Phenolic and flavonoid compounds from leaves and branches of Schotia brachypetala for the development of biofungicide for wood protection

Abstract

The extracts of Schotia brachypetala were tested against the molecularly identified fungi Alternaria alternata, Botrytis cinerea, and Fusarium oxysporum, which cause early blight of tomatoes, gray mold of cucumber immature fruits, and Fusarium wilt, respectively. Leaves and branches of S. brachypetala were extracted using acetone and bio-assayed for their antifungal activity at 2%, 4%, and 6% when applied to white mulberry wood samples. Using high-performance liquid chromatography analysis, the most abundant compounds in leaf extract were kaempferol (37900 µg/g extract) and gallic acid (7480 µg/g extract), and in branch extract were gallic acid (3120 µg/g extract) and chlorogenic acid (1320 µg/g extract). By increasing the extract concentration to 6%, the percentage inhibition of fungal mycelial was significantly increased compared to the positive (Cure-M) and negative control samples. This study indicates that extracts from leaves and branches of S. brachypetala can be effective as bio-based agents in wood protection and that they can prevent the growth of pathogenic fungi.

Full Article

Phenolic and Flavonoid Compounds from Leaves and Branches of Schotia brachypetala for the Development of Biofungicide for Wood Protection

Mohamed Z. M. Salem ,^a,* Nader A. EL-Shanhorey,^b Nashwa H. Mohamed,^c and Abeer A. Mohamed ^d

The extracts of Schotia brachypetala were tested against the molecularly identified fungi Alternaria alternata, Botrytis cinerea, and Fusarium oxysporum, which cause early blight of tomatoes, gray mold of cucumber immature fruits, and Fusarium wilt, respectively. Leaves and branches of S. brachypetala were extracted using acetone and bio-assayed for their antifungal activity at 2%, 4%, and 6% when applied to white mulberry wood samples. Using high-performance liquid chromatography analysis, the most abundant compounds in leaf extract were kaempferol (37900 µg/g extract) and gallic acid (7480 µg/g extract), and in branch extract were gallic acid (3120 µg/g extract) and chlorogenic acid (1320 µg/g extract). By increasing the extract concentration to 6%, the percentage inhibition of fungal mycelial was significantly increased compared to the positive (Cure-M) and negative control samples. This study indicates that extracts from leaves and branches of S. brachypetala can be effective as bio-based agents in wood protection and that they can prevent the growth of pathogenic fungi.

DOI: 10.15376/biores.20.1.1069-1087

Keywords: Schotia brachypetala; Leaf extract; Branch extract; Antifungal activity; HPLC; Phenolic compounds; Flavonoid compounds; Gray mold; Fusarium wilt; Early blight

Contact information: a: Forestry and Wood Technology Department, Faculty of Agriculture (El‑Shatby), Alexandria University, Alexandria 21545, Egypt; b: Botanical Garden Research Department – Horticultural Research Institute Agriculture Research Center, Alexandria, Egypt; c: Timber Trees Research Department, Horticulture Research Institute, Agriculture Research Center, Alexandria, Egypt; d: Plant Pathology Research Institute, Agricultural Research Center (ARC), Alexandria 21616, Egypt;

* Corresponding author: zidan_forest@yahoo.com

GRAPHICAL ABSTRACT

INTRODUCTION

Schotia brachypetala Sond. is a deciduous tree that grows to a medium to large size and belongs to the Caesalpinaceae family. It is native to southern Africa (Coates Palgrave 1997). Schotia brachypetala is one of four species and three hybrids of Schotia in southern Africa (Arnold and Wet 1993). Roasted seeds of all species are edible (Watt and Breyer-Brandwijk 1962). The plant, especially the bark and root, are used in traditional medicine and to treat diarrhea and dysentery (Gelfand 1985; Hutchings et al. 1996; Coates Palgrave 1997; McGaw et al. 2002a).

In an initial screening, the antibacterial activity of S. brachypetala leaves was found to be higher than that of the roots (McGaw et al. 2002a); thus, activity-directed fractionation from the leaf extract was carried out once more to specify the most bioactive compounds. The bark and roots of S. brachypetala are used for nervous conditions (Van Wyk and Gericke 2000). Acetylcholinesterase (AChE) inhibition was seen in the extracts from the roots and bark of S. brachypetala (Adewusi et al. 2011). The promising antityrosinase and antibacterial activities of S. brachypetala root bark extract, together with its low to moderate cytotoxicity against HaCat cells (a human epidermal keratinocyte line), were reported (Lall et al. 2019).

The dried leaves ethanol extract contained what are thought to be the active fractions: methyl-5,11,14,17-eicosatetraenoate and 9,12,15-octadecatrienoic (linolenic acid), both of which showed antibacterial activity against Staphylococcus aureus and Bacillus subtilis (McGaw et al. 2002b). Furthermore, at 0 and 24 h of bacterial development, the acetone extracts of S. brachypetala reduced the formation of biofilms from Enterococcus faecalis by 113% and 135%, respectively (Khunoana et al. 2019).

