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Research Article - (2018) Volume 9, Issue 10

Potential of Grape Epiphytic Antagonists to Biocontrol Aspergillus Transmission and Accumulation of Aflatoxin B1 and Ochratoxin A in Post Harvest Taify Table Grape

Abd El-Raheem R El-Shanshoury1, Yasser H El-Halmouch2,3*, Samia F Mohamed4 and Mervat F Fareed5
1Faculty of Science, Microbiology Division, Department of Botany, Bacteriology Unit, Tanta University, Tanta, Egypt
2Faculty of Science, Biotechnology department, Taif University, Taif, Kingdom of Saudi Arabia
3Faculty of Science, Department of Botany, Damanhour University, Damanhour, Egypt
4Department of Biochemistry, Animal Health Institute, Pharmacology and Toxicology Unit, Giza, Egypt
5Faculty of Specific Education, Department of Home Economics, Tanta University, Tanta, Egypt
*Corresponding Author: Yasser H El-Halmouch, Faculty of Science, Biotechnology department, Taif University, Taif 21974, P.O. Box 888, Kingdom of Saudi Arabia, Tel: +966 55 6382034, Fax: +966 27274299 Email:

Abstract

Taify grape is one of the important summer fruits in the Taif area in Saudi Arabia. A part of grape spoiled because by fungi and their toxins, influencing intersection processing, and export, alongside the quality of superior general welfare risk of exposure. The aim of this work was to check the ability of live cells and crude cell-free extracts of the grape epiphytic antagonists Pseudomonas aeruginosa, Bacillus vallismortis and B. amyloliquefaciens to control the transmission of Aspergillus niger, A. parasiticus and A. tubingensis to post-harvest Taify table grape berries (Vitis vinifera L.). Gathering of aflatoxin B1 (AFB1) and ochratoxin A (OTA) was also evaluated. Also, the activity of peroxidase enzyme, total phenol, and lipid peroxidation evaluated. The healthy and injured grape berry inoculated or suspended in the live bacterium cell suspension or bacteria free extract were challenged by the above fungi and stored at 5°C and 20°C for 28 and 50 days, individually. The treatments were effective for lessening the parasites misuse, spoil advance, and decay rate, and AFB1 and OTA aggregation. A pre-dousing of the in-place grape berries was prevalent. The peroxidase enzyme and the total phenol expanded further, slightness the lipid peroxidation, all reinforce the antagonists. The bacteria, their cell-free extracts, the incited peroxidase enzyme, total phenol and the brought down lipid peroxidation, made the medium ominous to the fungal establishment and further developing, and after that developing aggregate AFB1 and OTA ceased. Additional work expected to recognize the bacterial causal variable as well as the active metabolites.

Keywords: Aspergillus sp.; Antagonistic bacteria; Mycotoxins; Peroxidase enzyme; Total phenols; Lipid peroxidation

Introduction

Grapevine (Vitis vinifera L.) is one of the most important fruit crops worldwide. It has reached all countries but is successfully cultivated only in temperate climate regions with sufficient rain, warm and dry summers and relatively mild winters [1]. Genetic relationships between wild and domesticated grapevine distributed from Middle East countries to European rural areas was studied by many workers [2]. In the Taif area in Saudi Arabia, the grape taken as the unity of the important summer fruits. The significant portion of the grape is lost due to over-ripening, physical injuries, disorders, and diseases caused by fungi, bacteria and/ or viruses during pre and/or post-harvest periods, affecting production, processing, and export, along with fruit quality [3,4]. If efficient steps are not followed to prevent the spread of these microorganisms and their toxin [5], subsequent processes infected and affectedness a public health hazard [6]. Contamination of grapes with fungi was studied [7,8]. The main ochratoxingenic and/or aflatoxigenic fungi are much concerned with some members of black Aspergilli and Penicilli contaminating grape [8-11]. Mycotoxins were produced by the genera Aspergillus, Fusarium, and Penicillium, during crop growth, harvest or storage [12]. Among the most important and harmful mycotoxin in the world is ochratoxin A (OTA) [13]. High levels of OTA contamination were reported on dried vine fruits (e.g. Raisins) worldwide, showing frequently around 100% of the samples contaminated [14]. Damaging effects of chemical control to post-harvest fruit pathogens, mycotoxins, and the establishment of fruit pathogen resistance to fungicides encouraged scientists to look for alternative safe control methods. In this context, much success achieved [8,15-23]. Progress was attained from the reduction of the post-harvest diseases and the tale of their toxins by stress from natural flora by utilizing the epiphytic and endophytic microorganisms existing in the yield [8,24,25]. Ponsone and his research team [26] demonstrated the efficacy of two yeast strains of Kluyveromyces thermotolerant for reducing OTA accumulation (from 3% to 100%) and the growth rate of ochratoxigenic fungi (from 11% to 82.5%), in the field. Prevention and control terminal of black Aspergilli and to reduce mycotoxin hazard were reviewed by Sanzania and his coworkers [11]. OTA removal was also possible by using both dead and alive yeast strains [26-28]. Grapevine metabolites are extremely regarded by environmental or pathogen approach [1]. In this respect, plant resistance induction due to pathogens, the abiotic and biotic agents were described [29,30]. It was reported that various defense reactions such as hypersensitivity, the product of phytoalexins, antimicrobial proteins, and strengthen the cell walls, all initiated in plants when they infected with various pathogens [31]. Increased activeness of phenols, peroxidase, and lipid peroxidation, in response to infection by the pathogens, also reported [29-33]. Induced resistance enhances the plants to mobilize appropriate cellular defines responses before or during pathogen attack [34]. It is generally systemic and triggered by non-pathogenic bacteria [31,35]. Induced systemic resistance (ISR) varies in their effect against nonpathogenic and pathogenic organisms [30,31,34,35]. In plants exhibiting induced systemic resistance, beyond infection sites, deposition of callose, lignin, phenolics, and calcium reported [31] and activities of hydrolytic enzyme increased [32,36]. In view of the above, the objective of our study was to assess the ability of certain isolates of epiphytic and antagonistic Pseudomonas and Bacillus sp., isolated from grape berries, to prevent the transmission of the mycotoxigenic Aspergillus sp. to post-harvest Taify table grape berries (Vitis vinifera L.). Subsequently, accumulation of the aflatoxin B1 (AFB1) and the ochratoxin A (OTA) in the grape berries. The measurements of activities of the peroxidase enzyme, total phenols, and lipid peroxidation, in reaction to the fungal challenge and bacterial treatment, were also taken in this work.

