- Email: [email protected]
Revisiting the efficacy of hot water treatment in managing anthracnose and stem-end rot diseases of mango cv. ‘Carabao’
Crop Protection 67 (2015) 96e101
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Revisiting the efficacy of hot water treatment in managing anthracnose and stem-end rot diseases of mango cv. ‘Carabao’ Dionisio G. Alvindia a, b, *, Miriam A. Acda a a
Philippine Center for Postharvest Development and Mechanization (PhilMech), Central Luzon State University (CLSU), Compound 3120, ~ oz, Nueva Ecija, Philippines Science City of Mun b ~ oz, Nueva Ecija, Philippines Department of Biological Sciences, College of Arts and Sciences, CLSU, 3120, Science City of Mun
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 June 2014 Received in revised form 22 September 2014 Accepted 23 September 2014 Available online
This study was conducted to revalidate the efficacy of hot water treatment (HWT) as a standard protocol in managing postharvest disease in 'Carabao' mangoes. We elucidated the possible reasons for the inadequacy of HWT in management of anthracnose and stem-end rot. The effect of HWT on the cultures of anthracnose and stem-end rot-causing pathogens and on the overall quality of fruit was examined. The present investigation suggested 53 C for 20 min as optimal exposure for 'Carabao' mango. At this exposure however, the propagules of Colletotrichum gloeosporioides and Lasiodiplodia theobromae were not totally controlled. HWT was unstable in controlling C. gloeosporioides and L. theobromae as demonstrated by high standard deviation of radial growth. HWT manner of control is fungistatic rather than fungicidal as pathogens developed after treatment. Fungistatic activity of HWT was perhaps inadequate to protect the fruit from decay due to absence of residual action. There were no significant changes in the quality of 'Carabao' mangoes submerged in hot water at 53 C for 20 min whilst the severity of anthracnose was reduced by 48.71%e52.63% and stem-end rot by 48%e60.86%. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Mangifera indica Decay Heat treatment Fruit firmness Fruit color
1. Introduction Mango (Mangifera indica Linn.) is one of the most important fruit crops grown in the Philippines. ‘Carabao’ as the main variety, mango production during the first quarter of 2014 was 160,940 MT (http://www.bas.gov.ph/?ids¼fruitssituation). A significant amount however, is wasted, estimated at 2e33% because of fruit drop cracking, immaturity, and postharvest decay, mainly due to anthracnose and stem end rot (Nuevo and Apaga, 2010). Anthracnose is caused by Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (Fitzel and Peak, 1984; Jeffries et al., 1990; Dodd et al., 1997; Arauz, 2000) while stem-end rot is caused by Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (Prusky et al., 1997; Kobiler et al., 2001; Mascarenhas et al., 1995). Infection of fruit occurs during production and postharvest operations (Johnson and Hofman, 2009) and compromises storage life of the fruit (Dodd et al., 1997; Prusky et al., 1997). ‘Carabao’ is mainly traded as fresh produce. About 60e70% is exported to Japan while the remaining portion is shipped to Hong * Corresponding author. Philippine Center for Postharvest Development and Mechanization (PhilMech), Central Luzon State University (CLSU), Compound 3120, ~ oz, Nueva Ecija, Philippines. Tel.: þ63 44 4560 213. Science City of Mun E-mail address: [email protected] (D.G. Alvindia). http://dx.doi.org/10.1016/j.cropro.2014.09.016 0261-2194/© 2014 Elsevier Ltd. All rights reserved.
Kong, Singapore, Korea, China, US, Australia, Europe and New Zealand (Aveno and Orden, 2008). As postharvest protocol for export, mangoes are dipped in hot water at 52e55 C for 10 min to manage postharvest diseases (Buganic et al., 1997) with 10 min hydro-cooling, and 30 min drying prior to packing (Esguerra et al., 2006). Hot water treatment (HWT) in combination with fungicide is usually avoided due to strict requirements by importing countries on pesticide residues. HWT is widely used and currently the cheapest postharvest protocol available in the Philippines for export of ‘Carabao’ mangoes. There were reports however that incidence of anthracnose and stem-end rot is significant in Japan (Hilas Marketing Corporation, personal communication). Likewise, fruit grown in areas with prolonged wet periods as in Mindanao incur 20% loss upon reaching Hong Kong due to anthracnose and stem end rot (Esguerra et al., 2006). With the very strict phyto-sanitary requirement of importing countries, particularly Japan, revisiting HWT is a serious concern to sustain the marketability of ‘Carabao’ mangoes. The high incidences of postharvest diseases reported in Japan and Hong Kong may occur in other countries importing ‘Carabao’ mangoes with HWT as postharvest protocol. The present study investigated the inadequacy of HWT in managing anthracnose and stem-end rot. We examined the effect of HWT on the
D.G. Alvindia, M.A. Acda / Crop Protection 67 (2015) 96e101
cultures of anthracnose and stem-end rot-causing pathogens, and on the overall quality of fruit. The result of the present investigation would provide benchmark information to improve the performance of HWT in controlling postharvest diseases of ‘Carabao’ mangoes. 2. Material and methods 2.1. The fruit used Naturally infected mango fruit aged 110e115 days after flower was carefully harvested early in the morning at an orchard in Quezon, Nueva Ecija about 149 km N of Manila. Fruit were transported to the laboratory (25 km from the orchard) immediately after harvest. The working space in the laboratory was surface sterilized with chlorox solution (5e6% sodium hypochlorite) (Golden Bat, Manila, Philippines) a day before the fruit arrived. Fruit were washed in running tap water to remove impurities and air dried for 30 min before use in the succeeding experiments. Fruit were used for the experiments within 12 h after harvest. 2.2. The test pathogens Matured naturally infected fruit, yellow in color, obtained from the local market were the sources of anthracnose- (C. gloeosporioides) and stem end rot-causing (L. theobromae) pathogens. Using a sterile scalpel, five 1-cm2 of surface tissue from each disease symptom were excised from the fruit. Three fruit were used for each disease symptom. The tissue pieces were surface sterilized with chlorox solution for 30 s, rinsed twice in sterile water for 1 min and blotted dry on sterile filter paper. Five tissue pieces per plate were placed on fresh potato dextrose agar (PDA) plates and incubated for 3e7 days at 25 C. Fungal colonies clearly developing from the fruit tissues were isolated and identified by cultural and morphological methods. The pathogenicity of C. gloeosporioides and L. theobromae was established on naturally infected green matured mangoes. 2.3. Tolerance of pathogens to hot water An electric water bath (Memmert, Germany) of 22 L capacity with usable dimension of 350 290 220 mm (l w d) was used. The effect of different temperatures (48, 50, 52, 53, 55 C) and exposures (10, 20, 30, and 50 min) were tested on conidial germination and mycelial growth of the pathogens. C. gloeosporioides was grown on PDA plates at 25 C until conidial masses were visible for harvest. Spores were gently scraped-off with a sterile spatula and washed-off from culture plates with 10 mL sterile distilled water (SDW). The spore suspension was decanted and filtered through four layers of sterile cheesecloth to remove mycelium and agar pieces. The resulting filtrate was suspended in 100 mL SDW in a beaker and adjusted with a haemacytometer (Hausser, Horsham, PA, USA) to give a final count of 106 spores m L1. Ten milliliters of spore suspension was transferred separately in six (18 150 mm) sterile glass test tubes and dipped in hot water at the exposure times described above. Test tubes were hydro-cooled for 10 min and incubated at 25 C for 48 h, after which 2 mL of spore suspension from each test tube was separately poured in six 9 cm sterile watch glasses for microscopic examination. Five hundred spores were counted for each watch glass and the percent germination was estimated by dividing the number of germinated spores by the total number of spores counted. Percentage reduction in spore germination was evaluated by the difference of the spore germination of the control and the spore germination of the treatment divided by the spore
97
