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Variation in Fumonisin and Ochratoxin Production Associated with Differences in Biosynthetic Gene Content in Aspergillus niger and A. welwitschiae Isolates from Multiple Crop and Geographic Origins

View Article: PubMed Central - PubMed

ABSTRACT

The fungi Aspergillus niger and A. welwitschiae are morphologically indistinguishable species used for industrial fermentation and for food and beverage production. The fungi also occur widely on food crops. Concerns about their safety have arisen with the discovery that some isolates of both species produce fumonisin (FB) and ochratoxin A (OTA) mycotoxins. Here, we examined FB and OTA production as well as the presence of genes responsible for synthesis of the mycotoxins in a collection of 92 A. niger/A. welwitschiae isolates from multiple crop and geographic origins. The results indicate that (i) isolates of both species differed in ability to produce the mycotoxins; (ii) FB-nonproducing isolates of A. niger had an intact fumonisin biosynthetic gene (fum) cluster; (iii) FB-nonproducing isolates of A. welwitschiae exhibited multiple patterns of fum gene deletion; and (iv) OTA-nonproducing isolates of both species lacked the ochratoxin A biosynthetic gene (ota) cluster. Analysis of genome sequence data revealed a single pattern of ota gene deletion in the two species. Phylogenetic analysis suggest that the simplest explanation for this is that ota cluster deletion occurred in a common ancestor of A. niger and A. welwitschiae, and subsequently both the intact and deleted cluster were retained as alternate alleles during divergence of the ancestor into descendent species. Finally, comparison of results from this and previous studies indicate that a majority of A. niger isolates and a minority of A. welwitschiae isolates can produce FBs, whereas, a minority of isolates of both species produce OTA. The comparison also suggested that the relative abundance of each species and frequency of FB/OTA-producing isolates can vary with crop and/or geographic origin.

No MeSH data available.


Related in: MedlinePlus

(A) Comparison of the intact and deleted ota cluster region in selected isolates of A. niger and A. welwitschiae. Arrows and gene designations are as in Figure 1. An15g07870 and An15g07930 are genes flanking each side of the ota cluster; and the An15g07870 and An15g07930 designations are the original gene model designations for GenBank accessions for the genome sequence of A. niger strain CBS 513.88. (B) Maximum likelihood tree inferred from the An15g07870-An15g07930 region: i.e., the region spanned by the bracket at the bottom of (A) in strains that lack an ota cluster as well as the homologous sequences from strains that have an intact cluster. The scale at the upper right indicates the number of substitutions per site. On the JGI website, ATCC 13157 is classified as A. phoenicis. Although A. phoenicis is considered to be synonymous with A. welwitschiae (Hong et al., 2013), our analysis indicates that ATCC 13157 is a strain of A. niger sensu stricto (Figure S1).
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Figure 5: (A) Comparison of the intact and deleted ota cluster region in selected isolates of A. niger and A. welwitschiae. Arrows and gene designations are as in Figure 1. An15g07870 and An15g07930 are genes flanking each side of the ota cluster; and the An15g07870 and An15g07930 designations are the original gene model designations for GenBank accessions for the genome sequence of A. niger strain CBS 513.88. (B) Maximum likelihood tree inferred from the An15g07870-An15g07930 region: i.e., the region spanned by the bracket at the bottom of (A) in strains that lack an ota cluster as well as the homologous sequences from strains that have an intact cluster. The scale at the upper right indicates the number of substitutions per site. On the JGI website, ATCC 13157 is classified as A. phoenicis. Although A. phoenicis is considered to be synonymous with A. welwitschiae (Hong et al., 2013), our analysis indicates that ATCC 13157 is a strain of A. niger sensu stricto (Figure S1).

