Antifungal effects of phenolic extract from industrial residues of Aloe vera

This research is concerned with the fungicidal properties of the phenolic extract from industrial residues of Aloe vera used for antifungal treatment of various plant pathogens (Fusarium oxysporum f. sp. radicis-lycopersici, Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, and Sporisorium scitamineum). Six phenolic compounds were identified in this extract from A. vera cortex: aloesin, α-barbaloin, chromone X, isoaloeresin D, βbarbaloin and aloeresin E. Phenolic extract was added to PDA medium at 20 concentrations from 0.32% to 10% and the growth of four different plant pathogenic fungi was tested. Fungal inhibition was calculated in order to evaluate the antifungal efficacy of phenolic extract against pathogens. Inhibition of Sporisorium scitamineum hyphal growth was observed after treatment with the phenolic extract at concentrations higher than 2.5% and a fungistatic effect with a 58.2% mycelia growth inhibition was detected at 3% extract concentration. Inhibition of P. chlamydospora and P. aleophilum hyphal growth was observed at concentrations higher than 4% and 3% respectively. A fungistatic effect with a 71.65% and a 19.87% mycelia growth inhibition was detected at 4.5% and 3.5% extract concentration respectively. About F. oxysporum f. sp. radicis-lycopersici, inhibition of hyphal growth was observed at concentrations higher than 2.5% and a fungistatic effect with a 32.07% mycelia growth inhibition was detected at 3% extract concentration. The results indicate that the tested extract possess antifungal activities against these pathogens at various concentration levels and could be used as a potential natural fungicide in order to control fungi pathogens providing a new use for the A. vera industrial residues. Additional keywords: phytopathogenic fungi; phenolic compounds; fungicide. Abbreviations used: FORL (Fusarium oxysporum f. sp. radicis-lycopersici); LOEC (lowest-observed-adverse-effect-concentration); MGI (mycelia growth inhibition); MIC (minimum inhibitory concentration); NOAEC (no-observed-adverse-effect concentration); PAL (Phaeoacremonium aleophilum); PCH (Phaeomoniella chlamydospora); PDA (potato dextrose agar); SS (Sporisorium scitamineum). Authors ́ contributions: All authors conceived and designed the experiments, read and approved the final manuscript. Performed the experiments: BPL, AGG, LMC, EPUC, MLT, DPG. Analyzed the data: AGG, BPL, AEC, LMC. Contributed reagents/materials/ analysis tools: DPG, AEC, EPUC, MLT. Wrote the paper: AGG, BPL, LMC. Citation: Pintos, B.; Martín-Calvarro, L.; Piñón, D.; Esteban, A.; Tello, M. L.; Pérez-Urria, E.; Gómez-Garay, A. (2018). Antifungal effects of phenolic extract from industrial residues of Aloe vera. Spanish Journal of Agricultural Research, Volume 16, Issue 4, e1010. https://doi.org/10.5424/sjar/2018164-12480 Received: 26 Oct 2017. Accepted: 21 Nov 2018. Copyright © 2018 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC-by 4.0) License. Funding: INIA-Ministerio de Economía y Competitividad (RTA2010-00009-C03-02). Competing interests: The authors have declared that no competing interests exist. Correspondence should be addressed to Beatriz Pintos: bpintos@ucm.es


Introduction
Direct crop losses in the field are caused by plant pathogens by reducing crop yield and quality. The control of plant diseases in traditional agricultural production systems is mainly based on the application of fungicides. However, fungicides application has led to problems and constraints in the control of these diseases by a loss in efficiency, due to an increased resistance to active ingredients, ecological damage and a serious negative impact on the human health. An efficient alternative to inhibit the growth of several fungal pathogens and reach the control of crop diseases can be obtained by the use of bio-based products (Romanazzi et al., 2012;De Corato et al., 2016). Nevertheless, the commercial use of natural fungicides on the market is low, the 5th Annual Meeting of the Biological Control Industry reports a total of 55 biological fungicides registered in the US market and in the EU the registered biopesticides are much fewer: 21 fungicides for be used in Pome fruit, vines and tomato (Castillo et al., 2012).
