Initial In Vitro Assessment of the Antifungal Activity of Aqueous Extracts from Three Invasive Plant Species

Abstract

The development of new, safe, and effective methods of managing fungal pathogens is required. This study was conducted to perform an initial in vitro assessment of the antifungal activity of water-based plant extracts from three plants which are invasive in Egypt: Prosopis juliflora, Ipomoea carnea, and Leucaena leucocephala. These extracts were tested against three pathogenic fungi species that cause high crop losses in Egypt: Fusarium solani, Alternaria solani, and Colletotrichum circinans. Three extract concentrations, 10%, 20%, and 30%, were tested using a completely randomized design, with three replicates per treatment. Antifungal activity was determined based on the effects of plant extracts on fungal radial growth inhibition, average daily growth of fungi, spore formation, spore germination, and total biomass. Inhibition of the growth of fungal strains increased with increasing plant extract concentration, with the highest inhibitory rate at the 30% extract concentration. In addition, spore density, spore germination, and total biomass decreased significantly with increasing extract concentration. The three fungal pathogens differed in their inhibition and their response to these plant extracts. Prosopis juliflora had the highest inhibitory effect on the three fungal pathogens, compared to the extracts from the other two invasive plants. The results of this feasibility study indicate that P. juliflora extracts have high antifungal activity and follow-up in vivo assays should be conducted to determine their efficacy in the safe and sustainable management of these and other fungal pathogens.

1. Introduction

Across the globe, a multitude of plant pathogens attack agricultural crops, causing severe and damaging plant diseases. Of these pathogens, fungi are responsible for the greatest reductions in agricultural productivity. For example, 20–40% of all know plant diseases are caused by plant pathogenic fungi, leading to pre- and post-harvest crop losses [1]. Due to the amount of crop damage by fungal pathogens and their high economic costs [2], and the production of mycotoxins that are harmful to animals and human health [3,4], synthetic fungicides are widely used to control pathogenic fungi. Synthetic fungicides can build up in the soil, in plants, and in water, which can harm the environment and various components of the food chain. In addition to residue buildup, the widespread use of fungicides also has other problems including application costs, handling hazards, and threats to human health [5,6]. Also, the emergence of pathogenic fungi resistant to fungicides is concerning. Because of these problems and concerns, the development of fungicides that are effective and safe for human health and the environment is required.

Secondary metabolic pathways of plants produce numerous bioactive compounds [7,8,9]. These compounds have several functions, including inhibiting the growth of other plants (allelopathic effect) and protecting plants from pathogen and insect attack. A diverse array of bioactive compounds is found in plant extracts and essential oils and such compounds are now being used in the management of pathogens and insects [7,8,9,10,11,12,13,14,15]. These bioactive compounds include the following: alkaloids, coumarins, cyanogenic glycosides, flavonoids, flavonols, glucosinolates, lipids, phenolics, polyacetylenes, polythienyls, quinones, and terpenes. These compounds are also referred to as botanical extracts, botanical pesticides, or botanicals and are generally considered to be an eco-friendly alternative to the use of synthetic fungicides and pesticides [8,13,14].

Plant extracts and essential oils have been shown to inhibit a wide range of fungal pathogens through three modes of action: increased fungal mortality (fungicidal effect), inhibiting fungal growth and development (fungistatic effect), and/or improving plant growth by inducing the defense responses of infected plants [14,15]. Plant extracts can kill pathogenic fungi directly or can inhibit their growth and development because many of the compounds listed above possess antimicrobial activity [1,16]. The antimicrobial activity of these compounds can also reduce spore germination of pathogenic fungi [17]. Plant extracts might also control fungal pathogens because they can act as secondary messengers that enhance plant defense mechanisms [15,16]. Plant extracts can also increase the activity of peroxidase enzymes, increase the accumulation of phenolic compounds, and lead to an increase in the concentration of H₂O₂, all of which can reduce the severity of disease in infected plants [1,14,15,16].

Biological invasions occur when organisms are taken up in their native range, introduced into a new region, in which their descendants persist, proliferate, and spread [18,19]. Invasive plants can often outcompete native plant species because many invasive plants have traits contributing to invasiveness. As a result, invasive plants can be the dominant species in a diverse array of habitats [20,21,22]. As their populations occur at high densities [23], invasive plant species that produce compounds with antifungal activity are likely to be a rich source of tissue from which antifungal compounds can be extracted. The Novel Weapons hypothesis [24] posits that introduced species may become invasive if they produce secondary compounds that are novel to their new range and can inhibit native plant species. Such compounds may have antifungal activity capable of inhibiting plant pathogens. For example, Eloff [25] observed that Melianthus comosus, a weedy invasive plant, has secondary compounds with highly effective antifungal activity.

Many non-native plants have invaded large portions of Egypt; these include the woody invasive plant species Prosopis juliflora (Sw.) DC (mesquite), Ipomoea carnea Jacq. (pink morning glory), and Leucaena leucocephala (Lam.) de Wit (river tamarind) [26]. Many invasive species have also proliferated across Saudi Arabia, particularly in the southwest region of the country, where 74% of plant species occur [27]. These same woody plant species are the most important invasive plants in Saudi Arabia, growing in a variety of habitats and invading new areas [27].

