Exploring cold quarantine to mango fruit against fruit fly using artificial ripening

Mango quarantine is based mainly on heat treatment, with a possible deterioration of fruit quality. We studied the effects of cold quarantine (19 days storage at 2 °C) on fruit quality of commercial mango cvs. Keitt and Shelly for three consecutive years. Chilling injury (CI) occurs in mango fruit stored at temperatures lower than 12 °C. By reanalysing our previous transcriptome, we found that under sub-optimal temperature storage (5 °C), the fruit increases its ethylene biosynthesis and osmolarity by activating sugar metabolism, thereby probably reducing its freezing point. Similarly, ripe fruit with higher sugar concentration should be more resistant to cold-storage stress. Here, mango fruit was artificially ripened with 150 ppm ethylene. The control group, stored at 2 °C, suffered from severe CI, whereas the combined treatment of artificial ripening, modified atmosphere (fruit were enclosed in perforated bags) and subsequent low-temperature conditioning resulted in a significant reduction in CI to satisfactory levels for consumer acceptance (taste, aroma and texture). The combined treatment reduced lipid peroxidation and maintained flavour, leading to a novel cold-quarantine treatment for mango fruit. Thus, by reversing the supply chain and storing ripe and ready-to-eat fruit, cold quarantine was enabled for mango, and possibly other chilling-susceptible fruits.

Hence, in the current work, we integrated artificial ripening (AR) with LTC and MA to enhance mango fruit's resistance to cold. Remarkably, these combined treatments allowed the storage of mango fruit under cold quarantine of 2 °C for 19 days with the minimal development of CIs. Hence, AR combined with other treatments that are known to reduce CI enables the possibility of storing mango fruit at a temperature lower than 12 °C. By reversing the storage paradigm, cold-sensitive fruit can undergo cold-quarantine treatment during export of ready-to-eat fruit.

Progression in Ripening Parameters during Cold Quarantine.
Our previous results showed that to cope with storage at sub-optimal temperature, mango fruit activates sugar metabolism and degrades starch to mono-saccharides, thereby elevating osmolarity and reducing its freezing point 9 . In a similar manner, during fruit ripening, fruit degrade starch to mono-saccharides. Thus, in the current study, we artificially ripened fruits of mango cv. Keitt and Shelly and integrated the ripening with a MA and LTC. Several experiments were conducted on cv. Keitt during the years 2015 and 2017 and on cv. Shelly in 2016, with similar results. As expected, AR led to close to full ripening of the fruit with significant softening, change in peel colour, reduced acidity and increased Brix (Fig. 1, Table 1). Brix is commonly used for rough measurements of soluble sugars. Indeed, as the 'Keitt' fruit ripened in the AR + MA treatment, the Brix (% total soluble solids [TSS]) increased significantly in comparison to the control, from 8.67 to 12.97% (Fig. 1C). After ripening, the elevated levels of Brix (%TSS) in the AR treatment remained nearly constant whereas the fruit in the control group slowly accumulated sugars during storage (Fig. 1C). The increased fruit ripening in the AR + MA treatment was visible relative to controls as the firmness levels dropped swiftly from 106.5 to 10.0 N (before cold storage), whereas in the controls, the firmness decreased gradually, with the fruit remaining firm and not softening well during storage (Fig. 1A). Moreover, fruit treated with AR + MA had reduced acidity relative to controls, which supports fruit ripening (Fig. 1D). Similar results of increased ripening parameters (increased Brix, reduced acidity and firmness) after AR treatment were observed for the two cultivars (Keitt and Shelly) in different years (2015 and 2016, respectively) ( Table 1).
Further, AR + MA treatment ripened the fruit and the colour changed from green to orange as recorded for cv. Shelly in 2016, with hue number decreasing from 118.31 in controls to 92.46 after cold storage, and after shelf life, from 109.46 to 86.82 (Table 1), and yellowing index (1-10) decreasing from 1-1.5 (green) in the control to 8-8.6 (yellow) in the AR + MA treatment after cold storage and shelf life, respectively (Table 1). A similar but less pronounced increase in the yellowing index was observed for cv. Keitt (Fig. 1B).
