Abstract

Fumonisins produced by Fusarium constitute a concern group of mycotoxins contaminating food products. In Côte d'Ivoire, data on the sanitary quality of marketed Moringa powder remains unknown. This study was carried out to characterize molds of the genus Fusarium isolated from Moringa powder by detecting the presence of a gene encoding the biosynthesis of fumonisins. A total of 192 Moringa leaf powder samples of approximately 250g each were taken from various sales sites in Abidjan markets including Abobo, Adjamé, Koumassi and Yopougon. The isolation and purification of the Fusarium strains were carried out on Sabouraud’s medium supplemented with 10 μg / mL of chloramphenicol. The Polymerase Chain Reaction (PCR) method was used for molecular identification of Fusarium strains and the detection of the presence of the Fum13 gene involved in the fumonisins biosynthesis. The contamination rate of Moringa powder by Fusarium-type molds was 15.43%. The predominantly isolated species was Fusarium solani (11.43%) followed by Fusarium sp (4%). The presence of the Fum13 gene was detected in 62.96% (17/27) of the Fusarium strains isolated. The presence of molds of the genus Fusarium and the detection of a gene coding for the fumonisins biosynthesis, requires the implementation of health safety measures during the manufacturing process of Moringa powder.

1. Introduction

Fumonisins are toxic secondary metabolites produced by Fusarium verticcillioides, Fusarium proliferatum, and Fusarium species ¹. The International Agency for Research on Cancer classified fumonisins as Group 2B possibly carcinogenic to humans ²

Fumonisins are one of the most recently discovered cytotoxic and carcinogenic mycotoxins that can cause toxic effects in humans ³. Birth defects, alimentary canal irritation, vomiting, acute toxicosis, and a number of other diseases in humans and animals ².

Food contamination by Fusarium mycotoxins in human and animal health mentions with fumonisin, has been reported in several countries ⁴. Indeed, studies carried out in Africa, Asia, Europe and South America have shown that the incidence of fumonisins in maize samples can reach 100% in some cases with mean levels above the maximum limit recommended by the European Union for corn intended for direct human consumption (1000 µg/kg) ⁵.

In several countries of the world and particularly in Africa Moringa Oleifera has gone from an unknown plant to a new food and economic resource for the countries of the South in a decade ⁶. Known as the tree of many virtues, the leaves are recognized around the world as an excellent dietary supplement ⁷. Moringa leaves are an important food and nutrition in tropical countries ⁸. This plant is of fairly common use in traditional medicine and food in African and Asian societies. It constitutes a food supplement for the undernourished subjects because of its richness in proteins, vitamins and minerals.

This plant also plays an important role in the care of subjects living with HIV / AIDS as a fortifier and stimulant of their immune system ⁹. However, the production method does not always guarantee the safety of the commercialized Moringa powder ¹⁰. Indeed, the work of ¹⁰ on the evaluation of the sanitary quality of Moringa powder marketed for the benefit of people living with HIV in Cotonou (Benin) revealed fungal contaminants, including molds of the genus Fusarium, in Moringa leaf powder. In Côte d'Ivoire, data on the sanitary quality of commercialized Moringa powder and on the detection of genes involved in the biosynthesis of fumonisins produced by Fusarium remain unknown. The objective of this work was to characterize the Fusarium moulds isolated from Moringa powder by detecting the presence of the gene coding for the biosynthesis of fumonisins.

2. Materials and Methods

Moringa leaf powder is obtained after a artisanal processing which consists of drying the Moringa leaves on superimposed grids. The dried leaves are poured into a mill to obtain a really fine powder without grain. The powders are then bagged and put-on the market ¹¹. Sampling was carried out in the period from June to July 2019. Moringa powder samples were obtained aseptically from retail stalls in different large markets in the municipality of Abidjan, namely: Abobo, Adjamé, Koumassi and Yopougon. A total of 192 samples were collected and transported to the laboratory for mycological analysis.

