Antifungal, Antibacterial, and Antioxidant Activities of Acacia Saligna (Labill.) H. L. Wendl. Flower Extract: HPLC Analysis of Phenolic and Flavonoid Compounds

In this study, for the environmental development, the antifungal, antibacterial, and antioxidant activities of a water extract of flowers from Acacia saligna (Labill.) H. L. Wendl. were evaluated. The extract concentrations were prepared by dissolving them in 10% DMSO. Wood samples of Melia azedarach were treated with water extract, and the antifungal activity was examined at concentrations of 0%, 1%, 2%, and 3% against three mold fungi; Fusarium culmorum MH352452, Rhizoctonia solani MH352450, and Penicillium chrysogenum MH352451 that cause root rot, cankers, and green fruit rot, respectively, isolated from infected Citrus sinensis L. Antibacterial evaluation of the extract was assayed against four phytopathogenic bacteria, including Agrobacterium tumefaciens, Enterobacter cloacae, Erwinia amylovora, and Pectobacterium carotovorum subsp. carotovorum, using the micro-dilution method to determine the minimum inhibitory concentrations (MICs). Further, the antioxidant capacity of the water extract was measured via 2,2′-diphenylpicrylhydrazyl (DPPH). Phenolic and flavonoid compounds in the water extract were analyzed using HPLC: benzoic acid, caffeine, and o-coumaric acid were the most abundant phenolic compounds; while the flavonoid compounds naringenin, quercetin, and kaempferol were identified compared with the standard flavonoid compounds. The antioxidant activity of the water extract in terms of IC50 was considered weak (463.71 μg/mL) compared to the standard used, butylated hydroxytoluene (BHT) (6.26 μg/mL). The MIC values were 200, 300, 300, and 100 µg/mL against the growth of A. tumefaciens, E. cloacae, E. amylovora, and P. carotovorum subsp. carotovorum, respectively, which were lower than the positive control used (Tobramycin 10 μg/disc). By increasing the extract concentration, the percentage inhibition of fungal mycelial was significantly increased compared to the control treatment, especially against P. chrysogenum, suggesting that the use of A. saligna flower extract as an environmentally friendly wood bio-preservative inhibited the growth of molds that cause discoloration of wood and wood products.

Acacia saligna (Labill.) H. L. Wendl. (Acacia cyanophylla Lindl.), native to Western Australia and belonging to the family Fabaceae, has been planted in Egypt and other Mediterranean countries in Africa, become an invasive species, and is considered a fast-growing tree [41,42]. Different Acacia plants produce allelopathic materials as bioactive compounds [43,44]; where phenolics, tannins, flavonoids, phenols, and proanthocyanidins are the most common compounds identified in various parts of the Acacia species [45][46][47][48][49]. The methanol extract of flowers and leaves was observed to have allelopathic effects greater than those of an aqueous extract on the germination percentage of Hordeum murinum [50], which was caused by a variety of active components [51]. The ethyl acetate extract of Acacia saligna leaves was more effective against Staphylococcus aureus, S. pyogenes, Bacillus cereus, B. subtilis, and Candida albicans than methanolic and water extracts [52].
The aim of this study was to evaluate the bioactivity of a water extract from flowers of Acacia saligna in terms of the fungal resistance of wood treated with the water extract, antibacterial activity against some phytopathogenic bacteria, and antioxidation properties. Further, the main phenolic and flavonoid compounds in the water extract were analyzed using an HPLC method.

Isolated Fungi
The isolation trails and the ITS sequences revealed that the three fungal isolates were Fusarium culmorum, Rhizoctonia solani, and Penicillium chrysogenum. GenBank accession numbers of the isolated fungi are listed in Table 1. Table 1. Accession numbers of fungal isolates used for antifungal activity evaluation.

