Preliminary Studies on Suppression of Important Plant Pathogens by Using Pomegranate and Avocado Residual Peel and Seed Extracts

Abstract

Potential synergistic action of aqueous extracts of pomegranate peel (PP), avocado peel (AP), and avocado seed (AS) wastes isolated by microwave-assisted extraction were assessed in in vitro and in vivo assays as biocontrol agents against several plant pathogenic fungi. The study findings contribute to the utilization of a value-added industrial byproduct and provide significant value in advancing the development of new plant protecting compositions that benefit from the synergistic effects between two important plant species that contain several natural bioactive compounds. More specifically, the in vitro results proved that the use of 100%-pure (PP) extracted waste affected the mycelium growth of Penicillium expansum. Furthermore, mycelium growth of Aspergillus niger was decreased by 10.21% compared to control after 7 days of growth in medium agar containing 100% AP and extracted waste. Moreover, mycelium growth of Botrytis cinerea was affected by equal volume of avocado extraction wastes (50% peel and 50% seed) only at the first 3 days of the inoculation, while at the seventh day of the inoculation there was no effect on the mycelium growth. Equal volumes of the examined wastes showed decreased mycelium growth of Fusarium oxysporum f.sp. lycopersici by 6%, while Rhizoctonia solani mycelium growth was found to be the most sensitive in PP application. In addition, the in vivo assay shown that PP extract suppresses damage of tomato plants caused by R. solani followed by extracted wastes from AP. Based on the research findings, it can be argued that PP and AP extracts can be used as natural antifungals instead of dangerous synthetic antifungals to effectively treat phytopathogens that cause fruit and vegetable losses during cultivation.

1. Introduction

Due to the growing global population, which is estimated to reach 9.7 billion by 2050 [1,2], a significant increase in food supply is required. Other major challenges are the nearly 1 billion malnourished people worldwide [3], and climate change, which poses a serious threat to agricultural production stability [4]. Furthermore, other biotic and abiotic stresses, such as drought, salinity, host specialization, pests, diseases, and weed infestations, affect further crop production and are likely to increase in the future [5,6]. To address the growing demand for food, more attention is needed in order to increase yield and reduce losses. Thus, the Food and Agriculture Organization of the United Nations (FAO) stated that a minimum increase of 50% in agri-food production is required in a moderate economic growth scenario [7,8], while other models require a higher increase in agricultural production [6,9]. Therefore, a further increase in the effectiveness of plant protection is required [10].

Although the use of agrochemicals has prevailed in the control of plant pathogens, the issues caused by the use of chemicals have prompted many researchers to search for new alternatives for plant protection. More specifically, synthetic chemical fungicides pose significant risks to human health, also causing serious environmental effects to ecosystems [11], and therefore it is necessary to find alternative methods of controlling plant diseases. Thus, a large number of the pesticides used to control plant diseases are not accepted by the international community, as their residues can cause fruit discard.

Furthermore, despite alternatives such as the use of chemical substances, agricultural practices for managing important plant pathogens incorporation of organic amendments, etc., are also either environmentally unacceptable or technically demanding. Thus, the exploitation and the use of several natural compounds derived from plants and their wastes can potentially provide the most effective means of controlling invasion and distribution of important fungal plant pathogens [12]. Despite the abovementioned control methods, the use of biological control agents has also been recognized as one of the most effective biological control alternatives against soilborne pathogens, including root–knot nematodes [13,14].

Moreover, it has been agreed that nowadays, during processing of ready-to-eat fresh fruits, large amounts of peel and seeds, constituting about 50% of the total weight, are discarded as waste [15,16]. Thus, wastes of the agro-food industry are a constant threat to the environment and a severe operational issue for them. However, plant wastes contain bioactive compounds with several activities [17,18,19,20,21,22]. Thus, the extraction technologies for the production of bioactive compounds contained in plant species, such as pomegranate, have been highlighted [23]. Finally, according to Wu et al. [24] many compounds extracted from plant material have shown promise for inhibiting bacterial pathogens.

As mentioned above, despite synthetic chemicals used for crop protection, plants can protect themselves by producing natural metabolites used for antimicrobial control. Among these natural metabolites with the above characteristics are polyphenols, whose beneficial activity for human health has been commented on [25]. Furthermore, phenolic compounds, free fatty acids, and aromatic compounds have not only been detected in plant tissues but also in their residual byproducts. Several research studies have exhibited antimicrobial activity [12,26,27,28,29,30] controlling bacterial infestations [31] and fungi [32,33] after appropriate treatment [23,34]. Thus, this alternative action in the field of plant protection supports the fact that biological plant protection offers a strong alternative to disease control by chemicals [35,36].

The aim of this research study was the in vitro and in vivo assessment of different concentrations and mixtures of polyphenolic substances extracted from PP and AP and seeds used as potential plant protective material against economically important fungal plant pathogens.

2. Materials and Methods

Extraction method and conditions of AP, PP, and AS extracts used in this study are described in detail in previous research work [37,38]. In more detail, PP were collected as a byproduct, were frozen after juice processing, and the frozen peels were milled mechanically, using a commercial mill. Additionally, green skin avocado fruits (“Pinkerton” variety) were purchased from Crete Island in southern Greece. The AP and the AS were then separated and stored at −28 °C until further mechanical shivering was performed using a sphere mill in order to powder the samples. The final AP and AS powders were sealed in plastic bags, weighed at 2 kg, and kept frozen at −28 °C until the extraction procedure. In order to extract PP and AP, each sample (2 kg) was extracted using 20, 50, or 80 L of water, depending on the dilution ratio used, as proposed by the experimental design software. The extraction experiments were conducted in “Pella’s Nature P. Co.” facilities (Edessa, Greece), using an industrial-type MAC-75 multimode microwave extractor (Milestone Inc., Sorisole (BG), Italy). The experimental temperature was set at 40, 60, or 80 °C, the microwave power was set at 2000, 4000, or 6000 W, and the processing time was fixed at 10, 50, or 90 min, respectively, based on experimental design conditions. The extraction vacuum was set at 355 mbar for all samples. Each experiment was performed in triplicate. The obtained extracts were filtered and kept in a freezer at 20 °C until further analysis. The samples were centrifuged at 12,000 rpm for 10 min, and the obtained supernatant was used for further analyses.

2.1. In Vitro Studies

Important plant pathogens, such as Fusarium oxysporum f.sp., lycopersici (code: 2550), Botrytis cinerea (code: 1948), Rhizoctonia solani (code: 2531), Aspergillus niger (code: 1970), and Penicilium expansum (code: 1395), were used in this study in order to evaluate the antimicrobial activity of PP, AP, and AS extracts using the Well diffusion assay, according to the method described in a previous research study [39]. In more detail, before potato dextrose agar (PDA) application to the Petri dishes, 1.5 mL of the examined solutions were placed in them. Then, 20 mL of PDA nutrient substrate were applied to each plate with simultaneous stirring to incorporate the solution into the nutrient substrate. The plates were then allowed to stand for about 30 min to stabilize the nutrient substrates. Finally, a small well was then centrifuged in the center of the plate with the sterilized nutrient substrate, in which 25 μL of the test fungus was poured. For each treatment, five replicates were applied and the experiment was repeated twice.

All fungal pathogens used were obtained from Benaki Phytopathological Institute (B.F.I.) in Athens, Greece. Mixture combinations of the PP, AP, and AS extracts were determined using a mixture design expert.

Table 1. Concentrations and combinations of the examined extracts.

Number of Sample	Pomegranate Peel % (PP)	Avocado Peel % (AP)	Avocado Seed % (AS)
1	16.70	66.70	16.70
2	0	100	0
3	0	0	100
5	0	50	50
6	33.30	33.30	33.30
8	100	0	0
9	50	50	0
10	16.70	16.70	66.70
12	66.70	16.70	16.70
13	50	0	50

represents the concentrations and the combinations of liquid PP, AP, and AS waste extracts used in this study.

