Characterization of Phytoconstituents from Alcoholic Extracts of Four Woody Species and Their Potential Uses for Management of Six Fusarium oxysporum Isolates Identified from Some Plant Hosts

Background: Trees are good sources of bioactive compounds as antifungal and antioxidant activities. Methods: Management of six molecularly identified Fusarium oxysporum isolates (F. oxy 1, F. oxy 2, F. oxy 3, F. oxy 4, F. oxy 5 and F. oxy 6, under the accession numbers MW854648, MW854649, MW854650, MW854651, and MW854652, respectively) was assayed using four extracts from Conium maculatum leaves, Acacia saligna bark, Schinus terebinthifolius wood and Ficus eriobotryoides leaves. All the extracts were analyzed using HPLC-VWD for phenolic and flavonoid compounds and the antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and β-carotene-linoleic acid (BCB) bleaching assays. Results: In mg/kg extract, the highest amounts of polyphenolic compounds p-hydroxy benzoic, benzoic, gallic, and rosmarinic acids, with 444.37, 342.16, 311.32 and 117.87, respectively, were observed in C. maculatum leaf extract; gallic and benzoic acids with 2551.02, 1580.32, respectively, in A. saligna bark extract; quinol, naringenin, rutin, catechol, and benzoic acid with 2530.22, 1224.904, 798.29, 732.28, and 697.73, respectively, in S. terebinthifolius wood extract; and rutin, o-coumaric acid, p-hydroxy benzoic acid, resveratrol, and rosmarinic acid with 9168.03, 2016.93, 1009.20, 1156.99, and 574.907, respectively, in F. eriobotryoides leaf extract. At the extract concentration of 1250 mg/L, the antifungal activity against the growth of F. oxysporum strains showed that A. saligna bark followed by C. maculatum leaf extracts had the highest inhibition percentage of fungal growth (IPFG%) against F. oxy 1 with 80% and 79.5%, F. oxy 2 with 86.44% and 78.9%, F. oxy 3 with 86.4% and 84.2%, F. oxy 4 with 84.2, and 82.1%, F. oxy 5 with 88.4% and 86.9%, and F. oxy 6 with 88.9, and 87.1%, respectively. For the antioxidant activity, ethanolic extract from C. maculatum leaves showed the lowest concentration that inhibited 50% of DPPH free radical (3.4 μg/mL). Additionally, the same extract observed the lowest concentration (4.5 μg/mL) that inhibited BCB bleaching. Conclusions: Extracts from A. saligna bark and C. maculatum leaves are considered potential candidates against the growth of F. oxysporum isolates—a wilt pathogen—and C. maculatum leaf as a potent antioxidant agent.

The aim of the present study was to evaluate the biological activity of ethanol extracts four extracts of four plant species to control the wilt pathogen-Fusarium oxysporum. Phenolic and flavonoid compounds were also identified using HPLC-VWD, and the antioxidant activity was also reported.

Extraction of Plant Materials
Leaves of Conium maculatum L. were collected from the Garden of the Faculty of Agriculture, Alexandria University, Alexandria, Egypt, while Acacia saligna (Labill.) H.L.Wendl. (bark), Schinus terebinthifolius Raddi wood and Ficus eriobotryoides leaves collected from Antoniadis Gardens, Alexandria, Egypt, during June 2019, were used in the present study [8,27]. All the plant materials were identified by coauthor Dr. Mohamed Z.M. Salem at the Department of Forestry and Wood Technology, Faculty of Agriculture, Alexandria University. The plant materials were air-dried at room temperature until each of them could be transferred to powder using a small laboratory mill. After obtaining the powdered of all materials, 50 g from each plant material was extracted by soaking method [69,70], in 80% ethanol (150 mL) for one week, and then filtrated through cotton plug followed by filter paper (Whatman no. 1). The extracts were concentrated with evaporating the solvent using a rotary evaporator and poured in Petri dishes to complete the dryness. Three replicates for each extract were carried out. The afforded quantities of extracts were 4.45 ± 0.57, 9.57 ± 0.51, 7.06 ± 0.58, and 5.5 ± 0.81 g/100 g dry weigh from C. maculatum leaves, A. saligna bark, S. terebinthifolius wood, and F. eriobotryoides leaves, respectively. After that, the extracts were separately prepared in a stock solution of 200 mL as dissolved in 10% dimethyl sulfoxide (DMSO) and the following concentrations 500, 750, 1000 and 1250 mg/L were prepared.

