Volatile organic compounds of Hanseniaspora uvarum increase strawberry fruit flavor and defense during cold storage

Abstract Volatile organic compounds (VOCs) of antagonistic yeasts are considered as environmental safe fumigants to promote the resistance and quality of strawberry (Fragaria ananassa). By GC‐MS assays, VOCs of Hanseniaspora uvarum (H. uvarum) fumigated strawberry fruit showed increased contents of methyl caproate (5.8%), methyl octanoate (5.1%), and methyl caprylate (10.9%) in postharvest cold storage. Possible mechanisms of H. uvarum VOCs involved in regulations of the defense‐related enzymes and substances in strawberry were investigated during postharvest storage in low temperature and high humidity (2 ± 1°C, RH 90%–95%). Defense‐related enzymes assays indicated H. uvarum VOCs stimulated the accumulation of CAT, SOD, POD, APX, PPO, and PAL and inhibited biosynthesis of MDA in strawberry fruit under storage condition. Moreover, the expression levels of related key enzyme genes, such as CAT, SOD, APX42, PPO, and PAL6, were consistently increased in strawberry fruit after H. uvarum VOCs fumigation.

One of those antagonistic yeast strain that inhibits the growth of B. cinerea on strawberry fruit is H. uvarumi, probably by nutrients and space competition, host defense induction, morphology change of hyphae, and synthesis of secondary metabolites (Cai, Yang, Xiao, Qin, & Si, 2015;Qin et al., 2013). Preharvest application of H. uvarum on strawberry fruit was able to stimulate the enzymes associated with active oxygen metabolism, such as POD, polyphenol oxidase (PPO), SOD, phenylalanine ammonialyase (PAL), and CAT (Cai et al., 2015).
Although it is relatively efficient to control postharvest diseases by applying biocontrol yeast strains directly on strawberry fruit, BCAs treatment might increase the risk of microbe contamination during marketing stage. Thus, VOCs treatment has a broader prospect instead of directly spraying BCAs culture on strawberry fruit surface in postharvest stage. However, little is known about these specific mechanisms of controlling strawberry postharvest diseases of by VOCs.
In previous study, VOCs produced by H. uvarum showed effective inhibition to the growth of B. cinerea in vitro (Cai et al., 2015). Here, we explore the further functions of VOCs produced by H. uvarum by testing possible alterations in strawberry volatile emissions, as well as the variations of defense-related enzymes and substances in strawberry fruit during postharvest storage period.

| H. uvarum strain and culture
Hanseniaspora uvarum (Genbank accession number: JX125041) was isolated from the surface of strawberry and maintained in PDA medium (200 g/L potato, 20 g/L dextrose, 15 g/L agar).
Hanseniaspora uvarum strain was grown in PDB liquid medium (200 g/L potato, 20 g/L dextrose) at 28°C for 24 hr in a gyratory shaker at 200 g. The strain culture was centrifuged at 6,000 g for 15 min at 4°C, and the yeast strain precipitate was then adjusted to final concentration of 1 × 10 9 CFU/ml with sterile distilled water with 0.05% Tween-20. Five hundred microliter of yeast suspension was inoculated on the PDA dishes for the preparation of VOCs treatment.

| Fruit and VOCs treatment
Fruit of Fragaria ananassa "Hong Yan" was harvested in early morning from a farm in Yuhua district, Nanjing city, Jiangsu province, China.
The strawberry fruit was then transported to laboratory condition immediately within 2 hr. A total of 300 strawberry fruits in similar shape and size and without physical injuries or microorganisms infection were randomly selected.
Hanseniaspora uvarum VOCs treatment on strawberry fruit was conducted in sealed glass desiccators (9 cm by 27 cm, down diameter by up diameter, with a hollow clapboard inside) as previously described (Qin, Xiao, Cheng, Zhou, & Si, 2017). Eight uncovered Petri dishes with H. uvarum culture on PDA medium were placed on the bottom of the glass desiccator (use noninoculated PDA medium as negative control), and fresh strawberries were placed on the hollow clapboard. All desiccators were sealed by parafilm and incubated at 2°C for 3 days. Then, all treated strawberry fruits were placed in normal storage condition (2 ± 1°C, RH 90%-95%) with appropriate aeration system. Strawberry fruit samples were collected for further biochemical assessment after 0, 3, 8, 13, 18, and 25 days, respectively.

