BIO-CONTROL AND ULTRASTRUCTURE OF POST-HARVEST PATHOGENIC FUNGI OF APPLE FRUITS

S such as the increase of the electron density of the outer layer of the hyphal wall more than the control, also numerous big lipid bodies were almost occupied the cytoplasm. Eucalyptus citridora and Thymus capitatus had a potential as antifungal agent for biocontrol of post-harvest pathogenic fungi of tested apple fruits

Ismaelia, Egypt, were among the five fungal species tested for their antifungal activity Trichoderma herzianum was graciously provided by Plant Pathology Department, Faculty of Agriculture, ,Mansoura University.

Mixture of the best bio-control agents
E. citrodora and T. capitatus, the two most effective bio-control agents discovered, were put to the test singly or in combination; in the latter case, the two extracts were either given in half dose or full dose. The purpose of this experiment was to determine whether the combined effect of the two extracts is synergistic, additive, or antagonistic. It was decided to investigate the antifungal activity of five concentrations: 0, 1, 2, 3, and 5%.

Evaluation of antifungal activity In Vitro
According to Baka (2014), the food poisoning technique was applied with modificatin. The produced bio-control agents in various doses were tested against an isolate of Alternaria alternata found in apple fruits. For Alternaria alternata, the solidified extract-amended media in the Petri plates were inoculated, each alone at the centre with 7 mm inoculums disc of each tested fungus. The fungal growth's diameter (in cm) and the percentage of inhibition of fungal growth compared to the control were assessed. The fatal concentration that inhibits fungal growth by 50% was used to determine the relative efficacy of both plant extracts and microbial filtrates (LC50).

Antifungal activity of In Vivo
With slight adjustments, Badawy et al. (2012)'s method for evaluating the antifungal activity in vivo was used. Fresh apple fruits that were in good health were cleaned with tap water before being sterilised by immersion in 70% ethanol for 1 minute, followed by three times in sterile distilled water, and then allowed to dry. A 0.7 cm disc of the margins of recently developed A. alternata that was 7 days old and freshly grown was used to inoculate each treatment. Fruits that were roughly equivalent in weight and volume were randomly divided into 6 equal groups, each with 3 fruits. After 24 hours of A. alternata infection, six treatments were carried out as follows: spraying apple fruits with ethanol extracts of T. capitatus only at a concentration of 5%, E. citriodora only at a concentration of 10%, and a mixture of ethanol extracts of both E. citriodora and T. capitatus at a concentration of 2%. Two control sets were also prepared: a healthy set as a negative control, an infected Fruits that had been treated were placed in plastic bags and incubated for 28 days at 25°C and >85% RH. Every week, the effectiveness of the therapy was evaluated. There were three trials with three fruits each in each replication. By measuring the black zone's diameter and calculating the percentage of disease incidence compared to the infected control, researchers were able to assess the effectiveness of the treatment. Alternaria infection manifests as a black zone around the infected location.

Scanning Electron Microscopy (SEM)
According to Park et al., (2009) the samples were prepared for SEM observation. Fungal hyphae prior to sporulation were handled in the following manner in order to examine the impact of plant extract on the hyphae of both Alternaria alternata using SEM. First, colonies on both control and treatment plates had hyphal discs (diameter 1 cm) cut from the actively expanding margin. These discs were then fixed for two hours at room temperature with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH7.2). The fixed hyphal discs were then passed through a graduated ethanol series of 70, 80, and 90%, once for ten minutes at each concentration, before being washed twice for ten minutes each in the same buffer (three times; 30 min at each concentration). The samples were critical point dried with CO2 in a Polaron CPD 7501 critical point drying machine (VG Microtech, East Grinstead, UK). Then, using a sputter coater system in a high-vacuum chamber (Polaron SC7620, VG Microtech), the fixed material was mounted on stubs using double-sided carbon tape and coated with gold/palladium for 150 s at 9 mA. Using a JEOL model JSM-6510LV scanning electron microscope (JEOL Ltd., Tokyo, Japan), the samples were examined and digital pictures were recorded.

