amiri and Bompeix, 2011; Lobato et al., 2011; Miller et al., 2006; Rebollar-Alviter et al., 2007). Although phosphite has been widely used, most published works about direct antifungal activities of phosphite focused on the control efficacy. The knowledge of its potential targets and the action mode were still incomplete. Therefore, this study aimed to evaluate the effects of potassium phosphite (Phi) on growth and patulin production of P. expansum, as well as the control efficiency on blue mold in apple fruit. The response of P. expansum to exogenous Phi were then assessed at both protein and mRNA levels. The results will provide new insights for exploring the possible antifungal mechanisms of phosphite. 2. Materials and methods 2.1. Pathogen, fruit and preparation of Phi A filamentous fungal strain P. expansum link (CGMDD3.3703) was used in this study and cultivated for 10 days at 25 °C on potato dextrose agar (PDA) plates to enhance sporulation. PDA was prepared by boiling 200 g of sliced potatoes in1 L distilled water for 30 min, decanting the broth through cheesecloth, adding distilled water to obtain the total suspension volume of 1 L, adding 20 g dextrose and 20 g agar powder, and sterilizing by autoclaving at 121 °C for 15 min. Apple (Malus domestica Borkh. Cv. Red Fuji) fruits with similar size at commercial maturity were purchased from local market. Before the inoculation, apples were dipped in 2% sodium hypochlorite for 2 min, washed with tap water twice and then air-dried at room temperature. Phi was prepared from KOH and H3PO3 to have the same K2O:P2O5 ratio of 28:26 as described by Thao et al. (2008).

For antifungal assays of Phi in vivo, each apple fruit was wounded (3 mm deep and 3 mm wide) at its equator by a sterile borer and inoculated with 10 μL of the spore suspension at 1.0 × 105 spores/mL or sterile distilled water (as negative control). Before inoculation, spores were cultured under shaking conditions as described previously in PDB medium supplemented with 0 or 5 mmol/L Phi at 25 °C for 6 h, and washed twice by sterile distilled water. Disease incidence and lesion diameters were recorded daily. There were 10 fruits in each treatment with three replicates and the experiment was repeated. 2.3. Detection of patulin production and related-genes expression Spores of P. expansum were cultured statically in liquid PDB medium (1.0 × 106 spores/mL) supplemented with 0 or 5 mmol/L Phi at 25 °C for 12, 24, 48, 72 and 96 h. PDB mycelium filtrate was filtered through a 0.22 μm syringe filter (Albet, Spain) and transferred to an autosampler vial. Patulin content in filtrate was determined by injecting 10 μL of sample into a reversed-phase HPLC (high performance liquid chromatography) with UV detection (Waters, USA) as described by Morales et al. (2013). A Waters XTerra RP18 column (Waters, USA) was used with mobile phases (95% water and 5% acetonitrile). The operational parameters were as follows, Flow rate: 0.8 mL/min; column oven temperature: 18 °C; detector wave length: 276 nm. Under 0 or 5 mmol/L Phi stress, conidia of P. expansum were cultured under shaking conditions as described previously in PDB medium at 25 °C for 6, 12 and 18 h. The culture solutions were centrifuged at 10000 g for 20 min to separate mycelium and supernatant. The total RNA of harvested mycelium was extracted using TRIzol Reagent (Invitrogen, USA) in accordance with the manufacturer's instruction. Genomic DNA elimination and first strand cDNA synthesis were carried out by FastQuant RT Kit (Tiangen Biotech, China) according to the protocol of producer. The qRT-PCR (quantitative real-time polymerase chain reaction) was performed using 2 × Ultro SYBr mixture (CW Bio, China) in a CFX96-real Time System (Bio-Rad, USA) and primer pairs for the specific 15 genes involved in patulin biosynthesis were used as described by Tannous et al. (2014). The PCR conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 58 °C for 15 s and 72 °C for 20 s. The change in fluorescence of SYBR Green in every cycle was monitored by the system software, and the threshold cycle (Ct) over the background was calculated for each reaction. Sample were normalized using β-tubulin and the relative expression levels were measured using the 2(−△Ct) analysis method.

