The Penicillium digitatum antifungal protein PdAfpB shows high activity against mycobiota involved in sliced bread spoilage.

This study aimed to isolate and identify fungal species involved in sliced bread spoilage, and to evaluate their susceptibility to antifungal proteins of fungal origin (AFPs). Proteins include PdAfpB from Penicillium digitatum and PeAfpA, PeAfpB and PeAfpC from Penicillium expansum. Based on morphological criteria, a group of sixteen fungal isolates were selected and subsequently identified at the species level using sequence analysis. Penicillium species, the predominant mycobiota, were identified based on a combined phylogenetic analysis using ITS and ß-tubulin sequences, being P. roqueforti, P. brevicompactum, P. chrysogenum and P. crustosum the most abundant species. Aspergillus versicolor, Aspergillus niger and Bissochlamys spectabilis were also identified. Regarding the antifungal activity of AFPs, PdAfpB and PeAfpA were the most potent proteins since the growth of most of tested fungi was completely inhibited by concentrations ranging from 2 to 32 µg/mL. PeAfpB showed moderate antifungal activity, whereas PeAfpC was the least active protein. The best in vitro AFPs, PdAfpB and PeAfpA, were also evaluated in in situ protection assays against P. roqueforti. PdAfpB provoked a clear reduction of P. roqueforti growth in sliced bread samples, suggesting that this AFP has a protective effect on bread. This study underlines the potential of the AFPs tested, in particular PdAfpB, as alternative antifungal agents for extending sliced bread shelf life.

Development of a FungalBraid Penicillium expansum-based expression system for the production of antifungal proteins in fungal biofactories.

Fungal antifungal proteins (AFPs) have attracted attention as novel biofungicides. Their exploitation requires safe and cost-effective producing biofactories. Previously, Penicillium chrysogenum and Penicillium digitatum produced recombinant AFPs with the use of a P. chrysogenum-based expression system that consisted of the paf gene promoter, signal peptide (SP)-pro sequence and terminator. Here, the regulatory elements of the afpA gene encoding the highly produced PeAfpA from Penicillium expansum were developed as an expression system for AFP production through the FungalBraid platform. The afpA cassette was tested to produce PeAfpA and P. digitatum PdAfpB in P. chrysogenum and P. digitatum, and its efficiency was compared to that of the paf cassette. Recombinant PeAfpA production was only achieved using the afpA cassette, being P. chrysogenum a more efficient biofactory than P. digitatum. Conversely, P. chrysogenum only produced PdAfpB under the control of the paf cassette. In P. digitatum, both expression systems allowed PdAfpB production, with the paf cassette resulting in higher protein yields. Interestingly, these results did not correlate with the performance of both promoters in a luciferase reporter system. In conclusion, AFP production is a complex outcome that depends on the regulatory sequences driving afp expression, the fungal biofactory and the AFP sequence.

Antifungal Peptides and Proteins to Control Toxigenic Fungi and Mycotoxin Biosynthesis.

The global challenge to prevent fungal spoilage and mycotoxin contamination on food and feed requires the development of new antifungal strategies. Antimicrobial peptides and proteins (AMPs) with antifungal activity are gaining much interest as natural antifungal compounds due to their properties such as structure diversity and function, antifungal spectrum, mechanism of action, high stability and the availability of biotechnological production methods. Given their multistep mode of action, the development of fungal resistance to AMPs is presumed to be slow or delayed compared to conventional fungicides. Interestingly, AMPs also accomplish important biological functions other than antifungal activity, including anti-mycotoxin biosynthesis activity, which opens novel aspects for their future use in agriculture and food industry to fight mycotoxin contamination. AMPs can reach intracellular targets and exert their activity by mechanisms other than membrane permeabilization. The mechanisms through which AMPs affect mycotoxin production are varied and complex, ranging from oxidative stress to specific inhibition of enzymatic components of mycotoxin biosynthetic pathways. This review presents natural and synthetic antifungal AMPs from different origins which are effective against mycotoxin-producing fungi, and aims at summarizing current knowledge concerning their additional effects on mycotoxin biosynthesis. Antifungal AMPs properties and mechanisms of action are also discussed.

Potential of Antifungal Proteins (AFPs) to Control Penicillium Postharvest Fruit Decay.

