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RESEARCH ARTICLE BIOSCIENCE RESEARCH, 2019 16(2):2104-2118. OPEN ACCESS

First record of black spot disease infecting Guava fruits in Egypt and its pre and post-harvest management Mokhtar Mohamed Abdel-Kader, Nehal Samy El-Mougy and Mohamed Saied Ali Khlil Plant Pathology Dept., National Research Centre, Dokki, 12622, Giza , Egypt *Correspondence: [email protected] Accepted: 04 May 2019 Published online: 16 June. 2019

Occurrence of black spot on Guava fruits was observed at several commercial markets in Egypt. Isolation, identification and pathogenicity tests revealed that the causal fungus of black fruit spot disease of Guava is Guignardia mangiferae. Results of surveyed guava trees grown at different field localities of North Egypt, for fruit black spot disease incidence revealed that the maximum records of disease occurrence was recorded at some fields belong to Alexandria governorate followed by 3.7 and 3.3% at Kafer El-Sheekh and Beheira governorates, meanwhile lesser occurrence was observed as 2.6, 1.2% at Domiat and Qualiobia governorates, respectively. In vitro highly significant complete reduction (100%) in fungal growth at treatments of propolis dissolved in either acetic or lactic acids, chitosan dissolved in acetic acid, Beeswax suspended in water or ethanol and combined treatments of propolis and chitosan dissolved in acetic or lactic acid. Moderate significant inhibitor effect was observed at treatment of Chitosan dissolved in lactic acid and Saccharomyces cerevisiae treatments. The antioxidant salicylic and ascorbic acids revealed the lowest significant effect against the fungal mycelia growth. Under field conditions, treatments of S. cerevisiae, salicylic and ascorbic acids had announced protecting effect against black spot incidence whereas no disease symptoms on guava fruits, artificially infested with G. mangiferae, were observed at these treatments which used as pre-harvest application. Results for post-harvest test, under artificial infestation with G. mangiferae, showed that no disease symptoms was observed at treatments of propolis dissolved in acetic acid, chitosan dissolved in acetic acid, Beeswax dissolved in water or ethanol and combination between propolis plus chitosan dissolved in acetic acid. Meanwhile, 48.4% diseased guava fruits were recorded at Chitosan dissolved in lactic acid treatment followed by 35.1% at combined treatment of Propolis dissolved in latic acid plus Chitosan dissolved in lactic acid and 28.6% at Propolis dissolved in lactic acid treatment. This study showed the efficacy of antioxidants, natural products as well as bio-agent to control and reduce Guava fruit black spot caused by Guignardia mangiferae.

Keywords: ascorbic acid, beeswax, black spot disease, chitosan, Guava fruits, Guignardia mangiferae, pre-harvest, post-harvest, propolis, salicylic acid, Saccharomyces cerevisiae

INTRODUCTION Guava (Psidium guajava Linn.) is an essential

product of subtropical regions. It is solid harvest and is developed effectively even in poor soils. There are various pathogens, principally fungi,

bacteria, algal and some physiological disorders or deficiencies which affect the guava crop (Misra and Prakash, 1994). More than 90 pathogens are reported on fruits, 42 on foliage, 18 on twigs and 18 on roots. These cause various diseases viz. pre and postharvest spots of fruits, canker, wilt,

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Abdel-Kader, et al., Black spot disease on Guava fruits and its management in Egypt

Bioscience Research, 2019 volume 16(2): 2104-2118 2105

die-back, defoliation, twig drying, leaf spot, leaf blight, anthracnose, red rust, sooty mould, rust, seedling blight and damping off etc. Guava (Psidium guajava L.) is a fruit-bearing tree in the Myrtaceae plant family. Closely related genera include Eucalyptus L’Heritier and Syzygium R.Br. ex Gaertn. The tree is native to Mexico, the Caribbean and Central America, where it is an economically important crop (Morton, 1987) Leading producers of guava fruit are Brazil, India and Mexico.2 In South Africa, approximately 41000 tons of guava are harvested per annum for fresh sales and processing (Anonymous, 2009).

Guava gathers did not put resources into maintaining the plants they harvested, so fruit spot losses were not considered in the cost of producing guava products. With expansion of the market for guava products and changing land use patterns, commercial plantings are rapidly replacing wild plants as a major fruit source, and fruit disease losses are an important factor in determining profit ability.