Materials and Methods

Samples

Thirty Taify table grape berry samples (Vitis vinifera L.) were collected in 2016 from six private farms in Taif city, Saudi Arabia. Samples were harvested in July during the source of the grape harvest. Grape bunches (each roughly 1 kg) were gathered and invested in previously sterilized cardboard boxes and kept at 4°C until analysis.

Microorganisms used

Aspergillus niger BAVSH1, A. parasiticus BAVSH4, and A. tubingensis BAVSH5, previously isolated from contaminated grape berries and identified to the molecular level by El-Shanshoury and his coworkers [8] used for challenges in Taify table grape and to check OTA and AFB1 production. The antagonistic grape epiphytic bacteria Pseudomonas aeruginosa EBVHSH17, Bacillus vallismortis EBHVSH28 and B. amyloliquefaciens EBHVSH29, previously isolated and identified to the molecular level by El-Shanshoury and his coworkers [8] employed to prevent the Aspergillus transmission, control Aspergillus rot and aflatoxin B1, and ochratoxin A gathering in post-harvest Taify Table grape.

Detection and determination of aflatoxin B1 and ochratoxin A in Aspergillus sp.

Fungal isolates belonging to the genus Aspergillus were grown on Sabouraud’s agar medium [8] for five days. Agar plugs (5 mm diameter) taken from a growing edge of a 5-day-old test fungal colony and transferred to 500 ml Erlenmeyer flask containing sterile 100 ml Sabouraud’s liquid media. Erlenmeyer flasks containing inoculated media incubated for 10 days at 28°C in the dark. The content of each flask filtered through Whatman No. 4 filter paper and extracted in a separating funnel with chloroform (three times; each extraction, 5 ml). The organic extracts containing Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) combined, evaporated to dryness and suspended in 1 ml of chloroform and dried with steam bath (50°C). The dried extract kept in the refrigerator at 5°C for further analysis. Separation and detection of AFB1 and OTA in the sample extract carried out using thin layer chromatography (TLC) on pre-coated plastic sheets of silica gel 60 without fluorescence indicator (20 × 20 cm, layer thickness 0.2 mm, Merck) according to Makun et al. [37]. TLC performed on all samples by using chloroform: acetone (9:1, v/v) for AFB1 or benzene: Glacial acetic acid (9:1 v/v) for OTA as development solvents. The plates were examined under long wave UV light (365 nm). Blue fluorescence indicates that AFB1 is present, while the bluish-green fluorescence indicates the presence of OTA. For confirmation, a TLC plate sprayed with a fine mist of H2SO4, 50% (v/v) solution in water for AFB1, while spraying with an alcoholic solution of sodium bicarbonate (6.0 g of NaHCO3 in 100 ml H2O+20 ml ethanol) for OTA and let dry. Under longwave UV light, the blue fluorescence of AFB1 turns to yellow while the blue-green fluorescence of OTA turns to blue and becomes more intense. The quantities of AFB1 and OTA determined by reading the silica gel plates quantitatively by fluoro-densitometer (TLD-100 Vita- Torn) after the methods described elsewhere [38-42].

Grape berries, antagonists, and fungal pathogens

Taify table grape berries (Vitis vinifera L.) harvested at maturity and those having uniformity in size and ripeness and lack of any clear injuries or infection selected. Berries samples were surface-disinfected with sodium hypochlorite at 0.1% (v/v) for 1 min, rinsed with sterile tap water for three times, and allowed to air dry at room temperature (20°C). Inoculants suspensions and concentrations of the bacteria P. aeruginosa, B. vallismortis and B. amyloliquefaciens prepared and adjusted to 108 cells/ml colony forming Unit (CFU). Spore suspensions and concentrations of the pathogens A. niger, A. parasiticus and A. tubingensis were prepared and adjusted to 104 spores/ml CFU as before described by El-Shanshoury and his coworkers [8].

In vivo control of Aspergillus transmission and rot disease

For postharvest Aspergillus transmission and disease assays, the surface-disinfected grape berries injured in the middle with a sterile cylindrical tool (about 2 mm diameter and 3 mm deep) and the cut tissue removed. Into each wound, 30 μl of sterile water or living cell suspension in sterile distilled water by the antagonistic bacteria P. aeruginosa, B. vallismortis and B. amyloliquefaciens, adjusted at 1 × 108 cells/ml pipetted and inoculated. In place grape berries immersed and shaken with either the living bacterial cell suspension at 1 × 108 cells/ml or with crude cell-free extracts from freeze-dried and thawed bacterial cell suspension at 1 × 108 cells/ml, for 20 min at 120 rpm. After the suspensions were absorbed, the berries injured after 72 hours. After the proper interval, berries challenged with the pathogens A. niger, A. parasiticus or A. tubingensis. Berries injured and 1 × 104 spores/ml CFU were introduced in 30 μl. Injuries treated with sterile distilled water and with fungi spore suspension served as the inoculated positive disease controls. In each experiment, injuries treated with either sterile distilled water or antagonistic bacteria suspension, without pathogen inoculation used as negative controls. For all experiments, negative controls did not show disease symptoms. After air drying, the treated berries stored at 20 and 5°C in pre-disinfected, covered plastic containers to keep up a high relative humidity. The lesion diameters of the infected fruits examined daily and recorded every fourth day after inoculation, started after four and ten days for those incubated at 20°C and 5°C, respectively. Inhibition (%)=[(average lesion diameter of infected wounds in the control average lesion diameter of infected injuries in the treatment)/average lesion diameter of infected injuries in the control] × 100. Each experiment consisted of three replicates per treatment.

