Effect of pre-harvest calcium chloride applications on fruit calcium level and post-harvest anthracnose disease of papaya Babak Madani a, * , Mahmud Tengku Muda Mohamed a, ** , Alan R. Biggs c , Jugah Kadir b , Yahya Awang a , Amin Tayebimeigooni a , Taha Roodbar Shojaei d a Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia b Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia c West Virginia University, Tree Fruit Research and Education Center, Kearneysville, WV, USA d Institute of Advanced Technology (ITMA), Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia article info Article history: Received 18 June 2013 Received in revised form 3 October 2013 Accepted 4 October 2013 Keywords: Papaya Anthracnose Calcium Disease incidence Disease severity abstract Anthracnose disease of papaya, caused by Colletotrichum gloeosporioides Penz, can cause extensive postharvest losses. The goal of this research was to use pre-harvest calcium applications to reduce anthracnose disease. Six pre-harvest foliar calcium sprays were applied biweekly to papaya trees in experimental orchards at Universiti Putra Malaysia. Additional in vitro and in vivo tests were carried out to test the effect of calcium on fruit calcium content, spore germination, mycelial growth and disease severity. Calcium chloride at 1%, 1.5% and 2.0% concentrations significantly decreased spore germination. Calcium content of papaya fruit was significantly increased by calcium sprays at a concentration of 2.0% in 2012 and 2013. In vivo studies showed that increasing calcium content in fruit bycalcium sprays at 1.5 and 2.0% concentrations significantly reduced anthracnose incidence of fruits during five weeks storage at 12 2 C, and delayed initiation of disease symptoms by four weeks. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Papaya, Carica papaya L., is a large perennial plant with rapid growth (Paull and Duarte, 2011). It is a significant fruit in the Malaysian economy, ranking third after durian and banana. The Eksotika II cultivar is a high yielding F 1 hybrid with good quality that was released by the Malaysian Agricultural Research and Development Institute (MARDI). The cultivar has gained popularity in the local and export market (Shukor and Shokri, 1997); however, postharvest pathogens inflict significant losses in some years (Paull et al., 1997). Anthracnose, caused by Colletotrichum gloeosporioides Penz, is one of the most devastating pathogens in storage for papaya. Inoculum originates from drying, infected leaf petioles, which under favourable conditions (moist for a few hours) produce appressoria and subsequent infections that remain quiescent until ripening (Chau and Alvarez, 1983). The disease typically is managed with fungicides or hot water exposure, but hot water can damage fruits and the pathogen has become resistant to some of the fun- gicides currently in use (Djioua et al., 2010). With increased interest in non-fungicidal management approaches, researchers are looking for new ways to maintain disease-free fruit in the postharvest milieu (Ali et al., 2010). Calcium is a key plant nutrient that has a significant role in cell functions, including reducing softening and senescence of fruits (Barker and Pilbeam, 2007). Many disorders of crops, such as bitter pit in apple, cork spot in pear, and blossom end rot in tomato that are caused by calcium deficiency could be reduced by calcium spraying (Kader, 2002). However, there are few re- ports about effects of calcium on infection of tropical fruits by C. gloeosporioides. Ghani et al. (2011) found that calcium can increase anthracnose resistance in dragon fruit. In addition, cal- cium decreased susceptibility to anthracnose in banana (Chillet et al., 2000). In papaya, there are a few reports about the role of calcium in anthracnose disease, and they are mostly related to postharvest uses (Mahmud et al., 2008). There is some evidence that calcium is antagonistic to C. gloeosporioides and it may have potential use as an alternative method in integrated disease management (Biggs et al., 2000). The objective of this study was to examine the effect of pre-harvest applications of calcium chloride on anthracnose disease of papaya. * Corresponding author. Tel.: þ60 123338642. ** Corresponding author. Tel.: þ60 389474823. E-mail addresses: [email protected](B. Madani), mahmood@agri. upm.edu.my (M.T. Muda Mohamed). Contents lists available at ScienceDirect Crop Protection journal homepage: www.elsevier.com/locate/cropro 0261-2194/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cropro.2013.10.009 Crop Protection 55 (2014) 55e60
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Crop Protection 55 (2014) 55e60
Contents lists avai
Crop Protection
journal homepage: www.elsevier .com/locate/cropro
Effect of pre-harvest calcium chloride applications on fruit calciumlevel and post-harvest anthracnose disease of papaya
Babak Madani a,*, Mahmud Tengku Muda Mohamed a,**, Alan R. Biggs c, Jugah Kadir b,Yahya Awang a, Amin Tayebimeigooni a, Taha Roodbar Shojaei d
aDepartment of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, MalaysiabDepartment of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, MalaysiacWest Virginia University, Tree Fruit Research and Education Center, Kearneysville, WV, USAd Institute of Advanced Technology (ITMA), Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
a r t i c l e i n f o
Article history:Received 18 June 2013Received in revised form3 October 2013Accepted 4 October 2013
E-mail addresses: [email protected] (upm.edu.my (M.T. Muda Mohamed).
