Cultivar-Dependent Responses of Eggplant (Solanum melongena L.) to Simultaneous Verticillium dahliae Infection and Drought

Several studies regarding the imposition of stresses simultaneously in plants have shown that plant responses are different under individual and combined stress. Pathogen infection in combination with drought can act both additively and antagonistically, suggesting a tailored-made plant response to these stresses. The aforementioned combination of stresses can be considered as one of the most important factors affecting global crop production. In the present research we studied eggplant responses to simultaneous Verticillium dahliae infection and drought with respect to the application of the individual stresses alone and investigated the extent to which these responses were cultivar dependent. Two eggplant cultivars (Skoutari and EMI) with intermediate resistance to V. dahliae were subjected to combined stress for a 3-week period. Significant differences in plant growth, several physiological and biochemical parameters (photosynthesis rate, leaf gas exchanges, Malondialdehyde, Proline) and gene expression, were found between plants subjected to combined and individual stresses. Furthermore, plant growth and molecular (lipid peroxidation, hydrogen peroxide, gene expression levels) changes highlight a clear discrimination between the two cultivars in response to simultaneous V. dahliae infection and drought. Our results showed that combined stress affects significantly plants responses compared to the application of individual stresses alone and that these responses are cultivar dependent

Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive (<i>Olea europaea</i>) Twigs

Metabarcoding approaches for the identification of plant disease pathogens and characterization of plant microbial populations constitute a rapidly evolving research field. Fungal plant diseases are of major phytopathological concern; thus, the development of metabarcoding approaches for the detection of phytopathogenic fungi is becoming increasingly imperative in the context of plant disease prognosis. We developed a multiplex metabarcoding method for the identification of fungal phytopathogens and endophytes in olive young shoots, using the MinION sequencing platform (Oxford Nanopore Technologies). Selected fungal-specific primers were used to amplify three different genomic DNA loci (ITS, beta-tubulin, and 28S LSU) originating from olive twigs. A multiplex metabarcoding approach was initially evaluated using healthy olive twigs, and further assessed with naturally infected olive twig samples. Bioinformatic analysis of basecalled reads was carried out using MinKNOW, BLAST+ and R programming, and results were also evaluated using the BugSeq cloud platform. Data analysis highlighted the approaches based on ITS and their combination with beta-tubulin as the most informative ones according to diversity estimations. Subsequent implementation of the method on symptomatic samples identified major olive pathogens and endophytes including genera such as Cladosporium, Didymosphaeria, Paraconiothyrium, Penicillium, Phoma, Verticillium, and others

Método para mejorar la tolerancia de las plantas al estrés ambiental

Un ácido nucleico que codifica una proteína DBF1 de una planta o un fragmento de la misma capaz de enlazarse con una secuencia de ADN reguladora de cis en DRE seleccionada del grupo de: (a) ácido nucleico que contiene la secuencia de ADN como la presentada en la SEQ ID NO 2, (b) ácido nucleico que codifica una proteína que comprende una secuencia de aminoácidos como la presentada en la SEQ ID NO 3, (c) un fragmento del ácido nucleico de (a), que codifica una proteína de al menos 50 aminoácidos de longitud capaz de enlazarse con una secuencia de ADN reguladora de cis en DRE, y (d) un fragmento del ácido nucleico de (b) que codifica una proteína de al menos 50 aminoácidos de longitud capaz de enlazarse con una secuencia de ADN reguladora de cis en DRE.Peer reviewedConsejo Superior de Investigaciones CientíficasT3 Traducción de patente europe

Method for improving plant tolerance to environmental stress

Filing date: 2002-03-28 -- Priority data: EP 01870069.0 (2001-03-28) US 60/301,912 (2001-06-29)Provided are DNA sequences encoding a novel type of AP2 domain-containing transcription factor as well as methods for obtaining similar sequences. Also described are methods for obtaining plants with improved growth and enhanced stress tolerance, particularly tolerance to osmotic and dehydration stress, such methods comprising expression of such DNA sequences in a plant or parts thereof. Further described are diagnostic compositions comprising the aforementioned DNA sequences and the use of such sequences in plant breeding and/or agriculture.Peer reviewe

Monitoring the Temporal Expression of Genes Involved in Ochratoxin A Production of Aspergillus carbonarius under the Influence of Temperature and Water Activity