Fungal Isolate Accession Number
Fusarium culmorum MH352452 Rhizoctonia solani MH352450 Penicillium chrysogenum MH352451 Figure 1 shows that wood samples treated with water extracts of different concentrations (1, 2, and 3%) of A. saligna flower presented different degrees of inhibitions to fungal growth compared to the control treatment (wood samples treated with 10% DMSO). The water extract of flowers at 3% exhibited the highest inhibition percentage of mycelial growth of F. culmorum, P. chrysogenum, and R. solani, with values of 38.51%, 65.92%, and 41.48%, respectively, compared to the control. It can be seen from Table 2 that there is no significant difference among the three concentrations (1, 2, and 3%) of water extracts for the inhibition percentage of R. solani. However, with an increasing concentration, the percentage inhibition of fungal mycelia of P. chrysogenum was significantly increased. Furthermore, there is no significant difference between the concentrations 1% and 2% for the inhibition percentage of F. culmorum, but the concentration 3% showed the highest inhibition percentage with a significant value against the same fungus. Table 3 presents the antibacterial activity of the water extracts, where the MIC values in µg/mL of 200 (A. tumefaciens), 300 (E. cloacae), 300 (E. amylovora), and 100 (P. carotovorum subsp. carotovorum) were observed and all the values were lower than those reported from the positive control used (Tobramycin 10 µg/disc).  Table 3 presents the antibacterial activity of the water extracts, where the MIC values in μg/mL of 200 (A. tumefaciens), 300 (E. cloacae), 300 (E. amylovora), and 100 (P. carotovorum subsp. carotovorum) were observed and all the values were lower than those reported from the positive control used (Tobramycin 10 μg/disc).

Phytochemical Constituents and DPPH Activity of Extract
The HPLC chromatograms for the identified phenolic and flavonoid compounds are shown in Figures 2 and 3, respectively. Table 4 presents the phenolic and flavonoid compounds identified in the water extract of A. saligna flowers. The most abundant phenolic compounds were benzoic acid (161.68 mg/100 g), caffeine (100.11 mg/100 g), o-coumaric acid (42.09 mg/100 g), p-hydroxy benzoic acid (14.13 mg/100 g), and ellagic acid (12.17 mg/100 g); while the identified flavonoid compounds were naringenin (145.03 mg/100 g), quercetin (111.96 mg/100 g), and kaempferol (44.49 mg/100 g).     A spectrophotometric DPPH assay was used to evaluate the antioxidant properties of the flower water extract. Results of the antioxidant activity of the water extract from A. saligna flowers showed that with increasing concentration, the total antioxidant activity (TAA%) increased ( Figure 4). The results obtained show that the total flower extract presents a weak scavenging capacity. The measured antioxidant activity in terms of the IC 50 value (the concentration of the extract able to scavenge 50% of the DPPH free radical) was 463.71 µg/mL, which was lower than the value reported with BHT (6.26 µg/mL).     A spectrophotometric DPPH assay was used to evaluate the antioxidant properties of the flower water extract. Results of the antioxidant activity of the water extract from A. saligna flowers showed that with increasing concentration, the total antioxidant activity (TAA%) increased ( Figure 4). The results obtained show that the total flower extract presents a weak scavenging capacity. The measured antioxidant activity in terms of the IC50 value (the concentration of the extract able to scavenge 50% of the DPPH free radical) was 463.71 μg/mL, which was lower than the value reported with BHT (6.26 μg/mL).

