1. Introduction

It is well known that fruits constitute commercially and nutritionally important indispensable food commodity. Fruits play an important role in human nutrition by supplying the necessary growth factors such as vitamins and essential minerals in human daily diet and that can help to keep a good and normal health. Fruits are widely distributed in nature. Banana (Musa paradisiaca L.) is an edible fruit, botanically a berry [1]. Banana plant is the largest herbaceous flowering plant [2], and its above ground parts arise from a corm [3]. Normally the plants are tall and firm with a false stem that is often mistaken to be a tree. Bananas usually grow in soils that are at least \(60\text{cm}\) deep, have a good drainage and are not compacted [3].

The banana develops from a dense hanging cluster that is made of tiers. Each tier usually contains \(20\) fruits. The hanging cluster of \(3-20\) tiers called a bunch can weigh \(30-50\) kilograms \((66-110\text{lb})\). Individual banana fruit weigh approximately \(125\) grams \((0.276\text{lb})\) of which \(75\%\) is water and \(25\%\) dry matter. Fruits may differ in size, color and firmness but mostly it is curved and long containing highly starch rich fresh and green, yellow or brown rind when ripe. Cultivated bananas differ in size depending upon their variety and growing conditions mostly \(5\text{m}\ \ (16\text{ft})\) tall, ranging from Dwarf Cavendish plants which are about \(3\text{m}\ \ (10\text{ft})\) tall to Gros Michell that are \(7\text{m}\ \ (23 \text{ft})\) tall [4]. Cultivated bananas are parthenocarpic; because they lack viable seeds, propagation involves removing and transplanting of corm by farmers.

As banana is a non-seasonal crop it is available round the year. The majority of all edible varieties developed from specific (inter- and intra-) hybridization of two seeded diploid Musa species (M. accuminata and M. balbisiana) and subsequent selection of diploid and triploid seedless clones. Despite rich genetic and phenotypic diversity, only a few clones developed, over time, into global commodities either as desert bananas, such as triploid Cavendish clones, or as important staple foods such as cooking bananas and plantains. Currently bananas are widely grown in tropics and are consumed in nearly all countries around the world, providing crucial nutrition for millions of people [5].

Bananas to be exported are picked when green and are ripened in special rooms on their arrival to destination countries. The rooms are designed to be airtight and are filled with ethylene to induce ripening of fruits. The vivid yellow color normally relates with the supermarket bananas and it is caused by artificial ripening process [6]. Ripening temperatures also affects the flavor and texture of bananas. During transport, bananas are refrigerated at \(13.5^0\)c to \(15^0\)C or \(56.3^0\)F to \(59.0^0\)F. At temperatures lower than this banana turn gray because of permanent stallation of ripening resulting in the breakage of cell walls.

Bananas are the fourth most important staple food in the world, after rice, wheat, and maize with more than \(100\) million tons produced annually [7]. Although the majority of bananas produced are consumed locally, banana export is a multi-dollar business [8]. Banana is a major fruit crop of Pakistan. It is grown on \(34,800\) hectares with production of \(154,800\) tons. It is mainly grown in Sindh province where the soil and climatic conditions are favorable for its successful cultivation. The total share of Sindh province alone in its cultivation is about \(85\%\) to \(92\%\) in banana acreage and about \(90\%\) of production.

Bananas are exported over long distances from the tropics to world markets. To prolong the shelf life, fruit is harvested before it matures. Storage and transport of bananas requires careful handling, rapid transport to ports, refrigerated shipping and cooling. The major goal during storage and transport is to prevent the bananas from producing its natural ripening agent, ethylene. This allows the storage and transport to be elongated for \(3 - 4\) weeks at \(13^0\) C or \(55^0\) F. One of the limiting factors that influence the fruit's economic value is the relatively short shelf life period caused by pathogens attacked. It is estimated that about \(20\%-25\%\) of the harvested fruits are decayed by pathogens during post-harvest handling even in developed countries. In developing countries, post-harvest losses are often more severe due to inadequate storage and transportation facilities [9].

Banana fruit is attacked by various pathogens either bacteria, viruses or fungi during their growing seasons, harvesting, handling, transport and post-harvest storage and marketing conditions, or after purchasing by the consumer. Fruits contain high levels of sugars and nutrient elements and their low pH values make them particularly desirable to fungal decay. Toxigenic fungi have been isolated from spoiling fruits. During refrigeration, some moulds may produce mycotoxins. Pathogenic fungi on the other hand, could cause infections or allergies. Aspergillus spp. is known to produce several toxic metabolites such as malformins, naphthopyrones and they can produce ochratoxins (OTA).