The treated Petri dishes were incubated at 28 °C for up to various days depending on the mycelium growth of each fungal species. More specific final mycelium growth measurement for F. oxysporum f.sp., lycopersici, A. niger, B. cinerea, and P. expansum was made after 7 days of incubation at 28 °C, while for R. solani, the incubation time was extended to 12 days due to the slow mycelium growth.

Minimum inhibitory concentration (MIC) was also used in order to observe the potency of different mixtures and concentrations of the examined polyphenolic extracts against fungal plant pathogens in order to determine the major effect on inhibiting the mycelium growth. In this method, sterile test tubes containing 10 mL of potato dextrose broth (Oxoid, U.K.) (PDB) substrate were prepared as described in previous work [39]. The above tested fungal species were used in order to evaluate the inhibitory effects on the samples. Inoculation of the test tubes was followed by streak of five plant pathogenic fungi (0.1 mL of each phytopathogenic fungal suspension), separately for each test tube. The test tube containing spores and mycelium of each fungus was placed in the vortex apparatus for stirring before each use. The test tubes with the fungi were incubated at 22 °C for 7 days. After completion of the incubation, the results concerning the growth of mycelium in the solution (on the surface or bottom of the test tube) were recorded. Test tubes in which the pathogenic fungal mycelium had grown showed that the amount of liquid polyphenol was not sufficient to inhibit the growth of microorganisms and were labeled as “positive”. On the contrary, test tubes that did not show any mycelial growth of the tested fungus were considered to have inhibited its growth (“negative”).

In order to determine whether these amounts of polyphenols in the samples were capable of causing, in addition to inhibiting the fungi, lethal effect, tubes containing 5 mL of Maximum Recovery Diluent (Neogen) (MRD) were added with 0.1 mL of the sample to the test tube in which the fungus had not grown at all. The amount of this test tube was stirred well in the vortex and incubated at 27 °C for 48 h. In those tubes where mycelium growth was observed, it was concluded that the amount of liquid polyphenol had simply only inhibited the growth of the fungus. In contrast, in test tubes where no growth of mycelium was observed, the minimal amount of polyphenol added was also the lethal concentration.

2.2. In Vivo Studies

The in vivo antimicrobial activity of polyphenols was evaluated against F. oxysporum f.sp., lycopersici, P. expansum, and R. solani. The phytopathogenic fungi were grown in Petri dishes filled with PDA and kept at 4 °C. In order to evaluate the effect of PP, AP, and AS extract, in vivo studies were conducted on tomato plant growth in greenhouse conditions. Different concentrations and combinations of the examined polyphenolic extracts were used in this study. In more detail,

Table 2. Ratios of PP, AP, and AS polyphenol extracts.

Number of Sample	Pomegranate Peel (PP) (%)	Avocado peed (AP) (%)	Avocado Seed (AS) (%)
2	0	100	0
6	33.33	33.33	33.33
8	100	0	0
9	50	50	0
13	50	0	50

represents the ratios of PP, AP, and AS extracts.

The treatments used in this study were Fusarium fungus + 100% AP extract, Fusarium fungus + equal concentrations of PP, AP, and AS extracts; Fusarium fungus + 100% PP extract; Fusarium fungus + 50% PP and 50% AP extracts; Fusarium fungus + 50% PP and 50% AS extracts; Fusarium fungus used as control; Rhizoctonia fungus + 100% AP extract; Rhizoctonia fungus + equal concentrations of PP, AP, and AS extracts; Rhizoctonia fungus + 100% PP extract; Rhizoctonia fungus + 50% PP and 50% AP extracts; Rhizoctonia fungus + 50% PP and 50% AS extracts; Rhizoctonia fungus used as control; Penicillium fungus + 100% AP extract; Penicillium fungus + equal concentrations of PP, AP, and AS extracts; Penicillium fungus + 100% PP extract; Penicillium fungus + 50% PP and 50% AP extract; Penicillium fungus + 50% PP and 50% AS extracts; Penicillium fungus used as control; and treatments of extracts 100% AP; equal concentrations of PP, AP, and AS; 50% PP and 50% AP; and 50% PP and 50% AS used as control. Finally, there was a treatment where tomato plants were cultivated with no application of either fungal species or extracts.

2.3. Plant Preparation and Isolation

Twenty-day-old tomato plants, “Bella Donna” variety, were used to ascertain the effectiveness of the antimicrobial activity of different concentrations and combinations of the examined polyphenolic compounds against three important fungal pathogens on tomato plants. The possible phytotoxic effects of these extracts were also evaluated. The method followed and greenhouse conditions are described in previous work [39]. After 30 days of application, plants were harvested and fresh and dry plant weight, fresh and dry root weight, plant height, number of blossoms, and number of tomato fruits were measured. For the determination of dry root and plant weight, the tomato plants were dried at 50 °C for 4 days.

2.4. Statistical Analysis

A randomized complete block experiment design was selected for the study. Data are expressed as the means of four measurements. Statistical differences among the mean values were detected by ANOVA. Tukey’s pairwise comparison, Fisher’s, and Duncan’s tests were used in order to group the examined samples at 0.05 level. Minitab 17.1.0 software was used as a tool to perform the statistical analyses. The experiments were repeated twice.

3. Results and Discussion

3.1. Results on pH and Brix Measurements

The pH measurements ranged from 8.2 in sample 3 (100% AS extract) to 4.53 in sample number 6 (100% PP peel extract). However, values in the other mixing samples were usually in the range of 5.5–7.0. Similarly, Brix values ranged from 0.2 in sample 8 (16.7% PP, 6.7% AP, 66.7% AS extract) to 0.9 in samples 6 and 7 containing 100% PP extract and 50% PP and 50% AP extract, respectively. In more detail, pH and Brix measurements are presented in the following Figure 1 and Figure 2.

3.2. Results on Mycelium Growth Assay

According to

Table 3. Inhibition rate (%) of F. oxysporum f.sp., lycopersici compared to the control.

Treatments	Days of Incubation at 28 °C
	3 Days	5 Days	7 Days
(1) 16.7% PP, 66.7% AP, 16.7% AS	−10.49 ± 0.40%	−3.27 ± 0.20%	−3.30 ± 0.20%
(2) 100% AP	−12.54 ± 0.40%	0.61 ± 0.10%	0.98 ± 0.10%
(3) 100% AS	−27.40 ± 0.50%	−1.81 ± 0.20%	−1.66 ± 0.20%
(5) 50% AP, 50% AS	−6.67 ± 0.30%	−1.76 ± 0.20%	−1.04 ± 0.20%
(6) 33.3% PP, 33.3% AP, 33.3% AS	−30.07 ± 0.50%	−8.26 ± 0.30%	−6.48 ± 0.30%
(8) 100% PP	1.24 ± 0.20%	0.83 ± 0.10%	−3.45 ± 0.20%
(9) 50% PP, 50% AP	−21.08 ± 0.50%	−7.15 ± 0.30%	−6.42 ± 0.30%
(10) 16.7% PP, 16.7% AP, 66.7% AS	−15.12 ± 0.40%	−1.63 ± 0.20%	0.44 ± 0.20%
(12) 66.7% PP, 16.7% AP, 16.7% AS	−24.91 ± 0.50%	−8.17 ± 0.30%	−6.57 ± 0.30%
(13) 50% PP, 50% AS	−7.11 ± 0.30%	−3.09 ± 0.20%	−2.82 ± 0.20%

Measured values are medians of two repetitions ± standard deviation.

