Shelf Life of Fresh Pistachio (Pistacia vera L.) upon Foliar Application of Sulfur as Pesticide


 Application of sulfur as pesticide is a strategy in plant protection, but it is of great importance for a pesticide not to influence the quality of the product. In this study, movento (0.5 kg/1000 L), confidor (0.4 kg/1000 L) and refinery micronized (R) and mineral (M) sulfur (30 and 60 kg/1000 L in water) were sprayed on pistachio trees of cultivar ‘Ahmadaghaei’ twice (in middle May and after 50 days) for controlling the psylla. Pistachios were harvested in late September and stored in 4 °C for 25 and 50 days in order to evaluate the effect of pesticides on the quality of hulls and kernels. Weight, taste, aroma, appearance, respiration rate, firmness of hull and kernel, phenolic content of kernel, anthocyanin, chlorophyll, carotenoid, a*, b* and chroma of hull and kernel declined over time, while phenolic content of hull, L* and hue of hull and kernel displayed an increasing trend. Antioxidant activity of hulls and kernels and ethylene production remained stable over time. Chemical pesticides stimulated loss of weight and hull firmness. Refinery micronized sulfur at both concentrations caused higher wight loss, higher chlorophyll of hull and kernel, lower carotenoid of hull and kernel, higher L* and hue and lower a*, b* and chroma of hull and kernel (lighter and greener color). All sulfur treatments reduced firmness and antioxidant activity of hull and kernel, while elevated the respiration rate, hull phenolics and hull anthocyanin. R30 saved the taste and aroma after 50 days. R30 and M30 showed higher ethylene production.


Introduction
Pistachio (Pistacia vera L.) tree produces popular nuts rich in nutritional components including fatty acids, vitamins, sterols and phenolics. Pistachio is among the most important and valuable nuts that is mainly traded as dry, in-shell kernels (Tsantili et al. 2011). But drying leads to various degrees of loss in nutrients and antioxidant components of pistachio, therefore, there has been an increasing demand for fresh pistachios due to health bene ts (Sheikhi et al. 2019). Fresh pistachio can be stored in cold storage conditions for a short period of time, with different factors affecting its shelf life. Several fungi, bacteria and yeasts can infect the fresh pistachios causing postharvest spoilage, therefore, disinfection of fresh pistachios is a crucial step after harvest to reduce health risks and to prevent postharvest losses due to microbial decay (Sheikhi et al. 2020).
Application of different kinds of sulfur as a pesticide has been one of the strategies used in pistachio cultivation in the recent decades. Therefore, various forms of sulfur as a pesticide, especially against psylla, have been extensively produced and used in pistachio orchards. Sulfur in the commercial form is produced as a byproduct of coal, natural gas and petroleum re nement and mining process. Its effect is a non-systemic contact (Afrousheh and Hasheminasab 2018). Elemental sulfur is correlated with increased hydrogen sul de (H 2 S) and sulfurous off-aroma formation during fermentation, so is often used sparingly with long intervals. The advantages of sulfur over alternatives include its low cost, good e cacy, low risk of resistance development, and its acceptability within various production systems (Kwasniewski et al. 2014). The type, concentration and time of exposure should be determined for each crop in order to avoid the toxic and deleterious effects of sulfur. For example, fumigation of blueberries with high (250-350 µL L-1 h) concentration of SO 2 caused higher weight loss than control after 45 days of cold storage, but lower concentrations (50-200 µL L-1 h) had no adverse effects (Rivera et al. 2013).
The phenolic content of wheat plants increased by 0.06 ppm SO 2 fumigation for 8 h daily (Agrawal and Deepak 2003), but 28 nLs − 1 L − 1 SO 2 fumigation did not in uence the phenolic content of blueberries (Cantin et al. 2012).
Sulfur is used for several hundred kinds of foods and crops, ornamental plants, turfs, and residential sites. Besides pistachio psylla, sulfur is widely used for controlling brown rot of peaches (Holb and The e cacy of sulfur on controlling pistachio pests, especially psylla, has been con rmed (Bakhtiari et al. 2016). But it is essential for a pesticide not to affect the quality of the product. Fortunately, the edible part of pistachio is its kernel that is covered by the shell and hull, protecting it from sulfur penetration.
However, it is obvious that the kernels receive signals from their surrounding tissues and are under the in uence of the physiological status of hull and the whole plant. Therefore, in this study, mineral and re nery micronized sulfur were applied on pistachio fruits of cultivar 'Ahmadaghaei' twice with a 50-day interval for controlling psylla. Then, it was aimed to investigate the effect of sulfurs on the physiology and quality of fresh pistachios (hull and kernel, separately) at harvest and two periods (45 and 90 days) of cold storage. The effects were also compared with two chemical pesticides, movento and con dor, which were previously applied against psylla.

