Antifungal Potency of Amaranth Leaf Extract: An In Vitro Study

Plant diseases are a serious problem for agricultural crops, the food industry and human health. Significant efforts have been made in recent years to find natural products that could reduce the growth of plant pathogens and improve food quality. At present, there is an increased interest in plants as a source of biological active compounds that can protect crops from diseases. Important sources of these phytochemicals are lesser-known pseudocereals such as amaranth. The objective of this study was to determine the antifungal activity of leaf extracts of four amaranth species (A. cruentus, A. hypochondriacus × hybridus, A. retroflexus and A. hybridus). The antifungal potency of amaranth extracts was analyzed against selected strains of fungi. The results suggested that the antimicrobial properties of the tested extracts varied depending on the amaranth species and the fungal strain. The studied extracts inhibited the growth of Fusarium equiseti, Rhizoctonia solani, Trichoderma harzianum and Alternaria alternata. A lower inhibitory effect of the extracts was recorded against F. solani, while no inhibitory effect was observed against F. oxysporum and Colletotrichum coccodes.


Introduction
Food-borne illnesses are a major concern for consumers, the food industry and food safety authorities. Pathogenic microorganisms present on plants cause diseases, as well as deterioration in the quality of stored food products. Among these microorganisms are many polyphagous phytopathogenic fungi that contribute to food spoilage. These include, among others, fungi of the genus Fusarium, Botrytis or Alternaria. In addition to the losses they cause in crops, they are also a source of mycotoxins that accumulate in food. The genus Fusarium produces specific toxins whose profile and harmfulness depend on the species and even strain of the fungus. Fusarium species are dangerous pathogens with a high toxicity potential. Secondary metabolites of these fungi, such as deoxynivalenol, zearalenone and fumonisin B1, are among the five most important mycotoxins in Europe and the world [1][2][3]. Similarly, the genus Alternaria can produce under certain conditions secondary metabolites with toxic properties, known as Alternaria toxins [4]. Currently, scientists are emphasizing the importance of several toxins, including alternariol (AOH), alternariol monomethyl ether (AME), altertoxin (ATX), altenuene (ALT), tenuazonic acid (TeA), tentoxin (TEN) and AAL toxins that can be found as contaminants in agricultural crops [5]. Mycotoxins can spread from rotten plant tissue to surrounding areas; thus, they are also present in processed foods [6]. Therefore, many pathogenic microorganisms remain an important problem for agriculture and the food industry.
Consumers are concerned about synthetic preservatives used in food. As a result, there is a growing demand for natural products that can serve as an alternative to food

Polyphenol Content
The research object was the leaves of Amaranthus cruentus (PC), A. hypochondricus × hybridus (PH), A. retroflexus (TR) and A. hybridus (TH). Dried amaranth leaves were used as a material for the production of plant extracts, in which the total polyphenol content and antioxidant activity were determined using the synthetic DPPH radical. Table 1 presents the results of the total polyphenol content in the tested extracts obtained from amaranth leaves and the respective antioxidant activity values.
The highest concentration of total polyphenols in the initial extracts from amaranth leaves was recorded for A. hybridus (TH)-6.75 mg GAE/mL, while the lowest concentration of polyphenols was determined in the extract from A. cruentus (PC)-4.31 mg GAE/mL, which had an approx. 36% lower concentration of the tested active substances. It should be noted that extracts from plant material obtained from Turkish crops were characterized by significantly higher concentrations of polyphenols compared with amaranth extracts from Poland. The content of biologically active ingredients in plants is influenced by varietal factors, as well as cultivation factors (growing season, fertilization, soil, temperature, rainfall, etc.) [24]. Species of the genus Amaranthus are thermophilic plants. Turkey has more optimal growing conditions for this type of plant compared with Poland [25]. Optimal cultivation conditions promote more efficient synthesis of biologically active substances, which was confirmed by higher concentrations of polyphenolic compounds in the extracts of amaranth species cultivated in Turkey. The results of the antioxidant activity of the tested extracts corresponded with polyphenol levels. The highest antioxidant activity was observed in the extract obtained from the leaves of the species A. hybridus (TH) from Turkey (21.04 mM TE/mL), while extracts from Polish raw materials showed significantly lower antioxidant activity (13.84 mM TE/mL (PH) and 8.77 mM TE/mL (PC).