Several plant extracts have been used to manage fungal diseases that infect plants. Leucaena leucocephala (Lam.) is one such extract that is used to combat Rhizoctonia solani, Fusarium solani, and Alternaria solani (Elbanoby et al. 2024). Eucalyptus camaldulensis and Origanum majorana essential oils were tested against Fusarium oxysporum (Shakam et al. 2022). This study focused on three common plant pathogenic fungi, namely Alternaria alternata, Fusarium oxysporum, and Botrytis cinerea. One of the most prevalent fungi, B. cinerea, can cause gray mold in greenhouses and cause significant fruit losses in cucumber plants (Kumar et al. 2016; Ziedan et al. 2022). The tomato (Solanum lycopersicum L.) is particularly susceptible to several fungi that can cause significant reductions in tomato yield, such as fusarium wilt (F. oxysporum) (Sharma 2023). One of the pathogens that most drastically reduces agricultural productivity is Alternaria alternata, which causes tomato early blight disease and harms crops all over the world (Sharma et al. 2021).

Root rot disease, primarily caused by Fusarium spp. species, is a severe problem for the commercially important mulberry (Nguyen et al. 2019), which is grown for its nutritious leaves that are needed to generate the most valuable silkworm cocoons. According to Beevi and Qadri (2010), this disease diminishes acreage and produces significant damage. Fig fruit disease is caused by A. alternata (Anwaar et al. 2022). More than 200 plant species are susceptible to gray mold disease, which is caused by the fungus B. cinerea. According to AbuQamar et al. (2016), this fungus is one of the primary pathogenic fungi that can infect mulberries. These pathogens can be identified through Internal Transcribed Spacer (ITS) ribosomal RNA (rRNA) sequencing, molecular identification has emerged as one of the most significant and sophisticated techniques for fungus identification (Abd-Elkader et al. 2021; Ziedan et al. 2022).

Synthetic fungicides, which are the cornerstone of fungal disease control schemes, are the most effective way to minimize fruit fungus deterioration or stains. These days, growing worries about wood safety and environmental issues have led to the development of creative and eco-friendly control techniques, specifically involving natural extracts (Singh and Singh 2012; Broda 2020; Kirker et al. 2024; Taha et al. 2024), and their use for nanomaterials as antifungal agents (Borges et al. 2018; Salem 2021; Khadiran et al. 2023; Elshaer et al. 2024). Along with their protective qualities, the extract’s phenolic components helped slow down the aging process of the wood by lowering light-induced and other-caused deterioration (Alonso-Hurtado et al. 2024). The plant extracts were investigated for their effects on anatomical features and specific gravity of wood species (Atılgan et al. 2013; Atılgan 2023).

Mulberry wood’s ease of burnishing and varnishing and its flexibility and elastic properties when steam-cooked make it a valuable material for the sporting equipment sector. Mulberry wood produces most hockey sticks, tennis, and badminton rackets. Mulberry planks can also be used to make wood accessories, furniture, and beautiful veneers. Paper is made in China and Europe from the very fibrous stem bark of the white mulberry (Lochynska and Oleszak 2011). In Beni-Suef, Egypt, it is a year-round wild plant that grows in abundance (Hussein et al. 2010). According to reports, the wood is long-lasting (Se Golpayegani et al. 2010; Se Golpayegani 2011). The methanol extract from M. alba heartwood showed good inhibition against the growth of Aspergillus niger at a concentration of 32 μg/mL (Mansour et al. 2015a).

The authors’ ongoing work assesses the antifungal effect of extracts from S. brachypetala leaves and branches against the growth of three common mold fungi: Alternaria alternata, Botrytis cinerea, and Fusarium oxysporum. This was done as part of the ongoing study using natural products to develop an eco-friendly wood bio-preservative against mold fungi. Therefore, in the present work, the commonly used local wood species white mulberry was used to study the effect of extracts from leaves and branches of Schotia brachypetala as bio-fungicides. Furthermore, a high-performance liquid chromatography (HPLC) approach was used to assess the phenolic and flavonoid components present in the extracts.