0261-2194/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.cropro.2013.10.009
a b s t r a c t
Anthracnose disease of papaya, caused by Colletotrichum gloeosporioides Penz, can cause extensivepostharvest losses. The goal of this research was to use pre-harvest calcium applications to reduceanthracnose disease. Six pre-harvest foliar calcium sprays were applied biweekly to papaya trees inexperimental orchards at Universiti Putra Malaysia. Additional in vitro and in vivo tests were carried outto test the effect of calcium on fruit calcium content, spore germination, mycelial growth and diseaseseverity. Calcium chloride at 1%, 1.5% and 2.0% concentrations significantly decreased spore germination.Calcium content of papaya fruit was significantly increased by calcium sprays at a concentration of 2.0%in 2012 and 2013. In vivo studies showed that increasing calcium content in fruit by calcium sprays at 1.5and 2.0% concentrations significantly reduced anthracnose incidence of fruits during five weeks storageat 12 � 2 �C, and delayed initiation of disease symptoms by four weeks.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Papaya, Carica papaya L., is a large perennial plant with rapidgrowth (Paull and Duarte, 2011). It is a significant fruit in theMalaysian economy, ranking third after durian and banana. TheEksotika II cultivar is a high yielding F1 hybrid with good qualitythat was released by the Malaysian Agricultural Research andDevelopment Institute (MARDI). The cultivar has gained popularityin the local and export market (Shukor and Shokri, 1997); however,postharvest pathogens inflict significant losses in some years (Paullet al., 1997). Anthracnose, caused by Colletotrichum gloeosporioidesPenz, is one of the most devastating pathogens in storage forpapaya. Inoculum originates from drying, infected leaf petioles,which under favourable conditions (moist for a few hours) produceappressoria and subsequent infections that remain quiescent untilripening (Chau and Alvarez, 1983). The disease typically is managedwith fungicides or hot water exposure, but hot water can damage
B. Madani), mahmood@agri.
All rights reserved.
fruits and the pathogen has become resistant to some of the fun-gicides currently in use (Djioua et al., 2010). With increased interestin non-fungicidal management approaches, researchers are lookingfor new ways to maintain disease-free fruit in the postharvestmilieu (Ali et al., 2010).
Calcium is a key plant nutrient that has a significant role incell functions, including reducing softening and senescence offruits (Barker and Pilbeam, 2007). Many disorders of crops, suchas bitter pit in apple, cork spot in pear, and blossom end rot intomato that are caused by calcium deficiency could be reducedby calcium spraying (Kader, 2002). However, there are few re-ports about effects of calcium on infection of tropical fruits byC. gloeosporioides. Ghani et al. (2011) found that calcium canincrease anthracnose resistance in dragon fruit. In addition, cal-cium decreased susceptibility to anthracnose in banana (Chilletet al., 2000). In papaya, there are a few reports about the roleof calcium in anthracnose disease, and they are mostly related topostharvest uses (Mahmud et al., 2008). There is some evidencethat calcium is antagonistic to C. gloeosporioides and it may havepotential use as an alternative method in integrated diseasemanagement (Biggs et al., 2000). The objective of this study wasto examine the effect of pre-harvest applications of calciumchloride on anthracnose disease of papaya.