Analysis of the aflatoxin B1 and the ochratoxin A in fungi infected grape berries

Grape berries showed fungal infection freeze-dried, ground and subjected to aflatoxin B1 (AFB1) and ochratoxin A (OTA) determination. 5 grams of milled sample extracted according to the method of Golinski and Grabarkiewicz-Szezesna [43], using chloroform (three times; each extraction, 5 ml). The organic extracts containing AFB1 and OTA joined, evaporated to dryness and suspended in 1 ml of chloroform and dried with steam bath (50°C). The dried extract was kept in the refrigerator at 5°C for further analysis. Identification and estimation of AFB1 and OTA in the sample extract carried out as described above.

Grape berries, peroxidase, lipid peroxidation, and total phenols

The surface-disinfected grape berries injured and inoculated with living cells of antagonists. Intact fruits suspended in living bacterial cells or in cell-free extract and injured after 72 h. Injured fruits were then fugally challenged and stored in sterile covered plastic containers at 20°C for different time intervals, up to 36 h. Treated and untreated grape berries analysed for peroxidase activity, lipid peroxidation, and total phenols.

Extraction and assay of peroxidase

Grape berries (0.5 g fresh weight) collected at 4, 12, 24 and 36 h intervals of the fungal challenge extracted in 50 mM sodium phosphate buffer (pH 7.0) and assayed as described by Kato and Schimizu [44]. The reaction mixture (3 ml) consisted of 7.2 mM guaiacol, 11.8 mM hydrogen peroxide. Add about 0.1 ml of crude enzyme extract to start the reaction, which measured spectrophotometric ally (Genesys 10 views) at 470 nm. Enzyme activity expressed in terms of the change in absorbance per minute at 470 nm in the linear phase of the slope (Δ 470/min/g fresh weight) immediately after adding the substrate.

Extraction and determination of lipid peroxidation

Lipid peroxidation measured by the amount of malondialdehyde (MDA) as a product of peroxidation of unsaturated fatty acid (linolenic acid, 18:3). One gram of grape berries homogenized in three ml of 1.15M KCl. The homogenate filtered. MDA concentration measured by the method of Mesbah et al. [45]. Briefly, 0.5 ml of enzyme extract, 0.5 ml of TCA (20%) and 1 ml of TBA (0.67%) mixed together in centrifugation tubes and heated to 100°C for 15 min. After cooling, 4 ml butanol was added and the content was mixed by vortexing the tubes and centrifuged at 1500 rpm for 15 min. The absorbance of the supernatant measured at 530 nm in a spectrophotometer. The MDA activity expressed in nm/g fresh weight.

Extraction and estimation of total phenols

0.1 grams of dry grape berries powder kept in 10 ml ethyl alcohol (95%) in the dark for 24 hours and filtered through Whitman No 1 filter paper. Total phenols estimated by the method of Jindal and Singh [46]. Briefly, 1 ml of alcohol extract was pipetted in a graduated tube. Then 0.1 ml of Folin-ciacalteau reagent added followed by 1 ml of 20 present Na2CO3 solutions. The tube was shaken and heated in boiling water bath for 1min. The tube cooled under running tap water. The blue solution was diluted to 25 ml with distilled water and absorbance measured at 650 nm in a spectrophotometer. A control containing all reagents except fruit extract used to adjust the absorbance at zero. All treatments conducted at least three times per treatment for each time interval. The amount of phenolic content was expressed as phenol equivalents in mg/g-dry tissue.

Statistical analyses

All results subjected to one-way ANOVA and the means compared according to the Student–Newman–Keuls (SNK) multiple range tests (p ≤ 0.05).

Results

Detection and determination of ochratoxin A and aflatoxin B1

The results obtained from the analysis of ochratoxin A (OTA) and Aflatoxin B1 (AFB1) in Aspergillus species summarized in Table 1. 60% of the isolates belonged to A. niger presented detectable mean levels of 150 μg/g OTA. For AFB1, it occurred in 46 and 53% of A. parasiticus and A. tubingensis isolates, respectively. The mean values of positive samples were lower than OTA, ranging between 40 and 53 μg/g.

Fungus No isolates % positive isolates Average mycotoxins (µg/g)
Assayed Positive
AFB OTA AFB OTA AFB OTA AFB OTA
  1. niger BAVSH1
10 10 Nd 6 Nd 60 Nd 150
A. parasiticus BAVSH4 15 15 7 Nd 46 Nd 42 Nd
A. tubingensis BAVSH5 15 15 8 Nd 53 Nd 40 Nd
ND: Not detected under the experimental conditions

Table 1: The capacity of Aspergillus sp. isolated from Taify table grape to produce aflatoxin B1 (AFB) and ochratoxin A (OTA).