2. Objectives

The proposed objectives of the present study were as follows;

• 1. Exploring the mycoflora associated with banana fruit rot;
• 2. Morphological and molecular characterization of isolated fungal pathogens toprovide authentic identification of species using polyphasic taxonomic approach;
• 3. Detection of new and unreported fungal pathogen(s) from Pakistan;
• 4. Determination of phylogenetic relationships of different species belonging to the same or different taxonomic groups of fungi.

3. Materials and methods

3.1. Sampling of diseased Banana fruits

Infected banana fruits were selected for the present study. About \(10-15\) samples were collected randomly from local markets of Lahore during January-February, \(2017\). Initially, infected fruits were examined physically and then brought to the laboratory in pre-sterilized sealed polythene bags, and kept in the refrigerator for further study. For each sample, records of sampling sites and data were maintained.

3.2. Isolation and purification of Pathogens

Isolations of fungi from diseased fruits were carried out on Malt Extract Agar (MEA) medium. MEA was prepared by dissolving \(20\text{g}\) malt extract and \(20\text{g}\) agar in \(1000\text{mL}\) of distilled water. pH of fungal growth medium was adjusted to \(6.5\), followed by sterilization at \(121^0\)C and \(15lb/\text{inch}^2\) for \(15\) minutes. To the sterilized medium that was cooled to \(40- 45\), streptomycin at the rate of \(200\text{mg}\) per liter medium was added to avoid bacterial growth. Approximately, \(25\text{mL}\) medium was poured into each pre-sterilized petri plate. The medium in each petri plate was gently swirled and allowed to solidify at room temperature. Isolations from different samples were performed by either direct transfer of visible spores or transferring a small fragment of infected fruit part to the growth medium. Inoculated petri plates were incubated at \(25+2^0\)C for \(3-4\) days. Emerging colonies were purified by sub-culturing the spores from actively growing colonies onto the fresh MEA plates. Pure cultures of all the isolates were preserved for identification at \(4^0\)C.

3.3. Identification and characterization of pathogens

Isolates were initially identified morphologically. Such phenotypic identification was confirmed by nucleotide sequence analysis of Internal Transcribed Spacer (ITS) sequence of rDNA.

3.3.1. Morphological characterization

For morphology based identification macroscopic and microscopic observations were made using \(7\) days old pure fungus cultures grown at \(25+2^0\)C on MEA growth medium. Further to this, cultural and microphotographs were taken for the use of future reference and comparison in fungus identification. Macroscopic characters include colony color, size, shape, texture, margin zonation studied via naked eye and microscopic characters include conidiophores length, wall, vesicle size, and shape (if present), ornamentation of conidia, conidial size, shape and color etc. For microscopic examination of each fungal species, small pieces of fungal mycelium were placed in a drop of magnification of a calibrated compound microscope. Complete description of each isolate based on macro and micro morphological characters was prepared. Species were key out by comparing its description with published authentic literature [10,11,12,13].

3.3.2. Molecular characterization

In this study, amplification of Internal Transcribed Spacer (ITS) region of ribosomal RNA was carried out using universal primer pair. Amplified gene products were sent for nucleotide sequencing and resulting DNA sequence were analyzed using bio informational tools.

3.3.3. Genomic DNA extraction

Approximately \(250-300\text{mg}\) of freshly grown fungal cells was grounded into fine powder in a sterile pestle and mortar with the help of liquid nitrogen. The powdered cells were suspended in \(2\text{mL}\) Nucleon reagent \(\text{B}\)( \(400 \text{mM}\) tris \(\text{pH} 8\), \(120 \text{mM}\) EDTA \(\text{pH} 8, 150 \text{mM}\) NaCl and 1% (w/v) SDS containing \(0.5 \mu \text{L}\) of \(10 \text{mgmL}^{-1}\) RNase A in a sterile tube to nullify the RNA contamination in extracted DNA incubated at \(37^0\)C for \(30\) minutes. From the stock of \(5\text{M}\) Sodium perchlorate solution, \(0.5 \text{mL}\) was added to cell mixture and mixed thoroughly by inverting the tube several times. An aliquot of \(2 \text{ml}\) of ice chilled chloroform (at \(-20^0\)C) was added and mixed vigorously. The tube was then centrifuged at \(4000 \text{rpm}\) for \(5\) minutes. The supernatant was carefully transferred to a new sterile tube and \(2 \text{mL}\) of ice cold \(96\%\) ethanol was added. This tube was again centrifuged at \(4000 \text{rpm}\) for \(5\) minutes. Precipitated DNA pellets were collected and washed with \(70\%\) ethanol. Air dried DNA pellet was suspended in \(50 \mu \text{L}\) TE buffer (\(10\text{mM}\) tris, \(0.1\text{mM}\) EDTA, and \(\text{pH} 8\)) and incubated at \(65^0\)C for \(15\) minutes to inhibit the potential DNAse activity.