The biological compounds most useful in fungi control have different effects on the development of mycelia growth and the effect on sporulation rate and inhibition of germination ranging from a fungistatic effect to complete inhibition. Plant extracts applied as a crude state or as a fraction inhibit partially or totally fungal growth, affecting the development of colonies in laboratory tests when applied at low concentrations of bioactive compounds (Mahlo et al., 2010;Castillo et al., 2012;Cerqueira et al., 2016). Among plant metabolites, plant phenolics are therefore active in biological systems and probably the capacity or biological value explains its abundance in plant tissues (Meckes et al., 2004). Synthesis of aromatic substances is a major defense mechanism of plants. Biochemicals and their oxidation products are implicated in disease resistance (Sukand & Kulkarni, 2006;Patzke & Schieber, 2018). Phenolic compounds and related oxidative enzymes are considered as one of the most important biochemical parameters for disease resistance (Pradeep & Jambhale, 2002;Osorio et al., 2010;Patzke et al., 2017).
In natural plant defense, accumulation of phenolic compounds at the infection site has been correlated with the restriction of pathogen development, since such compounds are toxic to pathogens (Benhamou et al., 2000). This mechanism can be exploited in the fight against plant pathogens by the use of exogenous phenolics. The amphipathicity of these compounds can explain their interactions with the membrane and thus the antimicrobial activity (Veldhuizen et al., 2006). Phenolic extracts show, among other properties, antifungal activity by reducing or totally inhibiting fungal growth in a dose-response manner.
One of the plant species whose use has spread over in recent years for various purposes is the aloe. Aloe vera L. (also referred by Miller as Aloe barbadensis) has a wide spectrum healing qualities, which vary in terms of its components: enzymes, monosaccharides, polysaccharides, anthraquinones, amino acids, minerals and vitamins (Capasso et al., 1998;López et al., 2013). Although the main composition is water and aloe represents 99-99.5% of the plant, the remainder, between 0.5% and 1%, is a high concentration of sugars (up to 60% of dry matter), vitamins, proteins and amino acids, organic acids, minerals and phenolic compounds. Among these components, there are more than 150 that theoretically could have potential activity, of which 75 are biologically active compounds. Many A. vera active ingredients have been identified (Jasso de Rodríguez et al., 2005;Debnath et al., 2018).
Diverse extractives (including phenolics) of A. vera fresh leaves show antifungal activity against different plant pathogenic fungi (Jasso de Rodriguez et al., 2005;Subramanian et al., 2006;Nebedum et al., 2009;Nidiry et al., 2011;Zapata et al., 2013;Flores-López et al., 2016). However, these works were based on the application of A. vera gel, pulp and liquid fraction from leaves. In the present study, we have reported the efficacies of the phenolic extract obtained from leave residues resulted from the A. vera extractive industry.
In this work four plant pathogenic fungi were selected because these produce severe losses in crops. Sugarcane smut, caused by the fungus Sporisorium scitamineum, is considered the most severe disease of sugarcane in the world. Disease affects germination, tiller counts, smut incidence, plant girth, millable stalks per hectare and tons cane per hectare.
Fusarium crown and root rot is also a soil-borne disease, with the potential to limit productivity in glasshouse and field tomato crops. The causal agent is Fusarium oxysporum f. sp. radicis-lycopersici. Fungicides are of little use on most Fusarium diseases (Sierotzki & Ulrich, 2003).
Phaeomoniella chlamydospora and Phaeoacre monium aleophilum are both fungi frequently associated with esca and grapevine decline. Esca is a destructive disease that affects grapevines worldwide.
Processing of A. vera gel extraction leads to the formation of several by-products and residues that can have a nowadays not exploited economical potential. The aim of this study was the evaluation of the in vitro antifungal activity of phenolic extract from A. vera leaf residues against four different plant pathogenic fungi: Sporisorium scitamineum, Fusarium oxysporum f. sp. radicislycopersici, Phaeomoniella chlamydospora and Phaeoacremonium aleophilum.