, treatments 5 (contained equal volume of PP, AP, and AS), 7 (50% PP, 50% AP) and 9 (66.7% PP, 16.7% AP, 16.7% AS) exhibited the greatest inhibitory activity against mycelial growth of F. oxysporum f.sp., lycopersici after 3 days of incubation at 28 °C reducing mycelium growth by 30%, 21%, and 25% respectively. However, this inhibition did not occur after 7 days of incubation, and mycelium growth was reduced by only about 6.5% compared to the control.

All of the abovementioned treatments differed statistically significantly from the control which contains only the phytopathogenic fungus. It is noteworthy that there was no statistically significant difference between control with the extracts 2 (100% PP), 10 (16.7% PP, 16.7% AP, 66.7% AS), 3 (100% AS), 5 (50% AP, 50% AS) and 13 (50% PP, 50% AS). According to Table 3, inhibition of mycelium growth was observed during the first 3 days of the inoculation for the majority of the treatments tested. This suggests that the fungus may exceed the initial shock in expression of the tested samples experienced during the first days of incubation.

According to

Table 4. Inhibition rate (%) of A. niger compared to the control.

Treatments	Days of Incubation at 28 °C
	3 Days	5 Days	7 Days
(1) 16.7% PP, 66.7% AP, 16.7% AS	1.90 ± 0.20%	2.18 ± 0.20%	1.86 ± 0.20%
(2) 100% AP	−10 ± 0.40%	−10.65 ± 0.40%	−10.21 ± 0.40%
(3) 100% AS	−4.86 ± 0.20%	−3.37 ± 0.20%	−3.48 ± 0.20%
(5) 50% AP, 50% AS	−1.57 ± 0.20%	−2.03 ± 0.20%	−5.34 ± 0.30%
(6) 33.3% PP, 33.3% AP, 33.3% AS	7.03 ± 0.20%	3.02 ± 0.20%	2.61 ± 0.20%
(8) 100% PP	−3.65 ± 0.20%	−3.91 ± 0.20%	−3.92 ± 0.20%
(9) 50% PP, 50% AP	0.52 ± 0.10%	−1.68 ± 0.20%	−1.98 ± 0.20%
(10) 16.7% PP, 16.7% AP, 66.7% AS	−5.53 ± 0.30%	−5.75 ± 0.30%	−7.28 ± 0.30%
(12) 66.7% PP, 16.7% AP, 16.7% AS	−2.46 ± 0.20%	−0.89 ± 0.10%	−0.19 ± 0.10%
(13) 50% PP, 50% AS	−1.04 ± 0.20%	−1.78 ± 0.20%	−1.86 ± 0.20%

Measured values are medians of two repetitions ± standard deviation.

, treatments 2 (100% AP), 10 (16.7% PP, 16.7% AP, 66.7% AS), and 5 (50% AP, 50% AS) exhibited the greatest inhibitory activity against mycelial growth of A. niger after 7 days of incubation at 28 °C, reducing mycelium growth by 10.21, 7.28, and 5.34%, respectively. There was no statistically significant difference between treatments 1, 3, 6, 8, 9, 12, and 13. According to Table 4, inhibition of mycelium growth observed during the first 3 days of the inoculation was also observed after 7 days except treatment 5 (50% AP, 50% AS) where inhibition of mycelium was 1.57% in the first 3 days and raised to 5.34% after 7 days. Comparing our results with results from a previous study [40], it was noticed that the growth of fungus A. niger was affected by the addition of 100% PP, leading to the smaller size of mycelium at 3 d (4.37 mm), 5 d (10.32 mm), and 7 d (11.82 mm) of measurements. The second-most effective combination was the 50% PP + 50% AP which exhibited significantly lower growth at 5 d (6.35 mm) and higher at 3 d and 7 d (17.42 mm). Results obtained from Skenderidis et al. [40], based on the mixture design analysis, recorded that the optimum mixtures to achieve the best antimicrobial activity for A. niger, P. expansum, and the C. jejuni, individually for each microorganism, were 100% PP, 49.5% PP + 51% AP, and 65% PP + 35% AP.

According to

Table 5. Inhibition rate (%) of P. expansum compared to the control.

Treatments	Days of Incubation at 28 °C
	3 Days	5 Days	7 Days
(1) 16.7% PP, 66.7% AP, 16.7% AS	−2.26 ± 0.20%	1.22 ± 0.20%	0.66 ± 0.10%
(2) 100% AP	−7.00 ± 0.30%	−4.00 ± 0.20%	−0.66 ± 0.10%
(3) 100% AS	−1.32 ± 0.20%	−2.61 ± 0.20%	0.77 ± 0.10%
(5) 50% AP, 50% AS	−5.29 ± 0.30%	−2.04 ± 0.20%	1.59 ± 0.20%
(6) 33.3% PP, 33.3% AP, 33.3% AS	−3.96 ± 0.20%	−3.43 ± 0.20%	−2.99 ± 0.20%
(8) 100% PP	−5.29 ± 0.30%	−7.27 ± 0.30%	−11.26 ± 0.40%
(9) 50% PP, 50% AP	−0.18 ± 0.10%	−1.39 ± 0.20%	−3.29 ± 0.20%
(10) 16.7% PP, 16.7% AP, 66.7% AS	−7.75 ± 0.30%	−5.06 ± 0.03%	0.03 ± 0.10%
(12) 66.7% PP, 16.7% AP, 16.7% AS	0.18 ± 0.10%	0.81 ± 0.10%	−0.38 ± 0.10%
(13) 50% PP, 50% AS	−8.50 ± 0.30%	−6.78 ± 0.30%	−9.42 ± 0.40%

Measured values are medians of two repetitions ± standard deviation.

, treatments 8 (100% PP) and 13 (50% PP, 50% AS) exhibited the greatest inhibitory activity against mycelial growth of P. expansum after 7 days of incubation at 28 °C, reducing mycelium growth by 11.26%, and 9.42%, respectively. Furthermore, according to Table 5, inhibition rate in these two treatments was even lower in the beginning of the incubation at the third day (5.29% and 8.5%). There was no statistically significant difference between control and treatments 1 (16.7% PP, 66.7% AP, 16.7% AS), 2 (100% PP), 3 (100% AS), 5 (50% AP, 50% AS), 6 (33.3% PP, 33.3% AP, 33.3% AS), 9 (50% PP, 50% AP), 10 (16.7% PP, 16.7% AP, 66.7% AS), and 12 (66.7% PP, 16.7% AP, 16.7% AS). However, treatments 2 (100% PP), 5 (50% AP, 50% AS), and 10 (16.7% PP, 16.7% AP, 66.7% AS) showed a mycelium inhibitory effect during the first 3 days of incubation. This effect was eliminated on the seventh day of incubation, most probably due to the fast growth of the mycelium and the production of the asexual reproduction forms (conidiophores). Comparing our results with results from a previous study [40], it was noticed that 100% PP also showed inhibited fungus growth at 3 d (4.59 mm). Furthermore, Tayel et al. [41] documented that those extracts of solid pomegranate juice residues can also be used for plant protection.

Regarding inhibitory effect of the examined treatments on mycelium growth of R. solani, measurements were taken on the 5th, 8th, and 14th day after inoculation due to the slower mycelium growth on the tested medium. According to

Table 6. Inhibition rate (%) of R. solani compared to the control.

