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. White mycelium appears on the surface, and when the growing fungus is about 1 inch in diameter, olive green spores are produced. The entire fruit surface is rapidly covered with the olive green spores, which are easily spread if the fruit is handled or exposed to air currents (Figure 1- A.).

Cultural and morphological characteristics of the fungal isolates

On PDA, the fungus formed radial colonies of white airy mycelia, and the substrate mycelium was olive green with a clear zone observed from the bottom of the plate (Figure 1-B.).Conidiophores of the fungus are showing two-stage branching, cylindrical phialides and chains of single-celled conidia (Figure 1- C.). The isolate was confirmed by morphological and cultural characters which were Colony: Colour, shape, margins and pigmentation; Mycelium: Colour, 57

100 86 a 80 60 b 60 42.2 c 40

20 10 d 0 e 0 e

Mycelium growth (%) growth Mycelium 0 35 40 15 20 25 30

Temperature in ˚C

Figure 2. Effect of temperature on P. digitatum growth. Values of columns with different letters differ significantly at P<0.05

100 91.2 a 80 b 80 73.5 b 60 57.5 c 40 20 0 d

0 Mycelium growth (%) growth Mycelium 77 82 87 91 100 Relative humidity %

Figure 3. Effect of relative humidity on of P. digitatum growth. Values of columns with different letters differ significantly at P<0.05

shape, septation, branching; Conidia: Colour, shape, size and septation.Based on the symptoms, cultural and morphological description the fungus was identified as P. digitatum (Pitt, 1991;Frisvad and Samson, 2004).

Effect of temperature on fungal growth

The optimal temperature conditions for the growth of P. digitatum were assessed, and shown in (Figure 2).The isolates P. digitatum can grow on a wide range of temperatures from 15 to 30°C. However, it was observed that at 25°C the fungus attained maximum growth of 86% after 7 days of incubation. The growth of fungus was drastically reduced at 15°C and failed to grow at 35 – 40ºC.Temperature is most important physical environmental factor for regulating the growth and reproduction of fungi (Ibrahim et al. 2011). The results were supported by Larous et al. (2007), Kanan (2008) and Thiyam and Sharma (2014) who reported that 25°C was the optimum temperature for the growth of the genus of Penicillium spp. The optimum growth temperatures for the majority of fungi studied was found to fall between 25˚C to 30˚C and above 40˚C the growth was poor and in some cases mortality may occur (Sharma and Razak, 2003).Kausar et al. (2009) who reported that optimum temperature for the growth of the Fusarium solani and Lasiodiplodia theobromae fungi was 25ºC and also observed that the growth of both fungi was reduced at temperatures below and/or above 25ºC.

Effect of relative humidity on fungal growth

The effect of relative humidity on mycelial growth of P. digitatum was significant but varied with the %RH (Figure 3). All the relative humidity regimes tested supported the growth of the fungus. 58

7 days 3 days 100 a b a 80

60 c

40 a d

b a e 20 colony diameter (mm) diameter colony b e f 0 0 0.02 0.05 0.2 0.5 1

CaCl2 conctrations (%)

Figure 4. Effect of calcium chloride on P. digitatum linear growth. Significant differences (P < 0.05) between means were indicated by different letters above histogram bars.

However, at 87% RH was found to be most suitable for mycelial growth of P. digitatum that recorded 91.2% after seven days of incubation. Above this relative humidity 91-100% RH the growth of fungus was to be reduced. At 91%RH the growth of the fungus did not decrease so much and it was recorded 80%. This result was in agreement with those of El-Gali (1996) who reported that most of mold fungi were grown and sporulation in 80%RH and above. Larous et al. (2007) also obtained the maximum growth of P. expansum at 91% RH.

Effect of CaCl2 on fungal growth

Calcium chloride concentrations tested by agar diffusion plate method caused significant(P≥0.5) reduction in the growth of P. digitatum. The rate of growth reduction was directly proportional with the increase of concentration from 0.2- 1.0% in the medium at all days of incubation (Figure 4.).Slight rate of growth was observed in P. digitatum, at 1.0% of CaCl2 in the medium. This results were in harmony with those of Madani et al. (2014) who reported that concentrations of 1%, 1.5% and 2.0% of Calcium chloride significantly decreased spore germination of Colletotrichum gloeosporioides Penz., in another study, Ismail et al. (2010) reported that calcium chloride (2%) reduced the linear growth of Rhizopus stolonifer. The inhibition of linear growth may be due to fungistas is of spore pathogen led to reduction of pathogen conidia germination and germ tube elongation by limiting nutrients available to pathogens on the fruit surface (Moline, 1994). Under normal conditions, as fruit mature, there is increased availability of nutrients for pathogens on the surface of the fruit. However, calcium may enhance resistance of fruit to pathogens by interacting with cell wall components. Postharvest pathogens produce pectolytic enzymes, which cause softening of host tissues (Conway, et al., 1994). Calcium ions bind tightly to the pectins in the cell walls and produce cationic bridges between pectic acids, or between pectic acids and other acidic polysaccharides. These bridges make the cell walls less accessible to the action of pectolytic enzymes (Moline, 1994, Conway et al., 1994). Lower concentrations 0.02- 0.05% enhanced fungal growth (Figure 4.).Marin et al. (2002) found that suboptimal doses (0.03%) of all tested preservatives (potassium sorbate, calcium propionate and sodium benzoate) led to an enhancement of growth of Aspergillus and Penicillium isolates.