Antifungal Activity and Minimum Inhibitory Concentration (MIC) Assays of Four Plant Extracts
The antifungal activity of four plant extracts was assessed against six fungal isolates of Fusarium oxysporum F. oxy 1, F. oxy 2, F. oxy 3, F. oxy 4, F. oxy 5 and F. oxy 6, collected from different plant hosts of Peas (Pisum sativum L.), Zucchini (Cucurbita pepo L.), Egyptian Rice (Oryza sativa L.), Pepper (Capsicum annuum L.), Cape gooseberry (Physalis peruviana L.), and Bean (vicia faba L.) with their sequencing ITS regions submitted and registered to GenBank under the accession numbers MW854648, MW854649, MW854650, MW854651, MW854652, and MW854653, respectively. The plant extracts were prepared as mentioned above at the concentrations of 500, 750, 1000 and 1250 mg/L [74]. Carbendazim (reference chemical fungicide) prepared at concentrations of 200 mg/L were assessed using the broth dilution method according to Clinical and Laboratory Standards Institute (CLSI) [75]. F. oxysporum isolates were cultivated on a PDA medium. Then, a single 0.5 cm culture disk was taken from actively growing cultures and placed in the middle of the Petri dishes were with the different concentrations of plant extracts. The plates were incubated for 6 days at 28 • C, and three replications were used for each isolate [53,76,77]. The fungal inhibition percentage was calculated with the formula of inhibition percentage of fungal growth (IPFG) (%) = [DC-DT/DC] × 100, where DC and DT are the average diameters (mm) of fungal colonies under the control and experimental treatments, respectively. Three replicates were carried out for all of the treatments [52]. The minimum inhibitory concentrations (MIC) of the plant extracts prepared at concentrations of 64 to 1250 mg/L were assessed according to CLSI [75].

Antioxidant Activity of the Extracts
Free radical scavenging activity of the obtained four extracts was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) method (absorbance at 517 nm), along with the β-carotene-linoleic acid (BCB) bleaching assay [71,78,79]. The DPPH is a stable free radical alcohol soluble and the assay is based on its scavenging by the active principles of the extracts, while BCB assay is based on the bleaching inhibition of this system by the extract biocompounds. The concentration of extract or the references compounds ascorbic acid (AA) and butylated hydroxyl toluene (BHT)) responsible for 50% of inhibition of DPPH radical or BCB bleaching inhibition after 24 h of incubation was determined [80][81][82].