Plant material and extraction of phenols
Phenolic compounds were extracted from the dried residue of aloe cortex as follows: a mass of 150 g dried powder was placed in an Erlenmeyer flask with 750 mL of distilled water. The flask was covered with aluminum foil to avoid light exposure. This mixture was refluxed at 60 o C for 12 h. The pH of the sample was adjusted to 3.0 by adding glacial acetic acid for the stabilization of the phenolic compounds. After this process, the sample was filtered using Whatman filter paper no. 41 and centrifuged at 3500 rpm for 15 min (Osorio et al., 2010). The supernatant (phenolic extract) was used for further experiments.

HPLC analysis of phenolic compounds
HPLC analysis was carried out in a Beckman system (San Ramon, CA, USA) comprising a Programmable Solvent Module 126 pump, Scanning Detector Module 167 and manual injector. The data were processed with the GOLD system. Reverse-phase HPLC (RP-HPLC) of phenolic compounds was carried out following Esteban-Carrasco et al. (2001): the system operated at 45 °C on a Beckman ultrasphere 5 μm ODS column (25 cm × 4.6 mm I.D.) using a flow rate of 1 mL/min. Solvent A was 2.5% acetic acid in water and solvent B was acetonitrile. The elution conditions applied were a linear gradient from 12 to 26% in 30 min, from 26 to 70% in 15 min, and finally, washing and re-conditioning of the column. Monitoring was set at 290 nm for identification. In order to quantify phenolics compounds, peak heights were correlated with the concentration according to the calibration curve of aloin (β-barbaloine), which was purchased from Sigma (Saint Louis, MO, USA).

In vitro antifungal assay
The antifungal properties of phenolic extract were evaluated for assessing in contact phase effects towards mycelia growth. In vitro assay was performed in PDA media treated with different concentrations of phenolic extract. PDA medium was autoclaved at 121C for 15 min and cooled to 40 ± 2 o C. The control treatment contained only PDA culture medium. Different concentrations of phenolic extract (from 0.32 to 10%) were prepared by mixing in the flasks with warm sterile molten medium to obtain final concentrations. The PDA agar with phenolic extract was poured into sterile 50 mm Petri plates (6 mL/plate). Mycelial plugs (5 mm ø) obtained from the actively growing margin of four weeks-old cultures of each species were obtained and placed aseptically at the centre of each treatment Petri plate and cultivated in the dark at optimum temperature. In order to reach the minimum inhibitory concentration (MIC) each phenolic extract concentration was tested in triplicate in each assay and the assays were repeated to confirm results.
The antifungal activity of the phenolic extract was tested using radial growth technique. The mean radial mycelia growth of each fungus was determined by measuring the diameter of the colony in two directions. The growth was compared to the control plate 7 days after inoculation in SS and FORL and 14 days after inoculation in PCH and PAL. For each concentration, three replicate plates were used.

Data analysis
All experiments were performed with three replications of each phenolic extract concentration. The mean growth values were obtained and then the radial inhibition was calculated. The percentage of mycelia growth inhibition (MGI) in relation to the control treatment was calculated by using the formula MGI (%) = ((R-r)/R) × 100, where R is the radial growth of fungal mycelia on the control plate and r is the radial growth of fungal mycelia on the treated plate. Data were analyzed using a one-way analysis of variance at the significance level of p < 0.05 and the Duncan's test. The Dunnett's test was used to calculate the minimum difference between the control and the treatment means detected as being statistically significant. The no-observed-adverse-effect concentration (NOAEC), lowest-observed-adverse-effect-concentration (LOEC) and MIC for mycelial growth of the four plant pathogens were calculated. Significant difference was considered as that with a p value < 0.05 in all statistical analyses. All the statistical analyses were implemented using the statistical package Statistica v. 9.