2.2. Antimicrobial effect assays 2.4. Comparative proteomics and transcriptome analysis Conidial suspension was obtained by flooding the sporulating cultures of P. expansum with sterile distilled water containing 0.05% (v/v) Tween-20. Then, a suitable aliquot of fresh spore suspension was added to a 250 mL conical flask containing 100 mL potato dextrose broth (PDB), which is formulated identically to PDA without agar, to obtain a final concentration of 1.0 × 106 spores/mL with the aid of a hemocytometer. Phi was added to the medium with final concentrations 0, 1, 2.5, 5 or 7.5 mmol/L. During the incubation of 6 to 12 h at 25 °C on a rotary shaker at 200 rpm, the germination rate of at least 100 spores in each treatment was microscopically determined. To facilitate the evaluation on the morphology and vitality of fungal spores under 5 mmol/L Phi stress, the 4′,6′-diamidino-2-phenylindole (DAPI) and Mito Tracker Orange CM™ROS (Invitrogen, USA) were used at the concentration of 50 mg/L and 2 μmol/L respectively (Lai et al., 2015; Czarna et al., 2010). The dry weight of hypha was measured after centrifugation, repeated washing with distilled water and during at 60 °C in hot oven to a constant weight at the indicated time. The effect of Phi on the mycelial growth of P. expansum in vitro was assayed as follows: A mycelial agar disk (5 mm in diameter) was placed on the center of a 9 cm diameter petri dish containing 25 mL PDA supplemented with 0 or 5 mmol/L Phi. During 8 days of incubation at 25 °C, radial growth was measured daily by decussation method.

Spores of P. expansum were inoculated in PDB medium supplemented with 0 or 5 mmol/L Phi and cultured under shaking conditions as described previously for 6 h at 25 °C on a rotary shaker at 200 rpm. The harvested spores were quickly frozen with liquid nitrogen for the following experiments. Proteins preparation and iTRAQ (isobaric tags for relative and absolute quantitation) analysis were performed using a service from LC-Bio of LC Science (USA). Raw data files acquired from the orbitrap were converted in to MGF files using Proteome Discoverer 1.2 (PD 1.2 Thermo). Protein identification was performed by using mascot search engine (Matrix Science, UK, Version 2.3.02) with the following parameter setting, Type of search: MS/MS Ion search; Enzyme: Trypsin; Fragment Mass Tolerance: 0.1 Da; Mass Values: Monoisotopic; Variable medications: Gln- N pyro-Glu (N-term Q), Oxidation (M), iTRAQ8plex (Y); Peptide Mass Tolerance: 0.05 Da; Instrument type: Default; Max Missed Cleavages: 1; Fixed medications: Carbamidomethyl (C), iTRAQ8plex (N-term), iTRAQ8plex (K). The quantitative protein ratios were weighted and normalized the median ration in mascot. The proteins which ration with P-value b 0.05 and fold changes of N1.2 in two biological repeats between control and Phi-treated spores were considered as the differentially expressed proteins (DEPs). Functional annotations of the proteins were conducted using Blast2GO program against

