Antifungal activities of extracts from different parts of mulberry plant against Alternaria alternata and Fusarium sp.

Abstract

In the present study, we investigated the antifungal activity of methanol and ethanol extracts of different parts (leaves, twigs, and root bark) of mulberry plant against Alternaria alternata and Fusarium sp. Among them, the methanol and ethanol extracts of mulberry root bark exerted the highest inhibitory activity against the mycelial growth of A. alternata ($70.6{\pm}1.6$ to $80.8{\pm}6.7%$ and $58.7{\pm}0.0$ to $80.8{\pm}6.7%$, respectively) and Fusarium sp. ($15.5{\pm}2.7$ to $39.3{\pm}3.4%$ and $26.4{\pm}2.7$ to $47.6{\pm}4.8%$, respectively). In contrast, the methanol and ethanol extracts from mulberry leaves and twigs did not suppress the mycelial growth of these fungal species. Importantly, the methanol and ethanol extracts of mulberry leaves tended to even accelerate the mycelial growth of A. alternata and Fusarium sp. Therefore, the results of this study indicate that methanol and ethanol extracts of mulberry root bark can be used as control agents against A. alternata and Fusarium sp.

Introduction

Mulberry belongs to the Moraceae family and is widely distributed around the world (Wasano et al., 2009; He et al., 2013). In Korea and other Asian countries, different parts (fruits, leaves, twigs, and root bark) of Morus alba are commonly used as traditional medicines (Ju et al., 2018; Kim et al., 2017; Kwon et al., 2017; Wei et al., 2016). Extracts from various parts of mulberry have been reported to exert antibacterial and antifungal effects (Park et al., 2003; Sohn et al., 2004; Rao et al., 2012). Park et al. (2003) found that Kuwanon G, which was isolated from the methanol extract of mulberry root bark, had antibacterial activity against oral pathogens such as Streptococcus mutans, Streptococcus sanguis, Streptococcus sobrinus, and Porphyromonas gingivalis. Additionally, Sohn et al. (2004) discovered that mulberrofuran G and albanol B from mulberry root bark extract strongly inhibited the growth of Salmonella typhimurium, Staphylococcus epidermis, and Staphyloccoccus aureus. In addition, methanol, chloroform, and petroleum ether extracts of mulberry leaves strongly suppressed the development of pathogenic fungi such as Candida albicansand Aspergillus niger (Rao et al., 2012).

Pathogenic fungi are the main infectious agents in plants, causing injuries during various developmental stages, including harvesting, postharvest handling, storage, or distribution. In addition, more than 70% of all major crop diseases are caused by fungi, which also reduce the shelf life and storability of fresh produce (Agrios, 2005; Barth et al., 2009; Dellavalle et al., 2011; Akhtar et al., 2013). Various fungal species worsen the nutritional value organoleptic characteristics and limit the shelf life of fruit and vegetables, lowering their overall quality (Agrios, 2004) and causing significant economic losses in agriculture. Furthermore, the mycotoxins or allergens produced by some fungi can lead to toxic disorders or allergies in consumers (Dellavalle et al., 2011).

Alternaria and Fusarium are important plant pathogen species leading to significant agricultural productivity losses worldwide (Lira-De León et al., 2014). Alternaria alternata causes leaf spots and blight on a large variety of agricultural crops, such as apple (Malus domestica), beans (Pisum sativum), broccoli (Brassica oleracea; Botrytis Group), cabbage (Brassica oleraceavar. capitata), carrot (Daucus carota), cauliflower (Brassica oleracea var. botrytis), peach (Prunus persica), peppers (Capsicum annuum), potato (Solanum tuberosum), tomato (Lycopersicon esculentum), and citrus species (Rodino et al., 2014). Fusarium is also an important plant pathogen species causing diseases, such as crown rot, head blight, scab on cereal grains, root rots, and cankers (Booth, 1971). Furthermore, both Alternaria and Fusarium species can cause allergic rhinitis and asthma in immunodepressed patients (Stroud et al., 2001; Kuna et al., 2011).

In the present study, we investigated the antifungal activity from methanol and ethanol extracts of different parts (leaves, twigs, and rood bark) of mulberry plants against the mycelial growth of Alternaria alternata and Fusarium sp.

Materials and Methods

Plant materials

The different parts (leaves, twigs, and root bark) of mulberry plants were collected from the experimental fields of the Sericulture and Apiculture Division of the Department of Agricultural Biology, Rural Development Administration, Jeon-Ju, Republic of Korea.

Fungal strains

Alternaria alternata and Fusarium sp. used in this study were isolated from the spoilage fungi of mulberry fruits after harvest at the Sericulture and Apiculture Division (Department of Agricultural Biology, Rural Development Administration, Jeon-Ju, Republic of Korea) in 2018 and were identified by their ITS rDNA sequences (Kwon et al., 2018).

