The Potential Antibacterial and Antifungal Activities of Wood Treated with Withania somnifera Fruit Extract, and the Phenolic, Caffeine, and Flavonoid Composition of the Extract According to HPLC

In the present study, Melia azedarach wood blocks treated with different acetone extract concentrations from Withania somnifera fruits are assessed for their antibacterial and anti-fungal activities. Wood blocks of M. azedarach treated with W. somnifera fruit extract at concentrations of 0, 1, 2, and 3% are evaluated for in vitro antimicrobial activity against five genbank accessioned bacterial strains—Agrobacterium tumefaciens, Dickeya solani, Erwinia amylovora, Pseudomonas cichorii, and Serratia pylumthica—and two fungi, namely, Fusarium culmorum and Rhizoctonia solani. Through HPLC analysis we find that the most abundant quantified phenolic and flavonoid compounds of acetone extract (mg/100 g) are salicylic acid (9.49), vanillic acid (4.78), rutin (4702.58), and myricetin (1386.62). Wood treated with the extract at 2% and 3% show no growth of A. tumefaciens, E. amylovora, and P. cichorii. Use of the extract at 3% causes inhibition of fungal mycelia of F. culmorum and R. solani by 84.07% and 67.03%, respectively. In conclusion, potent antifungal and antibacterial activity against plant pathogens is found when an acetone extract of W. somnifera fruits is applied to wood samples. Record Type: Published Article Submitted To: LAPSE (Living Archive for Process Systems Engineering) Citation (overall record, always the latest version): LAPSE:2020.0237 Citation (this specific file, latest version): LAPSE:2020.0237-1 Citation (this specific file, this version): LAPSE:2020.0237-1v1 DOI of Published Version: https://doi.org/10.3390/pr8010113 License: Creative Commons Attribution 4.0 International (CC BY 4.0) Powered by TCPDF (www.tcpdf.org)


Introduction
Plant extract compounds and their application in food industries have great value in preventing growth of fungi or bacteria, and can be involved in possible processing technologies in which they can be exploited as ideal preservative solutions.
The highlighted benefits and challenges of plant-derived products need further research for green society implementation and governmental regulation [1,2]. Increasing regulatory restrictions and negative consumer responses to chemical compounds and to the use of antibiotics in agriculture have contributed to pressure for the development of alternative compounds for use as antimicrobial agents [3,4].
W. somnifera extracts have been demonstrated to possess strong antifungal and antibacterial activities [18][19][20][21][22]. Acetone root-bark extract from Salvadora persica has been shown to be very effective at stopping the bacterial growth of Agrobacterium tumefaciens and Dickeya solani [23], and leaf aqueous extract has been observed to inhibit the mycelial growth and spore germination of some plant pathogenic fungi [24].
During recent years, research manuscripts have introduced soft rot bacteria as a dangerous pathogen that could destroy many horticulture crops, and have developed methods to detect or characterize it, and even to provide control strategies [25,26]. Dickeya spp. strains have been isolated from diseased plants in Finland, Poland, France, the Netherlands, Switzerland, and other European countries, while Egypt and Israel have been associated with Serratia pylumthica [23,[27][28][29][30]. D. solani strains are considered more aggressive than other blackleg-causing bacteria [31]. Erwinia amylovora, a Gram-negative bacterium, is the causal agent of fire blight [32].
A 16S rRNA gene PCR-based assay has been developed as a fast-molecular diagnostic method to differentiate between phylogenetically closely related species, such as the Crown gall bacterial pathogen caused by Agrobacterium tumefaciens and other species causing plant bacterial diseases which have different symptoms, in order to identify and discriminate the strains belonging to all bacterial species [33,34].
Molds (Penicillium selerotigenum, Paecilomyces variotii, and Aspergillus niger) show different growths on Citharexylum spinosum and Morus alba woods, reflecting their natural durability [35]. On the other hand, and during storage conditions with moist and poor environments, wood, wood containers, and wood-boxes may deteriorate or become stained with the growth of molds [36], which colonize these wood types and use simple sugars and starches for growth [37,38]. Previous works have shown that extracts or essential oils have potential antimicrobial activities against several fungi and bacteria in treated wood of Pinus sylvestris, P. rigida and Fagus sylvatica, Leucaena leucocephala, Melia azedarach, and Acacia saligna [39][40][41][42][43][44][45][46].
Hence, in this work we assess the antimicrobial activity of Melia azedarach wood treated with W. somnifera fruit extract against some plant pathogenic bacteria and fungi. This study also analyzes the chemical compositions of phenolic and flavonoid compounds using HPLC analysis.