EXPERIMENTAL

Materials

The biomass parts (leaves and branches) of Schotia brachypetala Sond. were collected from Antoniadis Garden, Alexandria, Egypt. The plant was identified at the Timber Trees Research Department, Horticulture Research Institute, Agriculture Research Center, Alexandria, Egypt, by the coauthor Nashwa H. Mohamed and vouchered with number Z0013. Leaves and branches were first air-dried at room temperature and then ground to powder. The particles that were passed through a 0.4-mm sieve (40-mesh) and retained on a 0.27-mm sieve (60-mesh) were used for the extraction. Acetone solvent was used for the extraction, where about 100 g of each powdered material were soaked in 200 mL of acetone solvent (99.99%, technical grade-Merck) for three days, separately in a stoppered conical flask (500 mL). After this period, the extracted biochemical compounds were filtered with a cotton plug and Whatman filter paper No. 1 to afford the acetone extracts. The extracts were concentrated in a vacuum using a rotary evaporator. The extract percentage was calculated as follows: Extract percent = [extract amount (g)/air-dry leaf or branch sample amount (g)] × 100. The percentages of extracts were 1.23% and 2.5% from branches and leaves, respectively. The concentrated extracts were poured into Petri dishes and air-dried before further analysis (Elbanoby et al. 2024). The plant extracts were stored in a refrigerator at 4°C in airtight glass vials (Eldesouky et al. 2024).

Methods

Isolation of fungal pathogens

The pathogens of early blight and fusarium wilt of tomato and gray mold of cucumber fruits were isolated. To meet the development requirements of the organisms, the fungal pathogen was isolated from tissues using potato dextrose agar medium (PDA media) and incubated at 26 ± 1 °C for a week (Klug et al. 2024).

Molecular characterization of genomic DNA

Genomic DNA was extracted from three pure isolates of Botrytis spp., Fusarium spp., and Alternaria spp. grown on PDA, and their genomic DNA was extracted using the rapid micro preparation technique (Shakam et al. 2022). Using the universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGA TATGC-3′), the ITS DNA region of these isolates was amplified by polymerase chain reaction (PCR). The conserved ribosomal internal transcribed spacer (ITS) region was used to identify fungal cultures (Moore et al. 2011; Mohamed et al. 2021; Salem et al. 2021), and three isolates’ amplified ITS1-5.8s and ITS2 regions (500 to 700 bp) of selected Botrytis spp., Fusarium spp., and Alternaria spp. isolates were sent for sequencing (Macrogen, Scientific Services Company, Korea) (Kumar et al. 2016). Using the BLAST search on the National Centre for Biotechnology Information (NCBI), the sequences were compared to those in GenBank (http://www.ncbi.nlm.nih.gov).

Antifungal activity

First, the acetone extracts of S. brachypetala leaves and branches were prepared in concentrations of 2%, 4%, and 6% by dissolving the respective amounts (g) in dimethyl sulfoxide (10% DMSO) in stock solutions of 100 mL. Air-dried wood blocks of 2 (L) × 2 (R) × 0.7 cm³ from white mulberry (Morus alba L.) with a moisture content range from 8.12 to 11.2% were autoclaved at 121 °C for 20 min and left to cool (Abo Elgat et al. 2021). A culture of Alternaria spp., Botrytis spp., and Fusarium spp., each aged seven days, was cultivated on PDA medium. The PDA medium was added to the Petri dishes and left to settle in the incubator. The concentrations of acetone extracts (2%, 4%, and 6%) were applied to wood samples in conditions of surface sterilization using Laminar Flow Sterile Cabinets. Each wood sample was given 150 µL of the concentrated acetone extracts and positive and negative samples. After covering the PDA medium with all the treated wood blocks, each fungus was immediately inoculated onto a Petri dish using a disc that measured 5 mm in diameter. The dishes were then cultured for 10 days at 25 ± 1 °C. Every treatment was assessed three times. The samples were treated as a negative control sample with 10% DMSO and as a positive control with the commercial fungicide Cure-M 72% WP (Mancozeb 64%+ Metalaxyl 8%). The percentage of mycelial growth inhibition was measured with the following formula (Ashmawy et al. 2020; Shakam et al. 2022); MGI = [(A_c − A_t) / A_c] × 100; where MGI is the mycelial Growth reduction (%) and A_c and A_t are average diameters of the fungal colony of the control and treatment, respectively.

HPLC conditions

The HPLC analysis of the acetone extract was carried out using an Agilent 1260 series device. The separation was performed using a Zorbax Eclipse Plus C8 column (4.6 mm × 250 mm, id, 5 µm film thickness). The mobile phase consisted of water (A) and 0.05% trifluoroacetic acid in acetonitrile (B) at a flow rate of 0.9 mL/min. A mobile phase linear gradient program was implemented with a step size of 1 min and durations of 5, 8, 12, 15, 16, and 20 min, using (A) concentrations of 82, 80, 60, 60, 82, 82, and 82%, respectively. The multi-wavelength detector was monitored at 280 nm. The injection volume was 5 μL for each of the sample solutions (redissolved in acetone). The column temperature was maintained at 40 °C (Hamzah et al. 2024). Standard HPLC-grade phenolic and flavonoid compounds were used (Fig. 1), including gallic acid, chlorogenic acid, catechin, methyl gallate, caffeic acid, syringic acid, pyrocatechol, rutin, ellagic acid, p-coumaric acid, vanillin, ferulic acid, naringenin, rosmarinic acid, daidzein, quercetin, cinnamic acid, kaempferol, and hesperetin. The identification of compounds was confirmed by comparing their retention time with the standard one. All chemical standards (high-performance liquid chromatography (HPLC grade) were from Sigma‒Aldrich (St. Louis, MO, USA).

