Reduction of postharvest decay in organic citrus fruit by a
short hot water brushing treatment
Ron Porat, Avinoam Daus, Batia Weiss, Lea Cohen, Elazar Fallik,
Samir Droby *
Department of Posthar6est Science of Fresh Produce,ARO,The Volcani Center,P.O.Box6,Bet Dagan50250,Israel Received 2 June 1999; accepted 8 September 1999
Abstract
The marketing of organic citrus fruit has markedly increased during the last few years. However, these fruits are not treated with chemical fungicides and suffer from relatively high rates of decay. In this study, we examined the possible use of a new hot water brushing (HWB) treatment, to disinfect the fruits and reduce decay development during postharvest storage. Preliminary observations have shown that a minimum exposure period of 20 s at 56°C was required to inhibitPenicillium digitatum(Pers.: Fr.) Sacc spore germination in vitro. In vivo studies, carried out by rinsing and brushing the fruit 24 h after artificial inoculation with aP.digitatumspore suspension, indicated that HWB at 56, 59 and 62°C for 20 s, reduced decay development in the infected wounds to only 20, 5 and less than 1%, respectively, of that in untreated control fruits or fruits treated with tap water. The effects of HWB at 56, 59 and 62°C for 20 s on epiphytic microflora, was further confirmed by the reduction of the microbial counts (CFU) on the fruit surface to only 24, 12, and less than 1%, respectively, of those on fruit that had been rinsed and brushed with tap water. Scanning electron microscopy showed that HWB at 56°C for 20 s had smoothed the fruit epicuticular waxes and thus covered and sealed stomata and cracks on the fruit surface, which could have served as potential pathogen invasion sites. Postharvest storage experiments using various organically grown citrus cultivars such as ‘Minneola’ tangerines, ‘Shamouti’ oranges and ‘Star Ruby’ red grapefruit, showed that HWB at 56°C for 20 s reduced decay development by 45 – 55%. The HWB treatment at 56°C did not cause surface damage, and did not influence fruit weight loss or internal quality parameters. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Citrus; Decay; Hot water brushing;Penicillium digitatum; Postharvest
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1. Introduction
The marketing of organic farming products has markedly expanded because of increased con-sumer demand for healthy food products, which are free of synthetic chemical residues, and the resulting improvements in the production and dis-* Corresponding author. Tel.:+972-3-9683615; fax:+
972-3-9683856.
E-mail address:samird@netvision.net.il (S. Droby)
tribution systems (Sylvander, 1993). However, since organic fruits are not treated with chemical fungicides, they suffer from relatively high rates of decay, which develops during storage and shelf-life. The commercial loss from decay could reach 30 – 50% in some susceptible citrus cultivars, and this represents a major limitation to the further expansion of the organic fruit market.
Recently, we have developed a unique non-chemical method for rinsing and disinfecting fruit and vegetables with hot water and brushes (Fallik et al., 1999; Israeli patent 116965). This technique involves rinsing fruits with sprays of hot water as they move along a set of brush rollers, thus simultaneously cleaning and disinfecting the fruits, improving their general appearance and maintaining quality. This technique was designed to be a part of a commercial packing house sorting line.
In the present study, we examined the optimum temperatures and exposure periods required to disinfect organic citrus fruits and to reduce the development of the green mold pathogen Penicil -lium digitatum, which is the cause of most of the postharvest losses of citrus fruit (Eckert and Brown, 1986). Accordingly, we established a hot water brushing (HWB) treatment to clean and disinfect the fruits, thus reducing postharvest decay.
2. Materials and methods
2.1. Plant material and storage conditions
‘Minneola’ tangerines (C. reticulata Blanco), ‘Shamouti’ oranges (C.sinensisOsbeck) and ‘Star Ruby’ red grapefruit (C. paradisi Macf.) were obtained from local orchards and were used the day after harvest. For storage experiments, ‘Min-neola’ tangerines were kept at 5°C for 4 weeks; ‘Shamouti’ oranges at 5°C for 6 weeks; and ‘Star Ruby’ red grapefruit at 11°C for 6 weeks. After storage, the fruits were transferred to shelf-life conditions at 20°C for another week. The relative humidity was 90% in all storage rooms. Each treatment comprised five replicate boxes, each containing 40 fruits.
2.2. Posthar6est HWB treatments
HWB treatments at 56, 59 and 62°C were ap-plied as a rinse to fruits moving along a set of brush rollers as described by Fallik et al. (1999). The fruits were exposed to different HWB temper-atures for 20 s. As a control, fruit were rinsed and brushed with tap water (20°C).
2.3. Juice chemical determination
The total soluble solids (TSS) content in the juice was determined with a refractometer, and the acidity percentage was measured by titration with 0.1 N NaOH to pH 8.3, the results being expressed as citric acid.
