Antifungal activity of hydroxytyrosol enriched extracts from olive mill waste against Verticillium dahliae, the cause of Verticillium wilt of olive

Copyright: © 2021 M. I. Drais, E. Pannucci, R. Caracciolo, R. Bernini, A. Romani, L. Santi, L. Varvaro. This is an open access, peer-reviewed article published by Firenze University Press (http:// www.fupress.com/pm) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


INTRODUCTION
Verticillium wilt, caused by Verticillium dahliae, severely affects olive trees (Olea europaea L.), causing economic losses due to plant death (Jiménez-Díaz et al., 1998;López-Escudero and Mercado-Blanco, 2011). The soilborne fungus is considered one of the most serious threats to olive fruit and oil production, and the disease is widely distributed in all Mediterranean olive-growing regions (Jiménez-Díaz et al., 2012). Olive trees are highly sectored with direct vascular connections of specific roots and shoots (Lavee et al., 1996). Verticillium dahliae infects host plants through their roots, and colonizes vascular systems, blocking water flow and eventually inducing wilt symptoms (Van Alfen, 1989). This damage results in significant reductions of leaf transpiration, which lead to leaf chlorosis and defoliation. Severe attacks cause trees to eventually die.
Since no control measures have proved to be successful when individually employed, an integrated strategy is recommended for management of Verticillium wilt of olive (VWO) (López-Escudero and Mercado-Blanco, 2011). Several studies have reported the use of antagonistic microorganisms as biological control agents (BCAs) against V. dahliae in olive (Mercado-Blanco et al., 2004;Triki et al., 2012;Markakis et al., 2016;Varo et al., 2016;Mulero-Aparicio et al., 2019). In addition, natural compounds could be complementary for integrated ecofriendly management of this important disease.
Beside the well-known antifungal activity of plant derived essential oils, which has been demonstrated for V. dahliae (López-Escudero et al., 2007;Varo et al., 2017), other classes of natural products also hold promise for disease management. Phenolic compounds have demonstrated, in parallel with strong antioxidant activity, antimicrobial activity in general and antifungal activity in particular (Yangui et al., 2009;Pannucci et al., 2019). These compounds can be obtained from the waste products of olive oil production; the two most prominent of these compounds are oleuropein obtained from leaves, and hydroxytyrosol (HTyr) from drupes (Thielmann et al., 2017). For HTyr, recent studies have demonstrated bactericidal and fungicidal activities of olive mill waste products (OMWP) obtained by a proprietary, environmentally friendly membrane technology . Several studies have examined the phenolic components of OMWP for use as biopesticides for crop protection (Mekki et al., 2006a(Mekki et al., , 2006b(Mekki et al., , 2008Yangui et al., 2008Yangui et al., , 2010Larif et al., 2013;Lykas et al., 2014). These components would also fulfil the criteria for a sustainable economic development (Romani et al., 2016;Bernini et al., 2017).
The present study evaluated the effectiveness of two HTyr-enriched extracts from OMWP against V. dahlia using pure HTyr as standard. In vitro inhibitory effects on mycelium growth and conidium germination of a V. dahliae isolate were assessed. The aim was to determine the potential for using these extracts as part of integrated management of VWO.

Chemicals
All solvents and chemical used for extractions were of the highest analytical grade (Sigma-Aldrich). Pure HTyr used as standard in experiments was synthetized to purity >98%, using a proprietary procedure (Bernini et al., 2008;.

