Bananas, chilies and oranges are the most widely grown plants in Viet Nam. However, their fruits are severely affected by anthracnose disease caused by Colletotrichum gloeosporioides. Therefore, the objective of this study was to evaluate the effects of CaCl2 on invitro mycelial growth and in vivo lesion diameters on pisang awak bananas, oranges and chillies .
AGU International Journal of Sciences – 2019, Vol (3), 75 – 83 EFFECTS OF CALCIUM CHLORIDE TREATMENT ON SUPPRESSION OF FRUIT ANTHRACNOSE DISEASE CAUSED BY COLLETOTRICHUM GLOEOSPORIOIDES Le Thanh Toan1, Vo Thi Huong Duong2, Nguyen Thi My Linh1, Vo Trong Ky1, Trieu Phuong Linh1 Can Tho University An Giang University, VNU - HCM Information: Received: 25/02/2019 Accepted: 09/10/2019 Published: 11/2019 Keywords: Anthracnose, CaCl2, chili, orange, pisang awak banana ABSTRACT Bananas, chilies and oranges are the most widely grown plants in Viet Nam However, their fruits are severely affected by anthracnose disease caused by Colletotrichum gloeosporioides Therefore, the objective of this study was to evaluate the effects of CaCl2 on invitro mycelial growth and in vivo lesion diameters on pisang awak bananas, oranges and chillies Solution of CaCl2 at three concentrations of 20, 40 or 60 mM was mixed into medium of PDA at temperature of approximately 55-60ºC, gently shaken and poured into petri dish with a volume of approximately 10 mL per one petri A hyphal round slice of Colletotrichum was put at the center of each petri dish Diameter of colony was recorded at 2, and days after putting the fungal slice Then, fully matured fruits were inoculated with a spore suspension of C gloeosporioides at a density of 106 spore mL-1, treated with CaCl2 20mM by soaking the fruits for 30 seconds The results showed that the solution of CaCl2 at concentrations of 20, 40 and 60 mM highly inhibited hyphal development of Colletotrichum at in vitro conditions Moreover, the calcium chloride solution at 20 mM protected the fruits from anthracnose damage INTRODUCTION important and destructive disease in bananas, oranges and chillies On unripe fruits, the fungus forms quiescent infections During the ripening period, anthracnose symptoms appear, develop and fruits are rot quickly Fruits including bananas, oranges and chillies form an important part of daily food supply in all countries and contribute to balanced nutrition, health, stability and productivity The demand for daily consumption of those fruits results in an increase in their production Despite advances in fruit production and disease management, many challenges always face farmers cultivating bananas, oranges and chillies Disease-causing organnisms could reduce quality, yield, shelf-life and consumer satisfaction of these produce Anthracnose caused by Colletotrichum gloeosporioides is an Therefore, managing anthracnose disease is an essential task of all farmers on the production of bananas, chillies and oranges The cost of producing and protecting these produces continues to climb, placing an ever-increasing economic pressure on farmers Rising land use, water, equipments, fuel, fertilizers, pesticides and labor expenses force farmers to maximine yields and seek economical ways to control 75 AGU International Journal of Sciences – 2019, Vol (3), 75 – 83 diseases A current challenge is the worldwide trend of reducing the use of certain pesticides in agricultural production Due to environmental pollution and human health, some pesticides are no longer available for use on treating anthracnose on fruits Besides, consumers always want fewer chemicals on their agricultural products, but still demand high quality and a long shelf-life A disease control method that heavily depends on chemicals requires a revision and development of novel safe and integrated strategies Calcium chloride could be attributed to control anthracnose lesions and prolong shelf-life of these fruits treatments could be obtained for consumer health and environmental safety The effects of CaCl2 treatment were examined on anthracnose of dragon, apple and papaya fruits On red-flesh dragon fruits, a soaking application of CaCl2 solutions containing 1.0, 2.0, 3.0 and 4.0 gL-1 highly elevated the fruit Ca content in the fruit peels, but did not produce significant effect on anthracnose incidence (Awang et al 2011) In 2014, Stosic et al showed that solutions of CaCl2 at 1.5% and 2.0% significantly inhibited spore germination and hyphal growth of Colletotrichum acutatum and C gloeosporioides which isolated from apple anthracnose lesions On papaya fruits, a treatment of CaCl2 leads to a reduction of anthracnose lesion diameters in the infected ones (Madani et al 2016; Ayon-Reyna et al 2017) Tian et al (2002) showed that an addition of CaCl2 solution at 2% (w/v) significantly inhibited spore germination rates and growth of the pathogen Rhizopus stolonifer in vitro, as well as a lower disease incidence on peach and nectarine fruits A similar result was reported on R stolonifer and guava fruits with CaCl2 at a concentration of 4% (Hassanein et al 2018) Moreover, the efficacy of CaCl2 was evaluated on mycelial growth of the fungus Fusarium oxysporum f.sp cepae, the causal agent of onion rot (Turkkan, 2013), on fungal growth of Alternaria alternata, Alternaria solani, Aspergillus niger, Botrytis cinerea, Fusarium solani which isolated from guava fruits (Hassanein et al 2018), on growth of Lasiodiplodia theobromae from mango (Nur Fatimma et al 2018) Calcium chloride is a nontoxic mineral, even in high concentrations, and is very effective in detoxifying high concentrations of other mineral elements in plants After entering plants, the calcium content of plant tissues varies between 0.1-0.5% of dry weight depending on the growing conditions, plant species and plant organs Calcium readily enters the apoplasm and is bound in an exchangeable form to cell walls and at the exterior surface of the plasma membrane Most of its activity is related to its capacity for co-ordination by which it provides stable but reversible intermolecular linkages; predorminantly in the cell walls and the plasma membrane The proportion of Ca in the cell walls is an important factor for determining the susceptibility of the fruit tissues to Colletotrichum infection Calcium chloride treatments were studied on a reduction of biotic or abiotic stresses on some previous researches Ilker and Morris (1975) found that treatments with resolutions of calcium salts could support some protection against chilling injury in okra Similarly, the severity of chilling injury in squash (Cucurbita moschata) fruits, stored at oC, was reduced by dipping in 1% CaCl or 10 mM sodium benzoate at 20 oC for 30 (Lee & Yang 1999) The major advantage of CaCl2- The goal of this study is to assess efficacy of calcium chloride treatment on growth of Colletotrichum hyphae and diameter of anthracnose lesions on banana, orange and chilli fruits 76 AGU International Journal of Sciences – 2019, Vol (3), 75 – 83 MATERIALS AND METHODS The experiment was carried out in CRD with two treatments including CaCl2-treated and control treatments, with 12 replications, one fruit per one replication, four inoculated points per one orange fruit, one inoculated point per one banana or chili fruit 2.1 Materials Fungal source of Colletotrichum gloeosporioides and CaCl2.2H2O (Merck, Germany) were obtained from Department of Plant Protection, College of Agriculture, Can Tho University The fungi were prepared on Potato Dextrose Agar (PDA) at approximately 10-12 days before conducting experiments Experimental fruits in the experiment were uniform in color and had no damage on fruit peel The fruits were then washed on tap water, cleaned by ethanol at a concentration of 70% (v/v), air-dried at room temperature A bunch of four sterile needles (diameter of approximately mm) was used to create tiny holes with a depth of mm of peel After that, one mL of a Colletotrichum spore suspension at a density of 106 spore mL-1 was dropped on these tiny holes Inoculated fruits were kept at an incubation chamber at 25 oC with relative humidity approximately 98% for 24 h Later, inoculated fruits were soaked on a solution of CaCl2 approximately 30 s On the untreated control, the fruits were handled identically, but distilled water was used instead of CaCl2 Finally, inoculated fruits were put on transparent nilong bags with wet cotton inside, at room temperature (Cao et al., 2008; Hang, 2012; Talibi et al., 2012; Yu et al., 2012) 2.2 Methods 2.2.1 Assessment of calcium chloride efficacy on hyphal development of Colletotrichum gloeosporioides in vitro The experiment was carried out in completely randomized design (CRD), with four treatments including CaCl2 at three concentrations of 20, 40, and 60 mM, and a water control treatment, with six replications, one petri dish per one replication The solution of CaCl2 was filtered by Whatman papers with filter pores at approximately 0.2 µm The filtered solution was then mixed into the medium of PDA at temperature approximately 55-60 oC The mixture medium was gently shaken and poured into petri dishes with a volume approximately 10 mL per one petri After the medium hardened, a hyphal round slice of Colletotrichum at mm was put at the center of each petri dish (Dhinggra and Sinclair, 1995) Diameter of colony was recorded at 2, and days after putting fungal slice Length of anthracnose lesions were recorded at 4, 