Statistical Analysis
The results of the percentages of the fungal linear inhibition of six isolates of Fusarium oxysporuma as affected by four concentrations (500, 750, 1000 and 1250 mg/L) of the ethanol extract of C. maculatum leaves, A. saligna bark, S. terebinthifolius wood and Ficus eriobotryoides leaves were statistically analyzed with two-way analysis of variance (ANOVA) using SAS software (SAS Institute, Release 8.02, Cary, North Carolina State University, Raleigh, NC, USA) [83]. The means were compared against the control treatment according to Duncan's Multiple Range Test at a 0.05 level of probability. Table 1 presents the chemical compounds of the phenolic and flavonoid compounds as well as caffeine identified in the 80% ethanolic extracts from Conium maculatum leaves, Acacia saligna bark, Schinus terebinthifolius wood, and Ficus eriobotryoides leaves. Figure 1 shows the HPLC chromatograms of the identified compounds from studied extracts. The highest amounts (mg/kg extract) of chemical compounds p-hydroxy benzoic acid        Table 2 presents the antifungal activity of extracts against the growth of six isolates of F. oxysporum. The highest inhibition percentage of fungal growth (IPFG%) against the growth of F. oxy 1 was observed with extracts from A. saligna bark followed by C. maculatum leaves at 1250 mg/L with IPFG of 80%, and 79.5%, respectively, while F. eriobotryoides leaf extract showed good activity with IPFG of 73.1% at 1250 mg/L. However, these values are lower than the value from carbendazim (88.89%). Extract from A. saligna bark showed the potent antifungal activity against isolate F. oxy 2 with IPFG of 86.4% at 1250 mg/L, which higher than the values from carbendazim (85.2%). Furthermore, extracts from C. maculatum leaves, S. terebinthifolius wood and F. eriobotryoides leaves showed good activity against the growth of F. oxy 2 with IPFG values of 78.9, 73.5, and 66.6%, respectively, at the concentration of 1250 mg/L. Extracts from A. saligna bark (IPFG 86.4%), and C. maculatum leaves (IPFG 84.2%) showed the highest activity against the growth of F. oxy 3, which higher than the IPFG from carbendazim (79.2%). In addition, F. eriobotryoides leaf extract at the concentration of 1250 mg/L observed IPFG value of 86.4% against F. oxy 3. At concentration of 1250 mg/L, extract from A. saligna bark, C. maculatum leaves, and F. eriobotryoides leaves showed the highest activity against the growth of F. oxy 4 with IPFG values of 84.2, 82.1, and 76.6, respectively, and were higher than the values from carbendazim (75.8%). Extracts from A. saligna bark and C. maculatum leaves observed the highest activity against F. oxy 5 with IPFG values of 88.4% and 86.9%, respectively, and those values were higher than the reported from carbendazim (84.81%). In addition, F. eriobotryoides leaves extract at the concentration of 1250 mg/L showed good activity against F. oxy 5 with an IPFG value of 82.9%. Extracts from A. saligna bark and C. maculatum leaves showed a significant effect against F. oxy 6 with values of IPFG 88.9, and 87.1%,  Table 2 presents the antifungal activity of extracts against the growth of six isolates of F. oxysporum. The highest inhibition percentage of fungal growth (IPFG%) against the growth of F. oxy 1 was observed with extracts from A. saligna bark followed by C. maculatum leaves at 1250 mg/L with IPFG of 80%, and 79.5%, respectively, while F. eriobotryoides leaf extract showed good activity with IPFG of 73.1% at 1250 mg/L. However, these values are lower than the value from carbendazim (88.89%). Extract from A. saligna bark showed the potent antifungal activity against isolate F. oxy 2 with IPFG of 86.4% at 1250 mg/L, which higher than the values from carbendazim (85.2%). Furthermore, extracts from C. maculatum leaves, S. terebinthifolius wood and F. eriobotryoides leaves showed good activity against the growth of F. oxy 2 with IPFG values of 78.9, 73.5, and 66.6%, respectively, at the concentration of 1250 mg/L. Extracts from A. saligna bark (IPFG 86.4%), and C. maculatum leaves (IPFG 84.2%) showed the highest activity against the growth of F. oxy 3, which higher than the IPFG from carbendazim (79.2%). In addition, F. eriobotryoides leaf extract at the concentration of 1250 mg/L observed IPFG value of 86.4% against F. oxy 3. At concentration of 1250 mg/L, extract from A. saligna bark, C. maculatum leaves, and F. eriobotryoides leaves showed the highest activity against the growth of F. oxy 4 with IPFG values of 84.2, 82.1, and 76.6, respectively, and were higher than the values from carbendazim (75.8%). Extracts from A. saligna bark and C. maculatum leaves observed the highest activity against F. oxy 5 with IPFG values of 88.4% and 86.9%, respectively, and those values were higher than the reported from carbendazim (84.81%). In addition, F. eriobotryoides leaves extract at the concentration of 1250 mg/L showed good activity against F. oxy 5 with an IPFG value of 82.9%. Extracts from A. saligna bark and C. maculatum leaves showed a significant effect against F. oxy 6 with values of IPFG 88.9, and 87.1%, respectively, and these values were highest than the value of carbendazim (82.6%). The MIC values (mg/L) measured against the growth of six isolates from F. oxysporum are shown in Table 3. The range of these values were 32-125, 64-125, 125-250, and 125-250 mg/L, as the extracts from C. maculatum, A. saligna, S. terebinthifolius and F. eriobotryoides, respectively, were measured. Nevertheless, these values were lower than the reported from carbendazim (5-10 mg/L).    Table 4 presents the antioxidant activity of extracts from C. maculatum leaves, A. saligna bark, S. terebinthifolius wood and F. eriobotryoides leaves compared with those reported from the standards ascorbic acid (AA) and butylated hydroxyl toluene (BHT) as measured by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenges and β-Carotene-linoleic acid bleaching (BCB) assays. The lowest concentrations that inhibited 50% of DPPH free radicals were 3.4 µg/mL (C. maculatum leaves) and 5.12 µg/mL (S. terebinthifolius fruits) where they were lower than the value from AA (7.66 µg/mL) but higher than from BHT (2.4 µg/mL). Comparing with the other method, BCB, the lower values were reported as C. maculatum extract (4.5 µg/mL) was tested, which was lower than the value reported from AA (5.12 µg/mL) and higher than as found be BHT (2.78 µg/mL). It can be observed that the extract from A. saligna bark had weakened antioxidant activity as measured by DPPH and BCB methods.