Fungistatic Activity
Amaranth extracts exerted different effects on the growth of the tested fungal strains, depending on the fungal species, the type and concentration of the extract, and the duration of action of the biologically active ingredients (Tables 2 and 3, Figure 1).   The fungistatic effects of amaranth leaf extracts largely depended on the fungus species tested (Tables 2 and 3). The best results were recorded for F. equiseti, regardless of the concentration and type of extract (Table 3). Extracts from species cultivated in Poland such as A. cruentus (PC) and A. hypochondriacus × hybridus (PH) showed the strongest fungistatic effect, significantly inhibiting the surface growth of F. equiseti throughout the experiment (35.6-72.2%) ( Figure 1). A strong fungistatic effect was also exhibited by the 15% extract from the species cultivated in Turkey, such as A. retroflexus (TR15), and the inhibition of fungal growth compared with the control remained at a high level throughout the experiment, i.e., 53.9-61.9% (Figures 1 and 2). The extracts tested inhibited the growth of other species of the genus Fusarium to a low extent. The superficial growth of F. solani was most strongly inhibited by the 10% extract of A. cruentus (PC10), but only on day 4 of the experiment, and reached 49.4%. The remaining types and concentrations of extracts had no significant effect on F. solani growth inhibition; their antifungal activity in the first days of the experiment ranged from 1.3 to 26.5% and quickly decreased in the following days to a statistically insignificant level (2.4-11.8%), even contributing to a slight stimulation of the surface mycelium growth (TR10, TR15) ( Figure 1). Amaranth extracts did not show any fungistatic activity against F. oxysporum, and the highest recorded level of fungal growth inhibition was 2.9-5.0% (TR10). Extracts, mainly from A. hybridus (TH10), stimulated superficial colony growth by up to 30% compared with the control sample.    A strong fungistatic effect of amaranth extracts was recorded against Rhizoctonia solani, but only in the first days of the experiment (day 4) for all tested experimental combinations. Extracts from A. cruentus (PC15), A. hybridus and A. retroflexus (TH15, TR15) strongly reduced the surface growth of R. solani at the level of 66.3-80.8% (Figures 1 and 2). The remaining extracts inhibited the growth of R. solani by only 37.8-44.7%. On subsequent days, the size of R. solani colonies exceeded the plate diameter (90.0 mm); therefore, measurements for individual experimental combinations were not performed ( Figure 1). Amaranth extracts inhibited the growth of A. alternata only in the first days of the experiment, while they stimulated the growth of the fungus in the subsequent days. The strongest antifungal effect was recorded on day 4 of the experiment for extracts from species cultivated in Turkey: A. retroflexus (TH10-40.8%; TH15-55.4%) and A. cruentus (PC15-40.8%). Other concentrations of these extracts (PH, TR) inhibited A. alternata growth, but at a statistically insignificant level, i.e., 12.5-27.9%. In the consecutive days, the antifungal activity of the extracts decreased, and the surface growth of A. alternata was stimulated by more than 20% compared with the control sample (TR10) (Figure 1). Amaranth extracts, from species cultivated in both Poland and Turkey, showed very weak antifungal activity against C. coccodes. The highest degree of fungistatic effect was recorded only for the 15% A. cruentus extract at the beginning of the experiment (PC15-21.9%) and for the A. hybridus extract (TH10, TH15-20.4 and 26.9%, respectively), while their fungistatic effect in the following days rapidly decreased, even contributing to the stimulation of fungus growth. On the other hand, Amaranth extracts inhibited the surface growth of T. harzianum. Significant antifungal activity in the first days of the experiment was recorded for the following extracts: A. hypochondriacus × hybridus (PH10, PH15-37.4%; 37.8%) and A. hybridus (TH15-43.3%). In the following days of the experiment, the T. harzianum colony diameter exceeded the diameter of the plate (90.00 mm); therefore, measurements for individual experimental combinations were not performed (Figure 1) The conducted experiment also focused on changes in the morphology of the fungi under the influence of plant extracts (Figure 2, Table 4). Most common were changes in the mycelial structure and the coloration of the obverse and reverse of the colony. The addition of extracts to the medium caused changes in the mycelial structure. Fungi growing on the medium with the addition of extracts formed less fluffy aerial mycelium with more relaxed growth. In some species, the disappearance of aerial mycelium and the growth of substrate mycelium were even observed (C. coccodes, T. harzianum). The reverse of the test fungi had a more intense color than in the control samples (F. equiseti, F. oxysporum) ( Table 4). The addition of the extract to the medium caused deformation of conidia (A. alternata) and impaired sporulation.

