Molecular Investigations of Food-Borne <em><em>Cladosporium</em></em> and Fusarium Species from Nigeria

Research Article

J Bacteriol Mycol. 2016; 3(2): 1024.

Molecular Investigations of Food-Borne Cladosporium and Fusarium Species from Nigeria

Moore GG¹ and Fapohunda SO²*

¹Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, New Orleans, Louisiana, USA

²Department of Biosciences and Biotechnology, Babcock University, Ilishan-Remo, Nigeria

*Corresponding author: Stephen O. Fapohunda, Department of Biosciences and Biotechnology, Babcock University, Ilishan-Remo, Nigeria

Received: March 27, 2016; Accepted: May 06, 2016; Published: May 09, 2016


A sampling of contaminated foodstuffs throughout southwest Nigeria yielded three fungalisolates belonging to the genus Fusarium and two belonging to the genus Cladosporium. In this study we subjected these isolates to various molecular investigations. The morphological species identifications were confirmed or refined with BLAST queries for sequences from two genomic regions (translation elongation factor-1 α and ITS). BLAST results uncovered species identification inconsistencies for one Fusarium isolate, SRRC1606, and for both Cladosporium isolates based on the examined loci. Using additional species sequences obtained from Gen Bank, the phylogenetic associations for each genomic region were explored and observed species haplotype associations for the Fusarium sequences; however, this was not the case for the Cladosporium sequences. There was evidence of recombination in both loci for the Fusarium species, but only in the translation elongation factor locus for the Cladosporium sample population. Fusarium coalescent analyses for both loci inferred two lineages, one containing only F. oxysporum sequences and the other containing the remaining species examined. These same analyses for the Cladosporia inferred ancient segregation of one lineage, containing only the outgroup taxa, from a second lineage that exhibited recent divergence events among the other taxa examined. Mycological population dynamics and analyses can achieve a better understanding of interventions to protect consumers from contaminated foods.

Keywords: Fungal contaminants; Genotyping; Phylogenetics; Recombination; Speciation


The genus Fusarium includes species noted for the production of secondary metabolites and cause of diseases in both plants and animals [1,2], and Cladosporium species are most often associated with cutaneous and pulmonary infections [3]. They occupy very diverse habitats such as plumbing drains [4], tomato plants [5], cosmetics [6], and hospitals [7,8]. Previous research has emphasised the value of molecular techniques, beyond morphological investigation, as critical components for reliable fungal classification. For example, utilization of a molecular technique on Fusarium, known as PCR-ITS-RFLP, was able to further distinguish between strains within the Fusarium Solani Species Complex (FSSC) [9]. A newly developed species-specific primer, for the conserved regions of 28s-rDNA and the intergenic spacer, to genotype F. oxysporum f. sp.psidii was reported by Mishra and co-workers [10]. New genotypes and races of F. Oxysporum f. sp. vasinfectum were discovered, through comparison with previously described races, using sequences from translation elongation factor, phosphate permease, and beta-tubulin genes [11]. Similar attention has been focused on the use of molecular techniques to differentiate Cladosporium species [12-15]. The identities of two races of C. fulvum were recently confirmed by amplifying and sequencing 580bp of their respective ITS regions [5]. Molecular association of metabolite production with fungal genes has also been investigated such as in the FUM-1 (fumonisin) gene, and both TRI-13 and TRI-7trichothecene genes in F. proliferatum and F.verticillioides, respectively [16]. Also, molecular techniques were used to elucidate the genotypes of nonmycotoxigenic strains of F. proliferatum, F.verticillioides, and the F. graminearum species complex strains [17].

The aim of this preliminary study, not disregarding the sample size, was to analyze genomic loci in previously described Fusarium and Cladosporium isolates, sampled from contaminated foodstuffs, to observe species diversity and to assure the integrity of their taxonomic placements. The molecular analysis was to also designed to enhance in -depth fungal knowledge, which is a critical and strategic input in their control and management

Materials and Methods

Fungal identification

Fungal strains selected for this study, originally sampled from contaminated foods in Nigeria, were identified based on morphological characters as in Fapohunda et al. [18] and all isolates are currently housed in the Southern Regional Research Center (SRRC) fungal collection in New Orleans, Louisiana, USA. Three Fusarium isolates (SRRC1606, SRRC1630 and SRRC1633) were morphologically identified from among the collected samples. Also, two Cladosporium isolates were identified based on morphology (SRRC1616 and SRRC1634). In addition to morphology, BLAST queries of sequences from two genomic regions were performed to verify species identification for each isolate: the Internal Transcribed Spacer (ITS) region and the translation elongation factor-1 α (tef1α). The tef1α locus is considered a good delimiter of species for fungal genera such as Fusarium and Cladosporium [19, 20]. All isolates were PCR-amplified for ITS as in Fapohunda et al. [18]. Amplification of the tef1α locus involved primers reported in Carbone and Kohn [21]. Amplicons for each isolate were purified and sequenced, and the sequences were trimmed, cleaned, and aligned using Sequencher version 5.3 (Gene Codes Corporation). Individual sequences for each isolate/locus were BLAST queried to verify species associations previously determined by morphological observations. In this study we did not test any isolates for the presence of mycotoxins.

Comparative analyses

Sampling was done on less than four isolates per genus, and searches for each locus were performed using the NCBI database and additional species sequences from each genus were included in the respective sequence alignments. Whenever possible, the same accessioned isolate (or at least species) was used in alignments for the genomic loci examined. In addition to the various sequences acquired to build sample populations for each genus and locus, outgroup taxa were selected for each genus. Fusarium chlamydosporum [22] and Cladosporium salinae [23] have been reported as appropriate phylogenetic out group taxa for their respective genera, and; therefore, were chosen for this study. All sequence alignments were exported in nexus format for analyses in SNAP Workbench [24]. The first analysis component involved collapsing the sequences into haplotypes using SNAP: Map [25]. Resulting out files were examined for haplotype associations and evidence of the ancestral sequence. The ancestral sequence is evident when its nucleotide composition is identical to the consensus sequence. Next to be performed was the phylogenetic inference for each locus; additionally, bootstrap values and heuristics (e.g., consistency indices and numbers of most-parsimonious trees) were determined using PAUP* software [26]. Comparative analyses of recombination and coalescence were also performed for each locus and sample population with the recombination events for each locus/species group was performed using RecMin [27]. If evidence of recombination was observed, an Ancestral Recombination Graph (ARG) was inferred using the beagle algorithm with one million simulations [28]. Finally, to explore divergence patterns among the species for each locus, without the interference of recombination, coalescent analyses were performed which only consider mutational differences among isolates [29].

The Gen Bank accession numbers for the Fusarium isolates are KP771785-KP771787 (ITS) and KT950249-KT950251 (tef1α). The accession numbers for the Cladosporium isolates are KP771788- KP771789 (ITS) and KT950252-KT950253 (tef1α).


Molecular confirmation of species

Table 1 lists the sampled Nigerian isolates, the food stuffs from which they were sampled and their BLAST identification results.