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the non-redundant protein database (NR; NCBI). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.kegg.jp/kegg/ pathway.html) and the COG database (http://www.geneontology.org/) were used to classify and group these identified proteins. The total experiment was repeated once. The detail information of these DEPs was provided as Appendices Table A.1. For RNA-seq (RNA sequencing), sample preparation with a group of biological replicates was same as the previous description of iTRAQ. Total RNAs preparation and RNA-seq (RNA sequencing) were performed using a service from Shanghai Personal Biotechnology Co., Ltd. with the Illumina platform. Briefly, total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instruction. RNA quality was checked using agarose gel electrophoresis and spectrophotometry. High quality of total RNA with a 28S:18S ratio N 1.5 and absorbance 260/280 ratio between 1.7 and 2.0 was used for library construction and sequencing. The cDNA libraries were constructed using TruSeq RNA Sample Preparation Kits v2 (Illumina, USA), following the manufacturer's protocol. Magnetic beads with oligo-dT used to combine the poly-A of the mRNA for purifying the mRNA from the total RNA. Fragmentation buffer was added to cleave the mRNA into short fragments. Random hexamer primers were used to generate first-strand cDNA from the fragments, which was transformed into double stranded cDNA using RNase H and DNA polymerase I. A paired-end library was constructed from the cDNA synthesized using a Genomic Sample Prep Kit (Illumina, USA). Fragments of desirable lengths were purified using a QIA quick PCR Extraction Kit (Qiagen, USA), end-repaired, and linked with sequencing adapters. AMpure XP beads (Beckman Coulter, USA) were used to remove unsuitable fragments, and the sequencing library was then constructed using PCR amplification. PicoGreen staining (Quant-iT PicoGreen dsDNA Assay Kit, Invitrogen, USA) and fluorospectrophotometry (Quantifluor™-ST fluorometerE6090, Promega, USA) were used to check the library integrity and an Agilent 2100 Bioanalyzer quantified it (Agilent, USA). The multiplexed DNA libraries were then mixed in equal volumes at a normalized concentration of 10 mM. The library was then sequenced on the Illumina NextSeq™ 500 platform. Raw sequencing reads of all the samples were mix together to perform filtration using a stringent process and subsequent de novo assembly. The adaptor contamination was removed, the reads were screened from 3′ to 5′ to trim the bases with a quality score of Q b 20 by using 5 bp windows, and the reads with a final length of b 25 bp were removed. FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) were used to analyze the quality of data filtering. The clean reads were mapped to reference genome (http://www.ncbi.nlm.nih.gov/ Traces/wgs/?download=JHUC01.1.fsa:nt.gz) using bowtie2/tophat2 (http://tophat.cbcb.umd.edu/). During mapping, mismatches of no more than two bases were allowed in the alignment. RPKM (reads per kilo bases per million reads) was used to evaluate the expression level of each annotated gene in sample. HTSeq (http://www-huber.embl. de/users/anders/HTSeq/doc/overview.html) and NOISeq method (Tarazona et al., 2011) were used to search the differentially expressed genes (DEGs) between control and Phi-treated groups. The cut-off was a two-fold change with P-value b 0.05. Then GO and KEGG function enrichment analysis to the differentially expressed genes (DEGs) were performed as described previously. The normalized data table was provided as Appendices Table A.2. Comparison of RNA-seq and iTRAQ datasets was based on semantically similarity of their GO terms. The detailed information of common DEGs acquired from two approaches was presented by Fig. 6. VENNY 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/index.html) and HemI 1.0.1 were used to make Venn diagram and heatmap. 2.5. Statistical analysis Except for specified experiments, data were pooled across three independent repeat experiments and were performed with SPSS software (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was used to

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compare more than two means, and Duncan's multiple range test was used for means separations. Differences at P b 0.05 were considered to be significant. 3. Results 3.1. Effect of Phi on growth of P. expansum in vitro Effects of Phi at different concentrations on spore germination of P. expansum were first evaluated in this experiment. Results indicated that the antifungal ability of Phi improved with the increase of working concentration. When the concentration reached 5 mmol/L, Phi showed a stable inhibitory effect to P. expansum. After 12 h of incubation in PDB, germination rate of P. expansum spores was below 15% in 5 mmol/L Phitreated group, while it was over 80% in the control group (Fig. 1A). With extension of culturing time, mycelia biomass production of P. expansum in Phi-treated group was also significantly lower than that in control group (Fig. 1B). From Fig. 1C and F, it was observed that not only sporulation of P. expansum was defective, but also hyphal growth was slower in PDA supplemented with 5 mmol/L Phi. Meanwhile, colony color was canary yellow, compared to blue in the control (Fig. 1D). Therefore, the concentration of 5 mmol/L was used in the subsequent experiments. DAPI staining was then carried out to investigate the morphological changes of P. expansum spores under Phi stress. After 6 h of incubation, the cell nuclei had a distinct outline and distributed regularly in both control and treated spores. The average volume of the spores was obviously smaller in treated group than that in control group, and some control spores began to germinate (Fig. 1E). At the same time, Mito Tracker Orange CM™ROS, a mitochondrion-selective stain, was used for studying mitochondrial distribution and functionality in P. expansum spores. In Fig. 1F, red fluorescence was more intensive and extensive in most control spores, which suggested that mitochondria existed throughout the intracellular space. In contrast, the number of stained mitochondria in Phi-treated spores was reduced obviously, and even in some cases, all the mitochondria could not be observed. As the accumulation of the stain in cells is dependent upon membrane potential, which is one of the most important parameters indicating mitochondrial functionality, it can be deduced that the vitality of treated spores was less than control spores. 3.2. Effect of Phi on the control of blue mold in apple fruits The pH value of 5 mmol/L Phi was about 6.0 and this solution was also checked to ensure no surface injury to apple fruit. Then, after 7 days of inoculation, although the decay symptoms developed in both positive control group and treatment group, the disease incidence and lesion diameter in apple fruits inoculated with Phi-treated spores were significantly lower than those in control group (Fig. 2D and E). Through observation of the surface and cross section in artificially inoculated apples, the reduced infectivity of Phi-treated pathogen was visually perceived (Fig. 2A, B and C). These results demonstrated that the curative application of Phi had a potential to reduce postharvest decay caused by P. expansum in apple fruits. 3.3. Effect of Phi on patulin production and expression of genes involved in patulin biosynthesis Effect of Phi on patulin production was measured via HPLC approach. Results showed that patulin production significantly increased in control group with increasing of culturing time, especially after 72 h of culturing. However, comparing with control, patulin productivity was very inefficient in Phi-treated group. Meanwhile, patulin output was remarkably lower than that in control group during 96 h of cultivation (Fig. 3A). Meanwhile, the expression changes of 15 genes involved in patulin biosynthesis under Phi stress was analyzed using qRT-PCR (Fig. 3B to P). Overall, comparing with control, expressions of PatA, PatC, PatE,