3.3.4. Agarose gel electrophoresis for DNA quality analysis

Quality of extracted fungal DNA was analyzed through agarose gel electrophoresis. Agarose gel \((1\%)\) was prepared in 1X solution of TAE buffer from its \(50X\) stock (\(2\text{M} (242\text{g})\) of tris base in \(750\text{mL}\) of deionized water, \(1\text{M} (57.1\text{mL})\) of glacial acetic acid, \(100 \text{mL}\) of \(0.5 \text{M}\) EDTA by raising the volume \(1\) liter, \(pH 8.5\). Agarose \((1\text{g})\) was dissolved in \(100 \text{mL} 1\text{X}\) TAE by heating in a microwave oven. \(3\mu l\) Ethidium bromide from the stock of \(10 \text{mg}/\text{m}l\) was added to an agarose solution and mixed well. Gel was poured into the mold by avoiding air bubbles. Suitable comb was set to make wells for loading DNA samples. The gel was allowed to set completely for \(30-45\) minutes at room temperature. After solidification, the comb was removed carefully avoiding the tearing of wells. The gel casting tray was placed horizontally in an electrophoresis tank, containing an appropriate amount of \(1\text{X}\) TAE buffer. Gel loading dye (\(\mu \text{L}\) from \(6\text{X}\)) was added to each DNA sample. Samples were loaded carefully along with the DNA size marker. DNA samples were electrophoresed at \(100\) V for \(45\) minutes or until the dye migrated one third of the gel. DNA bands were visualized using a UV transilluminator and compared with the standard DNA size marker for quality estimation

3.3.5. Amplification of ITS region of rDNA

The internal transcribed coding region of genome was amplified by using the genomic DNA template. Taq polymerase with appropriate buffer was used for amplification in \(25 \mu \text{L}\) PCR reaction mixture (\(12.25 \mu \text{L ddH}_2O; 1.5 \mu \text{L}\) of \(15 \text{mM MgC}l2; 2.5 \mu \text{L}\) dNTPs; \(2.5 \mu \text{L} 10\text{X}\) Taq buffer; \(0.5 \text{mL}\) of each primer, \(ITS1\) forward (\(5\)'-TCC GTA GGT GAA CCT GCG G-\(3\)') and \(\text{ITS}4\) reverse primer (\(3\)'-TCC TCC GCT TAT TGA TAT GC-\(5\)'), \(0.25 \mu \text{L}\) of Taq polymerase and \(5 \mu \text{L}\) of genomic DNA per reaction.

The PCR reaction was carried out according to the following program. Amplification consisted of an initial denaturation step at \(94^0\)C for \(5\) minutes followed by \(35\) cycles each consisted of denaturation at \(94^0\)C for \(30\) seconds, annealing at \(55^0\)C for \(30\) seconds and amplification at \(72^0\)C for \(30\) seconds, with final extension at \(72^0\)C for \(10\) minutes. Amplified product was analysed on \(1\%\) agarose gel (\(5 \mu L\) of PCR product and \(1 \mu \text{L}\) of \(6\text{X}\) gel loading dye) by electrophoresis in \(1\text{X}\) TAE buffer and visualized under the UV transilluminator.

3.3.6. Analysis of nucleotide sequence by Bioinformatics tools

The nucleotide sequence of PCR product were analysed by Basic Local Alignment Search Tool (BLAST) on the NCBI and EBI (National Centre for Biotechnology Information and European Bioinformatics Institute respectively website and matched the similarity of amplified product nucleotides with NCBI and EBI data.

4. Results

In the present study, four fungal pathogens were isolated from the rotten fruits of banana. These pathogens include Aspergillus niger, Aspergillus fumigatus and Aspergillus flavus. These isolates were identified initially on the basis of their morphological characters and then genetic analysis was carried out to confirm morphology based identifications.

4.1. Morphology based Identifications

For morphological identifications, a comprehensive study of colonies as well as microscopic characteristics of all the strains was made and then compared with standard and authentic literature. All morphological observations were carried out on \(7\) days old pure fungus cultures grown at \(37^0\)C on MEA. Sporulation patterns, conidiophores and conidial morphology were examined under compound microscope (Labomed CX22; Labo America, Inc. USA).

4.1.1. Aspergillus niger [Nyongesa et al., Advances in microbiology, pp. 205-229, (2000)]

Macroscopic features

Colony on MEA was growing rapidly reaching \(3-3.5\) cm in diameter. Color of the colony was grey to black from the front while black at the reverse side. Colony texture was powdery due to heavy sporulation and distinct growth zones were present (Figure 1(B) &(C)). Visual examination of mycelia suggested that hyphae were initially yellow which turned to black with the formation of conidia or upon maturity of the colony. Under stereoscope conidial heads were strictly radiate (Figure 1(D)).