Isolation Procedure
Bacterial isolation trials were carried out on infected pear and cabbage leaves, guava root galls, and potatoes that showed symptoms collected from Beheira Governorate during 2018 in Egypt. The plant samples were rinsed with water, placed in 1% sodium hypochlorite solution for 3 min, washed in sterile distilled water, and left to dry on sterilized paper. The infected tissues were milled in a mortar with sterile NaCl 0.8% solution, and then 5% sucrose nutrient agar plates for the pear leaves bacteria and glycerol nutrient agar for the other studied bacteria were inoculated with a loop of the previous suspension [47]. Pure colonies were picked and reserved in a refrigerator until use. The two fungal cultures used in this study had been previously isolated and molecularly identified as Fusarium culmorum and Rhizoctonia solani, and were obtained from Dr. Said Behiry, Head of the Molecular Plant Pathology Laboratory, Agricultural Botany Department, Faculty of Agriculture Saba Basha, Alexandria University, Alexandria, Egypt [3]. All the isolates were reserved on glycerol nutrient agar (for bacteria) and potato dextrose agar (for fungi) for later use.

Bacterial Phenotypic Characterization
The morphological, physiological, and biochemical features of the bacterial isolates were characterized and described according to the Schaad et al. [47] laboratory guide.

DNA Extraction, PCR Analyses, and Sequencing
DNA isolation from the bacterial cultures was performed according to the protocol cited by Ausubel et al. [48].

Preparation of Wood Blocks
Air-dried Melia azedarach wood samples were prepared with dimensions of 1 × 1 × 0.5 cm at the Department of Forestry and Wood Technology in January 2018. The samples were autoclaved at 121 • C for 20 min and then cooled.

Extraction and Preparation of Concentrations of Acetone Extract from Withania somnifera fruits
Fruits of Withania somnifera (Alexandria, Egypt) were thoroughly washed using tap water, air-dried, and then crushed into small pieces using a small laboratory Willy mill. Each flask contained 200 mL of acetone solvent; 50 g were soaked for three days at room temperature [44] and then filtered using Whatman No. 1 filter paper and microfiltered using a Millipore (0.45 µm pore size membrane). The acetone solvent was evaporated using a rotary evaporator at 45 • C and then the crude acetone extract was stored in sealed vials at 4 • C until further use [49]. The extract was prepared at concentrations of 1%, 2%, and 3% (w/v) as dissolved in 10% dimethyl sulfoxide (Sigma-Aldrich, Darmstadt, Germany) prior to treatment of the wood samples. All the treatments were compared with a control treatment (10% DMSO).

Treatment of M. azedarach Wood using the Soaking Method
Each wood sample of M. azedarach received 100 µL of the concentrated extract, and the control received 100 µL of 10% DMSO. All the treated woods were kept for 6 h in flasks. Three wood samples were used for each treatment [50].

Antimicrobial Activity of Wood Treated with Acetone Extract
The antibacterial activity of wood treated with an acetone extract of W. somnifera was tested against Agrobacterium tumefaciens, Dickeya solani, Erwinia amylovora, Pseudomonas cichorii, and Serratia pylumthica, while the antifungal activity was evaluated using the fungal strains Fusarium culmorum, MH352452 and Rhizoctonia solani, MH352450.
The antibacterial activity of wood treated with an acetone extract of W. somnifera fruits was measured according to National Committee for Clinical Laboratory Standards (NCCLS) [51] with minor modifications, wherein the treated wood samples were placed over the inoculated medium with each bacterium at 30 • C for three days and compared with the control treatment. For antifungal activity, the fungal isolates were grown at 28 • C and the wood-treated samples were assayed according to previous studies [41][42][43]50], with measurement of the mycelial growth inhibition percentage performed. The inhibition zone (IZ) was measured from the outer margin of the IZ to the inner margin of the surrounding pathogens, including the treated wood blocks for bacteria, and the IZ was measured from the edge of the treated wood blocks to the outer margin of the surrounding pathogens for the fungi.

Analytical HPLC of Phenolic Compounds/Caffeine and Flavonoids for the Acetone Extract
The HPLC apparatus and conditions used (Table 1), as well as all the standards used, can be found in previous works [39,44,45,52]. The minimum concentration that the detector was able to measure was 0.1 µg/mL.

Statistical Analysis
The percentages of mycelial inhibition growth of the three fungi and two bacterial strains as affected by the three concentrations (1, 2, and 3%) of W. somnifera fruit extract were statistically analyzed and compared with the control treatment (10% DMSO) using one-way ANOVA [53]. Comparisons among means were performed using the least significant difference at the 0.05 level of probability (LSD 0.05 ) .

Bacterial Isolation and Characterization
Five bacterial isolates were isolated from infected cabbage, guava, pear, and potato plants. The morphological and biochemical characteristics and the partial sequence of 16S rRNA revealed that the isolates were Erwinia amylovora, Agrobacterium tumefaciens, Pseudomonas cichorii, Dickeya solani, and Serratia pylumthica ( Table 2).