Extraction from different parts of the mulberry plant

Leaves, twigs, and root bark of mulberry plants were thoroughly washed and oven-dried for 72 h at 40°C. After cutting and grounding each of the aforementioned parts to small pieces, a sample (10 g) of each part was immersed into 250 mL of each of the two solvents utilized (96% methanol and 96% ethanol) for 24 h at room temperature (from 24°C to 26°C). Then, these mixtures were sonicated for one hour at 50°C using a bath sonicator BRANSON 8210 (Branson Ultrasonic Corp., Danbury, CT, USA) at a power of 200 Watts and an output frequency of 44 kHz. Next, the samples were filtered through Whatman No. 1 filter paper. These processes were repeated three times. After evaporating the solvent in vacuo, all the extracts (adjusted to 100 mg/mL) were dissolved in 20% dimethylsulfoxide (DMSO) and were preserved at 4°C until the next experiment.

Antifungal activity using disc diffusion method

To determine the antifungal activity, test discs from different mulberry plant parts were treated with 30 μL of 100 mg/mL extract, after which they were allowed to dry for 10–15 min. Mycelial discs (8 mm) of each of the examined fungal species (A. alternata and Fusarium sp.) were placed at the center of plates with potato dextrose agar (PDA) medium (Difco Laboratories, Detroit, MI, USA). These discs and two control discs (treated 30 μL of double distilled water and 20% DMSO) were arranged at regular intervals around each of the fungus species (A. alternataand Fusarium sp.) on the PDA medium and incubated at 28°C for three days. The inhibitory effects of the treatments with extracts from each of the mulberry plant parts on the mycelial growth were evaluated visually and photographed.

Determination of antifungal activity from mulberry plant extracts

The antifungal activities of the extracts from each mulberry plant part (leaves, twigs, and root bark) on the mycelial growth of A. alternata and Fusarium sp. were determined by the poisoned food technique (Schmitz, 1930). PDA medium with 5% concentration (v/v) of each solvent extract (methanol and ethanol) were prepared for each of the different mulberry plant parts. Further, the media were poured into each Petri plate allowed to solidify. PDA medium with 5% concentration (v/v) of 20% DMSO solution was used as the control.

Then, mycelial discs with a diameter of 8 mm, taken from the margins of an actively growing culture of each fungus species (A.alternata and Fusarium sp.) were placed at the center of the petri plates and incubated at 26°C ± 2°C. The measurement of the mycelial growth of the fungus was carried out for 10 days until the mycelium in the control treatment reached the margins of the Petri plate (90 mm). The radius of the growing mycelia in each treatment was measured every two days and photographed. The experiment was performed in triplicate, and the data obtained were expressed as mean ± standard deviation (SD).

The percentage of the mycelial growth inhibition (MGI) of the different extract treatments compared to control was calculated by the formula: [MGI (%) = (1–Rt/Rc) × 100] (%), where Rcrepresents the average mycelial growth radius measured in the control with no treatment, and Rt denotes the average mycelial growth radius measured in the treated with plant extracts Petri plates.

Results and Discussion

To investigate the antifungal activity of methanol and ethanol extracts from different mulberry plant parts (leaves, twigs, and root bark) against A. alternata and Fusarium sp., we initially employed the disc diffusion method (Fig. 1A–E). The treatment of the discs with methanol and ethanol extracts from mulberry root bark (Fig. 1C and 1F) at 30 uL of 100 mg/mL exerted more intense antifungal activity against A. alternata and Fusarium sp. than the methanol and ethanol extracts from leaves (Fig. 1A and 1D) and twigs (Fig. 1B and 1E).

Fig. 1. Antifungal activity of the different part extracts of mulberry plant against the mycelial growth of A. alternata (A-C) and Fusarium sp. (D-F) by disc diffusion method. Control, treatment with 20% DMSO; DDW, double distilled water; MLME, mulberry leaf methanol extract; MLEE, mulberry leaf ethanol extract; MTME, mulberry twig methanol extract; MTEE, mulberry twig ethanol extract; MRME, mulberry root bark methanol extract; MREE, mulberry root bark ethanol extract.

We also investigated the inhibitory effects of the methanol and ethanol extracts from the different mulberry plant parts (leaves, twigs, and root bark) on the growth of A. alternata and Fusariumsp. in PDA medium with 5% concentration (v/v, each extract of 100 mg/mL) (Fig. 2–4). The measurements of the suppressive influence on the mycelial growth of A. alternata and Fusariumsp. are graphically represented in Figure 4. The mycelial growth inhibition was measured at intervals of two days for a period of 10 days. We found that the methanol and ethanol extracts of mulberry root bark suppressed the mycelial growth of A.alternata and Fusarium sp. by3.4- to 5.2-fold and from 2.4- to 3.6-fold, respectively, than control (treated with 20% DMSO, v/v). In contrast, the methanol and ethanol extracts from leaves and twigs of mulberry plant did not inhibit the mycelial growth of A.alternata and Fusarium sp.

Fig. 2. Antifungal activity of the different part extracts of mulberry plant against A. alternata.

Fig. 3. Antifungal activity of the different part extracts of mulberry plant against Fusarium sp.