Discussion
Amaranth is a plant originating from Central America and is now widely cultivated in the countries of the tropics. In many countries, including India and tropical countries, it is still used in folk medicine as a laxative, to heal purulent lesions, boils and burns, and as an anti-malarial agent [26]. It has been proven that Amaranthus, thanks to its rich chemical composition, also shows antibacterial and antioxidant properties [27]. The plant is described in the literature as an important source of bioactive compounds such as lectins, phenols and flavonoids [28][29][30]. Flavonoids are an important group of biologically active compounds commonly present in many plant species. These compounds, contained in green plan parts, are characterized by, e.g., antimicrobial, anti-inflammatory or antioxidant activity [31,32]. The presence of twenty-five flavonoid and phenolic acids, such as protocatechuic acid, vanillic acid, gallic acid, salicylic acid, gentisic acid, p-hydroxybenzoic acid, β-resorcylic acid, syringic acid, ellagic acid, m-coumaric acid, trans-cinnamic acid, caffeic acid, chlorogenic acid, ferulic acid, sinapic acid, p-coumaric acid, rutin, naringenin, kaempferol, myricetin, catechin, isoquercetin, apigenin, hyperoside and quercetin, was detected in the leaves of A. gangeticus. Among the compounds, seven were identified as cinnamic acids, nine as benzoic acids and nine as flavonoid compounds. With respect to the three major classes of phenolics, the most prominent compounds were identified in four advanced lines of A. gangeticus genotypes in the following order: benzoic acids, cinnamic acids, flavonoids [33]. Not only flavonoids, but also phenolic acids determine the antimicrobial effect of amaranth extracts. The chemical composition of extracts varies and depends, among others, on the geographical location, as well as the composition and quality of the soil during plant growth [24]. The high content of polyphenolic compounds in alcoholic plant extracts was shown to be correlated with their high antioxidant capacity [34]. This was confirmed in our study involving amaranth leaf extracts carried out using the DPPH free radical method.
The biologically active compounds contained in amaranth plants are characterized by significant antimicrobial activity and cytotoxicity [35]. The conducted research showed the varied fungistatic effects of amaranth extracts. The strongest antifungal activity was recorded for extracts from A. cruentus (PC), A. hypohondriacus (PH) and A. hybridus (TH), of which TH was characterized by the highest polyphenol contents. The extracts strongly inhibited the growth of F. equiseti, R. solani and T. harzianum, and their highest fungistatic effect was observed at the beginning of the experiment. Jadhav and Biradar [20] conducted a similar study investigating the effect of A. spinosus on Fusarium spp. and Aspergillus spp. They showed that ethanol extract of amaranth leaves, applied at a concentration of 1000 µg/mL, strongly inhibited the surface growth of F. oxysporum, while its lower concentrations in the medium (100 µg/mL and 500 µg/mL) had a fungistatic effect only against A. flavus and A. niger. According to the studies conducted by Akbar et al. [36], Amaranthus viridis leaf extract demonstrated potent antifungal activity against F. oxysporum, because they reduced the fungal growth up to 48% in F. oxysporum. Our research did not confirm these reports, as it indicated not only a lack of inhibition of the surface growth of F. oxysporum (regardless of extract concentration), but even its stimulation. Studies on the antifungal effect of various plant extracts against F. oxysporum have been conducted by many researchers [12,17] who showed that F. oxysporum was not very sensitive to the effects of extracts from medicinal plants. Kursa et al. [13] showed that 20% ethanol extracts of tansy, yarrow and horseradish, considered potent against many phytopathogens, inhibited the surface growth of F. oxysporum only at the level of 24.7-26.56%, and only in the first days of the experiment. The strong inhibitory effect of the amaranth extract was recorded against F. equiseti, mainly in relation to the extracts of A. retroflexus (TR) and A. hypochondriacus × hybrisus (PH). F. equiseti is an important pathogen of crops, recorded increasingly often in horticultural plants that have been shown to be highly pathogenic to seedlings, causing root rot and decay in horticultural plants [8,[37][38][39]. F. equiseti, as other species of the genus, produces toxins and antibiotics, especially trichothecenes and equisetin [1,40]. Therefore, in this regard, A. cruentus (PC), A. hypochondriacus × hybrisus (PH) and A. retroflexus (TR) can be considered as plants with significant antifungal potential. A study of Carminate et al. [41] confirmed the antifungal efficacy of Amaranthus viridis against Colletotrichum musae causing banana anthracnose. The present study showed no antifungal effect against C. coccodes, and a stimulating effect on mycelial surface growth was even observed. The growth of this species was also poorly inhibited by other plant extracts, including extracts from plants with high biocidal potential [13]. Carminate et al. [41] also indicated the great potential of Amaranthus viridis in controlling F. solani f. sp. piperis, responsible for fusariosis in black pepper. Similarly, our research demonstrated the possibility of using amaranth extracts (PC, PH, TH) to limit the development of F. solani. The strongest antifungal activity against the species was recorded for the A. cruentus (PC) extract at the beginning of the experiment. This raises the prospect of obtaining more interesting results using higher amaranth extract concentrations in the preventive plant protection against F. solani. Promising results were also obtained against Rhizoctonia solani. All the tested extracts strongly limited the surface growth of the fungus, but only in the first days of the experiment. Shirazi et al. [42] showed that Amaranthus viridis extract at concentrations of 1, 3 and 5% exhibited maximum inhibitory potential activity in the disc diffusion method against soil-borne pathogens R. solani, F. oxysporum and M. phaseolina. The authors reported that A. viridis could be used for the control of fungal diseases, particularly those caused by R. solani; promising results have also been obtained with A. alternata. Fungi of the genus Alternaria are pathogens of stored fruits and vegetables. They penetrate the plant through mechanical and enzymatic degradation of the cell wall, causing rotting in the stored crops. The destructive effect on the host plant is caused by the production of enzymes and specific (HST) and non-specific toxins (NHST) [6]. Higher concentrations of amaranth extracts (mainly TH and PC) already significantly inhibited the surface growth of A. alternata in the first days of the experiment, but their antifungal activity decreased over time. The obtained results were consistent with the study of Akbar et al. [36]. These authors showed that all organic solvent extracts from Amaranthus viridis significantly reduced the biomass of the tested fungi with increasing extract concentrations (5, 10, 15, 20 and 25 mg mL −1 ); strong activity was shown especially by ethyl acetate leaf fraction, resulting in reduced A. alternata growth by up to 44%.
Due to their cytotoxic properties, the biologically active compounds contained in plants have a direct effect on pathogen cells [35]. The antimicrobial activity of plant extracts rich in polyphenols causes many changes at the cellular level, e.g., damage of the microbial cell membrane through increased cell membrane permeability and, consequently, the leakage of cell contents [10]. Plant extracts from Amaranthus spp., in addition to inhibiting the growth of the tested fungi, caused changes in the color and structure of aerial mycelium, as well as sporulation. Kursa et al. [17] reported that the addition of plant extracts from tansy, sage and wormwood also caused changes in the color and structure of the aerial mycelium of the tested fungi. The results of the present experiment on amaranth extracts are a valuable source of information for further field research in plant protection against pathogens, and as an ingredient of biological products protecting agricultural crops from rotting.