Microscopic features

Conidiophores were hyaline, thick and smooth walled, long and globose at tip, usually \(100-135 \times 4-8 \mu m\) in size. Vesicles are sub-globose with an average diameter of \(10-25 \mu m\) (Figure 1 E & F). Conidial heads were biseriate with the phialides about \(8-10 \mu m\). Conidia were sub-globose to globose, and were greyish black as matured, produced in chains. Mature conidia ranged in size from \(3-5 \mu m\) (Figure 1(G)). The spore wall was smooth. Based on morphological characteristics, the pathogen was identified as Aspergillus niger.

4.1.2. Aspergillus fumigatus [Van Tieghem, in Ann. Sci. Nat. Botan., Ser. 5, 8: 240 (1867)]

Macroscopic Features

Colony on MEA spread rapidly and achieved a diameter of \(5-6\text{cm}\). Color of the colony was green at the front and off white to yellow at the reverse (Figure 2(B)&(C)). Margins of the colony were regular and no growth zones were observed. Texture of the colony was powdery due to extensive sporulation. Under stereoscope conidial heads were small and mostly radial (Figure 2(D)). At some positions conidial heads appeared columnar.

Macroscopic Features

Hyphae were septate and hyaline. Conidiophores were short, long and cylindrical, coarsely roughened with conical terminal vesicle and ranged in size from \(15-25 \mu \text{m}\) (Figure 2(E) &(F)). Conidiophores usually range in size from \(200-215 \times 3-7 \mu \text{m}\). Conidial heads were uniseriate which supported a single row of phialides about \(10-12 \mu \text{m}\) in size on the upper two thirds of the vesicle. Conidia were smooth walled, globose, green in color, with an average size of \(2-4 \mu \text{m}\) (Figure 2(G)). Based on morphological features, the species was identified as Aspergillus fumigatus.

4.1.3. Aspergillus flavus [Link, in Observations, pp. 16 (1809)]

Macroscopic features

Colony was growing rapidly attaining the diameter of \(5-6 c\text{m}\) when grown on MEA. Colony was heavily sporulated, powdery in texture, light to bright green in color and olive green on the reverse side (Figure 3(B)&(C)). Vegetative mycelia were white. Growth zones were absent. Under stereoscope conidial heads were prominent and radial (Figure (D)).

Macroscopic features

Hyphae were septate and hyaline that formed dense mycelia. Conidiophores were hyaline, coarsely roughened with an average size of \(170 \times 15 \mu \text{m}\). Conidial heads were uniseriate with phialides mostly \(10-18 \mu \text{m}\) size and born directly on the vesicle. Vesicles were hyaline, subglobose to globose with an average diameter of \(25-30 \mu \text{m}\) (Figure 3(E)&(F)). Conidia were sub-globose to globose and yellowish green in color that arranged in size from \(3-6 \mu \text{m}\) (Figure 3(G)). Based on morphological features, the species was identified as Aspergillus flavus.

4.1.4. Aspergillus fumigatus [Thom and Raper, Manual of the Aspergilli, pp. 148-151, (1945).]

Macroscopic features

Colony was bright green in color and pale green on the reverse side. Texture of the colony was powdery and diameter on MEA was \(4.5-5 \text{cm}\) (Figure 4(B)&(C)). Conidial heads were columnar when studied under stereoscope (Figure 4(D)).

Microscopic features

Conidiophores were hyaline, thick and smooth walled having an average size \(180 \times 17 \mu \text{m}\). Hyphae were septate and hyaline. Both uniseriate and biseriate conidial heads were present. Vesicles were sub-globose that ranged in size from \(11-15 \mu \text{m}\) (Figure 4(E) &(F)). Conidia were globose, pale green in color, \(2-4 \mu \text{m}\) in diameter. The wall of conidia was smooth however some geniculations were observed (Figure 4(G)). Based on morphological features the species was identified as Aspergillus fumigatus.

4.2. Molecular analysis

In the present studies, genetic analysis of isolated fungal species was performed by nucleotide sequence analysis of ITS region of rDNA.

4.2.1. Genomic DNA extraction of fungal species

Total four isolated fungal strains were subjected to DNA extraction. The isolated DNA samples were run on \(1\%\) agarose gel. For all strains, a compact single band of about \(13000 \text{bp}\) was observed on gel. The DNA bands were compared with standard DNA marker and equalized by eye for easy quantifications. Results of all the isolates are shown in Figure 5.