Vegetable crops are constantly under attack by a wide range of plant pathogens. Worldwide, cucumber (Cucumis sativus L.), tomato (Solanum lycopersicum L.), and onion (Allium cepa L.) are three of the most important vegetable crops, and all three are important crops in Egypt [28,29,30]. In the main agricultural areas of Egypt, approximately 186,000 ha is cultivated with tomatoes and annual production approaches seven million tons [29]. There are many insect pests and plant pathogens that attack and damage the quality and quantity of all three crop plants. Chief among these plant pathogens are pathogenic fungi.

The objective of this study is to evaluate the ability of different water-based plant extracts from three invasive plants, P. juliflora, I. carnea, and L. leucocephala, to inhibit mycelial growth and spore viability of three plant pathogenic fungi, Fusarium solani (Mart.) Sacc., Alternaria solani Sorauer, and Colletotrichum circinans (Berk.) Voglino, isolated from cucumber, tomato, and onion, respectively. To the best of our knowledge, no studies have been published using water-based plant extracts from these invasive plants to control these pathogenic fungi. However, previous research assessing the inhibition of F. solani using plant extracts from a variety of plant species reveals highly variable results of antifungal activity [31,32]. Because of this variability, this study was designed as an initial assessment of the inhibition of these fungal pathogens using an in vitro approach. Based on these results, in vivo experiments will be designed and conducted in the future.

2. Materials and Methods

2.1. Crop Plants, Pathogenic Fungi, and Culture Medium

The major agricultural regions in Egypt are in the Nile River Delta and the Nile River Valley. Cucumbers, tomatoes, and onions are grown in the Upper Nile region, near Qena, Egypt (26°11′00.6˝ N–32°44′46.2˝ E), and in this region these vegetable crops are attacked by a variety of plant pathogenic fungi. The fungi used in this study were obtained by collecting plant tissues from the three crop plants that showed disease symptoms and three fungal pathogen species were cultured and identified: Fusarium solani, Alternaria solani, and Colletotrichum circinans (

Table 1. List of the three pathogenic fungal species used in this study, the crop plant from which they were isolated, and their disease symptoms.

Fungal Species	Crop Plant	Disease
Fusarium solani	Cucumber	Root rot/wilt
Alternaria solani	Tomato	Early blight
Colletotrichum circinans	Onion	Anthracnose/onion smudge

).

Plant pathogenic fungi can be identified morphologically, using light microscopy, and/or using molecular data (DNA sequence data) [33]. We identified the three pathogenic fungi species using morphological characteristics of colonies, mycelium, and conidia using a key of imperfect fungi [34]. Microscopic observations were carried out from prepared specimens mounted in 3% KOH using a bright field and phase contrast Leica DM 300 microscope (Leica Microsystems GmbH, Wetzlar, Germany). The fungi were grown on Spezieller Nahrstoffarmer agar [35] at 25 °C with alternating light/darkness photoperiods (12/12 h) for 7 days.

All fungal isolates were maintained on potato dextrose agar (PDA), and these cultures were stored at room temperature. They were sub-cultured once a month. The fungi were allowed to grow for 7–10 days before being used in the plant extract trials.

2.2. Invasive Plants and Preparation of Plant Extracts

In June 2021, leaves from three invasive plants, P. juliflora, I. carnea, and L. leucocephala were collected from fields near Qena, Egypt. Taxonomic identification of these species was conducted at the Herbarium of the Botany and Microbiology Department, South Valley University, Qena, Egypt. Leaves from the three species were washed in distilled water to remove surface contamination and then chopped into small pieces. This leaf material was air-dried in the laboratory for five days 25 °C, and then ground to a fine powder using an electric grinder. Dried material is preferred when conducting large-scale extraction of plant leaf tissue [36]. Three water-based extracts concentrations (10%, 20%, and 30%) were prepared by grinding 10, 20, and 30 g of plant leaves in 100 mL distilled water, and the extracts were obtained following filtration, using Whatman No. 1 filter paper (to remove leaf material and plant debris).

2.3. Inhibition of Plant Extracts on Fungal Radial Growth

To evaluate the inhibition of mycelial growth by plant extracts we used a modification of the method described by Hendricks et al. [37]. The three fungal pathogens, F. solani, A. solani, and C. circinans, were grown on PDA for 5 days at 25 °C; after which, mycelia were harvested and used for the radial growth assays. Following this, 20 μL of each plant extract concentration treatment (10%, 20%, and 30% extracts from P. juliflora, I. carnea, or L. leucocephala) was placed in the center of a PDA plate (60 mm diameter). When the plant extracts absorbed (diffused) into the agar, a 5 mm diameter size plug from the PDA fungal cultures was added to the center of the plate. Each assay was replicated three times. The cultures were incubated for 5 and 8 d at 25 °C and exposed to white light. The radial mycelial growth was determined after 5 and 8 d of exposure by calculating the mean of two perpendicular mycelia-growth diameters for each replicate. The percent inhibition of radial growth (IR) was calculated according to the following formula [38]:

$$IR=\frac{dc-dt}{dc}\times 100$$

where IR is the percentage of mycelia growth inhibition, dc is the average diameter of the fungal mycelia-growth of the control (distilled water only), and dt is the average diameter of the fungal mycelia-growth treated with the different plant extracts.

