Bioactivity of Selected Phenolic Acids and Hexane Extracts from Bougainvilla spectabilis and Citharexylum spinosum on the Growth of Pectobacterium carotovorum and Dickeya solani Bacteria: An Opportunity to Save the Environment

: Phenolic acids and natural extracts, as ecofriendly environmental agents, can be used as bio bactericides against the growth of plant pathogenic bacteria. In this study, isolation trails from infected potato tubers and stems that showed soft rot symptoms in ﬁelds revealed two soft rot bacterial isolates and were initially identiﬁed through morphological, physiological, and pathogenicity tests. The molecular characterization of these isolates via PCR, based on the 16S rRNA region, was carried out by an analysis of the DNA sequence via BLAST and Genbank, and showed that the soft rot bacterial isolates belong to Pectobacterium carotovorum subsp. carotovorum (PCC1) and Dickeya solani (Ds1). The in vitro results of the tested phenolic acids against the cultured bacterial isolates proved that concentrations of 800, 1600, and 3200 µ g / mL were the most e ﬀ ective. Ferulic acid was the potent suppressive phenolic acid tested against the Ds1 isolate, with an inhibition zone ranging from 6.00 to 25.75 mm at di ﬀ erent concentrations (25–3200 µ g / mL), but had no e ﬀ ect until reaching a concentration of 100 µ g / mL in the PCC1 isolate, followed by tannic acid, which ranged from 7.00 to 25.50 mm. On the other hand, tannic acid resulted in a signiﬁcant decrease in the growth rate of the PCC1 isolate with a mean of 9.11 mm. Chlorogenic acid was not as e ﬀ ective as the rest of the phenolic acids compared with the control. The n -hexane oily extract (HeOE) from Bougainvillea spectabilis bark showed the highest activity against PCC1 and Ds1, with inhibition zone values of 12 and 12.33 mm, respectively, at a concentration of 4000 µ g / mL; while the HeOE from Citharexylum spinosum wood showed less activity. In the GC / MS analysis, nonanal, an oily liquid compound, was found ata percentage of 38.28%, followed by cis-2-nonenal (9.75%), which are the main compounds in B. spectabilis bark HeOE, and 2-undecenal (22.39%), trans -2-decenal (18.74%), and oleic acid (10.85%) were found, which are the main compounds in C. spinosum wood HeOE. In conclusion, the phenolic acids and plant HeOEs seem to raise the resistance of potato plants, improving their defense mechanisms against soft rot bacterial pathogens.

.Identification of the compounds was done by a comparison of their retention times, as well as the MS reported from the WILEY 09 and NIST 11 mass spectral databases [46]. The values of the standard index (SI) and reverse standard index (RSI) were also reported in order to confirm that all of the spectra were appended to the library [47,48].

Statistical Analysis
The data were analyzed statistically with a two-way analysis of variance (ANOVA) using SAS software (SAS Institute, NC, USA) [49]. The two factors that analyzed were phenolic and extracts, as well as their respective concentrations. The means of the treatments were compared with control treatment, according to the Duncan's Multiple Range Test at a 0.05 level of probability.

Isolation Trails of the Causal Bacterial Pathogens
The isolation trails of the soft rot and blackleg symptoms (Figure 1) collected from the El-Nubaria and Wadi Elnatron regions, Egypt, revealed two bacterial isolates PCC1 and Ds1 which belonging to Pectobacterium and Dickeya genera, respectively (Table 1).

Phenotypic and Molecular Identification of the Soft Rot Bacteria
Based on the morphological, biochemical, and physiological characteristics of the isolated soft rot bacteria, the bacterial isolates were identified as Pectobacterium carotovorum subsp. carotovorum (PCC1) and Dickeya solani (Ds1) ( Table 2). The identification of the isolates PCC1 and Ds1 was confirmed using the 16S rDNA sequences analysis, and was deposited in the GenBank database under accession numbers MN598002 and MN598003, respectively.

