Effects of calcium chloride treatment on suppression of fruit anthracnose disease caused by colletotrichum gloeosporioides

AGU International Journal of Sciences – 2019, Vol 7 (3), 75 – 83<br /> <br /> <br /> <br /> <br /> EFFECTS OF CALCIUM CHLORIDE TREATMENT ON SUPPRESSION OF FRUIT<br /> ANTHRACNOSE DISEASE CAUSED BY COLLETOTRICHUM GLOEOSPORIOIDES<br /> <br /> Le Thanh Toan1, Vo Thi Huong Duong2, Nguyen Thi My Linh1, Vo Trong Ky1, Trieu Phuong Linh1<br /> 1<br /> Can Tho University<br /> 2<br /> An Giang University, VNU - HCM<br /> <br /> Information: ABSTRACT<br /> Received: 25/02/2019<br /> Bananas, chilies and oranges are the most widely grown plants in Viet<br /> Accepted: 09/10/2019<br /> Nam. However, their fruits are severely affected by anthracnose disease<br /> Published: 11/2019<br /> caused by Colletotrichum gloeosporioides. Therefore, the objective of this<br /> Keywords: study was to evaluate the effects of CaCl2 on invitro mycelial growth and in<br /> Anthracnose, CaCl2, chili, vivo lesion diameters on pisang awak bananas, oranges and chillies .<br /> orange, pisang awak banana. Solution of CaCl2 at three concentrations of 20, 40 or 60 mM was mixed<br /> into medium of PDA at temperature of approximately 55-60ºC, gently<br /> shaken and poured into petri dish with a volume of approximately 10 mL<br /> per one petri. A hyphal round slice of Colletotrichum was put at the center<br /> of each petri dish. Diameter of colony was recorded at 2, 4 and 6 days after<br /> putting the fungal slice. Then, fully matured fruits were inoculated with a<br /> spore suspension of C. gloeosporioides at a density of 106 spore mL-1,<br /> treated with CaCl2 20mM by soaking the fruits for 30 seconds. The results<br /> showed that the solution of CaCl2 at concentrations of 20, 40 and 60 mM<br /> highly inhibited hyphal development of Colletotrichum at in vitro<br /> conditions. Moreover, the calcium chloride solution at 20 mM protected the<br /> fruits from anthracnose damage.<br /> <br /> <br /> 1. INTRODUCTION important and destructive disease in bananas,<br /> Fruits including bananas, oranges and chillies oranges and chillies. On unripe fruits, the<br /> form an important part of daily food supply in fungus forms quiescent infections. During the<br /> all countries and contribute to balanced ripening period, anthracnose symptoms appear,<br /> nutrition, health, stability and productivity. The develop and fruits are rot quickly.<br /> demand for daily consumption of those fruits Therefore, managing anthracnose disease is an<br /> results in an increase in their production. essential task of all farmers on the production<br /> Despite advances in fruit production and of bananas, chillies and oranges. The cost of<br /> disease management, many challenges always producing and protecting these produces<br /> face farmers cultivating bananas, oranges and continues to climb, placing an ever-increasing<br /> chillies. Disease-causing organnisms could economic pressure on farmers. Rising land use,<br /> reduce quality, yield, shelf-life and consumer water, equipments, fuel, fertilizers, pesticides<br /> satisfaction of these produce. Anthracnose and labor expenses force farmers to maximine<br /> caused by Colletotrichum gloeosporioides is an yields and seek economical ways to control<br /> <br /> <br /> 75<br /> AGU International Journal of Sciences – 2019, Vol 7 (3), 75 – 83<br /> <br /> diseases. A current challenge is the worldwide treatments could be obtained for consumer<br /> trend of reducing the use of certain pesticides in health and environmental safety.