About 20-25% of the harvested fruits are estimated as decayed by pathogens during post-harvest handling even in developed countries (Droby, 2006; Zhu, 2006). Fruits infected by fungi may occur during the growing season, harvesting, handling, transport and post-harvest storage and marketing conditions, as well as after purchasing by the consumer. When fruits contain high levels of sugars and nutrients, and their low pH makes them susceptible to fungal decaying (Singh and Sharma, 2007). Initially infected fruits remain attached to the tree until they are manually dislodged or fall in the course of natural maturation. As a result of this disease, yield of marketable fruits was significantly reduced in the field and in storage before processing.

Application of fungicides has been the most effective and widely used strategy and provides effective and reliable post-harvest disease control measure. However, their use is expensive and may also affect the populations of beneficial microorganisms. This problem has been additionally muddled by the improvement of fungicidal resistance, notwithstanding its negative impacts on public health and environmental balance. Therefore, fungicide alternatives for disease management, i.e. the use of biological control agents have become more attractive.

In this regards, propolis is a resinous product that bees collect from the buds of trees and which is subsequently treated with the addition of beeswax, pollen and enzymes. For its insulating properties it is used by bees as a glue to seal the

cell walls. For its antimicrobial, antiviral, antifungal and anti-inflammatory properties, its use in medicine dates back to ancient times (Wulandari et al., 2013; Freires et al., 2016).

Also, Beeswax is the substance that frames the structure of a honeycomb; the honey bees emit wax to assemble the honeycombs where to store nectar. As of late, the enthusiasm of scientists has moved even on antimicrobial properties of beeswax in spite of the fact that there are as yet few reports in the literature focused on the activity of beeswax. The few investigations demonstrated an antimicrobial viability of beeswax against overall Staphylococcus aureus, Salmonella enterica, Candida albicans and Aspergillus niger (Fratini et al., 2016).

Moreover, antioxidants represent a special important class of preservatives as, unlike bacterial or fungal spoilage (Iverson, 1995). They attributed the efficacy of antioxidant used in his study to its chemical constants of Vitamins used. In addition antioxidants are vital to raise host plant resistance; trigger innate immunity in plants to refine and promote methods of defense to the plant against the pathogens was reported by Madukwe et al., (2013).

Therefore, the aim of present work was designed to evaluate the effect of some antioxidants (salicylic and ascorbic acids), natural products extracts (honey bee wax and propolis), chitosan and the bioagent Saccaromyces cerevisiae against black spot disease incidence of guava when applied as fruits coating at pre-, and post-harvest stages. MATERIALS AND METHODS

Disease Survey and Detection In Egypt the estimated cultivated area of guava

trees about 38.873 feddan produced 314.438 tons fruits with the average of 9.49 tons/feddan (Anonymous, 2006). Survey of guava fruit black spot incidence was carried out at during the period of August to November, 2017 where Guava trees are cultivated at different field localities of North Egypt, Qualiobia, Beheira, Domeit, Kafer El-Sheekh and Alexandria governorates.

Disease symptoms on Guava fruits were initially appeared as small, circular, black spots developed on fruits. Later, the spots enlarged rapidly resulted in a sunken skinny feature decay shape of the fruit flesh (Fig. 1).



Isolation and identification of the causal fungus

Several samples of Guava fruits showing disease symptoms were collected from different local and central markets in Egypt and examined in the laboratory.

Infected areas of fruits were excised, surface sterilized in 2% sodium hypochlorite for one minute, placed on sterile filter paper to dry, and plated on autoclaved water agar. After incubation for approximately 3 days at 24°C, advancing mycelium was transferred to potato dextrose agar (PDA) by hyphal tip transfer and monoconidial cultures were prepared and retained for all future experiments. All of colony size, color, texture and color of conidial masses were examined microscopically after seven days incubation on PDA. The isolated fungus was identified based on morphological and cultural characteristics according to Gilman (1957) and Barnett & Hunter (1972).

Pathogenicity test The pathogenic ability of isolated fungus

(Guignardia mangiferae) was evaluated under laboratory conditions. The isolated fungus G. mangiferae was grown on potato dextrose broth (PDB) medium and incubated at 20±2°C for 14 days. The culture was filtered through sterile glass wool and the collected spores were adjusted to 106 spore/ml using haemaocytometer slide. Fruits of guava and Orange apparently healthy were surface disinfected with 70% ethyl alcohol, allowed to dry and then several wounds/fruit each (0.5-1.0 cm deep) were made up at the fruit body using sterile scalpel.

The wounded fruits were inoculated by spraying them with the prepared spore suspension of the tested fungus. For development of disease symptoms the guava fruits or Orange were placed in sterilized moist desiccators to maintain a high humidity, held for 7-10 days at 20°C and examined for typical black spot symptoms. A set of wounded fruits either Guava or Orange were sprayed with sterilized water and kept as control. Twenty fruits of Guava or Orange were used and the percentage of infected fruits was calculated. The fungus was re-isolated and identified to fulfill Koch’s postulates.