Effect of CaCl2 on fruit decay

This experiment was carried out to test the impact of calcium chloride concentrations on fruit decay. Results in (Figure 5) showed all treatments did not protect the fruits from rot infection. Fruit inoculated with 106 spores∙ml-1 of P. digitatum 59

120

100 a

80

60 b

40 c

Diseases severity % severity Diseases 20 d 0 0 2 4 6 CaCl2 concentration (%)

Figure 5. Effect of CaCl2 on green mold severity. Fruit were treated with of different concentrations of CaCl2, inoculated with P. digitatum and stored for 5 days at 25 °C. Significant differences (P < 0.05) between means were indicated by different letters above histogram bars

was completely decayed after 5 days of incubation. All concentrations gave less decay area due to P. digitatum than did the control treatment (0%). However, the size of lesion (infected symptom) on the inoculated fruits was reduced up to 90% as the CaCl2 concentration increased from 0 to 6 %.These results indicated that increased concentration of CaCl2 in the treatment solutions could inhibit disease development from the inoculation site, thus may reduce overall severity of the disease. Significant effects of post-harvest CaCl2 treatment were shown at higher concentrations rather than concentrations below 2 %. This result was in agreement with Conway et al.(1994) and Madani et al. (2014) who reported that the concentration 2% and above reduced decay on fruits. Similarly, Maouniet al. (2007) reported that in vitro, calcium chloride significantly reduced pear fruit decay caused by Alternaria and Penicillium expansum when used at 4 and 6%.The least disease incidence was found in those fruits infiltrated with 2.5% calcium. Hence, it can be concluded that postharvest infiltration of calcium at 2.5% has the potential to control disease incidence, prolong the storage life and preserve valuable attributes of postharvest papaya (Carica papaya L.), presumably because of its effects on inhibition of ripening and senescence process and loss of the fruit firmness of papaya (Mahmud et al., 2008). Awang et al. (2011) found that the disease severity of red-flesh dragon fruit (Hylocereus polyrhizus) soaked previously in solutions -1 containing 0, 1.0, 2.0, 3.0 and 4.0 g.L of CaCl2, then inoculated with spore suspension of Colletotrichum gloeosporioides deceased with increasing of Calcium concentration. Numerous investigations have suggested that many physiological and pathological disorders of apples are related to the content of calcium in their tissue (Fallahi et al. 1997; Naradisorn, 2013) and reduces decay caused by postharvest pathogens (Conway et al., 1991). Apple fruit cell walls with higher Ca concentrations are more resistant to cell wall degrading enzymes such as polygalacturonase and pathogens than cell walls with lower of calcium (Conway et al. 1994; Naradisorn, 2013). The role of calcium in maintaining firmness has been associated with plant disease resistance (Maas, 1998). It is suggested that addition of calcium to fruit can either enhance resistance of fruit to postharvest pathogens or reduce susceptibility to postharvest diseases and disorders.

CONCLUSION

Postharvest calcium treatment is a means of applying calcium directly to a fruit, and may be the best method of increasing flesh calcium content. This work suggests that Calcium is applied directly onto fruit by dipping in 6% CaCl2solution can be utilized in elevating Ca content in orange fruits and led to resist the infection by green mold. Low levels of calcium also tend to increase the chances of infection and growth of pathogen. A breeding program to develop calcium efficient cultivars may provide a long-term strategy for management of plant diseases. It is apparent from the 60

study that low temperature and low relative humidity do not support the growth of P. digitatum. It will provide the help to better understand the optimum conditions for the growth of associated pathogen. Therefore, it is highly recommended that fruits should be stored at low temperature and low relative humidity regimes to avoid infections due to P. digitatum and this may contribute significantly in controlling the heavy losses our peasant farmers incurred due to this fungus.

