Postharvest Hot Water Treatment For The Control Of

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Postharvest Biology and Technology 36 (2005) 323–327 Research note Postharvest hot water treatment for the control of Thielaviopsis black rot of pineapple R.S. Wilson Wijeratnam ∗ , I.G.N. Hewajulige, N. Abeyratne Post Harvest Technology Group, Agro Food Technology Division, Industrial Technology Institute, 363 Bauddhaloka Mawatha, Colombo 07, Sri Lanka Received 9 October 2004; accepted 13 January 2005 Abstract Black rot of pineapple (Chalara paradoxa (De Seyen.) Sacc.) is a common postharvest problem in many countries. Consumer resistance to the use of fungicides has precipitated the need for alternative means of controlling the disease. In vitro studies on heat-treated (50 ◦ C for 3 min) spores of the pathogen showed a mean colony count of 11 ± 0.7, following 48 h incubation at 28 ◦ C, while plates with heat-treated spores at 54 and 58 ◦ C showed a mean colony count of 1 ± 1.0 and 1 ± 0.7, respectively. Pineapples inoculated with 104 spores/ml, C. paradoxa, followed by a hot water dip treatment at 54 ◦ C for 3 min were free of disease when stored at 10 ◦ C for 21 days followed by 48 h at an ambient temperature (28 ± 2 ◦ C). Inoculated dip-treated fruit held at 28 ± 2 ◦ C for 6 days also remained healthy. However, characteristic symptoms of the disease were observed in fruit that were inoculated and held as controls under similar storage conditions, with no hot water dip treatment. No significant difference occurred between hot water dip-treated and untreated controls with respect to flesh and shell colour of fruit, ascorbic acid levels and titratable acidity. Mean ascorbic acid level was 18.8 mg/100 g in fruit stored at 10 ◦ C compared with 9.3 mg/100 g in fruit stored at 28 ± 2 ◦ C, irrespective of whether they were inoculated or non-inoculated fruit. A significant difference (p < 0.05), in total soluble solids (mean Brix of 14◦ ), occurred in hot water treated fruit compared with untreated fruit (mean Brix of 11.5◦ ), irrespective of storage temperature. © 2005 Elsevier B.V. All rights reserved. Keywords: Pineapple; Chalara paradoxa; Black rot; Hot water dip treatment 1. Introduction Phylogenetic and taxonomic evaluation of the pathogen Chalara paradoxa (De Seyen.) Sacc. (syn.) ∗ Corresponding author. Tel.: +94 11 2683128; fax: +94 11 2686567. E-mail address: [email protected] (R.S. Wilson Wijeratnam). 0925-5214/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2005.01.003 Thielaviopsis paradoxa (De Seyen.) H¨ohn., teleomorph: Ceratocystis paradoxa (Dade) C. Moreau has been conducted by Paulin-Mahady et al. (2002). The pathogen gains entry into host tissue through wounds and causes black rot disease in pineapple (Rohrbach and Phillips, 1990). In Sri Lanka, infection occurs after harvest, either in the field or during pack house operations via the cut end of the peduncle. The disease 324 R.S. Wilson Wijeratnam et al. / Postharvest Biology and Technology 36 (2005) 323–327 develops into a soft rot under warm humid conditions. Black rot of pineapple is controlled by the application of specific fungicides (Liu and Marcano, 1973; Sridhar, 1975; Cho et al., 1977). However, increasing consumer resistance and the restrictions imposed on the use of these chemicals, have created an urgent need for the development of safe and effective alternatives. Heat treatment technology is a safe and environmentally friendly procedure with increasing acceptability in commercial operations. It is used successfully, to control the incidence of postharvest disease in several commodities (Fallik, 2004). Pre-storage heat treatments to control decay, are applied for short periods of time (min), as target pathogens are present in the outer-most layers of host tissue. Water is the preferred medium of application as it is a more efficient medium of heat transfer than air (Lurie, 1998). Pre-storage hot water treatments, methods of hot water immersion and treatment duration have been reviewed by Fallik (2004). The thermal death point of C. paradoxa is recorded as 52.5–53 ◦ C (Ames, 1915). Hot water dip treatment at 53 ◦ C followed by TMTD (Thiram) resulted in a limited control of the pathogen in sugarcane propagules (Buergo et al., 1989). However, a hot water dip treatment of 1 min at 52 ◦ C slowed the rate of rot development in litchi (Olesen et al., 2004). Thus, the use of hot water dip treatments as a means of controlling incidence of black rot in Mauritius variety pineapples was investigated. 2. Materials and methods C. paradoxa was isolated from the stem end of a naturally infected pineapple showing characteristic symptoms of black rot disease. Pure cultures of the isolate were maintained on potato dextrose agar (PDA) at 6 ◦ C. Pathogenicity of the culture was maintained by inoculation and re-isolation of the pathogen at regular intervals. Spore suspensions containing 104 spores/ml were prepared from 5-day-old cultures of C. paradoxa. In each instance, the culture plate was flooded with 10 ml sterile distilled water and the surface gently agitated with a sterile bent glass rod. Concentration of conidia was determined using a haemocytometer after filtration through layered sterile muslin. A conidial suspension of 104 spores/ml was prepared by serial dilution and a separate set of three sterile screw cap test tubes, each containing 10 