Phenotypic and Molecular Identification of the Soft Rot Bacteria
Based on the morphological, biochemical, and physiological characteristics of the isolated soft rot bacteria, the bacterial isolates were identified as Pectobacterium carotovorum subsp. carotovorum (PCC1) and Dickeya solani (Ds1) ( Table 2). The identification of the isolates PCC1 and Ds1 was confirmed using the 16S rDNA sequences analysis, and was deposited in the GenBank database under accession numbers MN598002 and MN598003, respectively.

Pathogenicity Tests
The two tested bacterial isolates were pathogenic and produced soft rot symptoms on potato tubers. The PCC1 isolate showed a high disease index (86.04%), while the disease index of the isolate Ds1 was 71.62% (Table 1).

Influence of Some Phenolic Acids and Plant Oily Extracts on Growth of PCC1 and Ds1 Isolates
The data presented in Table 3 show the highly significant effects of the tested phenolic acids/oily extracts and their concentrations against the growth of PCC1 and Ds1. Table 4 shows that the different concentrations of the tested phenolic acids or the n-hexane oily extracts (HeOEs) from Bougainvillea spectabilis bark and Citharexylum spinosum wood caused different degrees of growth inhibition on the PCC1 and Ds1 isolates. It is evident that ferulic acid was the most suppressive to Ds1 isolate growth, with an inhibition zone (IZ) that ranged from 6 to 25.75 mm but had no effect on the PCC1 isolate growth until reaching a concentration of 100 µg/mL. On the other hand, tannic acid application decreased the growth rate of the PCC1 isolate with a mean of 9.11 mm. Finally, chlorogenic acid was less effective than all of the other phenolic acids used compared with the control. Significant differences were found among all phenolics at concentrations of 400 and 800 µg/mL. On the other hand, phenolic acid concentrations of 25 and 50 µg/mL had no noticeable effect on the two isolates, except for ferulic acid. Overall, the PCC1 isolate was more tolerant to all of the phenolic acids than the Ds1 isolate, and the applied concentrations of 800, 1600, and 3200 µg/mL were the most effective at inhibiting the two isolates. Additionally, from Table 4, the n-hexane oily extracts (HeOEs) from B. spectabilis bark and C. spinosum wood showed that with increasing the HeOE concentration, the IZ observed against the growth of PCC1 and Ds1was increased. The highest IZ (12 mm) against PCC1 was observed for B. spectabilis bark HeOE applied at a concentration of 4000 µg/mL, followed by the same HeOE with an IZ of 9.66 mm at a concentration of 2000 µg/mL. Furthermore, B. spectabilis bark HeOE at 4000, 2000, and 1000 µg/mL showed the highest IZs against the growth of Ds1, with values of 12.33, 11, and 10.33 mm, respectively. Furthermore, C. spinosum HeOE showed an IZ value of 10 mm against the growth of PCC1 at 4000 µg/mL level of concentration. Overall, the phenolic acids showed the highest activity against the growth of both of the bacteria, compared with the HeOEs.  The chemical compositions of the HeOE from C. spinosum wood are shown in Table 6. The abundant chemical constituents were 2-undecenal (22.39%), trans-2-decenal (18.74%), oleic acid (10.85%), nonanal (9.75%), 2-methylenecholestan-3-ol (6.01%), (Z)-2-tridecenal (4.03%), Z-(13,14-epoxy)tetradec-11-en-1-ol acetate (3.58%), 3-hydroxy-dodecanoic acid (3.34%), 9-hexadecenoic acid (2.3%), 1-dodecene (1.96%), (E)-2-nonenal (1.78%), octanal (1.72%), and 12,15-octadecadiynoic acid methyl ester (1.7%).