Plant material and treatments
This project began in middle May 2019 in the orchards of Anar city, Iran. Pistachio trees of cultivar 'Ahmadaghaei' were sprayed with movento (0.5 kg/1000 L), con dor (0.4 kg/1000 L) and two types of sulfur, including re nery micronized (30 and 60 kg/1000 L in water) and mineral sulfur (30 and 60 kg/1000 L in water). The concentrations were chosen based the suggestions of farmers around the area.
Control trees were not sprayed. The spraying was repeated after 50 days, in July. Each treatment consisted of three rows of trees, the replicates of which were selected in the middle row. Six neighboring trees were considered as one replication and the sampling was done from four sides of each tree. The spraying operation was carried out in the morning and ended before the weather warmed up. In late September and early October, at the time of pistachio harvest, the fruits were sampled and transferred to laboratory. A portion of fruits was used for obtaining the data of harvest time and the rest were kept in disposable tableware with closed lid and stored in 4°C. After 25 and 50 days, the fruits were analyzed again.

Moisture content
One kilogram of whole fruits of each replication was weighted at harvest, kept in disposable tableware with closed lid in 4°C and reweighted after 25 and 50 days. The weight loss was expressed as a percentage of the rst weight (Cantin et al. 2012).

Firmness
Tissue rmness was evaluated using a Digital Force Tester (Lutron fg5020, Taiwan), tted with a sharp 11 mm probe. Two different measurements were carried out on two opposite sides of the central zone of kernels and hulls. The values were expressed as kilogram-force (KgF).
Water activity One g of kernels or hulls were used as replicates. Water activity was directly measured by a water activity meter (Novasina, Switzerland) and expressed as a w .

Phenolic content
Total phenolic concentration was determined according to the Folin-Ciocalteu procedure, with absorbance measurement at 760 nm using gallic acid for preparing the standard curve. The results were expressed as mg of gallic acid in 100 g of fresh weight (Orthofer and Lamuela-Raventos 1999).

Antioxidant activity
Antioxidant activity was determined by the 2,2-diphenyl-1-picryl-hidrazil (DPPH) radical-scavenging method. The absorbance was measured at 517 nm, using a spectrophotometer (lambda-Elmer Perkin, American) and was expressed as the inhibition percentage of the DPPH radical (Serrano et al. 2005).

Anthocyanin
Anthocyanin content was determined using Wanger method with absorbance measurement at 550 nm and was expressed as µmol g − 1 of fresh weight (Wanger 1979).

Photosynthetic pigments
Chlorophyll and carotenoid content were measured according to Lichtenthaler's method with absorbance at 470, 646.8 and 663.2 nm using a UV-visible spectrophotometer. The concentrations were expressed as mg g − 1 of fresh weight (Lichtenthaler 1987).

Color indicators
Color values of hulls and kernels (with testa) were directly measured with a color meter (Minolta Chroma Meter Model CR-400, Minolta, Japan). The color was measured as the lightness (L*), red-green (a*) and blue-yellow (b*). The chroma value and hue angle were calculated by the fallowing equations (Equations 2 and 3) [24].