In vitro test The inhibitory effect of yeast Saccharomyces

cerevisiae, Salicylic acid, Ascorbic acid, as well as extracts of Propolis, beeswax and chitosan at different concentrations against the linear growth

of G. mangiferae was evaluated under laboratory conditions. Stock solutions of Propolis, beeswax and chitosan were prepared as follows:

Propolis and beeswax were purchased from local market. Prior to use, propolis samples were kept at room temperature in the dark. Crude propolis was grounded into powder and macerated in different solvents by shaking. The used solvents were acetic acid (4g propolis + 96 ml acetic acid) and lactic acid (4g propolis + 96 ml lactic acid).

After extraction, extracts of propolis were filtered through Whatman No. 1 filter paper to obtain a stock solution. Solutions are clear, yellow, viscous liquids and remain stable when stored, i.e. the color remains unchanged, no precipitate is observed and they do not turn white. Prior fruits coating a dilution of stock solution were prepared at concentration of 4% (4 ml stock solution: 96 ml water).

Meanwhile beeswax was melted at 65°C in hot water or ethanol, prior to use, at the rate of 5g beeswax: 95 ml water.

Chitosan stock solution was prepared by dissolving 2g powder into either acetic or lactic acid at the rate of 2% (2g chitosan: 98 ml acid). Chitosan solution was used at the rate of 2% (2ml of prepared solution: 98ml water).

Emulsified stocks at high concentration of tested materials were prepared by dissolving in sterilized distilled water. A few drops of the emulsifier Tween 80 (Sigma Co.) were added the prepared emulsifier volumes.

The tested concentrations of salicylic and ascorbic acids were 0.1, 0.2 and 0.4%, meanwhile the other prepared tested materials used at concentrations of 1.0, 2.0 and 4.0%. Cell suspension of Saccharomyces cerevisiae were prepared by adding 10g yeast to one liter of warm water.

Different volumes of the prepared emulsions were added to conical flasks containing 100 ml of sterilized PDA medium before its solidification to obtain the proposed concentrations. The supplemented media were poured into Petri-dishes (9 cm) about 20 ml each. PDA plates free of tested materials were also prepared as control. Five plates were used as replicates for each particular treatment as well as control. Discs (5 mm diameter) of cultures of G. mangiferae were placed on to the Centre of Petri dishes with PDA, incubated at 20+2oC. When the fungal growth in the control filled the whole Petri dish, then all treatments were examined and the mean linear fungal growth was measured and recorded.



Pre- and post-harvest studies (storage experiment)

Pre-harvest experiment To manage black spot disease of guava fruits,

the postharvest disease that causes losses during handling and storage, some foliar approaches were applied to guava trees three weeks before fruits picking for the purpose of pre-harvest treatment.

This experiment was initially carried out as foliar application with different treatments at certain private field located at Kafr El-Dawar district, Alexandria governorate. This field is one of the surveyed locations during the previous seasons which showed highly occurrence with black spot disease of guava fruits.

Under field experiment, certain guava trees were chosen in random with consideration of three replicate trees for each particular treatment. Guava trees were sprayed individually according to certain treatment three weeks before harvest. All trees include their fruits were sprayed with respect to the rain full point. The sprayed treatments were as follows:

(1) Yeast Saccharomyces cerevisiae (10 g/L) (2) Salicylic acid (2g/L) (3) Ascorbic acid (2g/L) (4) Untreated control

Then after the harvested treated guava fruits were subjected to artificial infestation black spot pathogens under in vivo conditions at laboratory of Plant Pathology Dept.

All the fruits were disinfected (Lopez-Reyes et al., 2010) in sodium hypochlorite solution (2%) for 2 min, then air dried. Guava fruits were arranged to four groups according to the previous field spray application treatments. Guava fruits were wounded (0.5 cm deep and 1.0 cm long-five wounds per fruit) using sterile scalpel. The wounds were inoculated with spore suspension (Lopez-Reyes et al., 2010).

Conidia of the fungal pathogen G. mangiferae were recovered from 2-week old cultures by adding 10 ml of sterile water to each plate. The conidia suspension was filtered through three layers of sterile cheesecloth. The concentration of the conidial suspension was adjusted to 106 conidia per ml and a drop of Tween 80 was added to the suspension. Then after, each fruit group was inoculated with G. mangiferae through spraying with the fungal suspension.

Post-harvest experiment

Fruit coating preparation and inoculation: The tested coating solutions were prepared as

mention before. Mature apparently healthy guava fruits of almost uniform sizes collected from different markets located at Giza, Qualubia and Monifia governorates were used for this experiment.