germination of the control multiplied by 100. The experiment was repeated once. Conidial germination test for L. theobromae was not done due to its poor sporulation on PDA. Mycelial discs (4 mm) from a 5-day-old pathogen in a PDA dish were transferred aseptically into a test tube with 15 mL SDW. The test tube was dipped in pre-heated water bath at the exposure times described above. After hot water dip, three mycelial discs were seeded equidistantly onto fresh PDA plates. Mycelial growth was measured after 7 days of incubation at 25 C. Mycelium discs seeded onto fresh PDA plates without exposure to hot water served as a control. Six plates were provided for each treatment and the experiment was repeated once. Percentage reduction in colony diameter was estimated by the difference in colony diameter of the control and the treatment divided by the colony diameter of the control, multiplied by 100. 2.4. Tolerance of fruit to hot water Naturally infected matured green fruit were placed in plastic mesh bags to avoid touching the walls of the water bath and for ease in handling during treatment. Each treatment used 20 fruit with exposure times as defined in Section 2.3. Thereafter, fruit were hydro-cooled for 10 min and air dried for 30 min prior to storage in an air conditioned room (18e20 C). Fruit were inspected for severe scalds (SS), localized light brown scalds (LLBS), and shriveling (SH) right after treatment, 7 days, and 14 days after treatment. The experiment was repeated once. 2.5. Pathogenicity of pathogens exposed to HWT Spore suspension and mycelium discs of C. gloeosporioides were exposed in hot water (53 C, 20 min) to examine capabilities to cause rotting on matured green naturally infected fruit. Fruit were dipped in chlorox solution for 5 s and immediately rinsed in SDW three times. One side of a fruit was marked with three squares (1 1 cm) well-spaced from each other with a black waterproof pen. Each square was inoculated with a 5-day-old mycelium disc (4 mm) or 0.5 mL spore suspension (106 spores mL1). The control fruit were inoculated with pathogen inocula without dipping in hot water. Fruit were kept in transparent plastic boxes lined with moist sterile paper towels in an air conditioned room and inspected after 7 days for development of disease symptons. Each treatment was replicated in three fruit and the experiment was repeated once. 2.6. Postharvest application An electric water bath, described in 2.3, was preheated before dipping the matured green naturally infected fruit at 53 C for 20 min. Immediately thereafter, fruit were hydro-cooled for 10 min, air dried for 30 min, and placed in a carton box lined with perforated plastic sheets. The box was kept in an air conditioned room for 14 days. Fruit immersed for 10 min in Mancozeb solution (2.5 g/ L of tap water) or SDW served as control checks. The experiment was repeated once with 20 fruit per treatment. 2.7. Assessment of fruit quality Weight loss (WL), fruit hardness (FH), pH, total soluble solid (TSS), pulp firmness (PF), titratable acidity (TA), color (b* value), and incidence of postharvest diseases were evaluated 14 days after treatment. For WL, the fruit were weighed on a Mettler Toledo New Classic ML Precision Balance (Cole Parmer, Vernon Hills, IL, USA) at the start and end of the experiment. WL was determined using the formula (WiWf)/Wi 100 ¼ %WL, where Wi represents the initial mass and Wf the final mass. PF was determined by measuring the
98
D.G. Alvindia, M.A. Acda / Crop Protection 67 (2015) 96e101
maximum force required to penetrate 10 mm into the fruit, using a texture analyzer (EZ-Test, Shimadzu, MD, USA) equipped with a 2 mm diam cylindrical penetrometer moving at 10 mm/min speed. Fruit hardness was measured in kg m/s. Color (b* value) was evaluated using a portable colorimeter BYK Gardner (MT, USA). Positive b* denotes yellowness while negative b* indicates blueness of the fruit. pH was measured by an Omega PHH224 Meter with pH TDS probe (Stamford, CT, USA). TSS content was determined by the index of refraction using a refractometer (Atago, WA, USA), and is referred to as degrees Brix. Brix is the percentage of sucrose (sugar) in the solution (60 Brix is equivalent to a sugar content of 60%). Titration was done with an alkaline solution-sodium hydroxide and the value represents the g of citric acid/100 mL. Ten readings were randomly taken for each fruit. Severity of anthracnose and stem end-rot in mango was evaluated using indexes described in Figs. 1 and 2. 2.8. Mycoflora of hot water treated fruit Mycoflora of matured green naturally infected fruit exposed to HWT (53 C, 20 min) was assessed after 14 days. Using a sterile scalpel, ten tissue squares (1 cm2) were randomly taken from the fruit skin. Tissue pieces were surface sterilized with chlorox solution for 30 s and rinsed three times in SDW. Five tissue pieces were planted on each of three PDA plates and incubated for 3e7 days at 25 C. Fungal colonies developing from the tissues were isolated and identified.
3. Results 3.1. Tolerance of pathogens to hot water Water temperature and dipping time considerably influenced mycelium development of pathogens (Table 1). L. theobromae was more sensitive to higher temperature and longer exposure than C. gloeosporioides. Mycelium growths of both pathogens were partially inhibited by HWT regardless of trial, temperature and dipping time. The maximum reduction in radial growth of C. gloeosporioides after 7 days was 79.33% at 52 C, 50 min whilst the highest decrease in mycelium development of L. theobromae was 90.5% at 53 C, 50 min. Reduction in spore germination of C. gloeosporioides after HWT is summarized in Table 2. Similarly, spore germination was not totally inhibited even with the highest temperature and exposure tested. Nevertheless spore germination significantly decreased as temperature increased. Analysis of variance (ANOVA) showed that temperature significantly (P ¼ 0.05) influenced the radial growth of C. gloeosporioides while trial and exposure had insignificant effect. The interaction effect of trial, temperature and exposure on colony diameter was insignificant, too. Meanwhile, temperature and exposure or their interactions considerably influenced the mycelium growth of L. theobromae. The interaction effect of trial, temperature and exposure was not significant on colony development of L. theobromae. Meanwhile, temperature and exposure or their combinations had significant effect on spore germination of C. gloeosporioides.
2.9. Statistical analysis 3.2. Tolerance of fruit to hot water All data were subjected to analysis of variance (ANOVA) and the significant differences among treatments were separated by Tukey's test at P ¼ 0.05 using SPSS Statistics version 16.0 for Windows (IBM, Armonk, NY, USA).
HWT at 48e50 C, 10e50 min and 52 C, 10e20 min was not injurious to ‘Carabao’ mangoes after 14 days. Fruit dipped at 52 C for 30e50 min had no injury immediately after treatment but LLBS
Fig. 1. Severity index of anthracnose of mango on a scale from 0 to 5, where 0 ¼ no visible spots, 1 ¼ one depressed spot, dark in color with 1e5 mm in diameter on the epidermis of the fruit, 2 ¼ 2e3 depressed spots, dark in color with 1e5 mm in diameter, 3 ¼ 2e3 depressed spots, dark in color with more than 5 mm in diameter, 4 ¼ more than 3 depressed spots, dark in color with more than 5 mm in diameter, 5 ¼ depressed spots merged.
D.G. Alvindia, M.A. Acda / Crop Protection 67 (2015) 96e101
99
Fig. 2. Severity index of stem-end rot of mango on a scale from 0 to 5, where 0 ¼ no discoloration of the stem-end, 1 ¼ discoloration limited at the stem-end, 2 ¼ 10% discoloration of the fruit surface area initiated by stem-end rot, 3 ¼ 11e30% discoloration of the fruit surface area initiated by stem-end rot, 4 ¼ 31e50% discoloration of the fruit surface area initiated by stem-end rot, 5 ¼ more than 51% discoloration of the fruit surface area initiated by stem-end rot.
were noticed after 7 days (Table 3). Hot water dips at 53 C for 10e20 min was not deleterious to mangoes but more than 20 min would cause LBS after 14 days. HWT at 55 C for 10e50 min yielded fruit with LLBS, SS and SH after 7 days. The optimum exposure for ‘Carabao’ mango was 53 C, 20 min. 3.3. Pathogenicity of pathogens after HWT C. gloeosporioides formed chocolate brown and depressed spots on fruit, while L. theobromae formed brown to black lesions of irregular shape after 7 days of artificial inoculation. C. gloeosporioides and L. theobromae were isolated from their respective lesions.