2.4. Fungal cultures
P. digitatum was obtained from an infected grapefruit and cultured on Potato Dextrose Agar (Difco, Detroit, MI). Spore suspensions were pre-pared by removing the spores from the sporulat-ing edges of a 2 – 3-week-old culture with a bacteriological loop, and suspending them in ster-ile distilled water. Spore concentration was deter-mined with a hemocytometer and adjusted as required.
2.5. Effects of heat treatments on P. digitatum spore germination in 6itro
Sterile glass tubes containing 1.8 ml distilled water were placed in water baths at 56, 59, and 62°C, and allowed to equilibrate for 30 min. Afterwards, 0.2 ml of a concentrated P.digitatum spore suspension was added to the tubes, to achieve a final concentration of 2×105 spores ml−1
. After 10, 15 and 20 s, tubes were removed from the water baths and placed immediately on ice. Aliquots (50ml) of the spore suspensions were
transferred to wells of tissue culture clusters (Corning Costar Corporation, Cambridge, MA) containing 450 ml of 10% Potato Dextrose Broth
(Difco, Detroit, MI). Samples of these solutions (30 ml drops), were placed on ethanol-washed
and incubated for 24 or 48 h at 25°C in darkness. Spore germination was measured in three micro-scope fields, each containing 40 – 50 spores, under a light microscope.
2.6. Effects of HWB on infection of P. digitatum in6i6o
‘Star Ruby’ red grapefruits were wounded with a dissecting needle (1 – 2 mm deep) at three sites around the stem end. The wounded sites were inoculated with 20 ml of a P. digitatum spore
suspension (104
spores ml−1
), and the fruits were kept for incubation in plastic trays, at 24°C under humid conditions. After 24 h, the fruit were rinsed and brushed with hot water for 20 s, and kept for incubation under the same conditions. The per-centage of infected wounds was determined 4 days after inoculation. Twenty fruits were used for each treatment (total of 60 wounds).
2.7. Effects of HWB on epiphytic microbial population
After HWB treatments, ‘Star Ruby’ red grape-fruits and ‘Minneola’ tangerines were placed in 200 ml of sterile distilled water in sealed auto-claved beakers, and were incubated for 1 h on an orbital shaker (200 rpm). The fruit washings were serially diluted, and 100 ml of each dilution was
plated on Potato Dextrose Agar (Difco, Detroit, MI). The number of colony-forming units (CFU) was determined after 3 days of incubation at 25°C. For each treatment, the microflora of three separate fruits were evaluated. Fruit treated with tap water served as a control.
2.8. Scanning electron microscope (SEM) analysis
‘Minneola’ tangerine peel disks excised from control and hot-water-brushed fruits, were imme-diately frozen by placing them on a copper block cooled with liquid nitrogen. The frozen samples were further dried by sublimation in a high-vac-uum device as described by Newbury et al. (1986). The samples were analyzed by a JEOL GSM-T300A SEM at 10 and 15 kV, 0 and 30° tilt and 10 mm working distance. Samples from five fruits of each treatment were analyzed.
3. Results
To establish a postharvest HWB treatment that is efficient in disinfecting organically grown citrus fruits, we first examined the effects of various heating periods on the in vitro spore germination of the green mold pathogen P. digitatum. Short exposures of 10 or 15 s at 56°C only delayed spore germination, but a longer exposure of 20 s at the same temperature markedly inhibited spore ger-mination to zero after 24 h and 32% after 48 h (Fig. 1). Heating at 59 or 62°C was more effective in inhibiting P. digitatum spore germination than heating at 56°C; a short exposure of 10 s at 59°C reduced spore germination to only 10% after 48 h, whereas longer exposures of 15 or 20 s at the same temperature, or only 10 s at 62°C, com-pletely inhibited spore germination (Fig. 1).
To evaluate the effects of HWB treatments on eradication of established infections, ‘Star Ruby’ red grapefruit were wound-inoculated with a P. digitatum spore suspension, and treated with HWB after a 24-h incubation period. The results showed that rinsing and brushing the fruit with non-heated tap water was ineffective in reducing decay, whereas after HWB at 56, 59 and 62°C only 20, 5 and less than 1%, respectively, of the infected wounds developed decay (Fig. 2).
The effect of the HWB treatment on epiphytic microflora of ‘Star Ruby’ red grapefruit was also evaluated. Rinsing and brushing the fruit with tap water reduced the naturally occurring epiphytic microflora population on the fruit surface two-fold to only 1.4% of that on control unwashed fruit (Fig. 3). HWB treatments at 56, 59 and 62°C resulted in a further reduction in microbial counts (CFU) to only 24, 12 and less than 1%, respec-tively, of those observed on tap water washed fruit (Fig. 3). Similar results were observed with ‘Minneola’ tangerines (data not shown).