Impact of the antagonists on Aspergillus establishment, transmission, and rot development

Suspension of intact Taify table grape berries in live cells and crude cell-free extracts of the antagonistic epiphytic bacteria P. aeruginosa, B. vallismortis and B. amyloliquefaciens reduced Aspergillus infection to hundred percentages of those in the inoculated controls (data not presented). Application of living cells of the antagonist’s reduced transmission thus induced rot resistance on grape berries, during the stages of 28 and 50-day incubation period at 20°C and 5°C, respectively (Figure 1). The results showed the relationship of average lesion diameter inhibition percentage of injuries treated with living cells of different bacteria and the incubation time after pathogen inoculation. The efficacy of bacterial treatments in inhibiting the lesion size was closely correlated with the type of bacterial cells used and incubation time. After sixteen days of inoculation, the lesion inhibition percentages reached a hundred percentages compared to the inoculated controls. This was clear in injuries treated with living cells of P. aeruginosa, B. vallismortis and B. amyloliquefaciens and infected with A. niger at 20°C (Figure 1A1), injuries treated with living cells of B. amyloliquefaciens and infected with A. parasiticus at 20°C (Figure 1B1), injuries treated with living cells of P. aeruginosa and B. vallismortis and infected with A. tubingensisat 20°C (Figure 1C1). Also, complete inhibition of injuries infected with A. tubingensis at 5°C was detected by B. vallismortis, B. amyloliquefaciens and P. aeruginosa after 10, 18 and 22 days, respectively (Figure 1C2). While, a relative low inhibition (less 50%) was observed by the tested bacteria against both A. niger and A. parasiticus (Figures 1A2 and 1B2, respectively). In case of A. parasiticus infection at 5°C, it noted that although living cells were effective in inhibiting the early stages of infection, it decreased as incubation time after pathogen inoculation bypassing (Figure 1B2).

plant-pathology-microbiology-injured-Taify

Figure 1: The effect of inoculating injured Taify table grape berries with living bacterial cells on Aspergilli rot disease development (lesion inhibition%), over time intervals at 20°C and 5°C. Grapefruits were injured and inoculated with bacterial cells at 1 × 108 cells/ml, 72 h prior to challenge with Aspergillus niger BAVSH1 (A1, A2), A. parasiticus BAVSH4 (B1, B2) and A. tubingensis BAVSH5 (C1, C2) at 1 × 104 spores/ml.

Peroxidase, lipid peroxidation, and total phenols

The changes in peroxidase, lipid peroxidation, and total phenols recorded in pathogens inoculated, bacteria-treated, grape berries along with their respective non-inoculated controls. The samples collected at different time intervals from 4 to 36 h after the challenge. The activity of peroxidase enzyme and total phenols were higher in infected and bacteria-treated, grape berries than in uninfected or no bacteria treated ones. The vies was in the case of lipid peroxidation.

Peroxidase activity

The data in Table 2 showed that the peroxidase (PO) activity of the grape berries, treated with different antagonistic bacteria, was higher than that of the untreated berries, after times from application. Fungal challenged grapes caused higher PO activity that changed according to the fungus than the non-inoculated fruits, whether treated or untreated, particularly after 4 h of treatment. Pre-immersion of intact berries, either in living cells or crude cell-free extracts recorded higher enzyme activity than in wounded fruits. For all fungi, the induction of PO by all tested bacteria varied significantly compared to its control (Table 2). The highest levels of PO determined 4 h after treatment, then decreased and re increased with time in all cases. Control samples recorded the lowest activity of 9.33 U at 12 h after challenge with A. niger, with the maximal activity of 80 U at 4h. While the highest activity of PO for the infected control was at 4h of A. parasiticus infection. In berries suspended in living bacterial cells of P. aeruginosa and challenged with A. parasiticus, PO activity reached the peak at 4 h after fungal challenge with 193.31U. It was about 250% higher than the infested control. Grapevine treatment by living cells of B. vallismortis and B. amyloliquefaciens was the best inducer for PO activity against A. tubingensis at 4 h. They were 166.70 and 166.70U, respectively. It was about 28% higher than the infested control (Table 2). In case A. niger infection, PO activity induced by all tested bacteria was higher than the infested control and varied insignificantly. Moreover, more 100% of PO activity was induced using the crude cell-free extract of B. vallismortis.