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Fig. 1. The inhibitory effects of Phi on P. expansum growth in vitro. Spore germination (A), hypha production (B) and mycelia expansion (C) of P. expansum were assayed at indicated time under Phi stress. The colonial morphology of P. expansum under 5 mmol/L Phi stress in PDA medium after 8 days of incubation (D). Microscopic observation of P. expansum spores stained by DAPI (E) and Mito Tracker Orange CM™Ros (F) after 6 h of incubation in PDB medium supplemented with 0 or 5 mmol/L Phi. Bars represent standard deviation of the means of three independent experiments. Lower case letters indicated significant differences at P b 0.05 at each time point. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

PatJ, PatK and PatO were down-regulated or showed a decreasing trend with culturing time increasing; meanwhile, expressions of PatD, PatI, PatL and PatN was up-regulated or presented an increasing trend with culturing time extension. Expressions of PatF, PatG, PatH and PatM first increased and then decreased. The expression of PatB was not influenced under Phi stress. 3.4. Identification and functional classification of DEPs and DEGs by iTRAQ and RNA-seq To explore the potential inhibitory mechanism of Phi on P. expansum growth, an iTRAQ-based quantitative proteomic analysis was utilized to gain a global view of proteome alteration of Phi-treated P. expansum spores. Among different culturing time, we chose 6 h to perform subsequent analysis because of similar morphology and different viability between control and Phi-treated spores at this time point. According to the criteria, a total of 3459 proteins were identified in two independent biological replicates. A total of 582 proteins were confirmed in both two biological replicates and considered as significant changes in abundance (the cut-off value was 1.2) under Phi stress. Among them, expressions of 374 proteins were down-regulated and of 208 proteins were up-regulated. The detailed information of these identified proteins was summarized in Appendices Table A.1 with the ratio of iTRAQ reporter ion intensities. The functions of majority DEPs were involved in macromolecular complex, membrane-bounded organelle, intracellular nonmembrane-bounded organelle, cellular component biogenesis,

carbohydrate metabolic process, organelle lumen and intracellular organelle lumen. The DEPs involved in glycerol ether metabolic process and ether metabolic process possessed the maximum rich factor value (Fig. 4A). According to the pathway enrichment statistics of DEPs, most of DEPs participated in carbohydrate metabolism, amino acid metabolism, lipid metabolism and genetic information processing. The number of DEPs involved in biosynthesis of secondary metabolites was the largest and the DEPs involved in synthesis and degradation of ketone bodies and proteasome possessed the maximum rich factor value (Fig. 4B). Via RNA-seq, averagely 60.4 million and 41.3 million raw reads, containing 16.9 million kb and 11.6 million kb, were generated from control and treatment samples respectively. After verifying the quality of the reads and removing adaptor sequence, about 14.3 million kb and 9.8 million kb clean data were obtained. About 93 million reads and 58 million reads were aligned to reference sequences, and mapped percent of useful reads were 89.61% in control group and 80.52% in treatment group. A total of 177 genes were considered as significant changes in abundance under Phi stress. Among them, expressions of 38 DEGs were down-regulated and of 139 DEGs were up-regulated. The detailed information of all DEGs was summarized in Appendices Table A.2 and the biological functions and intracellular distribution of these DEGs were represented by Fig. 5A. Through pathway classification, these DEGs participated in a variety of biological processes and the majority got involved in amino acid metabolism (35%), carbohydrate metabolism (22%), energy metabolism (11%) and lipid metabolism (5%) (Fig. 5B).