Transmission Electron Microscopy (TEM)
samples (1 mm3) of a fungal culture were evaluated and processed by TEM using Hayat's method (2000) using extracts of a combination of Eucalyptus citriodora and Thymus capitatus (0.3%, 1.5%) for A. alternata. The samples were initially submerged for two hours at four degrees Celsius in a solution of 3% (v/v) glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.0. They were then washed in the same buffer and post-fixed in 1% (w/v) OsO4. They were then imbedded in Spurr's resin after being dehydrated using a graduated series of ethanol solutions. On Formvar-coated copper grids, ultrathin slices were obtained, stained with uranyl acetate (UA) and lead citrate (LC), and then analysed using a JEOL (JEM-2100) transmission electron microscope (JEOL Ltd., Tokyo, Japan).

Statistical analysis
The inhibition zone of fungal growth estimated as percentage of the control was arcsine transformed before performing statistical analysis to ensure homogeneity of variance. Data were analyzed using SPSS version 22. Main separation was performed using the Duncan's multiple range tests at p˂0.05.

Isolation and identification of fungal pathogens
Three fungal infections were found to be infecting the apple fruit in the local markets of New Damietta, according to the preliminary examination into the occurrence of post-harvest deterioration of the fruit ( Table 2). The following isolated fungi attacked the apple fruits used for testing. Aspergillus niger, Penicillium expansum, and Alternaria alternata were discovered to be the three most prevalent fungus species (Table 2).

Antifungal activity of plant extracts
Five different concentrations (0, 1, 3, and 10%) of aqueous and ethanol extracts from eleven different plant species were used to investigate A's susceptibility to fungus development.A alternata. Table 3 showed that plant species and extract type had an impact on fungal growth as well as a highly significant variance in fungus susceptibility.  The concentration-response correlations of Figure 1 were used to compute the relative potency of the aqueous and ethanol extracts of the studied plant species on fungus growth in Table 5. In general, the LC50 value of the various species' ethanol extracts was significantly lower than that of the aqueous extract. The amount of fungal growth inhibition caused by the aqueous extract in the majority of A. alternata was too small to allow for the determination of the LC50. Only the most potent plannt species (Thymus capitatus), Eucalyptus citriodora, and Schinus terebinthifolius' LC50 of the aqueous extract could be estimated.