Resistance to CI during Cold Quarantine. Cold quarantine requires 18 days of storage at 2.2 °C to eradicate the Mediterranean fruit fly from various fruit types 7 . In the current experiment, we stored the fruit at 2 °C   Table 1, Supplementary Fig. S1). Black spots and pitting were significantly decreased when treated by AR + MA in comparison to the control after storage (Fig. 3, Table 1). For example, black spots (index 0-10) were lowered from very severe (8.2 or 9.9) in the control to mild (2 or 1.2) and acceptable in the AR + MA treatment after shelf-life storage (Fig. 3A,C). Pitting (index 0-10), which is a deterioration of the black spots, was very mild (0.4 or 0) in AR + MA compared to very severe (8.6 or 10) in the control group after shelf life (Fig. 3B,D). These results highlight the importance of AR + MA for maintaining mango quality with close to no CI during cold quarantine. Similar results of reduced black peel spots and pitting were recorded in 'Keitt' and 'Shelly' (Table 1). Indeed, representative pictures of treated and untreated fruits of 'Keitt' (Fig. 2) and 'Shelly' (Supplementary Fig. S1) after cold quarantine and an additional 4 days of shelf-life storage clearly show a significant reduction in CI on the AR + MA fruit compared to the untreated fruit, resulting in good-quality ready-to-eat fruit after cold storage.
Post-harvest Decay during Cold Quarantine. After cold storage, the AR + MA-treated fruit was ready to eat and no decay was observed. However, the minor decay of 2.8% and 17.50% was observed after the additional 4 days of storage at 20 °C in the combined AR + MA treatment for cvs. Keitt and Shelly, respectively (Table 1). In contrast, in the control of Shelly cultivar had 100% of very severe side decay (Table 1). Stem-end rot incidence and severity were quite minor after 4 days of shelf-life storage for all treatments (data not shown). Indeed, representative pictures of treated and untreated 'Keitt' and 'Shelly' mango fruit after cold quarantine and an additional 4 days of shelf-life storage clearly show a significant reduction in decay in the ripe fruit compared to the untreated fruit ( Fig. 2 and Supplementary Fig. S1), with the ready-to-eat fruit remaining in good condition for up to 4 or 5 days. However, it is worth mentioning that after 7 days of shelf-life storage of ready-to-eat fruit, all treatments showed severe decay (data not shown).
Lipid Peroxidation during Cold Quarantine. Luminescence of mango fruit from the control group was high 4 days after storage at 2 °C (Fig. 4B), whereas a significant reduction was recorded after 19 days of cold storage ( Supplementary Fig. S2). However, fruit from AR treatment showed lower lipid peroxidation and luminescence after 4 days of storage at 2 °C, whereas those from the combined AR + MA treatment had very low lipid peroxidation after 4 days and after 19 days of storage at 2 °C ( Fig. 4B; Supplementary Fig. S2), which indicate a lower CIs in the AR + MA treatments after cold-quarantine. Lipid peroxidation could also generate secondary compounds, including the volatile compounds, which could cause off-flavors in fruits. GC-MS analysis of lipid peroxidation related volatiles showed that hexanal, nonanol and isohexanol, which are the linolenic acid degradation product, were accumulated by 2.27 and up to 20-fold in the fruit peel of the control compared to the combined treatment. Additionally, ethanol and acetaldehyde was significantly induced in the controlled fruit in comparison to the combined treatments (data not shown).