3.4. Effects of treatments on quality changes of fruit Effect of treatments on quality changes in mangoes after 14 days is presented in Table 4. Loss in mass in hot water dipped fruit was higher (14.38%e15.74%) as compared to those treated with fungicide (12.95%e14.17%) and distilled water (13.16%e14.16%). Yet the difference in the loss in mass among the treatments was insignificant. Similarly, fruit hardness, pH, TSS, TA, and color were not significantly affected by the treatments. Conversely, the severity of fruit decay due to anthracnose and stem-end rot was significantly affected by the treatments. The severity of anthracnose (0.90e1.10) and stem-end rot (0.50e0.60) after 14 days was least on fruit treated with the fungicide. This was followed by hot water dipped
Table 1 Reduction (%) in colony growth of Colletotrichum gloeosporioides and Lasiodiplodia theobromae 7 days after dipping in hot water at various temperatures and exposure times. Water temp ( C)
% Reduction in colony diameter after 7 days (n ¼ 6)a 10 min Trial 1
Colletotrichum gloeosporioides 48 52.67 50 68.50 52 75.00 53 77.67 55 75.33 Lasiodiplodia theobromae 48 37.83 50 71.83 52 74.67 53 82.17 55 81.67 a
20 min
30 min
50 min
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
a b b b b
54.67 76.00 75.17 70.17 72.67
ab b ab ab ab
68.50 75.17 74.67 70.00 74.67
a a a a a
68.00 71.33 73.83 76.33 77.67
a a a a a
69.67 73.17 74.00 76.17 75.17
a a a a a
67.50 69.83 71.50 69.83 71.17
a a a a a
74.17 74.00 74.83 75.50 76.00
a a a a a
73.17 75.33 79.33 73.50 73.00
a a a a a
a b b b b
37.00 70.67 76.83 81.83 79.67
a b b b b
36.17 76.50 75.50 84.83 81.33
a b b b b
36.17 76.00 75.17 80.00 82.67
a b b b b
30.83a 68.00 b 84.17 c 87.50 c 90.00 c
34.17 69.83 90.17 72.00 81.17
a b c c c
44.17 74.00 85.67 90.50 87.67
a b c c c
42.83 75.33 83.50 78.00 84.67
a b b b b
Means within a column followed by the same letter are not significantly different at P ¼ 0.05 according to Tukey's test.
100
D.G. Alvindia, M.A. Acda / Crop Protection 67 (2015) 96e101
Table 2 Reduction (%) in spore germination of Colletotrichum gloeosporioides after 48 h of dipping in hot water at various temperatures and exposure times. Water temp ( C)
% Reduction in spore germination (n ¼ 6)a 10 min
48 50 52 53 55 a
20 min
30 min
50 min
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
18.33 52.83 70.00 63.83 67.00
20.00 47.83 73.33 64.67 71.17
50.83 57.50 69.33 71.83 72.83
57.50 55.00 72.67 74.67 68.00
55.83 54.17 67.50 72.33 74.00
56.67 56.33 60.83 74.33 75.17
53.33 69.50 76.50 71.33 76.33
55.83 73.17 75.17 75.50 76.83
a b c bc c
a b c c c
a bc bc c c
bc a bc c bc
ab a abc bc c
a a a b b
a b b b b
a b b b b
Means within a column followed by the same letter are not significantly different at P ¼ 0.05 according to Tukey's test.
Table 3 Effect of hot water dips on the physical appearance of mango cv. ‘Carabao’ with various temperatures, exposure times and time after treatment.a Temp ( C)
After treatment 10
20
30
50
10
20
30
50
10
20
30
50
48 50 52 53 55
CC CC CC CC CC
CC CC CC CC CC
CC CC CC CC CC
CC CC CC LBS SS
CC CC CC CC SH
CC CC CC CC SH
CC CC LBS LBS SS
CC CC LBS LBS LBS
CC CC CC CC SH
CC CC CC CC SH
CC CC LBS LBS SS
CC CC LBS LBS LBS
After 7 d
After 14 d
a SS-severe scalds, LBS-light brown scalds, SH-shriveling, CC-comparable with the control.
fruit with anthracnose severity index of 1.80e2.0 and 0.90‒1.20 for stem-end rot. Fruit receiving distilled water (untreated control) had the highest indexes of fruit rot attributable to anthracnose (3.80e3.90) and stem-end rot (2.30e2.50). The efficiency of postharvest treatments, calculated as the % reduction in severity of anthracnose or stem-end rot by a treatment as compared to the untreated control, showed that fungicide controlled anthracnose by 71.79%e79.31% and stem-end rot by 73.91%e80%. HWT inhibited anthracnose by 48.71%e52.63% and stem-end rot by 48%e60.86%.
3.5. Mycoflora of fruit after storage Fusarium verticillioides, Penicillium spp., Aspergillus spp., Pestalotiopsis spp., L. theobromae, C. gloeosporioides and Acremonium spp. were isolated on fruit tissues after 14 days. F. verticillioides, the dominant fungus, was present in almost all tissues analyzed. C. gloeosporioides and L. theobromae were particularly present on epidermis with symptoms of anthracnose and stem-end rot, respectively. The fungal population of each fungus, however, was not determined.
4. Discussion Most overseas markets are no longer permitting chemical treatments, such as fungicidal dips, for fruit entering their countries (Jacobi and Giles, 1997). HWT is a widely used postharvest treatment for export of mangoes as it does not leave chemical residues. HWT of mangoes for disease control involves a 10 min dip at 52e55 C (Quimio and Quimio, 1974; Lizada et al., 1986; Buganic et al., 1997). We were prompted to revisit HWT, however, due to the significant amount of decay in ‘Carabao’ caused by anthracnose and stem end-rot in export markets. The severity of anthracnose and stem-end rot in ‘Carabao’ mangoes was reduced by 83% and 100%, respectively by hot water dips at 52e55 C for 10 min (Buganic et al., 1997). Yet the high degree of postharvest disease control in this report may not be attributed exclusively to HWT as cultural management was involved. Fruit were wrapped with paper two months after flower induction until harvest. This practice may have protected the developing fruit from attack by pathogens present in the canopy of mango trees at all times, hence, fruit may have limited latent infection at harvest. Fungal populations established during fruit development are responsible for postharvest diseases such as anthracnose and stem-end rot (Prusky et al., 1983). Obviously, cultural management such as fruit bagging should be implemented as the extent of disease control could be influenced by the degree of disease pressure during production. Furthermore, HWT at 52e55 C for 10 min did not completely inhibit fungal inoculum which may develop progressively overtime to decay the fruit as shown in the present study. Fruit wrapping is an essential component of good agricultural practice for ‘Carabao’ mango particularly for fruit intended for the export market. The high cost of labor and materials to be used have limited the ability of some growers to implement fruit bagging. During times of high demand and limited supply of ‘Carabao’ mangoes, however, some unwrapped fruit are exported. Exporters should strictly implement a ‘no wrapping no export’ policy.
Table 4 Effect of various treatments on fruit quality after 14 days of storage at 18e20 C.a Treatment
Weight loss (%)
Hardness (N)
pH
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
HWT (53 C, 20 min) Mancozeb (2.5 g/L) Untreated (Distilled water)
14.38 a 12.95 a 13.16 a
15.74 a 14.17 a 14.16 a
4.24 a 3.24 bc 3.67 ab
3.38 a 3.72 a 3.58 a
6.47 a 6.45 a 6.52 a
6.20 ab 6.63 b 6.44 a
5.70 a 6.92 b 6.82 b
6.68 a 8.40 a 7.20 a
1.50 a 1.46 a 2.09 a
a b c d
TSS
Color (b* value)b
Anthracnosec
Stem-end rotd
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
1.54 a 1.85 a 2.20 a
45.34 a 53.63 b 52.48 b
51.92 a 51.43 a 50.64 a
2.00 ab 1.10 a 3.90 b
1.80 a 0.90 a 3.80 b
1.30 a 0.50 a 2.50 b
0.90 a 0.60 a 2.30 b
TA
Means within a column followed by the same letter are not significantly different at P ¼ 0.05 according to Tukey's test. Negative b* indicates blueness while positive b* denotes yellowness of the fruit. See Fig. 1 for visual characterization of anthracnose severity disease index. See Fig. 2 for visual characterization of stem-end rot severity disease index.