Sensory evaluation
The sensory analysis included a panel test constituted of ten semi-trained panelists. The panelists evaluated the taste, aroma, appearance and general acceptance of fruit. Excellent was shown with ve, very good with four, good with three, moderate with two, poor with one (Wang et al. 2006).
Ethylene production and respiration rate 15 g of whole pistachio fruits was placed in 1.6 L airtight respiration jars at 20°C and allowed to equilibrate for 2 h. 1 mL of head-space atmosphere was withdrawn using plastic syringes and injected into a gas chromatograph (Agilent 7890B, USA) equipped with a packed column and a thermal conductivity detector (TCD) for quanti cation of CO 2 or C 2 H 4 . Injector, detector and oven temperatures of gas chromatograph were set at 100, 120, 80°C respectively. Nitrogen was used as the carrier gas at the ow rate of 73.7 mL min − 1 . The respiration rate and ethylene production were expressed as ng kg − 1 s − 1 ).

Statistical analysis
The study was a factorial experiment based on a completely randomized design with four replications. Sources of variation were storage time (0, 25 and 50 days), treatments (control, movento, con dor, R30, R60, M30, and M60) and their interaction. Mean values were calculated and reported as the mean ± standard error of means. Data were analyzed by SAS 9.1 statistical software package, and the least signi cant difference (LSD) test at P = 0.01 probability was used to compare the means. Where the interaction of factors was signi cant, the data was presented in graphs, and where the simple effect of factors was only signi cant, the data was presented in tables. The graphs were plotted in MS-Excel software package.

Weight loss
The interaction of storage time and treatments had signi cant (P = 0.01) effect on weight loss. This trait increased by time. Two chemical pesticides caused higher weight loss at both storage times comparing control. Re nery micronized sulfur at both concentrations (R30 and R60) caused higher wight loss than control on 25th day but was similar to it after 50 days (Fig. 1). Research shows that sulfur may have adverse effects on fruit weight loss, which is concentration dependent. For example, in blueberry cultivars 'Brigitta' and 'ONeal', fumigation with high (250-350 µL L − 1 h) concentration of SO 2 caused higher weight loss than control after 45 days of cold storage, but lower concentrations (50-200 µL L − 1 h) were similar to control (Rivera et al. 2013). In this study, 30 and 60 kg/1000 L of re nery micronized sulfur showed adverse effects on weight loss but mineral sulfur does not. Mineral sulfur at its lower concentration (M30) even showed less weight loss than control on day 50. This result may be due to the longer persistence of re nery micronized sulfur on fruit surface. In a study on grapes, residue concentrations generally were lower for a wettable powder of elemental sulfur versus a micronized formulation applied at the same rate and timing of the last application before harvest (Kwasniewski et al. 2014). On the other hands, our results indicated that the negative effects of sulfur on water loss was eliminated sooner than that of movento and con dor. It is well known that chemical pesticides have varied degrees of phytotoxicity along with their bene ts. They can destruct the plant cell walls and membranes leading to elevated transpiration and water loss (Parween et al. 2014).