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Fig. 2. Effects of Phi on controlling blue mold rot caused by P. expansum in apple fruits. The surfaces and cross sections of apple fruits inoculated by P. expansum spores with 0 (A) or 5 mmol/L Phi (B) treatment were shown after 7 days of storage at 25 °C, and the sterile H2O was as a negative control (C).·The disease incidences (D) and lesion diameters (E) of P. expansum in apple fruits were detected during 7 days of storage at 25 °C. There were 10 fruits in each treatment with three replicates and the experiment was repeated. Bars represent standard deviation of the means of three independent experiments. Lower case letters indicated significant differences at P b 0.05 at each time point. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

Fig. 3. Effects of Phi on patulin production (A) and expression of 15 genes potentially related to patulin biosynthesis in P. expansum (B–P). Relative expression of PatA to PatO genes was determined by qRT-PCR at indicated time. The β-tubulin housekeeping gene was used as the internal control. Bars represent standard deviation of the means of three independent experiments. Lower case letters indicated significant differences at P b 0.05 at each time point.

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Fig. 4. Scatter plots of top 20 Gene Ontology (A) and KEGG (B) enrichment of DEPs in P. expansum spores under 5 mmol/L Phi stress after 6 h of incubation. The deeper color in the color code represented the higher confidence for biological process. Rich factor: the number of DEPs in one GO or KEGG/the number of total identified proteins in the same GO or KEGG. Higher value indicated higher enrichment level. The detailed information of these DEPs was listed in Appendices Table A.1. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

Fig. 5. GO functional classification (A) and pathway enrichment analysis (B) of DEGs in P. expansum spores under 5 mmol/L Phi stress after 6 h of incubation. (C) Venn diagram showing the overlap of DEPs from iTRAQ and RNA-seq datasets. The cut-off was a 1.2 fold-change for iTRAQ data and a two-fold change for RNA-seq data. The detailed information of these DEGs was listed in Appendices Table A.2.

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3.5. Comparison of the iTRAQ and RNA-seq datasets After combination and simplification of the results according to homologies and biological function annotations, 414 DEPs from iTRAQ and 110 DEGs from RNA-seq results were remained to perform correlation analysis. Finally, a total of 39 candidates were obtained in the overlap of iTRAQ and RNA-seq datasets which get involved in amino acid metabolism, carbohydrate metabolism, lipid metabolism, xenobiotics biodegradation and metabolism, energy metabolism, environmental information processing, protein folding, sorting and degradation, nucleotide metabolism and others pathways (Fig. 5C and Fig. 6). Unexpectedly, under Phi stress, expressions of majority of 39 candidates were downregulated at translation level but up-regulated at transcription level. 4. Discussion 4.1. Inhibitory effect of Phi on the growth of P. expansum The ongoing diminishing of the armoury of synthetic fungicides, through increasing regulatory restrictions on their use, reflects the growing public consensus for reducing pesticide inputs to safeguard public health and environment. The phosphite had a lower phytotoxicity risk when it was applied at doses less 5 g/L or 36 kg/ha. Therefore, it could be considered as another option to be included in integrated disease management program as well as production costs (Deliopoulos et al., 2010). The mode of action of phosphite against fungi has been the topic of controversy among researchers for a long time and there were mounting evidences that phosphite could rapidly and strongly activate plant defence system (Eshraghi et al., 2011; Massoud et al., 2012; Olivieri et al., 2012). Meanwhile, direct inhibition of fungal sporulation and development were also reported (Bock et al., 2012; Ogoshi et al., 2013; Shearer and Crane, 2012; Silva et al., 2011). Nevertheless, the inhibitory effect of phosphite on the growth of P. expansum, which severely threatened postharvest storage and processing of fruits and vegetables, was rarely mentioned. Especially, the overall response at molecular level in P. expansum under exogenous phosphite stress has