Treatment
Challenge Time Control P. aeruginosa EBVHVSH17 B. vallismortis EBHVSH28 B. amyloliquefaciens EBHVSH29
Living cells+ Living cells++ Crude cell-free extract+++ Living cells+ Living cells++ Crude cell-free extract+++ Living cells+ Living cells++ Crude cell-free extract+++
Non-infected control  4 h  93.33a  24.02c  80.11a 43.32b  80.33a  17.44e  20.33d  80.46a 76.79 a  33.34c
12 h  30.33b  14.02cd  5.73d 36.71b  9.32cd  16.73cd  49.79a  6.33d  20.34c  40.32b
24 h  13.32c  17.32c  11.73c 75.37a  16.71 c  18.32c  42.33b  13.33c  43.33b  74.37a
36 h  15.84e  19.71de  38.04bc 72.39a  22.73c  44.35b  74.78a  70.77a  34.36bcd  80.63a
 4 h  54.00f  93.31e 193.31a 130.13c  13.42h  32.32g 146.70b 127.30c 103.30d 151.72b
A. parasiticus BAVSH4 12 h  16.70d  33.33c  10.18d 94.71a  10.29d  25.71c  10.36d  10.38d  6.23e  60.44b
24 h  24.00d 124.03b 121.32b 17.32cd  81.32c 122.71b  23.32d  12.33d  13.31d 151.39a
36 h  22.01cd  11.12d  20.12cd 11.39d  77.11b  81.31b  19.72cd  28.42c 147.65a 151.35a
 4 h 130.00bc 126.72c 100.22b 130.23b 100.21d 166.70a 150.11a 133.31b 166.72a 140.20b
A. tubingensis BAVSH5 12 h  73.31cd  73.31cd  63.32d 10.10f 135.33b  10.32f  40.02e  12.72f 149.79a  90.33c
24 h  12.33 c  13.00c  17.72c 18.72c 170.44a  13.11c  14.31c 167.31a 134.39a  57.35b
36 h  26.32b  22.30b  15.33b 5.11c 134.37a 144.17a 140.39a 133.77a 136.72a 130.77a
 4 h  80.00b 146.70a 150.08a 146.77a 140.28a 140.37a 160.44a 133.35a 123.37a 136.76a
12 h  9.33e  76.72bc  61.71cd 80.38b  76.75bc  9.34 e  13.33e  18.12e 147.78a  57.04d
A. niger BAVSH1 24 h  19.71d  18.73d  11.35d 29.31c  58.72b  40.72bc  74.36a  71.38a  77.23a  70.33 a
36 h  39.00d 145.70a 145.77a 23.32d  66.31c  68.73c  63.79c  70.72c 108.79b 111.33b
Means with different letters within the same row differ significantly at p ≤ 0.05,+Injuried berries inoculated with the living bacterial cells at 1 × 108 cells/ml followed by challenge with different Aspergillus sp. at 1 × 104 spores/ml after 72 h and kept at 20°C, ++ Intact berries suspended in the living bacterial cells at 1 × 108 cells/ml, injurieed and challenged with different Aspergillus sp. at 1 × 104 spores/ml after 72 h and kept at 20°C, +++ In place berries suspended in the crude cell-free extract from freeze-dried and thawed bacterial cell suspensions at 1 × 108 cells/ml, injuried and challenged with different Aspergillus sp. at 1 × 104 spores/ml after 72 h and kept at 20°C.

Table 2: Peroxidase activity (enzyme unit/g fresh weight/min) of the Taify table grape berries treated or untreated with the antagonistic epiphytic bacteria over different time intervals of the challenge with different Aspergillus sp.

Lipid peroxidation activity

The results indicated that the levels of MDA activity gradually decreased in all treatments with increasing the time after infection and then increased after 36h. MDA levels induced by all tested bacteria for infected berries that varied significantly compared to its control (Table 3). Fungal-infected and bacteria-free treated samples showed low MDA activity of 23.91 at 24 h, after the challenge with A. tubingensis. The highest value (103.39) was obtained at 4h for non-infected control. The activity was maximal at 36 h by the living cells of P. aeruginosa with A. parasiticus and A. tubingensis. It was 156.71 and 171.79, respectively. Also, it was detected by the living cells of B. vallismortis at 36 h against A. niger infection. In berries treated with bacteria, MDA levels were markedly decreased, as compared with non-infected and infected controls. Generally, bacteria cell-free extracts recorded lower MDA than living cells-treated wounds fruits, except those of B. amyloliquefaciens against A. niger infection at 24 h of infection. Crude cell-free extract of B. amyloliquefaciens increased MDA by 1.8 times, over the infested control.

Treatment
Challenge Time Control P. aeruginosa EBVHVSH17 B. vallismortis EBHVSH28 B. amyloliquefaciens EBHVSH29
Living cells+ Living cells++ Crude cell-free extract+++ Living cells+ Living cells++ Crude cell-free extract+++ Living cells+ Living cells++ Crude cell-free extract+++
Non-infected control  4 h 103.39c 109.37c 116.33c  88.33c 116.42c  89.86c  80.75c 141.51b 167.78a  72.91c
12 h  40.67f  88.40a  58.90c  30.19 g  56.54d  37.95fg  34.40fg  52.74e  68.89b  39.10fg
24 h  52.33b  27.14cd  21.85d  33.71c  36.07c  20.48d  28.03cd  35.67c  73.11a  20.58d
36 h  94.37c 198.76a 127.69bc 117.21c  88.00 c  98.98c 123.86bc  96.14c 124.26bc 134.16b
A. parasiticus BAVSH4  4 h  80.06b 104.86b 106.79b  67.91b  79.58b 108.09b 101.92b 116.22a  91.63b 110.71b
12 h  34.79d  64.03b 109.27a  43.12c  57.72b  48.31b  59.29b  8.92f  49.29b  20.39e
24 h  43.71ab  42.53abc  39.30abc  50.18a  40.86abc  20.78 d  36.36abcd  40.28abc  22.93cd  26.17bcd
36 h  58.21e 108.19bc 156.71a  77.52d 117.62b 101.14bc 101.72bc  89.95cd 116.01b 120.15b
 4 h  73.01cd 122.21a 101.14bc 108.29b  50.18d  90.36c  75.27cd 101.92bc 101.33bc 114.76a
A. tubingensis BAVSH5 12 h  35.28 c  25.49 d  47.13b  22.74d  54.68a  35.97c  33.31c  31.93 c  20.68d  49.88b
24 h  23.91e  70.26b  47.33bc  48.70bc  48.80bc  46.85bc  26.98d  83.04a  52.82b  29.60c
36 h  93.52d 116.23cd 171.79a  49.98e 102.93d 135.53b  51.25e  83.59d 165.03a 117.66c
 4 h  98.33c  78.20cd  94.96c  77.13cd 124.17b 143.47a  75.85cd  78.50cd  60.37 d  58.93d
A. niger BAVSH1 12 h  53.51b  17.25d  54.73b  28.52c  80.07a  24.01cd  23.92cd  29.01c  54.40b  24.72cd
24 h  24.52d  46.55b  40.57c  30.38cd  21.98d  26.75d  20.39d  10.58e  30.18cd  68.20a
36 h  84.09c  76.83c  24.94e  38.92d 147.11a 151.11a 120.82b  84.67c  42.53d 105.35bc
Means with different letters within the same row differ significantly at p ≤ 0.05,+Injuried berries inoculated with the living bacterial cells at 1 × 108 cells/ml followed by challenge with different Aspergillus sp. at 1 × 104 spores/ml after 72 h and kept at 20°C, ++ Intact berries suspended in the suspension of living bacterial cells at 1 × 108 cells/ml injuried and challenged with different Aspergillus sp. at 1 × 104 spores/ml after 72 h and kept at 20°C, +++ Intact berries suspended in the crude cell extract from freeze-dried and thawed bacterial cell suspensions at 1 × 108 cells/ml injuriedand challenged with different Aspergillus sp. at1 × 104 spores/ml after 72 h and kept at 20°C.