Firmness
The interaction of storage time and treatments had signi cant (P = 0.01) effect on hull rmness. This trait decreased by time. Movento and all sulfur treatments showed a reduction in hull rmness comparing control at harvest time, but not during storage (Fig. 2). The simple effects of treatments and storage time were signi cant on kernel rmness (P = 0.01). Day 50 and sulfur treatments caused a reduction in kernel rmness (Tables 1 and 2). Firmness is a major quality indicator for fruit marketability. Normally, when fruit water content decreases over time, a reduction in rmness is expectable. The lower rmness of sulfur-treated samples at harvest time indicates the lower water content comparing control. Comparably, application of sulfur (100-150 µL L − 1 h) on blueberry through SO 2 pads inside the packaging, resulted in lower rmness than control (Rodrigues and Zoffoli 2016).  Water activity The simple effect of storage time was only signi cant of the water activity of hulls and kernels. This trait displayed a reduction on 50th day comparing control ( Table 2). The growth of pathogens in foods is markedly affected by water activity (Romero et al. 2007). The water activity of both hulls and kernels was above 0.9 a w which is high and suitable for the growth of most pathogens. The treatments did not in uence the water activity because it is mostly dependent on temperature (Slade et al. 1991). All the samples had relatively the same temperature at harvest time and also at cold storage. Although the water content of a food does not necessarily determine the water activity, the severe water loss on 50th day resulted in a reduction in water activity.

Phenolic content
The interaction of storage time and treatments was signi cant (P = 0.01) on the content of phenolics of hull. In control, this trait remained statistically stable, but sulfur-treated samples revealed an increasing trend over time so that R30, R60 and M60-treated samples had 57.3%, 38.4% and 40.3% higher phenolic content of hull respectively, on 50th day comparing harvest time (Fig. 3). Pistachio hulls are a rich source of phenolic compounds being ranked among the rst 50 food products highest in antioxidant potential The simple effect of storage time was signi cant (P = 0.01) on phenolic content of kernels. The phenolic content of kernels was totally lower than that of hulls which has been reported in previous studies (Tomaino et al. 2010). Phenolic content of kernels declined at both storage times comparing harvest time, which indicates the difference in metabolic status between hulls and kernels ( Table 2). In another study, the phenolic content of pistachio kernels measured by DPPH method, showed a 12.2% decrease after high water loss (Tsantili et al. 2011).

Antioxidant activity
The interaction of storage time and treatments was signi cant (P = 0.01) on the antioxidant activity of hulls and kernels. Pesticides-treated hulls were similar to control, however, sulfur-treated hulls had lower antioxidant activity than control at all storage times (Fig. 4a). In kernels, sulfur treatments had lower antioxidant activity than control, while movento showed a more similar effect with control (Fig. 4b).
These results are not in agreement with the results of phenolic content and imply that in sulfur treatments, a signi cant decrease must have occurred in the content of other molecules with antioxidant properties, probably antioxidant enzymes, vitamins or carotenoids (carotenoid content was lower in R30 and R60 sulfur treatments (Fig. 7)). In another study, the antioxidant activity of 10 different fruit extracts was mostly correlated to the content of vitamin C and carotenoids rather than phenolics (Olsson et al. 2004). Similar to hulls, the antioxidant activity of kernels remained stable over time for most samples except the samples that were treated with con dor and R30, in which a decrease was observed on 50th day comparing harvest time. Another study indicated that the content of anthocyanins in longan pericarp decreased after SO 2 fumigation (Han et al. 2001). The highest anthocyanin content of kernels was related to harvest time for control. Some treatments revealed a decreasing trend over time (Fig. 5b). Most of the kernel anthocyanins are present in its testa which is a thin layer on the green seed. The testa dries rapidly by time and the anthocyanins get out of the vacuoles and may be degenerated by polyphenol oxidase.

Chlorophyll
The interaction of storage time and treatments was signi cant (P = 0.01) on chlorophyll content of hulls and kernels. Re nery micronized sulfur at both concentrations (R30 and R60) showed the highest chlorophyll content than other samples. Chlorophyll content of some samples had a slight decrease during storage (Fig. 6a,b). Since all the pistachios were completely ripe at harvest time, the high chlorophyll content in sulfur treatments is not due to the difference in ripening process. Chlorophylls are responsible for harvesting light quanta and transferring energy during photosynthesis. The way that the plants response to SO 2 stress depends on the speci c physiological state of them and their need to energy (Li and Yi 2020). Usually, the studies report the negative effect of SO 2 on photosynthesis and chlorophyll content, but some reveal the stimulation of chlorophyll synthesis under SO 2 treatments. For example, the total chlorophyll content was signi cantly increased in the leaves of Arabidopsis plants exposed to 30 mg/m 3 SO 2 for 72 h (Li and Yi 2020). In green gram, 200 µg (active ingredient) kg − 1 of sulfosulfuron stimulated the chlorophyll synthesis in fresh leaves (Khan et al. 2006).

