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Podosphaera xanthii is the main causal agent of powdery mildew (PM) disease for Cucurbita pepo. Disease control is attained principally by applications of chemical fungicides, along with parallel use of tolerant crop varieties and alternate application of elicitors to control development of disease resistance. To get insight into C. pepo molecular responses to P. xanthii infection and elicitor treatment we studied the proteomic profile differences at the phyllosphere of a zucchini cultivar susceptible to PM, at the onset of P. xanthii (PX) infection and after application of Reynoutria sachalinensis (RS) plant extract, respectively, using a nano-LC-HRMS/MS, Q-Exactive-Orbitrap approach. Analysis of peptide sequences regarding four treatment groups (Control; PX; RS; and RSPX (PX-infected priorly treated with RS)) resulted in 2070 CuGenDB annotations. Three comparisons (treatments vs. Control) encompassed most of the Differentially Expressed Proteins (DEPs). In these three comparisons, KEGG and Gene Ontology functional analyses highlighted unique differentially enriched pathways—some of which included highly expressed proteins—in PX-related (proteasome, pentose phosphate pathway, and carbon fixation), RS-related (biosynthesis of secondary metabolites, flavonoids, and starch and sucrose metabolism), and RSPX-related (pyruvate metabolism and polycomb repressive complex) comparisons, respectively, suggesting distinct mechanisms of early plant responses modulated by PX and RS. Furthermore, in four out of six comparisons the thiamine metabolism pathway was found to be enriched, suggesting a pivotal role in PX-induced responses. [ABSTRACT FROM AUTHOR]

Powdery mildew (PM) disease, caused by the obligate biotrophic fungal pathogen Podosphaera xanthii, is the most reported and destructive disease on cultivated Cucurbita species all over the world. Recently, the appearance of highly aggressive P. xanthii isolates has led to PM outbreaks even in resistant crops, making disease management a very difficult task. To challenge this, breeders rely on genetic characteristics for PM control. Analysis of commercially available intermediate resistance courgette (Cucurbita pepo L. var. cylindrica) varieties using cytological, molecular, and biochemical approaches showed that the plants were under a primed state and induced systemic acquired resistance (SAR) responses, exhibiting enhanced callose production, upregulation of salicylic acid (SA) defense signaling pathway genes, and accumulation of SA and defense metabolites. Additionally, the intermediate resistant varieties showed an altered epigenetic landscape in histone marks that affect transcriptional activation. We demonstrated that courgette plants had enriched H3K4me3 marks on SA-BINDING PROTEIN 2 and YODA (YDA) genes of the Pm-0 interval introgression, a genomic region that confers resistant to Cucurbits against P. xanthii. The open chromatin of SA-BINDING PROTEIN 2 and YDA genes was consistent with genes' differential expression, induced SA pathway, altered stomata characteristics, and activated SAR responses. These findings demonstrate that the altered epigenetic landscape of the intermediate resistant varieties modulates the activation of SA-BINDING PROTEIN 2 and YDA genes leading to induced gene transcription that primes courgette plants. [ABSTRACT FROM AUTHOR]

BACKGROUND Aspergillus is a diverse genus of fungi with high economic and social impact. Various species that belong to section Nigri (black aspergilli) are common agents of grape spoilage and potent producers of ochratoxin A (OTA), a mycotoxin associated with various nephrotoxic and immunotoxic effects in humans. Black aspergilli are difficult to classify following only phenotypic criteria; thus chemotaxonomic and molecular methods are employed in parallel with phenotypic ones for species characterization. These approaches, though accurate and replicable, require more than one individual step and are to a certain extent laborious when a rapid identification of these species is required. RESULTS: The aim of this study was to develop a high‐resolution melting polymerase chain reaction (HRM‐PCR) assay as a rapid method for identification of Aspergillus spp. section Nigri isolates and their detection in grape samples. Melt curve analysis of amplicons originating from the internal transcribed spacer 2 (ITS2) ribosomal region generated species‐specific HRM curve profiles, enabling the accurate differentiation of the analyzed genotypes. Furthermore, the assay was able to identify A. carbonarius, A. tubingensis, A. niger, A. ibericus and A. japonicus in grape samples artificially inoculated with conidia of these fungi. CONCLUSION: To our knowledge this is the first report on the development of an HRM‐PCR assay for the identification of black Aspergillus species in grape samples. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

In this work, a SYBR Green I real-time PCR method has been developed for the detection and quantification of Aspergillus carbonarius in grapes by targeting the veA gene with a primer pair (veAF4/veAR4) that specifically amplifies a 91-bp PCR product. The quantification of the fungal DNA was performed by generation of standard curves for two A. carbonarius strains, using spectrophotometrically measured DNA quantities (Log) with a linearity range from 50 to 5 X 10-4 ng of DNA. A high positive correlation (R² > 0.99) between exponential increases of DNA and real-time PCR threshold cycles showed a high amplification efficiency for the assay (E values 100.06 and 101.51%, respectively). Quantification of the fungal genomic DNA in grape samples artificially inoculated with A. carbonarius conidia was successfully performed with a minimum threshold of 104 conidia per g of grape berry. The assay developed would allow reliable, specific, and efficient detection and quantification of A. carbonarius in grapes. [ABSTRACT FROM AUTHOR]