Table 3: Lipid peroxidation activity (MDA in nmol/g fresh weight) formed in Taify table grape berries treated or untreated with antagonistic epiphytic bacteria over different time intervals of the challenge with different Aspergillus sp.

Total phenol content

The total phenols measured in the fungal inoculated and noninoculated grape berries treated and untreated with the kinds of antagonistic bacteria (Table 4). Total phenol content was considerably increased simultaneously in infected berries, which increased with the increase in the period of infection, then diminished, compared to the dominance. It was obvious that fungi non-inoculated intact fruits and immersed in the crude cell-free extract and the living cells have considerable amounts of phenol compounds. Bacteria pre-treated, grape berries and challenged with the pathogens showed more levels of total phenols. The accumulation of phenols in the bacteria treated berries was relatively increased than in the non-inoculated fruits, during all periods of work. In the case of A. niger infection, total phenol content was maximal (37.33) at 4 h from the application by the cell-free extract of P. aeruginosa. Treatment by living cells of P. aeruginosa showed a high total phenols production for A. tubingensis infection at 24 h. While, crude cell-free extract induced a high total phenols production in case of A. parasiticus and A. niger at 24 and 4 h, respectively. After 12 h of infection a relatively high total phenols production recorded in grapes fruits, using crude cell free extract of B. amyloliquefaciens as a bio control agent to A. tubingensis and A. niger (Table 4). Generally, bacteria pre-treated, grape berries and challenged with the pathogens showed more levels of total phenols. The accumulation of phenols in the bacteria treated berries was highly increased than in the noninoculated fruits, during all periods of work. Total phenol content was exhibited at the 4th h from the application, in the bacteria-treated and fungi-inoculated fruits, and then decreased progressively.

Treatment
Challenge Time Control P. aeruginosa EBVHVSH17 B. vallismortis EBHVSH28 B. amyloliquefaciens EBHVSH29
Living cells+ Living cells++ Crude cell-free extract+++ Living cells+ Living cells++ Crude cell-free extract+++ Living cells+ Living cells++ Crude cell- free extract+++
Non-infected control 4h 3.43c 6.91b 3.48c 16.63a 6.85b 11.11ab 8.91b 7.39b 11.46ab 9.23b
12h 4.43c 16.93a 7.03c 9.27bc 9.43bc 5.13c 8.67c 9.17bc 7.84c 11.22b
24h 1.07b 13.33a 1.41b 4.17b 3.53b 0.31c 2.93b 2.83b 1.03b 4.53b
A. parasiticus BAVSH4 36h 18.57ab 18.9ab 14.03bc 21.63a 14.53bc 11.57c 18.77ab 20.07a 10.93c 11.93c
4h 12.17bc 12.87b 9.07c 14.53b 15.93ab 14.12b 10.77bc 11.43bc 10.67bc 10.63bc
12h 13.77a 12.86a 7.23c 17.4a 6.93c 14.13a 8.73b 7.53c 10.63a 10.27a
24h 4.53bc 4.87bc 10.11ab 12.17a 9.87ab 6.22b 2.27c 2.27c 2.47c 2.43c
A. tubingensis BAVSH5 36h 16.43b 12.07b 11.71c 10.92c 20.47a 15.43bc 13.62bc 11.43c 12.6bc 0.67d
4h 23.83a 14.63c 12.03c 10.73c 13.93c 12.91c 7.73d 17.42b 9.44c 13.03c
12h 10.63a 11.23a 8.87a 11.27a 11.37a 11.27a 8.97a 11.27a 8.87a 12.48a
A. niger BAVSH1 24h 2.31c 24.87a 2.43c 2.33c 2.12c 1.33c 2.73c 2.33 c 2.23c 18.57b
36h 20.37a 7.73b 7.07b 10.22b 5.97b 5.77b 6.93b 6.43b 10.33b 10.93b
4h 13.17b 8.27bc 7.53c 37.33a 8.13bc 11.63bc 9.57bc 8.13bc 10.77bc 0.74d
12h 7.33bc 7.27bc 7.23bc 9.37a 6.13c 7.43c 5.13c 14.13a 7.83b 11.33a
24h 1.07d 0.51e 2.62b 2.13e 1.67c 2.13c 1.33c 0.73e 6.97a 1.95c
36h 8.77abc 15.47a 6.83bc 11.87ab 12.43ab 12.55ab 3.63c 8.97abc 4.53c 6.53bc
Means with different letters within the same row differ significantly at p ≤ 0.05;+Injuried fruits inoculated with the living bacterial cells at 1 × 108 cells/ml followed by challenge with different Aspergillus sp. at 1 × 104 spores/ml after 72 h and kept at 20°C, ++ The intact berries suspended in living bacterial cells at 1 × 108 cells/ml injuried and challenged with different Aspergillus sp. at 1 × 104 spores/ml after 72 h and kept at 20°C, +++ The intact berries suspended in the crude cell-free extract from freeze-dried and thawed bacterial cell suspensions at 1 × 108 cells/ml injuried and challenged with different Aspergillus sp. at 1 × 104 spores/ml after 72 h and kept at 20°C.