ml of the spore suspension was used for each temperature tested. Temperature regulated water baths (Grant W6–KD) were pre-heated where necessary and held at 28 ◦ C (ambient temperature), 50, 54 and 58 ◦ C, respectively. Sets of spore suspensions were held in respective water baths for 5 min including the 2 min required for the 10 ml suspension to reach the required temperature. Each set of tubes was removed from the water bath after the prescribed heat treatment and cooled in cold water (20–22 ◦ C). Spore germination on PDA Petri plates after heat treatment was determined via a spread plate technique for each treatment temperature. Colony counts were recorded 48 h after incubation at 28 ◦ C. Spore suspensions (104 spores/ml) from plates of controls, as well as heat-treated spores showing sporulation and mycelial growth were prepared. Respective spore suspensions were inoculated onto the peduncles of healthy pineapples. Post-heat treatment virulence of the pathogen was tested by examining nine pineapples inoculated with 0.1 ml of 104 spores/ml of the control spore suspension. Further sets of nine pineapples were similarly inoculated with cultures of the respective heat-treated spores. Incidence of disease on fruit was recorded after 6 days incubation at 28 ◦ C. For in vivo experiments, the inoculum of C. paradoxa spores was prepared as described previously in the in vitro studies. Concentration of inoculum for in vivo studies was determined by preparing spore concentrations in sterile distilled water, ranging from 10 to 106 spores/ml of C. paradoxa. Peduncles of nine fruit were inoculated for each spore concentration. A set of nine non-inoculated fruit served as controls. Peduncles were trimmed to a length of 3–4 cm before inoculation. Fruit were incubated at 28 ± 2 ◦ C for 6 days, and examined for incidence and severity of disease. The experiment was repeated twice. Results presented in Fig. 1(a and b), confirmed the findings of Cho et al. (1977). Thereafter, 0.1 ml of the spore suspension (104 spores/ml) was used to inoculate the peduncles soon after trimming. With non-inoculated treatments, 0.1 ml of sterile distilled water was placed on the cut surface of the peduncle. All fruit were incubated at room temperature for 3 h, before application of respective dip treatments. Dip treatments were conducted on Mauritius variety pineapples at 20% yellow stage of maturity within 12 h R.S. Wilson Wijeratnam et al. / Postharvest Biology and Technology 36 (2005) 323–327 Fig. 1. (a and b) Development of internal and external symptoms of black rot in Mauritius variety pineapples in relation to inoculum concentration (N.B. non, no infection; stage 1, minimal infection; stage 2, extensive infection). after harvest. Eight treatments were tested with 36 fruit per treatment, and six fruit packed into each carton. In both inoculated and non-inoculated fruit, the hot water dip treatment was conducted at 54 ◦ C and ambient temperature dip treatments in tap water at 28 ◦ C. One set of 36 fruit from each of the respective inoculated and non-inoculated hot water and tap water treatments were stored at 10 ◦ C for 21 days followed by 48 h at ambient temperature (28 ± 2 ◦ C). The other set of diptreated, inoculated and non-inoculated fruit were held at 28 ± 2 ◦ C for 6 days. The hot water dip treatment was carried out in a 70 cm × 145 cm rectangular metal tank of depth 45 cm, fitted with a detachable rectangular rack with circular rings of 10 cm diameter. Pineapples placed on the rings in the crown up position in batches of 18 fruit, were subjected to the 3 min dip treatment. The detachable rack ensured that only peduncles of fruit were exposed 325 to hot water and prevented scorch damage to other areas of the fruit during the dip treatment. The water in the tank was preheated and maintained at 54 ◦ C via a programmable thermostat. Temperature gradients were avoided by means of a 0.5 hp pump, which ensured circulation of water inside the tank. Water temperature was monitored using a temperature probe (Omega USA, Type T, Model EP1 6114). Water in the tank was changed after each treatment. Pineapples used as controls were dipped in tap water at 28 ◦ C for 3 min. The above experiment was repeated twice. Severity of black rot disease (non, no symptoms of disease; stage 1, symptoms of disease on stem only; stage 2, symptoms of disease on stem and flesh) and external and internal incidence, observed via longitudinal dissection of the fruit, were scored after periods of storage. Total soluble solids (Brix measured using an ‘Erma’ 0–25% range refractor meter), titratable acidity, by titration with 0.1N NaOH using phenolphthalein as indicator and ascorbic acid levels (Rangana, 1977) were recorded from six randomly selected fruit for each treatment tested. Similarly, shell colour of pineapples was recorded using a 0–5 scale (0, full green and 5, full yellow), while flesh colour was recorded using a 0–3 scale (0, pale yellow; 1, yellow; and 3, golden yellow). A completely randomized design was adopted for all in vivo treatments. Results from fruit quality trials were subjected to analyses of variance and mean separations conducted via DMRT at the 5% level of significance. Statistical analysis of data was carried out with the Statistical Analysis System (SAS) computer package– Version 6. 