| Detection of defense-related enzymes in strawberry fruit
The enzyme activities of strawberry fruit were measured following the methods described in previous experiment with some modifications (Cai et al., 2015). Strawberry fruit samples (2.0 g for each treatment) were homogenized in 8 ml ice-cold phosphate buffer (50 mmol/L, pH 7.8, containing 1% polyvinylpyrrolidone [PVP]) and then centrifuged at 12,000 g for 20 min at 4°C, and supernatant was collected as crude extract. Each strawberry fruit treatment had three parallel lines, and the test was repeated twice.
For POD activity measurement, reaction buffer (1 ml 0.1 mol/L acetic acid buffer, 1 ml crude extract, 1 ml 2-methoxyphenol, 0.4 ml 0.75% H 2 O 2 ) was mixed with crude extract of strawberry fruit. Each variation (0.001) of the mixture absorbance at 460 nm within 2 min represents 1 unit of the enzyme activity. The specific activity was expressed as units per gram of fresh weight.
The SOD activity of strawberry fruit was evaluated by following procedures. The reaction buffer (2.0 ml 50 mmol/L, pH 7.8 phosphate buffer, 0.2 ml 30 mmol/L L-methionine, 0.2 ml 750 mol/L nitrotetrazolium blue chloride, 0.3 ml 20 µmol/L riboflavin) was gently mixed with 0.5 ml crude extract, and reaction mixture was then exposed by fluorescent lamp (400 lx) for 10 min. Each enzyme unit of SOD activity was defined as 50% inhibition rate of the photochemical reaction displayed by absorbance at 560 nm, and the reaction mixture without exposure treatment was considered as blank. The specific activity was expressed as units per gram of fresh weight.
For CAT activity measurement, reaction buffer (3 ml sodium phosphate buffer, 0.4 ml 0.75% H 2 O 2 ) was mixed with 0.2 ml crude extract. Each variation (0.001) of the mixture absorbance at 240 nm within 2 min represents 1 unit of the CAT enzyme activity. The specific activity was expressed as units per gram of fresh weight.
For APX activity measurement, reaction buffer (3 ml sodium phosphate buffer, 0.2 ml 0.01 mol/L L-ascorbic acid, 0.2 ml 0.75% H 2 O 2 ) was gently mixed with 0.2 ml crude extract. Each variation (0.001) of the mixture absorbance at 290 nm within 2 min represents 1 unit of the APX enzyme activity. The specific activity was expressed as units per gram of fresh weight.
For activity of PAL and PPO, content measurement of MDA, and superoxide anion in strawberry fruit, the procedures were as described in previous study (Cai et al., 2015).

| Volatile compound analysis on strawberry fruit
Top parts of three strawberry fruits for each treatment were collected and placed in headspace sampling tubes. GC-MS analysis was then conducted as following steps. A 2-cm fused-silica fiber coated with divinylbenzene/carboxen/polydimethylsiloxane 50/20 μm (DBV/CAR/PDMS) was inserted into the side of the sampling tubes via a silicone septum after 15 min of headspace equilibration.
Volatile compounds were collected from headspace sampling tubes for 30 min and then desorbed into GC injector for 5 min at 250°C.
Separation was achieved on an HP-Innowas fused-silica capillary column. The GC oven temperature program consisted of 40°C for 2.5 min, raised from 40 to 200 at 5°C/min, and then to 240°C for 5 min at 10°C/min. Helium was used as carrier gas with a constant column flow rate of 0.004 mol/hr. Compounds exiting the column were ionized via electron impact at 70 eV and scanned with a quadrupole mass spectrometer with a m/z range between 30 and 300 Th.
The volatile profile of each sample was reported as absolute peak area.

| Real-time PCR analysis of mRNA abundance
Fresh strawberry fruit for defense relevant gene test was treated with H. uvarum VOCs for 1, 2, and 3 days, and then, strawberry samples (0.1 g for each) were collected and ground into powder with liquid N 2 . All the microbe treatments followed the method described in fruit and VOCs treatment section. Total RNA was extracted using 1 ml TRIZOL reagent (Invitrogen Co.) and was treated with RNase-free DNAse (Takara Co.) to remove genomic DNA. Reverse transcription of RNA was performed with 1 μg of total RNA using M-MLV reverse transcriptase, according to the manufacturer's protocol (Invitrogen Co.). The primers used were listed in Table 1. A constitutively expressed gene (18S rRNA in Fragaria x ananassa) was a reference gene in quantitative real-time PCR analysis. Three replicates were performed for each treatment.

| Statistical analysis
All statistical analyses were performed in the SAS Software (version 8.2; SAS Institute). Comparison of means was performed by Duncan's multiple range tests. Statistical significance was assessed at the level of p < 0.05.