Plant Materials
The leaves of fully matured Amaranthus spp. (BBCH19), A. cruentus (PC) and A. hypochondriacus × hybridus (PH) species from a cultivation located in Bodaczów near Zamość (south-eastern Poland) and A. retroflexus (TR) and A. hybridus (TH) from plantations located in the province of Düzce (north-western Turkey) were collected in 2019. The collected materials were dried at room temperature (in the shade) and subsequently stored in polyethylene bags at 4 • C.

Extract preparation
Dried amaranth leaves were ground to a homogeneous fraction using an A11 Basic laboratory mill (IKA).

Extraction
Crushed plant material (100.0 g) was weighed into round bottom flasks and then 1000 mL of 70% ethanol was added. Extraction was carried out for 6 h under reflux condenser at the boiling point of ethanol. The obtained extract was filtered through a sterile 22 µm filter (AlfaChem) and concentrated to 100 mL (1:1 extract) using a rotary evaporator (Heidolph Instruments, Schwabach, Germany). The final extract did not contain ethyl alcohol.

Total Polyphenol Analysis
The concentration of total polyphenols in the tested extracts was determined using the spectrophotometric method (λ = 725 nm) with the Folin -Ciocalteau reagent, according to the modified method of Singelton and Rossi [43]. Phenol content results are expressed in gallic acid equivalents (GAE) (Sigma-Aldrich, St. Louis, MO, USA, ACS reagent ≥ 98.00%). The results were calculated based on the equation of the calibration curve prepared for gallic acid standards in the concentration range of 0.01-1 mg/mL (0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 and 1.00 mg/mL). Each sample, depending on the initial concentration, was diluted according to the range on the standard curve [17]. All analyses were performed in triplicate.