Discussion
Bioactive compounds extracted from plant materials are recognized to be the first step for the utilization of phytochemicals in dietary supplement preparation, as well as of food ingredients, pharmaceutical products, and antimicrobial agents [2]. Several studies have examined the antimicrobial activity of water extracts from Acacia species. The aqueous extract of A. cyanophylla leaves showed different levels of activity against the growth of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Xanthomonas, Escherichia coli, and Citrobacter [53]. The extract obtained with A. saligna leaves was effective against the tested bacteria B. subtilis, E. coli, Klebsiella pneumoniae, P. aeruginosa, S. aureus, and Micrococcus luteus (Gumgumjee and Hajar, 2015). The ethanol extract of A. saligna showed good activity against A. niger, A. fumigatus, A. flavus, and C. albicans at a concentration of 200 mg/mL [54]. To the best our knowledge, no studies have been carried out to study the effects of flower extracts against the phytopathogenic bacteria. Other studies showed that alkaloidal extracts from Conocarpus lancifolius leaves had MICs value of 20-50 µg/mL and >200 µg/mL against the growth of A. tumefaciens and E. amylovora, respectively [55]; while it was 125, 250, 125, and 16 µg/mL against P. carotovorum subsp. carotovorum when treated with acetone extract of Callistemon viminalis flower, n-butanol extract of C. viminalis flower, essential oil from the aerial parts of Conyza dioscoridis, and n-butanol extract from the bark of Eucalyptus camaldulensis, respectively; and against A. tumefaciens with values of 16, 250, >4000, and 250 µg/mL with the same extracts [3]. The leaf ethanol extract of A. cyanophylla showed activity against Aspergillus niger, A. fumigatus, A. flavus, and Candida albicans, where A. fumigatus was the most susceptible fungi and A. niger the most resistant [56].
Phenolic and flavonoid compounds extracted from different plant materials have shown potential antioxidant properties, i.e., flavonoids from Larix decidua [62] and Abies spectabilis [63] bark extracts. The catechol group presented in the quercetin derivative is known to strongly increase activity in the DPPH assay [64]. On the other hand, moderate and lower antioxidant activity than standard antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and ascorbic acid), was found with ethanolic extracts of Salvia microstegia, S. brachyantha, and S aethiopis, while the main phenolic and flavonoid compounds were kaempferol, rosmarinic acid, apigenin, luteolin, p-coumaric acid, and chlorogenic acid [65]. In the present study, we found weak antioxidant activity, which could be the result of the poor solubility of polyphenols in the water extract [66]. For example, the S. aethiopis water extract and S. microstegia ethanol extract presented an activity similar to ascorbic acid, but the lowest activity was observed in the S. microstegia water extract [65].
Quercetin-7-O-diglucoside isolated from stem bark and wood extracts of Terminalia brownii possessed significant antifungal activity against Aspergillus and Fusarium strains [67]. In addition, quercetin and its derivatives have been reported to have good antifungal activities with low MIC values [68][69][70][71]. The growth of Fusarium spp. has been suppressed by dihydroquercetin isolated from barley [72]. Naringenin and its derivatives demonstrated some antifungal and antibacterial activity [73].
It should be mentioned that, while the flavonoid compounds relies on the comparison of their retention times with standard compounds, there still the biggest peaks in the chromatograms at 2.97 and 5.35 min which could be contributed properly the assessed biological activities, especially the identified flavonoids seem to be minor components in the water extract of A. saligna. These results supported the limitations of the HPLC analysis when using the available standard compounds.
Overall, it can be concluded from the current study that the water extract from flowers, containing various phenolic compounds, can be used as a bio-fungicide against certain wood-staining molds.

Plant Material and Preparation of the Extract
Flowers of Acacia saligna were collected from Alexandria, Egypt. The extraction procedure was carried out according to Salem et al. [74], with some modification, where approximately 25 g of the flowers were extracted with distilled water (200 mL) for 3 h under heat using a water bath at 50 • C. The extract was filtered with cotton plugs and then with filter paper (Whatman No. 1) (Mumbai, India) and concentrated to a small volume using a rotary evaporator (Rotavapor RII, Cole-Parmer GmbH, BucHI, Essen Germany). The water extract (7.45% w/w of fresh weight) was stored in a brown vial prior to chemical and bioactivity analyses.

Fungal Isolation, DNA Extraction, PCR, and Sequencing
During 2017, the tissues of infected Citrus sinensis L. trees showing root rots, cankers, and green fruit rot symptoms (Beheira, Egypt) were collected from Beheira governorate in Egypt and sent to the Plant Pathology laboratory of the Agricultural Botany Department, Alexandria University, Alexandria, Egypt, for isolation of the causal agents. The fungal isolates were isolated, kept on potato dextrose agar (PDA) plates, and incubated for seven days. The cultural and morphological characteristics of the isolated fungi were used for identification to the genus level, and for further molecular characterization, the DNA extraction was carried out on freshly growing mycelium using the GenElute™ Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer's instructions. The PCR was carried out using primer pairs (ITS1/ITS4) to amplify the internal transcribed spacer (ITS) region of the rDNA [75,76].
The reaction was carried out in a Techne Prime thermal cycler (Techne, Cambridge, United Kingdom). The reaction cycle consisted of denaturation for 4 min at 95 • C; followed by 40 cycles of 1 min at 94 • C, 1 min at 50 • C, and 1 min at 72 • C; and a final extension of 8 min at 72 • C. Amplification products were separated in 1.2% (w/v) agarose gel (iNtRON Biotechnology, Inc., Seongnam, South Korea), and pre-stained with red safe (iNtRON Biotechnology, Inc., Seongnam, South Korea) along with a 100 bp plus ladder at 70 V. The DNA bands were observed under a UV transilluminator. Purified fragments of ITS were sequenced by Macrogen, Inc., Seoul, Korea. The generated sequences were deposited in GenBank under the accession numbers presented in Table 1.