Results and discussion
In this work, the phenolic extract obtained from industrial residues of A. vera was tested against important plant pathogens. Six phenolic compounds were identified in this extract (Fig. 1, Table 1): aloesin, α-barbaloin, chromone X, isoaloeresin D, β-barbaloin and aloeresin E. These compounds have also been identified in previous studies in methanolic extracts from A. vera cortex (Esteban- Carrasco et al., 2001). Anthraquinones are the phenolic compounds present in our extract, the most prominent being chromone X (0.119 mM) and aloesin (0.108 mM). The aloin/bar baloin (10-β-D-glucopyranosyl-1, 8-dihydroxy-3-hydroxymethyl-9) is considered to be the most important phytoconstituents found in aloe species (Tyler, 1994;Dubey, 2015). It is one of the main biologically active constituents of aloe which is found in nature as a mixture of two diastereosiomers, aloin A (10R) and aloin B (10S). Aloin is generally contained in the exudate seeping out from freshly cut leaves, whilst very low amounts of aloin exist in Aloe gel obtained from the internal mass of aloe leaf (Fanali et al., 2010). Aloin is a good antifungal agent; previous reports demonstrate such effect (Zapata et al., 2013;Dubey, 2015).
Results of biological activity of this extract against four fungal strains showed a strong fungicide effect on the growth of SS, PCH, PAL and FORL. For all four phytopathogenic fungi a total inhibitory concentration was reached, while lower concentrations showed a fungistatic activity of the extract. Fungicide activity was considered when no fungal growth was observed in the plates and fungistatic activity was considered when fungal growth was delayed.
The contact phase effects of different concentrations of phenol extract on the mycelia growth of SS, PCH, PAL and FORL strains are shown in Table 2. The antifungal effects of the extract were diverse for the four tested fungi. These results showed that phenolic extract of A. vera industrial residue was highly inhibitory to mycelia growth of SS among the pathogens tested. Inhibition of SS hyphal growth was observed after treatment with the phenolic extract at concentrations higher than 2.5% (Table 3). For SS a fungistatic effect with a 58.2% MGI was detected at 3% extract concentration (LOEC, Table 3) while complete inhibition (100%) was observed at 3.5% extract concentration (MIC, Table 3 and Fig. 2A). The sensitivity or resistance of sugarcane to smut can be related to changes in sugarcane leaf levels of phenolic compounds. Thus an increase in both caffeic and syringic acids was detected in the resistant cultivar Mayarí 55-14 when it was inoculated with SS (Santiago et al., 2010). Thus, SS is susceptible to phenolic compounds, but not only to sugarcane ones because present results show the efficiency of A. vera phenolic compounds against SS too.
On the esca and grapevine decline pathogens PCH and PAL different results were obtained. An effective concentration range was obtained narrower in PCH (between 4 and 5%), while in PAL it ranged from 3% to 9% (Table 3). Inhibition of PCH and PAL hyphal growth was observed following treatment with the phenolic extract at concentrations higher than 4% and 3% respectively (Table 2). For PCH a fungistatic effect with Figure 1. Profile of RP-HPLC phenolics compounds from the extract of the industrial residues of A. vera. 1, Aloesin; 2, α-Barbaloin; 3, Cromone X; 4, Isoaloeresin D; 5, β-Barbaloin and 6, Aloeresin E. Table 1. Identification of phenolic compounds from the extract of the industrial residues of Aloe vera by HPLC. The retention times (R t ) and peak wavelength of each compound are shown. The concentration was calculated from the aloine (β-barbaloine) calibration curve.   , Table 3), while complete inhibition (100%) was observed at 5% extract concentration (MIC , Table 3 and Fig. 2B). For PAL a fungistatic effect with a 19.87% MGI was detected at 3.5% extract concentration (LOEC ,  Table 3) while complete inhibition (100%) was observed at 9% extract concentration (MIC, Table 3 and Fig. 2C). It has been described an increase in phenolic compounds in esca-diseased plants (Amalfitano et al., 2000;Agrelli et al., 2009;Lima et al., 2010). Nevertheless, there are Vitis vinifera susceptible varieties to esca disease, which are able to activate phenolic production as a defense response, but this may be not sufficient to successfully fight the disease (Lima et al., 2010). In this way, the phenolic extract studied in this work offers a new possibility in order to act against these both pathogens, PCH and PAL. About the other studied pathogen, FORL, a fungistatic effect with a 32.07% MGI was detected at 3% extract concentration (LOEC, Table 3) while complete inhibition (100%) was observed at 5.5% extract concentration (MIC , Table 3 and Fig. 2D). Total phenol accumulations in the host tomato plant induced by the plant pathogen or by applying chemical inducers as salicylic acid and biocontrol agents as Trichoderma harzianum play an important role in resistance and defense against F. oxysporum (Ojha & Chatterjee, 2012). The current study indicates that applying phenolic extract from A. vera industrial residue can be a novel strategy in plant disease management.