4.2.2. Results of internal transcribed spacer (ITS) sequencing

The consensus primers \(\text{ITS}1\) and \(\text{ITS}4\) were used for the amplification of Internal Transcribed region. Fungus-specific universal primer pairs (\(\text{ITS}1\) and \(\text{ITS}4\)) were able to successfully amplify the \(\text{ITS}1-5.8\text{S}\) rDNA-\(\text{ITS}4\) region of all the tested isolates, providing a single PCR product of about \(500 \text{bp} - 650 \text{bp}\) (Figure 6). Purified PCR products yielded sequences of \(500-595 \text{bp}\) in length. Using the National Centre for Biotechnology Information (NCBI) and European Bioinformatics Institute (EBI) bioinformatics websites DNA sequences were BLAST. Fungal species were selected after DNA sequence blast with maximum similarity percentage \((95-100\%)\).

3. Blast results

The ITS sequence alignment of two different isolates of Aspergillus fumigatus showed \(99\%\) homology to \(004\ \ (\text{KU}321562.1),\ \ \text{SK}1 \ \ (\text{KM}207771.1),\ \ \text{CD}1621 \ \ (\text{JX}092088.1),\ \ \text{DS-C}3\ \ (\text{HQ}285554.1),\ \ \text{LR}7 \ \ (\text{KM}520022.1)\) and \(98\%\) identity to \(\text{AHBR}16\ \ (\text{KF}305755.1),\ \ \text{SF}8\ \ (\text{KX}011021.1)\) (Figures 7 and 8).

The BLAST results revealed \(99\%\) identity of Aspergillus fumigatus to the strains \(004\ \ (\text{K}4321562.1),\ \ \text{SK}1\ \ (\text{KM}207771.1),\ \ \text{Cd}1621\ \ (\text{JX}092088.1)\) and \(\text{DS-C}3\ \ (\text{HQ}285554.1)\).

The BLAST results revealed \(99\%\) identity of Aspergillus fumigatus to the strains \(\text{LR}7\ \ (\text{KM}520022.1)\) and \(98\%\) to the strains \(\text{SK}1\ \ (\text{KM}207771.1),\ \ \text{AHBR}16 \ \ (\text{KF}305755.1),\ \ \text{Sf}8\ \ (\text{KX}011021.1)\).

The present study was aimed to isolate and identify economically important fungal strains using polyphasic approaches comprising of morphology and genetics. Aspergillus species are known to cause infection in variety of fruits in addition to banana i.e., citrus fruits, tomato, peach, mango etc. Aspergillus flavus and Aspergillus fumigatus causing tomato spoilage was also investigated by [15]. Similarly, peach and oranges had been studied for fungal decay in storage and a number of Aspergillus species including Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus candidus had been isolated [16]. Aspergillus niger was also isolated from spoiled ripe mango by Palwjwala et al., [17].

The consensus primers \(\text{ITS}1\) forward (\(5’-\text{TCC GTA GGT GGA CCT GCG G-}3’\)) and \(\text{ITS}4 \ \ (5’\text{- TCC TCC GCT TAT TGA TAT GC - }3’)\) were used for the amplification of Internal transcribed region. The small variations in the band size probably makes ITS an unreliable parameter for separating fungal species [18]. The uniformity of ITS size in different fungal groups makes nucleotide sequencing of ITS fragment necessary to reveal interspecific and in some cases, also intraspecific variations.

Resulting nucleotide sequences were analyzed using National Centre for Biotechnology Information (NCBI) and European Bioinformatics Institute (EBI) bioinformatics websites. The fungal species finalized after DNA sequence blast with maximum similarity percentage \((97-100\%)\). Two species of Aspergillus fumigatus has been \(98\%\) and \(99\%\) similarity with DNA sequence when blasted and pre-identified on the basis of morphology.

Fungal isolations are very helpful in the fields of fungal biotechnology that plays a major role for many industries including food and feed, pharma, paper and pulp etc. For example, the worldwide production of citric acid produced using filamentous fungi Aspergillus niger by far exceeds the production of any other organic acid made by microbial fermentation [19].

The identification of these isolates was not an easy task due to morphological similarities amongst the different isolates of a genus. Molecular tools like ITS provided further authenticity to the identification. Therefore, polyphasic taxonomic approaches adopted in current investigation facilitated to achieve a reliable identification of four isolates with different sections of fungi that will indeed aid great help in different areas of research and teaching.

5. Conclusion

Four strains of fungi were isolated from rotten banana fruits and were preliminary identified on morphological basis. Identified species were two of Aspergillus fumigatus, one each of Aspergillus flavus and Aspergillus niger.

BLAST results using ITS sequence of all identified strains showed \(97-100\%\)identity with many of their respective strains deposited to GenBAnk. Phylogenetic analysis of ITS based results revealed lack of clear distinction amongst phylogenetically similar isolates.