D.G. Alvindia, M.A. Acda / Crop Protection 67 (2015) 96e101
We showed that 53 C for 20 min was the optimum exposure for ‘Carabao’ mangoes and the best combination in controlling culture growth of pathogens. In vivo application at this exposure, however, did not yield significant reduction in anthracnose (48.71%e52.63%) and stem-end rot (48%e60.86%) after 14 days. The present study demonstrated that even the optimum exposure for ‘Carabao’ mangoes was inadequate in giving a high degree of postharvest disease control. A considerable level of latent infection may have been present on fruit surfaces as unwrapped fruit were used in our experiments. The poor efficiency of HWT could be attributed to its fungistatic action leaving active propagules after treatment which develop overtime and cause decay on fruit. Moreover, the inconsistent performance seen, for example variation in disease severity within a treatment, has to be addressed. Severity indexes of anthracnose and stem-end rot showed standard deviations ranging from ±1.05 to ±2.98, remarkably higher than those treated with fungicide (±0.69 to ±0.99). The volatility of HWT in practical use agreed with the in vitro result of the present study i.e., high variation in radial growth and spore germination of C. gloeosporioides and L. theobromae. A correct setting of water temperature is important as fruit absorb heat. For example, a 22 L water bath preheated at 80 C fell to 51.6 C with 20 fruit immersed. The temperature increased to 53 C after 3e5 min. This scenario would significantly affect the performance of HWT to destroy latent infections since the desired exposure is not achieved. To address the problem, the personnel in charge must be knowledgeable in setting temperature ranges to attain the correct exposure. Upgrading water baths with a precise heating and control system would also help address the problem. Recycling of water should be considered as fresh water is becoming limited and this treatment will have an economic impact in the future. HWT at 53 C for 20 min had no negative effect on the overall quality of fruit as fruit firmness, TSS, TA, color, and weight loss were statistically comparable with the untreated controls after 14 days. In ordinary conditions, firmness of healthy ‘Carabao’ mangoes decreases as the fruit ripen which could be attributed to the activity of enzymes that degrade pectic substances (Pantastico et al., 1984). Yet heat treatment inhibits solubilization of the carbonate soluble pectin fraction which is one of the main factors contributing to firmness retention (Lizada, 1991). Fruit firmness is one of the most widely used indicators of fruit quality. The HWT technology is a more cost effective alternative to vapor heat treatment and has added benefits such as ease of use, less time consuming, reliable, and it gives fruit surface sanitation to exclude plant debris and disease control (Jacobi et al., 2001). For the time being, HWT will continue as a popular postharvest protocol for ‘Carabao’ mangoes in response to export requirements imposed specifically by Japan. Conversely, HWT efficiency has to be improved as an effective quarantine procedure in preventing anthracnose and stem end-rot on harvested mangoes. Biological control agents (BCA) and/or generally regarded as safe (GRAS) compounds may be integrated with HWT. Such a temperature and exposure time as in this study could, however, retard the development of the pathogen in the fruit, and thus create a vacuum that could be exploited by BCA and/or GRAS. Naturally occurring antagonistic bacteria or fungi and application of GRAS compounds
101
such as sodium carbonate, sodium bicarbonate and sodium hypochlorite reduced postharvest diseases on fruit (Alvindia et al., 2004; Alvindia and Natsuaki, 2007, 2008, 2009).
References Alvindia, D.G., Kobayashi, T., Natsuaki, K.T., Tanda, S., 2004. Inhibitory influence of inorganic salts on banana postharvest pathogens: preliminary application to control crown rot. J. Gen. Plant Pathol. 70, 61e65. Alvindia, D.G., Natsuaki, K.T., 2007. Control of crown rot-causing fungal pathogens of banana by inorganic salts and a surfactant. Crop Prot. 26, 1667e1673. Alvindia, D.G., Natsuaki, K.T., 2008. Evaluation of fungal epiphytes isolated from banana fruit surfaces for biocontrol of banana crown rot disease. Crop Prot. 27, 1200e1207. Alvindia, D.G., Natsuaki, K.T., 2009. Biocontrol activities of Bacillus amyloliquefaciens DGA14 isolated from banana fruit surface against banana crown rot-causing fungi. Crop Prot. 28, 236e242. Arauz, L.F., 2000. Mango anthracnose: economic impact and current options for integrated management. Plant Dis. 84, 600e611. Aveno, J.L., Orden, M.M., 2008. Hot Water Treatment of Mango: a Study of Four Export Corporations. Accessed from. http://www.docstoc.com/docs/2388169/ HOT-WATER TREATMENTeOFeMANGO-A-STUDYeOFeFOUR-EXPORTCORPORATIONS (Date downloaded 05.11.13.). Buganic Jr., R.D., Lizada, M.C.C., De Ramos, M.B., 1997. Disease control in Philippine 'Carabao' mango with preharvest bagging and postharvest hot water treatment. Acta Hortic. 455, 797e804. Dodd, J.C., Prusky, D., Jeffries, P., 1997. Fruit diseases. In: Litz, R.E. (Ed.), The Mango: Botany, Production and Uses. CAB Int., Wallingford, pp. 257e280. Esguerra, E.B., Chavez, S.M., Traya, R.V., 2006. A modified and rapid heat treatment for the control of postharvest diseases of mango (Mangifera indica Linn. cv. Carabao) fruits. Philipp. Agric. Sci. 89, 125e133. Fitzel, R.D., Peak, C.M., 1984. The epidemiology of anthracnose disease of mango: inoculum sources, spore production and dispersal. Ann. Appl. Biol. 104, 53e59. Jacobi, K.K., Giles, J.E., 1997. Quality of 'Kensington' mango (Mangifera indica Linn.) fruit following combined vapour heat disinfestation and hot water disease control treatments. Postharvest Biol. Technol. 12, 285e292. Jacobi, K.K., MacRae, E.A., Hetherington, S.E., 2001. Postharvest heat disinfestation treatments of mango fruit. Sci. Hortic. 89, 171e193. Jeffries, P., Dodd, J.C., Jeger, M.J., Plumbley, R.A., 1990. The biology of Colletotrichum species on tropical fruit and vegetables. Plant Pathol. 39, 343e366. Johnson, G.I., Hofman, P.J., 2009. Postharvest technology and quarantine treatments. In: Litz, R.E. (Ed.), The Mango: Botany, Production and Uses. CAB Int., Wallingford, pp. 528e605. Kobiler, I., Shalom, Y., Roth, I., Akerman, M., Vinokur, Y., Fuchs, Y., Prusky, D., 2001. Effect of 2,4-dichlorophenoxy acetic acid on the incidence of side and stem end rots in mango fruits. Postharvest Biol. Technol. 23, 23e32. Lizada, M.C., 1991. Post-harvest physiology of the mango e a review. Acta Hortic. 291, 437e453. Lizada, M.C.C., Agravante, J.U., Brown, E.O., 1986. Factors affecting postharvest disease control in ‘Carabao’ mango subjected to hot water treatment. Philipp. J. Crop Sci. 11, 153e161. Mascarenhas, P., Behere, A., Sharma, A., Padwal-desa, S.R., 1995. Postharvest spoilage of mango (Mangifera indica) by Botryodiplodia theobromae. Mycol. Res. 100, 27e30. Nuevo, P.A., Apaga, A.R.M., 2010. Technology reducing postharvest losses and maintain quality of fruits and vegetables (Philippines). In: Huang, C.C., Yang, C.M., Ou, S.K., Chen, J.J. (Eds.), Technology Reducing Postharvest Losses and Maintain Quality of Fruits and Vegetables, Taiwan Agr. Res. Inst., Spec. Publ. No., vol. 147, pp. 154e167. Pantastico, E.B., Lam, P.F., Ketsa, S., Yuniarti, Kosittrakul, M., 1984. Postharvest physiology and storage of mango. In: Mendoza Jr., D.B., Wills, R.B.H. (Eds.), Mango: Fruit Development, Postharvest Physiology and Marketing in ASEAN. Association of South East Asian Nations (ASEAN) Food Handling Bureau, pp. 39e52. Prusky, D., Falik, E., Kobiler, I., Fuchs, Y., Zauberman, G., Pesis, E., Roth, I., Weksler, A., Akerman, M., Ykutiety, O., Waisblum, A., Keinan, A., Ofek, G., Lavi, U., 1997. Hot water bruch anem method for control of postharvest diseases caused by Alternaria rot in mango fruits. Acta Hortic. 455, 780e785. Prusky, D., Fuchs, Y., Yanko, U., 1983. Assessment of latent infections as a basis for control of post-harvest disease of mango. Plant Dis. 67, 816e818. Quimio, A.J., Quimio, T.H., 1974. Postharvest control of Philippine mango anthracnose by hot water treatment. Philipp. Agric. 58, 138e146.