The evaluated treatments for the purpose of post-harvest experiment were as follows:

Saccharomyces cerevisiae (1.0 %) Propolis dissolved in acetic acid (4%) Propolis dissolved in lactic acid (4%) Chitosan dissolved in acetic acid (4%) Chitosan dissolved in lactic acid (4%) Bee wax suspended in water (4%) Bee was suspended in ethanol (4%) [Propolis dissolved in acetic acid (4%)] +

[Chitosan dissolved in acetic acid (4%)] [Propolis dissolved in latic acid (4%)] +

[Chitosan dissolved in lactic acid (4%)] Untreated control

All the fruits were disinfected, wounded as previously described. Coating was applied manually on the dried guava fruits, in respect to each treatment, by using a commercial sponge of 4 cm x 4 cm x 4 cm size. Excess coating was allowed to drain off. The wounded treated guava fruits were inoculated with the fungal suspension as mentioned above.

All treated fruits were air dried, after each individual treatment, for 2 hours in a laminar flow. The inoculated treated fruits were placed into carton box (52x23x28 cm) with a capacity of 36 fruit/box and stored at 20±2oC for 2 weeks. Three boxes were used as replicates for each particular treatment. Percentage of infected fruits was calculated after the storage period.

Statistical analysis Tukey test for multiple comparisons among

means was utilized as described by Neler et al. (1985).

RESULTS AND DISCUSSION

Disease Survey and Detection Results of surveyed guava trees grown at

different field localities of North Egypt, Qualiobia, Beheira, Domiat, Kafer El-Sheekh and Alexandria governorates for fruit black spot disease incidence are shown in Table (1). Presented data revealed that the maximum records of disease occurrence



was recorded at some fields belong to Alexandria governorate followed by (3.7 and 3.3% at Kafer El-Sheekh and Beheira governorates, respectively. Meanwhile lesser occurrence of black spot was observed as 2.6, 1.2% at Domiat and Qualiobia governorates, respectively.

Table (1): Survey of black spot of guava fruits

at different locations at five Governorates located at North Egypt

Governorates (localities)

Black spot of guava fruit (%)

Qualiobia 1.2* e

Beheira 3.3 cd

Domiat 2.6 d

Kafer El-Sheekh 3.7 ab

Alexandria 4.8 a * These percentages are the mean of surveyed fields

Figures with the same letters in each column are not significantly differed (P≤0.05)

The favorable conditions are apparently required for fruit spots. For example, the severely affected orchard is situated in a small valley with a rapidly flowing stream running through it. Air drainage is poor, resulting in high humidity and persistent dew well into the day (Srivastava and Tandon, 1969). In addition, infection courts were probably provided by insects, because what appeared to be oval position sites were almost always associated with the disease. Fruits are visited by a variety of insects, including fruit flies. The conditions for disease development and involvement of insects in the disease are being investigated (Ooka, 1980). In the present study, the surveyed governorates characterize with humidity and dew drops throughout the day and the insect fruit flies as well.

Isolation, identification and pathogenic ability of the causal fungus

Isolation trails revealed a fungus identified as Guignardia mangiferae. The Guignardia Genus (Kingdom Fungi, Phylum Asco- mycota, Class Dothideomycetes, Order Botryosphaeria- les, Family Botryosphaeriaceae) encompasses around 330 known species, many of them with unknown anamorphic phase (Crous et al., 2006). Many species considered plant endophytic fungi are classified in this family and genus, among them, the causal agents of Citrus Black Spot (Sutton and Waterston, 1966) and G. mangiferae the causal agents of Guava black spot (Wickert et al., 2009).

There are two forms of the fungus Guignardia spp. associated with citrus plants: G. citricarpa and G. mangiferae (Baayen et al., 2002). Both forms of Guignardia belong to distinct species, with the endophytic symptomless type being classified as G. mangiferae (Baayen et al., 2002). Kotzé (1988) and Baayen et al., (2002) stated that the colonies of G. citricarpa and G. mangiferae have totally different morphologies on potato dextrose agar (PDA). Truter (2010) reported that two main Guignardia species are morphologically similar occurring on Citrus, G. citricarpa, causing black spot on Citrus, and G. mangiferae, showed no symptoms to Citrus, causing only symptomless infections that remains latent (Meyer et al., 2001; Baayen et al., 2002; Bonants et al., 2003). These species showed difference in culture characteristics as well as host range. Isolates of G. citricarpa can be distinguished from G. mangiferae by a combination of several characteristics. Guignardia mangiferae fast growing on PDA medium, its colony is dark brown, with black pigment and produce both pycnidia with pycnidiospores and pseudothecia with ascospores, disease symptoms appear as spots on guava and mango fruits. Meanwhile, G. citricarpa slow growing on PDA medium, its colony is dark brown with a wider translucent outer zone and lobate margin, Produce pycnidia and pycnidiospores and rarely infertile pseudothecia (never ascospores), disease symptoms appear as spots on fruit, leaves and twigs of citrus only (McOnie, 1964a &b; Korf, 1998; Baayen et al., 2002; Baldassari et al., 2008). Isolates of G. mangiferae produces both pycnidiospores and ascospores in culture, although not all isolates formed fertile pseudothecia (Baayen et al., 2002; Baldassari et al., 2008).