Antifungal Activity of Wood Treated with Water Extract
The water extract from A. saligna flowers was prepared at concentrations of 0%, 1%, 2%, and 3% by dissolving the extract in 10% dimethyl sulfoxide (DMSO). A total of 36 wood samples of Melia azedarach with dimensions of 0.5 cm × 1 cm × 2 cm, air-dried, and autoclaved at 121 • C for 20 min, were used for the antifungal activity test ( Figure 5). Three wood samples were used for each concentration. The antifungal activity was evaluated against the linear growths of Fusarium culmorum, Rhizoctonia solani, and Penicillium chrysogenum. The inhibition percentage of mycelial growth was calculated using the following equation: Mycelial growth inhibition (%) = [(A C − A T )/A C ] × 100 [77], where A C and A T are average diameters of the fungal colony of the control and treatment, respectively. Wood samples soaked only with 10% DMSO were used as the control.

Antibacterial Activity
The minimum inhibitory concentrations (MICs) of water extracts from A. saligna flowers were evaluated against the four phytopathogenic bacteria Agrobacterium tumefaciens, Enterobacter cloacae, Erwinia amylovora, and Pectobacterium carotovorum subsp. carotovorum using the micro-dilution method with serial concentrations of 4-350 μg/mL [78], and compared with the positive control (Tobramycin 10 μg/disc).

Determination of Antioxidant Activity
The antioxidant capacity was assessed by the 2,2′-diphenylpicrylhydrazyl (DPPH) assay [79] in terms of IC50 (the concentration that caused a 50% inhibition of growth compared with control) using the calibration curve compared with butylated hydroxytoluene (BHT).

Statistical Analysis
The results of the inhibition percentage of mycelial growth of the three fungi as affected by the four concentrations (0, 1, 2, and 3%) of the A. saligna flower water extract were statistically analyzed using one-way analysis of variance (ANOVA) SAS software (SAS Institute, Release 8.02, Cary, North

Antibacterial Activity
The minimum inhibitory concentrations (MICs) of water extracts from A. saligna flowers were evaluated against the four phytopathogenic bacteria Agrobacterium tumefaciens, Enterobacter cloacae, Erwinia amylovora, and Pectobacterium carotovorum subsp. carotovorum using the micro-dilution method with serial concentrations of 4-350 µg/mL [78], and compared with the positive control (Tobramycin 10 µg/disc).

Determination of Antioxidant Activity
The antioxidant capacity was assessed by the 2,2 -diphenylpicrylhydrazyl (DPPH) assay [79] in terms of IC 50 (the concentration that caused a 50% inhibition of growth compared with control) using the calibration curve compared with butylated hydroxytoluene (BHT).

Statistical Analysis
The results of the inhibition percentage of mycelial growth of the three fungi as affected by the four concentrations (0, 1, 2, and 3%) of the A. saligna flower water extract were statistically analyzed using one-way analysis of variance (ANOVA) SAS software (SAS Institute, Release 8.02, Cary, North Carolina State University, Raleigh, NC, USA), and the means were compared against the control treatment.

Conclusions
In the present study, the water extract of A. saligna flowers at 3% shows moderate antifungal properties against the three mold species tested (F. culmorum, R. solani, and P. chrysogenum). Therefore, it is possible to assert that there are some potential applications of this extract for wood protection. The water extract showed lower antibacterial and antioxidant activities than the standards used, Tobramycin and butylated hydroxytoluene (BHT), respectively. Overall, the presence of the activity of the water extract could be related to the presence of some phenolic and flavonoid compounds.