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Revisiting the efficacy of hot water treatment in managing anthracnose and stem-end rot diseases of mango cv. ‘Carabao’ Dionisio G. Alvindia a, b, *, Miriam A. Acda a a
Philippine Center for Postharvest Development and Mechanization (PhilMech), Central Luzon State University (CLSU), Compound 3120, ~ oz, Nueva Ecija, Philippines Science City of Mun b ~ oz, Nueva Ecija, Philippines Department of Biological Sciences, College of Arts and Sciences, CLSU, 3120, Science City of Mun
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 June 2014 Received in revised form 22 September 2014 Accepted 23 September 2014 Available online
This study was conducted to revalidate the efficacy of hot water treatment (HWT) as a standard protocol in managing postharvest disease in 'Carabao' mangoes. We elucidated the possible reasons for the inadequacy of HWT in management of anthracnose and stem-end rot. The effect of HWT on the cultures of anthracnose and stem-end rot-causing pathogens and on the overall quality of fruit was examined. The present investigation suggested 53 C for 20 min as optimal exposure for 'Carabao' mango. At this exposure however, the propagules of Colletotrichum gloeosporioides and Lasiodiplodia theobromae were not totally controlled. HWT was unstable in controlling C. gloeosporioides and L. theobromae as demonstrated by high standard deviation of radial growth. HWT manner of control is fungistatic rather than fungicidal as pathogens developed after treatment. Fungistatic activity of HWT was perhaps inadequate to protect the fruit from decay due to absence of residual action. There were no significant changes in the quality of 'Carabao' mangoes submerged in hot water at 53 C for 20 min whilst the severity of anthracnose was reduced by 48.71%e52.63% and stem-end rot by 48%e60.86%. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Mangifera indica Decay Heat treatment Fruit firmness Fruit color
1. Introduction Mango (Mangifera indica Linn.) is one of the most important fruit crops grown in the Philippines. ‘Carabao’ as the main variety, mango production during the first quarter of 2014 was 160,940 MT (http://www.bas.gov.ph/?ids¼fruitssituation). A significant amount however, is wasted, estimated at 2e33% because of fruit drop cracking, immaturity, and postharvest decay, mainly due to anthracnose and stem end rot (Nuevo and Apaga, 2010). Anthracnose is caused by Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (Fitzel and Peak, 1984; Jeffries et al., 1990; Dodd et al., 1997; Arauz, 2000) while stem-end rot is caused by Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (Prusky et al., 1997; Kobiler et al., 2001; Mascarenhas et al., 1995). Infection of fruit occurs during production and postharvest operations (Johnson and Hofman, 2009) and compromises storage life of the fruit (Dodd et al., 1997; Prusky et al., 1997). ‘Carabao’ is mainly traded as fresh produce. About 60e70% is exported to Japan while the remaining portion is shipped to Hong * Corresponding author. Philippine Center for Postharvest Development and Mechanization (PhilMech), Central Luzon State University (CLSU), Compound 3120, ~ oz, Nueva Ecija, Philippines. Tel.: þ63 44 4560 213. Science City of Mun E-mail address: [email protected] (D.G. Alvindia). http://dx.doi.org/10.1016/j.cropro.2014.09.016 0261-2194/© 2014 Elsevier Ltd. All rights reserved.
Kong, Singapore, Korea, China, US, Australia, Europe and New Zealand (Aveno and Orden, 2008). As postharvest protocol for export, mangoes are dipped in hot water at 52e55 C for 10 min to manage postharvest diseases (Buganic et al., 1997) with 10 min hydro-cooling, and 30 min drying prior to packing (Esguerra et al., 2006). Hot water treatment (HWT) in combination with fungicide is usually avoided due to strict requirements by importing countries on pesticide residues. HWT is widely used and currently the cheapest postharvest protocol available in the Philippines for export of ‘Carabao’ mangoes. There were reports however that incidence of anthracnose and stem-end rot is significant in Japan (Hilas Marketing Corporation, personal communication). Likewise, fruit grown in areas with prolonged wet periods as in Mindanao incur 20% loss upon reaching Hong Kong due to anthracnose and stem end rot (Esguerra et al., 2006). With the very strict phyto-sanitary requirement of importing countries, particularly Japan, revisiting HWT is a serious concern to sustain the marketability of ‘Carabao’ mangoes. The high incidences of postharvest diseases reported in Japan and Hong Kong may occur in other countries importing ‘Carabao’ mangoes with HWT as postharvest protocol. The present study investigated the inadequacy of HWT in managing anthracnose and stem-end rot. We examined the effect of HWT on the
D.G. Alvindia, M.A. Acda / Crop Protection 67 (2015) 96e101
cultures of anthracnose and stem-end rot-causing pathogens, and on the overall quality of fruit. The result of the present investigation would provide benchmark information to improve the performance of HWT in controlling postharvest diseases of ‘Carabao’ mangoes. 2. Material and methods 2.1. The fruit used Naturally infected mango fruit aged 110e115 days after flower was carefully harvested early in the morning at an orchard in Quezon, Nueva Ecija about 149 km N of Manila. Fruit were transported to the laboratory (25 km from the orchard) immediately after harvest. The working space in the laboratory was surface sterilized with chlorox solution (5e6% sodium hypochlorite) (Golden Bat, Manila, Philippines) a day before the fruit arrived. Fruit were washed in running tap water to remove impurities and air dried for 30 min before use in the succeeding experiments. Fruit were used for the experiments within 12 h after harvest. 2.2. The test pathogens Matured naturally infected fruit, yellow in color, obtained from the local market were the sources of anthracnose- (C. gloeosporioides) and stem end rot-causing (L. theobromae) pathogens. Using a sterile scalpel, five 1-cm2 of surface tissue from each disease symptom were excised from the fruit. Three fruit were used for each disease symptom. The tissue pieces were surface sterilized with chlorox solution for 30 s, rinsed twice in sterile water for 1 min and blotted dry on sterile filter paper. Five tissue pieces per plate were placed on fresh potato dextrose agar (PDA) plates and incubated for 3e7 days at 25 C. Fungal colonies clearly developing from the fruit tissues were isolated and identified by cultural and morphological methods. The pathogenicity of C. gloeosporioides and L. theobromae was established on naturally infected green matured mangoes. 2.3. Tolerance of pathogens to hot water An electric water bath (Memmert, Germany) of 22 L capacity with usable dimension of 350 290 220 mm (l w d) was used. The effect of different temperatures (48, 50, 52, 53, 55 C) and exposures (10, 20, 30, and 50 min) were tested on conidial germination and mycelial growth of the pathogens. C. gloeosporioides was grown on PDA plates at 25 C until conidial masses were visible for harvest. Spores were gently scraped-off with a sterile spatula and washed-off from culture plates with 10 mL sterile distilled water (SDW). The spore suspension was decanted and filtered through four layers of sterile cheesecloth to remove mycelium and agar pieces. The resulting filtrate was suspended in 100 mL SDW in a beaker and adjusted with a haemacytometer (Hausser, Horsham, PA, USA) to give a final count of 106 spores m L1. Ten milliliters of spore suspension was transferred separately in six (18 150 mm) sterile glass test tubes and dipped in hot water at the exposure times described above. Test tubes were hydro-cooled for 10 min and incubated at 25 C for 48 h, after which 2 mL of spore suspension from each test tube was separately poured in six 9 cm sterile watch glasses for microscopic examination. Five hundred spores were counted for each watch glass and the percent germination was estimated by dividing the number of germinated spores by the total number of spores counted. Percentage reduction in spore germination was evaluated by the difference of the spore germination of the control and the spore germination of the treatment divided by the spore
97