In Vivo activity of plant extracts on fruits
The most promising In Vitro results were used to guide the use of plant extracts in vivo, which was done to see whether there was a difference between the two types of research. Following infection by 24 hours, six treatments were administered, each lasting for 21 days, or until deterioration of the whole fruits in the infected control was noticed. According to Table (6), the type of treatment, the length of storage, and their interaction all had a highly significant impact on the fungal susceptibility. The diameter of the infected area, which is an indication to disease spreading, increased by extending of storage period. Furthermore, the results indicated that all the treatments significantly decreased the infected area during the storage period (21 days), compared with the untreated control. Trails of spraying the apple fruits with fungicide, E. citriodora only, T. capitatus only and the mixture of both showed a significant reduction in the infection of the fruit with different magnitude (Figure 2).    A and B) showed that the untreated A. alternata hyphae as observed by SEM appeared normal, with no deformity and normal conidia were observed with verruculose surface ornamentation. A filiform peak with smooth surface was also noticed (Plates 4B and C). In contrast, A. alternata hyphae treated with ethanolic extract of the mixture of both E. citriodora and T.capitatus revealed a strong detrimental effect of the extract on both the hyphal and spore morphology. Deformity in hyphae was observed in the form of flattened hyphae, in addition affected and abnormal spores were noted (Plates 5 A, B and C). Furthermore, the ultrastructure of A. alternata hyphae and conidia without treatment (control) as observed by TEM showed normal hyphae enclosed by a wall composed of three layers in which the middle layer is more electrondenser than the outer and inner layers. An intact plasma membrane was also observed. In addition, an electron-dense material was observed at the tip of the hyphae. The cytoplasm contained several organelles such as the nucleus, lipid bodies and vacuoles (Plates 6 A and B). On the other hand, the hyphae of A. alternata treated with the ethanolic extracts of the mixture of E. citriodora and T. capitatus exhibited many dramatic changes as noted in both T.S and L.S such as the increase in the electron density of the outer layer of the hyphal wall more than the control, also numerous big lipid bodies were almost occupied the cytoplasm. In addition, vacuoles were filled with small particles were also observed (Plates 7 A, B and C).  (W). Note that the hyphal wall became more electron-denser than that of the control. Note numerous lipid bodies (L) and electron-dense vesicles (VS) inside the hyphal cytoplasm. A septum (S) can also be seen. Bar = 2 µm. (B). Showing two hyphae (T. S. and L.S). Note granulated cell walls (W) and electron-dense layer (arrows) deposited on the outer surface of the wall. Note also big lipid bodies (L) are almost occupy the electrondense cytoplasm (CY). A hyphal septum can also be seen. Bar = 2 µm. (C). Showing two hyphae with thick granulated wall (W). Note an electron-dense layer deposited on the outer surface of the wall (arrows). Note also lipid bodies (L) and vacuoles (V) filled with small particles. Bar = 2 µm.

Post-harvest loss of fruits due to the fungal infection is considered a severe global problem in particular the developing countries (Baka et al., 2015)
In support to the present results, Amiri and Bompeix (2005) reported numerous Penicillium spp. associated with post-harvest fruit spoilage. Of these species, Penicillium expansum, P. digitatum, P. crustosum, and P. solitum had been recognized as the most frequent causative agents of apple spoilage (Kim et al.,  2005). Alternaria spp. is also a major fungal pathogen, which infect various local fruits such as apple. Several factors control fruit invasion by fungal pathogens especially after harvest. The traditional measure to limit this problem is the use of chemical fungicides to increase the shelf life time of fruits. But, due to their dangerous consequences on human health, biological control of fruit spoilage is the current trend to solve this problem (Tsoho, 2004; Enyiukwu et al., 2014). Many