Mariarosaria Vergara

Effects of anoxia on sucrose degrading enzymes in cereal seeds

Summary We report here data on the effects of anaerobic conditions on the status of cytosolic sucrose degrading enzymes. The activities of sucrose synthase (EC 2.4.1.13) and cytosolic alkaline invertase (EC 3.2.1.26) were studied in rice ( Oryza sativa L.), maize ( Zea mais L.), wheat ( Triticum aestivum L.), and barley ( Hordeum vulgare L.) seeds and seedlings subjected to air-anoxia transition or directly sown under anaerobic conditions. The activity of alcohol dehydrogenase (EC 1.1.1.1) is also reported, to allow comparison with a typical anaerobic polypeptide. The data, discussed and compared with the other available reports on this subject, indicate sucrose synthase induction at the transcriptional level also in maize seedlings, and suggest that sucrose synthase is the major sucrose-degrading enzyme in cereal seeds directly sown under anaerobic conditions.

A comparative study of Neogymnomyces virgineus, a new keratinolytic species from dung, and its relationships with the Onygenales

Isolations of onygenalean fungi were made recently from different dung samples from Italy. A striking snow-white species with gymnothecial ascomata, developed in damp chamber on dormouse dung collected in a cave, was subjected to keratinolytic tests and morphological, cultural, and phylogenetic studies. The keratinolytic ability of this species, associated with a Chrysosporium anamorph and a sexual state of appendiculate reticuloperidia and oblate ascospores, allows it to be accomodated in Onygenaceae. White ascomata, blunt or subcapitate peridial appendages, pitted ascospores, and tuberculate conidia suggest it to be a new Neogymnomyces, and this was confirmed by parsimony analyses of LSU and ITS nrDNA sequences. Following recent phylogenetic analyses, the morphological and physiological features of order Onygenales and its families are re–examined and discussed. After the introduction of a new species, Neogymnomyces is reviewed and compared with all other genera in Onygenaceae. The Chrysosporium imperfect state of Neogymnomyces virgineus is described and compared to the anamorph of N. demonbreunii. It is also compared to the atypical Chrysosporium merdarium and to several other Chrysosporium species with echinulate to verrucose–tuberculate conidia, isolated from guano, dung, and nitrogen–rich soils in caves. The onygenalean fungi isolated from any kind of dung are discussed and their facultative coprophily ascribed to variable faecal contents of keratin or other degradable substances. A key to the families and genera of the Onygenales is provided.

A Coding Region in Diaporthe Helianthi Reveals Genetic Variability Among Isolates of Different Geographic Origin

A set of Diaporthe helianthi isolates collected in different geographic areas was studied in order to examine whether different genetic biotypes could be responsible for epidemiological differences shown by sunflower stem canker. D. helianthi causes serious losses in France and in former Yugoslavia, while the pathogen is only sporadically recorded in Italy in spite of conducive pedoclimatic conditions. Variability of a D. helianthi coding genomic region, evaluated by means of polymerase chain reaction (PCR), Southern blot hybridisation and restriction fragments length polymorphism (RFLP), showed a conserved homogeneous pattern shared by French and Yugoslavian isolates compared with the heterogeneous pattern of Italian isolates. These results are consistent with other investigations (IGS and ITS region variability) performed on the same set of isolates, allowing a correlation between D. helianthi biotypes, their geographic origin and sunflower stem canker epidemiology.

Plasmid distribution in European Diaporthe helianthi isolates

Diaporthe helianthi is the causal agent of sunflower stem canker, a serious pathogen of sunflower in Europe, which has been sporadically recorded in Italy. A collection of 26 Diaporthe helianthi isolates deriving from different geographic origins was analysed in order to determine the presence of extra-chromosomal genetic determinants and their molecular diversity. Extra-chromosomal bands in total genomic DNAs were identified in every French and the Yugoslavian isolate and in only one Italian isolate, while no Romanian and Argentinean isolates resulted to host any plasmids. When tested for their chemicophysical nature, they were recognised as linear plasmids sized about 2.3xa0Kb. A more detailed analysis was performed on a plasmid purified from a French isolate (plasmid F). Its intracellular localisation resulted as mitochondrial. Plasmid F was also exploited as a probe in Southern hybridisation experiments, in which it recognised only plasmids present in the genomes of French and Yugoslavian isolates (countries were the disease has a heavy incidence) indicating a strong correlation to geographic origin. An RFLP hybridisation analysis performed on genomic DNAs revealed a homogeneous restriction pattern in all French and Yugoslavian isolates, suggesting molecular homology among plasmids present in those isolates.

