Quantification of patulin in fruit leathers by ultra-high-performance liquid chromatography-photodiode array (UPLC-PDA)

<div><p>Patulin is a mycotoxin commonly found in certain fruit and fruit products. For this reason many countries have established regulatory limits pertaining to, in particular, apple juice and apple products. Fruit leathers are produced by dehydrating fruit puree, leaving a sweet product that has a leathery texture. A recent report in the literature described the detection of patulin at substantial levels in fruit leathers. To investigate this further, an ultra-high-performance liquid chromatography-photodiode array (UPLC-PDA) method was developed for the sensitive detection of patulin in fruit leathers. Investigations were also made of the suitability of direct analysis in real time-mass spectrometry (DART-MS) for detection of patulin from the surface of fruit leathers. Results indicated DART-MS was insufficiently sensitive for quantification from the surface of home-style apple leathers, although patulin spiked onto the surface of leather or peel could be detected. The UPLC-PDA method was used to determine the fate of patulin during the preparation of home-made fruit leathers. Interestingly, when a home-style process was used, the patulin was not destroyed, but rather increased in concentration as the puree was dehydrated. The UPLC-PDA method was also used to screen for patulin in commercial fruit leathers. Of the 36 products tested, 14 were above the limit of detection (3.5 μg kg^–1) and nine were above the limit of quantification (12 μg kg^–1). Positive samples were confirmed by UPLC-MS/MS. Only one sample was found above the US regulatory limit for single-strength apple juice products (50 μg kg^–1). These results suggest patulin can be concentrated during preparation and can be found in fruit leathers. The limited survey suggests that patulin is fairly prevalent in such commercial products, but that the levels are usually low.</p></div

Evolution of structural diversity of trichothecenes, a family of toxins produced by plant pathogenic and entomopathogenic fungi

<div><p>Trichothecenes are a family of terpenoid toxins produced by multiple genera of fungi, including plant and insect pathogens. Some trichothecenes produced by the fungus <i>Fusarium</i> are among the mycotoxins of greatest concern to food and feed safety because of their toxicity and frequent occurrence in cereal crops, and trichothecene production contributes to pathogenesis of some <i>Fusarium</i> species on plants. Collectively, fungi produce over 150 trichothecene analogs: i.e., molecules that share the same core structure but differ in patterns of substituents attached to the core structure. Here, we carried out genomic, phylogenetic, gene-function, and analytical chemistry studies of strains from nine fungal genera to identify genetic variation responsible for trichothecene structural diversity and to gain insight into evolutionary processes that have contributed to the variation. The results indicate that structural diversity has resulted from gain, loss, and functional changes of trichothecene biosynthetic (<i>TRI</i>) genes. The results also indicate that the presence of some substituents has arisen independently in different fungi by gain of different genes with the same function. Variation in <i>TRI</i> gene duplication and number of <i>TRI</i> loci was also observed among the fungi examined, but there was no evidence that such genetic differences have contributed to trichothecene structural variation. We also inferred ancestral states of the <i>TRI</i> cluster and trichothecene biosynthetic pathway, and proposed scenarios for changes in trichothecene structures during divergence of <i>TRI</i> cluster homologs. Together, our findings provide insight into evolutionary processes responsible for structural diversification of toxins produced by pathogenic fungi.</p></div

Biosynthetic pathways for the trichothecene analogs deoxynivalenol and T-2 toxin in <i>Fusarium graminearum</i> and <i>F</i>. <i>sporotrichioides</i>, respectively.

<p>The inset at the top right shows the structure and numbering systems for trichodiene and 12,13-epoxytrichothec-9-ene (EPT).</p

Representative diversity of trichothecene structures in fungi other than <i>Fusarium</i>.

<p>See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006946#ppat.1006946.g001" target="_blank">Fig 1</a> for examples of <i>Fusarium</i> trichothecenes.</p

Functions of trichothecene biosynthetic genes.

<p>Functions of trichothecene biosynthetic genes.</p

Functional analysis of <i>TRI3</i> in <i>Trichoderma arundinaceum</i>.

<p>(<b>A-C</b>) High performance liquid chromatograms showing harzianum A (HA) production by: (<b>A</b>) wild-type progenitor strain Ta37; (<b>B</b>) <i>tri3</i> mutant strain tri3.1; and (<b>C</b>) strain tri3.1.C1, <i>tri3</i> mutant strain tri3.1 complemented with a wild-type copy of <i>TRI3</i>. (<b>D</b>) Quantification of HA production in the wild type and <i>tri3</i> deletion mutant strains tri3.1, tri3.30, tri3.33, and tri3.48. (<b>E</b>) Quantification of HA production in the wild type, <i>tri3</i> mutant strain tri3.1, and three tri3.1-derived strains that were complemented with a wild-type copy of <i>TRI3</i> (strains tri3.1.C1, tri3.1.C4 and tri3.1.C5). (<b>F</b> and <b>G</b>) Total ion chromatograms from gas chromatography-mass spectrometry analysis of culture extracts of the (<b>F</b>) wild-type strain and (<b>G</b>) <i>tri3</i> mutant strain tri3.1. The peaks labeled 4OH and ISD are for trichodermol (4-hydroxy EPT) and isotrichodiol, respectively.</p

Comparison phylogenetic tree inferred from concatenated sequences of <i>TRI3</i>, <i>TRI5</i> and <i>TRI14</i> (left) and a species phylogeny inferred from concatenated sequences of 20 housekeeping genes (right).

<p>Numbers near branch nodes are bootstrap values from 1000 pseudoreplicates. Only bootstrap values greater than 70% are shown. The housekeeping genes used in this analysis are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006946#ppat.1006946.s003" target="_blank">S3 File</a>.</p

<i>TRI</i> gene content of fungi examined in the current study.

<p>A gray box indicates that a <i>TRI</i> gene is present in the genome of a fungus, while a white box indicates the gene was not detected. Numbers within boxes indicate the number of paralogs. The Greek letter ψ indicates that a large portion of the gene is present but that it is a pseudogene.</p

Maximum likelihood trees inferred from predicted amino acid sequences of Tri17 (top) and Tri101 (bottom) and related homologs from trichothecene-nonproducing fungi.

<p>The chemical structures shown to the right of the Tri17 tree are predicted structures of polyketides synthesized by the different Tri17 homologs. Asterisks (*) indicate species/strains that produce trichothecenes or are predicted to produce trichothecene based the presence of <i>TRI</i> genes. In the <i>TRI101</i> tree, the gray boxes indicate the strains/species that have a <i>TRI101</i> gene that functions or is likely to function in trichothecene biosynthesis. Numbers near branch nodes are bootstrap values based on 1000 pseudoreplicates. Strain designations are shown only for species with two or three strains included in a tree.</p