B. Madani et al. / Crop Protection 55 (2014) 55e6056
2. Materials and methods
2.1. Trial description
Papaya trees (cultivar Eksotika II) in an orchard at Agro-techUnit,University Agriculture Park (TPU), Universiti Putra Malaysia, Ser-dang, Selangor were selected for calcium application experiments.Trees were eight months old, approximately 2.2 m tall. The treeswere spaced 3 m by 3 m with two plants in each 3 � 3 m plot.Commercial fertilization rates (12:12:17:2 N:P:K:Mg) were useduniformly each month around the canopy periphery for all treat-ments according to Malaysia Ministry of Agriculture recommenda-tions (Basir, 2005). Irrigation was accomplished with overheadsprinklers at approximately 4-day intervals. Weeds were controlledby mowing as needed. The experiment was carried out in 2012 andrepeated in 2013. In 2012, solutions of 0, 0.5, 1.0, 1.5 and 2.0% (w/v)calcium chloride (CaCl2$2H2O (99% CC, SYSTERM�, Malaysia) with0.03% v/v Tween 20 as surfactant were sprayed 21 days after floweranthesis to the fruits and leaves of papaya trees until run-off (threepositions from top to bottomand three positions frombottom to thefruits and leaves (approximately 1.5 L per tree) using a 16 L knapsacksprayer (Jun Chong�, Malaysia, with a solid cone nozzle, nozzle cap,0.70e0.85 L/min). Spray applications began in June and wererepeated every twoweeks for six times, with the final spray appliedfour days before fruit harvesting (13 August). Fruits of uniform sizeand shape were harvested at index 2 (green with trace of yellow),washedwithwater and allowed to air dry. Then, theywere assignedrandomly to five different treatments. After that, fruits in eachtreatment were packed in commercial boxes and stored at 12� 2 �Cand 85e90% relative humidity for five weeks. In 2013, the experi-mentwas carried outwith 0,1.5 and 2% calcium chloride to the fruitsand leaves of eightmonth old papaya inTPU. Sprayswere applied on15 January, 2013, with the final spray applied two days before fruitharvesting (28 March). In all other respects the 2013 trial wasmanaged using the same procedures as in the previous season.
2.2. Calcium measurement in fruit
In both years, samples of peel were taken with a metal bladescalpel from the middle part of fruits and dried at 60 �C in an air-circulating oven. Once dried, 0.25 g of the peel was digested in5 ml 98% H2SO4 on a hot plate at 280 �C in a fume chamber for7 min. Then, 10 ml H2O2 was added into the mixtures and theheating was continued for another 4 min. The solution mixtureswere brought up to100 ml with distilled water. Calcium ion con-centration was measured with an atomic absorption spectropho-tometer (Perkin Elmer, Model AAS 3110, Palo Alto, California, USA),and results were expressed as mg calcium g�1 D.W. For calciummeasurements in the peel, four replications with two fruits perreplication arranged in randomized complete block design wereused in both years.
2.3. In vitro evaluation of fungicidal activity of calcium
2.3.1. C. gloeosporioides isolate and culture conditionsC. gloeosporioides was isolated from infected papaya fruit. Eight
small parts of the peel were sterilised with sodium hypochloriteand washed three times in distilled water, then dried and placed inPetri dishes that contained potato dextrose agar (PDA), and incu-bated at ambient temperature (28 � 2 �C). When mycelial growthwas observed, colonies were reisolated on to fresh PDA to obtainpure cultures (Ali et al., 2010). The isolates were identified bymorphological and cultural characters based on a publisheddescription by Barnett and Hunter (1972) and confirmed at theDepartment of Plant Protection, Faculty of Agriculture, Universiti
Putra Malaysia. Identified C. gloeosporioideswasmaintained on PDAslants for usage.
2.3.2. In vitro mycelial growthAn agar disk (5 mm diameter) from a pure culture of
C. gloeosporioideswas placed in the center of PDA dishes containingdifferent concentrations of calcium chloride (0, 0.5, 1.0, 1.5 and 2.0%w/v). The control dishes only contained PDA. Then, Petri disheswere placed at ambient temperature (28� 2 �C) for 7 days, at whichtime the fungus reached the edge of dishes.