Characterization of HTE1 and HTE2
HTE1 and HTE2 were characterized using High Performance Liquid Chromatography/Diode Array Detector (HPLC/DAD) and Nuclear Magnetic Resonance ( 1 H NMR), and the analytical profiles were compared with HTyr. The HPLC/DAD analyses were carried out using an HP 1200 liquid chromatograph (Agilent Technologies), equipped with an analytical column (Lichrosorb RP18 250 × 4.60 mm i.d, 5 μm; Merck). The eluents were H 2 O adjusted to pH = 3.2 with HCOOH (solvent A) and CH 3 CN (solvent B). A four-step linear solvent gradient was used, starting from 100% of solvent A up to 100% of solvent B, for 88 min at a flow rate of 0.8 mL min -1 (Romani et al., 2016;Bernini et al., 2017). Phenolic compounds found in the extracts were identified by comparing retention times and UV/Vis spectra with those of the authentic standards. Each compound was characterized using a fi ve-point regression curve built with the available standards. Analytical data are reported in Figure 1.
1 H NMR spectra were recorded using a 400 MHz Nuclear Magnetic Resonance Spectrometer Avance III (Bruker). Chemical shift s were expressed in parts per million (δ scale) and referred to the residual protons of the solvent. Samples were prepared solubilizing 20-30 mg of each sample in methanol-d4. NMR spectra are shown in Figure 2.

Preliminary activity screening using HTyr against selected fungus isolates
Six fungus isolates were used (Table 1). Synthesized HTyr was used as standard for preliminary testing eff ects on the fungi, and to identify an active concentration of the compound to be used as reference for comparisons with HTE1 and HTE2. Verticillium dahliae and B. sorokiniana were inhibited by HTyr. Th e V. dahliae pathogen from olive was selected and additional tests were carried out to confi rm the antifungal activity of HTyr and the two extracts. Th e other fungus isolates showed high variability and low susceptibility to the compounds and were not investigated further.   Disc diffusion assays. These were used to evaluate the antifungal activity of the HTyr standard against the V. dahliae isolate. The antifungal test was carried out by placing a mycelium plug in the centre of each PDA Petri dish, and at 3 cm from a paper disk (Oxoid) to which 1 mg of HTyr had been applied. After 72 h incubation at 25°C, the distance (mm) between the edge of the resulting fungus mycelium and the edge of the disk was measured (Balouiri et al., 2016). Data are expressed as means of three independent replicas per treatment.
Modified top agar assays. These were used to evaluate the antifungal activity of HTyr, HTE1 and HTE2 obtained from OMWP against V. dahliae. HTyr was tested at concentrations of 0.25, 0.5 or 1 mg mL -1 . HTE1 was tested at 7.6, 15.2 or 30.4 mg mL -1 , and HTE2 was tested at 2.15, 4.3 or 8.6 mg mL -1 . These concentrations correspond to 0.25, 0.5 or 1 mg mL -1 of the corresponding HTyr content for each extract. The Top Agar was obtained by incorporating HTyr or the extracts at different concentrations into a final volume of 5 mL of PDA. The substrate obtained was poured into Petri dishes on top of 20 mL of previous solidified PDA. Solidified Top Agar was inoculated in the centre of each Petri dish with a mycelium plug of the V. dahliae isolate. Negative experimental controls were prepared by replacing the volume of samples with the same volume of sterile distilled water.
At 3, 5 and 7 days post-inoculation the diameter of mycelium growth in each Petri dish was measured (Figure 3), and these data were subsequently analyzed to calculate the percentage of mycelium growth inhibition (MGI%), using the following formula: where C = diameter of mycelium growth in the experimental control, and T= diameter of mycelium growth treated with HTyr or the extracts.
Antifungal activity of HTyr, HTE1 or HTE2 on Verticillium dahliae conidium germination Effects of HTyr on conidium germination were evaluated by placing 5 μL of V. dahliae conidium suspension (10 5 conidia mL -1 ) on a thin layer of water agar on a glass microscope slide. The water agar amended with 1 mg mL -1 of HTyr, HTE1 or HTE2. The Microscope slides were placed in Petri dishes lined with moist filter papers (100% RH.), and were incubated for 24 h at 25°C. Germinated conidia were counted after 6 h and 24 h, using a minimum of 100 conidia per replicate, with four replicates accessed (Khalil et al., 1985). The results were expressed as percentages of inhibition of germination in relation to experimental controls, as follows: where C = conidium germination in the control, and T = conidium germination in treatment with HTyr or the extracts.

Data analyses
Differences (at P < 0.05) between means of the parameters measured were determined by analysis of variance (ANOVA) and Tukey's (HSD) multiple range test. IBM SPSS Statistics for Windows, Version 22.0, was used for these statistical analyses.

