Multiple lysine substitutions in the peptaibol trichogin GA IV enhance the antibiotic activity against plant pathogenic Pseudomonas syringae.

Plant diseases caused by Pseudomonas syringae are essentially controlled in the field with the use of copper-based products and antibiotics, raising environmental and safety concerns. Antimicrobial peptides (AMPs) derived from fungi may represent a sustainable alternative to those chemicals. Trichogin GA IV, a non-ribosomal, 11-residue long AMP naturally produced by the fungus Trichoderma longibrachiatum has the ability to insert into phospholipidic membranes and form water-filled pores, thereby perturbing membrane integrity and permeability. In previous studies, peptide analogs modified at the level of specific residues were designed to be water-soluble and active against plant pathogens. Here, we studied the role of glycine-to-lysine substitutions and of the presence of a C-terminal leucine amide on bioactivity against Pseudomonas syringae bacteria. P. syringae diseases affect a wide range of crops worldwide, including tomato and kiwifruit. Our results show that trichogin GA IV analogs containing two or three Gly-to-Lys substitutions are highly effective in vitro against P. syringae pv. tomato (Pst), displaying minimal inhibitory and minimal bactericidal concentrations in the low micromolar range. The same analogs are also able to inhibit in vitro the kiwifruit pathogen P. syringae pv. actinidiae (Psa) biovar 3. When sprayed on tomato plants 24 h before Pst inoculation, only tri-lysine containing analogs were able to significantly reduce bacterial titers and symptom development in infected plants. Our results point to a positive correlation between the number of lysine substitutions and the antibacterial activity. This correlation was supported by microscopy analyses performed with mono-, di- and tri-Lys containing analogs that showed a different degree of interaction with Pst cells and ultrastructural changes that culminated in cell lysis.

Effect of potassium sorbate (E-202) and the antifungal PgAFP protein on Aspergillus carbonarius growth and ochratoxin A production in raisin simulating media.

BACKGROUND: Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium fungi. The presence of OTA in raisins is mainly related to black Aspergillus spp. contamination. This toxin poses risks to human and animal health due to its high toxicity and carcinogenicity. New strategies to avoid the risk associated with OTA are therefore necessary. RESULTS: A comparison was made between the effects of the antifungal protein PgAFP and potassium sorbate (E-202) on the growth of Aspergillus carbonarius, biosynthetic- and stress-related gene expression and its OTA production at two water activity (aw ) levels, 0.95 and 0.93 aw . The results showed that PgAFP successfully controlled OTA production, whereas E-202, despite being able to reduce Aspergillus carbonarius growth, caused a significant increase in OTA production by the fungus. CONCLUSION: PgAFP protein, a biological compound with an antifungal activity, is safer to use than E-202 and may be proposed as a food preservative and a useful strategy to control ochratoxigenic A. carbonarius in raisins. © 2018 Society of Chemical Industry.

LAMP-based group specific detection of aflatoxin producers within Aspergillus section Flavi in food raw materials, spices, and dried fruit using neutral red for visible-light signal detection.

Aflatoxins can be produced by 21 species within sections Flavi (16 species), Ochraceorosei (2), and Nidulantes (3) of the fungal genus Aspergillus. They pose risks to human and animal health due to high toxicity and carcinogenicity. Detecting aflatoxin producers can help to assess toxicological risks associated with contaminated commodities. Species specific molecular assays (PCR and LAMP) are available for detection of major producers, but fail to detect species of minor importance. To enable rapid and sensitive detection of several aflatoxin producing species in a single analysis, a nor1 gene-specific LAMP assay was developed. Specificity testing showed that among 128 fungal species from 28 genera, 15 aflatoxigenic species in section Flavi were detected, including synonyms of A. flavus and A. parasiticus. No cross reactions were found with other tested species. The detection limit of the assay was 9.03pg of A. parasiticus genomic DNA per reaction. Visual detection of positive LAMP reactions under daylight conditions was facilitated using neutral red to allow unambiguous distinction between positive and negative assay results. Application of the assay to the detection of A. parasiticus conidia revealed a detection limit of 211 conidia per reaction after minimal sample preparation. The usefulness of the assay was demonstrated in the analysis of aflatoxinogenic species in samples of rice, nuts, raisins, dried figs, as well as powdered spices. Comparison of LAMP results with presence/absence of aflatoxins and aflatoxin producing fungi in 50 rice samples showed good correlation between these parameters. Our study suggests that the developed LAMP assay is a rapid, sensitive and user-friendly tool for surveillance and quality control in our food industry.