Treatments	Days of Incubation at 28 °C
	5 Days	8 Days	14 Days
(1) 16.7% PP, 66.7% AP, 16.7% AS	−20.18 ± 0.50%	−16.23 ± 0.40%	−11.16 ± 0.40%
(2) 100% AP	−7 ± 0.30%	−5 ± 0.30%	0%
(3) 100% AS	−11.33 ± 0.40%	−13.10 ± 0.40%	−7.09 ± 0.30%
(5) 50% AP, 50% AS	−29.97 ± 0.50%	−21.74 ± 0.50%	−14.73 ± 0.40%
(6) 33.3% PP, 33.3% AP, 33.3% AS	−18.15 ± 0.50%	−19.18 ± 0.50%	−14.73 ± 0.40%
(8) 100% PP	−4.97 ± 0.20%	−0.76 ± 0.10%	−0.24 ± 0.10%
(9) 50% PP, 50% AP	−16.68 ± 0.40%	−11.91 ± 0.40%	−4.27 ± 0.20%
(10) 16.7% PP, 16.7% AP, 66.7% AS	−20.73 ± 0.50%	−20.27 ± 0.50%	−16.26 ± 0.40%
(12) 66.7% PP, 16.7% AP, 16.7% AS	−22.94 ± 0.50%	−21.32 ± 0.50%	−17.88 ± 0.40%
(13) 50% PP, 50% AS	−100.00 ± 0.50%	−55.12 ± 0.5%	−23.77 ± 0.50%

Measured values are medians of two repetitions ± standard deviation.

, it was observed that all treatments inhibited mycelium growth of R. solani during the first 5 days of the incubation time. In more detail, treatment 13 (50% PP, 50% AS) inhibited 100% of the mycelium growth of the tested fungus followed by treatments 5 (50% AP, 50% AS), 12 (66.7% PP, 16.7% AP, 16.7% AS), 10 (16.7% PP, 16.7% AP, 66.7% AS), 1 (16.7% PP, 66.7% AP, 16.7% AS), 6 (33.3% PP, 33.3% AP, 33.3% AS), and 9 (50% PP, 50% AP) inhibited mycelium growth by 30%, 22.94%, 20.73%, 20.18%, 18.15%, and 16.68%, respectively. However, inhibition rate did not remain stable in these levels after 14 days of incubation, and mycelium growth was reduced by 23.77% in treatment 13 (50% PP, 50% AS), 17.88% in treatment 12 (66.7% PP, 16.7% AP, 16.7% AS), 16.26% in treatment 10 (16.7% PP, 16.7% AP, 66.7% AS), 14.73% in treatments 5 and 6, 11.16% in treatment 1 (16.7% PP, 66.7% AP, 16.7% AS), and 7.09% in treatment 3 (100% AS). According to Table 6, there was no statistically significant difference between control and treatments 2 (100% AP), 8 (100% PP), and 9 (50% PP, 50% AP).

Regarding inhibition effect of the examined treatments against mycelium growth of B. cinerea, it was observed that after 7 days of incubation there was no inhibitory effect, and mycelium covered all the area of the medium contained on Petri dishes. However, B. cinerea mycelium growth was inhibited on the first 3 days of incubation time and mycelium growth was reduced in treatments 5 (50% AP, 50% AS), 13 (50% PP, 50% AS), and 8 (100% PP) by 27.78%, 19.09%, and 12.66%, respectively (

Table 7. Inhibition rate (%) of B. cinerea compared to the control.

Treatments	Days of Incubation at 28 °C
	3 Days	5 Days	7 Days
(1) 16.7% PP, 66.7% AP, 16.7% AS	5.61 ± 0.30%	6.49 ± 0.30%	0
(2) 100% AP	1.90 ± 0.20%	13.13 ± 0.40%	0
(3) 100% AS	−1.71 ± 0.20%	9.96 ± 0.40%	0
(5) 50% AP, 50% AS	−27.78 ± 0.50%	4.54 ± 0.20%	0
(6) 33.3% PP, 33.3% AP, 33.3% AS	4.07 ± 0.20%	8.74 ± 0.30%	0
(8) 100% PP	−12.66 ± 0.40%	12.35 ± 0.40%	0
(9) 50% PP, 50% AP	1.71 ± 0.20%	10.54 ± 0.40%	0
(10) 16.7% PP, 16.7% AP, 66.7% AS	8.14 ± 0.03%	9.27 ± 0.40%	0
(12) 66.7% PP, 16.7% AP, 16.7% AS	5.24 ± 0.30%	10.83 ± 0.40%	0
(13) 50% PP, 50% AS	−19.09 ± 0.50%	9.42 ± 0.40%	0

Measured values are medians of two repetitions ± standard deviation.

). Furthermore, Li Destri Nicosia et al. [42] also evaluated PP extract as a natural antifungal preparation for the control of post-harvest rot from fungi, such as B. cinerea, P. digitatum, and P. expansum.

3.3. MIC Assay

The results of the effect of different concentrations and mixtures of the extracts tested by the method of MIC are described in

Table 8. MIC test of pomegranate peel and avocado seed and avocado peel extracts on five plant pathogens.

		F. oxysporum f.sp., lycopersici	A. niger	P. expansum	R. solani	B. cinerea
16.7% PP, 66.7% AP, 16.7% AS (Treatment 1)	0%	+	+	+	+	+
	2.5%	+	+	+	+	+
	5%	+	+	+	+	+
	7.5%	+	+	+	+	+
	10%	+	+	+	+	+
100% AP (Treatment 2)	0%	+	+	+	+	+
	2.5%	+	+	+	+	+
	5%	+	+	+	+	+
	7.5%	+	+	+	+	+
	10%	+	+	+	+	+
100% AS (Treatment 3)	0%	+	+	+	+	+
	2.5%	+	+	+	+	+
	5%	+	+	+	+	+
	7.5%	+	+	+	+	+
	10%	+	+	+	+	+
50% AP, 50% AS (Treatment 5)	0%	+	+	+	+	+
	2.5%	+	+	+	+	+
	5%	+	+	+	+	+
	7.5%	+	+	+	+	+
	10%	+	+	+	+	-
33.3% PP, 33.3% AP, 33.3% AS (Treatment 6)	0%	+	+	+	+	+
	2.5%	+	+	+	+	+
	5%	+	+	+	+	+
	7.5%	-	+	+	+	+
	10%	-	+	+	+	+
100% PP (Treatment 8)	0%	+	+	+	+	+
	2.5%	+	+	+	+	+
	5%	+	+	+	+	+
	7.5%	+	+	-	+	+
	10%	+	+	-	+	+
50% PP, 50% AP (Treatment 9)	0%	+	+	+	+	+
	2.5%	+	+	+	+	+
	5%	+	+	+	+	+
	7.5%	-	+	+	+	+
	10%	-	+	+	+	+
16.7% PP, 16.7% AP, 66.7% AS (Treatment 10)	0%	+	+	+	+	+
	2.5%	+	+	+	+	+
	5%	+	+	+	+	+
	7.5%	+	+	+	+	+
	10%	+	+	+	+	+
66.7% PP, 16.7% AP, 16.7% AS (Treatment 12)	0%	+	+	+	+	+
	2.5%	+	+	+	+	+
	5%	+	+	+	+	+
	7.5%	+	+	+	+	+
	10%	-	+	+	+	+
50% PP, 50% AS (Treatment 13)	0%	+	+	+	+	+
	2.5%	+	+	+	+	+
	5%	+	+	+	+	+
	7.5%	-	+	-	-	-
	10%	-	+	-	-	-

(+) Mycelium growth or sediment; (-) No mycelium or sediment on the tested tubes.