Mentions: To further evaluate whether the amplification Patterns o-1 and o-2 were consistent with the presence and absence of ota genes, we examined the genome sequences from a subset of one A. niger and seven A. welwitschiae isolates for which amplification-pattern data were determined. This analysis revealed that the three A. welwitschiae isolates (ITEM 4552, ITEM 6142, and ITEM 7097) that exhibited Pattern o-1 had an intact ota cluster; i.e., they had all five ota genes, and the genes were apparently functional. Further, the sequence analysis indicated that the one A. niger isolate (ITEM 10355) and the four A. welwitschiae isolates (ITEM 7468, ITEM 10353, ITEM 10932, and ITEM 11209) with Pattern o-2 did not have any of the ota genes, except for an approximately 1100-nucleotide region near the 3′ end of ota1. These results indicate that the ota amplification patterns were consistent with the presence and absence of ota genes. Furthermore, the results of both the PCR and genome sequence data provide evidence that the OTA-nonproduction phenotype in the majority of isolates of both A. niger and A. welwitschiae results from the absence of ota genes in the isolates. Examination of previously generated genome sequences for the ota cluster confirmed that the cluster was intact in A. niger strain CBS 513.88 and absent in A. niger strain ATCC 1015 as previously reported (Andersen et al., 2011). This analysis also revealed that an intact ota cluster was present in A. niger strains ATCC 13157 and ATCC 13496, but not in A. niger strain ITEM 10355 as well as A. welwitschiae strains ITEM 7468 and ITEM 11945 (Figure 5A). Further examination of both previously generated genome sequence data and sequence data generated during this study (20 genome sequences in total) revealed that all strains examined have homologs of the ota cluster flanking genes An15g07870 and An15g07930 regardless of species and whether the strains have an intact cluster (Figure 5A). In both A. niger and A. welwitschiae sequences that lack the ota cluster, the An15g07870-An15g07930 intergenic region ranges in length from 2508 to 2800 nucleotides. 94% of this aligned intergenic sequence was homologous to three sequence elements in A. niger and A. welwitschiae strains with an intact ota cluster: 18% of the region was homologous to the sequence immediately 5′ to the An15g07870 start codon; 41% was homologous to sequence near the 3′ end of ota1; and 35% was homologous to sequence immediately 5′ to the An15g07930 start codon (Figure 5A). The identity of homologs of the three sequence elements ranged from 79 to 92% in strains with an intact vs. a deleted ota cluster. In strains that had a deleted cluster, the sequence corresponding to ota1 was 1127–1178 nucleotides in length and was homologous to nucleotides 7012–8126 of the intact ota1 coding region (with introns included). Variation in the length of the ota1-fragment homologs resulted from deletions and insertions within the homologs. Regardless of species, the ota1-fragment homologs began and ended at the same positions, nucleotides 7012 and 8126 respectively, of the intact ota1 coding region. The homology of the three sequence elements within the An15g07870-An15g07930 intergenic region facilitated alignment and phylogenetic analysis of the sequences. The tree inferred from the analysis indicated that, regardless of species, sequences from strains with the ota cluster deletion are more closely related to one another than to sequences from strains with the intact ota cluster (Figure 5B).