Table 4: Total phenols (mg/g dry weight) formed in Taify table grape berries treated or untreated with the antagonistic epiphytic bacteria over different time intervals of the challenge with different Aspergillus sp.

Ochratoxin A and aflatoxin B1 in grape berries

The grape samples before treated with the antagonistic bacteria and kept at 20°C and 5°C, that still showing fungal contamination, after 28 and the 50-day challenge, were subjected for determination of OTA and AFB1. Suspension of in place grapes in living cells and crude cell extracts of the epiphytic antagonistic bacteria P. aeruginosa, B. vallismortis and B. amyloliquefaciens do contain neither AFB1 nor OTA that inhibited to 100 percentages of the inoculated controls (data not presented). Application of living cells of the bacteria reduced accumulation of OTA and AFB1 (Table 5). All the same, the inhibition percentages of the mycotoxins were more pronounced at 20°C torn in 5°C. Levels of OTA and AFB1 were nil and the toxin inhibition percentages were a hundred percentages of the inoculated controls. This phenomenon was clear in the following treatments: wounds treated with living cells of P. aeruginosa, B. vallismortis, and B. amyloliquefaciens, infected with A. niger and incubated at 20°C; injuries treated with living cells of P. aeruginosa at 5°C and 20°C and B. vallismortis at 20°C, infected with A. parasiticus; injuries treated with living cells of P. aeruginosa at 5°C and B. amyloliquefaciens at 5°C and 20°C and infected with A. tubingensis. Other treatments showed detectable amounts of mycotoxins and thus the percentage of inhibition varied according to the treatment and the storage temperature. Referable to the treatments with the antagonistic bacteria, the present of inhibition of OTA ranged between 11-100 percentages and the inhibition percentage of AFB1 ranged between 38% to 100%.

Treatment
Toxins of tested fungi Control P. aeruginosa B. vallismortis B. amyloliquefaciens
EBVHVSH17 EBHVSH28 EBHVSH29
5°C 20°C 5°C 20°C 5°C 20°C 5°C 20°C Range of inhibition %
A. niger BAVSH1 (OTA) 3.13 4.36 2.81 0 0.79 0 0.38 0 (11-100)
A. parasiticus BAVSH4 (AFB1) 4.52 7.62 0 0 3.25 0 2.81 2.86 (38-100)
A. tubingensis BAVSH5 (AFB1) 3.78 37.61 0 17.48 1.63 2.81 0 0 (55-100)

Table 5: Levels of aflatoxin B1 (AFB1) and ochratoxin A (OTA) (ug/Kg dry weight), and inhibition percentage (%) in Taify table grape berries showed Aspergilli rot symptoms after injury treatment with living cells of the antagonistic epiphytic bacteria and challenge with different Aspergillussp.