<br /> agricultural production. Due to environmental The effects of CaCl2 treatment were examined<br /> pollution and human health, some pesticides are on anthracnose of dragon, apple and papaya<br /> no longer available for use on treating fruits. On red-flesh dragon fruits, a soaking<br /> anthracnose on fruits. Besides, consumers application of CaCl2 solutions containing 1.0,<br /> always want fewer chemicals on their 2.0, 3.0 and 4.0 gL-1 highly elevated the fruit Ca<br /> agricultural products, but still demand high content in the fruit peels, but did not produce<br /> quality and a long shelf-life. A disease control significant effect on anthracnose incidence<br /> method that heavily depends on chemicals (Awang et al. 2011). In 2014, Stosic et al.<br /> requires a revision and development of novel showed that solutions of CaCl2 at 1.5% and<br /> safe and integrated strategies. Calcium chloride 2.0% significantly inhibited spore germination<br /> could be attributed to control anthracnose and hyphal growth of Colletotrichum acutatum<br /> lesions and prolong shelf-life of these fruits. and C. gloeosporioides which isolated from<br /> Calcium chloride is a nontoxic mineral, even in apple anthracnose lesions. On papaya fruits, a<br /> high concentrations, and is very effective in treatment of CaCl2 leads to a reduction of<br /> detoxifying high concentrations of other anthracnose lesion diameters in the infected<br /> mineral elements in plants. After entering ones (Madani et al. 2016; Ayon-Reyna et al.<br /> plants, the calcium content of plant tissues 2017). Tian et al. (2002) showed that an<br /> varies between 0.1-0.5% of dry weight addition of CaCl2 solution at 2% (w/v)<br /> depending on the growing conditions, plant significantly inhibited spore germination rates<br /> species and plant organs. Calcium readily enters and growth of the pathogen Rhizopus stolonifer<br /> the apoplasm and is bound in an exchangeable in vitro, as well as a lower disease incidence on<br /> form to cell walls and at the exterior surface of peach and nectarine fruits. A similar result was<br /> the plasma membrane. Most of its activity is reported on R. stolonifer and guava fruits with<br /> related to its capacity for co-ordination by CaCl2 at a concentration of 4% (Hassanein et al.<br /> which it provides stable but reversible 2018). Moreover, the efficacy of CaCl2 was<br /> intermolecular linkages; predorminantly in the evaluated on mycelial growth of the fungus<br /> cell walls and the plasma membrane. The Fusarium oxysporum f.sp. cepae, the causal<br /> proportion of Ca in the cell walls is an agent of onion rot (Turkkan, 2013), on fungal<br /> important factor for determining the growth of Alternaria alternata, Alternaria<br /> susceptibility of the fruit tissues to solani, Aspergillus niger, Botrytis cinerea,<br /> Colletotrichum infection. Calcium chloride Fusarium solani which isolated from guava<br /> treatments were studied on a reduction of biotic fruits (Hassanein et al. 2018), on growth of<br /> or abiotic stresses on some previous researches. Lasiodiplodia theobromae from mango (Nur<br /> Ilker and Morris (1975) found that treatments Fatimma et al. 2018).<br /> with resolutions of calcium salts could support The goal of this study is to assess efficacy of<br /> some protection against chilling injury in okra. calcium chloride treatment on growth of<br /> Similarly, the severity of chilling injury in Colletotrichum hyphae and diameter of<br /> squash (Cucurbita moschata) fruits, stored at 4 anthracnose lesions on banana, orange and<br /> oC, was reduced by dipping in 1% CaCl or 10<br /> 2 chilli fruits.<br /> mM sodium benzoate at 20 oC for 30 min (Lee<br /> & Yang 1999). The major advantage of CaCl2-<br /> <br /> <br /> 76<br /> AGU International Journal of Sciences – 2019, Vol 7 (3), 75 – 83<br /> <br /> 2. MATERIALS AND METHODS The experiment was carried out in CRD with<br /> 2.1 Materials two treatments including CaCl2-treated and<br /> control treatments, with 12 replications, one<br /> Fungal source of Colletotrichum<br /> fruit per one replication, four inoculated points<br /> gloeosporioides and CaCl2.2H2O (Merck,<br /> per one orange fruit, one inoculated point per<br /> Germany) were obtained from Department of<br /> one banana or chili fruit.