3. Results Following 48 h incubation at 28 ◦ C, plates with heattreated spores at 50 ◦ C showed a mean colony count of 11 ± 0.7, while plates with heat-treated spores at 54 ◦ C and 58 ◦ C showed a mean colony count of 1 ± 1.0 and 1 ± 0.7, respectively. Colony counts were not possible in control plates spread with non heat-treated spores as plates were covered with mycelium. All fruit inoculated with spores harvested from cultures that survived the heat treatments and spores from non heat-treated cultures of C. paradoxa, showed internal and external symptoms of black rot disease when 326 R.S. Wilson Wijeratnam et al. / Postharvest Biology and Technology 36 (2005) 323–327 observed after 6 days incubation. Fruit maintained as controls showed no symptoms of the disease. The results from the in vivo experiment to determine the effective concentration of spores for development of both internal and external symptoms of black rot in pineapples are shown in Fig. 1 (a and b). All fruit showed internal and external infection, when inoculated with the pathogen at a spore concentration of 104 spores/ml and this concentration was used as inoculum thereafter. There was no evidence of disease on fruit that were not inoculated indicating that if the pathogen was present in the plantation, the concentration of spores carried into storage was not sufficient to cause infection. No infection was observed in both inoculated and non-inoculated fruit subjected to the hot water dip treatment at 54 ◦ C, irrespective of the storage temperature or duration of storage (data not shown). However, 100% external and internal infection was observed in the fruit inoculated with the pathogen. No significant difference occurred between hot water dip treated and untreated controls with respect to flesh and shell colour of fruit, ascorbic acid levels and titratable acidity. However, an ascorbic acid level of 18.8 mg/100 g was observed in fruit stored at 10 ◦ C for 17 days followed by 48 h at 28 ± 2 ◦ C, compared with 9.3 mg/100 g in fruit stored at 28 ± 2 ◦ C (p < 0.05). A significant difference in total soluble solids (p < 0.05) occurred in hot water treated fruit (mean Brix value of 14), compared with untreated fruit (mean Brix value of 11.5), irrespective of storage temperature. ling the pathogen either in the field or during postharvest operations. The results from this study indicate that the 3 min hot water dip treatment at 54 ◦ C, applied to the trimmed peduncle of pineapple was very effective in controlling the incidence of black rot. As with chemical treatments (Rohrbach and Phillips, 1990), the hot water dip treatment was carried out 6–12 h after harvest. These findings are of particular interest to situations such as in Sri Lanka, where the main site of pathogenic infection is the peduncle. Application of hot water treatment would be a safe and environmentally friendly method of controlling black rot disease of pineapple for domestic as well as overseas markets, and could be adopted by the trade as a viable alternative to fungicide treatments. The thermal death point of T. paradoxa is between 52.5 ◦ C and 53 ◦ C. We chose to maintain a 3-min treatment temperature of 54 ◦ C to protect quality of the fruit and allow for marginal fluctuations in temperature during commercial operations. The specially designed metal frame on which fruit were placed, protected them from exposure to hot water, but permitted the stems to be immersed for the treatment. When whole pineapples were immersed in hot water at 54 ◦ C for 3 min (data not presented), crowns were discoloured and premature change in shell colour of fruit was observed. A low level of T. paradoxa spore survival was observed at 58 ◦ C. This emphasizes the importance of preventing spore build up in hot water treatment systems in commercial operations, where care must be taken to change water in treatment tanks before the spore count of the pathogen reaches critical levels. 4. Discussion The black rot pathogen is of economic importance to the pineapple industry, when inoculum build up occurs. The effective concentration of spores for development of disease in Mauritius variety pineapples was confirmed as 104 spores/ml from the in vivo experiment, and was in accordance with results reported by Cho et al. (1977). The application of fungicides continues to be the means of controlling black rot disease in pineapples (Cho et al., 1977; Rohrbach and Phillips, 1990; Hernadez and Mayato, 1990). Yeast isolates identified as microbial antagonists of T. paradoxa, (Rheys et al., 2004) could prove to be an effective means of control- Acknowledgements This study was part of a research project funded by the National Research Council, Sri Lanka. The authors wish to acknowledge the assistance of Ms. Anoja Fernando, Ms. Amutha Rasalingam and Ms. Shiranthi Perera with conducting this study. References Ames, A., 1915. The temperature relations of some fungi causing storage rots. Phytopathology 5, 10–19. R.S. Wilson Wijeratnam et al. / Postharvest Biology and Technology 36 (2005) 323–327 Buergo, J.A., Herrera, J., Gonyalez, E., 1989. Physico-chemical method for the control of Ceratocystis paradoxa in sugar cane propagules. Revistande-Prot.-Vegelal 4, 57–61. Cho, J.J., Rochrbach, K.G., Apt, W.J., 1977. Induction and chemical control of rot caused by Ceratocystis paradoxa on pineapples. Phytopathology 67, 700–703. Fallik, E., 2004. 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