In Vitro Visual Observations of the Antibacterial Activity of Extract-Treated Wood
The antibacterial activity of the treated wood with acetone extract from W. somnifera fruits is shown in Table 3 and Figure 1. No growth of A. tumefaciens, E.a amylovora, and Pseudomonas cichorii was found when using the extract at 2% and 3%, and the inhibition zone (IZ) reached 90 mm. No significant differences among the three concentrations of extract were shown against the growth of D. solani, where the IZ reached 31.66 mm and was higher than the value for the control treatment. The IZ values found against the growth of S. pylumthica were 76 mm and >90 mm for wood treated at 2% and 3% of the extract, respectively.    Table 4 and Figure 2 present an antifungal bioassay of wood treated with acetone extracts of W. somnifera fruits against two fungi (Fusarium culmorum and Rhizoctonia solani). Compared to control treatments with complete growth of fungi and when increasing the concentrations of the extract, fungal mycelia inhibition (FMI) was observed. At 3% of the extract, the FMI reached 84.07% and 67.03% for growth of F. culmorum and R. solani, respectively. On the other hand, the lowest values of 49.62% and 49.25% were observed at the concentration of 1% against the growth of F. culmorum and R. solani, respectively.

Phenolic/Caffeine and Flavonoid Compounds of the Acetone Extract
HPLC chromatograms of the phenolic/caffeine and flavonoid compounds in the acetone extract of W. somnifera fruits (Table 5) are presented in Figure 3a,b, respectively. In the mg/100 g extract, the most abundant phenolic compounds were found to be salicylic acid (9.49), vanillic acid (4.78), and o-coumaric acid (1.22), while the identified flavonoid compounds were found to be rutin (4702.58), myricetin (1386.62), and kaempferol (8.29).

Discussion
In this work, acetone extracts of W. somnifera fruits applied to M. azedarach wood showed potent activity against A. tumefaciens, E. amylovora, and P. cichorii, as well as mycelia inhibition of F. culmorum and R. solani.
The aqueous fruit extract of W. somnifera at 2% has exhibited potential antifungal action against F. oxysporum f. sp. radicis-lycopersici [58]. Phenolic compounds, flavonoid compounds, glycoside, fixed oils, tannins, alkaloids, and saponins, as well as withaferin A, ascorbic acid, and anthocyanin as polar compounds, are responsible for the potential antifungal activity of the extracts from this plant [18,59].
The black pointed disease caused by A. alternata in Triticum aestivum has been found to be reduced significantly when an aqueous extract of W. somnifera is begun to be used [60]. Methanol extract from fruit and leaf of W. somnifera has been observed to decrease Ascochyta rabiei biomass, the cause of chickpea blight disease [61]. Additionally, F. oxysporum f. sp. cepae biomass has been seen to be decreased by 93% when root methanolic extracts of W. somnifera are added [62].
Our previous works which have carried out studies of the effects of soaked or treated wood samples with natural products on the growth of pathogenic fungi and bacteria have shown some promising results. Acacia saligna wood treated with a combination treatment of Paraloid B-72 and Cupressus sempervirens wood methanolic extract has been found to have potent biocide activity against the mold fungus Trichoderma harzianum [40].
Several vital species of the pathogens Fusarium, Phaeoacremonium, Phytophthora, and Uromycladium are associated with diseases occurring in woody plant tissues. The best known of these include twig dieback in citrus, dieback and cankers on fruit trees (Petri and esca diseases), and collar rots and rusts of Acacia [63][64][65]. Species from many families are responsible for severe damage diseases of trees; for example Diaporthales and Botryosphaeriaceae cause diseases such as stem cankers, shoot, and twig blight of Eucalyptus [66,67].
Several studies have shown that wood can be protected by biopreservation chemicals using extracts or essential oils; for instance, the extent of mycelia of A. alternata, F. subglutinans, Chaetomium globosum, A. niger, and Trichoderma viride have been found to decrease or have their growth prevented on the surfaces of some woods treated with extracts from Pinus rigida (heartwood essential oil and extract), Eucalyptus camaldulensis (leaf extract and essential oil), and rhizomes of Costus speciosus (extract) [42,43]. Leucaena leucocephala wood treated with a combination of concentrations of extracts from the inner and outer bark of A. saccharum var. saccharum with citric acid has shown good inhibition against the growth of T. viride, F. subglutinans, and A. niger [44], which is related to the presence of phenolic compounds such as p-hydroxy benzoic acid, gallic acid, salicylic acid, and caffeine. M. azedarach wood treated with A. saligna flowers has shown antifungal and antibacterial activities with a high content of quercetin, benzoic acid, naringenin, caffeine, o-coumaric acid, and kaempferol [45]. Additionally, M. azedarach wood samples treated with peel extracts of Musa paradisiaca L. in the presence of ellagic acid, gallic acid, rutin, myricetin, and naringenin have shown good antifungal activity against R. solani and F. culmorum, as well as antibacterial activity against A. tumefaciens [39].

Conclusions
The potent antifungal and antibacterial activities exhibited by W. somnifera extract might be attributed to the presence of either the single or synergetic effects of more than one compound, or the high amount of flavonoid compounds, and may help us to discover new antibiotic substances that could serve as alternative treatments for plant diseases and their control. Hence, W. somnifera extract might be used as a bioactivity agent (antibacterial or antifungal) against plant pathogens. Further field studies are required to generalize the activity of this plant extract in treating various plant diseases.