Phenolic profiles of HTE1 and HTE2
The qualitative and quantitative HPLC profiles of HTE1 have been previously described by Pannucci et al. (2019). In this extract, the main compound found was HTyr at 32.83 mg g -1 , representing the 64.7% w/w of the total phenols (50.7 mg g -1 ). Minor components in this extract were verbascoside (12.9 mg g -1 , 25.4% w/w), tyrosol, gallic acid and verbascoside derivatives, which in total constituted the 9.6% w/w of total phenols. This is the first description of HTE2. The chromatographic profile indicated that HTE2 contained 149.7 mg g -1 of polyphenols. The main compound in the extract was HTyr (115.2 mg g -1 , 77% w/w), and the secondary components were HTyr glycol and tyrosol (34.5 mg g -1 , 23% w/w).
For effects of different concentrations of each extract on growth of V. dahliae, from HTE1, after 3 d, there was no difference between the 0.25 mg mL -1 (mean = 27.7, SD = 2.66) and 0.5 mg mL -1 (mean = 40.9, SD = 4.69) treatments, but a significant difference was detected between the 1 mg mL -1 (mean = 58.7, SD = 12.56) and the 0.5 and 0.25 mg mL -1 treatments. Similarly, after 5 and 7 d of incubation, there was a difference in inhibition of V. dahliae from the greatest concentration of HTE1. For HTE2, there were no statistically significant differences in inhibition from the three different concentrations, after 3, 5 or and 7 d incubation.