Author Contributions

All authors contributed equally to the writing of this paper. All authors read and approved the final manuscript.

Conflicts of Interest

The Authors declare that there is no conflict of interest.

1. Introduction

The impact of helminth infections associated with the lack of efficacy of the major commercial compounds and reports of anthelmintic resistant strains worldwide [1,2], has raised the need to find alternative methods for the control of parasites of equids. Moreover, the low probability of obtaining new synthetic anthelmintic drugs [3], and the increasing demand for organically produced food [4], represent additional factors that further increase the need for sustainable methods for parasite control.

More recently, new approaches have been widely studied for the control of equid nematode infections. These include pasture management [5,6], the selection of adapted/rustic and resistant breeds [7], biological control methods [8,9], and the offering of nutraceuticals and other natural therapies [10,11,12]. The latter are considered particularly promising for the control of gastrointestinal nematodes (GIN) in livestock [10,13]. In fact, natural compounds can be the source of new anthelmintic molecules or can be directly used for the formulation of food supplements [14].

People in most regions, have always used plants and other natural compounds as treatment for various ailments and diseases, including those caused by parasites [15,16]. Among the countless natural compounds known for their benefits, marine compounds have also shown a wide range of activities such as anthelmintic, antibacterial, anticoagulant, antifungal, antiprotozoal, and antiviral [17,18]. Due to their nutritional value and the content of bioactive molecules, especially marine macroalgae (seaweeds) are often used as food, herbal medicines, and dietary supplements [19]. Seaweeds, comprising thousands of species, are vegetal organisms empirically distinguished in red, brown and green algae belonging to three phyla: Rhodophyta, Ochrophyta and Chlorophyta, respectively. Such phyla, however, differs each other in addition to pigmentation, more substantially in thallus structure, biochemical characteristics including photosynthetic pigments, reproductive structures, ultrastructure of chloroplasts, storage compounds, etcetera. Red algae are known not only for their use in food and cosmetic productions [20], but also for their numerous biological activities, including anticancer [21], antidiarrheal [22], antimicrobial and anthelmintic activities [17,22,23,24]. The latter activity has been known since ancient times in Eastern countries and around the Mediterranean area including Italy, where red algae were used as an anthelmintic in popular medicine [25,26].

The anthelmintic activity of red algae was previously reported in Italy, such as Ellisolandia elongata (syn. Corallina elongata), taken as decoction in the Tuscany Archipelago [27], and Hypnea musciformis eaten fresh for the same purpose used in the coastal of Abruzzo [25,26]. In Sicily, many species of red algae are also well known for their medicinal property [28]. During an ethnobotanical survey carried out in the Sicilian Egadi Islands, several reports of the anthelmintic use of an aqueous extract (about at \(10%\)) made with a mixture of red algae gathered on the rocks in intertidal zone, which was called "semenza de vermi", were collected. This aqueous extract was widely used in the past mainly to treat worm infections in children, and it was even marketed out of the islands until the post-war period (Laura Cornara, unpublished data).

Considering the ethnobotanical relevance of this remedy used for the treatment of human worm infections in the Italian folk medicine, this study was aimed to assess in vitro the effective anthelminthic properties of this red algal aqueous extract on donkey nematodes. Another objective was to identify the components of the red algal species that composed the mixture of this remedy. In fact, the confirmation of the anthelminthic properties of this extract could be extremely useful for its possible use as a new tool for the control of nematode infections in donkeys, and possibly be used in other mammals.

2. Materials and methods

2.1. Red algae collection, identification, and extract preparation

We collected a mixture of red algae on sea rocks in the intertidal zone, following the method used by elderly people from Egadi Islands to prepare anthelmintic remedies. The material was collected at Favignana island, Egadi islands, Sicily, Italy, in January 2017. The algal species in the mixture were identified according to Furnari et al., [29], and Serio et al., [30] description keys\(’\) (see Figure 1).

From this mixture, a \(10%\) aqueous extract (red algae extract, RAE) was obtained. For this, \(50\) g of dried algae were macerated in \(500\) ml of distilled water for \(3\) h, followed by sonication for \(30\) min. The crude extract was filtered (\(0.45 \mu m\) filter), frozen and lyophilised (yield of \(75.6%\) on a dry matter basis). The dried extract (\(10\) g) was solubilized in distilled water (\(100\) ml). The obtained solution was frozen until it was used for the in vitro activity tests.

2.2. Recovery, suspension, and purification of gastrointestinal strongyles (GIN) eggs

Individual faecal samples were taken from the rectal ampoule of naturally infected donkeys and analysed using a McMaster method with a sensitivity of 50 eggs per gram of faeces (EPG) [31]. Only samples with more than 1000 EPG were pooled and used in the study. A previously reported technique was used for the concentration and purification of the GIN eggs [13]. Purified eggs were diluted in saline solution, counted, and used for the in vitro evaluation for the RAE anthelmintic test, using a minimum of 100 eggs in each repetition.