2.3.3. In vitro conidial germinationTo test the effect of calcium on spore germination, conidia were
harvested by scraping them off the agar with a glass rod and sterile,distilled water. Then, the slurry was filtered through 4 layers ofcheesecloth and adjusted to 20 ml. The number of conidia per mlwas determined with a haemocytometer and adjusted to 5 � 105
conidia/ml (Obagwu and Korsten, 2003). One hundred microlitersof this suspension were pipetted to PDA agar in dishes containingcalcium chloride (0, 0.5, 1.0, 1.5 and 2.0%) and kept in the dark for7 h at 28 � 2 �C. The control dishes contained only PDA. Germi-nation of 100 spores in 10 microscopic fields with magnification at40� were used to calculate percent germination. A conidium wasconsidered germinated when the germ tube was longer thanconidium (El Ghaouth et al., 1992). Six replications of five petridishes arranged in a completely randomised design were used forin vitro experiments, which were conducted twice.
2.4. In vivo assay of calcium against C. gloeosporioides
2.4.1. Anthracnose disease incidence and severityIn both years, to determine disease incidence and severity, fruits
were put in commercial export boxes (EXOTIC STAR�, Kajang,Selangor, Malaysia) and stored at 12 � 2 �C and 85e90% RH for fiveweeks. Disease incidence data were expressed as percentage offruits showing anthracnose out of the total number of fruits in eachtreatment (Ali et al., 2010). Disease severity was measured as per-centage of fruit surface with anthracnose disease. For diseaseincidence and severity, four replications with six fruits per repli-cation arranged in a randomized complete block design with twofactors (days in storage and calcium chloride concentrations), wereused in both years.
2.4.2. Inoculation tests for lesion diameter of anthracnose in fruitsIn 2012, after harvest, papaya fruit were washed with sodium
hypochlorite (0.01%) for 3 min, rinsed with distilled water and airdried at ambient temperature (28 � 2 �C). Fruits were wounded onthree sides to a depth of threemm in diameter, immersed for 30 s ina conidial suspension of 105 conidia per millimeter and drained andplaced at ambient temperature. Lesion diameter of fruits wasmeasured after four and eight days. Lesion diameter per fruit wasexpressed as the average of the three values from each fruit. Forlesion diameter, four replications with three fruits per replicationarranged in randomized complete block design were used.
2.5. Microscopy observations
Small samples (1.5 mm3) of the mid-region of papaya fruit peel,were cut and fixed in 4% glutaraldehyde at 4 �C for 24 h. Sampleswere washed in 0.1 mol/L sodium cacodylate buffer (pH ¼ 7.6) forthree times, and post fixed in 1% (w/v) osmium tetraoxide for 2 h.Then, they were rinsed again in 0.1 mol/L sodium cacodylate buffer(pH ¼ 7.6). After that, tissues were dehydrated in graded series ofacetone at 35e100%. Finally, samples were embedded in beamcapsules and polymerized at 60 �C for 2 days. Ultra-thin sections
Fig. 2. Effect of calcium on in vitro conidial germination of C. gloeosporioides. Meanswith different letters were significantly different according to the WallereDuncan k-ratio t-test (p ¼ 0.05). The vertical bars represent the standard error of means for sixreplications.
B. Madani et al. / Crop Protection 55 (2014) 55e60 57
(60e90 nm) of tissues were cut and mounted onto copper grids.Sections were stainedwith saturated uranyl acetate and lead citrate(Reynolds, 1963) and were examined under a transmission electronmicroscope (TEM) (Hitachi, H-7100, Japan).
2.6. Statistical analysis
Data were subjected to analysis of variance (ANOVA) usingstatistical analysis system (SAS) version 8.2 (SAS Institute Inc., Cary,NC, USA). The means were separated using the WallereDuncan k-ratio t-test at a significance level of p ¼ 0.05.
3. Results
3.1. In vitro evaluation of fungicidal activity of calcium
Calcium chloride at 0.5 and1% stimulated mycelial growth ofC. gloeosporioides relative to the control (Fig. 1). Calcium at con-centrations of 1.5 and 2.0% showed similar mycelial growth to thecontrol (Fig. 1).