| Effects of H. uvarum VOCs on gray mold decay of strawberry during cold storage
Hanseniaspora uvarum is an effective BCA in preventing postharvest diseases (Cai et al., 2015), and its VOCs also showed TA B L E 1 Sequence of the primers used for genes expression in strawberry    In assays of APX activity in strawberry fruit, the APX enzyme activities of H. uvarum VOC-treated fruit were slightly higher than nontreatment group on the 3, 8, 13, and 25 dpt. The peak value of APX enzyme activity was observed on day 13 in H. uvarum VOC-treated fruit which is approximately 600 U/(min g kg −1 ), and the APX activity of treated fruit was significantly higher (p < 0.05) than that of the nontreatment group on 18 dpt ( Figure 3a). As shown in Figure 3b, under the cold storage condition, SOD accumulation peaked at 13 dpt in strawberry fruit of both H. uvarum VOCs treatment and nontreatment groups, and the SOD enzyme activity of H. uvarum VOC-treated fruit was significantly higher (p < 0.05) than nontreatment group at 3 and 13 dpt. In the strawberry fruit during storage period, POD activity reached a maximum at 8 dpt in both H. uvarum

| Effects of H. uvarum
VOCs treatment and nontreatment groups, and the POD activity of treatment group was induced approximately 1.2-fold than that of the nontreatment group (Figure 3c). In Figure 3d, it was shown that the CAT activity of H. uvarum VOC-treated fruit was significantly higher (p < 0.05) than that of the control group from 3 to 25 dpt, and the CAT activity in H. uvarum VOCs treatment group has been increased by 16.18% compared with nontreatment group at the end of storage. Relative abundance

| Effects of H. uvarum VOCs on expression levels of defense-related enzyme genes in strawberry
Expression levels of defense-related enzyme genes were analyzed in strawberry fruit on 1, 2, and 3 days during H. uvarum VOCs fumigation. In this study, relative expression levels of key enzyme genes involved in biosynthesis of APX, SOD, CAT, PPO, and PAL in strawberry fruit were shown in Figure 5.  Our previous data indicated that volatile emissions of "Hong Yan" mostly were methyl caproate, methyl butyrate, ethyl butyrate, and butyl acetate, which is similar with presented results in  (Gill & Tuteja, 2010). Collectively, the levels of SOD, CAT, POD, APX, and PAL activities are the physiological characteristics to analyze and quantify the strawberry host resistance against pathogen infection.
Our results proved that VOCs produced by H. uvarum could significantly induce the accumulation of defense-related enzymes, such as SOD, POD, CAT, APX, PAL, and PPO. Importantly, the ability to improve contents of defense-related enzymes is normally related to induction of system resistance in plants (Zhu & Ma, 2007), which suggests that H. uvarum VOCs could also be a potential microbeassociated molecular patterns and function in the early perception status of the ISR of H. uvarum.
As an elicitor for improving the resistance of strawberry fruit by inducing the activities of antioxidant enzymes, our study also manifested that H. uvarum VOCs could reduce the contents of MDA in strawberry fruit throughout the storage period ( Figure 4a), whereas the content of superoxide anion production rate was merely lower than nontreated strawberry fruit at the end of storage period (25 dpt, Figure 4b). MDA is the product of lipid peroxidation in plant cell membrane, as well as the generated rate of superoxide anion, which directly reacts with the degree of fruit ripening. Therefore, these two parameters partially represent the degree of damage to plant tissue (Macarisin, Droby, Bauchan, & Wisniewski, 2010 (Huang et al., 2011). Moreover, they investigated the specific species of C. intermedia VOCs, such as esters, alcohols, alkenes, alkanes, alkynes, organic acids, ketones, and aldehydes, in which 1,3,5,7-cyclooctatetraene and 3-methyl-1-butanol were the most abundant.
In another study, a total of 28 species were detected in H. uvarum VOCs, in which ethyl acetate and 1,3,5,7-cyclooctatetraene were the most abundant (Qin et al., 2017 F I G U R E 5 Effect of fumigation with H. uvarum volatile organic compounds (VOCs) on expression levels of defense-related enzyme genes. Expression levels of SOD (a), CAT (b), PAL (c), PPO (d), and APX42 (e) were detected in strawberry fruit during cold storage. Each value is the mean for three independent replicates. Asterisks indicate statistical differences compared to control according to Duncan's multiple range test at p ≦ 0.05 level. The vertical bar indicates the standard error, and three biological replicates were performed for each assay with similar results

| CON CLUS IONS
The present study showed that treatment with VOCs produced by the antagonist H. uvarum increased specific esters in strawberry volatile emissions during postharvest storage. Furthermore, the study indicated that treatment with VOCs can enhance the activity of defense-related enzymes and inhibit the accumulation of MDA content and the production rate of superoxide anion. These results also showed that VOCs maintain the strawberry quality via inducing the expression of defense-related enzyme genes. The molecular mechanism of VOCs treatment to increase strawberry resistance has not been elucidated yet. The future work should pay attention to investigate which signal pathway was induced on strawberry fruit treated with H. uvarum VOCs.

CO N FLI C T O F I NTE R E S T
It should be understood that none of the authors have any financial or scientific conflict of interest with regard to the research described in this manuscript.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.