Kotzé (1988) reported among the 36 isolates grown on PDA, G. citricarpa were more compact, with differentiated regions of yellow pigmentation.

The colony edges of G. citricarpa did not show invaginations, meanwhile colonies of G. mangiferae had a spongy appearance, with homogeneous mycelial growth and invaginating round edges with dark pigmentation.

All previous reports confirmed that the isolated fungus in the present study is Guignardia mangiferae. Colonies of G. mangiferae display a more ‘spongy’ shape, with invaginations causing them to be irregular with differentiated regions of dark pigmentation as shown in Fig. (1, E).



Figure. (1) Black spot symptoms of Guava and Orange fruits (A: artificially infested Guava fruits with G. mangiferae; B: Un-infested healthy Guava fruits; C: artificially infested Orange

fruits with G. mangiferae revealed no disease symptoms; D: Un-infested healthy Orange fruits and E: Growth of G. mangiferae the causal of Guava fruit black spot).



The tested pathogenic ability of isolated fungus revealed typical symptoms of black spot disease on Guava fruits artificially infested with Guignardia mangiferae which can be seen in Figure (1, A). Disease symptoms were visible on the skin of young fruits which progressed as fruits got larger. All guava fruits artificially infested with spore suspension of G. mangiferae revealed black spot incidence (data not shown). On the other hand no symptoms were observed on Orange artificially infested with the same fungus.

Wickert et al., (2014) tested the pathogenic ability of G. citricarpa and G. mangiferae for inducing black spot on either guava or citrus fruits. They found that G. citricarpa caused citrus black spot symptoms on citrus fruits, but it did not produce any symptoms on guava fruits meanwhile, G. mangiferae isolates were able to cause black spot symptoms on guava fruits, but they have not produced any symptoms on citrus fruits.

This report support our results in pathogenicity tests that the isolated fungus has pathogenic ability to induce black spot disease on guava fruits

only and symptomless on orange fruits (Fig. 1 A and C).

In vitro test Some antifungal products were evaluated

against the linear growth of Guignardia mangiferae under in vitro conditions. Data presented in Table (2) and Figure (2) revealed highly significant complete reduction (100%) in fungal growth at treatments of propolis dissolved in either acetic or lactic acids, chitosan dissolved in acetic acid, Beeswax suspended in water or ethanol and combined treatments of propolis and chitosan dissolved in acetic or lactic acid. Moderate significant inhibitor effect was observed at treatment of Chitosan dissolved in lactic acid whereas the mycelia growth recorded 15.0, 8.0, 0.0 mm at concentrations of 1.0, 2.0 and 4.0%. Also, mycelia growth recorded 30.0, 18.0, 12.0 mm at concentrations of 0.125, 0.25, 0.500 g/100 ml at Saccharomyces cerevisiae treatment. The antioxidant salicylic and ascorbic acids revealed the lowest significant effect against the fungal mycelia that the linear fungal growth was recorded as 65.0, 22.0, 8.0 mm and 80.0, 73.0, 48.0 mm, in respective order.

Table (2): Inhibitory effect of some antifungal products on the linear growth of G. mangiferae in vitro

Treatment

Concentration (g/100ml)

0.125 0.250 0.500

1- Saccharomyces cerevisiae 30 d 18 e 12 e

Treatment

Concentration (ml/100ml

0.1 0.2 0.4

2- Salicylic acid 65 bc 22 de 8 f

3- Ascorbic acid 80 b 73 b 68 bc

Treatment

Concentration (ml/100ml)

1.0 2.0 4.0

4- Propolis dissolved in acetic acid 0 g 0 g 0 g

5- Propolis dissolved in lactic acid 0 g 0 g 0 g

6- Chitosan dissolved in acetic acid 0 g 0 g 0 g

7- Chitosan dissolved in lactic acid 15 e 8 f 0

8- Beeswax suspended in water 0 g 0 g 0 g

9- Beeswax suspended in ethanol 0 g 0 g 0 g

10- [Propolis dissolved in acetic acid] + [Chitosan dissolved in acetic acid] 0 g 0 g 0 g