. The evaluation of the results obtained by determining the MIC assay demonstrated that a concentration of 7.5% and 10% of treatment 13 (50% PP, 50% AS) affected mycelium growth of F. oxysporum f.sp., lycopersici, P. expansum, R. solani, and B. cinerea. Furthermore, application of treatments 5 (50% PP, 50% AS), 6 (33.3% PP, 33.3% AP, 33.3% AS), and 12 (66.7% PP, 1 6.7% AP, 16.7% AS) showed a moderate inhibition effect on mycelium growth and spore germination of F. oxysporum f.sp., lycopersici. In addition, P. expansum growth was inhibited when at least 7.5% of 100% PP (treatment 8) was applied. Finally, MIC results on fungi B. cinerea, showed moderate sensitivity to the tested extracts of treatments 5 (50% AP, 50% AS) and 13 (50% PP, 50% AS), while at least 10% of the concentration was needed to be applied in order to inhibit mycelium growth and spore germination. Thus, it is believed that in most cases, an equal mixture of 50% PP and 50% AS (treatment 13) could be used as an alternative biocontrol agent against the four tested plant pathogens, directly on plant tissues and infected stems of the crops, preventing and suppressing the development of the plant pathogens. Comparing the results of this study with the results obtained from a previous study [40], it was found that 100% PP and 50% PP + 50% AS inhibit the growth of all microorganisms (except the P. expansum and P. putida) in the higher examined concentration, having an MIC from 25 to 50 mg/mL based on the tested bacteria.

Moreover, another study investigated the effectiveness of methanol extract from PP to control the growth of Fusarium sambucinum in vitro [43]. The methanol extract showed 75.5% inhibition of mycelial growth of F. sambucinum and complete inhibition of spore germination of the pathogen at a concentration of 20 mg/mL. The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of the extract were determined to be 20 and 120 mg/mL, respectively. Observation under the electron microscope revealed morphological modification in the textures of F. sambucinum, such as rotation, twisting, and collapse of the mycelium. Alterations including degradation of cytoplasmic organelles were also observed. In in vivo experiments, the methanol extract from PP caused a significant reduction in the development of the disease in potato tubers.

3.4. Plant Growth Study (In Vivo)

Different concentrations and combinations of polyphenolic compounds of PP, AP, and AS were tested against plant-pathogen-affected tomato plants. In order to evaluate the polyphenolic mixtures and the possible phytotoxic effect on tomato plants, plant height (expressed in cm), fresh and dry stem and root weight (expressed in grams), and number of blossoms and fruits were measured.

As is represented in

Table 9. Growth effect of tomato plants infected with F. oxysporum f.sp., lycopersici to which different concentrations and combinations of PP, AP, and AS polyphenolic extracts were applied.

Treatments	Plant Height (cm)	Fresh Plant Weight (g)	Dry Plant Weight (g)	Fresh Root Weight (g)	Dry Root Weight (g)	Num. of Blossoms	Num. of Tomato Fruits
F. oxysporum f.sp., lycopersici + 100% AP (sample 2)	25.30 ± 2.62bc	70.60 ± 2.80c	10.13 ± 0.58d	16.78 ± 0.15c	1.23 ± 0.56c	3.30 ± 2.89d	-
F. oxysporum f.sp., lycopersici + 33.3% PP, 33.3% AP, 33.3% AS (sample 6)	25.30 ± 1.52bc	85.60 ± 7.96bc	11.59 ± 1.09cd	19.88 ± 0.88bc	1.45 ± 0.16c	5.30 ± 0.57bcd	-
F. oxysporum f.sp., lycopersici + 100% PP (sample 8)	27.30 ± 0.57bc	90 ± 4.36b	12.37 ± 0.50bcd	20.75 ± 1.59bc	1.68 ± 0.27c	6.60 ± 1.15b	-
F. oxysporum f.sp., lycopersici + 50% PP, 50% AP (sample 9)	26.60 ± 1.15bc	77 ± 4.58bc	10.45 ± 0.31cd	18.54 ± 1.60bc	1.38 ± 0.13c	6.30 ± 1.15b	-
F. oxysporum f.sp., lycopersici + 50% PP, 50% AS (sample 13)	26.60 ± 0.57bc	84.30 ± 3.51b	11.56 ± 0.51cd	19.83 ± 0.63bc	1.43 ± 0.17c	6.60 ± 1.52b	-
100% AP (sample 2) control extract	24.60 ± 2.93bc	93 ± 6.08b	12.42 ± 0.45bc	19.24 ± 0.49bc	1.37 ± 0.36c	5.60 ± 0.57bc	-
33.3% PP, 33.3% AP, 33.3% AS (sample 6) control extract	25.30 ± 2.31bc	98.30 ± 6.2ab	12.51 ± 0.37bc	19.45 ± 0.16bc	1.38 ± 0.05c	5.03 ± 0.57bcd	-
100% PP (sample 8) control extract	28 ± 1abc	105.06 ± 6.66ab	14.31 ± 0.84ab	22.74 ± 1.17b	1.74 ± 0.16bc	6.60 ± 1.52b	0.30 ± 0.30b
50% PP, 50% AP (sample 9) control extract	27.60 ± 0.57bc	111.30 ± 2.37a	15.08 ± 0.37a	28.36 ± 0.59a	2.45 ± 0.12a	6.30 ± 1.15b	0.30 ± 0.30b
50% PP, 50% AS (sample 13) control extract	29 ± 1ab	109 ± 4.85ab	15.06 ± 0.31a	28.32 ± 0.59a	2.39 ± 0.12ab	6.30 ± 1.15b	0.30 ± 0.30b
F. oxysporum f.sp., lycopersici (Control fungus)	25.30 ± 0.57bc	72.30 ± 6.03c	11.40 ± 1.82cd	18.16 ± 2.63c	1.35 ± 0.11c	4 ± 1cd	-
Tomato plant (Control plant)	30 ± 1a	110 ± 1.3a	15.40 ± 0.58a	28.38 ± 3.47a	2.84 ± 0.03a	10.30 ± 0.57a	1 ± 0.30a

Measured values are medians of two repetitions ± standard deviation. Means that do not share a letter are significantly different in p ≤ 0.05.

,

Table 10. Growth effect of tomato plants infected with R. solani to which different concentrations and combinations of PP, AP, and AS polyphenolic extracts were applied.

Treatments	Plant Height (cm)	Fresh Plant Weight (g)	Dry Plant Weight (g)	Fresh Root Weight (g)	Dry Root Weight (g)	Num. of Blossoms	Num. of Tomato Fruits
R. solani + 100% AP (sample 2)	24.30 ± 1.15bc	92.30 ± 2.52bc	12.02 ± 0.38bcd	19.79 ± 2.69bc	1.43 ± 0.11b	5 ± 0bc	-
R. solani + 33.3% PP, 33.3% AP, 33.3% AS (sample 6)	26 ± 1bc	89 ± 8.81bc	10.99 ± 0.67de	16.37 ± 2.57cd	1.28 ± 0.02bc	4.30 ± 0.57cd	-
R. solani + 100% PP (sample 8)	27.60 ± 0.57bc	86 ± 2.65bc	10.92 ± 1.15de	15.42 ± 3.44cd	1.26 ± 0.15bc	4.30 ± 0.57cd	-
R. solani + 50% PP, 50% AP (sample 9)	27.30 ± 0.57bc	90.60 ± 1.15bc	11.28 ± 0.20cde	17.53 ± 1.16cd	1.30 ± 0.06bc	5.30 ± 0.57cd	-
R. solani + 50% PP, 50% AS (sample 13)	27 ± 1bc	81.30 ± 2.31c	10.45 ± 0.36e	14.84 ± 2.99d	1.19 ± 0.14bc	5.30 ± 0.57cd	-
100% AP (sample 2) control extract	24.60 ± 2.93bc	93 ± 6.08bc	12.42 ± 0.45bc	19.24 ± 0.49bc	1.37 ± 0.36b	5.60 ± 0.57bc	-
33.3% PP, 33.3% AP, 33.3% AS (sample 6) control extract	25.30 ± 2.31bc	98.3 ± 6.20ab	12.51 ± 0.37bc	19.45 ± 0.16bc	1.38 ± 0.05b	5.30 ± 0.57bcd	-
100% PP (sample 8) control extract	28 ± 1abc	105.60 ± 6.66ab	14.31 ± 0.84ab	22.74 ± 1.17b	1.74 ± 0.17b	6.60 ± 1.52b	0.30 ± 0.30b
50% PP, 50% AP (sample 9) control extract	27.60 ± 0.57bc	111.30 ± 2.37ab	15.08 ± 0.37a	28.36 ± 0.59a	2.45 ± 0.12a	6.30 ± 1.15b	0.30 ± 0.30b
50% PP, 50% AS (sample 13) control extract	29 ± 1ab	109 ± 4.85ab	15.06 ± 0.31a	28.32 ± 0.59a	2.39 ± 0.13a	6.30 ± 1.15b	0.30 ± 0.30b
R. solani (Control fungus)	11.60 ± 2.58d	45.30 ± 3.78d	6.61 ± 1.49f	8.69 ± 6.78e	0.70 ± 0.48c	2.60 ± 2.52d	-
Tomato plant (Control plant)	30 ± 1a	110 ± 1.3ab	15.4 ± 0.58a	28.38 ± 3.47a	2.84 ± 0.03a	10.30 ± 0.57a	1 ± 0.30a