<br /> Plant Protection, College of Agriculture, Can<br /> Tho University. The fungi were prepared on Experimental fruits in the experiment were<br /> Potato Dextrose Agar (PDA) at approximately uniform in color and had no damage on fruit<br /> 10-12 days before conducting experiments. peel. The fruits were then washed on tap water,<br /> cleaned by ethanol at a concentration of 70%<br /> 2.2 Methods<br /> (v/v), air-dried at room temperature. A bunch of<br /> 2.2.1 Assessment of calcium chloride efficacy four sterile needles (diameter of approximately<br /> on hyphal development of 3 mm) was used to create tiny holes with a<br /> Colletotrichum gloeosporioides in vitro depth of 3 mm of peel. After that, one mL of a<br /> The experiment was carried out in completely Colletotrichum spore suspension at a density of<br /> randomized design (CRD), with four treatments 106 spore mL-1 was dropped on these tiny holes.<br /> including CaCl2 at three concentrations of 20, Inoculated fruits were kept at an incubation<br /> 40, and 60 mM, and a water control treatment, chamber at 25 oC with relative humidity<br /> with six replications, one petri dish per one approximately 98% for 24 h. Later, inoculated<br /> replication. fruits were soaked on a solution of CaCl2<br /> The solution of CaCl2 was filtered by Whatman approximately 30 s. On the untreated control,<br /> papers with filter pores at approximately 0.2 the fruits were handled identically, but distilled<br /> µm. The filtered solution was then mixed into water was used instead of CaCl2. Finally,<br /> the medium of PDA at temperature inoculated fruits were put on transparent nilong<br /> approximately 55-60 oC. The mixture medium bags with wet cotton inside, at room<br /> was gently shaken and poured into petri dishes temperature (Cao et al., 2008; Hang, 2012;<br /> with a volume approximately 10 mL per one Talibi et al., 2012; Yu et al., 2012).<br /> petri. After the medium hardened, a hyphal Length of anthracnose lesions were recorded at<br /> round slice of Colletotrichum at 5 mm was put 4, 5, 6 and 7 days after inoculation (DAI)<br /> at the center of each petri dish (Dhinggra and (banana fruits), 4, 6, 8 and 10 DAI (orange and<br /> Sinclair, 1995). Diameter of colony was chilli fruits). Separated experiment on each kind<br /> recorded at 2, 4 and 6 days after putting fungal of fruits was repeated 3 times.<br /> slice. 2.3 Statistical analysis<br /> Each experiment was performed 3 times. Based All experiments were repeated three times, with<br /> on the results of the experiment, one effective similar results in all replications. Data were<br /> concentration of CaCl2 was chosen to conduct analyzed and subjected to analysis of variance<br /> following experiments on pisang awak banana, by SPSS software, version 16. The significance<br /> orange and chili fruits. of treatments was determined by the magnitude<br /> 2.2.2 Assessment of treating CaCl2 at an of F-value at P = 0.05. Treatment means were<br /> effective concentration on separated by Duncan’s Multiple Range Test<br /> banana/orange/chili fruits after an (DMRT) and t-test.<br /> inoculation with C. gloeosporioides<br /> <br /> <br /> 77<br /> AGU International Journal of Sciences – 2019, Vol 7 (3), 75 – 83<br /> <br /> 3. RESULTS AND DISCUSSION respectively, statistically different to those of<br /> 3.1 Efficacy of CaCl2 on Colletotrichum control treatment at 1.27 cm. At day 4 and 6,<br /> hyphal development in vitro only the treatment of 20 mM CaCl2 maintained<br /> a high efficacy on inhibitting hyphal<br /> The three concentrations of CaCl2 present<br /> development of C. gloeosporioides. Among all<br /> different colonial diameters at 2, 4 and 6 days<br /> calcium chloride concentrations tested, 20 mM<br /> after putting fungal slices (Table 1). At the first<br /> CaCl2 gave the best efficacy and was chosen to<br /> observation time point at day 2, two<br /> conduct following experiments on fruits of<br /> concentrations at 20 and 40 mM of CaCl2 had<br /> pisang awak banana, chilli and orange.