REFERENCES

Awang Y., AbdulGhani MA., Sijam K and Mohamad RB (2011). Effect of calcium chloride on anthracnose disease and postharvest quality of red-flesh dragon fruit (Hylocereus polyrhizus). African J. of Microbiol. Res., 5(29): 5250- 5259. Barkai-Golan R (2001). Postharvest diseases of fruits and vegetables. Development and control. 1st. Edn. Elsevier Science, Amsterdam, Netherlands. pp. 418. Biggs AR (2004). Effect of inoculum concentration and calcium salts on infection of apple fruit by Botryosphaeria dothidea.Plant Dis., 88: 147-151. Biggs AR., El-Kholi MM., El-Neshawy S and Nickerson R (1997). Effects of calcium salts on growth, polygalacturonase activity and infection of peach fruit by Monilinia fructicola. Plant Dis., 81: 399-403. Bracanto FP and Golding NS (1953). The diameter of the mold colony as a reliable measure of growth. Mycologia, 45: 848 Conway WS., Sams CE., Abbott JA and Bruton BD (1991). Postharvest calcium treatment of apple fruit to provide broad-spectrum protection against harvest pathogens. Plant Dis., 75:620-622. Conway SW., Sams CE., Wang CY and Abbott JA (1994). Additive effects of postharvest calcium and heat treatments on reducing decay and maintaining quality in apples. J. Am. Soc. Hort. Sci., 119(1): 49-53. Conway WS., Janisiewicz WJ., Klein JD and Sams CE (1999). Strategy for combining heat treatment, calcium infiltration, and biological control to reduce postharvest decay of ‘Gala’ apples. Hort Science, 34:700-704 Dhingra OD and Sinclair JB (1985). Basic plant pathology methods. CRC. Press, Inc. pp. 355. Eckert JW(1990) Recent development in chemical control of postharvest diseases. Acta Horticulture, 296:477-494. El-Gali ZI (1996). Aflatoxin contamination of some seeds crops in Al-Jabale Al-Akhtar area. MSc. Thesis. Omer Al- Mukhtar University. Plant Protection Department. El-Gali ZI (2008). Control of decay of apple fruits used by Calcium and Sodium chloride salts. Al-Mukhtar J. of Sci., 20:97- 111. El-Ghaouth A., Smilanick JL and Wilson CL (2000). Enhancement of the performance of Candida sitoana by the addition of glycolchitosan for the control of harvest decay of apple and citrus fruit. Postharvest Bio. and Technol.,19:103-110. Eryani-Raqeeb AA., Mahmud TM and Syed OS (2009). Effects of calcium and chitosan treatments on controlling anthracnose and postharvest quality of papaya (Carica papaya L.). Int. J. Agric. Res., 4: 53-68. Fallahi E., Conway WS., Hickey KD and Sams CE (1997). The role of calcium and nitrogen in postharvest quality and disease resistance of apple. Hort. Science, 32(5): 831-835. Frazier WC and Westhofe DC (1978). Contamination, preservation and spoilage of vegetables and fruits. Food Microbiology. New York. McGraw Hill Book Company, 3rd edition 12:194-214. Frisvad JC and Samson RA (2004). Polyphasic taxonomy of Penicillium subgenus Penicillium: A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins. Studies in Mycology, 49: 1-174. Ho WC and Ko WH. (1997). A simple method for obtaining single-spore isolates of fungi. Bot. Bull. Acad. Sin., 38:41- 44. Holmes GJ and Eckertm JW (1999). Sensitivity of Penicillium digitatum and P. italicum to postharvest citrus fungicides in California. Phytopathol., 89: 716- 721. Ibrahim M., Rabah AB., Liman B and Ibrahim NT (2011). Effect of temperature and relative humidity on growth of Helminthosporium flavum. Nigerian J. of Basic and App. Sci.. 19(1):127- 129. Ismail OM., Abd El-Moniem EA., Abd-Allah AS and El-Naggar MA (2010). Influence of some post-harvest treatments on guava fruits. Agric. Biol. J. N. Am.,1(6): 1309-1318. Janisiewicz WJ., Usall J and Bors B (1992). Nutritional enhancement of biocontrol of blue-mold on apples. Phytopathol., 82: 1364- 1370. Kamilova F., Kravchenko LV., Shaposhnikov AI., Makarova N and Lugtenberg B (2006). Effects of the tomato pathogen Fusarium oxysporum f.sp. radicis-lycopersici and of the biocontrol bacterium Pseudomonas fluorescens WCS365 on the composition of organic acids and sugar in tomato root exudates. Amer. Phytopathol. Soc., 19:1121-1126. Kanan GJ (2008). In vitro evaluation of Penicillium digitatum Sacc. Strains sensitivity to various fungicides from Jordan. Pakistan J. of Biol. Sci., 11(12):1517- 1529. Kausar P., Chohan S and Paveen R (2009). Physiological studies on Lasiodiplodia theobromae and Fusarium solani, the cause of shesham decline. Mycopath. 7(1): 35-38. Larous L., Hendel N., Abood JK and Ghoul M (2007). The growth and production of patulinmycotoxin by Penicillium expansum on apple fruits and its control by the use of propionic acid and sodium benzoait. Arab J. of Plant Protect., 61