germination of the control multiplied by 100. The experiment was repeated once. Conidial germination test for L. theobromae was not done due to its poor sporulation on PDA. Mycelial discs (4 mm) from a 5-day-old pathogen in a PDA dish were transferred aseptically into a test tube with 15 mL SDW. The test tube was dipped in pre-heated water bath at the exposure times described above. After hot water dip, three mycelial discs were seeded equidistantly onto fresh PDA plates. Mycelial growth was measured after 7 days of incubation at 25 C. Mycelium discs seeded onto fresh PDA plates without exposure to hot water served as a control. Six plates were provided for each treatment and the experiment was repeated once. Percentage reduction in colony diameter was estimated by the difference in colony diameter of the control and the treatment divided by the colony diameter of the control, multiplied by 100. 2.4. Tolerance of fruit to hot water Naturally infected matured green fruit were placed in plastic mesh bags to avoid touching the walls of the water bath and for ease in handling during treatment. Each treatment used 20 fruit with exposure times as defined in Section 2.3. Thereafter, fruit were hydro-cooled for 10 min and air dried for 30 min prior to storage in an air conditioned room (18e20 C). Fruit were inspected for severe scalds (SS), localized light brown scalds (LLBS), and shriveling (SH) right after treatment, 7 days, and 14 days after treatment. The experiment was repeated once. 2.5. Pathogenicity of pathogens exposed to HWT Spore suspension and mycelium discs of C. gloeosporioides were exposed in hot water (53 C, 20 min) to examine capabilities to cause rotting on matured green naturally infected fruit. Fruit were dipped in chlorox solution for 5 s and immediately rinsed in SDW three times. One side of a fruit was marked with three squares (1 1 cm) well-spaced from each other with a black waterproof pen. Each square was inoculated with a 5-day-old mycelium disc (4 mm) or 0.5 mL spore suspension (106 spores mL1). The control fruit were inoculated with pathogen inocula without dipping in hot water. Fruit were kept in transparent plastic boxes lined with moist sterile paper towels in an air conditioned room and inspected after 7 days for development of disease symptons. Each treatment was replicated in three fruit and the experiment was repeated once. 2.6. Postharvest application An electric water bath, described in 2.3, was preheated before dipping the matured green naturally infected fruit at 53 C for 20 min. Immediately thereafter, fruit were hydro-cooled for 10 min, air dried for 30 min, and placed in a carton box lined with perforated plastic sheets. The box was kept in an air conditioned room for 14 days. Fruit immersed for 10 min in Mancozeb solution (2.5 g/ L of tap water) or SDW served as control checks. The experiment was repeated once with 20 fruit per treatment. 2.7. Assessment of fruit quality Weight loss (WL), fruit hardness (FH), pH, total soluble solid (TSS), pulp firmness (PF), titratable acidity (TA), color (b* value), and incidence of postharvest diseases were evaluated 14 days after treatment. For WL, the fruit were weighed on a Mettler Toledo New Classic ML Precision Balance (Cole Parmer, Vernon Hills, IL, USA) at the start and end of the experiment. WL was determined using the formula (WiWf)/Wi 100 ¼ %WL, where Wi represents the initial mass and Wf the final mass. PF was determined by measuring the
98
D.G. Alvindia, M.A. Acda / Crop Protection 67 (2015) 96e101
maximum force required to penetrate 10 mm into the fruit, using a texture analyzer (EZ-Test, Shimadzu, MD, USA) equipped with a 2 mm diam cylindrical penetrometer moving at 10 mm/min speed. Fruit hardness was measured in kg m/s. Color (b* value) was evaluated using a portable colorimeter BYK Gardner (MT, USA). Positive b* denotes yellowness while negative b* indicates blueness of the fruit. pH was measured by an Omega PHH224 Meter with pH TDS probe (Stamford, CT, USA). TSS content was determined by the index of refraction using a refractometer (Atago, WA, USA), and is referred to as degrees Brix. Brix is the percentage of sucrose (sugar) in the solution (60 Brix is equivalent to a sugar content of 60%). Titration was done with an alkaline solution-sodium hydroxide and the value represents the g of citric acid/100 mL. Ten readings were randomly taken for each fruit. Severity of anthracnose and stem end-rot in mango was evaluated using indexes described in Figs. 1 and 2. 2.8. Mycoflora of hot water treated fruit Mycoflora of matured green naturally infected fruit exposed to HWT (53 C, 20 min) was assessed after 14 days. Using a sterile scalpel, ten tissue squares (1 cm2) were randomly taken from the fruit skin. Tissue pieces were surface sterilized with chlorox solution for 30 s and rinsed three times in SDW. Five tissue pieces were planted on each of three PDA plates and incubated for 3e7 days at 25 C. Fungal colonies developing from the tissues were isolated and identified.
3. Results 3.1. Tolerance of pathogens to hot water Water temperature and dipping time considerably influenced mycelium development of pathogens (Table 1). L. theobromae was more sensitive to higher temperature and longer exposure than C. gloeosporioides. Mycelium growths of both pathogens were partially inhibited by HWT regardless of trial, temperature and dipping time. The maximum reduction in radial growth of C. gloeosporioides after 7 days was 79.33% at 52 C, 50 min whilst the highest decrease in mycelium development of L. theobromae was 90.5% at 53 C, 50 min. Reduction in spore germination of C. gloeosporioides after HWT is summarized in Table 2. Similarly, spore germination was not totally inhibited even with the highest temperature and exposure tested. Nevertheless spore germination significantly decreased as temperature increased. Analysis of variance (ANOVA) showed that temperature significantly (P ¼ 0.05) influenced the radial growth of C. gloeosporioides while trial and exposure had insignificant effect. The interaction effect of trial, temperature and exposure on colony diameter was insignificant, too. Meanwhile, temperature and exposure or their interactions considerably influenced the mycelium growth of L. theobromae. The interaction effect of trial, temperature and exposure was not significant on colony development of L. theobromae. Meanwhile, temperature and exposure or their combinations had significant effect on spore germination of C. gloeosporioides.
2.9. Statistical analysis 3.2. Tolerance of fruit to hot water All data were subjected to analysis of variance (ANOVA) and the significant differences among treatments were separated by Tukey's test at P ¼ 0.05 using SPSS Statistics version 16.0 for Windows (IBM, Armonk, NY, USA).
HWT at 48e50 C, 10e50 min and 52 C, 10e20 min was not injurious to ‘Carabao’ mangoes after 14 days. Fruit dipped at 52 C for 30e50 min had no injury immediately after treatment but LLBS
Fig. 1. Severity index of anthracnose of mango on a scale from 0 to 5, where 0 ¼ no visible spots, 1 ¼ one depressed spot, dark in color with 1e5 mm in diameter on the epidermis of the fruit, 2 ¼ 2e3 depressed spots, dark in color with 1e5 mm in diameter, 3 ¼ 2e3 depressed spots, dark in color with more than 5 mm in diameter, 4 ¼ more than 3 depressed spots, dark in color with more than 5 mm in diameter, 5 ¼ depressed spots merged.
D.G. Alvindia, M.A. Acda / Crop Protection 67 (2015) 96e101
99
Fig. 2. Severity index of stem-end rot of mango on a scale from 0 to 5, where 0 ¼ no discoloration of the stem-end, 1 ¼ discoloration limited at the stem-end, 2 ¼ 10% discoloration of the fruit surface area initiated by stem-end rot, 3 ¼ 11e30% discoloration of the fruit surface area initiated by stem-end rot, 4 ¼ 31e50% discoloration of the fruit surface area initiated by stem-end rot, 5 ¼ more than 51% discoloration of the fruit surface area initiated by stem-end rot.
were noticed after 7 days (Table 3). Hot water dips at 53 C for 10e20 min was not deleterious to mangoes but more than 20 min would cause LBS after 14 days. HWT at 55 C for 10e50 min yielded fruit with LLBS, SS and SH after 7 days. The optimum exposure for ‘Carabao’ mango was 53 C, 20 min. 3.3. Pathogenicity of pathogens after HWT C. gloeosporioides formed chocolate brown and depressed spots on fruit, while L. theobromae formed brown to black lesions of irregular shape after 7 days of artificial inoculation. C. gloeosporioides and L. theobromae were isolated from their respective lesions.