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not been described. In the present study, H3PO3 was neutralized with potassium hydroxide (KOH) to Phi which containing potassium dihydrogen phosphite (KH2PO3) or dipotassium hydrogen phosphite (K2HO3). And the antifungal activity of Phi against the P. expansum was demonstrated in vitro and in vivo. Results showed that Phi treatment could significantly inhibit spore germination, mycelia accumulation, colonial expansion, and patulin production of P. expansum. Furthermore, Phi showed a good efficacy on controlling the blue mold rot of apple. 4.2. Effect of Phi on expression of genes responsible for patulin biosynthesis Secondary metabolism in pathogenic fungi was usually strong controlled by environmental factors (Menniti et al., 2010; Tannous et al., 2015; Zong et al., 2015), and obvious decrease of patulin production was observed when P. expansum was under Phi stress in this study. As the most common mycotoxin produced by P. expansum, patulin is a cultivar-dependent factor favoring the colonization of hosts (Barad et al., 2014; Kumar et al., 2016; Sanzani et al., 2013; Snini et al., 2016); meanwhile, exposure of humans to patulin via consumption of contaminated fruit and fruit products may result in severe toxicosis (Barad et al., 2016; Glaser and Stopper, 2012; Puel et al., 2010). In recent years, biosynthetic pathway of patulin including about 10 enzymatic reactions (Moake et al., 2005; Sekiguchi et al., 1983) and one gene cluster containing 15 genes (PatA-PatO) putatively responsible for patulin biosynthesis in P. expansum has been reported (Artigot et al., 2009; Li et al., 2015; Puel et al., 2010; Tannous et al., 2014). In this work, expressions of most of the 15 genes (except PatB encoding a carboxylesterase) demonstrated an explicit association with patulin production under Phi stress at transcriptional level. Among these genes, PatA encoding a putative acetate transporter possibly participated in a subcellular compartmentalization of patulin biosynthesis pathway (Barad et al., 2016). The positive role of PatA in patulin biosynthesis was obvious since the first step of synthesis is a condensation of one acetate and three malonate units. As was well known, the conversion of acetyl CoA and malonyl-CoA to 6-

Fig. 6. Expression changes of overlapped candidates from iTRAQ and RNA-seq datasets in protein and mRNA levels. Each row in the color heat map indicated a single protein. The annotation and involved metabolic pathway of each protein were shown. The color changing from blue to red in color code indicated down- to up-regulation of candidates in Phitreated P. expansum spores. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

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methylsalicylic acid was catalyzed by a methylsalicylic acid synthase (6MSAS) encoded by PatK (Beck et al., 1990; Wang et al., 1991). Then, the transformation of 6-methylsalicylic acid, the stable patulin precursor, into m-cresol has been identified as a 6-methylsalicylic acid decarboxylase encoding by PatG (Snini et al., 2014). It was strongly expressed during patulin production whereas it was weakly expressed in non-patulin permissive condition (Robellet et al., 2008). PatH and PatI encoding cytochromes P450s (m-cresol hydroxylase and m-hydroxybenzyl alcohol hydroxylase) were responsible for hydroxylation of m-cresol to mhydroxybenzyl alcohol and of m-hydroxybenzyl alcohol to gentisyl alcohol (Artigot et al., 2009; White et al., 2006). As expected, the expressions of these genes all involved in early steps of patulin biosynthesis pathway were down-regulated under Phi stress. Exceptionally, the increasing expression of PatI was probably because of its versatility including catalyzing the conversion of hydrophobic intermediates and detoxifying environmental pollutant (Črešnar and Petrič, 2011). The PatN gene encoded a NADP-dependent isoepoxydon dehydrogenase which catalyzed the interconversion of isoepoxydon to phyllostine (Puel et al., 2007; White et al., 2006). The expression of PatN increased in the presence of Phi. This result was in agreement with report that single applications of quercetin and umbelliferone up-regulate PatN a medium-low level (Sanzani et al., 2009). PatL encoded a transcription factor containing a Zn(II)2Cys6 binuclear cluster DNA binding motif (Li et al., 2015). Previous research indicated that expression of PatL was not totally suppressed under patulin-restrictive conditions (Tannous et al., 2014). Snini et al. (2016) found that the mutant strain Pe△PatL presented morphological changes, then proposed that PatL affected the expression of numerous genes outside of the patulin biosynthetic cluster. Thereby, we speculated that a higher expression of PatL under Phi stress was probably due to the feedback of other affected genes In addition, although the exact functions of PatC, PatE, PatF, PatJ, PatM and PatO (encoding MFS transporter, glucose-methanol-choline oxidoreductase, hypothetical protein 1, hypothetical protein 2, ABC transporter and putative isoamyl alcohol oxidase) were unclear (Li et al., 2015), their expressions were also lower than those in control with culturing time extension. These were very similar to observation by Tannous et al. (2014) that the expressions of most genes involved in patulin biosynthetic pathway were significantly down-regulated under patulin restrictive conditions. Based on these results, it could be inferred that Phi created a patulinrestrictive condition and reduced patulin accumulation by acting on the transcription level of genes involved in the early steps of patulin synthesis. 4.3. A global view of proteome alteration of P. expansum spores under Phi stress Proteins are the substance basis of life and the chief actors within the cell which perform a vast array of functions. Therefore, proteomic analysis has been proven to be a powerful method for offering a more direct analysis of cellular response (Wiese et al., 2007). In this study, an iTRAQbased quantitative proteomic analysis was performed to acquire a global view of proteome alteration of P. expansum spores during 6 h of Phi stress. Through the analysis of 582 DEPs, the broad effects of Phi on proliferative status of P. expansum spores were revealed. Via GO enrichment statistics, a large number of DEPs were involved in composition and biogenesis of cellular organelles (including ribosome, proteasome, nucleolus, membrane and cell wall) and macromolecular complex. These were agreed with the retarded growth of P. expansum spores under Phi stress. In addition, DEPs, which correlated cell wall (P5, P105, P114, P227, P342, P348, P405, P493, P558, P566 and P573), ether or glycerol ether metabolic process (P42, P240 and P309), were noticed for higher rich factors in GO enrichment statistics. Fungal cells grow as separate tubular cells (hyphae) that extend apically and regularly branch by forming new hyphal apices. The possession of