25(1): 123-128. Lawal TE., Owoseni A., Atobatele OE., Ademola SG and Akomolafe DO (2013). Evaluation of the Nutritive Value of Citrus pulp Degraded with Penicillium notatum and Penicillium citrinum. Am. J. of Res. Comm., 12:1-11. Maas JL (1998). Compendium of strawberry diseases. 2nd ed.St. Paul, Minnesota, USA, APS Press.98 pp. Madani B., Mohamed MT., Biggs AR., Kadir J., Awang Y., Tayebimeigooni A and Shojaei TR (2014) Effect of pre- harvest calcium chloride applications on fruit calcium level and post-harvest anthracnose disease of papaya. Crop Protect., 55: 55- 60. Mahmud TM., Eryani-Raqeeb AL., Syed OS., Mohamed ZA and Al Eryani A (2008). Effects of Different Concentrations and Applications of Calcium on Storage Life and Physicochemical Characteristics of Papaya (Carica Papaya L.). Am. J. of Agric. and Bio. Sci., 3(3): 526-533 Manner HI., Buker RS., Smith VE., Ward D and Elevitch CR (2006)Citrus species (citrus). [Online] Available: http://www.Traditionaltree.Org. Maouni A., Lamarti A., Aidoun A., Khaddor M and Badoc A (2007) Effect of benzimidazolefungicides and calcium chloride on Alternaria and Penicillium expansum rot during storage of pears. Afr. J. Biotech. 6: 1289–1292. Marin S., Guynot ME., Sanchis V and Arbones J (2002) Aspergillus flavus , Aspergillus niger and Penicillium corylophilum spoilage prevention of bakery products by means of weak acid preservations. J. of Food Sci., 67: 2271- 2277. Moline HE (1994). Preharvest management for postharvest biological control. Pages 57-62 in: Biological Control of Postharvest Diseases - Theory and Practice. C. L. Wilson and M. E. Wisniewski, eds. CRC Press, Boca Raton, Florida, the United States of America. Naradisorn M (2013). Effect of calcium nutrition on fruit quality and postharvest diseases. International Journal of Science Innovations and Discoveries, 3(1): 8-13. Palou L, Smilanick JL, Usall J andViñas I(2001) Control of postharvest decay blue and green molds of oranges by hot water, sodium carbonate and sodium bicarbonate. Plant Dis., 85: 371-376. Pitt JI (1991). Laboratory guide to common Penicillium Species. Commonwealth Scientific and Industrial Research Organization. Food Research laboratory, N.S.W. Australia. Plaza P., Usall J., Teixide N and Vinas I (2003). Effect of water activity and temperature on germination and growth of Penicillium digitatum, P. italicum and Geotrichum candidum. J. Applied Microbiol., 94: 549-554. Plaza P., Sanbruno A., Usall J., Lamarca N., Torres R., Pons J and Vinas I (2004). Integration of curing treatments with decreeing to control the main postharvest diseases of Calementine mandarins. Postharvest. Boil. Technol., 34: 29-37. Prusky D., McEvoy JL., Levrentz B and Conway WS (2001). Local modulation of host pH by Colletotrichum species as a mechanism to increase virulence. Mol. Plant-Microbe Inter., 14:1105- 1113. Sharma R and Razak RC (2003). Keratinophilic fungi: Natural Keratin Degrading Machines; Their Isolation, Identification and Ecological role. Resonance, 28-30. Skaria M., Eayre CG, Miao H, Solis-Gracia N and Mackey B (2003). Effect of packing on rot and fruit damage in Rio Red grapefruit. Subtropical Plant Sci., 55: 75-78. Spiegely-Roy P and Goldschmidt EE (1996). Aspects of cultivated citrus: Rootstocks. Pages 127-129 in: Biology of citrus. Spiegel-Roy, P., Goldschmidt, E.E.; Eds. Cambridge University Press, Cambridge. Thiyam B and Sharma GD (2014). In vitro impact of temperature on the radial growth of pathogenic fungi. Indian Journal of Applied Research, 4(2): 4-5.