Measured values are medians of two repetitions ± standard deviation. Means that do not share a letter are significantly different in p ≤ 0.05.

and

Table 11. Growth effect of tomato plants infected with P. expansum to which different concentrations and combinations of PP, AP, and AS polyphenolic extracts were applied.

Treatments	Plant Height (cm)	Fresh Plant Weight (g)	Dry Plant Weight (g)	Fresh Root Weight (g)	Dry Root Weight (g)	Num. of Blossoms	Num. of Tomato Fruits
P. expansum + 100% AP (sample 2)	25 ± 2cd	88 ± 10.15cd	11.27 ± 1.55bc	20.32 ± 0.59bcd	1.59 ± 0.02cde	5.6 ± 1.15b	0.3 ± 0.3b
P. expansum + 33.3% PP, 33.3% AP, 33.3% AS (sample 6)	26.60 ± 1.15bcd	77 ± 1d	9.26 ± 1.27d	16.71 ± 1.75ef	1.28 ± 0.04e	7 ± 1.73b	-
P. expansum + 100% PP (sample 8)	28.60 ± 0.57ab	92.60 ± 17.5bcd	11.96 ± 0.56b	21.71 ± 0.89bc	1.70 ± 0.04cd	6.3 ± 1.15b	0.3 ± 0.3b
P. expansum + 50% PP, 50% AP (sample 9)	27.60 ± 0.57abc	76.60 ± 2.31d	9.78 ± 0.80d	16.15 ± 1.23f	1.28 ± 0.06e	7 ± 1b	-
P. expansum + 50% PP, 50% AS (sample 13)	28.60 ± 1.15ab	77.30 ± 7.23d	10.2 ± 0.82cd	17.20 ± 1.93def	1.31 ± 0.03de	7 ± 1b	-
100% AP (sample 2) control extract	24.60 ± 2.93d	93 ± 6.08bc	12.42 ± 0.45b	19.24 ± 0.49cde	1.37 ± 0.36de	5.60 ± 0.57b	-
33.3% PP, 33.3% AP, 33.3% AS (sample 6) control extract	25.30 ± 2.31cd	98.30 ± 6.2ab	12.51 ± 0.37b	19.45 ± 0.16cd	1.38 ± 0.05cde	5.30 ± 0.57b	-
100% PP (sample 8) control extract	28 ± 1ab	105.60 ± 6.66ab	14.31 ± 0.84a	22.74 ± 1.17b	1.74 ± 0.16c	6.60 ± 1.52b	0.3 ± 0.3b
50% PP, 50% AP (sample 9) control extract	27.60 ± 0.57bcd	111.30 ± 2.37a	15.08 ± 0.37a	28.36 ± 0.59a	2.45 ± 0.12ab	6.30 ± 1.15b	0.3 ± 0.3b
50% PP, 50% AS (sample 13) control extract	29 ± 1ab	109 ± 4.85ab	15.06 ± 0.31a	28.32 ± 0.59a	2.39 ± 0.12b	6.30 ± 1.15b	0.3 ± 0.3b
P. expansum (Control fungus)	26 ± 1bcd	77.60 ± 3.79d	10.03 ± 0.30cd	16.30 ± 4.50ef	1.27 ± 0.12e	6.60 ± 0.57b	-
Tomato plant (Control plant)	30 ± 1a	110 ± 1.30a	15.40 ± 0.58a	28.38 ± 3.47a	2.84 ± 0.03a	10.30 ± 0.57a	1 ± 0.3a

Measured values are medians of two repetitions ± standard deviation. Means that do not share a letter are significantly different in p ≤ 0.05.

, average height of tomato plants infected with F. oxysporum f.sp., lycopersici, R. solani, and P. expansum differed statistically significantly from the average height of tomato plants treated as control. From the examined treatments, only tomato plants treated with sample 3 (50% PP, 50% AS) and 100% PP (sample 8) were almost near the plant height of treatment where tomato plants were used as control, measuring 29 ± 1 and 28 ± 1 cm, respectively. Furthermore, in Table 9, Table 10 and Table 11, it is observed that growth of tomato plants infected with the Fusarium, Rhizoctonia, or Penicillium fungus and treated with the examined combinations and different concentrations of the tested extracts could not compare with the growth observed in uninfected and untreated plants (control), and was lower for any case. The same results were observed for fresh plant weight, where control did not differ statistically from treatments where 50% PP, 50% AS and 50% PP, 50% AP were applied as control extract in order to determine the possible phytotoxic effect of the examined extracts on tomato plants. However, Table 9 shows that in treatments where 100% PP (sample 8), equal ratio of PP, AP, and AS (33.3% PP, 33.3% AP, 33.3% AS; sample 6), and 50% PP, 50% AS (sample 13) were applied, it was found that fresh plant weight was improved compared with the treatment where tomato plants received no treatment. Dry weight of untreated tomato plants (control plant) was measured at 15.4 ± 0.58 g, while average dry weight of tomato plants infected with the Fusarium fungus was 11.4 ± 1.82 g.

However, the lowest average dry plant weight (10.13 ± 0.58 g) was observed in treatment where F. oxysporum f.sp., lycopersici was applied with 100% AP (sample 2), due to the possible phytotoxic effect of the high dose of the extract. Likewise, the majority of the extracts applied for the control of Fusarium fungus did not provide sufficient plant protection (average dry plant weight varies from 10.13 ± 0.58 g to 12.37 ± 0.5 g, much lower compared with the average dry plant weight of the untreated tomato plants (15.4 ± 0.58 g)). Fresh and dry root weight of the untreated control plants was almost 40% lighter than the treated with the Fusarium fungus treatments. However, for sample 8 (100% PP), it is believed that it could add some protection to the tomato plants, while fresh and dry weight of the root system for sample 2 (100% AP) weighed 16.78 ± 0.15 and 1.23 ± 0.56 g. Regarding the number of flowers, it could be said that uninfected plants appeared to form the largest number of flowers/plant (10.3 ± 0.57) and differed statistically significantly from all other treatments. The lowest number of blossoms was observed in the treatment where F. oxysporum f.sp., lycopersici + 100% AP (sample 2) was applied and in the treatment where Fusarium fungus was applied to tomato plants used as control fungus. Finally, Table 9, Table 10 and Table 11 show that untreated and uninfected tomato plants used as control formed at least 1 tomato fruit/plant while all the other treatments did not form tomato fruits, except treatments with 100% PP (sample 8) used as control extract, 50% PP, 50% AP (sample 9) control extract, and 50% PP, 50% AS (sample 13) control extract, where an average of 0.3 tomato fruit was formed/plant. Therefore, it is believed that none of the examined polyphenolic extracts can affect the growth of F. oxysporum f.sp., lycopersici on tomato plants, while the examined extract combinations and various concentrations could not significantly reduce the losses caused by the presence of the pathogen in this crop compared with the untreated control plants. However, 100% PP appears to show the most promising results in suppressing losses of tomato plant growth, compared with the control fungus.