Fungal growth diameter was recorded at the beginning of the experiment, before adding plant extracts, then the growth diameter was recorded at eight days of exposure. The Average Daily Growth (ADG) was calculated by dividing the difference of growth at the beginning and at eight days of exposure to the plant extracts by the number of days the experiment was conducted (eight days).

2.4. Inhibition of Plant Extracts on Fungal Spore Density

Spores of F. solani, A. solani, and C. circinans were harvested using sterile distilled water from a culture maintained in PDA slant-tube culture. To obtain the spores, the suspension was sieved using filter paper (Whatman No. 2). A 100 µL spore suspension (10⁵ spores/mL) was added to 10 mL PDA in test tubes containing the three concentrations of leaf extracts from P. juliflora, I. carnea, and L. leucocephala: 10%, 20%, and 30% (w/v). The cultures were maintained in the dark for five days at room temperature (28 ± 2 °C). The number of spores was counted using a hemocytometer under a light microscope. The inhibitory activity on spore formation was determined using the following formula [38]:

$$IS=\frac{dc-dt}{dc}\times 100$$

where IS is the inhibition of spore formation, dc is the spore density of the control (distilled water only), and dt is the spore density after treatment with plant extracts.

2.5. Inhibition of Plant Extracts on Spore Germination

Spores of the three pathogenic fungi were extracted from cultures grown in PDA slant tubes using sterile distilled water. The suspension was sieved through Whatman No. 2 filter paper to separate the spores from fungal mycelia or hyphae. A 100 μL spore suspension (10⁵ spores/mL) was inoculated into 10 mL PDB medium in test tubes with the three concentrations of P. juliflora, I. carnea, and L. leucocephala extracts (10%, 20%, and 30%, w/v). Three tubes were prepared for each concentration. These cultures were incubated in the dark, at room temperature (28 ± 2 °C), for 12 h. Using a hemocytometer, the number of germinated spores was visualized using a light microscope. The inhibition of leaf extracts on spore germination was calculated using a modification of the method described by Gemeda et al. [39]:

$$IG=\frac{GC-GT}{GC}\times 100$$

where IG is the inhibition of spore germination; GC is the germination of spores under the control (distilled water only); and GT is the germination of spores after treatment with plant extracts.

2.6. Inhibition of Plant Extracts on Fungal Biomass

A 100 mL potato dextrose broth (PDB) medium was placed in 200-mL Erlenmeyer flasks. The three concentrations of leaf extract from P. juliflora, I. carnea, and L. leucocephala, 10%, 20%, and 30% (w/v), were added to the flasks. The medium was inoculated with 1 mL of a spore suspension (10⁵ spores/mL). The final volume of the culture was 100 mL. Three flasks were prepared for each concentration. The culture was incubated in the dark at room temperature (28 ± 2 °C) for eight days. Fungal biomass was harvested through centrifugation at 5000 rpm for five minutes. The fungal biomass was placed on glass filter paper and dried in an oven at 60 °C, until a constant weight was achieved. The inhibition of fungal biomass was determined according to the following formula [38]:

$$IB=\frac{WC-WT}{WC}\times 100$$

where IB is the inhibition of fungal biomass, WC is the dry weight of biomass in the control (distilled water only), and WT is the dry weight of fungal biomass following treatment with plant extracts.

2.7. Phytochemical Analysis

Liquid chromatography-mass spectrometry (LC-MS) was used to identify the major chemical compounds in leaf extracts. The chromatographic analysis of extracts was carried out by reverse phase elution (Waters Symmetry LC18 column 250 × 4.6 mm, 5 μm) using Agilent 6500 Series Accurate-Mass Quadrupole Time-of-Flight (Q-TOF; Agilent, Santa Clara, CA, USA) LC-MS system with Agilent 1200 Series Diode Array Detector (module G1315B; detection type: 1024-element photodiode array; light source: deuterium and tungsten lamps; wavelength range 190–950 nm). The mobile phase used for the run consisted of (A) formic acid (0.1%, v/v) and (B) acetonitrile + 0.1% formic acid. The gradient for solvent B is: (i) 20%, from 0 to 20 min, (ii) 95%, from 20 to 27 min, and (iii) 35%, at 27–30 min, of the total run time. Other configuration settings included a flow rate 0.2 mL/min, injection volume 3 L, ESI parameters: both negative and positive ion mode, mass range 100–1200 m/z, spray voltage 4 kV, gas temperature 325 °C, and gas flow 10 L/min). Liquid chromatography-mass spectrometry was analyzed using Agilent technologies Mass Hunter software.

2.8. Statistical Analysis

Statistical analyses were performed using SPSS version 23 software (San Francisco, CA, USA). One-way analysis of variance (ANOVA and post hoc Bonferroni tests were used to determine significant differences between plant extracts based on their antifungal properties. In addition, Pearson’s squared correlation coefficient (r²) was calculated to measure pairwise associations among variables.