Effects of Cold Quarantine on Aroma Volatiles and Acceptance. Fruit volatiles were characterized
in the peel of control fruit and in the AR and AR + MA treatments (Supplementary Info S1). Interestingly, several volatiles that are known to contribute to the general aroma of mango fruit were induced in the AR + MA treatment in comparison to controls: α-Terpinen (4.0-fold), 1R-α-Pinene (1.7-fold), β-Pinene (2.6-fold), Limonene (2.4-fold), Terpinolen (3.3-fold) and 3-Carene (1.7-fold) (Fig. 5), which supported better acceptance and taste among the sensory panel (Fig. 6). During fruit ripening, acids are degraded and sugars increase, leading to a higher sugar-to-acid ratio-the key parameter determining acceptance and taste among consumers. In our experiment, AR + MA had 4.50and 2.60-fold higher acceptance and taste index values compared to controls, respectively, after cold quarantine (Fig. 6). Thus, fruit treated with AR + MA showed good quality, taste and aroma, while presenting resistance to the cold-quarantine treatment.

Discussion
Quarantine management must eradicate all fruit fly stages (eggs, larvae and mature insects). Cold quarantine against fruit fly for various fruit types, including mango, consists of storage at 2.2 °C for 18 days 8 . To develop cold quarantine for cold-susceptible fruit, it is mandatory to reduce CI or induce cold-resistance. Thus, cold quarantine for citrus, bell pepper, pomegranate and avocado includes treatments that reduce CI and induce fruit resistance to cold, such as an initial heat treatment, LTC or a combination of jasmonic acid, MA and conditioning 17,[20][21][22] . However, as a tropical fruit, mango is one of the most susceptible commodities to chilling 23,24 . Mango fruit is commercially stored at 12 °C, and storage below this temperature leads to CI 9 . Therefore, cold quarantine at 2 °C has never been considered for this fruit, and its quarantine is based mainly on heat treatments and to a minor extent on radiation 6 . This study challenges the main paradigm of storing unripe fruit in the cold by proposing storage of ripe fruit, which is much more resistant to cold. Previous unpublished results showed that a combination of treatments, such as jasmonic acid, MA and LTC, which allowed cold quarantine for avocado fruit 17 , reduced CI in unripe mango and enabled fruit storage at 7-8 °C, whereas storage at the lower temperature required for cold quarantine led to substantial CI (data not shown).
In climacteric fruit, including mango, sugar metabolism is coordinated by ethylene, which triggers the onset of ripening 25 . In the present study, exogenous application of ethylene triggered fruit ripening, which was accompanied with elevated brix, reduced acidity, fruit softening and change of color. By re-analysing our 'Keitt' mango fruit transcriptome in response to storage at optimal temperature at 12 °C and sub-optimal at 5 °C 9 , and not as was performed at this experiment were the fruit were stored at 2 °C, we found that mango fruit significantly activate the ethylene biosynthesis and signaling in response to sub-optimal storage at 5 °C temperature. Seven transcripts for ethylene biosynthesis were activated: s-adenosylmethionine synthase 2 (metK 2), three isoforms of s-adenosylmethionine synthase (speD), two isoforms of s-adenosylmethionine decarboxylase (ACS), and ACC oxidase (ACO) (Fig. 7A and Supplementary Info S2), along with seven transcripts for ethylene signal transduction: three isoforms of EIN 3 (i-iii), ERF 1 and three isoforms of ERF 2 (i-iii) (Fig. 7B, Supplementary Info S2). The synthesis and signal transduction of ethylene are known to activate fruit ripening and sugar metabolism 26 . Indeed, by re-analysing our previous RNA-seq of mango fruit response to storage at sub-optimal temperature, the pathway of sugar metabolism was strongly activated, including 25 transcripts that led to the degradation of starch to mono-and di-saccharides and ethanol formation (Fig. 7C, Supplementary Info S2). Thus, in response to sub-optimal storage at 5 °C temperature mango fruit activated, ethylene biosynthesis and signal transduction and sugar metabolism (Fig. 7). We hypothesize that these transcripts are triggered in response to fruit storage at a sub-optimal temperature to better cope with cold stress.