Conidial germination tests were conducted to test the potentialfor calcium chloride at different concentrations to inhibit germi-nation of C. gloeosporioides. Spore germination was decreasedsignificantly for calcium concentrations of 1%,1.5% and 2.0% relativeto the control (Fig. 2), whereas 0.5% was not significantly differentfrom the control (Fig. 2).
3.2. Calcium content in fruit
In 2012 and 2013, pre-harvest calcium applications resulted inincreased calcium in the peel, except for the 0.5% treatment in 2012,which was similar to the control. There were significant differencesbetween the control and 1.5 and 2.0% calcium chloride for calciumcontent in the peel for both years. The highest calcium content wasseen in the 2.0% calcium treatment with calcium concentrationsapproximately 130 and 78% higher relative to the control in 2012and 2013, respectively (Table 1).
3.3. In vivo fruit assay
3.3.1. Anthracnose disease incidence and severityThe in vivo assay of calcium against C. gloeosporioides showed a
significant interaction between calcium concentrations and days instorage for disease incidence and severity in both years (Tables 2and 3). For 2012, disease incidence was reduced significantly with
Fig. 1. Effect of calcium on mycelial growth of C. gloeosporioides after 7 days incubationat 28 � 2 �C. Means with different letters were significantly different according to theWallereDuncan k-ratio t-test (p ¼ 0.05). The vertical bars represent the standard errorof means for six replications.
1.5% and 2.0% in comparison with the control and lower concen-tration treatments (0.5 and 1.0%) after five weeks of storage at12 � 2 �C (Table 2). Disease incidence increased significantly withincreasing days in storage and reached maximum in the controlafter three weeks (100%). In 2013, disease incidence was decreasedsignificantly with 1.5% and 2.0% compared with the control afterfiveweeks of storage at 12� 2 �C (Table 3). The incidence increasedsignificantly with increasing days in storage and reachedmaximum(100%) in the control after three weeks. Furthermore, diseaseincidence in the 1.5 and 2.0% concentration treatments wassignificantly lower than in the control in the second, third andfourth weeks. However, there were no significant differences indisease incidence between the 1.5 and 2.0% concentration treat-ments after five weeks in storage in 2012 and 2013.
For disease severity, maximum protection to the fruit wasreached with the 1.5 and 2.0% calcium chloride concentrationtreatments after five weeks in storage. In the first week there wereno signs of disease on the fruits. Also, in the second week of storagecontrol fruits displayed significantly increased symptoms. Diseaseseverity was 6.1% in the 2.0% concentration treatment and 8.8% inthe 1.5% concentration treatment after four weeks in storage(Table 2). In 2013, maximum protection to the fruit was achieved inthe 1.5 and 2.0% concentration treatments after five weeks instorage (Table 3). In the first and second weeks there were no signsof disease in the fruits. However, in both years, disease severityreached the maximum in the control in the fifth week. There wereno significant differences in disease severity between the 1.5 and2.0% concentration treatments after four and five weeks in storagein 2012 and 2013. Also, there were no significant differences in
Table 1Effect of pre-harvest calcium chloride on calcium content in the peel of papaya fruitat harvest in 2012 and 2013.
Season Calcium chloride concentrations (%) Calcium in peel (mg g�1 D.W.)
2012 0 11.4a db
0.5 14.0 cd1.0 17.2 bc1.5 20.2 b2.0 26.1 a
2013 0 16.7 c1.5 24.2 b2.0 29.6 a
a Each value is the mean of 8 observations.b Means followed by the same letter in the same column are not significantly
different according to the WallereDuncan k-ratio t-test (p ¼ 0.05).
Table 2Effect of pre-harvest calcium chloride treatments and days in storage on diseaseincidence and severity (%) in papaya fruits during storage at 12 � 2 �C in 2012.
a Small letters in columns (a, b, c, d) show the mean comparison among con-centrations of calcium chloride. Means with the same letter are not significantlydifferent according to the WallereDuncan k-ratio t-test (p ¼ 0.05).
b Small letters in rows (w, x, y, z) show the mean comparison among days instorage. Means with the same letter are not significantly different according to theWallereDuncan k-ratio t-test (p ¼ 0.05).
c Each value is the mean of 24 observations.