3. Results

The results of the in vitro assays (three concentrations, from three invasive plant species) show that water-based plant extracts had varying levels of growth inhibition for the three pathogenic fungal species we tested (

Table 2. Diameter of mycelial growth and percent of radial growth inhibition of Fusarium solani, Alternaria solani, and Colletotrichum circinans after 5 and 8 days of treatment, under in vitro conditions. Letters indicate statistically significant treatment effects after post-hoc Bonferroni tests.

		Fusariumsolani		Alternariasolani		Colletotrichumcircinans
Invasive Plant	Conc. (%)	Diameter Mean (mm)	% Inhibition	Diameter Mean (mm)	% Inhibition	Diameter Mean (mm)	% Inhibition
Prosopis juliflora	After 5 days
	C	0.03 ± 0.00 d	0.0 ± 0.00 d	0.01 ± 0.00 d	0.0 ± 0.00 c	0.00 ± 0.00 d	0.0 ± 0.00 d
	10	1.93 ± 0.03 c	39.2 ± 2.50 c	1.12 ± 0.02 c	57.5 ± 2.31 b	0.63 ± 0.03 c	24.0 ± 1.20 c
	20	2.70 ± 0.01 b	48.0 ± 2.20 b	1.58 ± 0.04 b	58.9 ± 1.04 b	0.70 ± 0.14 b	40.0 ± 0.98 b
	30	4.30 ± 0.1 a	59.0 ± 2.10 a	2.42 ± 0.07 a	64.0 ± 2.63 a	0.83 ± 0.13 a	66.0 ± 0.54 a
Ipomoea carnea	C	0.02 ± 0.00 d	0.0 ± 0.00 d	0.03 ± 0.01 d	0.0 ± 0.00 d	0.00 ± 0.00 d	0.0 ± 0.00 d
	10	2.56 ± 0.10 c	2.4 ± 0.23 c	3.75 ± 0.63 c	1.8 ± 0.14 c	0.87 ± 0.02 c	6.7 ± 0.52 c
	20	5.58 ± 0.11 b	11.5 ± 1.00 b	4.20 ± 0.52 b	6.0 ± 0.17 b	1.20 ± 0.01 b	16.0 ± 1.62 b
	30	7.38 ± 0.09 a	20.0 ± 0.17 a	6.25 ± 0.41 a	7.8 ± 0.26 a	1.17 ± 0.52 a	27.2 ± 1.87 a
Leucaena leucocephala	C	0.04 ± 0.01 d	0.0 ± 0.00 d	0.03 ± 0.01 d	0.0 ± 0.00 d	0.00 ± 0.00 d	0.0 ± 0.00 d
	10	4.82 ± 0.93 c	3.4 ± 0.73 c	2.88 ± 0.23 c	11.5 ± 0.43 c	0.66 ± 0.66 c	7.6 ± 0.13 c
	20	5.45 ± 0.50 b	3.6 ± 0.42 b	3.40 ± 0.12 b	16.7 ± 0.47 b	1.33 ± 0.61 b	26.7 ± 0.55 b
	30	7.50 ± 0.43 a	9.1 ± 1.42 a	6.47 ± 0.72 a	18.7 ± 1.72 a	1.68 ± 0.04 a	34.7 ± 2.42 a
Prosopis juliflora	After 8 days
	C	0.01 ± 0.00 d	0.0 ± 0.00 d	0.01 ± 0.00 d	0.0 ± 0.00 c	0.00 ± 0.00 d	0.0 ± 0.00 d
	10	2.47 ± 0.15 c	46.0 ± 1.03 c	1.40 ± 0.09 c	58.3 ± 1.01 b	0.75 ± 0.03 c	33.0 ± 1.10 c
	20	3.38 ± 0.07 b	49.9 ± 1.05 b	2.38 ± 0.03 b	59.0 ± 0.38 b	0.85 ± 0.0 b	51.0 ± 1.00 b
	30	5.02 ± 0.23 a	61.0 ± 1.47 a	2.02 ± 0.20 a	72.0 ± 1.14 a	0.97 ± 0.02 a	68.0 ± 2.10 a
Ipomoea carnea	C	0.04 ± 0.00 d	0.01 ± 0.00 d	0.03 ± 0.00 d	0.0 ± 0.00 d	0.01 ± 0.00 d	0.0 ± 0.00 d
	10	4.88 ± 0.45 c	10.5 ± 0.43 c	3.83 ± 0.76 c	3.8 ± 0.26 c	1.48 ± 0.08 c	11.0 ± 0.63 c
	20	7.30 ± 0.13 b	11.6 ± 0.86 b	5.38 ± 0.06 b	7.7 ± 0.58 b	1.78 ± 0.28 b	27.2 ± 1.38 b
	30	8.35 ± 0.73 a	48.0 ± 1.32 a	6.58 ± 0.36 a	18.1 ± 1.01 a	1.47 ± 0.03 a	39.5 ± 1.51 a
Leucaena leucocephala	C	0.05 ± 0.00 d	0.0 ± 0.00 d	0.01 ± 0.00 d	0.0 ± 0.00 d	0.00 ± 0.00 d	0.0 ± 0.00 d
	10	5.18 ± 0.45 c	3.6 ± 0.35 c	3.17 ± 0.44 c	12.8 ± 0.78 c	1.42 ± 0.05 c	19.7 ± 1.05 c
	20	7.97± 0.23 b	7.3 ± 0.17 b	6.42 ± 0.11 b	17.8 ± 0.82 b	1.45 ± 0.03 b	33.0 ± 0.73 b
	30	8.53 ± 0.24 a	14.4 ± 0.53 a	6.83 ± 0.93 a	26.0 ± 1.03 a	1.97 ± 0.12 a	40.8 ± 1.05 a

Conc. = Concentration; C represented the control treatment, distilled water only.