2.3. Faecal cultures and identification of GIN genera

The identification of GIN genera was carried out through the setting up of faecal cultures from the same pooled samples used to obtain the eggs. More specifically, about \(20\) grams of pooled faeces were placed on a gauze stretched inside a plastic cup containing about \(20\) ml of water and covered with the bottom of a perforated plastic cup to allow proper oxygenation. Faecal cultures were incubated at \(26-28^0\)C for \(7\) days [32]. During the incubation period, faecal cultures were sprayed with water daily, to ensure adequate humidity conditions. After this period, \(L3\) were obtained by using the Baermann technique and were morphologically identified at the genus/species level according to reference keys [32,33].

2.4. Egg hatch test (EHT)

The EHT was used to evaluate the in vitro anthelmintic efficacy of the RAE. This test consists in evaluating the ability of a substance to inhibit the hatch of the egg, impeding the development of the L1 [34]. As previously described [13,35], \(0.5\) ml of saline containing about \(100\) purified eggs were placed inside each well (\(24\)-flat bottom cell plates) (Sarstedt, Verona, Italy), together with \(0.5\) ml of the \(10%\) extract at different dilutions \((100, 50, 5.0, 1.0%)\) in distilled water. The anthelmintic efficacy of each concentration of the REA was compared to untreated controls, containing about \(100\) purified eggs in \(0.5\) ml of saline \(+ 0.5\) ml of distilled water, or treated with \(0.5\) ml of the reference anthelmintic drug thiabendazole (TBZ) diluted at \(1%\) in distilled water (Sigma Aldrich, Milan, Italy). All tests were performed in triplicate and in three independent assays. Plates were incubated for \(48\) h at \(24^0\)C, in the dark with \(90%\) relative humidity. After this time, the plates were observed under a Leica \(M165\) C (Leica Microsystems, Milan, Italy) stereoscopic microscope and the number of larvae and eggs present in each plate was counted. The percentage of egg hatch reduction at each concentration was calculated using the following formula ([n. larvae/n. larvae \(+\) n. eggs]\(\times 100\)) [11].

2.5. Statistical analysis

Statistical analysis was performed by using one-way ANOVA with a significance of \(5% (P < 0.05)\). Significant data were further tested with the Tukey post-hoc test. Statistical analysis was carried out by using the Statistical Analysis System (SAS) program.

3. Results

In the red algae mixture, Palisada tenerrima, Laurencia intricata and Laurencia spp. were identified (see Figure 1). Moreover, in the collected materials, P. tenerrima, L. intricata and Laurencia spp. were found in the proportion \(2:1.5:0.5\), respectively.

The pure RAE was able to cause \(100%\) inhibition of egg hatch, showing an efficacy comparable to TBZ \(1% (98.7%)\) (see Figure 2). Furthermore, the anthelmintic efficacy of the extract was found to have a concentration-dependent effect, with an equation of \(y = 26.35x – 27.99\) and the correlation coefficient \((R2)\) of \(0.968\). In fact, at all other dilutions (\(50, 5\) and \(1%\)), the RAE tested in the study showed a decreasing efficacy of \(89.5, 43.7\), and \(23.4%\), respectively. Moreover, the two lowest dilutions of the extract showed a significant \((P < 0.05)\), lower activity than the pure extract and the reference drug.

The identification of GIN genera from the pooled faecal cultures revealed a high prevalence \((>90%)\) of cyathostomins. More specifically, we found species belonging to the genera Cylicocyclus and Cylicostephanus. The remaining \(10%\) of the identified larvae were identified as Triodontophorus.

4. Discussion

We have demonstrated the effect of RAE against GIN eggs of donkeys. Red algae are a group of eukaryotic organisms also known as rhodophytes, most of which live in warm seas [36]. About \(4000\) different species of red algae have been identified [37]. The alimentary use of red and other algae is particularly widespread in Eastern countries, where they are considered a gastronomic delicacy and a food source with excellent nutritional qualities (i.e. proteins, vitamins) [38,39]. Algae are also used as stabilizers, thickeners, emulsifiers, and gelling agents, and as adjuvants in the treatment of obesity, cellulite, and related diseases [18,20]. However, nowadays green, brown, and red marine algae are also considered an important source of bioactive compounds that can be used to inhibit pathogens, such as bacteria, viruses, and parasites [17,40,41,42,43], including helminth infections [17,41].

