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Control of Penicillium Digitatum on Orange Fruits with Calcium Chloride Dipping

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Control of Penicillium Digitatum on Orange Fruits with Calcium Chloride Dipping Journal of Microbiology Research and Reviews Vol. 2(6): 54-61, September, 2014 ISSN: 2350-1510 www.resjournals.org/JMR Control of Penicillium Digitatum on Orange Fruits with Calcium Chloride Dipping Zahra Ibrahim El-Gali Department of Plant Protection, Faculty of Agriculture, Omer Al-Mukhtar University, El-Beida Libya. Email for Correspondence: [email protected] ABSTRACT Citrus is one of the most widely grown plants in the world; however, it is affected by many types of fruit rot diseases after harvesting. Green mold disease of orange, caused by Penicillium digitatum Sacc, can cause extensive postharvest losses. The goal of this research was to use postharvest calcium applications to reduce fruit rot disease by soaking in solutions of CaCl2 and to test the effect of calcium on mycelial growth. Application of calcium chloride at 0.2%, 0.5% and 1.0% concentrations significantly decreased mycelial growth. In vivo studies showed fruit treatment by calcium soaking at 2% , 4% and 6% concentrations significantly reduced rots incidence of fruits. The optimal conditions of fungal growth were 25ºC and 87% relative humidity. Keywords: Citrus, green mold, calcium chloride, post-harvest treatment, control. INTRODUCTION Citrus is the second largest fruit crop worldwide (Spiegel-Roy and Goldschmidt, 1996) and it is one of the foremost export fruit Crops in Libya and in the rest of the world. It is primarily valued for its fruit, which is either consumed (sour orange, sweet orange, tangerine, grapefruit, etc.) or used in processing industry. All species have traditional medicinal value. Citrus has many other uses including animal fodder and craft and fuel wood (Manner et al., 2006). In addition, their essential oils are used in the cosmetic and pharmaceutical industries (Frazier and Westhofe, 1978) and a highly fermentable energy source with sweet taste and aroma (Lawal et al., 2013). Postharvest diseases play a major role in reducing the quantity and quality of citrus. Postharvest fungal decay may cause significant losses to the citrus industry worldwide (Holmes and Bckertn, 1999; Barkai-Gohan, 2001; Plaza et al., 2003). Injuries on citrus fruit caused during harvest, provide entries to wound pathogens, including Penicillium digitatum Sacc. and P. italicum Wehmer, causal agents of green and blue mold respectively. The mold invades the fruit much more rapidly and predominates in mixed infections, causing approximately 60-80% of decay (Palou et al., 2001; Skaria et al., 2003; Plaza et al., 2004). The growers normally use chemical fungicides to reduce the problem (Eckert, 1990). Attempts to find alternatives to chemical control were still ongoing because many fungi have become resistant to commonly used fungicides, so there is in creasing concern in respect to chemical residues on fruit products regarding health and environmental issues. However, alternative measures are generally less effective than fungicides. Simultaneous application of several physical and chemical methods could provide more effective means of control and consistent results than that of one approach alone. Some physical treatments and exogenous substances, such as chitosan, amino acids, antibiotics, calcium salts, and carbohydrates have also been used to enhance bio control of antagonists against fungal pathogens (Conway et al., 1999; El-Ghouthet al., 2000). Some studies revealed that application of CaCl2 at postharvest stage was also successful in controlling fungal infection. Calcium (Ca) dip was effective in inhibiting spore germination, thus providing a good control of C. gloeosporiodes infection on papaya (Eryani-Raqeeb et al., 2009) and on red-flesh dragon fruit (Hylocereus polyrhizus) 55 (Awang et al., 2011). Similar effect of Ca was reported to inhibit the growth of Botryosphaeria dothidea (Biggs et al., 1997) Botrytis cinerea (El-Gali, 2008) on apple and Monilinia fructicola on peach (Biggs, 2004). As the pathogenic fungi use carbon from sugars and acids of the host for their growth and development (Kamilova et al., 2006), and at the same time secreting cell wall degrading enzymes (Prusky et al., 2001), the quality of fungal infected fruit is expected to be reduced. With the possible role of Ca in inhibiting the growth of the fungus, fruit containing high concentration of Ca may be less susceptible to the fungal infection (Naradisorn, 2013).This study was conducted to determine the effects of postharvest application of different concentrations of calcium chloride on the occurrence of green mold of orange fruits and on mycelial growth of P. digitatum. The effect of different temperature and relative humidity on fungal growth was also studied in this work. MATERIALS AND METHODS Fruit source and isolation, identification of fungi Orange (Citrus sinensis L.) fruit both fresh and those found with symptoms of fungal infection were purchased from different markets located in