). Values of the diameter of mycelial growth (mm) and percent growth inhibition of the three fungal species was highest for the 30% extract concentration treatments, and less for the two lower concentrations (Table 2). Not surprisingly, percent growth inhibition for all three fungi was higher after eight days of exposure to plant extracts, compared to the percent growth inhibition after five days of exposure. Extracts of P. juliflora had the highest inhibitory effect on fungal growth. After five and eight days of exposure to the 30% P. juliflora extract concentration, F. solani experienced 59 and 61% inhibition, respectively; A. solani experienced 64 and 72% inhibition, respectively; and C. circinans experienced 66 and 68% inhibition, respectively. In general, a comparison of the extracts from all three invasive tree species and across all three extract concentrations, C. circinans exhibited the highest level of mycelial growth inhibition (Table 2).

The average daily growth of the three fungal species treated with three different concentrations of invasive plant leaf extracts indicates a reduction in growth with increasing concentration of plant extracts (

Table 3. Average daily growth (mm) of Fusarium solani, Alternaria solani, and Colletotrichum circinans, under in vitro conditions. These pathogenic fungi were treated with three concentrations of plant extracts (10%, 20%, and 30%), from the three invasive plant species.

Pathogenic Fungi	C	10% Conc.	20% Conc.	30% Conc.
	Prosopis juliflora
Fusarium solani	4.42	2.54	2.02	1.84
Alternaria solani	3.23	1.34	1.29	1.23
Colletotrichum circinans	1.17	0.67	0.46	0.51
	Ipomoea carnea
Fusarium solani	4.42	4.38	4.29	3.33
Alternaria solani	3.23	3.68	3.36	3.07
Colletotrichum circinans	1.17	0.97	0.98	0.79
	Leucaena leucocephala
Fusarium solani	4.42	2.54	2.27	2.04
Alternaria solani	3.23	1.34	1.88	1.73
Colletotrichum circinans	1.17	0.67	0.86	0.65

Conc. = Concentration; C represented the control treatment, distilled water only.

). Across all three pathogenic fungal species, extracts from P. juliflora and L. leucocephala had the greatest inhibitory effect on average daily growth, whereas extracts from I. carnea had the least effect.

Spore density and spore germination values for all three fungal species decreased with increasing extract concentration, regardless of which invasive plant species extract they were exposed to (

Table 4. Inhibitory effects of three concentrations of leaf extracts from the three invasive plants on spore density, spore germination, and biomass of the three fungal species, Fusarium solani, Alternaria solani, and Colletotrichum circinans, under in vitro conditions.