A Protocol for Nonradioactive Differential Display Tested on Carrot Auxin-Resistant Mutants

A new protocol for nonradioactive differential display visualized by ethidium bromide staining was developed for carrot total RNA. The procedure was used to identify genes specifically expressed in carrot auxin-resistant mutants compared to their wild-type. Different populations of total RNAs were reversely transcribed using three anchored oligo-dT primers. PCR amplification of relative cDNAs was carried out in combination with five arbitrary primers. New polyacrylamide gel properties (lower matrix concentration, higher thickness, nondenaturing gel and ethidium bromide staining) were combined to provide a simplified protocol that can resolve amplified transcripts over a large range of molecular weights. Several specific transcripts were successfully identified by this procedure.

Polyketide synthases of Diaporthe helianthi and involvement of DhPKS1 in virulence on sunflower

BackgroundThe early phases of Diaporthe helianthi pathogenesis on sunflower are characterized by the production of phytotoxins that may play a role in host colonisation. In previous studies, phytotoxins of a polyketidic nature were isolated and purified from culture filtrates of virulent strains of D. helianthi isolated from sunflower. A highly aggressive isolate (7/96) from France contained a gene fragment of a putative nonaketide synthase (lovB) which was conserved in a virulent D. helianthi population.ResultsIn order to investigate the role of polyketide synthases in D. helianthi 7/96, a draft genome of this isolate was examined. We were able to find and phylogenetically analyse 40 genes putatively coding for polyketide synthases (PKSs). Analysis of their domains revealed that most PKS genes of D. helianthi are reducing PKSs, whereas only eight lacked reducing domains. Most of the identified PKSs have orthologs shown to be virulence factors or genetic determinants for toxin production in other pathogenic fungi. One of the genes (DhPKS1) corresponded to the previously cloned D. helianthi lovB gene fragment and clustered with a nonribosomal peptide synthetase (NRPS) -PKS hybrid/lovastatin nonaketide like A. nidulans LovB. We used DhPKS1 as a case study and carried out its disruption through Agrobacterium-mediated transformation in the isolate 7/96. D. helianthi DhPKS1 deleted mutants were less virulent to sunflower compared to the wild type, indicating a role for this gene in the pathogenesis of the fungus.ConclusionThe PKS sequences analysed and reported here constitute a new genomic resource that will be useful for further research on the biology, ecology and evolution of D. helianthi and generally of fungal plant pathogens.

The Constitutive Endopolygalacturonase TvPG2 Regulates the Induction of Plant Systemic Resistance by Trichoderma virens

Trichoderma spp. are opportunistic fungi some of which are commonly present in the rhizosphere. Several species, such as T. virens, are also efficient biocontrol agents against phytopathogenic fungi and exert beneficial effects on plants. These effects are the consequence of interactions between Trichoderma and plant roots, which trigger enhanced plant growth and induce plant resistance. We have previously shown that T. virens I10 expresses two endopolygalacturonase genes, tvpg1 and tvpg2, during the interaction with plant roots; tvpg1 is inducible while tvpg2 is constitutively transcribed. Using the same system, the tomato polygalacturonase-inhibitor gene Lepgip1 was induced at the same time as tvpg1. Here we show by gene disruption that TvPG2 performs a regulatory role on the inducible tvpg1 gene and in triggering the plant immune response. A tvpg2-knockout strain fails to transcribe the inducible tvpg1 gene in neither in vitro in inducing media containing pectin or plant cell walls, nor during the in vivo interaction with tomato roots. Likewise, the in vivo induction of Lepgip1 does not occur, and its defense against the pathogen Botrytis cinerea is significantly reduced. Our data prove the importance of a T. virens constitutively produced endopolygalacturonase in eliciting plant induced systemic resistance against pathogenic fungi.