Penicillium phytopathogenic species provoke severe postharvest disease and economic losses. Penicillium expansum is the main pome fruit phytopathogen while Penicillium digitatum and Penicillium italicum cause citrus green and blue mold, respectively. Control strategies rely on the use of synthetic fungicides, but the appearance of resistant strains and safety concerns have led to the search for new antifungals. Here, the potential application of different antifungal proteins (AFPs) including the three Penicillium chrysogenum proteins (PAF, PAFB and PAFC), as well as the Neosartorya fischeri NFAP2 protein to control Penicillium decay, has been evaluated. PAFB was the most potent AFP against P. digitatum, P. italicum and P. expansum, PAFC and NFAP2 showed moderate antifungal activity, whereas PAF was the least active protein. In fruit protection assays, PAFB provoked a reduction of the incidence of infections caused by P. digitatum and P. italicum in oranges and by P. expansum in apples. A combination of AFPs did not result in an increase in the efficacy of disease control. In conclusion, this study expands the antifungal inhibition spectrum of the AFPs evaluated, and demonstrates that AFPs act in a species-specific manner. PAFB is a promising alternative compound to control Penicillium postharvest fruit decay.

Differential susceptibility of mycotoxin-producing fungi to distinct antifungal proteins (AFPs).

The global challenge to prevent fungal spoilage and mycotoxin contamination on foods and feeds require the development of new antifungal strategies. Filamentous fungi encode diverse antifungal proteins (AFPs), which offer a great potential for the control of contaminant fungi. In this study, four AFPs from Penicillium digitatum (PdAfpB) and Penicillium expansum (PeAfpA, PeAfpB and PeAfpC) belonging to classes A, B and C, were tested against a representative panel of mycotoxin-producing fungi. They included a total of 38 strains representing 32 different species belonging to the genera Alternaria, Aspergillus, Byssochlamys, Fusarium and Penicillium. PeAfpA exhibited a potent antifungal activity, since the growth of all tested fungi was completely inhibited by concentrations ranging from 0.5 to 16 µg/mL. PdAfpB and PeAfpB, although less effective than PeAfpA, showed significant activity against most of the mycotoxigenic fungi tested. Importantly, PeAfpC previously described as inactive, showed a powerful inhibition against B. spectabilis strains, which are important spoilage and mycotoxin fungi in pasteurized foods. Although less effective than in liquid media, AFPs affected fungal growth on solid media. This study also underlines the potential of these AFPs, in particular PeAfpA, as future antifungal agents for applications in foods, on growing crops or during postharvest storage.

Proteomic Analysis of Saccharomyces cerevisiae Response to Oxidative Stress Mediated by Cocoa Polyphenols Extract.

The present study addressed the protective effects against oxidative stress (OS) of a cocoa powder extract (CPEX) on the protein expression profile of S. cerevisiae. A proteomic analysis was performed after culture preincubation with CPEX either without stress (-OS) or under stress conditions (+OS) (5 mM of H2O2). LC-MS/MS identified 33 differentially expressed proteins (-OS: 14, +OS: 19) that were included By Gene Ontology analysis in biological processes: biosynthesis of amino acids, carbohydrate metabolism and reactive oxygen species metabolic process. In a gene-knockout strains study, eight proteins were identified as putative candidates for being involved in the protective mechanism of cocoa polyphenols against OS induced by H2O2. CPEX was able to exert its antioxidant activity in yeast mainly through the regulation of: (a) amino acids metabolism proteins by modulating the production of molecules with known antioxidant roles; (b) stress-responsive protein Yhb1, but we were unable to fully understand its down-regulation; (c) protein Prb1, which can act by clipping Histone H3 N-terminal tails that are related to cellular resistance to DNA damaging agents.

Multiplex Detection of Aspergillus Species.

Multiplex real-time polymerase chain reaction (PCR) provides a fast and accurate DNA-based tool for the simultaneous amplification of more than one target sequence in a single reaction. Here a duplex real-time PCR assay is described for the simultaneous detection of Aspergillus carbonarius and members of the Aspergillus niger aggregate, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the beta-ketosynthase and the acyl transferase domains of the polyketide synthase of A. carbonarius and the A. niger aggregate, respectively.Besides, a rapid and efficient fungi DNA extraction procedure is described suitable to be applied in wine grapes. It includes a pulsifier equipment to remove conidia from grapes which prevents releasing of PCR inhibitors.

Effect of oxidant stressors and phenolic antioxidants on the ochratoxigenic fungus Aspergillus carbonarius.