Invasive Plant	Conc. (%)	Fusarium solani			Alternaria solani			Colletotrichum circinans
		Spore Density (Spores/mL × 105)	Spore Germination (Spores/mL × 105)	Biomass (g/100 mL)	Spore Density (Spores/mL × 105)	Spore Germination (Spores/mL × 105)	Biomass (g/100 mL)	Spore Density (Spores/mL × 105)	Spore Germination (Spores/mL × 105)	Biomass (g/100 mL)
Prosopis juliflora	C	5.59 ± 0.03 a	2.80 ± 0.01 a	0.50 ± 0.01 a	3.25 ± 0.02 a	2.30 ± 0.01 a	0.33 ± 0.00 a	5.75 ± 0.05 a	3.75 ± 0.03 a	1.00 ± 0.01 a
	10	4.37 ± 0.09 b	2.60 ± 0.12 b	0.44 ± 0.04 b	3.17 ± 0.09 ab	2.13 ± 0.09 b	0.29 ± 0.03 b	5.50 ± 0.15 b	3.50 ± 0.12 b	0.95 ± 0.01 b
	20	2.50 ± 0.15 c	1.66 ± 0.11 c	0.20 ± 0.10 c	1.73 ± 0.09 c	1.42 ± 0.06 c	0.10 ± 0.01 c	3.40 ± 0.12 c	2.13 ± 0.09 c	0.74 ± 0.06 c
	30	0.11 ± 0.01 d	0.04 ± 0.03 d	0.10 ± 0.01 d	0.01 ± 0.00 d	0.01 ± 0.00 d	0.03 ± 0.01 d	1.20 ± 0.06 d	0.87 ± 0.03 d	0.60 ± 0.04 d
Ipomoea carnea	C	2.63 ± 0.03 a	2.01 ± 0.02 a	0.48 ± 0.00 a	3.45 ± 0.02 a	2.70 ± 0.02 a	1.52 ± 0.01 a	5.55 ± 0.05 a	3.15 ± 0.03 a	1.72 ± 0.02 a
	10	2.43 ± 0.09 b	1.90 ± 0.06 b	0.38 ± 0.03 b	3.20 ± 0.12 b	2.50 ± 0.06 b	1.33 ± 0.09 b	5.00 ± 0.25 b	3.07 ± 0.07 b	1.57 ± 0.18 b
	20	1.40 ± 0.12 c	1.43 ± 0.12 c	0.18 ± 0.02 c	2.00 ± 0.15 c	1.80 ± 0.06 c	0.78 ± 0.03 c	2.32 ± 0.03 c	2.30 ± 0.12 c	0.94 ± 0.02 c
	30	0.14 ± 0.08 d	0.03 ± 0.01 d	0.07 ± 0.03 d	0.43 ± 0.05 d	0.37 ± 0.12 d	0.58 ± 0.20 d	1.33 ± 0.09 d	1.07 ± 0.06 d	0.81 ± 0.09 d
Leucaena leucocephala	C	4.35 ± 0.03 a	2.97 ± 0.02 a	1.72 ± 0.02 a	5.83 ± 0.08 a	3.63 ± 0.03 a	2.01 ± 0.02 a	2.33 ± 0.01 a	1.75 ± 0.03 a	1.00 ± 0.01 a
	10	4.17 ± 0.12 b	2.77 ± 0.13 b	1.20 ± 0.35 b	5.28 ± 0.32 b	3.40 ± 0.15 b	1.77 ± 0.13 b	2.20 ± 0.06 b	1.50 ± 0.12 b	0.68 ± 0.29 b
	20	2.93 ± 0.15 c	1.88 ± 0.10 c	0.78 ± 0.15 c	4.53 ± 0.20 c	1.70 ± 0.06 c	0.97 ± 0.03 c	1.93 ± 0.09 c	1.17 ± 0.09 c	0.58 ± 0.15 c
	30	0.15 ± 0.02 d	0.50 ± 0.35 d	0.20 ± 0.06 d	1.07 ± 0.52 d	0.80 ± 0.21 d	0.37 ± 0.09 d	0.70 ± 0.20 d	0.16 ± 0.03 d	0.12 ± 0.04 d

Conc. = Concentration; C represented the control treatment, distilled water only. Letters indicate statistically significant treatment effects after post-hoc Bonferroni tests.

). The lowest values of spore density and spore germination (i.e., highest antifungal activity) were observed with the 30% P. juliflora extract treatment for F. solani and A. solani, with A. solani exhibiting the lowest values. Treatment with the 30% P. juliflora extract did not produce the same reduction in spore density and spore germination in C. circinans, as observed for the other two fungal species. Rather, extracts from L. leucocephala had the highest inhibitory effect on C. circinans spore density and spore germination. In general, spore density and spore germination in F. solani was most inhibited by extracts from I. carnea (Table 4).

Like spore density and spore germination, values of fungal biomass for all three fungal species decreased with increasing extract concentration, regardless of which invasive tree species extract they were exposed to (Table 4). Across all three fungal genera, P. juliflora extracts had the highest inhibitory effect on fungal biomass, and this is especially true for 30% P. juliflora extract concentrations. Prosopis juliflora extracts had the highest inhibitory effect on the biomass of A. solani, although these extracts were also effective in reducing the biomass of F. solani. Ipomoea carnea extracts had the highest inhibitory effect on F. solani; while extracts derived from L. leucocephala produced the greatest reduction in the biomass of C. circinans (Table 4).

LC-MS was used to identify bioactive compounds in the extracts of P. juliflora, I. carnea, and L. leucocephala leaves (

Table 5. LC-MS analysis of crude leaf extracts of Prosopis juliflora, Ipomoea carnea and Leucaena leucocephala.

Peak	RT (min)	Measured m/z	Formula	Proposed Compound	Library Score %
Prosopis juliflora
11	1.15	110.0575	C6H7NO	Hydroxymethylpyridine	28.6
25	1.16	118.0832	C5H11NO2	Betaine	100.0
34	2.89	122.0933	C8H11N	2-Phenethylamine	99.8
35	2.16	123.0521	C6H6N2O	Nicotinamide	88.4
41	2.13	136.0580	C5H5N5	Adenine	98.4
45	2.75	138.0878	C8H11NO	Apophedrin	92.4
128	3.03	180.0978	(C5H8O2)n	Poly (methyl methacrylate) PMMA	29.2
130	3.05	170.0773	C8H11NO3	Pyridoxine	84
131	3.28	170.1136	C9H15NO2	Aceclidine	50.4
379	5.35	275.1937	C18H26O2	Nandrolone	25.6
546	3.42	268.0981	C10H13N5O4	Adenosine	93.2
612	3.92	279.1732	C15H22N2O3	Tolycaine	60.5
Ipomoea carnea
20	1.88	74.0965	C4H11N	2-Butanamine	97.7
38	4.47	309.1308	C19H16S	Triphenylmethanethiol	99.4
49	4.47	273.1080	C11H16N2O6	4’-Methoxythymidine	97.3
50	4.53	303.1036	C8H18N2O10	Azane	95.4
119	4.94	60.0808	C3H9N	Trimethylamine	97.8
169	7.38	287.1615	C14H18N6O	Abacavir	99.8
203	8.75	597.4358	C34H60O8	Sorangiolide B	97.1
211	8.81	287.0563	C16H6N4O2	Dicyanoanthraquino Nediimine	99.8
238	9.53	107.0855	C8H10	O-Xylene	99.3
292	10.50	449.1102	C23H20N4O2S2	Glioclatine	95.5
Leucaena leucocephala
34	4.55	300.0896	C17H15N3	Yellow OB	97.0
118	4.97	58.0651	C3H7N	Propyleneimine	98.9
119	4.96	60.0808	C3H9N	Trimethylamine	95.9
203	8.76	597.4360	C34H60O8	Sorangiolide B	99.5
343	11.47	287.0565	C16H6N4O2	Dicyanoanthraquino Nediimine	97.3
522	15.67	341.1973	C19H24N4O2	Pentamidine	95.2
533	15.90	286.1396	C11H15N3O4	Pyricarbate	96.3
699	18.31	279.1450	C13H18N4O3	Pentoxifylline	95.8
707	18.21	335.2427	C17H34O6	Undecyl Glucoside	95.3