5, and days after inoculation (DAI) (banana fruits), 4, 6, and 10 DAI (orange and chilli fruits) Separated experiment on each kind of fruits was repeated times 2.3 Statistical analysis Each experiment was performed times Based on the results of the experiment, one effective concentration of CaCl2 was chosen to conduct following experiments on pisang awak banana, orange and chili fruits All experiments were repeated three times, with similar results in all replications Data were analyzed and subjected to analysis of variance by SPSS software, version 16 The significance of treatments was determined by the magnitude of F-value at P = 0.05 Treatment means were separated by Duncan’s Multiple Range Test (DMRT) and t-test 2.2.2 Assessment of treating CaCl2 at an effective concentration on banana/orange/chili fruits after an inoculation with C gloeosporioides 77 AGU International Journal of Sciences – 2019, Vol (3), 75 – 83 RESULTS AND DISCUSSION respectively, statistically different to those of control treatment at 1.27 cm At day and 6, only the treatment of 20 mM CaCl2 maintained a high efficacy on inhibitting hyphal development of C gloeosporioides Among all calcium chloride concentrations tested, 20 mM CaCl2 gave the best efficacy and was chosen to conduct following experiments on fruits of pisang awak banana, chilli and orange 3.1 Efficacy of CaCl2 on Colletotrichum hyphal development in vitro The three concentrations of CaCl2 present different colonial diameters at 2, and days after putting fungal slices (Table 1) At the first observation time point at day 2, two concentrations at 20 and 40 mM of CaCl2 had shorter diameters at 0.65 cm and 0.58 cm, Table Efficacy of CaCl2 on colonial diameter (cm) of C gloeosporioides Days after putting fungal slices Treatment 1/ 1/ 1/ Solution of CaCl2 at 20mM 0.65±0.07 c 2.30±0.06 c 3.08±0.06 c Solution of CaCl2 at 40mM 0.58±0.08 c 2.62±0.07 b 3.58±0.07 b Solution of CaCl2 at 60mM 1.08±0.09 b 2.58±0.07 b 3.68±0.07 b Control with distilled water 1.27±0.08 a 3.65±0.07 a 4.13±0.09 a * * * 13.63% 9.05% 5.64% Significance Coefficient of variance (%) 1/ Mean ± SE (standard error) followed by the same letter not differ significantly according to DMRT at P ≤ 0.05 *: significant at p ≤ 0.05 3.2 Efficacy of treating CaCl2 after an inoculation of a Colletotrichum suspension on pisang awak banana fruits Efficacy of CaCl2 at 20 mM on anthracnose on pisang awak fruits is shown in Table During four observation time points at 4, 5, and DAI, CaCl2-treatment had high efficacy on inhibiting the development of anthracnose disease, resulted in shorter lesion length, compared to those of the control treatment (Figure 1) Table Length of anthracnose lesions (mm) caused by C musae on pisang awak banana fruits Days after inoculation Treatment CaCl2.2H2O at 20 mM Non-treated control Significance Coefficient of variance (%) 1/ 1/ 1/ 1/ 6.41±1.5 b 11.00±2.4 b 18.50±3.5 b 24.42±4.7 b 15.58±2.5 a 23.83±5.6 a 29.75±3.6 a 10.42±2.1 a * * * * 14.61 12.15 13.42 8.72 78 AGU International Journal of Sciences – 2019, Vol (3), 75 – 83 1/ Mean ± SE (standard error) followed by the same letter not differ significantly according to DMRT at P ≤ 0.05 *: significant at p ≤ 0.05 CaCl2 Ctr Figure Efficacy of CaCl2 at a concentration of 20 mM on anthracnose lesions of pisang awak banana fruits at DAI 3.3 fficacy of treating CaCl2 after an inoculation of a Colletotrichum suspension on chili fruits Length of anthracnose lesions of calcium chloride treatment were 0.79, 1.39 and 1.82 cm at 4, and DAI, significantly lower than those of control treatment (Table and Figure 2) Table Length of anthracnose lesions caused by C gloeosporioides on chili fruits Days after inoculation Treatment 1/ 1/ 1/ CaCl2 at 20mM 0.79±0.21 b 1.39±0.31 b 1.82±0.38 b Non-treated control 1.82±0.27 a 1.97±0.28 a 2.18±0.32 a Significance Coefficient of variance (%) 1/ Mean * * * 16.12% 14.58% 11.43% ± SE (standard error) followed by the same letter not differ significantly according to DMRT at P ≤ 0.05 *: significant at p ≤ 0.05 79 AGU International Journal of Sciences – 2019, Vol (3), 75 – 83 Figure Efficacy of CaCl2 at a concentration of 20 mM on anthracnose lesions at DAI 3.4 Efficacy of treating CaCl2 after an inoculation of a Colletotrichum suspension on orange fruits Calcium chloride treatment protected orange fruits only at DAI At this time point, the length of anthracnose lesion of calcium chloride treatment was 30.42 mm, significantly different to that of control treatment at 40.25 mm However, the efficacy of calcium chloride treatment was not prolonged (Table and Figure 3) Table Length of anthracnose lesions caused by C gloeosporioides on orange fruits Days after inoculation