3.4. Effects of treatments on quality changes of fruit Effect of treatments on quality changes in mangoes after 14 days is presented in Table 4. Loss in mass in hot water dipped fruit was higher (14.38%e15.74%) as compared to those treated with fungicide (12.95%e14.17%) and distilled water (13.16%e14.16%). Yet the difference in the loss in mass among the treatments was insignificant. Similarly, fruit hardness, pH, TSS, TA, and color were not significantly affected by the treatments. Conversely, the severity of fruit decay due to anthracnose and stem-end rot was significantly affected by the treatments. The severity of anthracnose (0.90e1.10) and stem-end rot (0.50e0.60) after 14 days was least on fruit treated with the fungicide. This was followed by hot water dipped
Table 1 Reduction (%) in colony growth of Colletotrichum gloeosporioides and Lasiodiplodia theobromae 7 days after dipping in hot water at various temperatures and exposure times. Water temp ( C)
% Reduction in colony diameter after 7 days (n ¼ 6)a 10 min Trial 1
Colletotrichum gloeosporioides 48 52.67 50 68.50 52 75.00 53 77.67 55 75.33 Lasiodiplodia theobromae 48 37.83 50 71.83 52 74.67 53 82.17 55 81.67 a
20 min
30 min
50 min
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
a b b b b
54.67 76.00 75.17 70.17 72.67
ab b ab ab ab
68.50 75.17 74.67 70.00 74.67
a a a a a
68.00 71.33 73.83 76.33 77.67
a a a a a
69.67 73.17 74.00 76.17 75.17
a a a a a
67.50 69.83 71.50 69.83 71.17
a a a a a
74.17 74.00 74.83 75.50 76.00
a a a a a
73.17 75.33 79.33 73.50 73.00
a a a a a
a b b b b
37.00 70.67 76.83 81.83 79.67
a b b b b
36.17 76.50 75.50 84.83 81.33
a b b b b
36.17 76.00 75.17 80.00 82.67
a b b b b
30.83a 68.00 b 84.17 c 87.50 c 90.00 c
34.17 69.83 90.17 72.00 81.17
a b c c c
44.17 74.00 85.67 90.50 87.67
a b c c c
42.83 75.33 83.50 78.00 84.67
a b b b b
Means within a column followed by the same letter are not significantly different at P ¼ 0.05 according to Tukey's test.
100
D.G. Alvindia, M.A. Acda / Crop Protection 67 (2015) 96e101
Table 2 Reduction (%) in spore germination of Colletotrichum gloeosporioides after 48 h of dipping in hot water at various temperatures and exposure times. Water temp ( C)
% Reduction in spore germination (n ¼ 6)a 10 min
48 50 52 53 55 a
20 min
30 min
50 min
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
18.33 52.83 70.00 63.83 67.00
20.00 47.83 73.33 64.67 71.17
50.83 57.50 69.33 71.83 72.83
57.50 55.00 72.67 74.67 68.00
55.83 54.17 67.50 72.33 74.00
56.67 56.33 60.83 74.33 75.17
53.33 69.50 76.50 71.33 76.33
55.83 73.17 75.17 75.50 76.83
a b c bc c
a b c c c
a bc bc c c
bc a bc c bc
ab a abc bc c
a a a b b
a b b b b
a b b b b
Means within a column followed by the same letter are not significantly different at P ¼ 0.05 according to Tukey's test.
Table 3 Effect of hot water dips on the physical appearance of mango cv. ‘Carabao’ with various temperatures, exposure times and time after treatment.a Temp ( C)
After treatment 10
20
30
50
10
20
30
50
10
20
30
50
48 50 52 53 55
CC CC CC CC CC
CC CC CC CC CC
CC CC CC CC CC
CC CC CC LBS SS
CC CC CC CC SH
CC CC CC CC SH
CC CC LBS LBS SS
CC CC LBS LBS LBS
CC CC CC CC SH
CC CC CC CC SH
CC CC LBS LBS SS
CC CC LBS LBS LBS
After 7 d
After 14 d
a SS-severe scalds, LBS-light brown scalds, SH-shriveling, CC-comparable with the control.
fruit with anthracnose severity index of 1.80e2.0 and 0.90‒1.20 for stem-end rot. Fruit receiving distilled water (untreated control) had the highest indexes of fruit rot attributable to anthracnose (3.80e3.90) and stem-end rot (2.30e2.50). The efficiency of postharvest treatments, calculated as the % reduction in severity of anthracnose or stem-end rot by a treatment as compared to the untreated control, showed that fungicide controlled anthracnose by 71.79%e79.31% and stem-end rot by 73.91%e80%. HWT inhibited anthracnose by 48.71%e52.63% and stem-end rot by 48%e60.86%.
3.5. Mycoflora of fruit after storage Fusarium verticillioides, Penicillium spp., Aspergillus spp., Pestalotiopsis spp., L. theobromae, C. gloeosporioides and Acremonium spp. were isolated on fruit tissues after 14 days. F. verticillioides, the dominant fungus, was present in almost all tissues analyzed. C. gloeosporioides and L. theobromae were particularly present on epidermis with symptoms of anthracnose and stem-end rot, respectively. The fungal population of each fungus, however, was not determined.
4. Discussion Most overseas markets are no longer permitting chemical treatments, such as fungicidal dips, for fruit entering their countries (Jacobi and Giles, 1997). HWT is a widely used postharvest treatment for export of mangoes as it does not leave chemical residues. HWT of mangoes for disease control involves a 10 min dip at 52e55 C (Quimio and Quimio, 1974; Lizada et al., 1986; Buganic et al., 1997). We were prompted to revisit HWT, however, due to the significant amount of decay in ‘Carabao’ caused by anthracnose and stem end-rot in export markets. The severity of anthracnose and stem-end rot in ‘Carabao’ mangoes was reduced by 83% and 100%, respectively by hot water dips at 52e55 C for 10 min (Buganic et al., 1997). Yet the high degree of postharvest disease control in this report may not be attributed exclusively to HWT as cultural management was involved. Fruit were wrapped with paper two months after flower induction until harvest. This practice may have protected the developing fruit from attack by pathogens present in the canopy of mango trees at all times, hence, fruit may have limited latent infection at harvest. Fungal populations established during fruit development are responsible for postharvest diseases such as anthracnose and stem-end rot (Prusky et al., 1983). Obviously, cultural management such as fruit bagging should be implemented as the extent of disease control could be influenced by the degree of disease pressure during production. Furthermore, HWT at 52e55 C for 10 min did not completely inhibit fungal inoculum which may develop progressively overtime to decay the fruit as shown in the present study. Fruit wrapping is an essential component of good agricultural practice for ‘Carabao’ mango particularly for fruit intended for the export market. The high cost of labor and materials to be used have limited the ability of some growers to implement fruit bagging. During times of high demand and limited supply of ‘Carabao’ mangoes, however, some unwrapped fruit are exported. Exporters should strictly implement a ‘no wrapping no export’ policy.
Table 4 Effect of various treatments on fruit quality after 14 days of storage at 18e20 C.a Treatment
Weight loss (%)
Hardness (N)
pH
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
HWT (53 C, 20 min) Mancozeb (2.5 g/L) Untreated (Distilled water)
14.38 a 12.95 a 13.16 a
15.74 a 14.17 a 14.16 a
4.24 a 3.24 bc 3.67 ab
3.38 a 3.72 a 3.58 a
6.47 a 6.45 a 6.52 a
6.20 ab 6.63 b 6.44 a
5.70 a 6.92 b 6.82 b
6.68 a 8.40 a 7.20 a
1.50 a 1.46 a 2.09 a
a b c d
TSS
Color (b* value)b
Anthracnosec
Stem-end rotd
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
Trial 2
1.54 a 1.85 a 2.20 a
45.34 a 53.63 b 52.48 b
51.92 a 51.43 a 50.64 a
2.00 ab 1.10 a 3.90 b
1.80 a 0.90 a 3.80 b
1.30 a 0.50 a 2.50 b
0.90 a 0.60 a 2.30 b
TA
Means within a column followed by the same letter are not significantly different at P ¼ 0.05 according to Tukey's test. Negative b* indicates blueness while positive b* denotes yellowness of the fruit. See Fig. 1 for visual characterization of anthracnose severity disease index. See Fig. 2 for visual characterization of stem-end rot severity disease index.