Our previous study showed that in mango fruit stored at sub-optimal temperature, starch is degraded to soluble sugars 9 . Degradation of starch to mono-and di-saccharides such as sucrose, fructose, and glucose increases the osmolarity, and these compounds act as cryo-protectants to reduce the freezing point 27 . Indeed, mango fruit that is harvested during the late season have increased TSS (osmolarity) and can be stored at lower temperatures 19 . In the current study, mango fruit was artificially ripened before storage. Remarkably, ripe fruit (TTS over 13%) were much more resistant to cold storage and had less CI when stored at 2 °C (Figs 2, 3, Table 1 and Supplementary  Fig. S1). To eliminate the minor CI that remained after the ripening (AR) treatment, we integrated it with MA and LTC, which are known to reduce CI in various post-harvest commodities 17,22 . MA leads to a reduction of O 2 and elevation of CO 2 , thus inhibiting respiration leads to increase in mango fruit tolerance to chilling 10 . Thus, ripening by ethylene followed by reduction of respiration by MA resulted in a further reduction in CI to a level that was acceptable to consumers (Figs 2, 6, Table 1 and Supplementary Fig. S1).
CI is known to be strongly correlated with elevated production of reactive oxygen species 28 , which lead to the activation of linolenic acid pathway, lipid peroxidation and degradation 9,29 . The detection of fruit luminescence by in-vivo imaging systems can non-destructively pinpoint early lipid peroxidation 9 . In the present study, the integrated treatments of AR + MA had lower luminance, indicating reduced lipid peroxidation (Fig. 4) and CI. This lower lipid peroxidation was also expressed in reduced degradation of linolenic acid and reduced oxylipin C6 and C9 volatiles such as hexanal, isohexanol and nonanol (Fig. 4), which are associated with linolenic acid pathway, lipid peroxidation and degradation in response to chilling stress in mango fruit 9,29 . Thus, this combination of treatments opens the way for cold-quarantine application with cold-susceptible fruit such as mango.
Apart from reducing fruit CI, the most critical aspect of mango fruit storage is retention of fruit flavour, aroma and taste. While some reports have suggested that long heat-quarantine applications can reduce fruit quality 6,30-32 , the cold-quarantine application does not. Indeed, GC-MS analysis of the known mango aroma volatiles α-Terpinen, 1R-α-Pinene, β-Pinene, Terpinolen and 3-Carene 29,33 after cold quarantine of AM + MA-treated fruit showed their increase in the peel (Fig. 5). Those results were correlated to the results that cold quarantine of AM + MA-treated fruit showed reduced CI, good pulp taste and increased aroma volatiles in the peel, leading to high levels of consumer acceptance. The results presented here show that after cold storage, the fruit was ready to eat. These ready-to-eat fruit could be held at 20 °C for up to 4 days with the maintenance of relatively good quality and low decay.

Conclusions
Quarantine is vital for global trade 3 . Cold quarantine is easy to apply as it is performed during export shipment. Cold quarantine is applied mainly to chilling-resistant fruits. In this manuscript, we show that by inverting the cold chain and storing ripe and ready-to-eat fruit together with MA and LTC -chilling-susceptible as tropical fruit can be subjected to cold-quarantine treatment while maintaining their fruit quality. Post-harvest and Cold-quarantine Treatments. To reduce CI and maintain mango fruit quality, the fruit were treated with (1) AR -harvested fruit were artificially ripened with 150 ppm ethylene for 24 hours followed by 2 days of storage at 18 °C; (2) MA -fruit were enclosed in low-density-perforated (30 holes of 0.5 mm) polyethylene bags (StePac, Israel), removed from the bag after 1 day to avoid condensation (97-98% relative  Five cardboard boxes, containing 9 fruits ('Keitt') or 10 fruits ('Shelly'), were used for each treatment replication. The room temperature was monitored by DAQ tool -double strand wire logger/data acquisition control system (T.M.I Barak Ltd., Israel). Fruits core temperature was monitored using a MicroLite data Logger LITE5032P-EXT-A (Fourier technologies, Israel) by inserting its probe 3 cm deep, near to the mango stone.