The objective of this study was to investigate the effect of environmental factors, namely temperature and water activity, on genes involved in the regulation of ochratoxin A (OTA) production over time. For this purpose, the previously characterized toxigenic Aspergillus carbonarius Ac29 isolate from Greek vineyards and the A. carbonarius ITEM 5010 reference strain were subjected to combined temperature and water activity (aw) treatments to study OTA production and relative gene expression. The fungal isolates were grown on a synthetic grape juice liquid medium (SGM) under different temperature (20 °C, 25 °C and 30 °C) and aw (0.94 and 0.98) regimes. The expression of the AcOTApks, AcOTAnrps, and laeA OTA related genes was investigated using real time PCR. Gene expression was monitored at the same time points, along with fungal biomass and OTA accumulation at three, six and nine days of incubation. In gene expression analysis, stimulation of the biosynthetic genes was observed a few days before any toxin could be detected. This fact may underline a possible early indicator of potential toxin contamination of grapes. However, the transcript levels varied with respect to the different combinations of ecophysiological conditions and time, highlighting a complex regulation of OTA related gene expression of A. carbonarius in the specific medium

Recent Advances in Mycotoxin Analysis and Detection of Mycotoxigenic Fungi in Grapes and Derived Products

Mycotoxins are secondary metabolites of filamentous fungi that can cause toxic effects in human and animal health. Most of the filamentous fungi that produce these mycotoxins belong to four genera, namely, Aspergillus, Penicillium, Fusarium, and Alternaria. Mycotoxigenic fungi, along with mycotoxins, create a constant and serious economic threat for agriculture in many terms, counting product losses due to crop contamination and food spoilage, as well malnutrition when considering nutritional quality degradation. Given the importance of robust and precise diagnostics of mycotoxins and the related producing fungi in the grape food chain, one of the most important agricultural sectors worldwide, the present review initially delivers a comprehensive presentation of mycotoxin reports on grape and derived products, including a wide range of commodities such as fresh grapes, raisins, wine, juices, and other processed products. Next, based on worldwide regulations’ requirements for mycotoxins, and referring to the relative literature, this work presents methodological approaches for mycotoxin determination, and stresses major methods for the detection of fungal species responsible for mycotoxin production. The principle of function and basic technical background on the available analytical and molecular biology techniques developed—including chromatography, mass spectrometry, immunochemical-based assays, biosensors, and molecular assays—is briefly given, and references for their application to grape and derived product testing are highlighted

Biodiversity and ITS-RFLP characterisation of Aspergillus section Nigri isolates in grapes from four traditional grape-producing areas in Greece.

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

Maize DBF1-interactor protein 1 containing an R3H domain is a potential regulator of DBF1 activity in stress responses

The maize dehydration-responsive element (DRE)-binding factor, DBF1, is a member of the Apetala 2/Ethylene Response Factor transcription factors family and is involved in the regulation of the ABA-responsive gene rab17 through the DRE in an ABA-dependent pathway. In this study we analysed the functionality of DBF1 in abiotic stress responses and found that Arabidopsis plants over-expressing DBF1 were more tolerant to osmotic stress than control plants. In yeast two-hybrid analyses, DBF1 interacted with DBF1-interactor protein 1 (DIP1), a protein containing a conserved R3H single-strand DNA-binding domain. Subcellular localization of DIP1 showed that the protein fusion DIP1–Red Flourescent Protein (RFP) was mainly localized in the cytoplasm. However, after co-transformation of DBF1–GFP and DIP1–RFP, both proteins co-localized in the nucleus. Interestingly, when the N-terminal DBF1–GFP was co-expressed with DIP1–RFP, both proteins co-localized predominantly in the cytoplasmic speckles observed for N-terminal DBF1–GFP fusion protein. These results clearly show in vivo interaction of DBF1 with DIP1 in the cell and that this interaction is necessary for the nuclear localization of DIP1 protein. Analysis of the regulatory effect of the DBF1 and DIP1 interaction on the maize rab17 promoter activity indicated that co-transfection of DBF1 with DIP1 enhances promoter activity in the absence of ABA treatment. We suggest that the regulated association of DBF1 and DIP1 may control the levels of target gene expression during stress conditions.This work was supported by grants BIO2003-01133 from MCYT (Spain) and the ROST project QLK5-CT-2002-00841 from EU to MP. CL was supported by a Generalitat de Catalunya fellowship and ED-P by ROST.Peer reviewe

Identification, origin and OTA production of <i>A</i>. section <i>Nigri</i> isolates.

<p>Species identification and origin of <i>A</i>. section <i>Nigri</i> spp. isolated from the present study and their ochratoxigenic potential assayed by HPLC and ELISA methods.</p><p>Limit of Quantification (LOQ) 2 ng OTA g⁻¹ CYA and Limit of Detection (LOD) 1 ng OTA g⁻¹ CYA.</p