11- [Propolis dissolved in lactic acid] + [Chitosan dissolved in lactic acid] 0 g 0 g 0 g

12- Untreated control 90 a 90 a 90 a

Figures with the same letters in each column are not significantly differed (P≤0.05)



Figure. (2): Inhibition of the linear growth of G. mangiferae in response to some tested antifungal products in vitro



Pre-, and post-harvest studies (storage experiment)

A significant number of post-harvest diseases are economically controlled through postharvest handling procedures and using of fungicides. However, it will be beneficial if a suitable fungicide applied on trees before harvest can also reduce postharvest diseases incidence and severity. So also, it was accounted for that, successful pre-harvest fungicides with proceeding with action that endures to give postharvest decay control after harvest would be a helpful control tool for fruit cultivators (Ritenour, et al., 2004; Yildiz et al. 2005).

Also, pre-harvest spray application to Valencia orange trees with yeast and/or some food preservatives was found to be an effective approach for controlling the postharvest diseases (El-Mougy et al., 2015).

In this regards, the present study conducted with evaluation of re- and post-harvest treatments. Foliar guava trees spray three weeks before harvesting against black spot disease incidence was applied as pre-harvest treatment. The harvested Guava fruits as well as the other fruits collected from different markets (post-harvest) were artificially inoculated individually with G. mangiferae pathogens under laboratory conditions. Black spot incidence was recorded after two weeks of incubation. Presented data in Table (3) revealed that all applied treatments were

significantly differed than control whereas all guava fruits were infected (100%) with black spot disease. Treatments of S. cerevisiae, salicylic and ascorbic acids had announced protecting effect against black spot incidence.

No disease symptoms on guava fruits, artificially infested with G. mangiferae, were observed at these treatments which used as pre-harvest application.

As for post-harvest test, under artificial infestation with G. mangiferae, presented data in Table (3) showed that no disease symptoms was observed at treatments of propolis dissolved in acetic acid, chitosan dissolved in acetic acid, Beeswax dissolved in water or ethanol and combination between propolis plus chitosan dissolved in acetic acid.

Furthermore, 48.4% diseased guava fruits were recorded at Chitosan dissolved in lactic acid treatment followed by 35.1% at combined treatment of Propolis dissolved in latic acid plus Chitosan dissolved in lactic acid and 28.6% at Propolis dissolved in lactic acid treatment.

In the present study, application of the yeast S. cerevisiae, antioxidants, salicylic & ascorbic acids and natural products, chitosan, propolis and beeswax had drastic effect against the linear growth of G. mangiferae in vitro (Table, 2) and suppressed the black spot disease incidence on Guava fruits in vivo when used as pre-, and post-harvest treatments.

Table (3): Efficacy of Pre-, and Harvest treatments on black spot incidence of Guava fruits under artificial infection with G. mangiferae

Treatment Disease incidence (%)

Pre-

harvest

Saccharomyces cerevisiae 0.0 e

Salicylic acid 0.0 e

Ascorbic acid 0.0 e

Post-harvest

Propolis dissolved in acetic acid 0.0 e

Propolis dissolved in lactic acid 28.6 d

Chitosan dissolved in acetic acid 0.0 e

Chitosan dissolved in lactic acid 48.4 b

Beeswax suspended in water 0.0 e

Beeswax suspended in ethanol 0.0 e

[Propolis dissolved in acetic acid] + [Chitosan dissolved in acetic acid]

0.0 e

[Propolis dissolved in latic acid] + [Chitosan dissolved in lactic acid]

35.1 c

Saccharomyces cerevisiae 0.0 e

Control 100 a Figures with the same letters in each column are not significantly differed (P≤0.05)



