Evaluate the Efficiency of Gamma Irradiation and Chitosan on Shelf-Life of Strawberries Fruits

Chitosan play an important role as an antifungal against Botrytis cinerea and the effect was a concentration dependent. The obtained results of in vitro experiment demonstrated that chitosan (4%) decreased radial growth of B. cinereato 2 %. Invivo the severity of infection reduced from 59.8 and 100.0 to 9.7, 33.8 and 40.1 in first, second and third week's storage periods at 13C, respectively. Also, chitosan coating (4%) significantly caused an increase in fruit firmness whereas TSS was decreased with an increase by increasing in storage time. However,Vitamin C gave fluctuated results by increasing storage time. Gamma irradiation at 2.5 KGy reduced severity (%) of infected fruits from 55.5, 100 and 100 to 31.7, 45.9 and 49.9 and in healthy fruits severity (%) reduced from 48.9, 100 and 100 to 23.3, 25.1 and 29.1 in different storage periods 1, 2 and 3 weeks, respectively. Similarly, chitosan as well as gamma irradiation combination induced a significant increase of peroxidase enzyme (POD) activity. Induced changes in surface morphology and damage of cell structure caused by using chitosan shown by scanning electron microscopy. Also, gamma irradiation causes changes in hyphea structure and in surface morphology but combination of gamma irradiation with chitosan was more effective in altering fungus morphology and cell structure damage and no spore forming. This providing the efficiency of combination on reducing disease severity (%) of strawberry.

INTRODUCTION Strawberries (Fragaria x ananassa Duch.) was a highly perishable fruit in a postharvest stage due to fungal infections. The shelf-life of fresh fruits at low temperature (0-4 o C) was around 5 days.
Braun and Sutton (1987) showed the postharvest decay represent major losses in horticultural industry. Losses during storage and shipment of fruits by Botrytis cinerea and Rhizopus stolonifer caused gray mould and soft rot, diseases, respectively.
Application of fungicides is most effective method to control postharvest disease. However, chemical control program face imminent problem first there are reports of on increasing number of fungicide-resistant strains of postharvest fungi and second due to health risk concerns. Thus, there is a growing need to one tactic that is being actively pursued involves: the use of bio-active substances (Tarek 2004).
Chitosan, a high molecular weight cationic polysaccharide has been shown to be fungicidal against several fungi (El-Ghaouth et al., 1990).
Vargas et al., (2006) found that, chitosan treatment of strawberry fruits delayed the occurrence of fungal infections compared with the uncoated fruits which started to decay from the beginning of storage.
Gianfranco Romanazzi, et al. (2013) found that the commercial chitosan formulation was effective in the control of gray mold and Rhizopus rot of strawberries when immersed in this solution and preserved for 4 days at 20±1C°.Shiekh, et al. (2013) confirmed that the chitosan is edible active coatings, maintain the quality and expand shelf-life of fresh fruits and prevent microbial damage.
Milena Petriccione et al. (2015); Reported that chitosan coating significantly reduced water loss and delayed the qualitative changes in color,titratable acidity and ascorbic acid content of strawberry also chitosan coating enhanced the activity of some antioxidant enzymes, preventing flesh browning and reducing membrane damage.
Chu et al. (2015); gamma irradiation was evaluated for its in vitro and in vivo antifungal activity against Botrytis cinerea on cut rose varieties. The irradiating dose required to reduce the population by 90% was 0.99 kGy. Gamma irradiation showed complete inhibition of spore germination and mycelia growth of B. cinerea especially 4.0 kGy in vitro.

Isolation purification in vitro antifungal activity of chitosan:
The antifungal activity of chitosan against Botrytis cinerea were determined using PDA plates amended with (1˛2 and 4%) chitosan. The PDA plates were prepared then inoculated with disks (3mm diameter) of fungal growth taken from 7 days old culture of Botrytis cinerea. The linear growth of the fungus was measured when control plates reached full growth.

Preparation of inoculums
Botrytis cinerea was isolated from infected Strawberries and maintained on Potato dextrose agar (PDA). Condia of B. cinerea were recovered by filtering the mycelial suspension of 2 weeks old culture through 3 layers of sterile cheese cloth. The concentration of the conidial suspension was adjusted to 2 x 10 5 conidia per mL.