DISCUSSION
The increasing interest of the use of natural products in agricultural includes research on plant derived compounds for pest and disease management. This aims to meet the regulatory demands for reduction in the use of synthetic pesticides, to provide environmentally friend-ly approaches. Several reports have described effects of plant extracts on fungal plant pathogens. Phenolic compounds derived from OMWP have been shown to hold promise as natural fungicides against crop pathogens, including Alternaria solani, Botrytis cinerea and Fusarium culmorum (Winkelhausen et al., 2005). In particular, HTyr is well-known and of interest to the pharmaceutical industry, because of the antioxidant, anti-inflammatory (Bernini et al., 2015) and antimicrobial properties (Robles-Almazan et al., 2018;Pannucci et al., 2019) of this compound. OMWP enriched in HTyr, is a resource for agricultural applications. We have evaluated HTE1 and HTE2, obtained by OMWP, through a sustainable pilot process (Romani et al., 2016;Bernini et al., 2017) based on membrane technologies which mainly enrich HTyr, together with other low molecular weight phenols, as has been shown using HPLC and NMR analyses.
In the present study, the capacities have been demonstrated for HTyr, HTE1 and HTE2 to affect mycelium growth and conidium germination of V. dahliae, the cause agent of Verticillium wilt of olive trees. In vitro inhibitory effects of different concentrations were assessed. Inhibition of conidium germination is important because conidia are important for propagation of the disease.
HTE1 was the most effective treatment against mycelium growth and conidium germination of V. dahliae. The diameters of V. dahliae colonies decreased with increasing concentrations of this extract. At the tested concentrations, low antifungal effects of HTyr were detected, but greater inhibition was detected from HTE1 and HTE2 at the same relative concentrations of HTyr. HTE1 at 1 mg mL -1 gave greater inhibition of fungal growth than HTE2 or HTyr. However, even greater inhibition was achieved for V. dahliae conidium germination. Applying 1 mg mL -1 of HTyr, differences between HTE1 and HTE2 were detected. Similarly, significant differences between HTyr and HTE1 were observed for inhibition of conidium germination., However, no differences were noted between HTE2 and HTyr. The effects of 1 mg mL -1 of HTyr on conidium germination gave better results than detected for inhibition of growth of fungal colonies.
The results are similar to those of previous studies on HTyr enriched extracts from olive mill wastewater (OMWW) that were tested against the olive bacterium pathogens Pseudomonas savastanoi pv. savastanoi (Pss) and Agrobacterium tumefaciens (At) Pannucci et al., 2019). In those studies, HTE1 was also the most active extract, which completely inhibited the growth of Pss and at 0.5 mg mL -1 and at 1.0 mg mL -1 , compared to untreated controls. In contrast, HTyr . HTyr and HTE extracts mean inhibition (%) on Verticillium dahliae conidium germination. HTyr standard was tested at 1 mg mL -1 and HTE1 and HTE2 were tested at 1 mg mL -1 referred to the HTyr content in each extract. Germinated conidia were counted after 6 and 24 h incubation at 25°C in the dark, and means are for 100 conidia per replicate. Bars indicate standard errors, and different letters indicate significant differences (P < 0.05), Tukey's (HSD) multiple range test. at 1.0 mg mL -1 only reduced bacterium growth. We have verified that HTyr and HTE1 as antifungal agents produced similar results. This provides results that can be explored in the future, which may provide mechanism of action relating to interactions of these compounds with specific bacterium or fungus cell membranes. Yangui et al. (2010) observed a severe reduction numbers of viable conidia of V. dahliae by at least 15 g L -1 of HTyrrich OMWW or HTyr-rich extract with contact times greater than or equal to 30 min, or at 12.5 g L -1 with contact time of 60 min. Differently in the present study, we evaluated the inhibitory effects on V. dahliae mycelium growth and conidium germination, and the present results were obtained for HTE1 and HTE2 extracts from Italian olive cultivars from different geographical origins (Apulia and Sicily). Furthermore, a different extraction procedure based on membrane technology was used, which gave rise to a different phenolic content profile in the extracts.
For HTyr, HTE1 and HTE2, the greatest inhibition of V. dahliae mycelium growth was observed after the first 72 h of incubation. With longer periods of incubation, growth inhibition was less than that observed after 72 h. Decreasing growth inhibition with increasing incubation time possibly indicates that the active compounds were being metabolized by the fungus. Loss in inhibitory activity, and possibly stimulation of mycelium growth, is consistent with results for other fungi, such as Aspergillus sp. when growing on media containing rutin or quercetin, where the fugus produced an extracellular enzyme that degrades these glycosides (Westlake et al., 1959). In the present study, the extracts may not have inhibited mycelium growth during long incubation periods because of breakdown by V. dahliae enzymes.
Concerning the stronger activity of HTE1 and HTE2 observed compared to HTyr, this could be due to a central role of the minor phenolic components in the extracts. Some of these have been shown to have antimicrobial activity when tested singularly. Gallic acid possesses a high antifungal activity against Fusarium solani; the hyphae became collapsed and shrunken after 24 h incubation (Nguyen et al., 2013). Enriched, purified, but still complex mixtures of phenols could possibly provide multiple modes of action giving rise to synergistic antifungal effects.
Several mechanisms of action have been proposed for the antimicrobial activities of phenolic compounds. Their potency may result from the ability to compromise cell functions and membrane integrity, behaving as surface-active compounds (Yangui et al., 2008). Therefore, alteration of microbe membrane permeability, with the consequent loss of cytoplasmic constituents, could explain phenolic activity against pathogenic fungi (Yangui et al., 2009). The mechanisms by which polyphenols act are not entirely understood. However, results from the present study give evidence that the observed antifungal effects was directly related to the chemical composition of HTE1 and HTE2, and mainly to HTyr content of these extracts.
The present study showed that HTE1 and HTE2 have antifungal activity against V. dahliae. However, we consider that they are preliminary, since further research is required, including assessments on more pathogen isolates. In addition, studies of olive trees under field conditions are required to extend knowledge of management of this pathogen with these identified extract compounds. Some researchers have suggested that incorporation of OMWW into soil could be an eco-friendly alternative to soil fumigants for crop protection against V. dahliae (El-Abbassi et al., 2017). Nevertheless, safe use of OMWP for efficient plant disease control, without negative effects on cultivated crops and soils, remains a challenge. It is also necessary to demonstrate that the phenolic contents of OMWP retains biocidal activity after large-scale applications, allowing sustainable agroeconomic development.