Digenea spp. red algae are in fact known for more than a thousand years for their anthelmintic properties against intestinal ascarid worms [44,45]. Kainic acid is considered the active compound responsible for the deworm activity of this group of algae and it is probably due to its ability to induce a neuromuscular paralysis, which interrupts the peristaltic movements of the intestine of the parasites [23]. Kainic acid belongs to the family of kainoids that include also domoic acid, another neurotoxic compound produced mainly by marine planktonic diatom algae [46]. The similar activity of domoic and kainic acids is due to their related structure with glutamic acid, which alters the function of some ion channels [45,47]. At the dose of \(5\) to \(10\) mg, the domoic and kainic acids can permit the expulsion of roundworms without causing any side-effects in the infected hosts [23,48]. Domoic acid may be concentrated in marine seafood, such as oysters, mussels and crustaceans. Sea-food poisoning can be related to domoic acid to fish and humans [49,50]. The ingestion of marine organisms containing high levels of domoic acid, may even cause the death of fish and other marine mammals.

A further major class of metabolites produced by marine algae are represented by terpenes [43]. The Rhodophyceae are characterized by a high content in polyhalogenated monoterpenes showing potent and wide antimicrobial activities [51,52]. In this study, the \(10%\) aqueous extract from P. tenerrima, L. intricata and Laurencia spp. red algae showed a high anthelmintic efficacy against GIN of donkeys, comparable to that of the reference drug (TBZ \(1%\)). In addition, the in vitro anthelmintic efficacy showed a concentration-dependent effect against GIN eggs. Even the efficacy of RAE at \(50%\) was high, with an egg hatch reduction of about \(90%\).

This is the first study in which the anthelmintic activity of a RAE was tested using the EHT against nematode eggs of GIN of donkeys. It is interesting to note that the anthelmintic properties of algal species of the genus Laurencia were already reported [41,53]. Indeed, among \(11\) sequiterpenes and a long chain aldehyde from Laurencia dendroidea (syn. Laurencia scoparia), a halogenated \(\beta\)-bisabolene sesquiterpenoid showed anthelmintic properties against Nippostrongylus brasiliensis [41], a gastrointestinal nematode of rats with a life cycle and morphology similar to the human hookworms [54]. Moreover, very similar results to those obtained in the present study were reported when evaluating the anthelmintic activity against Meloidogyne javanica, an important plant root-knot nematode species. The methanolic and aqueous extracts of several seaweed species were tested using the EHT and the larval mortality test [55]. More specifically, in the previous study, a \(10%\) aqueous extract of the algal species Sargassum tenerrimum, Padina tetrastromatica and Melanothamnus afaqhusainii were able to cause a reduction of about \(96%\) of the egg hatch of M. javanica [55]. However, in the study of Kha et al., [55], the \(10%\) aqueous extract from the algal species, including Laurencia pinnatifida (now Osmundea pinnatifida), showed a lower anthelmintic activity in the EHT when compared to the \(10%\) aqueous extract examined in the present study.

Nevertheless, the phytochemical profile of the RAE tested in this study was not performed, and further studies aimed to identify the different components present in the extract are needed. In fact, the knowledge of the main bioactive components of this aqueous red algal extract could allow to estimate its potential toxic effects to mammals and, therefore, its effective potential as an anthelmintic of seaweed origin. Nevertheless, it is plausible to hypothesize that the anthelmintic activity of the RAE can probably be ascribed to the presence of kainic acid, as reported in other species of red algae [44,45]. Other compounds may also be important (i.e. sesquiterpenoids, \(1\)-aminocyclopropane-\(1\)-carboxylic, and acrylic acids) to red algae, which in previous studies were found to possess anthelmintic properties [53,55].

Further studies are needed to evaluate the anthelmintic activity of each of the three algal species contained in the mix of algae tested in this study. In fact, these latter studies may permit to assess whether there is a synergism among these species, or if one species has higher activity than the others. Finally, although in previous studies nematodes have been found to be highly tolerant to salinity [56,57], the potential anthelmintic effects of the content in sodium chloride of the tested algal extract cannot be excluded.

In conclusion, results from this study seem to confirm the anthelmintic properties previously reported in the Italian folk medicine for the red algae extract. Further studies based on the bio-guided purification of the red algae extract are encouraged.

Acknowledgments

Authors thank Egadi islands (Sicily, Italy) informants and in particular Mr. Santo Grammatico, for their help in collecting red algae. Authors thank Dr. Elena Hobart for her kind help in revising the English language.

Author Contributions

Conceptualization (LC, SP, MM); red algae collection (LC); red algae identification (GF and DS); extract preparation (FSRC); experimental (MM, MBM, SP); data analysis (SM, MM); writing the original draft (MM, SP). All authors revised and edited the final version of the manuscript.

Conflicts of Interest

The Authors declare that there is no conflict of interest.