Discussion

The transmission of pathogenic microorganisms to table grapes during storage could be reduced by several treatments. Among these treatments, less of storage time and discarding visibly rotted bunches. The incidence of in post-harvest fruits could be reduced with sulfur dioxide in cold storage at 0°C [47]. Attempts to cut fungal colonization and mycotoxin content of grapes by agronomic practices, chemical, and biological treatments are sometimes controversial and have varying degrees of success [11]. Biocontrol agents (BCAs) as a tool to cut grape fruit berries diseases are reported, yeasts followed by bacteria and then fungi are the most potential BCAs [26,48,49]. Recently, in vitro studies conducted by El-Shanshoury and his coworkers [8] showed inhibition of Aspergilli sp., isolated from Taify table grape, with three epiphytic bacteria. In this study, preventive treatment of Taify table grape berries with P. aeruginosa, B. vallismortis and B. amyloliquefaciens, resulted in a dramatic reduction of Aspergillus niger, A. parasiticus and A. tubingensis infection and the rate of berries. The data show that the antagonistic bacteria interfered with berries to prevent the establishment and growth of Aspergillus sp., thus preventing transmission and progress of rot disease. Exist of any one of the three biocontrol agents, in the three, applied forms, resulted in lower or zero disease incidences, compared to the control. This was clear when these bacteria introduced; 3 days before the pathogens and the suppression continued until 28 days at 20°C or 50 days at 5°C. These points and storage degrees desirable for the bacteria to synthesize significant amounts of antimicrobial compounds to prevent the disease progression. Some researchers argued that carbohydrates and proteins implicated in the adhesion reaction of the bacteria [50,51]. Similarly, in this study, the inhibition of Aspergillus rot by P. aeruginosa could be due to the association of the bacterium and its products to the challenge fungi and the plant cells at the wound site containing the sugars. This concurs with the facts that Pseudomonas species promote biofilms [52] and adhere to plant cells [53,54] and fungi [55]. The inhibitory compounds produced by B. vallismortis and B. amyloliquefaciens seem bactericidal rather than bacteriostatic, by the outcomes received by Ranjbariyan et al. [56]. Populations of many BCAs that introduced into artificial environments decline with time [57]. Thus, the necessary quantities of antimicrobial agents may be lost with the resulting cessation in the synthesis of inhibitory compounds as observed in our study. Agree with studies conducted by Bruce and West [58], Sticher et al. [59] and Bakker et al. [34], the growth in the peroxidase activity in reaction to infection by the pathogens and bacterial treatments accounted in this work. The increased levels of total phenols in grape berries treated with different varieties of bacteria may offer an adequate substrate for oxidative reactions consuming oxygen [60] and producing fungi toxic for compounds, make the medium unfavourable to the fungal establishment, further growth of pathogens and disappearance of Aspergillums rot disease. Peroxidase production is stimulated by biotic and non-pathogenic bacteria, as documented in this study, and as one of the major mediators for oxidation of phenols to highly toxic compounds with antifungal potential [34]. This phenomenon supports our finding concerning total phenols and peroxidase in grape berries. Likewise, in a survey led by Mohamed et al. [61], the enzyme peroxidase is significantly induced by both biotic and abiotic factors that helped in controlling downy mildew of cucumber. Important metabolites from grapevine reviewed with their role against certain stress factors in grapevine physiology [1]. In this connection, several abiotic and biotic agents induce and enhance plant resistance to pathogens, mediated by stimulating the biosynthesis of phenol and phytoalexin inhibitory substances, PR-proteins and defines-related oxidative enzymes [29,30,36,60-62]. Bacterial determinants such as lipopolysaccharides [63] and lipopeptides [64], pathogen-resistant genes or pathways as well [65] or an enhancement of the bioactive secondary metabolites such as salicylic acid [66], siderophores [67], apigenin-7-Oglucoside [68]. In Taify table grape berries, significant amounts of the total phenols and the peroxidase enzyme were detected, that increased by epiphytic bacterial treatments. Lipid peroxidation was reduced, time after infection by fungi and with bacterial treatments, compared to control. Lipid peroxidation and lipid damage may be partly responsible for some of the cell changes and probably affect membrane function. When plants are subjected to pathogen attack, the equilibrium between productions and scavenging of ROS is broken, resulting in oxidative damage o for proteins, DNA and lipids. Malondialdehyde (MDA) content (a characteristic of lipid peroxidation) released from cellular membranes of tissues and is made by the reaction of ROS (HO or/and O•-) with lipid molecules [60,69]. Nevertheless, the reduction in lipid peroxidation in infected plants might be connected to the high activity of antioxidant enzymes, preventing accumulation of free radicals, and consequently membrane damage, in conformity with a study conducted by El- Khallal [70]. The outcomes described in this survey indicate that the increased total phenols and the peroxidase enzyme may have more antibacterial activity. The decrease in lipid peroxidation in infected and treated grapes might be linked to preventing membrane damage. The antimicrobial effect of the antagonists supported by the antibacterial activities of total phenols and the peroxidase enzyme. In increase, preventing membrane damage, all helped grape berries to prevent the formation of the mycotoxigenic fungi, further development, berry infection and rot disease development. Bearing out this idea, the role of defense-associated enzymes and total phenols in the resistance mechanisms of plants against fungal pathogens and phytophagous insects has been reviewed by Lattanzio et al. [71]. The subsequent products have been demonstrated to possess antibacterial properties as documented by Croft et al. [72] and signaling function as recorded by Melan et al. [73]. Preformed anti-fungal phenolic compounds are present in healthy plants at levels that are expected to be antimicrobial. Their grades may increase further in response to the challenge by pathogens. In accordance with the conclusion of Khan et al. [74], high phenol production in Taify table grape may have loss of virulence in the pathogens and induced resistance by bacterial cell determinants or cell-free extract may cause higher phenolic contents in the grapes and subsequently have deleterious effects on the pathogens. In accord with the last reported by Ciconova and his coworkers [27] and Somma et al. [75], the answer recorded in the present study indicated exist of OTA and AFB1 in Aspergillus sp.- infected Taify table grapes. Some treatments of grape berries lowered the levels of OTA and AFB1 to nil. Suspension of Taify table grapes in living cells or crude cell extracts of the isolated antagonists was superior in this respect. Other treatments showed detectable amounts of the mycotoxins. The percentage of inhibition varied according to the treatment and the storage temperature. The stop of aggregation of OTA and AFB1 in the treated Taify table grape berries ascribable to the result of the bacterial antagonists, supported by the raised peroxidase enzyme and total phenols, in gain to lower lipid peroxidation in bacteria-treated groups. All shared grapes for destruction the establishment and developing of Aspergillus spp, thus preventing fungal transmission and gathering of OTA and AFB1. Other schemes prepared to minimize the levels of pre-or postharvest mycotoxins in different plants [23,76-80].

Conclusion

In this discipline, the antagonistic bacteria Pseudomonas aeruginosa, Bacillus vallismortis and B. amyloliquefaciens, their cellfree extracts, the incited peroxidase enzyme, total phenol and the brought down lipid peroxidation, made the medium ominous to the fungal establishment and further developing, and after that developing aggregate AFB1 and OTA ceased. Hence, the assemblage of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in post-harvest Taify table grape was ceased. As pre-immersion treatment is potentially the most effective, this highlights its grandness to prevent the infection by the mycotoxigenic Aspergillus species and AFB1 and OTA assemblage. It suggests that P. aeruginosa, B. vallismortis and B. amyloliquefaciens could be held up as a surface treatment during the storage periods of grapes. It could be held up as the potential eco-friendly biocontrol agent for works protective cover strategy and recommended over other antagonistic organisms for field level management. This is because they well organized in big quantities in simple media and in very little time. The antagonistic bacteria, used in this study, may have certain determinants or synthesize certain compounds with antimicrobial activities. Therefore, further work needed to find the active agents and possible identification.

References

Citation: El-Shanshoury AER, El-Halmouch YH, Mohamed SF, Fareed MF (2018) Potential of Grape Epiphytic Antagonists to Biocontrol Aspergillus Transmission and Accumulation of Aflatoxin B1 and Ochratoxin A in Post-Harvest Taify Table Grape. J Plant Pathol Microbiol 9: 454.

Copyright: © 2018 El-Shanshoury AER, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.