D.G. Alvindia, M.A. Acda / Crop Protection 67 (2015) 96e101
We showed that 53 C for 20 min was the optimum exposure for ‘Carabao’ mangoes and the best combination in controlling culture growth of pathogens. In vivo application at this exposure, however, did not yield significant reduction in anthracnose (48.71%e52.63%) and stem-end rot (48%e60.86%) after 14 days. The present study demonstrated that even the optimum exposure for ‘Carabao’ mangoes was inadequate in giving a high degree of postharvest disease control. A considerable level of latent infection may have been present on fruit surfaces as unwrapped fruit were used in our experiments. The poor efficiency of HWT could be attributed to its fungistatic action leaving active propagules after treatment which develop overtime and cause decay on fruit. Moreover, the inconsistent performance seen, for example variation in disease severity within a treatment, has to be addressed. Severity indexes of anthracnose and stem-end rot showed standard deviations ranging from ±1.05 to ±2.98, remarkably higher than those treated with fungicide (±0.69 to ±0.99). The volatility of HWT in practical use agreed with the in vitro result of the present study i.e., high variation in radial growth and spore germination of C. gloeosporioides and L. theobromae. A correct setting of water temperature is important as fruit absorb heat. For example, a 22 L water bath preheated at 80 C fell to 51.6 C with 20 fruit immersed. The temperature increased to 53 C after 3e5 min. This scenario would significantly affect the performance of HWT to destroy latent infections since the desired exposure is not achieved. To address the problem, the personnel in charge must be knowledgeable in setting temperature ranges to attain the correct exposure. Upgrading water baths with a precise heating and control system would also help address the problem. Recycling of water should be considered as fresh water is becoming limited and this treatment will have an economic impact in the future. HWT at 53 C for 20 min had no negative effect on the overall quality of fruit as fruit firmness, TSS, TA, color, and weight loss were statistically comparable with the untreated controls after 14 days. In ordinary conditions, firmness of healthy ‘Carabao’ mangoes decreases as the fruit ripen which could be attributed to the activity of enzymes that degrade pectic substances (Pantastico et al., 1984). Yet heat treatment inhibits solubilization of the carbonate soluble pectin fraction which is one of the main factors contributing to firmness retention (Lizada, 1991). Fruit firmness is one of the most widely used indicators of fruit quality. The HWT technology is a more cost effective alternative to vapor heat treatment and has added benefits such as ease of use, less time consuming, reliable, and it gives fruit surface sanitation to exclude plant debris and disease control (Jacobi et al., 2001). For the time being, HWT will continue as a popular postharvest protocol for ‘Carabao’ mangoes in response to export requirements imposed specifically by Japan. Conversely, HWT efficiency has to be improved as an effective quarantine procedure in preventing anthracnose and stem end-rot on harvested mangoes. Biological control agents (BCA) and/or generally regarded as safe (GRAS) compounds may be integrated with HWT. Such a temperature and exposure time as in this study could, however, retard the development of the pathogen in the fruit, and thus create a vacuum that could be exploited by BCA and/or GRAS. Naturally occurring antagonistic bacteria or fungi and application of GRAS compounds
101
such as sodium carbonate, sodium bicarbonate and sodium hypochlorite reduced postharvest diseases on fruit (Alvindia et al., 2004; Alvindia and Natsuaki, 2007, 2008, 2009).
References Alvindia, D.G., Kobayashi, T., Natsuaki, K.T., Tanda, S., 2004. Inhibitory influence of inorganic salts on banana postharvest pathogens: preliminary application to control crown rot. J. Gen. Plant Pathol. 70, 61e65. Alvindia, D.G., Natsuaki, K.T., 2007. Control of crown rot-causing fungal pathogens of banana by inorganic salts and a surfactant. Crop Prot. 26, 1667e1673. Alvindia, D.G., Natsuaki, K.T., 2008. Evaluation of fungal epiphytes isolated from banana fruit surfaces for biocontrol of banana crown rot disease. Crop Prot. 27, 1200e1207. Alvindia, D.G., Natsuaki, K.T., 2009. Biocontrol activities of Bacillus amyloliquefaciens DGA14 isolated from banana fruit surface against banana crown rot-causing fungi. Crop Prot. 28, 236e242. Arauz, L.F., 2000. Mango anthracnose: economic impact and current options for integrated management. Plant Dis. 84, 600e611. Aveno, J.L., Orden, M.M., 2008. Hot Water Treatment of Mango: a Study of Four Export Corporations. Accessed from. http://www.docstoc.com/docs/2388169/ HOT-WATER TREATMENTeOFeMANGO-A-STUDYeOFeFOUR-EXPORTCORPORATIONS (Date downloaded 05.11.13.). Buganic Jr., R.D., Lizada, M.C.C., De Ramos, M.B., 1997. Disease control in Philippine 'Carabao' mango with preharvest bagging and postharvest hot water treatment. Acta Hortic. 455, 797e804. Dodd, J.C., Prusky, D., Jeffries, P., 1997. Fruit diseases. In: Litz, R.E. (Ed.), The Mango: Botany, Production and Uses. CAB Int., Wallingford, pp. 257e280. Esguerra, E.B., Chavez, S.M., Traya, R.V., 2006. A modified and rapid heat treatment for the control of postharvest diseases of mango (Mangifera indica Linn. cv. Carabao) fruits. Philipp. Agric. Sci. 89, 125e133. Fitzel, R.D., Peak, C.M., 1984. The epidemiology of anthracnose disease of mango: inoculum sources, spore production and dispersal. Ann. Appl. Biol. 104, 53e59. Jacobi, K.K., Giles, J.E., 1997. Quality of 'Kensington' mango (Mangifera indica Linn.) fruit following combined vapour heat disinfestation and hot water disease control treatments. Postharvest Biol. Technol. 12, 285e292. Jacobi, K.K., MacRae, E.A., Hetherington, S.E., 2001. Postharvest heat disinfestation treatments of mango fruit. Sci. Hortic. 89, 171e193. Jeffries, P., Dodd, J.C., Jeger, M.J., Plumbley, R.A., 1990. The biology of Colletotrichum species on tropical fruit and vegetables. Plant Pathol. 39, 343e366. Johnson, G.I., Hofman, P.J., 2009. Postharvest technology and quarantine treatments. In: Litz, R.E. (Ed.), The Mango: Botany, Production and Uses. CAB Int., Wallingford, pp. 528e605. Kobiler, I., Shalom, Y., Roth, I., Akerman, M., Vinokur, Y., Fuchs, Y., Prusky, D., 2001. Effect of 2,4-dichlorophenoxy acetic acid on the incidence of side and stem end rots in mango fruits. Postharvest Biol. Technol. 23, 23e32. Lizada, M.C., 1991. Post-harvest physiology of the mango e a review. Acta Hortic. 291, 437e453. Lizada, M.C.C., Agravante, J.U., Brown, E.O., 1986. Factors affecting postharvest disease control in ‘Carabao’ mango subjected to hot water treatment. Philipp. J. Crop Sci. 11, 153e161. Mascarenhas, P., Behere, A., Sharma, A., Padwal-desa, S.R., 1995. Postharvest spoilage of mango (Mangifera indica) by Botryodiplodia theobromae. Mycol. Res. 100, 27e30. Nuevo, P.A., Apaga, A.R.M., 2010. Technology reducing postharvest losses and maintain quality of fruits and vegetables (Philippines). In: Huang, C.C., Yang, C.M., Ou, S.K., Chen, J.J. (Eds.), Technology Reducing Postharvest Losses and Maintain Quality of Fruits and Vegetables, Taiwan Agr. Res. Inst., Spec. Publ. No., vol. 147, pp. 154e167. Pantastico, E.B., Lam, P.F., Ketsa, S., Yuniarti, Kosittrakul, M., 1984. Postharvest physiology and storage of mango. In: Mendoza Jr., D.B., Wills, R.B.H. (Eds.), Mango: Fruit Development, Postharvest Physiology and Marketing in ASEAN. Association of South East Asian Nations (ASEAN) Food Handling Bureau, pp. 39e52. Prusky, D., Falik, E., Kobiler, I., Fuchs, Y., Zauberman, G., Pesis, E., Roth, I., Weksler, A., Akerman, M., Ykutiety, O., Waisblum, A., Keinan, A., Ofek, G., Lavi, U., 1997. Hot water bruch anem method for control of postharvest diseases caused by Alternaria rot in mango fruits. Acta Hortic. 455, 780e785. Prusky, D., Fuchs, Y., Yanko, U., 1983. Assessment of latent infections as a basis for control of post-harvest disease of mango. Plant Dis. 67, 816e818. Quimio, A.J., Quimio, T.H., 1974. Postharvest control of Philippine mango anthracnose by hot water treatment. Philipp. Agric. 58, 138e146.