Fig. 1. Effects of various heat exposures onP.digitatumspore germination in vitro.P.digitatumspore suspensions were dipped in hot water for various periods, and the percentage of germination was measured after 24 and 48 h at 25°C. Values are means of three replications per treatment, each containing 40 – 50 spores.
removed fungus spores and hyphae from its sur-face (Fig. 4A – B). Moreover, we found that the HWB treatment smoothed the fruit epicuticular waxes, so that it covered and sealed the stomata (Fig. 4C – D) and microscopic cracks on the fruit surface (Fig. 4E – F).
In storage experiments with ‘Minneola’ tanger-ines, ‘Shamouti’ oranges and ‘Star Ruby’ red grapefruit, the HWB treatment at 56°C for 20 s reduced decay development to only 45, 55 and 52%, respectively, of that on fruit from commer-cial organic packing houses, which was not treated with HWB (Fig. 5). In all cultivars tested, the HWB treatment at 56°C for 20 s did not cause any damage, and did not affect fruit weight loss and internal quality parameters, such as the per-centage of TSS in the juice, and the juice acidity (data not shown). Moreover, the HWB treatment markedly improved fruit appearance, making them cleaner and glossier.
4. Discussion
Postharvest heat treatments have been used for
many years to control fungal diseases in fruits and vegetables (Couey, 1989; Barkai-Golan and Phillips, 1991; Lurie, 1998). During the past few years, these heat treatments have attracted
in-Fig. 2. Effects of HWB treatments on green mold decay development after artificial inoculation of ‘Star Ruby’ red grapefruit. Surface wounds were inoculated with a P. digi
-tatumspore suspension (104spores ml−1), and after 24 h the
Fig. 3. Effects of HWB treatments on the epiphytic microflora of ‘Star Ruby’ red grapefruit. Fruits were rinsed and brushed at three different temperatures for 20 s and their surfaces were then washed with distilled water and plated on Petri dishes. CFUs were determined after 3 days of incubation at 25°C. Values are means9S.E. of three fruits per treatment.
(Fig. 2), and reduced the total amount of micro-bial population on the fruit surface by 76% (Fig. 3), as compared with that on fruit rinsed and brushed with tap water. HWB treatments at higher temperatures, of 59 and 62°C, were more effective in cleaning the fruit surface (Figs. 1 – 3). However, since they may cause heat damage in some citrus cultivars, we chose to focus our stud-ies on the HWB treatment at 56°C. Nevertheless, it should be mentioned that in other citrus culti-vars, such as ‘Star Ruby’ red grapefruit, even the higher temperature of 62°C did not cause any heat damage. Therefore, the optimum HWB tem-perature should be determined for each citrus cultivar separately.
Cleaning and disinfection of the fruit surface by the HWB treatment was further confirmed by SEM analysis (Fig. 4A, B). Another effect of the HWB treatment was that it melted the fruit epicu-ticular waxes and thus covered and sealed the stomata and cracks on the fruit surface; these could have served as potential pathogen invasion sites (Fig. 4C – F). A similar effect of the HWB treatment on sealing cracks on the fruit surface has also been reported in sweet pepper (Fallik et al., 1999), and following a hot water dip in ‘For-tune’ mandarins (Schirra and D’hallewin, 1997).
Postharvest storage experiments showed that the HWB treatments, without addition of any fungicides, reduced decay development by 45 – 55%, as compared with fruit from commercial organic packing houses (Fig. 5). Although this was a significant reduction in decay development, the influence of the HWB treatment on decay control under storage conditions was less effective as compared with previous laboratory tests (Figs. 1 – 3). This is probably since the HWB treatment effectively disinfected the fruit from pathogenic fungi immediately after harvest. However, it could not completely prevent further infection during the later storage and shelf-life periods.
In addition to decay control, the HWB treat-ment further cleaned the fruit and improved its general appearance, without affecting weight loss or TSS and acidity levels (data not shown). Simi-lar findings of improved general appearance of the fruits without alteration of their internal quality parameters have been reported previously (Fallik creasing interest as a result of the growing
de-mand to reduce the postharvest use of chemical fungicides.