studies had reported the use of bio-control agents for post-harvest fruits diseases (Manjula et al., 2005). The eleven plant species tested in the present study exhibited diverse antifungal activities which varied according to the fungal species, plant species and type of solvent. In general, Eucalyptus citriodora and Thymus capitatus exhibited the most effective effect against A. alternata whereas Nicotiana glauca was the least effective. The differences in toxicity recorded between extracts are likely to be influenced by several factors such as the method of extraction, type of extracting solvent (the efficiency of the solvent to extract bioactive substances, variation in quantity of the active constituents and the difference in bioactive constituents between plants. The composition of bioactive compounds in turn vary from species to species, climatic conditions, and the physiological stage of plant development (Pandey, 2007). In this study, ethanol had a greater capability for the extraction of active substances from tested plants than did water which is in agreement with the results obtained by Stephan et al. (2005) and the postulation of Pandey (2007) that the type of solvent and the ability of the solvent in extraction affect the inhibitory activity of the plant extracts. The antifungal activity of Eucalyptus citroidora and Thymus capitatus can be related to the unique secondary bioactive compounds produced by the two species. In this respect, Lee (2007) reported the occurrence of several active antifungal compounds, like citronellal and isopulegol in Eycalyptus citriodora essential oil and ρ-cymene, γ-terpinene and thymol in Thymus capitatus. These active substances, because of their considerable lipophilicity, are subjected to extraction by ethanol to a greater extent than by water, which can partially explain the stronger antifungal efficiency of the ethanolic extract. In agreement with Shagal et al. (2012) reported that aqueous extracts and ethanolic of Eucalyptus spp. share some components, but differ in others. Both the aqueous and ethanolic extracts contain high amounts of saponins, while the aqueous extract contains tannins, saponins, glycosides, steroids and anthraquinones but no alkaloids, flavonoids and terpenoids; however, the ethanolic extract contains tannins and steroids but no glycosides and anthraquinone. The presence of these phytochemicals in Eucalyptus spp. justifies manipulation of the plant in the management and bio control of various diseases or spoilage fruits. Likewise, it had been reported that Thymus capitatus has a powerful antifungal activity by virtue of its high content of a wide range of bioactive compounds like essential oils which can act as biogenetic precursors of phenolic compounds such as ρ-cimene, γ-terpinene, and β-cariophyllene; in addition to its high content of phenols such as carvacrol (Mariateresa et al., 2013). The mechanism of action of carvacrol and thymol as fungicides appears to be through the inhibition of ergosterol biosynthesis and disruption of membrane integrity of the fungus as reported by In addition, phytochemical screening of Thymus capitatus revealed the presence of saponins, resins, flavonoids, essential and fixed oils; compounds of profound inhibitory effect fungi (Kandil et al., 1994). Effective bioefficiency of thyme essential oils against B. cinerea as post-harvest fungi on apple fruits (Banani et al., 2018). Seed extract of Moringa oleifera showed pronounced inhibition of linear growth of Alternaria alternata, A. solani, Fusarium oxysporum, F. solani and F. chlamydosporum, Rhizoctonia solani, Sclerotium rolfsii and Macrophomina phaseolina (Anwar et al., 2015). The inhibitory activity of aqueous extracts of Cyperus rotundus rhizomes, Melia azedrach leaves and Lantana camara leaves against Alternaria brassicae. aqueous extracts of ginger, turmeric, and garlic have been effective in reducing growth of A. alternata growth and disease.The major chemical components of C. rotundus are essential oils, flavonoids, terpenoids, sesquiterpenes, acopaene, cyprotene, cyperene, aselinene, rotundene, valencene, cyperol, gurjunene, trans-calamenene, decadinene, gcalacorene, cadalene, amuurolene, gmuurolene, cyperotundone, mustakone, isocyperol and acyperone (Imam et al.