cell wall allows fungi to generate turgor. This property, in combination with the excretion of the wall and substrate-digesting enzymes at growing hyphal apices, enables hyphae to penetrate solid organic substrates by tunneling their way through digestible solids (Wessels, 1994). Therefore, the acquired DEPs related to cell wall, most of which were down-regulated under Phi stress, would provide some valuable information for exploring action mode of Phi on growth and help to partly explain the reasons of the attenuated pathogenicity of Phi-treated P. expansum spores. All of DEPs involved in ether or glycerol ether metabolic process were identified as thioredoxin which was a class of small redox proteins and participated in regulating DNA synthesis, gene transcription, cell growth and apoptosis. It was a major carrier of redox potential in cells as it acted as a cofactor for essential enzymes and was involved protein repair via methionine sulphoxide reductase as well as the reduction of protein disulphides (Lu and Holmgren, 2014). In present study, we speculated that the decreased expression of thioredoxin in Phi-treated P. expansum spores weaken the antioxidant system which regulated the expression of many stress defence enzymes and protected cells against oxidative stress. Most of structures that make up pathogen are made from carbohydrates, lipids and proteins. Both making these molecules during the construction of cells and using them as a source of energy by their digestion are crucial for the development of pathogen (Black and Black, 2012). Via pathway enrichment analysis of DEPs, 16 of top 20 pathways were relate to metabolism of these three basic classes of molecules, which means that the metabolism of carbohydrates, lipids and proteins (or amino acids) of P. expansum were greatly affected under Phi stress. Then, the corresponding phenotypes of P. expansum were developmental delay and vitality reduction. In addition, proteasome with a higher richer factor value was conspicuous in pathway enrichment analysis. The function of proteasome was to degrade unneeded or damage proteins by proteolysis, a chemical reaction that breaks peptide bonds. It was part of a major mechanism by which cells regulated the concentration of particular proteins and degraded misfolded proteins (Enenkel, 2014). In this research, 20 DEPs (proteasome B-type subunit, peptidase T1A, 26S proteasome complex ubiquitin receptor, Winged helix-turnhelix transcription repressor DNA-binding motif, proteasome endopeptidase complex, proteasome beta subunit, 26S proteasome non-ATPase regulatory subunit, proteasome A-type subunit, ATPase and some hypothetical proteins) related to proteasome were identified and their expressions were all declined significantly under Phi stress. Inactivity of the proteasomal degradation pathway will disturb the turnover of specific, short-lived regulatory proteins that control the activity of many metabolic and developmental processes. Furthermore, DEPs involved in other metabolic pathways, such as biosynthesis of secondary metabolites and ribosome, also provided additional insights of possible targets sites of Phi. 4.4. Comparison based on the iTRAQ and RNA-seq datasets To analyze the continually changing of cellular transcriptome in P. expansum spores under Phi stress and evaluate the correlation between mRNA and protein levels, an RNA-seq and comparison analysis between iTRAQ and RNA-seq data were performed. A total of 177 DEGs were acquired and most of their encoding products either possessed binding activity (ion or DNA) or acted as structural molecules. Through widespread distribution in subcellular level, they were involved in many biological processes, especially in transporting, small molecule metabolic process, redox balance, cellular nitrogen compound metabolic progress and biosynthetic progress. Pathway classification was compared between DEGs from RNA-seq and DEPs from iTRAQ analysis, the similar results were that both of the majority of DEGs or DEPs were involved in carbohydrates, lipids and proteins metabolism. However, DEGs involved in energy metabolism occupies a higher proportion in total DEGs. After correlation analysis based on semantically similarity of their GO terms, a total of 39