The explanation of the antifungal activity of this extract can be the capacity of phenols to form complexes with polysaccharides and proteins of external layers of the fungal cells destabilizing the functions of cell walls and membranes, causing the death of the microorganism (Aguilar et al., 2007). The hydrophilic part of the molecule interacts with the polar part of the membrane, while the hydrophobic benzene ring and the aliphatic side chains are buried in the hydrophobic inner part of the membrane (Viuda-Martos et al., 2008). Furthermore, the hydroxyl group in the formation of hydrogen bonds Table 3. The no-observed-adverse-effect concentration (NOAEC, %), lowest-observed-adverse-effect-concentration (LOEC, %) and minimum-inhibitory-concentration (MIC, %) for mycelial growth of four plant pathogens: Fusarium oxysporum f. sp. radicis-lycopersici (FORL), Phaeomoniella chlamydospora (PCH), Phaeoacremonium aleophilum (PAL), and Sporisorium scitamineum (SS) exposed to phenolic extract of Aloe vera industrial residues. SS <2.5 3 3.5 a Dunnett's procedure with p < 0.05. and the acidity of these phenolics compounds may have other possible explanations (Cristani et al., 2007). Thus, a factor to be considered is the slightly acidic nature of phenols and the possible relevance of pH effects in regulating the growth of these fungi. Growth may be profoundly affected by a number of physical factors like temperature, pH, light, aeration, pressure, etc. Most fungi generally prefer slightly acidic conditions for their growth (Maharshi & Taker, 2012). To study the possible pH impact over the fungi growth produced by the addition of the phenolic extract to PDA media, the pH of the media at the MICs were determined. PDA media were elaborated at the same MIC determined pH by adding HCl (1N) and the fungi were grown on this medium. Although a slight reduction was detected in the rate of growth (data not shown), in any case there was total inhibition thereof. We have not found any data on the effect of pH over the in vitro mycelia growth of SS. The growth of PCH and PAL varies at pH between 4.1 and 8.0 but in any case it results totally inhibited (Valtaud et al., 2009) and it is reported that acidic pH favors growth of all Fusarium sp. although the fungus could grow and sporulate under a wide range of pH from 4.0 to 8.0 (Agarwal & Sarbhoy, 1978;Gupta et al., 2010).
In summary, this study has clearly showed potential use of this extract on diseases suppression in vitro providing a new use for the A. vera industrial residues. The inclusion of such natural products as this phenolic extract in crop protection strategies, as alternative to synthetic fungicides, will help to maintain the balance of agroecosystems and the safety of the harvested products. Figure 2. Effect of phenol extract on radial hyphal growth on PDA medium in three replicates against four fungal strains: a) Sporisorium scitamineum, 7 days after inoculation; b) Phaeomoniella chlamydospora, 14 days after inoculation; c) Phaeoacremonium aleophilum, 14 days after inoculation; and d) Fusarium oxysporum f. sp. radicis-lycopersici, 7 days after inoculation.