In this concern, pre-harvest application with different salts and acids reduce green and blue molds of Valencia orange fruits El-Mougy et al., (2015). They attributed this effect to the increasing numbers of the yeast S. cerevisiae persistence on the orange fruit’s surface and/or synergistic effect between the yeast and/or applied salts and acids which may probably occur. Similar conclusions regarding these results are reported. Antagonistic efficacy for controlling postharvest diseases, including nutrient competition, antibiotic production, enzymes acting on fungal cell wall components such as chitinases and ß-1,3 glucanase and induced host resistance have been postulated and demonstrated as numerous modes of actions (Droby and Chalutz, 1994; Burkhead, 1995). Biological activity of antagonistic yeasts may involve nutrient competition (Droby et al., 1989), site exclusion (Droby and Chalutz, 1994), direct parasitism, and perhaps induced resistance (Wisniewski et al., 1988, 1991). Furthermore, a variety of fruit had been protected against postharvest pathogens by several antagonistic yeasts (Chang-Goyal and Spotts, 1996; Ortu, et al., 2003). Also, (Scherm et al., 2003) reported that two yeast strains of Candida guillermondii and Saccharomyces cerevisiae had remarkable antagonistic properties against P. expansum on apple. (Lui et al., 2017) tested the positive effect of three strains of S. cerevisiae for antagonizing Colletotricum gloeosporioides and were further evaluated for their mechanisms as biological control agents. They found that all three S. cerevisiae isolates produced antifungal compounds, β-1,3-glucanase and chitinase which able to inhibit conidia germination of C. gloeosporioides. They added that when all tested isolates of S. cerevisiae, artificially inoculated on grape berry, were able to colonize grape berry in large quantities and controlled C. gloeosporioides. They suggested that the antagonistic isolates of S. cerevisiae could be considered as an important biological control agent against anthracnose of grape caused by C. gloeosporioides.

In addition, ( Lopes et al., 2015) found that six S. cerevisiae isolates produced antifungal compounds, competed for nutrients, inhibited pathogen germination, and produced killer activity and hydrolytic enzymes when contacted with the fungus wall Colletotrichum acutatum, the causal agent of post-bloom fruit drop that occur in pre-harvest citrus. These isolates were able to control the disease when detached flowers were artificially inoculated, both preventively and curatively. They attributed the bio control mode of

action to the mechanisms such as production of antifungal compounds, nutrient competition, detection of killer activity, and production of hydrolytic enzymes of the isolates of S. cerevisiae.

Despite the fact that the utilization of antagonistic yeasts to control postharvest diseases have been shown with a few items, their commercialization will be rely upon whether they are able to do successfully controlling rot of fruits from various areas with variable inoculum loads, types of disease, and dimensions of mechanical damage.

In general, utilization of biological control has shown potential as an alternative against plant pathogens instead of synthetic chemical fungicides (Droby et al., 1989). In order to minimize the use of synthetic fungicides, more environmentally friendly and harmless compound (s) should be explored to improve the activity of the antagonist. Certainly, range of control methods that differ in their effectiveness, duration and cost could suppress plant pathogens (Shtienberg, 2000). Therefore, the present study conducted using the foliar spray containing the bioagent S. cerevisiae with several antioxidants, salicylic and ascorbic acids as fungicide alternatives for controlling Guava fruits black spot disease incidence under natural field infection.

The present study has demonstrated that all treatments tested using antioxidants have antifungal potential activities and could be useful against black spot disease. The efficacy of antioxidant used in present study could be attributed to its vital to raise host plant resistance, trigger innate immunity in plants to refine and promote methods of defense to the plant against the pathogens (Madukwe et al., 2013).

The newest research is confirming the hypothesis that salicylic acid and ascorbic acid (vitamin C) serve healing purposes for the plants that produce them. Furthermore, chemical elicitors (inducers) appear to incline the first protection components in plants against infections or create some new mixtures supporting it. Numerous others (Dmitrier et al., 2003; Achuo, et al., 2004) utilized a few chemical or natural mixtures known to induce plant resistance including salicylic, benzoic, citric, ascorbic and oxalic acids. Some chemical such as salicylic acid have been reported for inducing resistance as well as be active as antimicrobial against several soil-borne plant pathogens, i.e. fungal root rot and wilt pathogens (Chen- Chunquan et al., 1999), as well as fungal foliar diseases (Walters et al., 1993;

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Sathiyabama and Balasubramanian, 1999; Cameron, 2000). Also, salicylic acid at levels higher than 200 μg / ml inhibited the growth of Pythium aphnidermatum, the causal of cucumber root rot (Chen-Chunquan et al., 1999).

Chitosan, propolis and beeswax were found, in the present study, to have antifungal effect when applied either in vitro as growth suppressive (Table 2) or in vivo as protecting factor against fungal invasion to Guava fruits (Table 3).

Potentials for prolong storage period and control decay of many fruits, including strawberries have been reported (Hernández-Muñoz, et al., 2006). Chitosan has a wide application as edible coating material because of its good biocompatibility, biodegradability, antibacterial and antifungal activity, membrane forming capacity and safety. Chitosan exhibits a variety of antimicrobial activities and has been one of the most promising coating materials for fruits and vegetables because of its good film-forming property, broad antimicrobial activity, and excellent compatibility with other substances (Shahidi et al. 1999, Meng et al., 2010).