A study on the occurrence of Aspergillus section Nigri species on grapes from four traditional grape-producing areas in Greece during the 2011/2012 vintage, and their capability to produce OTA was conducted. One hundred and twenty-eight black aspergilli isolates were characterised at the species level initially by the use of morphological criteria in accordance with appropriate keys, followed by molecular characterisation performed with Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) of the 5.8 ribosomal RNA gene Internal Transcribed Spacer region (5.8 rRNA ITS). Restriction enzyme digestion of the ITS amplicons using the HhaI, HinfI and RsaI, endonucleases distinguished eleven different patterns of restriction fragment length polymorphism (RFLP), four for each of the HhaI and RsaI digests and three for HinfI. From a total number of 128 individual isolates, 124 were classified into four Aspergillus species corresponding to A. carbonarius, A. tubingensis, A. japonicus and A. ibericus, and the remaining 4 were classified as members of the A. niger aggregate. A. carbonarius and A. tubingensis being the main representative species were equally counted, with higher geographical representation of the former in southern and the latter in northern regions, respectively. All isolates were tested for their ochratoxigenic potential by use of High Performance Liquid Chromatography (HPLC) and Enzyme Linked Immuno Sorbent Assay (ELISA), resulting in significant interspecies differences in OTA production. [ABSTRACT FROM AUTHOR]

Tudor-SN (TSN) copurifies with the RNA-induced silencing complex in animal cells where, among other functions, it is thought to act on mRNA stability via the degradation of specific dsRNA templates. In plants, TSN has been identified biochemically as a cytoskeleton-associated RNA binding activity. In eukaryotes, it has recently been identified as a conserved primary target of programmed cell death–associated proteolysis. We have investigated the physiological role of TSN by isolating null mutations for two homologous genes in Arabidopsis thaliana. The double mutant tsn1 tsn2 displays only mild growth phenotypes under nonstress conditions, but germination, growth, and survival are severely affected under high salinity stress. Either TSN1 or TSN2 alone can complement the double mutant, indicating their functional redundancy. TSN accumulates heterogeneously in the cytosol and relocates transiently to a diffuse pattern in response to salt stress. Unexpectedly, stress-regulated mRNAs encoding secreted proteins are significantly enriched among the transcripts that are underrepresented in tsn1 tsn2. Our data also reveal that TSN is important for RNA stability of its targets. These findings show that TSN is essential for stress tolerance in plants and implicate TSN in new, potentially conserved mechanisms acting on mRNAs entering the secretory pathway. [ABSTRACT FROM AUTHOR]

In order to examine the genetic variation of clinical and food isolates of Listeria monocytogenes in Greece, a total of 61 L. monocytogenes non-duplicate isolates, recovered from clinical specimens (n=19) and food (n=42), were serotyped and genotyped using two different Random Amplification of Polymorphic DNA (RAPD) protocols and Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Serotype group 4b, 4d, 4e prevailed (39.4%), among both clinical and food isolates, followed by serotype group 1/2a, 3a (23.0%), which nevertheless was detected only among food isolates. The most discriminatory typing protocol was MLVA, which grouped four isolates into two pairs, while the remaining isolates produced unique fingerprints. Similar results were obtained when taking into account the combination of the two RAPD protocols (Simpson index 0.999); six isolates were grouped into three pairs, two of which were the pairs that were identified also by MLVA. Single use of each RAPD protocol resulted in inferior discrimination (Simpson index 0.978 and 0.997, respectively). In conclusion, the two molecular procedures, MLVA, and the combined RAPD protocols, produced similar results, showing that L. monocytogenes isolates from clinical and food specimens were highly heterogenous and that clustering was very uncommon.

Tudor-SN (TSN) copurifies with the RNA-induced silencing complex in animal cells where, among other functions, it is thought to act on mRNA stability via the degradation of specific dsRNA templates. In plants, TSN has been identified biochemically as a cytoskeleton-associated RNA binding activity. In eukaryotes, it has recently been identified as a conserved primary target of programmed cell death-associated proteolysis. We have investigated the physiological role of TSN by isolating null mutations for two homologous genes in Arabidopsis thaliana. The double mutant tsn1 tsn2 displays only mild growth phenotypes under nonstress conditions, but germination, growth, and survival are severely affected under high salinity stress. Either TSN1 or TSN2 alone can complement the double mutant, indicating their functional redundancy. TSN accumulates heterogeneously in the cytosol and relocates transiently to a diffuse pattern in response to salt stress. Unexpectedly, stress-regulated mRNAs encoding secreted proteins are significantly enriched among the transcripts that are underrepresented in tsn1 tsn2. Our data also reveal that TSN is important for RNA stability of its targets. These findings show that TSN is essential for stress tolerance in plants and implicate TSN in new, potentially conserved mechanisms acting on mRNAs entering the secretory pathway.