With citrus fruits, hot water dip treatments were reported to control postharvest decay several decades ago (Fawcett, 1922; Smoot and Melvin, 1965). Several recent studies have confirmed that hot water dips, usually for 2 – 3 min at 53°C, with or without the addition of fungicides, were capa-ble of reducing decay in a wide variety of citrus cultivars (Rodov et al., 1995; Schirra and Mulas, 1995; Schirra and D’hallewin, 1997; Schirra et al., 1997). In the present study, we examined the possible use of a recent improvement of the hot water treatment, which has been developed to combine a hot water rinsing and brushing treat-ment (Fallik et al., 1999). The advantages of this technique are that it simultaneously cleans and disinfects the fruit, it fits into the packing house sorting line, and it requires a much shorter expo-sure time (10 – 30 s) than conventional hot water dip treatments, which usually require a few min-utes. This HWB treatment has already been com-mercially adopted to clean and disinfect peppers (Fallik et al., 1999), mangoes (Prusky et al., 1999), and corn and melons (Fallik, personal communication).
et al., 1999; Prusky et al., 1999). Similarly, no measurable effects on fruit quality parameters were reported after hot water dips (Schirra and Mulas, 1995; Schirra et al., 1997).
In conclusion, our results show that HWB treatments can be used as non-chemical alterna-tive postharvest treatments for cleaning and
dis-infecting organic citrus fruit. In fact, these treatments have already been adopted in some of the organic packing houses in Israel. In the future, we intend to improve the HWB treatment by combining biological control agents and other natural antifungal compounds.
Fig. 5. Effects of HWB on incidence of postharvest decay in various organic citrus cultivars. Fruits from commercial or-ganic packing houses served as controls, or were rinsed and brushed at 56°C for 20 s. ‘Minneola’ tangerines were kept at 5°C for 4 weeks; ‘Shamouti’ oranges at 5°C for 6 weeks; and ‘Star Ruby’ red grapefruit at 11°C for 6 weeks. Decay was evaluated after an additional week of simulated shelf life at 20°C. Values are means9S.E. of five replications per treat-ment, each containing 40 fruits.
Eckert, J.W., Brown, G.E., 1986. Postharvest citrus diseases and their control. In: Wardowski, W.F., Nagy, S., Grier-son, W. (Eds.), FrCitrus Fruits. AVI Publishing, Westport, CT, p. 315.
Fallik, E., Grinberg, S., Alkalai, S., Yekutieli, O., Wiseblum, A., Regev, R., Beres, H., Bar-Lev, E., 1999. A unique rapid hot water treatment to improve storage quality of sweet pepper. Postharvest Biol. Technol. 15, 25 – 32. Fawcett, H.S., 1922. Packinghouse control of brown rot. Calif.
Citrog. 7, 232 – 254.
Lurie, S., 1998. Postharvest heat treatments of horticultural crops. Hortic. Rev. 22, 91 – 121.
Newbury, D.E., Joy, D.C., Echlin, P., Fiori, C.E., Goldstein, J.I. (Eds.), 1986. Advances in Scanning Electron Mi-croscopy and X-Ray Microanalysis. Advances in Specimen Preparation for Biological SEM. Plenum, New York, pp. 325 – 365.
Prusky, D., Fuchs, Y., Kobiler, I., Roth, I., Weksler, A., Shalom, Y., Fallik, E., Zauberman, G., Pesis, E., Aker-man, M., Yekutieli, O., Wiseblum, A., Regev, R., Artes, L., 1999. Effect of hot water brushing, prochloraz treat-ment and waxing on the incidence of black spot decay caused byAlternaria alternatain mango fruits. Postharvest Biol. Technol. 15, 165 – 174.
Rodov, V., Ben-Yehoshua, S., Albagli, R., Fang, D.Q., 1995. Reducing chilling injury and decay of stored citrus fruit by hot water dips. Postharvest Biol. Technol. 5, 119 – 127. Schirra, M., D’hallewin, G., 1997. Storage performance of
Fortune mandarins following hot water dips. Postharvest Biol. Technol. 10, 229 – 238.
Schirra, M., Mulas, M., 1995. Improving storability of ‘Tarocco’ oranges by postharvest hot-dip fungicide treat-ments. Postharvest Biol. Technol. 6, 129 – 138.
Schirra, M., Agabbio, M., D’hallewin, G., Pala, M., Ruggiu, R., 1997. Response of Tarocco oranges to picking date, postharvest hot water dips, and chilling storage tempera-tures. J. Agric. Food Chem. 45, 3216 – 3220.
Smoot, J.J., Melvin, C.F., 1965. Reduction of citrus decay by hot-water treatment. Plant Dis. Rep. 49, 463 – 467. Sylvander, B., 1993. Conventions on quality in the fruit and
vegetables sector: results on the organic sector. Acta Hor-tic. 340, 241 – 246.
Acknowledgements
We thank Erwin Fischer for his assistance with the SEM analysis. Contribution from the Agricul-tural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 410/99.
References
Barkai-Golan, R., Phillips, D.J., 1991. Postharvest heat treat-ments of fresh fruits and vegetables for decay control. Plant Dis. 75, 1085 – 1089.
Couey, H.M., 1989. Heat treatment for control of postharvest diseases and insect pests of fruits. HortScience 24, 198 – 202.