Table 10 represents the growth effect of tomato plants infected with R. solani to which different concentrations and combinations of PP, AP, and AS polyphenolic extracts were applied. Average height of tomato plants infected with R. solani differed statistically significantly from the average height of tomato plants treated as control, with 11.6 ± 2.58 and 30 ± 1 cm, respectively. Regarding plant height, fresh and dry plant weight, fresh and dry root weight, number of blossoms, and number of tomato fruits on tomato plants treated with the polyphenolic PP, AP, and AS extracts in different combinations and concentrations, it was observed that although plant growth was lower than the growth observed in control plants, it was also different statistically significantly to the growth observed on tomato plants infected only with the Rhizoctonia fungus and used as control fungus. Among the treatments used, application of R. solani + 100% PP (sample 8) resulted in tomato plants with height of 27.6 ± 0.57 cm, followed by treatment R. solani + 50% PP, 50% AP (sample 9), where average tomato plant height was 27.3 ± 0.57 cm. The lowest average plant height (24.3) was observed in the treatment where R. solani + 100% AP (sample 2) extract was applied, and this could be the result of a possible phytotoxic and fungicidal effect of the application on tomato plants. Regarding fresh plant weight, all treatments differed statistically significantly from treatments where fungus was applied as control. Among the treatments tested, application of R. solani + 100% AP (sample 2) resulted in tomato plants with fresh weight of 92.3 ± 2.52 g followed by treatment of R. solani + 50% PP, 50% AP (sample 9), where average tomato plant fresh weight was 90.6 ± 1.15 g. The lowest average dry plant weight (6.61 ± 1.49 g) was observed in treatment where R. solani was applied as control fungus. However, the majority of the extracts applied for the control of Rhizoctonia fungus could provide sufficient plant protection against the fungus infection, with average dry plant weight varying from 10.45 ± 0.36 g to 12.02 ± 0.38 g higher than that observed on the infected plants (6.61 ± 1.49 g), although differentiated from average dry plant weight of the untreated tomato plants (15.4 ± 0.58 g). Regarding fresh and dry root weight, it was observed that R. solani affected root growth of tomato plants inoculated only, with the phytopathogenic fungus weighing 8.69 ± 6.78 and 0.70 ± 0.48 (Table 10). On the contrary, in treatments where R. solani and 100% AP (sample 2) were applied, fresh and dry root weight was 19.79 ± 2.69 and 1.43 ± 0.11 g. Regarding the number of flowers observed in treatments where Rhizoctonia fungus and the examined polyphenolic extracts were applied, it could be said that uninfected plants appeared to form the largest number of flowers/plant (10.3 ± 0.57) and differed statistically significantly to all other treatments. The lowest number of blossoms was observed in the treatment where R. solani was applied to tomato plants as control fungus (2.6). The number of blossoms measured in treatments applied with any of the tested extract concentrations and combinations was between 5.3 ± 0.57 (in treatment 33.3% PP, 33.3% AP, 33.3% AS—sample 6—control extract) and 6.6 ± 1.52 (in treatment 100% PP (sample 8); control extract). However, number of blossoms was lower in treatments where R. solani fungus was applied, even when the tested extracts were applied, and varied from 4.3 ± 0.57 (R. solani + 33.3% PP, 33.3% AP, 33.3% AS—sample 6, and R. solani + 100% PP—sample 8) to 5.3 ± 0.57 in treatment R. solani + 50% PP, 50% AS (sample 13). Finally, untreated and uninfected tomato plants used as control formed at least 1 tomato fruit/plant, while all the other treatments did not form tomato fruits except treatments with 100% PP (sample 8) used as control extract, 50% PP, 50% AP (sample 9) control extract, and 50% PP, 50% AS (sample 13) control extract where an average of 0.3 ± 0.3 tomato fruit was formed/plant. Therefore, it is believed that although tomato plant growth was not the same as the growth observed on untreated tomato plants (control plant), the extract’s application could provide sufficient plant protection against R. solani infection.

Table 11 represents the growth effect of tomato plants infected with P. expansum to which different concentrations and combinations of PP, AP, and AS polyphenolic extracts were applied. Tomato plants treated with P. expansum appeared to be less affected by the fungus, compared with the other fungal species used in this study. More specifically, average height of tomato plants infected with P. expansum, although differing statistically significantly from the average height of tomato plants treated as control (26 ± 1 and 30 ± 1 cm respectively), the difference was not as expanded as on the other fungal species. Regarding plant height, fresh and dry plant weight, fresh and dry root weight, number of blossoms, and number of tomato fruits on tomato plants treated with the polyphenolic PP, AP, and AS extract in different combinations and concentrations, it was observed that although plant growth was lower than the growth observed in control plants, it also differed statistically significantly to the growth observed on tomato plants infected only with the Penicillium fungus and used as control fungus. Among the treatments used, treatments where P. expansum was applied with 100% PP (sample 8) and P. expansum + 50% PP, 50% AS (sample 13) resulted in tomato plants with height of 28.6 ± 0.57 cm, followed by treatment with P. expansum + 50% PP, 50% AP (sample 9), where average tomato plant height was 27.6 ± 0.57 cm. The lowest average plant height (25 ± 2 cm) was observed in the treatment where P. expansum + 100% AP (sample 2) extract was applied, and this could be the result of a possible phytotoxic and fungicidal effect of the application on tomato plants. Regarding fresh plant weight, all treatments were different statistically significantly to treatment where fungus was applied as control. Among the treatments used, application of P. expansum + 100% PP (sample 8) resulted in tomato plants with fresh weight of 92.6 ± 17.5 g, followed by treatment of P. expansum + 100% AP (sample 2), where average tomato plant fresh weight was 88 ± 10.15 g. Moreover, regarding dry plant weight, it was found that the lowest average dry plant weight (9.26 ± 1.27 g) was observed in treatment where P. expansum was applied with equal dose of PP, AP, and AS (sample 6). However, only treatments P. expansum + 100% PP (sample 8) and P. expansum + 100% AP (sample 2) could provide sufficient plant protection against the fungus infection, with average dry plant weight varying from 11.27 ± 1.55 g and 11.96 ± 0.56 g, respectively, higher to that observed on the infected plants (10.03 ± 0.3 g), although different to average dry plant weight of the untreated tomato plants (15.4 ± 0.58 g). Regarding fresh and dry root weight, it was observed that P. expansum affected root growth of tomato plants inoculated only with the phytopathogenic fungus, weighing 16.30 ± 4.5 and 1.27 ± 0.12 (Table 11). On the contrary, in treatments where P. expansum and 100% PP (sample 8) and 100% AP (sample 2) were applied, fresh and dry root weight was 21.71 ± 0.89; 20.32 ± 0.59 and 1.70 ± 0.04; 1.59 ± 0.02, respectively. Regarding the number of flowers, it could be said that uninfected plants appeared to form the largest number of flowers/plant (10.3 ± 0.57) and differed statistically significantly from all other treatments. Treatments where P. expansum was applied as control fungus resulted in the average formation of 6.6 ± 1.52 blossoms/plant. However, the lowest number of blossoms was observed in treatments where 33.3% PP, 33.3% AP, 33.3% AS (sample 6—control extract), and 100% AP (sample 2, control extract) was applied. The number of blossoms measured in treatments applied with any of the tested extract concentration and combination was between 5.3 ± 0.57 (in treatment 33.3% PP, 33.3% AP, 33.3% AS—sample 6—control extract) and 7 ± 1 in treatments with P. expansum + 50% PP, 50% AP (sample 9) and P. expansum + 50% PP, 50% AS (sample 13). Therefore, although tomato plant growth was not the same as the growth observed on untreated tomato plants (control plant), the extract’s application could affect tomato growth severely and it could provide sufficient plant protection against infection of P. expansum. However, it is believed that due to weak infestation ability of P. expansum on the tested tomato plants, application of the tested polyphenolic extracts needs to be studied further.