El-Beida city, Libya, and brought to the Microbiology laboratory at Agriculture college University, Omer, Al-Mukhtar, for further studies. A total of 90 randomly selected fungal infected fruits and 90 unblemished, healthy and clean looking fruits were purchased. They were washed with water, their surface were sterilized by exposing them in 1% sodium hypochlorite and then rinsed three times in sterile distilled water. Segments (3-5 mm) of tissues from the margins of the rotted areas were cut out with a sterile scalpel and placed on previously prepared potato dextrose agar (PDA) in Petri dishes and incubated at 25 ± 1oC for 5 days under 12 h photoperiod. The fungus was purified by single spore isolation technique (Ho and Ko, 1997) and the purified isolates were maintained on PDA slants for further studies. The pathogen that appeared was primarily identified up to species level using cultural and morphological features under the light microscopic(Pitt, 1991;Frisvad and Samson, 2004). Optimal growth conditions of fungus Effect of Temperature Petri plates (9cm) containing 20 ml of PDA medium were inoculated with 5mm mycelia disc from ten day old culture of fungal isolate. The inoculated plates were incubated at different temperature: 15, 20, 25, 30, 35 and 40°C. Three replications were maintained for each treatment. Seven days after inoculation, the radius of each growing colony was measured in two directions at right angles to each other and the percentage in fungal growth was calculated in relative to its plate radius as described earlier (Bracanto and Golding, 1953). Effect of Relative Humidity (RH) The effect of relative humidity (RH) on the growth of pathogen was studied following the method described by Dhingra and Sinclair (1985) using PDA medium.5mm discs from the actively growing ten day old culture of the pathogen were placed in the center of the Petri plates. The inoculated plates were placed in the upper chambers of the desiccators, to prepare the required relative humidity regimes. Then 250ml each of distilled water, Ammonium chloride, Potassium bromide, Sodium carbonate, and Potassium nitrate were placed in the lower chambers of the desiccators and allowed to stand for 4hours to attain the relative humidity regimes of 100%, 91%, 87%, 82% and 77.5% respectively. Desiccators were kept at 25ºC. Three replications were maintained for each treatment. The radial growth was measured as described above. Effect of CaCl2 on fungal growth To assess the influence of CaCl2on mycelium growth of P. digitatum. it was directly added to PDA medium. Different concentrations:0.02, 0.05, 0.2, 0.5 and 1.0% (gm/ml) were solubilized in the PDA medium. 20 ml of each of the mixture from liquid media and CaCl2 were poured into Petri-dishes, then inoculated with the test fungus. PDA plates free of salt were used as control treatment. Three replications were maintained for each treatment. All plates were incubated at 25±1°C and the mean of the fungal colonies was measured at the end of 3 and 7 days and the standard deviation of 56 Figure 1. (A) Green mold on orange, (B) Culture of P. digitatum and (C) Conidiophores and conidia of P. digitatum. mean was also calculated. Effect of CaCl2 on fruit decay Fruits Preparation A total of 40 fruits used in the study chosen in the same size , free from diseases, micro cracks and without wounds were obtained from local market commercial in El-Beida city, Libya. The fruits were washed using tap water, dried at 22°C and then soaked accordingly in five concentrations of CaCl2 solutions (0, 2, 4and 6%) for 30 min. After the treatment, the fruits were left overnight in an open shelf at room temperature (22°C). The fruits were then washed again with distilled water and then rinsed. Fruits inoculation The fruits surface were sterilized by exposing them in 1% sodium hypochlorite and artificially wounded (1 mm wide and 2 mm deep) with sterile needles in the middle of each fruit and inoculated with 20 µl of P. digitatum (106 spores mL- 1).Control treatment were inoculated with distilled water. The inoculated fruits were placed on tray packs in plastic boxes containing moistened filter paper, covered with cling-films, and stored at 25˚C with two replicates of 5 fruits for each treatment. After 5 days, theradii of rotted symptoms were measured from inoculation site on the fruits surface (Janisiewicz et al. 1992). Experimental design and statistical analysis. All experiments were conducted on the basis of completely randomized designs (CRD). Each treatment was replicated at least three times. For statistical analysis, data were subjected to the analysis of variance (ANOVA) using Co Stat Program. Angular transformed values were used for data analysis. Statistical comparisons among means were performed using Duncan’s multiple range test (DMRT). RESULTS AND DISCUSSION Isolation and identification of the pathogen Symptoms The initial symptom of green mold is the appearance of a soft, watery and slightly discolored spot from 1/4 to 1/2 inch (2.5 cm) in diameter. This spot enlarges to 1 to 2 inches in diameter after 1 to 1.5 days at 24±1ºC.
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