Discussion
Soft rot disease causes huge economic losses, estimated to be between 40% to 80% depending on climatic conditions, and Pectobacterium carotovorum subsp. carotovorum (PCC1) and Dickeya solani (Ds1) are the causal agents of soft rot disease in potato tubers in stores and in the field, where the early decay of mother tubers or seed tuber pieces may occur [13,14,[50][51][52]. The pathological behavior of the isolated bacterial cultures, as well as their cultural, morphological, and physiological characters conform to those known for all soft rot bacteria. On the basis of the obtained data, we could identify these isolates as PCC1 and Ds1, in the same way as many other researchers have in previous works [4,11,12,40,[53][54][55].
Nowadays, the major objective of modern Egyptian agriculture is to offer a strategy that would lead to minimizing the use of chemical pesticides, at the same time increasing the economic yield of crops. Therefore, much attention has been given to hinder the severity and spread of plant diseases, especially bacterial plant pathogens, by using all possible non-polluting methods of plant disease control. The objective of this research was to describe the tolerance of isolates PCC1 and Ds1 to phenolic acids. The findings in the present work showed that ferulic and tannic acids had a substantial inhibitory impact on the growth of Ds1 and PCC1 isolates. A mixture of caffeic and chlorogenic acids could prevent bacterial soft rot infection from occurring, and the major phenolic acids detected in the tuber peels that had soft rot antimicrobial effects were chlorogenic, caffeic, and ferulic acids [21,22].
Tannic acid inhibited the growth of certain bacterial strains [56], while tannic and gallic acids inhibited the growth and protease or pectatelyase enzyme activities of the soft rot isolate D. solani [23]. A more pronounced antimicrobial impact at different concentrations was found for tannic acid. The size difference and percentage of oHgroups between ferulic and tannic acids can explain this varied response against soft rot bacterial pathogens [57]. Both phenolic acids can affect pathogen growth by contact with the produced protease and pectate lyase enzymes, the effective mechanism could be described as protein inhibitors by modifying their stability and losing cellular permeability, or by reducing the substrate availability or chelating the metal co-factor, as the tannic acid can fix the iron metal [58][59][60][61][62][63]. In this study, both isolates (PCC1 and Ds1) were growth inhibited by the examined polyphenols, and we suggest that the mode of action could interact and inactivate the enzyme active sites, which leads to precipitating the enzymatic proteins. This is in agreement with several authors who have talked about the mechanisms of tannic acid, polyphenol compounds, and their significant biological impacts, for example as bactericidal, antiviral, or fungal repressors [64,65].
Citharexylum spinosum has been reported to have some biological isolated compounds, such as 5-deoxy pulchelloside, 8-epiloganin, iridoid glucoside, lamiidoside, duranterectoside C, and the lignan glucoside [36,72]. Flower essential oil and extracts exhibited antibacterial and antioxidant activities [33,73,74]. At 8 µg/mL of concentrated methanol extract of C. spinosum wood, there was a potent inhibition against the growth of P. variotii seen [75]. The B. spectabilis extract was more effective than C. spinosum extract, and this may be because it contains aldehydes and huge amounts of volatile compounds, such as nonanal, which was found in the phytochemical analysis at a percentage of 38.28%. The biological activities of nonanal have only been reported in a few publications, as it significantly inhibits the mycelia growth of P. cyclopium [76].
The inhibitory effect of B. spectabilis extract could correlate with the concentration of nonanal versa C. spinosum wood extracts, and these results are the same as other reports [77][78][79]. Inhibition against A. niger and P. selerotigenum growth was found with minimum inhibitory concentration (MIC) values of 250 µg/mL and 500 µg/mL, respectively [76]. Chloroform leaf extract from C. spinosum showed a weak activity against P. carotovorum subsp. carotovorum, P. atrosepticum and D. solani, [80]. The stem-bark ethyl acetate extract of C. spinosum showed the presence of vanillic acid [38]. ρ-coumaric acid, salicylic acid, and hispidulin were identified in the Citharexylum genus to have a good antimicrobial activity [81]. The n-butanol extract and essential oil (EO) of the cones of Pinus halepensis had a great antibacterial effect against the soft rot bacteria D. solani and P. atrosepticum [82].