The method described by ¹² was used for the isolation of fungal flora. Moringa powder samples were analyzed after a series of decimal dilutions (The dilutions 10^-1, 10^-2 and 10^-3) were retainedfor each sample. The inoculations were carried out on Sabouraud's medium supplemented with 10 μg/mL of chloramphenicol, and incubated at 30°C for 72 hours. A few isolated characteristic Fusarium colonies were subcultured. The subculturing was carried out by placing a portion of colony in the center of a new Petri dish containing Sabouraud medium with chloramphenicol. The pure strains obtained were identified according to morphological and cultural criteria as described by ¹³.

Two sets of primers were used in this study (Table 1). One primer set based on the conserved region of the ITS DNA specific to the genus Fusarium ¹⁴ and another primer set specific to the fumonisin biosynthesis gene were used ¹⁵. For this study the Fum 13 gene was chosen because inactivation of Fum 13 results in more than 90% inhibition of Fumonisin B1, FB2, FB3, and FB4 production and accumulation of their homologs. Deletion of this gene results in a total reduction of fumonisin production in mutants without accumulation of intermediate molecules. Hence the importance of searching for this gene. Genomic DNA isolated from Fusarium sp fungal isolates was used for PCR analysis. The first PCR was performed using ITS primers specific to the genus Fusarium and the second PCR was performed with the Fum13 gene for fumonisin production biosynthesis.

The DNA of each fungal isolate was extracted according to the method described by ¹⁶ with some modifications. DNA was extracted with 0.5 g of fungal mycelia harvested from freshly grown pure cultures on Sabouraud chloramphenicol medium. The mycelium was ground properly in a mortar and then transferred to phosphate buffered saline (PBS) for a series of three cycle of freezing/thawing in liquid nitrogen. Subsequently 500 µL of the fungal suspension was added to 500 µL of lysis buffer (1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCL) and incubated at 55°C for 3 hours in a dry bath. To the lysate, 500 µL of phenol-chloroform-alcohol-isoamyl (25-24-1) was added. The tubes were centrifuged at 13000 rpm for 10 minutes at 4 °C. The upper aqueous phase was transferred to in a new sterile 2 ml Eppendorf tube. To the upper aqueous phase, 1/10th of 3M sodium acetate and 500 µl of ice-cold absolute ethanol were added and stored at -80°C for 2 hours. The tubes were centrifuged at 13000 rpm for 20 min at 4 °C. Subsequently, 1 ml of ice-cold 70% ethanol was added to the pellet. The tubes were centrifuged at 13000 rpm for 10 min at 4°C. The pellet was dried in a dry water bath at 56°C for 2 h. The resulting DNA was dissolved in 50 µL of nuclease free water and stored at -20°C.

The first PCR was performed in a final volume reaction of 25μL containing 10.75µL nuclease-free water (Ambion), 5 µL PCR buffer (5X), 1.5µL Magnesium Chloride (MgCl2) (25 mM) (Promega Corporation, Madison, USA), 0.5µL Deoxynucleotide Triphosphates (dNTPs) (10 mM), 1μL of each primer (20mM), 0.25 µL Go Tag®G2 Flexi DNA polymerase 5 U/µL (Promega Corporation, Madison, USA) and 5 μL of template DNA. The PCR method was performed according to the following program: initial denaturation (94°C, 4 minutes), 40 cycles each composed of initial denaturation (94°C, 1 minute), primer annealing (58°C, 1 minute) and extension (72°C, 1 minute) and a final extension (72°C, 10minutes). The second PCR was performed to detect fumonisin biosynthetic gène in the same conditions than the first PCR. The difference was the PCR program: initial denaturation (94°C, 5 minutes), 40cycles each composed of initial denaturation (94°C, 1 minute), primer annealing (55 °C, 1 minute) and extension (72°C, 1 minute) and a final extension (72°C, 8 minutes). All PCR amplification products were revealed on a gel Doc EZ® imager (Bio-Rad) after electrophoresis in 2% agarose gel containing Syber safe (Invitrogen).