Assessment of Extract Antioxidant Activities
The antioxidant activity of plant extracts was determined using the modified method of Brand-Willams et al. [44] using the synthetic radical DPPH (1,1-diphenyl-2-picrylhydrase1 Sigma) converted to mM Trolox [45]. The inhibition of the DPPH radical by the extract sample was calculated according to the following formula: inhibition % = 100 (A 0 − A 1 )/A 0 , where A 0 is the absorbance of the control, and A 1 is the absorbance of the sample. Each extract sample was diluted appropriately to the range of the standard curve prepared for Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) standards. All analyses were performed in triplicate. Concentration values were based on the standard Trolox curve (0.2-1.2 mM) and expressed as millimoles of Trolox equivalents (TE) per ml of extract.

Fungal Cultures
Amaranth leaf extracts were individually tested against pathogenic fungi, such as Fusarium solani (ARIR14), Fusarium oxysporum (ECER4), Fusarium equiseti (ERIS8), Alternaria alternata (PCL10), Colletotrichum coccodes (P74/2), Rhizoctonia solani (TB71) and Trichoderma harzianum (A8/10). Fungal cultures were obtained in 2017-2020 as a result of plant mycological analyses (roots and aerial parts) of tomato (Lycopersicon esculantum L.) and pepper (Capsicum annuum L.) grown in the field. The fungal inoculum was derived from 10-day-old single-spore colonies grown on glucose-potato agar (PDA Difco, Becton, Dickinson and C., France), stored in the fungal collection of the Department of Plant Protection, University of Life Sciences in Lublin. Confirmation of strain species was carried out on the basis of microscopic analysis of each isolate/strain (spore structure and size, colony color) using appropriate mycological keys.
The study evaluated the effects of ethanol extracts of amaranth leaves (Polish and Turkish species: PC, PH, TR, TH) at concentrations of 10% and 15% on the linear growth of the test fungi. The method of poisoned substrates was used in the study, which is recommended to test chemical agents under laboratory conditions [13]. The method consisted of adding the test substance to sterile potato dextrose agar (PDA) medium cooled to 50 • C and inoculating the fungus species on the solidified medium. Medium and Amaranthus spp. extracts were poured into sterile Petri dishes of 90 mm and, subsequently, medium surface was inoculated with fungi colonies with a diameter of 3 mm. The control (CE) consisted of fungal colonies growing on potato dextrose agar (Difco PDA) with 10 and 15% residue after evaporation of the extractant used in the experiment (70% ethanol; a total volume of 1000 mL was evaporated to 100 mL in a rotary evaporator under the same conditions as in plant extract preparation). Five replicates of the tested extracts prepared at specific concentrations and added to each fungus were considered as objects. The plates prepared in this way were kept in an incubator for 12 days at 25 • C. After 4, 8 and 12 days, the diameter of fungal colonies was measured. The measure of antifungal activity was the inhibition of mycelial growth on medium enriched with Amaranthus spp. extract relative to growth on control medium. The antifungal efficacy of Amaranthus spp. extract was calculated from the Abbott formula: where: I-linear growth inhibition index of the fungus (percentage), C-diameter of the fungus colony in the control combination, T-diameter of the fungus colony in the combination containing the test substance concentration in the agar [13].

Statistical Analysis
Values are given as means ± standard deviation (SD) of each measurement. Where appropriate, the data were analyzed by analysis of variance (Duncan's test) at the 5% significance level using the SAS statistical software (SAS Version 9.1, SAS Inst., Cary, NC, USA).

Conclusions
The use of natural compounds to control pathogens is very attractive and creates new opportunities for biological plant protection. The results of the present experiment demonstrated a very diverse effect of amaranth leaf extracts (A. cruentus, A. hypochondriacus × hybridus, A. retroflexus, A. hybridus) on selected phytopathogenic and antagonistic fungi. The extracts were fungistatic only against F. equiseti, R. solani and A. alternata, and showed activity only during the first days of the experiment, which proved their selective and short-term antifungal effect. The strongest fungicidal effect was recorded for species cultivated in Turkey such as A. hybridus (TH), which, due to having the highest content of polyphenols and high antioxidant activity, can be recommended for limiting the growth of some phytopathogenic fungi, also causing spoilage of agricultural crops. Due to the lack of a strong fungistatic effect, it can be used as an ingredient in plant preparations. In addition to direct antifungal activity, plant extracts act as elicitors of defense reactions in the plant and as biostimulants. The effect of amaranth leaf extracts as plant biostimulants requires further in-depth laboratory and field studies.