In vivo antifungal activity of chitosan
Strawberries were immersed in a conidial suspension of B. cinerea containing 0.1% tween 80 and allowed to air dry at room temperature for 2 hrs. in order to fixed fungal infection. Different concentrations (0, 1, 2 and 4%) of chitosan were added individually to Erlenmeyer flasks (250ml capacity). Each contain 100 ml sterilized potato dextrose agar (PDA) media. The prepared media were poured in sterilized Petri dishes.
After solidification, the dishes were inoculated singly at the center with equal discs (3 mm diameter) of fungal growth taken from 10 days old culture grown on PDA medium incubated at 20 C. The linear growth of tested fungi was measured when the control plates reached full growth and the percentage of growth inhibition (%) calculated .Three replicates were used for each treatment.
After treated healthy and infected strawberries with chitosan or with gamma irradiation Strawberry fruits were examined for diseases assessment (Severity %) through different storage periods (weeks) under 13C.
Radiation: Strawberry fruits were exposed to different gamma irradiation doses 1.0, 1.5 and 2.5 KGy in Indian Co 60 gamma cell at the dose rate was 2.45kGy/hr at the time of experiment. Each treatment was replicated three times, each replicate contain 15 fruits. All treatments fruits and control were packed in perforated plastic containers and stored the Strawberry fruits were examined for disease assessment at different storage periods.
Chitosan treatment: chitosan solutions were prepared by dissolving 1, 2 and 4 gm of chitosan in 100 mL of distilled water with 2 mL acetic acid. Then heating with constantly agitation for 24 h. The obtained solution was adjusted to pH 5.5 by sodium hydroxide 0.1N; than 0.1 mL of tween 80 was added (El-Ghaouth et al., 1991). Sprays of the different coating chitosan concentrations were applied and then stored the treated fruits . Quality parameters: 1-Total soluble solids (TSS): TSS content expressed in 0 (Brix) was determined using a ago (Japan) NI refractometer according to Kader (1991). 2-Firmness: Firmness (Firm) was measured as the maximum penetration force reached during tissue breaking of each fruit with hand penetrometer equipped with 1-9 mm diameter plunger (g/Cm 2 ) according to Kader (1991).

3-Ascorbic acid (Vitamin C): Ascorbic acid content
was determined by titration in the presence of 2.6 dichlorophenol-indophenol dye as an indicator against 2% oxalic acid solution as substrate. Ascorbic acid was calculated as milligram L -ascorbic acid per 100 mL of juice as described by Lucoss (1994).

Determination of peroxidase activity:
Samples of infected strawberry fruits treated with each antioxidant at 8 g/L, caraway oil at 700 µl/L and 2.5 kGy radiation dose, were collected after 10 days storage at 13C for peroxidase activity assay. Also, infected fruits without treatment were used as control. Enzyme extract was obtained by grinding fruits tissues (2 ml/g fruits tissue) in 0.1 M sodium phosphate buffer at pH (7.1) in a porcelain mortar and extracted. The extracted tissues were strained through four layers of cheesecloth. Filtrates were centrifuged at 3000 rpm for 20 min. at 6C. The clear supernatants were collected and considered as crude enzyme extract. Peroxidase activity was expressed as changes in absorbance/min at 425 nm according to the method of Allam and Hollis (1972).Determination of peroxidase enzyme was conducted in Central Lab. of Biotechnology, Plant Pathology Research Institute, Agricultural Research Centre, Egypt. Scanning electron microscopy: Mycelia of B. cinerea grown in PD broth medium treated with chitosan 4 % and that from non-treated (control) were fixed in 2.5% glutaraldhyde at 4C for 24 hr and post-fixed in 1.0% osmium tetraoxide for one hr at room temperature (Harley and Fergusen, 1990). The specimens were then dehydrated with ascending concentrations of acetone, critical point dried, and finally sputter coated with gold. The examination and photographing was done through Joel Scanning Electron Microscope (JSM -1200 EX).