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candidates, whose expressions showed a significant change in both protein level and mRNA level, were screened out from iTRAQ and RNA-seq dataset. As the narrowing of the scope and reducing of the number, the remaining candidates probably could afford more precise and valuable information to explore the inhibitory mechanism of Phi on P. expansum. Attention still need to be paid on that, DEGs did not matched well with our iTRAQ data either in correlation or changing trends of expression. From our point of view, the number of candidate genes from RNAseq was limited by rigorous filter criteria which lead to lower correlations. Meanwhile, the discrepancy between the expression level of mRNA and the abundance of the corresponding protein meant that, on the one hand, the abundance of a proteins depended not only on transcription rate of the gene but also on posttranscriptional regulation and posttranslational modifications; on the other hand, the detection of up-regulated expression in transcriptional level yet down-regulated expression in protein implied there was a kind of compensatory response of P. expansum spores under Phi stress. In summary, exogenous Phi could significantly inhibit the growth and patulin production of P. expansum and possess good controlling effects against blue mold on apple fruits. Through qRT-PCR detection results, we speculated that the regulation of Phi at transcriptional level mainly acted at the early steps of patulin biosynthesis. In addition, a global view of proteome and transcriptome alteration of P. expansum spores under Phi stress was acquired by iTRAQ and RNA-seq approaches. These findings would provide useful information to better understand the inhibitory mechanism of Phi on growth and patulin production of P. expansum, and be beneficial to develop effective controlling approaches based on Phi. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijfoodmicro.2016.12.017.

Acknowledgements Ting Zhou was funded by a China Scholarship Council (CSC) overseas research grant (No. 201608330061). This work was supported by the National Natural Science Foundation of China (NSFC31501810, NSFC31401926), the Hangzhou Science and Technology Planning Project (Specific Funds of Agricultural Scientific Research 20140432B02), and the Zhejiang Provincial Natural Science Foundation (LQ14C200004). References Abouraïcha, E., Alaoui-Talibi, Z.E., Boutachfaiti, R.E., Petit, E., Courtois, B., Courtois, J., Modafar, C.E., 2015. Induction of natural defense and protection against Penicillium expansum and Botrytis cinerea in apple fruit in response to bioelicitors isolated from green algae. Sci. Hortic. 181, 121–128. Amiri, A., Bompeix, G., 2011. Control of Penicillium expansum with potassium phosphite and heat treatment. Crop. Prot. 30, 222–227. Artigot, M.P., Loiseau, N., Laffitte, J., Mas-Reguieg, L., Tadrist, S., Oswald, I.P., Puel, O., 2009. Molecular cloning and functional characterization of two CYP619 cytochrome P 450s involved in biosynthesis of patulin in Aspergillus clavatus. Microbiology 155, 1738–1747. Ballester, A.R., Marcet-Houben, M., Levin, E., Sela, N., Selma-Lázaro, C., Carmona, L., Wisniewski, M., Droby, S., González-Candelas, L., Gabaldón, T., 2015. Genome, transcriptome, and functional analyses of Penicillium expansum provide new insights into secondary metabolism and pathogenicity. Mol. Plant-Microbe Interact. 28, 232–248. Barad, S., Horowitz, S.B., Kobiler, I., Sherman, A., Prusky, D., 2014. Accumulation of the mycotoxin patulin in the presence of gluconic acid contributes to pathogenicity of Penicillium expansum. Mol. Plant-Microbe Interact. 1, 66–77. Barad, S., Sionov, E., Prusky, D., 2016. Role of patulin in post-harvest disease. Fungal Biol. Rev. 30, 24–32. Basak, S., Guha, P., 2015. Modelling the effect of essential oil of betel leaf (Piper betle L.) on germination, growth, and apparent lag time of Penicillium expansum on semi-synthetic media. Int. J. Food Microbiol. 215, 171–178. Beck, J., Pipka, S., Siegner, A., Schiltz, E., Schweizer, E., 1990. The multifunctional 6methylsalicylic acid synthase gene of Penicillium patulum its gene structure relative to that of other polyketide synthases. Eur. J. Biochem. 192, 487–498. Black, J.G., Black, L.J., 2012. Microbiology: Principle and Explorations. eighth ed. (Hoboken, New Jersey). Bock, C.H., Brenneman, T.B., Hotchkiss, M.W., Wood, B.W., 2012. Evaluation of a phosphite fungicide to control pecan scab in the southeastern USA. Crop. Prot. 36, 58–64.

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