Discussion
The results of the present work show that the extracts of C. maculatum leaves, A. saligna bark, S. terebinthifolius wood and F. eriobotryoides leaves possessed a remarkable and potential antifungal activity against the six F. oxysporum isolates as well as antioxidant properties. These activities could be related to the presence of the identified several phenolic and flavonoid compounds in their extracts.
p-Hydroxy benzoic acid, benzoic acid, gallic acid, rosmarinic acid, vanillic acid and p-coumaric acid were observed as the abundant compounds in C. maculatum leaf ethanolic extract. Previously, total phenolic compounds were presented in C. maculatum 33.28 mg GAE/g DW [84]. Coumarins, umbelliferone and scopoletin compounds isolated from C. maculatum extract showed inhibitory effects on Alternaria, and Bipolaris species spore germination, which were greater than those of xanthotoxin, furanocoumarins, bergapten and angelicin [85]. The tested furanocoumarins were most effective for inhibiting mycelial growth of Fusarium spp. than Alternaria and Bipolaris [85]. Leaf extract of C. maculatum showed weak activity against Phytophthora infestans [86]. At the concentration of 50%, C. maculatum roots ethanolic extract showed the maximum inhibition of mycelia growth and conidial germination of the Fusarium pallidoroseum [87].
In the present study and for the first time, we identified the polyphenolic compounds from Ficus eriobotryoides leaves, where rutin, o-coumaric acid, p-hydroxy benzoic acid, resveratrol, and rosmarinic acid were identified as the main compounds in the ethanol extract. Phenolic compounds such as furanocoumarins (psoralen and bergapten), ferulic acid, gallic acid, chlorogenic acid, and flavonoids like rutin identified from some Ficus plants have been recognized for their pharmacological properties [41,[113][114][115]. The strong antioxidant and antibacterial activities of F. microcarpa bark extract have been attributed to its high level of phenolic compounds such as catechol, vanillin, syringaldehyde, ppropylphenol, p-vinylguaiacol, and syringol [116]. Rutin, and chlorogenic acid, present in F. carica, and F. elastica extracts have been promised as potent antioxidant activity [117].
Phenolic and flavonoid compounds found in plants with different quantities depending on the plant part and the extraction process have great effects as antimicrobials and antioxidants [3,71,118]. Dihydroquercetin isolated from barley showed to suppress the growth of Fusarium spp. [119], while naringenin and its derivatives were displayed potential antimicrobial activities [120]. The methanol extract with its main compound rutin extracted from peels of Musa paradisiaca showed potential wood-biofungicide against the growth of Fusarium culmorum and Rhizoctonia solani [9]. Flower extract of A. saligna flower extract with its main phenolic and flavonoid compounds (o-coumaric acid, benzoic acid, quercetin, naringenin, and kaempferol) showed good antifungal activity against Penicillium chrysogenum [3]. Rutin from Polygala paniculata possessed good activity against Sporothrix schenckii and Cryptococcus gattii [121], while the extract from Phaleria macrocarpa fruit showed the presence of myricetin, naringin, and rutin, which could responsible for the bioactivity [122,123]. Quercetin, which was identified in S. terebinthifolius wood and F. eriobotryoides leaves, has shown antifungal and antioxidant activities [124]. Three flavonoids and two esters of gallic acid isolated from S. terebinthifolius leaves were observed for their antiradical potential [103].

Conclusions
This study provides the potential use of four extracts from Conium maculatum leaves, Acacia saligna bark, Schinus terebinthifolius wood and Ficus eriobotryoides leaves for the antifungal and antioxidant properties. Phytochemical investigations of the ethanolic extracts identified several phenolic and flavonoid compounds, where the most abundant compounds were p-hydroxy benzoic acid, benzoic acid, and gallic acid in C. maculatum leaf, gallic and benzoic acids in A. saligna bark, quinol, naringenin, rutin, catechol, benzoic acid, and quercetin in S. terebinthifolius wood, and rutin, o-coumaric acid, p-hydroxy benzoic acid, resveratrol, and rosmarinic acid in F. eriobotryoides leaves. The extracts showed promising antifungal and antioxidant properties. Extracts from A. saligna and C. maculatum showed the highest activity against all the studied six isolates from F. oxysporum. Among the four extracts, C. maculatum leaf extract showed promising antioxidant activity compared to standard antioxidant compounds. Therefore, the phenolic and flavonoid compounds as well as caffeine present in the four plants were identified as a promising natural source to control and manage the growth of Fusarium oxysporum isolates as well as for antioxidant activity.