Carotenoid
The interaction of storage time and treatments was signi cant (P = 0.01) on carotenoid content of hulls and kernels. Re nery micronized sulfur treatments (R30 and R60) showed a lower carotenoid content of hull at harvest time. The carotenoid content of hulls showed a decreasing trend over time for most samples, fallowed by a slight increase on 50th day (Fig. 7a). Similar to hulls, R30 and R60 caused a lower carotenoid content of kernels at harvest time. The carotenoid content of kernels remained stable during storage for some samples while some others showed a slight decreasing trend (Fig. 7b). Movento-treated hulls had the highest carotenoid content at harvest which can be related to the higher water loss and more phytotoxic effect of this pesticide.

Color indicators
The interaction of storage time and treatments was signi cant (P = 0.01) on the color indicators of hull. At harvest time, re nery micronized sulfur-treated samples (R30 and R60) had higher L* value but lower a* and b* values of hull than others (Fig. 8a,b,c). This means that R30 and R60 treated hulls were lighter and greener than others. In coordination, these two treatments had different hue (basic color) and chroma (color intensity) values (Fig. 8d,e). Lower hue again shows the greener color and less red and yellow pigments, while higher chroma value reveals more intense color of these two treatments. This is coordinated with the results of chlorophyll and carotenoid content. Similarly, SO 2 treatment caused a greener color in Arabidopsis leaves (Li and Yi 2020). Totally, L* value of hull showed an increasing trend in 25th day fallowed by a slight decrease in some samples. This increasing trend can be due to the gradual synthesis of cuticle on hull epidermal cells and also the gradual removal of sulfur particles. Likewise, fresh cut apples treated with 50% sodium sul te were lighter than control after 5 days of cold storage (Ortzi et al. 2018). a* and b* values revealed a decreasing trend during storage which is in agreement with the decreasing trend of carotenoids. In a similar way, the hue value of most samples increased on 50th day which shows the gradual loss of red and yellow pigments. The chroma value revealed a reduction over time that again shows the loss of pigments and loss of color intensity.
The interaction of storage time and treatments was also signi cant (P = 0.01) on the color indicators of testa of kernel. The results of testa were very similar to the results of hulls. So that, R30-and R60-treated samples had higher L* and hue values but lower a*, b* and chroma values of testa at harvest time ( Fig. 8f,g,h,i,j) which means that they were lighter and greener with less red and yellow pigments. These treatments provided a more intense color. L* and hue values showed increasing but a*, b* and chroma values showed decreasing trends over time. We are of this opinion that sulfur particles do not penetrate to the kernel if the hull is intact. But the kernels are certainly in uenced by the physiological status of hulls and get signals from them through metabolic pathways. This can be the reason why the kernels changed their color in a way that the hulls did.