Table 4Effect of pre-harvest calcium chloride treatments on anthracnose lesion diameter(mm) on papaya fruits after four and eight days at ambient temperature in 2012.
a Small letters in columns (a, b, c, d) show the mean comparison among con-centrations of calcium chloride. Means with the same letter are not significantlydifferent according to the WallereDuncan k-ratio t-test (p ¼ 0.05).
b Each value is the mean of 12 observations.
B. Madani et al. / Crop Protection 55 (2014) 55e6058
disease severity between the fourth and fifth week in the controltreatment in 2012 and 2013.
3.3.2. Inoculation tests for lesion diameter of anthracnose on fruitsA significant decrease in lesion diameter was observed on cal-
cium chloride-treated fruits in comparison to non-sprayed fruitsfour and eight days after incubation (Table 4). The 1.5% and 2.0%concentration treatments resulted in the lowest lesion diameterafter four and eight days at ambient temperature relative to thecontrol. There were no significant differences in lesion diameterbetween the 1.5 and 2.0% concentration treatments; however, bothwere significantly different from the control.
3.3.3. TEM observationsCell wall structure of TEM images of fruit peel showed better
preservation of themiddle lamella in calcium chloride-treated fruit.This structure had completely disappeared in untreated fruit afterthree weeks in storage compared with harvesting day (Fig. 3A, B),while it had been preserved in both 1.5% and 2% calcium treatmentsthree weeks after storage (Fig. 3C, D).
Table 3Effect of pre-harvest calcium chloride treatments and days in storage on diseaseincidence and severity (%) in papaya fruits during storage at 12 � 2 �C in 2013.
a Small letters in columns (a, b, c, d) show the mean comparison among con-centrations of calcium chloride. Means with the same letter are not significantlydifferent according to the WallereDuncan k-ratio t-test (p ¼ 0.05).
b Small letters in rows (x, y, z) show the mean comparison among days in storage.Means with the same letter are not significantly different according to the WallereDuncan k-ratio t-test (p ¼ 0.05).
c Each value is the mean of 24 observations.
4. Discussion
The primary goal of this research was to determine effects ofpre-harvest calcium chloride application on incidence and severityof anthracnose caused by C. gloeosporioides on papaya fruits,thereby providing growers with a practical tool for anthracnosedisease management. This study revealed that pre-harvest calciumchloride application can reduce disease incidence and severity, andalso reduce growth and spore germination in vitro of the anthrac-nose pathogen. Control of postharvest diseases is necessary whenlong transit times are needed for exporting papaya.
The effects of calcium in reducing spore germination wereprobably due to toxicity, with high concentrations likely affectingthe osmotic balance in fungal cells (Arras et al., 1998). Reducedspore germination shows that the pathogen may be more sensitiveto calcium at the conidial stage relative to the mycelial growthstage. The mechanism or mechanisms by which calcium inhibitsgerm tube growth have not been described. One hypothesis is thathigh concentration of extracellular calciummay increase calcium inthe cytosol to toxic levels (Droby et al., 1997).With aminimum levelof calcium ion concentration being necessary for normal cellgrowth, any limitation in regulation of intracellular calcium levelmay result in reduced organism development (Droby et al., 1997).Our findings are in agreement with those of Eryani-Raqeeb et al.(2009) who demonstrated that high concentration of calciumreduced spore germination of the papaya anthracnose pathogen.Our results are similar those of Droby et al. (1997) who showed thatPenicillium digitatum spore germination was reduced in the pres-ence of calcium in grapefruit.
Pre-harvest spray applications of calcium chloride increased thecalcium content in the peel of fruits that had been treated withcalcium.Marked increases in calcium concentration in the fruit peelsuggest that exogenous calcium penetrates the fruit epidermis andbecomes incorporated into the compound cell wall complex. Ap-plications with higher concentrations of calcium chloride spraysresulted in proportional increases of fruit calcium content. The in-crease of calcium in the peel is in agreement with other researcherswho found that exogenous applications of calcium increased cal-cium in dragon fruit and peach (Ghani et al., 2011; Elmer et al.,2007).