Other post-harvest parameters were measured using instruments. Firmness was tested via the peel using an 11-mm probe penetrometer (LT-Lutron FG-20 kg, Indonesia) at two points on each fruit (10 fruit per treatment). Fruit colour (hue) was measured by chromometer (Minolta LR-400/410) at two points on each fruit (10 fruit per treatment). TSS was measured by a digital refractometer (ATAGO PR-1, Japan) and presented in percent Brix (%TSS). Acidity concentration was measured by automatic titrator (Metrohm, 719 S Titrino, Switzerland) and calculated based on percent citric acid.
RNA-Seq Analysis. RNA-Seq of Sivankalyani et al., (2016) was re-analysed. Briefly, samples were extracted from mango fruit peel, from fruit that was stored for 2, 7 and 12 days at sub-optimal temperature of 5 °C and optimal (12 °C) temperatures (and not at 2 °C temperature as quarantine requires). Library construction and RNA-Seq analysis were carried out as described previously 9 . Up-regulated transcripts of clusters 2, 4 and 5 were annotated with Kyoto Encyclopedia of Genes and Genomes (KEGG) 34 and analysed for biosynthesis pathways and signal transduction. Heat maps were prepared for up-regulated transcripts with Heatmapper 35 . Identification and Quantification of Volatiles. Aroma and lipid-peroxidation volatiles were identified and quantified by gas chromatograph 7890 A and mass chromatograph 5975 C (Agilent Technologies Inc., USA). After cold-quarantine treatment, mango peel samples (1 g) were collected randomly from five fruit of each replicate in three biological repeats for cv. Keitt in 2017. Samples were immediately stored with 2 mL NaCl (20% w/v) to stop further enzymatic activity in 20-mL dark-coloured glass bottles (LaPhaPack, Germany) with a tight seal. S-2-octanol (Sigma-Aldrich) was used as the internal standard. In each run, NaCl without sample was used as a control. Analytical conditions for the GC-MS run were adjusted as described by 29 . Volatile identification was based on NIST mass spectral database version 5. Identified volatiles were quantified based on internal standard linear retention indices and expressed as µg/kg FW.

Fruit Organoleptic Characterization.
To determine the sensory attributes of 'Keitt' mango fruit after cold quarantine, untrained tasting panel of 15 people evaluated the acceptance and taste using an index of 1-10. These sensory attributes were judged for control, MA, AR and AR + MA fruit based on ranked general remarks on impression, sweetness, sourness and off flavours.
Lipid peroxidation and Luminescence. A pre-clinical in-vivo imaging system (Perkin Elmer, USA) and a highly sensitive charge-coupled camera (CCD) were used to detect alterations in the cellular membrane caused by lipid peroxidation. Fruits of cv. Keitt after cold quarantine was re-acclimatized for 2 h in the dark prior to in-vivo imaging system evaluation. Oxidative degradation of lipids was recorded at 640-770 nm wavelength emitted for 20 min as proposed by 29,36 . Data were recorded for three biological replications, and one representative picture is presented.
Statistical Analysis. Data from three different experiments are presented as average ± SE. One-way ANOVA was used to compare means of treatments to controls with JMP Pro 13.0 statistics software and data were subjected to Duncan's multiple-range tests. Differences at P < 0.05 were considered significant. Indices for black spot (scale 0-10), pitting (scale 0-10), acceptance (scale 1-10), taste (scale 1-10), firmness (scale 1-10), yellowing (scale 1-10) and side-decay severity (scale 0-10) were calculated with the formula: Respective Number of fruit present in that level Total number of fruit in the treatment scale