Sensory evaluation
The interaction of storage time and treatments was signi cant (P = 0.01) on taste, aroma and appearance of pistachios. As expected, all three parameters displayed a decreasing trend during storage, but in some cases, this decrease was not statistically signi cant. R30 treatment could maintain the taste and aroma of pistachios even after 50 days (Fig. 9a,b). Sulfur, in certain concentrations and application times, is applied as a preservative in food technology because it keeps the quality of food constant, meeting market requirements (Rossello et al. 1994). For example, in wine industry, small doses of SO 2 is required to extend the sensory quality (Wyk et al. 2018). Herein, the panelists did not record any off-avor for the treatments. Comparably, no off-avor was developed after SO 2 fumigation in blueberries (Cantin et al. 2012). All sulfur treatments showed a higher degree of appearance at harvest time (Fig. 9c), which is related to the antimicrobial effect of sulfur, keeping the fruit surface safe from disease damages. The lighter and more intense color of sulfur-treated hulls were other reasons for the higher degree of appearance in these treatments Ethylene production and respiration rate The interaction of storage time and treatments was signi cant (P = 0.01) on both ethylene production and respiration rate of pistachios. All sulfur treatments had higher respiration rate than control, which might be the result of high energy consumption for protection mechanisms activation under SO 2 stress. The respiration rate declined by time as the result of lowered metabolism under cold temperatures (Fig. 10a). The respiration rate of wheat plants increased by 0.06 ppm SO 2 fumigation for 8 h daily. It was claimed that the detoxi cation reactions against SO 2 are ATP mediated which is provided by respiration (Agrawal and Deepak 2003). In the present study, a probable toxi cation to hulls resulted in respiration elevation.
Sulfur treatments at lower concentration (R30 and M30) showed higher ethylene production than others. After 25 days, no signi cant difference was observed between the samples (Fig. 10b). Tomato plants fumigated by SO 2 for 1 h showed enhanced ethylene production and when the treatment prolonged for 4 h, ethylene production was also enhanced. It was concluded that ethylene might play an important role in SO 2 -induced plant injuries at relatively short terms of SO 2 fumigation (Gong Young et al. 1995). Alfalfa plants fumigated by 0.7 µL/L SO 2 for 8 h, increased ethylene production 10 times greater than control plants (Peiser and Yang 1979). Ethylene production of bean plants treated with elemental sulfur increased before full bloom and before fruit set, and owering occurred earlier because a slow oxidation of elemental sulfur in air produced SO 2 which enhances ethylene evolution from leaves (Recalde-Manrique and Diaz-Miguel 1981).

Conclusion
Practical evidence shows that sulfur application on pistachios is very effective on controlling the pests such as psylla that cause huge losses to the yield annually. At the same time, it is important to optimes the type and concentration of sulfur to prevent the in uence on product quality. The kernel which is the edible part of pistachio is covered by the shell and hull, so sulfur cannot easily penetrate to the kernel. However, the physiological status of hull and the whole plant may in uence the kernels and the yield. Yield weight, rmness, color, taste and aroma are some indicators for marketability and they were declined over time. Some sulfur treatments, especially re nery micronized type, provided lighter and greener color and could preserve the traste and aroma of pistachios over time, but at the same time, they reduced the weight, rmness and carotenoid content. Therefore, the gardeners should take this into account that besides the bene ts on controlling the pest, sulfur may have some effects on the product quality. Carotenoid content of (a) hulls and (b) kernels of pistachio cultivar 'Ahmadaghaei' treated preharvest with movento (0.5 kg/1000 L), con dor (0.4 kg/1000 L), re nery micronized sulfur at two concentrations (30 and 60 kg/1000 L) (R30 and R60) and mineral sulfur at two concentrations (30 and 60 kg/1000 L) (M30 and M60) during storage Figure 8 Color indicators of (a-e) hulls and (f-j) kernels of pistachio cultivar 'Ahmadaghaei' treated preharvest with movento (0.5 kg/1000 L), con dor (0.4 kg/1000 L), re nery micronized sulfur at two concentrations (30 and 60 kg/1000 L) (R30 and R60) and mineral sulfur at two concentrations (30 and 60 kg/1000 L) (M30 and M60) during storage Figure 9 Sensory indicators of pistachio cultivar 'Ahmadaghaei' treated preharvest with movento (0.5 kg/1000 L), con dor (0.4 kg/1000 L), re nery micronized sulfur at two concentrations (30 and 60 kg/1000 L) (R30 and R60) and mineral sulfur at two concentrations (30 and 60 kg/1000 L) (M30 and M60) during storage