DNA sequences of the PCR products of Fusarium isolates was determined using the primers ITSF and ITSR and the BigDye terminator Sequencing Kit (version 3.1, Applied Biosystems) following the manufacturer's instructions in the ABI 3500 Genetic Analyzer. Sequences informations were assembled and edited using the Bio Edit Sequence Alignment Editor. The Nucleotide Basic Local Alignment Search Tool (BLAST) was used to compare the DNA sequences obtained with the sequences in the National Center for Biotechnology Information (NCBI) database.

3. Results

Mycological analysis of 192 Moringa powder samples sold in informal markets showed that 175 (91.14%) were contaminated, and of these 175 contaminated Moringa samples, 27 (15.43%) samples had Fusarium isolates. The macroscopic and microscopic characteristics of these Fusarium isolates are shown in Figure 1.

All 27 fungal isolates were confirmed as belonging to the genus Fusarium by detection of the Its gene (Figure 2). The 431 bp fragments characteristic of the Its gene of the genus Fusarium were observed in all 27 isolated strains.

Among the 27 Fusarium strains, 17 (62.93%) were confirmed by sequencing as belonging to the species Fusarium solani and 10 (37.04%) belonging to the genus Fusarium sp. The isolates were found to be closely related to Fusarium solani strain Jfs-2 (accession number MT002830) and Fusarium sp strain NH12 (accession number MN602837). Figure 2 and Figure 3 represent a phylogenetic trees showing the genetic similarity between some isolates of Fusarium solani and Fusarium sp identified during this study (CI-007 Fus. solani; CI-008 Fus. solani; CI-014 Fus. solani; CI-015 Fus. solani; CI-01 Fus. sp; CI-019 Fus. sp; CI-009 Fus. sp etc…) and others found on the GenBank database. Evolutionary distances were calculated using the p-distance method using MEGA7 software (Version 7.0.26.).

Among the 27 Fusarium strains isolated, the 982 bp fragments (Figure 4) characteristic of the Fum13 gene for determining the potential of fumonisin production in the isolates was observed in 17 strains (62,96%), including 14 strains of Fusarium solani (51,85%) and 3 strains (11.11%) of Fusarium sp.

4. Discussion

Mycological analysis of Moringa powder samples revealed the presence of Fusarium strains in these samples. These fungal genera are present in the majority of poorly stored or insufficiently dried dry foods. Similar studies conducted in India by ¹⁷ reported the presence of fungal strains belonging to the genera Fusarium at a frequency of 12.5% in a staple food for human consumption. According to these authors, the presence of Fusarium strains could be explained by the unfavorable conditions during the harvesting, processing and storage of this food.

Indeed, observations made in the field have shown that washing the leaves of Moringa oleifera harvested by manufacturers would probably be insufficient or unsuitable to ensure complete decontamination.

The environment of drying, grinding, sieving powders and packaging could be a source of contamination of the finished Product ¹⁰. Similar results were also obtained by ¹⁸ who identified and characterized in their work 27 Fusarium spp isolated from 58 samples of sorghum seeds collected from all over India.

In addition, these strains harbor certain genes responsible for the production of fumonisins. These fumonisins are toxic secondary metabolites produced by certain species of Fusarium. This study also demonstrated the presence of the Its gene in 27 isolates of Fusarium. This result corroborates with those of ¹⁹ who revealed in their work the presence of Its gene in 64 isolates of Fusarium sp. This remark was also made by ¹⁴ in their work on the development of a multiplex PCR assay for the detection of fumonisin-producing species in Fusarium strain isolated from maize meal.