<br /> shorter diameters at 0.65 cm and 0.58 cm,<br /> Table 1. Efficacy of CaCl2 on colonial diameter (cm) of C. gloeosporioides<br /> <br /> Days after putting fungal slices<br /> Treatment<br /> 2 1/ 4 1/ 6 1/<br /> Solution of CaCl2 at 20mM 0.65±0.07 c 2.30±0.06 c 3.08±0.06 c<br /> Solution of CaCl2 at 40mM 0.58±0.08 c 2.62±0.07 b 3.58±0.07 b<br /> Solution of CaCl2 at 60mM 1.08±0.09 b 2.58±0.07 b 3.68±0.07 b<br /> Control with distilled water 1.27±0.08 a 3.65±0.07 a 4.13±0.09 a<br /> Significance * * *<br /> Coefficient of variance (%) 13.63% 9.05% 5.64%<br /> 1/<br /> Mean ± SE (standard error) followed by the same letter do not differ significantly according to<br /> DMRT at P ≤ 0.05<br /> *: significant at p ≤ 0.05<br /> 3.2 Efficacy of treating CaCl2 after an inoculation of a Colletotrichum suspension on pisang<br /> awak banana fruits<br /> Efficacy of CaCl2 at 20 mM on anthracnose on pisang awak fruits is shown in Table 2. During four<br /> observation time points at 4, 5, 6 and 7 DAI, CaCl2-treatment had high efficacy on inhibiting the<br /> development of anthracnose disease, resulted in shorter lesion length, compared to those of the<br /> control treatment (Figure 1).<br /> Table 2. Length of anthracnose lesions (mm) caused by C. musae on pisang awak banana fruits<br /> <br /> Days after inoculation<br /> Treatment<br /> 4 1/ 5 1/ 6 1/ 7 1/<br /> CaCl2.2H2O at 20 mM 6.41±1.5 b 11.00±2.4 b 18.50±3.5 b 24.42±4.7 b<br /> Non-treated control 10.42±2.1 a 15.58±2.5 a 23.83±5.6 a 29.75±3.6 a<br /> Significance * * * *<br /> Coefficient of variance (%) 14.61 12.15 13.42 8.72<br /> <br /> <br /> <br /> 78<br /> AGU International Journal of Sciences – 2019, Vol 7 (3), 75 – 83<br /> 1/<br /> Mean ± SE (standard error) followed by the same letter do not differ significantly according to DMRT at P ≤ 0.05<br /> *: significant at p ≤ 0.05<br /> <br /> <br /> <br /> <br /> CaCl2 Ctr<br /> <br /> <br /> Figure 1. Efficacy of CaCl2 at a concentration of 20 mM on anthracnose lesions of pisang awak banana fruits<br /> at 7 DAI.<br /> <br /> 3.3 fficacy of treating CaCl2 after an inoculation of a Colletotrichum suspension on chili fruits<br /> Length of anthracnose lesions of calcium chloride treatment were 0.79, 1.39 and 1.82 cm at 4, 6 and<br /> 8 DAI, significantly lower than those of control treatment (Table 3 and Figure 2).<br /> Table 3. Length of anthracnose lesions caused by C. gloeosporioides on chili fruits<br /> <br /> Days after inoculation<br /> Treatment<br /> 4 1/ 6 1/ 8 1/<br /> CaCl2 at 20mM 0.79±0.21 b 1.39±0.31 b 1.82±0.38 b<br /> Non-treated control 1.82±0.27 a 1.97±0.28 a 2.18±0.32 a<br /> Significance * * *<br /> Coefficient of variance (%) 16.12% 14.58% 11.43%<br /> 1/ Mean ± SE (standard error) followed by the same letter do not differ significantly according to DMRT at P ≤ 0.05<br /> *: significant at p ≤ 0.05<br /> <br /> <br /> <br /> <br /> 79<br /> AGU International Journal of Sciences – 2019, Vol 7 (3), 75 – 83<br /> <br /> <br /> <br /> <br /> Figure 2. Efficacy of CaCl2 at a concentration of 20 mM on anthracnose lesions at 8 DAI.<br /> <br /> 3.4 Efficacy of treating CaCl2 after an inoculation of a Colletotrichum suspension on orange<br /> fruits<br /> Calcium chloride treatment protected orange fruits only at 4 DAI. At this time point, the length of<br /> anthracnose lesion of calcium chloride treatment was 30.42 mm, significantly different to that of<br /> control treatment at 40.25 mm. However, the efficacy of calcium chloride treatment was not<br /> prolonged (Table 4 and Figure 3).