Extending fruits shelf life and preventing fungal decay during storage as a result of using propolis were reported (Çandir, et al., 2009; Özdemir, et al., 2010). The propolis contains about 50% resins, 30% wax, 10% essential oils, 5% pollen and 5% other organic compounds (Falcão et al., 2010). Curifuta, et al., (2012) found tested four concentrations (0.5, 1.0, 2.5 and 5.0 %) of propolis ethanoic extract (PEE) that showed significant antifungal inhibition effects to Penicillium expansum on agar medium. Matny, et al., (2014) reported that snap been treated with 5% of ethanol extract propolis (EEP) prevent white mold Sclerotinia sclerotiorum in storage conditions for more than two weeks. Also, Matny, et al., (2015) reported that propolis may be containing many and high concentration of active ingredients that control the pathogen, according on the types of trees that grow in the region of propolis collected. Bankova, (2005) and Hegazi., et al., (2014) stated that the chemicals compounds in propolis and its biological activities depend on different factors, i.e. geographical regions, collection time and plant source. Antimicrobial activity of bee propolis was being mention by many studies (Matny et al., 2014; Matny, 2015). Treatment with ethanol-extracted propolis was effective in preventing fungal decay in cherries fruits stored for 4 weeks (Candir, et al., 2009). The EEP increasing phytoalexin levels in soybean cotyledons in parallel to increasing the

applied concentration of EEP. The application of ethanol-extracted propolis (EEP) inhibited Penicillium digitatum growth in vitro (Soylu, et al., 2004, 2008) and limited the growth of Botrytis cinerea on strawberry (La Torre, et al., 1990). The presence of phenolic compounds, waxes, vitamins and essential oils could have the most significant biological action of propolis in inhibiting the microbial activity (Burdock, 1998). The application of ethanolic extracted propolis (EEP) as a fruit coating for different fruits were evaluated (Ali, et al., 2014, 2015).

Furthermore, in the present study, beeswax revealed antifungal effect against G. mangiferae growth in vitro as well as its invasion to Guava fruits in vivo. These results are confirmed by several reports. In this concern, there are some records about the application of beeswax based edible films and coatings in the preservation of fruits and vegetables (Navarro-Tarazaga et al., 2006; Galič, 2009). However, according to our knowledge, there are not many papers about the incorporation of natural biologically active compounds in the beeswax coating. It was reported that beeswax is a complex mixture (more than 300 components) of hydrocarbons, free fatty acids, esters of fatty acids and fatty alcohol, di-esters and exogenous substances (Tulloch, 1980). Crude beeswax showed antifungal (Al-Waili, 2004) and antibacterial activity against Gram-positive and Gram-negative bacteria (Ghanem, 2011). Also, Fagundes et al., (2015) recorded that edible composite coating of sodium benzoate based on either hydroxypropyl methylcellulose or beeswax was the most effective than sodium methyl paraben and sodium ethyl paraben against Alternaria alternata black spot on artificially inoculated cherry tomatoes.

CONCLUSION Fruit decay diseases caused by fungi are a

large problem in marketing and storage stage in the world wide, and the use of chemicals for the purpose of control fruit decay losses could cause a negative effect on the health of the consumers. New natural products and new methods are considered new methods for controlling post-harvest diseases in addition to its safe on consumer’s health. This study showed the efficacy of antioxidants, natural products as well as bio-agent to control and reduce Guava fruit black spot caused by Guignardia mangiferae. The present findings demonstrate that the applied treatments may have important implications for the future use a commercial scale for controlling such post-

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harvest diseases. Truly, these results showed to have a pronounced antifungal effect, which is a very important characteristic for application of this kind of biologically active edible coatings in the prevention of the fungal fruit decay, hence, the arget of this investigation was to decide whether the applied treatments could give improvement impact against fruit disease. Considering their attribute and broad-spectrum activities, fruitful improvement of such materials as antifungal would not just give a strong tool for controlling post-harvest diseases, yet in addition could guarantee achievement in multipurpose biorational options in contrast to traditional fungicides for the management of other plant diseases.

CONFLICT OF INTEREST The authors declared that present study was

performed in absence of any conflict of interest.

ACKNOWLEGEMENT The authors thank the owner of the private

field located at Kafr El-Dawar district, for his help to perform pre-harvest treatments of Guava trees.

AUTHOR CONTRIBUTIONS MMA designed and performed the field

experiments and also wrote the manuscript. NSE performed field experiment, fellow isolation and bio-assay Lab. experiments, and data analysis. MSAK and MMA performed field survey, diseased samples collection, field experiment. NSE, MMA and MSAK shared in designed experiments and reviewed the manuscript. All authors read and approved the final version.

Copyrights: © 2019 @ author (s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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