Fig. 4. Effects of the treatment with methanol and ethanol extracts from different parts of mulberry plant on the mycelial growth of A. alternata (A) and Fusarium sp. (B). Control, treatment with 20% DMSO (v/v); MLME, mulberry leaf methanol extract; MLEE, mulberry leaf ethanol extract; MTME, mulberry twig methanol extract; MTEE, mulberry twig ethanol extract; MRME, mulberry root bark methanol extract; MREE, mulberry root bark ethanol extract. Data are expressed as means ± SD (n = 3).

Rodino et al. (2014) examined the inhibitory activity on the mycelial growth of Alternaria alternata of treatments with ethanolic and aqueous extracts of four plants: aerial parts of Artemisia absinthium aerial part and Rosmarinus officinalis; leaves and fruits of Datura stramonium; and fruitsof Xanthium strumariu. They confirmed that the antifungal activity of the ethanolic extracts used in three concentrations (10%, 5%, and 2.5%) of the four tested plants had been higher (from 50.00% to 86.67%) than that of their aqueous extracts (from 16.67% to 60.00 %). In another study, Lira-De León et al. (2014) established that the root extracts of Adenophyllum aurantium (100 mg/mL, methanol and ethyl acetate extract) and Tournefortia densiflora(70 mg/mL, methanol extract) exerted maximal inhibitory effects on the mycelial growth of Fusarium solani (56.17% and 52.42%, respectively) and Alternaria alternata (68.64% and 69.07%, respectively). In addition, their found that eight extracts, including those from aerial parts of Acalypha cuspidata(methanol), Echeveria acutifolia (methanol), Prostechea varicosa bulb (methanol), and Salpianthus arenarius (methanol), as well as those from the seeds of Heliocarpus terebinthinaceus(aqueous and hexane) and the leaves of Ipomoea murucoides(ethyl ether). These extracts accelerated the mycelial growth of A. alternata (from –11.86 ± 3.94 to –41.44 ± 3.18%) and one of them (Prostechea varicosa bulb methanol extract) stimulated F.solani (–5.25 ± 2.58%). Similarly, methanol and ethanol extracts of mulberry leaves in the result of the present study tended to accelerate the mycelial growth of A. alternata and Fusarium sp. (Fig. 4 and 5).

Fig. 5. Inhibitory influence (%) of the treatments with methanol and ethanol extracts from different parts of mulberry plant on the mycelial growth of A. alternata (A) and Fusarium sp. (B). MLME, mulberry leaf methanol extract; MLEE, mulberry leaf ethanol extract; MTME, mulberry twig methanol extract; MTEE, mulberry twig ethanol extract; MRME, mulberry root bark methanol extract; MREE, mulberry root bark ethanol extract. Data are expressed as means ± SD (n = 3).

The highest suppressive effect on the mycelial growth of A. alternata and Fusarium sp. was established in the methanol and ethanol extracts from mulberry root bark (Fig. 5). The inhibition activity of A. alternata by the methanol and ethanol extracts of mulberry root bark was found to be 70.6 ± 1.6 to 80.8 ± 6.7% and 58.7 ± 0.0 to 80.8 ± 6.7%, respectively. In addition, the inhibition activity against Fusarium sp. exerted by the methanol and ethanol extracts of mulberry root bark was 15.5 ± 2.7 to 39.3 ± 3.4% and 26.4 ± 2.7 to 47.6 ± 4.8%, respectively. The mulberry root bark methanol extracts were more effective in the inhibition of the mycelial growth of A. alternata, whereas the mulberry root bark ethanol extracts suppressed more effectively the mycelial growth in Fusarium sp.

The root bark of Morus plants are commonly called "Sang-Beak-Pi" in Korea, "Sang-Bai-Pi" in China (Wei et al., 2016), and "Sohakuhi" in Japan (Nomura et al., 2009). The antimicrobial activity of the mulberry root bark firstly tested by Nomura et al. (1978). Many compounds in mulberry root bark have been isolated, several of which have been reported to have antimicrobial activity. Such compounds isolated from mulberry bark are kuwanon [C], mulberrofuran [G], albanol [B], morusi, and sanggenons [B, D], which have been found to effectively suppress the growth of various pathogenic bacteria and fungi, including Candida albicans, Saccharomyces cerevisiae, Salmonella typhimurium, Staphylococcus epidermis, and S. aureus (Sohn et al., 2004). In addition, mulberrofuran [A] was reported to exert antimicrobial activity against Staphylococcus aureus and Fusarium roseum (Nomura et al., 1978). Kuwanon [L] and sanggenons [B, C, D, E, G, and O] were confirmed to have inhibitory effects against Venturia inaequalis development (Rollinger et al., 2006).

The findings of the present study confirmed that the methanol and ethanol extracts of mulberry plant root bark had the most potent antifungal activity against A. alternata and Fusarium sp. among all plant part extracts examined. the methanol and ethanol extracts of mulberry root bark showed optimistic results in terms of their potential application as effective control agents against A. alternata and Fusarium sp. However, little is known about the specific, most active inhibitory compounds in the mulberry plant and their precise mechanisms of action against A. alternataand Fusarium sp. growth. Therefore, further research is highly required to evaluate comprehensively their antifungal activities.