In recent years, there has been a growing interest in the isolation, testing, and utilization of agricultural wastes [44] or other sources of plant tissues rich in polyphenols [27,45,46].

Regarding the antifungal activity of pomegranate extracts, according to Rosas-Burgos [47], the methanolic extracts of PP showed significant inhibition of the fungi A. flavus, F. verticillioides, A. alternata, and B. cinerea. More specific solid residues of PP of sour varieties are believed to be a significant source of natural antifungal and antibacterial substances that can be used as substitutes for synthetic chemicals. In addition, in vitro studies revealed strong fungicidal activity against the germination of conidia of the above phytopathogenic fungi. At concentrations of 1.2 and 12 g/L, complete inhibition of the infestations was achieved in the majority of pathogenic host combinations. In addition, PP extract was effective against Monilia laxa and B. cinerea in cherries and lemons. Tehranifar et al. [48] also investigated the antioxidant and antifungal properties of pomegranate against Penicillium italicum, Rhizopus stolonifer, and B. cinerea, fungal species that cause post-harvest fruit rot. From the results of their study, it was concluded that the peels and the extracts of the seeds of pomegranate fruit had a greater inhibitory effect than the extract of the leaves. The phenolic content of the peel extract was also calculated to be 2.8 times higher than that of the pomegranate leaf extract, and the antioxidant capacity of the peel of the pomegranate, seed, and leaf extracts was 55.3%, 35.7%, and 16.4%, respectively. A similar study was conducted by Glazer et al. [49], who studied the antifungal activity of solid pomegranate juice residue extracts in a number of known phytopathogenic fungi that are responsible for the deterioration of fruits and vegetables during storage, and in particular against A. alternata, Stemphylium botryosum, and Fusarium spp. According to their study, the action of these fungal pathogens was found to be significantly inhibited by the extract.

For more efficient application of these substances, an encapsulating agent is believed to play an important role. For example, Kaderides et al. [50] studied the microencapsulation of polyphenolic substances as food additives. In another study, Balooch et al. [51] compared fruit peel extract with various nanoparticle composites. The antifungal activity of these composites in post-harvest apple fruit rot from B. cinerea was tested in vitro and in vivo, and HPLC analysis and FTIR spectroscopy showed that the nanoparticles absorbed the phenolic compounds from the extract selectively. Kharchoufi et al. [52,53] also investigated the possible use of two edible coatings, chitosan (CH) and locust bean gum (LBG), which were incorporated in PP extract and in the biological control agent (BCA) Wickerhamomyces anomalus, in order to control the growth of the decomposition of oranges in post-harvest conditions. The study findings contribute to the utilization of a value-added industrial byproduct and provide significant value in advancing the development of new food-protecting compositions that benefit from the synergistic effects between biological control agents and natural bioactive compounds.

Besides fungicidal action, the antibacterial potential of extracts and residues from pomegranate and other exotic plants has also been examined for phytopathological bacteria [54]. For example, Quattrucci et al. [55] studied the antibacterial activity of ethanol peel extracts of Punica granatum against the bacterium Pseudomonas syringae pv. tomato both in vitro and in vivo. The minimum in vitro inhibition reached 0.5% of the concentration. The in vivo antibacterial activity of this natural substance lasted at least 15 days.

Regarding the antifungal activity of avocado, it has been found that avocado tissues contain specialized oil cells that contain various alkaloids, sesquiterpene hydroperoxides, and possibly terpenes [56]. The most active ingredient, called “persin” [57], is described as Z,Z-1-acetoxy-2-hydroxy-4-oxo-heneicosa 12,15-diene [58]. This antifungal diene has many other biological properties as it is an inhibitor of the growth of pests [59] and mammals [60]. Furthermore, Xoca-Orozco et al. [61] evaluated the avocado-derived extracts and concluded that chitosan showed an increase in antifungal activity by inhibiting mycelial growth and reducing sporulation as well as the germination of Colletotrichum gloeosporioides. These results are in accordance with results obtained from the study of Sivanathan and Adikaram [62], and Domergue et al. [63], where, besides persin, several components inhibited the in vitro growth of C. gloeosporioides. In addition, research completed by Xoca-Orozco et al. [61] studied the expression of genes associated with phenylpropanoids and the biosynthesis of the antifungal compound 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene. The results of this study showed increased gene expression of genes during normal fruit ripening, while other genes, such as flavonol synthase (Fls), increased only in samples taken from chitosan-treated fruits. These results reveal a new molecular mechanism in which chitosan induces specific accumulation of phenylpropanoids and antifungal diene, explaining the avocado’s resistance to pathogenic fungi. Therefore, secondary metabolites and, especially, phenols play an important role in the defense system of avocados.

A new, innovative approach is the study of Rajeshkumar and Rinitha [64], in which the biosynthesis of copper nanoparticles with AS extract was examined. Copper nanoparticles have promising antifungal and antibacterial activity and it is proved that when combined with AS extracts, they can control serious plant and food pathogens such as A. niger, A. fumigatus, and F. oxysporum. According to Zulfiqar et al. [65], future agriculture is focusing on reducing dependence on agrochemical products and/or increasing the effectiveness of their use on specific targets. This reduction can also be achieved through the micro-encapsulation of natural antimicrobials, which is a promising alternative application method.

Polyphenolic-rich plant tissues and agro-industrial residues such as pomegranate and avocado are in great demand in the world market because of their high nutritional value and their antioxidant and antimicrobial activity against several microorganisms, and, thus, their use in cosmetic, pharmaceutical, food, and plant protection industries are of great importance. Regarding plant production, although the number of biological control products marketed is increasing worldwide, their widespread acceptance is rare, mainly due to their limited mode of action and the limited effectiveness and several factors that affect farmers’, consumers’, and future scientists’ decisions in using organic plant-protective materials and alternative crops [66,67,68]. Thus, further efforts and research studies must be focused on the use of natural plant materials and their effects on alternative plant-protection methods not only for health issues, but also for possessing climate change effects.

4. Conclusions

The use of byproducts derived from plant tissue processing can offer new, natural products of high added value, but at the same time they can reduce environmental risks and pollution levels. The findings of this study demonstrate the in vitro stimulation and the potential synergistic activity of aqueous extracts derived from PP, AP, and AS wastes. These extracts had clear antimicrobial activity by controlling different plant pathogens in in vitro and in vivo test. Regarding the in vitro tests, it is believed that in most cases, an equal mixture of 50% PP and 50% AS could be used as an alternative biocontrol agent against the four tested plant pathogens, directly on plant tissues and infected stems of the crops, preventing and suppressing the development of the plant pathogens. Regarding the results obtained from the in vivo tests, it is believed that none of the examined polyphenolic extracts can affect the growth of F. oxysporum f.sp., lycopersici on tomato plants, while the examined extract combinations and various concentrations could not significantly reduce the losses caused by the presence of the pathogen in this crop compared with the untreated control plants. However, 100% PP appears to offer the most promising results in terms of suppressing losses of tomato plant growth compared with the control fungus. On the other hand, although tomato plant growth was not the same as the growth observed on untreated tomato plants (control plant), the extract’s application could affect tomato growth severely, and it could provide sufficient plant protection against infection of P. expansum and R. solani. However, further studies are needed in order to evaluate this effectiveness in more details.