<br /> Table 4. Length of anthracnose lesions caused by C. gloeosporioides on orange fruits<br /> <br /> Days after inoculation<br /> Treatment<br /> 4 1/ 6 1/ 8 1/ 10 1/<br /> <br /> CaCl2 at 20 mM 30.42±2.67 b 68.25±7.22 111.83±8.31 154.17±11.22<br /> <br /> Non-treated control 40.25±2.92 a 65.00±6.93 100.50±9.18 146.00±13.25<br /> <br /> Significance * ns ns ns<br /> <br /> Coefficient of variance (%) 19.27 21.57 22.25 23.47<br /> 1/ Mean ± SE (standard error) followed by the same letter do not differ significantly according to DMRT at P ≤ 0.05<br /> *: significant at p ≤ 0.05<br /> <br /> <br /> <br /> <br /> 80<br /> AGU International Journal of Sciences – 2019, Vol 7 (3), 75 – 83<br /> <br /> <br /> <br /> <br /> Figure 3. Efficacy of CaCl2 at a concentration of 20 mM on anthracnose lesions at 10 DAI.<br /> <br /> The results showed that CaCl2 could be used on host epidermis and weeken the activity of<br /> controlling post-harvest anthracnose on pisang pathogen enzymes (Easterwood, 2002;<br /> awak banana, chilli and orange fruits. Our Netravati et al., 2018). The proportion of Ca in<br /> results are in line with previous studies of the fruit cell walls is important for determining<br /> Awang et al. (2011), Stosic et al. (2014), the susceptibility of the tissue of fungal<br /> Madani et al. (2016) and Ayon-Reyna et al. infections. On fruits, the Ca2+ content of the cell<br /> (2017). At in vitro conditions, the solution of walls increases to the fully-grown immature<br /> CaCl2 at concentrations of 1-2% reduced stage but this is followed by a drop in the<br /> mycelial growth and germination of C. content just before the onset of ripening. The<br /> gloeosporioides approximately 10-50% at 7 fundamental role of Ca2+ in membrane stability<br /> days after putting fungal slices (Stosic et al. and cell integrity in fruits is reflected in various<br /> 2014; Ayon-Reyna et al. 2017). These results ways. Calcium stabilizes cell membranes by<br /> are in accordance with in vitro result of this bridging phosphate and carboxylate groups of<br /> study, at approximately 11-25%. At in vivo phospholipid and protein, preferentially at<br /> conditions, CaCl2 1-4 gL-1 could not decrease membrane surfaces (Rose et al., 2003). The<br /> anthracnose incidence on red-flesh dragon membrane protecting effect of Ca2+ is most<br /> fruits. However, CaCl2 34, 67 and 100 mM prominent under various stress conditions such<br /> could reduce anthracnose lesion diameters on as Colletotrichum infection and low<br /> papaya fruits (Madani et al. 2016). The temperature (Reyes-Medina et al., 2017; Harris<br /> concentrations of CaCl2 in the research of et al., 2017). The degradation of pectates in cell<br /> Madani et al. were higher than the effective walls is mediated by polygalacturonase, which<br /> concentration of CaCl2 of this study. is drastically inhibited by high Ca2+<br /> Post-harvest pathogens including concentrations (Rose et al., 2003). A shift in the<br /> Colletotrichum spp. secrete enzymes such as binding stage of Ca2+ occurs in which water<br /> polygalacturonase, chitinase and pectate soluble Ca2+ in favoured over wall bound Ca2+,<br /> transeliminate to degrade host epidermis during keeping the adequate calcium level required for<br /> the period of pathogen infection. Calcium the maintenance of membrane integrity (Gayed<br /> chloride treatment enforces the thickness of et al., 2017; Netravati et al., 2018).<br /> <br /> <br /> <br /> 81<br /> AGU International Journal of Sciences – 2019, Vol 7 (3), 75 – 83<br /> <br /> Consequently, anthracnose lesions on calcium Dhinggra O.D. & J.B. Sinclair. (1995). Basic<br /> treatment develop slower than those of the non- plant pathology methods (2nd edition). New<br /> treated control. The result on this research York: CRC Press.<br /> demonstrated that CaCl2-treament limits a Easterwood, G. W. (2002). Canxi’s role in<br /> disinfection of cell walls and a collapse of the plant nutrition. Tampa, Florida, USA:<br /> affected tissues around Colletotrichum- Hydro Agri North America Inc.<br /> inoculated lesions on fruits.<br /> Gayed, A. A. N. A., S. A. M. A. Shaarawi, M.<br /> 4. CONCLUSION A. Elkhishen & N. R. M. Elsherbini. (2017).<br /> CaCl2 well inhibited the development of C. 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