Nonanal, the main oily compound found B. spectabilis bark HeOE, a saturated fatty aldehyde, arises from a reduction in the carboxy group of nonanoic acid. The unexplainable phenomena were not noted in nonanal alone, suggesting that aldehyde hydrocarbons are much more effective in managing postharvest diseases than alcohols and olefine [83]. Octanal and nonanal showed medium activity among the aldehyde constituents [84].
The prospective concepts underlying the antimicrobial activity of aldehyde and terpenes are not fully realized, but a number of possible strategies have been proposed. It is recognized that Gram-negative bacteria are more resistant than Gram-positive bacteria to EOs components [85,86]. Unsaturated aldehydes such as (E)-2-hexenal, (E)-2-octenal, and (E)-2-nonenalhave been shown a noticeable activity against several fungal and bacterial isolates [87,88]. Thus, these aldehydes might be good compounds for playing a reserving role against human diseases caused by bacteria or as food preservatives, or might be a good alternative to other highly toxic disinfectants for hospital equipment. Recently, Pinus halepensis branch HeOE showed the presence of 2-undecenal, (Z)-2-decenal, nonanal, (2E)-2-decenal, and decadienal as main compounds, with a good antifungal activity against B. oryzae and F. oxysporum [89].
In the present study, in vitro antibacterial activity has encouraged us to assume that the potential antibacterial activity of nonanal, an essential compounds from hydrophobic oil, against P. carotovorum subsp. carotovorum, and D. solani could be closely correlated with the physiology of the membrane [90][91][92]. In addition, fatty acid methyl esters or aldehydes are able to penetrate the hydrophobic regions of the membranes, and the carboxyl groups pass through the cell membrane, causing the lowering of the internal pH and denaturing of proteins inside the cell [93][94][95][96].
The most bioactive molecules found naturally in plants are phenolics, such as tannins and lignans. The hydroxycinnamic (a) and the hydroxybenzoic (b) acids, are two main groups of phenolics; (a) group contains caffeine, ferulic and p-coumaric acids, but the (b) group contains gallic, protocatechic acids [97,98]. p-Coumaric acid is the stepping stone in synthesis process of caffeic, chlorogenic and ferulic acids, and these phenolics lead to have an antimicrobial and antiviral effects in different mode of actions as it could kill the fungal and bacterial cells by breakdowns and ruptures the plasma membrane [99][100][101][102][103]. In another study, the cinnamic acid proved to be effective in suppressing the virulent species of Pectobacterium spp. by blocks the quorum sensing molecules [22,104]. Several studies documented the strong antibacterial activity of the commercial form of caffeic, chlorogenic, and p-coumaric acids against multi bacterial isolates such as E. coli, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Salmonella typhimurium, with a minimum inhibitory concentration (MIC) values ranging from 8-1000 µg/mL [105][106][107][108]. While Wang et al. [109], confirmed the broad spectrum of antibacterial activity of the ferulic acid sourced from Halimodendron halodendron (Pall.) plant material towards the plant bacterial strains Agrobacterium tumefaciens, Pseudomonas syringae pv. lachrymans, Xanthomonas campestris pv. vesicatoria [109].

Conclusions
In the present study, isolates from Pectobacterium carorovorum subsp. carotovorum and Dickeya solani were conventionally and molecularly identified, and were proven to be pathogenic and cause potato soft rot. Oily extracts of Bougainvillea spectabilis bark (Ca. 4000, 2000, and 1000 µg/mL) at phenolic acid concentrations of 800, 1600, and 3200 µg/mL were the most effective against bacterial isolate growth. Our present study suggests that phenolics and plant extracts might be used as bactericides to fight against soft rot bacterial diseases.