Fig. 1. HPLC chromatograms of the standard phenolic and flavonoid compounds

Statistical Analysis

The inhibition of fungal growth measured from the treated wood samples with the acetone extracts from S. brachypetala leaves and branches at 2%, 4%, and 6% was statistically analyzed. The statistical method was performed using analysis of variance (ANOVA) and SAS software (SAS Institute, Release 8.02, Cary, NC, USA), and the means were compared against the control treatments using Duncan’s Multiple Range Test at the 0.05 level of probability.

RESULTS AND DISCUSSION

Phytochemical Compounds by HPLC

Table 1 shows the polyphenolic compounds identified in the leaves and branches of S. brachypetala extracts (in the solid form). The total identified phenolic and flavonoid compounds were 10500 µg/g extract and 43200 µg/g extract, respectively from the acetone extract of leaves. In the branch acetone extract, the total identified phenolic and flavonoid compounds were 6370 µg/g extract and 2130 µg/g extract, respectively.

The most abundant phenolic compounds in leaf acetone extract (Fig. 2) were gallic acid (7480 µg/g extract), pyrocatechol (907 µg/g extract), methyl gallate (548 µg/g extract), chlorogenic acid (541 µg/g extract), and rosmarinic acid (456 µg/g extract). The concentrated flavonoid compounds in the leaf acetone extract were kaempferol (37900 µg/g extract), rutin (1710 µg/g extract), naringenin (1660 µg/g extract), and catechin (1550 µg/g extract).

The most abundant phenolic compounds in branch extract (Fig. 3) were gallic acid (3120 µg/g extract), chlorogenic acid (1320 µg/g extract), vanillin (633 µg/g extract), rosmarinic acid (589 µg/g extract), syringic acid (403 µg/g extract), and ellagic acid (174 µg/g extract). The abundant flavonoid compounds in the acetone extract from branches were catechin (1030 µg/g extract), naringenin (647 µg/g extract), and quercetin (325 µg/g extract). The acetone extracts from the leaves and branches of S. brachypetala contained a total of 53700 and 8500 µg/g extracts of recognized phenolic and flavonoid components. These findings imply that phenolic chemicals found in naturally occurring antifungal drugs may be useful in the management of plant pathogens.

Table 1. Phenolic and Flavonoid Compounds Identified in the Extracts of Schotia Brachypetala Leaves and Branches

mAU: milli-Absorbance Units

Fig. 2. HPLC chromatograms of the phenolic and flavonoid compounds from leaf extract of Schotia brachypetala

Fig. 3. HPLC chromatograms of the phenolic and flavonoid compounds from branch extract of Schotia brachypetala

Molecular Characterization of Genomic DNA

Alternaria spp., Botrytis spp., and Fusarium spp. isolates were identified through ITS ribosomal RNA (rRNA) sequencing. The ITS sequences were submitted to GenBank. The sequence data alongside BLAST tool search showed the identities to be Botrytis cinerea (accession no. PP758475), Fusarium oxysporum (accession no. PP737874), and Alternaria alternata (accession no. PP737870). The DNA sequence obtained for each fungal isolate revealed 100% similarity with the isolates listed in GenBank.

Antifungal Activity of Wood Treated with Leaf and Branch Extracts

The antifungal activity of wood treated with acetone extracts from S. brachypetala leaves and branches was visually apparent (Fig. 4) against the growth of three mold species: A. alternata, B. cinerea, and F. oxysporum compared with the positive control. Furthermore, the three molds’ fungal mycelia growth was seen on the 10% DMSO-treated wood.

The wood blocks that were exposed to Cure-M 72% positive controls exhibited the highest percentage of inhibition against the growth of all three molds. The treated wood with acetone extracts showed distinct inhibitory zones. After treating wood blocks, the extracts and their concentrations demonstrated a notable suppression of the growth of A. alternata, B. cinerea, and F. oxysporum fungi. Furthermore, fungal linear growth inhibition increased with an increase in the concentration of acetone extract.

Note that each wood sample was treated with 150 µL of the concentrated extract before being positioned over the substrate that has been inoculated with fungi. As a result, the inhibition of fungal growth may be caused by the leaching or spreading of dissolved extracts. As shown in Fig. 4, the three fungi grew over the media, but neither growth nor inhibition was observed over the treated wood with 2% extract, suggesting that the extract is protecting the wood from mold growth when compared to the negative control.