Variation in Fumonisin and Ochratoxin Production Associated with Differences in Biosynthetic Gene Content in Aspergillus niger and A. welwitschiae Isolates from Multiple Crop and Geographic Origins
(A) Comparison of the intact and deleted ota cluster region in selected isolates of A. niger and A. welwitschiae. Arrows and gene designations are as in Figure 1. An15g07870 and An15g07930 are genes flanking each side of the ota cluster; and the An15g07870 and An15g07930 designations are the original gene model designations for GenBank accessions for the genome sequence of A. niger strain CBS 513.88. (B) Maximum likelihood tree inferred from the An15g07870-An15g07930 region: i.e., the region spanned by the bracket at the bottom of (A) in strains that lack an ota cluster as well as the homologous sequences from strains that have an intact cluster. The scale at the upper right indicates the number of substitutions per site. On the JGI website, ATCC 13157 is classified as A. phoenicis. Although A. phoenicis is considered to be synonymous with A. welwitschiae (Hong et al., 2013), our analysis indicates that ATCC 13157 is a strain of A. niger sensu stricto (Figure S1).
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Figure 5: (A) Comparison of the intact and deleted ota cluster region in selected isolates of A. niger and A. welwitschiae. Arrows and gene designations are as in Figure 1. An15g07870 and An15g07930 are genes flanking each side of the ota cluster; and the An15g07870 and An15g07930 designations are the original gene model designations for GenBank accessions for the genome sequence of A. niger strain CBS 513.88. (B) Maximum likelihood tree inferred from the An15g07870-An15g07930 region: i.e., the region spanned by the bracket at the bottom of (A) in strains that lack an ota cluster as well as the homologous sequences from strains that have an intact cluster. The scale at the upper right indicates the number of substitutions per site. On the JGI website, ATCC 13157 is classified as A. phoenicis. Although A. phoenicis is considered to be synonymous with A. welwitschiae (Hong et al., 2013), our analysis indicates that ATCC 13157 is a strain of A. niger sensu stricto (Figure S1).
Mentions: To further evaluate whether the amplification Patterns o-1 and o-2 were consistent with the presence and absence of ota genes, we examined the genome sequences from a subset of one A. niger and seven A. welwitschiae isolates for which amplification-pattern data were determined. This analysis revealed that the three A. welwitschiae isolates (ITEM 4552, ITEM 6142, and ITEM 7097) that exhibited Pattern o-1 had an intact ota cluster; i.e., they had all five ota genes, and the genes were apparently functional. Further, the sequence analysis indicated that the one A. niger isolate (ITEM 10355) and the four A. welwitschiae isolates (ITEM 7468, ITEM 10353, ITEM 10932, and ITEM 11209) with Pattern o-2 did not have any of the ota genes, except for an approximately 1100-nucleotide region near the 3′ end of ota1. These results indicate that the ota amplification patterns were consistent with the presence and absence of ota genes. Furthermore, the results of both the PCR and genome sequence data provide evidence that the OTA-nonproduction phenotype in the majority of isolates of both A. niger and A. welwitschiae results from the absence of ota genes in the isolates. Examination of previously generated genome sequences for the ota cluster confirmed that the cluster was intact in A. niger strain CBS 513.88 and absent in A. niger strain ATCC 1015 as previously reported (Andersen et al., 2011). This analysis also revealed that an intact ota cluster was present in A. niger strains ATCC 13157 and ATCC 13496, but not in A. niger strain ITEM 10355 as well as A. welwitschiae strains ITEM 7468 and ITEM 11945 (Figure 5A). Further examination of both previously generated genome sequence data and sequence data generated during this study (20 genome sequences in total) revealed that all strains examined have homologs of the ota cluster flanking genes An15g07870 and An15g07930 regardless of species and whether the strains have an intact cluster (Figure 5A). In both A. niger and A. welwitschiae sequences that lack the ota cluster, the An15g07870-An15g07930 intergenic region ranges in length from 2508 to 2800 nucleotides. 94% of this aligned intergenic sequence was homologous to three sequence elements in A. niger and A. welwitschiae strains with an intact ota cluster: 18% of the region was homologous to the sequence immediately 5′ to the An15g07870 start codon; 41% was homologous to sequence near the 3′ end of ota1; and 35% was homologous to sequence immediately 5′ to the An15g07930 start codon (Figure 5A). The identity of homologs of the three sequence elements ranged from 79 to 92% in strains with an intact vs. a deleted ota cluster. In strains that had a deleted cluster, the sequence corresponding to ota1 was 1127–1178 nucleotides in length and was homologous to nucleotides 7012–8126 of the intact ota1 coding region (with introns included). Variation in the length of the ota1-fragment homologs resulted from deletions and insertions within the homologs. Regardless of species, the ota1-fragment homologs began and ended at the same positions, nucleotides 7012 and 8126 respectively, of the intact ota1 coding region. The homology of the three sequence elements within the An15g07870-An15g07930 intergenic region facilitated alignment and phylogenetic analysis of the sequences. The tree inferred from the analysis indicated that, regardless of species, sequences from strains with the ota cluster deletion are more closely related to one another than to sequences from strains with the intact ota cluster (Figure 5B).

View Article: PubMed Central - PubMed

ABSTRACT

The fungi Aspergillus niger and A. welwitschiae are morphologically indistinguishable species used for industrial fermentation and for food and beverage production. The fungi also occur widely on food crops. Concerns about their safety have arisen with the discovery that some isolates of both species produce fumonisin (FB) and ochratoxin A (OTA) mycotoxins. Here, we examined FB and OTA production as well as the presence of genes responsible for synthesis of the mycotoxins in a collection of 92 A. niger/A. welwitschiae isolates from multiple crop and geographic origins. The results indicate that (i) isolates of both species differed in ability to produce the mycotoxins; (ii) FB-nonproducing isolates of A. niger had an intact fumonisin biosynthetic gene (fum) cluster; (iii) FB-nonproducing isolates of A. welwitschiae exhibited multiple patterns of fum gene deletion; and (iv) OTA-nonproducing isolates of both species lacked the ochratoxin A biosynthetic gene (ota) cluster. Analysis of genome sequence data revealed a single pattern of ota gene deletion in the two species. Phylogenetic analysis suggest that the simplest explanation for this is that ota cluster deletion occurred in a common ancestor of A. niger and A. welwitschiae, and subsequently both the intact and deleted cluster were retained as alternate alleles during divergence of the ancestor into descendent species. Finally, comparison of results from this and previous studies indicate that a majority of A. niger isolates and a minority of A. welwitschiae isolates can produce FBs, whereas, a minority of isolates of both species produce OTA. The comparison also suggested that the relative abundance of each species and frequency of FB/OTA-producing isolates can vary with crop and/or geographic origin.

No MeSH data available.


Related in: MedlinePlus