BACKGROUND: There are few studies dealing with the relationship between oxidative stress and ochratoxin A (OTA) biosynthesis. In this work, we analyzed the effect of the oxidant stressor menadione and the antioxidants 3,5-di-tert-butyl-4-hydroxytoluene (BHT), catechin, resveratrol and a polyphenolic extract on growth, generation of reactive oxygen species (ROS), OTA production and gene expression of antioxidant enzymes of Aspergillus carbonarius. RESULTS: Exposure to menadione concentrations higher than 20 µmol L(-1) led to increases in ROS and OTA levels and a decrease in growth rate. Exposure to 2.5-10 mmol L(-1) BHT also led to higher ROS and OTA levels, although growth rate was only affected above 5 mmol L(-1). Naturally occurring concentrations of catechin, resveratrol and polyphenolic extract barely affected growth rate, but they produced widely different effects on OTA production level depending on the antioxidant concentration used. In general, gene expression of antioxidant enzymes superoxide dismutase (SOD) and peroxiredoxin (PRX) was downregulated after exposure to oxidant and antioxidant concentrations that enhanced OTA production level. CONCLUSION: Aspergillus carbonarius responds to oxidative stress, increasing OTA production. Nevertheless, the use of naturally occurring concentrations of antioxidant phenolic compounds to reduce oxidative stress is not a valid approach by itself for OTA contamination control in grapes.

Real-time PCR based procedures for detection and quantification of Aspergillus carbonarius in wine grapes.

Aspergillus carbonarius is the main species responsible for ochratoxin A accumulation in wine grapes and consequently, its rapid and sensitive detection is increasingly investigated. A new real-time PCR (RTi-PCR) based procedure was developed for the rapid and specific detection and quantification of A. carbonarius in wine grapes. The procedure includes the use of the pulsifier equipment to remove conidia from grapes which prevents releasing of PCR inhibitors, and DNA extraction with the EZNA Fungal DNA kit. It reduced the time for A. carbonarius DNA extraction from grapes to 30 min. Two specific primers (AcKS10L/AcKS10R) delimiting a 161 bp fragment, and a probe were designed and directed to the beta-ketosynthase domain of a polyketide synthase from A. carbonarius. Specificity was confirmed by testing primers towards purified DNA from 52 fungal strains, including reference and food isolates. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated conidial suspensions from A. carbonarius. The SYBR-Green I and TaqMan RTi-PCR approaches established were able to detect at least 2.4 and 24 genomic equivalents, respectively, using purified DNA. Results obtained from conidial suspensions, after DNA extraction, showed that at least 5 conidia per reaction should be present for a positive result with SYBR-Green I and 50 in the case of TaqMan. The quantification of fungal genomic DNA in artificially inoculated wine grapes performed successfully, with a minimum threshold of 10(3) conidia mL(-1) for accurate quantification. The developed RTi-PCR assay is a promising tool in the prediction of potential ochratoxigenic risk, even in the case of low-level infections, and suitable for a rapid, automated and high throughput analysis.

Genetic structure and evolution of natural populations of viruses causing the tomato yellow leaf curl disease in Spain.

The population structure and genetic variation of two begomoviruses: tomato yellow leaf curl Sardinia virus (TYLCSV) and tomato yellow leaf curl virus (TYLCV) in tomato crops of Spain were studied from 1997 until 2001. Restriction digestion of a genomic region comprised of the CP coat protein gene (CPR) of 358 TYLC virus isolates enabled us to classify them into 14 haplotypes. Nucleotide sequences of two genomic regions: CPR, and the surrounding intergenic region (SIR) were determined for at least two isolates per haplotype. SIR was more variable than CPR and showed multiple recombination events whereas no recombination was detected within CPR. In all geographic regions except Murcia, the population was, or evolved to be composed of one predominant haplotype with a low genetic diversity (<0.0180). In Murcia, two successive changes of the predominant haplotype were observed in the best studied population. Phylogenetic analysis showed that the TYLCSV sequences determined clustered with sequences obtained from the GenBank of other TYLCSV Spanish isolates which were clearly separated from TYLCSV Italian isolates. Most of our TYLCV sequences were similar to those of isolates from Japan and Portugal, and the sequences obtained from TYLCV isolates from the Canary island of Lanzarote were similar to those of Caribbean TYLCV isolates.