2014)
The present work revealed that Nicotiana gluaca exhibited the least antifungal activity against A. alternata. The low activity of N. gluaca was well-demonstrated by Ochoa Fuentes et al. (2012).The antifungal activity of plant extracts may be related to the presence of many bioactive compounds such as flavonoids, terpenoids, alkaloids, tannins, steroids, glycosides and phenolics The function of phenolics is due to their amphipathicity which facilitate their interactions with biomembrane and thus induce the antimicrobial activity. The antifungal activity of alkaloids was already reported in several studies including different plant extracts (Veldhuzien et al. , 2006)). Results indicated that the values of LC50 of the ethanolic extracts are more frequent and of lower magnitude than those of the aqueous extracts and that Eucalyptus citriodora and Thymus capitatus yielded the lowest LC50 among the studied species. Normally, the lower the LC50 the more potent is the antifungal activity of the extract; and whenever an extract has no value for LC50 this means that the antifungal activity of this extract is too weak to the extent that the relative inhibition of fungal growth never attained 50% even at the top concentration used (10% w/v). The difference among pathogens in response to treatment with plant extracts may be attributed to their genetic or physiological differences The severity of disease in In Vivo can be reduced by combination of both chemical and bio-control agents as stated by Droby et al (1998) who found that tests in citrus packinghouses indicated that bio-control alone cannot provide adequate control and must be combined with diluted fungicides or other methods to control postharvest infection. The In Vivo test to control Alternaria rot disease in apple, demonstrated that the infection was in the form of lesions. This is in agreement with Vilanova et al.
(2012) who found that infection was in the form of lesions on fruits infected by Penicillium digitatum and lesions were not developed beyond the initial infection site. It was clear that all the treatments used in the present work showed a significant reduction in the infection of the fruits with different magnitude without completely destroying the pathogen. It is clear that, in general, the mixture of E. citriodora and T. capitatus was more potent. As far as the author is aware very little is known about the use of Penicillium roqueforti as a bio-control agent for the fungal post-harvest diseases of fruits. The antifungal activity of P. roqueforti against A. alternata explained on the basis of its ability to produce volatile terpenes such as, limonene, β-elemene and β-caryophyllene RI1494 (Baka , 2015). When the antifungal activity of ethanolic extracts of E. citriodora and T. capitatus were tested against A. alternata, either solitary or in combination at half or full dose, it was noticed that the full dose was more potent than the half one on A. alternata. Results indicated that the effect of the mixture of E. citriodora and T. capitatus was more effective on A. alternata. Furthermore, an increase in the density of the outer layer of the hyphal cell wall and appearance of numerous lipid bodies occupying the cytoplasm was observed in A. alternata. In addition, vacuoles filled with small particles were observed, but highly vacuolation and collapsed cytoplasm were observed in A. alternata. These findings are in agreement with those of Baka (2014) who referred to the inhibitory activity of plant extracts on the late blight disease of tomato to the reduction of mycelial growth and inhibition in spore germination of the pathogen in varying degrees and to the increase of vacuolization and lipid contents with consequent reduction of cytoplasm and alteration of cell wall and plasma lemma. The examination made with SEM are in accordance with those of Soylu et al. (2006) who verified that plant extracts caused the morphological alterations on the fungal hyphae of other plant pathogenic fungi. Generally, changes in the morphology of the hyphae could also be due to the loss of integrity of the cell wall. Consequently, plasma membrane permeability might be affected, which could explain the changes in the morphology and size of the internal organelles as suggested by Nakamura et al. (2007). Several studies attributed these abnormalities to the phenolic compounds since the amphipathicity of these compounds can explain their interactions with biomembrane and thus lead to the antimicrobial activity (Veldhuizen et al., 2006). Possible action mechanisms by which mycelial growth may be reduced or totally inhibited have been proposed. It is commonly accepted that the toxic effects of essential oils components of extracts on the functionality and structure of the cell membrane are responsible for the aforesaid activity (Sikkema et al., 1995). Omidbeygi et al. (2007) suggested that components of the essential oils and extracts cross the cell membrane, interacting with the enzymes and proteins of the membrane, and producing a flux of protons towards the cell exterior which induces changes in the cells and, ultimately, their death. In line with this, Soylu et al. (2006) and Cristani et al. (2007) reported that such antimicrobial activity is related to ability of terpenes to affect not only permeability but also other functions of cell membranes, these compounds might cross the cell membranes, thus penetrating into the interior of the cell and interacting with critical intracellular sites. Similarily, Lucini et al. (2006) indicated that mycelial growth inhibition is caused by the monoterpenes present in essential oils. These components would increase the concentration of lipidic peroxides such as hydroxyl, alkoxyl and alkoperoxyl radicals and so bring about cell death. The cyto-morphological modifications, particularly, the accumulation of lipid bodies and thickening of cell wall induced by the mixture of T. capitatus and E. citriodora extracts, is similar to those produced by some synthetic fungicides and other plant extracts (Bianchi et al., 1997). Increase in the size and number of vacuols along with other alternations might also, in turn, modify the activity of membrane enzymes involved in the formation of cell wall, causing anomalous development. However, the response to extracts seems to be different depending on the target agent used and this was clear A. alternata.

CONCLUSION
In conclusion, the antifungal activity of aqueous and ethanollic extracts of some medicinal plants was tested. Out of the tested medicinal plants the T. capitatus and E. citriodora where the most effective in the antifungal test. The obtained results revealed that this technique is eco-friendly and cheap in cost might be applied to control the post-harvest pathogenic fungi of apple.