). Our findings indicated that extracts of P. juliflora contain a high amount of Betaine (library score = 100%), 2-Phenethylamine (99.8%), Adenine (98.4%), Adenosine (93.2%), Apophedrin (92.4%), Pyridoxine (84%), and Nicotinamide (88.4%). Ipomoea carnea extract contains large quantities of Triphenylmethanethiol, Abacavir, Dicyanoanthraquino Nediimine, and O-Xylene (~99.5%), and 2-Butanamine, 4’-Methoxythymidine, Trimethylamine, Sorangiolide B were found in large quantities (~97.5%). In addition, Azane and Glioclatine were also detected in relatively large amounts (95.5%). Leucaena leucocephala contain Sorangiolide B (99.5%), Propyleneimine (98.8%), Dicyanoanthraquino Nediimine (97.3%), Yellow OB (97%), Pyricarbate (96.3%), Trimethylamine, Pentamidine, Pentoxifylline, and Undecyl Glucoside (95%). These results indicate the presence of diverse and complex chemical compounds in the leaf tissue of all three invasive plants species.

4. Discussion

Collectively, pathogenic fungi are the largest and most important group of plant pathogens. Many fungi species are capable of damaging crop plants by attacking them at different stages of their life cycle [1,35]. Fusarium solani forms a species complex of at least 26 closely related filamentous fungi in the Ascomycota, family Nectriaceae [40]. It is a common soil fungus and attacks a diverse array of crops [41], including cucumbers in Egypt [34]. Fusarium solani causes root rot of host plants and causes soft rot of plants by penetrating plant cell walls and destroying xylem tissues [42]. In Egypt, A. solani is a fungal pathogen that causes early blight disease in tomato and potato plants [43]. The pathogen produces distinctive “bullseye” pattern leaf spots and can also cause stem lesions and fruit rot of tomatoes and tuber blight of potatoes. If uncontrolled, early blight can cause significant yield reductions [44]. Colletotrichum circinans is an ascomycete pathogen of Allium, and this fungus attacks leeks, onions, garlic, and shallots in Egypt [30,45].

Previously published research reveals that a variety of natural plant extracts play an essential role in reducing the severity of plant diseases by acting either directly or indirectly to inhibit the growth and reproduction of plant pathogenic fungi [7,8,9,10,11,12,13,14,15,16]. Another report indicates that plant extracts are effective management tools against other plant pathogens, such as bean common mosaic virus [46].

In this study, we evaluated the antifungal activity of plant extracts from three invasive plant species (P. juliflora, I. carnea, and L. leucocephala), applied at three different concentrations, against three fungal pathogens, F. solani, A. solani, and C. circinans, under in vitro conditions. As has been reported in other studies [7,8,9,10,11,12,13,14,15,16], our results revealed differences in the responses of these three fungal species to the extracts from the three invasive plant species. This was clearly indicated by differences in the diameter of mycelial growth (mm) and the percent of radial growth inhibition (Table 2). Values of both parameters were highest for the 30% extract concentration, especially after eight days of exposure: with extracts from P. juliflora having the highest inhibitory effect.

Similarly, values of average daily growth for the three fungal pathogens reveal that the greatest reduction in daily growth occurred at the highest extract concentration (Table 3). Extracts from P. juliflora and L. leucocephala had similar, and the largest, inhibitory effect on average daily growth of the fungi. Colletotrichum circinans seems to be more sensitive to these plant extracts, compared to the other two fungal pathogens; it had the lowest values of average daily growth. These extracts did not only inhibit/reduce fungal mycelial growth, but they also reduced fungal spore density, spore germination, and fungal biomass (Table 4). Lowest values were observed with the 30% P. juliflora extract treatments with F. solani and A. solani.

Results of this study are generally consistent with the those described in reviews assessing the ability of various plant extracts and essential oils to inhibit plant pathogens, especially plant pathogenic fungi: Zaker, 2016 [7], Borges et al., 2018 [8], and Choudhury et al., 2018 [9]. These reviews indicate highly variable responses concerning the inhibition of plant pathogenic fungi; different plant extracts inhibit fungi taxa differentially, i.e., fungal mycelium growth and spore viability were reduced to varying degrees. Similar results have been reported in studies assessing the inhibitory effects of different plant extracts on F. solani [12,16,31,32] and A. solani [29,43].