In both years of this study, calcium applications resulted indecreased anthracnose disease incidence and severity on papayafruits. Calcium chloride at 1.5 and 2.0% concentrations also signif-icantly delayed disease onset. Calcium may decrease the incidenceand severity of disease directly by inhibition of spore germinationand by inhibiting cell wall degrading enzymes that are produced bythe pathogen (Wisniewski et al., 1995). Indirect effects of calciumon the host/pathogen interaction may also be expressed on thelevel of host cell wall integrity (Biggs, 1999). In addition, higherconcentrations of cytosolic calcium have been shown to induce
Fig. 3. AeD. Transmission electron microscopy images of papaya peel receiving no additional calcium (A ¼ at harvest, B ¼ 3 weeks after harvest) and papaya peel treated 6 times inthe pre-harvest setting with 1.5% calcium chloride (C) and 2% calcium chloride (D) at 3 weeks after harvest. Note thickened middle lamallae (ML) in C and D relative to the thin ML inA and no ML in B (arrows). Bar ¼ 1 mm.
B. Madani et al. / Crop Protection 55 (2014) 55e60 59
endogenous resistance mechanisms, through the synthesis ofphytoalexins and phenolic compounds that decrease the activity ofpathogen pectolytic enzymes (Miceli et al., 1999). Calcium defi-ciency could increase cell wall membrane permeability and lead toincrease in the activity of enzymes like pectin methylesterase andpolygalacturonase in fruit (Deytieux-Belleau et al., 2008). Conwayet al. (1987) concluded that calcium decreased Glomerella cingu-lata in apple fruit by inhibition of polygalacturonase from thepathogen. Our results are in agreement with those of other re-searchers who demonstrated that pre-harvest applications of cal-cium chloride significantly decreased postharvest rotting in grapeand strawberry (Nigro et al., 2006; Wojcik and Lewandowski,2003).
In addition to reducing disease incidence, calcium supplemen-tation can also result in reduced disease severity. For example,Biggs et al. (1997) studied the effects of different calcium sources onthe lesion diameter of peach fruits. They showed that all calciumsalts reduced lesion diameter compared to the control. Moreover,Manganaris et al. (2005) used two different calcium salts toinvestigate the effects of calcium on fungistatic protection. Theyconcluded that calcium salts decreased brown rot symptoms inpeach compared to non-treated fruits. In another study, Biggs(1999) examined the effects of calcium salt solutions on two Col-letotrichum species and the lesion diameter of apple fruits followingcontrolled inoculations. The results verified the effect of calciumsalts on reduction of disease severity, expressed as lesion diameter.
In the case when a calcium salt solution could be utilized as afungistatic protection compound, it could provide the requiredtime for the host to enhance its own natural defense mechanisms(Conway and Sams, 1984).
Ripening-related softening of fruit is commonly related to thedissolution of the middle lamella and with modifications in thecomposition, structure and linkages between cell wall poly-saccharides (Vicente et al., 2007). Anthracnose symptoms are nor-mally not apparent at the time of harvest, but appear when thefruits are ripening or have ripened (Rohani, 1994). Calcium acts asan intermolecular binding agent that stabilizes pectineproteincomplexes of the middle lamella (Dey and Brinson, 1984). Cell walldegradation results in softening of fruit (Payasi et al., 2009). In thepresent study, preservation of structural integrity of cell walls mayhave arisen at least partly from calcium deposition in pectin poly-saccharides (Ortiz et al., 2011). This effect of calcium resulted inhigher firmness in papaya fruit (data not shown) which resulted inlower anthracnose disease incidence and severity.
Calcium is a safe and applicable method for postharvest diseasecontrol in papaya. Calcium salts may also have a significant role inthe future in disease management of papaya fruit during storage,especially for fruit subject to long distance transportation. Futurestudies are needed to explore pre-harvest calcium applicationsusing 1.5 and 2.0% concentrations in combination with postharvestdipping and infiltration of calcium to determine their combinedeffectiveness for managing papaya anthracnose.
B. Madani et al. / Crop Protection 55 (2014) 55e6060
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