The results of molecular phylogeny show an affinity between the majority of the isolated Fusarium solani, Fusarium sp species and other Fusarium strains found in the GenBan database with high phylogenetic affinity. On the basis of the Its gene, two phylogenetic trees comprising respectively the Fusarium solani and Fusarium sp strains isolated from Moringa powder sold in Abidjan were constructed.

These trees confirm the phylogenetic identification of the 27 Fusarium isolates isolated in this study. In the present study, phylogenetic analyzes showed that Fusarium strains isolated from Moringa powder sold in Abidjan had the same phylogenetic clusters as those isolated in other countries. This contact was also established by ²⁰ who also observed a strong phylogenetic affinity of Fusarium solani strains isolated from potato tubers with other Fuarium isolates found in the GenBan database.

The results obtained in this study indicated that Fusarium solani was one of the most isolated (62.93%) in the Moringa leaf powder analyzed. This result could be explained by the fact that fungi belonging to the genus Fusarium sp are very frequently found in soil, plants, air and water. Their geoclimatic distribution is also very diverse because these filamentous fungi exist in both temperate and tropical regions ²¹. Moreover, recently a team of researchers has shown the presence of several species of Fusarium (Fusarium oxysporum, Fusarium solani, Fusarium equiseti, Fusarium dimerum and Fusarium proliferatum) in atmospheric dust and rainwater. These data explain the sometimes long dispersal of Fusarium solani by rain and wind ²².

Moringa powder processing, which goes through a stage of washing the leaves after harvest, could therefore explain the presence of this fungus in Moringa powder. This study showed that among 27 Fusarium isolates, 20 (74.07%) strains harbored the Fum 13 gene which is responsible for the biosynthesis of fumonisin, this present result is in agreement with those of ²³ who found that among 85 isolates of Fusarium isolated from rice, 20 strains showed a positive amplification for the Fum13 gene encoding the biosynthesis of fumonisins. In this study, the presence of the Fum 13 gene was revealed in 14 of the 17 strains of Fusarium solani identified and confirmed by sequencing. This detection of the Fum 13 gene in Fusarium solani strains could be one of the first studies showing the presence of a fumonisin biosynthesis gene in Fusarium solani strains isolated from Moringa powder.

The same observation was made by ²⁴ who for the first time detected a gene coding for the biosynthesis of fumonisins (Fum1) in strains of Fusarium proliferatum isolated from onion plants and seeds in Japan. In fact, fumonisin is mainly produced by species such as Fusarium verticillioides ¹⁷ but also by Fusarium moniliforme, Fusarium prolératum and Fusarium culmorum ²⁵. The study of ¹⁸ also revealed the specificity of the primers for the fumonisin-producing isolates of Fusarium verticillioides in a multiplex PCR. PCR in full here based on the search for Fum13 has already been developed by several authors, but the Fum13 gene responsible for the biosynthesis of fumonisins has been detected in Fusarium isolates such as Fusarium anthoplhilum, Fusarium Proliferatum and Fusarium verticillioides ^{15, 19, 23}. Unfavorable conditions during harvesting, processing and storage are factors that may favor the fungal contamination of Moringa powder used as a food supplement.

5. Conclusion

This study revealed the presence of potentially Fumonisin-producing Fusarium molds in Moringa leaf powder. The identity of the species was established by the ITS sequences, and the fumonisin production potential of the identified Fusarium strains was based on the crucial Fum13 gene. The presence of the Fum13 gene was detected in strains of Fusarium solani and Fusarium sp. The presence of molds of the genus Fusarium in Moringa leaf powder, which is commonly consumed by immunocompromised people, indicates a potential risk to the health of the consumer. Therefore, the detection and control of potentially mycotoxic molds are crucial steps in preventing toxins from entering the food chain in order to safeguard the health of the consumer.

Acknowledgements

We would like to thank all the sellers of Moringa oleifera powder in the various informal markets of Abidjan. We are also grateful to the laboratory of Mycology and Parasitology of the Institut Pasteur of Cote d'Ivoire.

References