Conclusion
This study demonstrated that chitosan play an important role as an antifungal against Botrytis cinerea . Also, chitosan coating (4%) significantly caused an increase in fruit firmness whereas TSS was decreased with an increase by increasing in storage time. However ,Vitamin C gave fluctuated results by increasing storage time. Gamma irradiation at 2.5 KGy reduced severity (%) . but combination of gamma irradiation with chitosan was more effective in altering fungus morphology and cell structure damage and no spore forming. This providing the efficiency of combination on reducing disease severity (%) of strawberry.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
There is no Fund but This work was carried out in Food irradiation department, National center for Radiation Research and Technology, Atomic Energy Authority .and Mythological Res. and Plant Dis. Survey Dept., Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt

Experimental design and statistical analysis:
All treatments in this study were arranged in complete randomized design. The obtained data were subjected to analysis of variance using the general linear module procedure of SAS (1985), where appropriate treatment means were separated using Duncan's multiple range test (Duncan 1955) and all percentages were transferred to angles before statistical analysis.
The obtained data show that as chitosan % increased the severity % decreased. The lowest severity % obtained at 4% chitosan. Also as the storage period increase the severity % increased. Moreover, as storage period increase the severity (%) increased, and different doses of gamma ray decreased the severity (%) and at 2.5 KGy is the effective dose decrease severity (%) in different storage periods.

Effect of chitosan treatments concentrations, storage time (weeks) and Botrytis cinerea infection on some strawberries quality parameters.
Data in Table (3) show that interaction between storage time and chitosan treatments on quality parameters of strawberry fruits, Data indicate that, treating strawberries with chitosan significantly decreased the values of TSS by increasing storage time (1, 2, 3 weeks) while an opposite effect was obtained in firmness which increased by using chitosan coating at different concentrations (0, 1, 2 and 4%), since at 4% chitosan give the highest values of firmness at different storage periods. Vitamin gave fluctuated values by increasing storage time.  (4) show that combination effect of gamma ray (2.5 KGy) and chitosan (4%) on severity (%) of gray mold on strawberry fruits. The combination between gamma ray (2.5 KGy) and chitosan (4%) was more effective to reduce severity (%) as compared when we used chitosan (4%) alone or when used gamma rays at (2.5 KGy) alone, since combination reduced severity (%) from 55.5, 48.9 to 8.5, 2.1 for infected and healthy fruits respectively at first week, from 100.0, 100.0 to 19.9, 8.7 for infected and healthy fruits respectively at second week and at third week severity (%) of infected and healthy fruits decreased from 100.0, 100.0 to 24.7, 18.9 respectively.

Effect of gamma irradiation (2.5 kGy), chitosan (4% ) and combination between gamma irradiation and chitosan on peroxidase enzyme activity in strawberry fruits infected with B. cinerea and stored for one week
Results in Fig(2) Show that strawberry fruits inoculated with B.cinerea treated with combination of chitosan (4%) and gamma irradiation 2.5kGy induce higher activity of peroxidase (POD) enzyme. followed by chitosan(4%) and gamma irradiation 2.5 kGy irrespectively as compared with control fruits after one week storage periods .

Fig.2: Effect of gamma irradiation (2.5 kGy),chitosan (4%) and combination between gamma irradiation and chitosan on peroxidase enzyme activity in strawberry fruits infected with B. cinerea and stored for one week
Scanning electron microscopy Fig. (3) showed the morphological changes occurred in hyphae and conidiophores of B. cinerea treated with chitosan(4%) , gamma irradiation 2.5 kGy and combination between chitosan (4%) and gamma irradiation2.5kGy irrespectively It was found that control fungus B. cinerea have normal hyphea, sporangium, sporangiophore and normal cell wall and spore (Fig. 3A).
Chitosan treatment(4%) induced changes in surface morphology and cause damage to cell structure of B.cinerea and sporangiophore without spore (Fig. 3B).
Gamma irradiation induced changes in surface morphology and cause damage to hypha also an affected sporangiophore (Fig. 3C).
The combination effect of chitosan(4%) and gamma irradiation2.5kGy on B. cinerea show more destructive effect in surface morphology and more effective damage to cell structure , corrugate surface and no spore found (Fig. 3D).