Plants can synthesize aromatic secondary metabolites such as alkaloids, coumarins, cyanogenic glycosides, flavonoids, flavonols, glucosinolates, lipids, phenolics, polyacetylenes, polythienyls, quinones, and terpenes, which can have potent antifungal effects [7,8,9,10,11,12,13,14,15,16]. Prosopis juliflora is a member of Leguminosae and is one of the most widespread invasive species around the world, where it is used as a significant source of fuel and fodder [47,48]. Prosopis juliflora has been used as a traditional medicinal plant to treat catarrhal, colds, diarrhea, dysentery, flu, hoarseness, inflammation, measles, sore throat, and hepatic and ocular problems [49,50]. In addition, analysis of extracts from other parts of P. juliflora have revealed distinct secondary metabolites: tannins, phenolics, flavonoids, alkaloids, terpenes, and steroids, with some of these compounds potentially possessing therapeutic antibacterial activity [51,52]. This complex set of secondary metabolites may explain why extracts of P. juliflora had the highest inhibitory levels on all three fungal pathogens included in this study, compared to the inhibition effects of the extracts of the other two invasive plants (Table 2, Table 3 and Table 4).

Likewise, DART-MS analysis showed that P. juliflora leaves are a rich source of piperidine alkaloid and contain two diverse groups of alkaloids, one with an indolizidine ring in the center of the molecule (viz., juliprosopine, juliprosine and juliprosinine) and the other without an indolizidine ring (viz., julifloridine, projuline and prosafrinine). Among them, juliprosopine and julifloridine were present in high concentrations [51]. For example, juliprosinene and juliflorinine isolated from P. juliflora exhibit an antibacterial effect on bacteria such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Shigella sonnei [52]. The phytochemical analysis of P. juliflora reported here includes a diverse array of bioactive compounds that likely played a role in the inhibition of the three pathogenic fungi tested in this study (Table 5).

Ipomoea carnea is an ornamental shrub belonging to the Convolvulaceae [53]. It is used in folk medicine for healing wounds, skin infections, and leucoderma [54], and as a topical antiseptic and antirheumatic remedy [55]. Extracts and polar fractions of I. carnea leaves exhibited significant antioxidant activity, as they are rich in natural antioxidants, and the plant may also be a valuable sources of other bioactive molecules [56,57,58]. In addition, an acetone extract of I. carnea leaves showed antimicrobial activity against two species of bacteria, Proteus vulgaris and Salmonella typhimurium [58].

Leucaena leucocephala (Leguminosae) is one of the fastest-growing leguminous trees in drought-prone and semi-arid habitats [59]. Various parts of L. leucocephala have been reported to possess medicinal properties ranging from the control of stomach diseases to having contraception and abortion properties. The seed gum has been a binder in tablet formulation [60,61]. Sulfated glycosylated polysaccharides from the seeds possess significant cancer chemopreventive and antiproliferative properties [62]. Also, mimosine, an amino acid from its seeds, was reported to contain anticancer activity and the ability to inhibit hair growth [63,64]. Other studies on the extracts of the seeds reveal they possess varying biological activities, including depressing the central nervous system, anthelmintic properties, and antidiabetic properties [65,66,67]. Recently, seed oil from L. leucocephala was used to engineer a novel bio-device used in bio-membrane modelling in lipophilicity determination of drugs and xenobiotics [68].

The LC-MS analysis of crude extracts of P. juliflora, I. carnea, and L. leucocephala revealed great differences in their secondary metabolites (Table 5). This may explain the different antifungal effects of extracts from the three invasive plant species tested in this study. For instance, the highest inhibitory effect of extracts from P. juliflora was on A. solani, followed by F. solani and C. circinans. Whereas the highest inhibitory effects of extracts from I. carnea was on F. solani, followed by C. circinans and A. solani. Finally, the highest inhibitory effect of extracts from L. leucocephala was on C. circinans, followed by A. solani and F. solani. With the inhibitory effect of the plant extracts, our tests against the three fungal species varied; overall, P. juliflora appeared to have the highest inhibitory effect across all three fungal species.

5. Conclusions

The results of this feasibility study indicate that the extracts of all three invasive plant species have antifungal activity. Future in vivo assays should be conducted to determine their efficacy in the safe and sustainable management of these and other fungal pathogens. This is especially true for P. juliflora extracts, which had the greatest inhibitory effects. In addition, to fine-tune this method for controlling plant pathogenic fungi, future research should identify the specific chemical compounds, from these and other plants, which elicit the greatest inhibitory effect against one or more pathogenic fungi. The application/utilization of these plant extracts will not only result in safe and effective antifungal treatments; it will be an eco-friendly alternative to synthetic fungicides and likely increase the growth and yield of the vegetable crops treated with these extracts. Finally, if invasive plant species are used as the sources from which antifungal extracts are prepared, harvesting this plant tissue would reduce their biomass and thus potentially reduce some of their negative ecological consequences.