Treatment 1/ 1/ 1/ 10 1/ CaCl2 at 20 mM 30.42±2.67 b 68.25±7.22 111.83±8.31 154.17±11.22 Non-treated control 40.25±2.92 a 65.00±6.93 100.50±9.18 146.00±13.25 * ns ns ns 19.27 21.57 22.25 23.47 Significance Coefficient of variance (%) 1/ Mean ± SE (standard error) followed by the same letter not differ significantly according to DMRT at P ≤ 0.05 *: significant at p ≤ 0.05 80 AGU International Journal of Sciences – 2019, Vol (3), 75 – 83 Figure Efficacy of CaCl2 at a concentration of 20 mM on anthracnose lesions at 10 DAI The results showed that CaCl2 could be used on controlling post-harvest anthracnose on pisang awak banana, chilli and orange fruits Our results are in line with previous studies of Awang et al (2011), Stosic et al (2014), Madani et al (2016) and Ayon-Reyna et al (2017) At in vitro conditions, the solution of CaCl2 at concentrations of 1-2% reduced mycelial growth and germination of C gloeosporioides approximately 10-50% at days after putting fungal slices (Stosic et al 2014; Ayon-Reyna et al 2017) These results are in accordance with in vitro result of this study, at approximately 11-25% At in vivo conditions, CaCl2 1-4 gL-1 could not decrease anthracnose incidence on red-flesh dragon fruits However, CaCl2 34, 67 and 100 mM could reduce anthracnose lesion diameters on papaya fruits (Madani et al 2016) The concentrations of CaCl2 in the research of Madani et al were higher than the effective concentration of CaCl2 of this study Post-harvest pathogens including Colletotrichum spp secrete enzymes such as polygalacturonase, chitinase and pectate transeliminate to degrade host epidermis during the period of pathogen infection Calcium chloride treatment enforces the thickness of host epidermis and weeken the activity of pathogen enzymes (Easterwood, 2002; Netravati et al., 2018) The proportion of Ca in the fruit cell walls is important for determining the susceptibility of the tissue of fungal infections On fruits, the Ca2+ content of the cell walls increases to the fully-grown immature stage but this is followed by a drop in the content just before the onset of ripening The fundamental role of Ca2+ in membrane stability and cell integrity in fruits is reflected in various ways Calcium stabilizes cell membranes by bridging phosphate and carboxylate groups of phospholipid and protein, preferentially at membrane surfaces (Rose et al., 2003) The membrane protecting effect of Ca2+ is most prominent under various stress conditions such as Colletotrichum infection and low temperature (Reyes-Medina et al., 2017; Harris et al., 2017) The degradation of pectates in cell walls is mediated by polygalacturonase, which is drastically inhibited by high Ca2+ concentrations (Rose et al., 2003) A shift in the binding stage of Ca2+ occurs in which water soluble Ca2+ in favoured over wall bound Ca2+, keeping the adequate calcium level required for the maintenance of membrane integrity (Gayed et al., 2017; Netravati et al., 2018) 81 AGU International Journal of Sciences – 2019, Vol (3), 75 – 83 Consequently, anthracnose lesions on calcium treatment develop slower than those of the nontreated control The result on this research demonstrated that CaCl2-treament limits a disinfection of cell walls and a collapse of the affected tissues around Colletotrichuminoculated lesions on fruits Dhinggra O.D & J.B Sinclair (1995) Basic plant pathology methods (2nd edition) New York: CRC Press Easterwood, G W (2002) Canxi’s role in plant nutrition Tampa, Florida, USA: Hydro Agri North America Inc Gayed, A A N A., S A M A Shaarawi, M A Elkhishen & N R M Elsherbini (2017) Pre-harvest application of calcium chloride and chitosan on fruit quality and storability of ‘Early Swelling’ peach during cold storage Ciencia e Agrotecnologia ,2, 220223 CONCLUSION CaCl2 well inhibited the development of C gloeosporioides in vitro At in vivo condition, calcium treatment protected pisang awak banana, chili fruits from anthracnose damage However, in fruit production, the farmers need a highly efficient treatment in controlling anthracnose Therefore, soaking fruits in CaCl2 20 mM for 30 s can further studied as a postharvest tool to manage anthracnose Hang L.T.T (2013) Diagnosis of post-harvest fungal pathogens on banana fruits (Musa sapientum L.) 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Colletotrichum suspension on orange fruits Calcium chloride treatment protected orange fruits only at DAI At this time point, the length of anthracnose lesion of calcium chloride treatment was 30.42... that of control treatment at 40.25 mm However, the efficacy of calcium chloride treatment was not prolonged (Table and Figure 3) Table Length of anthracnose lesions caused by C gloeosporioides on