IV. DISCUSSION
Several studies have been performed to extend strawberry fruits shelf-life, using alternative methods rather than chemicals to avoid residues such as fungicide residues for the fruit itself (Peng and sutton, 1991) and to avoid pathogen populations from developing resistance to pesticides (Bakkali et al., 2008).
Chitosan, a high molecular weight cationic polysaccharide, has been shown to be fungicidal against several fungi (El-Ghouth et al., 1990).
The obtained results show that chitosan (4%) reduced the severity % of gray mold on different storage period and these results are in agreement with Li and Yu (2000). Confirmed the potential effect of chitosan to protect postharvest brown rot of peach caused by M. fructicola by decreasing the incidence, prolonging the incubation period and reducing of brown rot is correlated with chitosan induction of defence response, in addition to its antifungal property. Romanazzi et al. (2000) reported that strawberries dipped in 1% and 0.5% chitosan decreased the gray mold infection from natural inoculum after 10-days storage at 0C. Followed by 4 days shelf-life. Casariego(2004) confirmed that chitosan films were also reported to inhibit the growth of fungi and yeasts in the area of contact, forming a halo of inhibition on the inoculated plates.
Atiaet al., (2005)suggested that the mechanism by which chitosan coating reduced that decay of strawberries appear to be related to its fungistatic property rather than to its ability to induce defense enzymes such as chitinase, chitosanase and -1,3-glucanase.and its capacity to stimulate plant defence mechanisms ( Aziz, et al.,2006) Ribeiro et al. (2007)explained that strawberry in non-climacteric fruits, but has a high postharvest respiration rate, which leads to a rapid deterioration at room temperature, coating with 1% chitosan reduced the growth rate of microorganisms in strawberries.
Romanazzi (2010)confirmed that pre-harvest and postharvest chitosan treatments of table grapes, strawberries and sweet cherries reduce their decay under field and during storage.
Besides its antifungal activity, chitosan also has the potential for inducing defenserelated enzymes (Bautista -Bonas et al.,2006) and phenolics in plants (Benhamou,1996 al. (2000)used chitosan as a semi-permeable coating and found that it can maintain the qualities of the treated fruit and prolong its storage life, chitosan slows down the aging process of peaches by decreasing respiration rate and ethylene production, reducing malondialldehyde(MDA) production, stimulate superoxide dismutase(SOD) activity and maintaining membrane integrity.
Chitosan has a double mechanism of action: it reduces the growth of decay causing fungi, and it induces resistance responses in host tissues. With this double effectiveness chitosan can be considered as the first compound of a new class of plant protection products (Atia et al., 2005). Hernandez-Lauzardoetal. (2011) demonstrate the mode of action of chitosan on different fungal pathogen . They reported that molecules of chitosan can penetrate the intracellular level and interact with intracellular structure and cause damage. Greater effects of chitosan to inhabit the growth of B. cinarea and cause serious damage to cell structure as well as the ability to form an impervious layer around the cell, therefore, chitosan could be considered as a potential alternative for synthetic fungicides (Silva Junior et  al.,2014). SEM show that chitosan causing changes on morphology of B. cinerea and cause damage to cell structure also gamma irradiation cause changes in surface morphology and cause damage to hypha also effect on sporangiophore but combination between chitosan(4%) and gamma irradiation 2.5 kGy show more destructive effect in surface morphology and more damage to cell structure. these result arein agreement with Swelim (2004) who confirmed that scanning electron microscope showed that the decrease in sporulation and morphology abnormalities of Fusarium solani were occurred after irradiation with 6 , 8 and 10 kGy. Meanwhile low dose levels of 1, 2 and 3 kGy cause malformation and compactness of mycelia as well as absence of sporulation in F. verticillioides.
Our results indicated that treating strawberries with chitosan significantly decreased the value of TSS by increasing storage time( weeks) while an opposite effect was obtained in firmness which increased by chitosan coating but vitamin C would not be detected in clear level of amounts. These results are in agreement with El -Gaouth (1991) and Luna et al., (2001) who reported that greater firmness of fruits such as strawberries, tomatoes and peaches were obtained when fruits coated with a chitosan. Also, Dam and Nguyen 2011 suggested that, all chitosan treatments enhanced the firmness of strawberries fruits compared to untreated fruits.
Gamma irradiation doses reduced the severity (%) of strawberry fruits in our obtained results and 2.5 kGy doses was the most effective doses decreased the severity % these obtained results are in agreement with Shadia and Ehab (2011) who confirmed that gamma radiation decreased the percentage of infection of strawberry fruits artificially inoculated with B. cinerea and naturally infected at 2.5 KGy compare with control.
The combination of chitosan and gamma radiation indicated that this treatment was the more effective in reducing severity (%) as compared when use every one alone.