Maria Aragona

Improvement of grain legumes general part: diseases

Abstract Grain legume yield is reduced in many growing areas by diseases affecting the roots and the aerial parts of the plants. Correct disease diagnosis, and precise pathogen identification are the prerequisites for the application of control measures, among which resistance is of paramount importance. To achieve effective and stable resistance the strategies used must be based on at least some knowledge of the pathogen populations, and suitable screening methods must be developed. Diagnostic techniques can be divided into: (i) ‘conventional’, including light and electron microscopy, use of selective media and of traditional serological techniques, and (ii) ‘innovative’, based on the analysis of proteins, nucleic acids, and fatty acids. Although the use of innovative methods, namely nucleic-acid based techniques, is rapidly expanding, there is still need to use both techniques, sometimes jointly, to achieve the most reliable results.

Molecular and physiological characterization of Italian isolates of Pyrenochaeta lycopersici

Corky root of tomato caused by Pyrenochaeta lycopersici is a disease of concern in Italy and for many tomato growing areas in the world. Isolates of the fungus were characterized at both the physiological and molecular level. The optimal in vitro growth temperature for all isolates was 23 degrees C. All Italian isolates of P. lycopersici showed similar RAPD and esterase banding patterns. No relevant polymorphisms were detected after enzymatic digestion of PCR-amplified ITS and IGS regions. The overall results indicate a low degree of genetic variability within a collection of 43 Italian isolates. These data are of interest in breeding programs for resistance against corky root of tomato and they provide useful information for the development of molecular diagnostic tools for the rapid identification and detection of P. lycopersici.

De novo genome assembly of the soil-borne fungus and tomato pathogen Pyrenochaeta lycopersici

BackgroundPyrenochaeta lycopersici is a soil-dwelling ascomycete pathogen that causes corky root rot disease in tomato (Solanum lycopersicum) and other Solanaceous crops, reducing fruit yields by up to 75%. Fungal pathogens that infect roots receive less attention than those infecting the aerial parts of crops despite their significant impact on plant growth and fruit production.ResultsWe assembled a 54.9Mb P. lycopersici draft genome sequence based on Illumina short reads, and annotated approximately 17,000 genes. The P. lycopersici genome is closely related to hemibiotrophs and necrotrophs, in agreement with the phenotypic characteristics of the fungus and its lifestyle. Several gene families related to host–pathogen interactions are strongly represented, including those responsible for nutrient absorption, the detoxification of fungicides and plant cell wall degradation, the latter confirming that much of the genome is devoted to the pathogenic activity of the fungus. We did not find a MAT gene, which is consistent with the classification of P. lycopersici as an imperfect fungus, but we observed a significant expansion of the gene families associated with heterokaryon incompatibility (HI).ConclusionsThe P. lycopersici draft genome sequence provided insight into the molecular and genetic basis of the fungal lifestyle, characterizing previously unknown pathogenic behaviors and defining strategies that allow this asexual fungus to increase genetic diversity and to acquire new pathogenic traits.

Molecular and functional characterization of an endoglucanase in the phytopathogenic fungus Pyrenochaeta lycopersici

Many fungal plant pathogens secrete an array of cell wall degrading enzymes mainly involved in the pathogenesis. In this work, a cDNA clone encoding an extracellular endo-1,4-β-glucanase (named PlEGL1) from the causal agent of the Corky Root Rot of tomato, Pyrenochaeta lycopersici, was isolated and characterized, in order to understand its putative role in the pathogenesis and its mechanism of action. Multiple alignment of the deduced amino acidic sequence shows a high homology with other endoglucanases from different phytopathogenic fungi and detects a well-defined conserved domain of the Glycosyl Hydrolase family 61 (GH61). In vitro, Plegl1 gene transcription is correlated to a cellulolytic activity of the fungus, regulated, in its turn, by the presence of sugar and/or cellulose in the culture medium. In the infected plants, expression level of Plegl1 is positively correlated to the development of the disease. PlEGL1 was heterologously expressed in Escherichia coli and the recombinant protein was purified and tested for its cellulolytic ability, showing a very weak activity, in agreement with all the endoglucanases belonging to GH61 family. The finding in this paper will provide the basis for further determination of biochemical properties of the PlEGL1 protein and its possible involvement in the host–pathogen interaction.

Electrophoretic karyotypes of the phytopathogenic Pyrenophora graminea and P. teres

Contour-clamped homogeneous electric field has been applied to analyse genome size and organisation of two important pathogens of barley, Pyrenophora graminea and P. teres. Electrophoretic karyotypes of four P. graminea isolates resolved 6–8 chromosomal bands, depending on the isolate, while the one P. teres isolate analysed contained six chromosomal bands. Chromosome length polymorphism was observed among P. graminea isolates. For both fungi the chromosomes ranged from 1.6 to more than 6 Mb, with chromosomes larger than 6 Mb migrating as a single band within the compression zone of the gel.

Genetic transformation of the tomato pathogen Pyrenochaeta lycopersici allowed gene knockout using a split-marker approach

Pyrenochaeta lycopersici, as other soil-transmitted fungal pathogens, generally received little attention compared to the pathogens affecting the aerial parts of the plants, although causing stunt and important fruit yield reduction of agronomic relevant crops. The scope of this study was to develop a system allowing to investigate the functional role of P. lycopersici genes putatively involved in the corky root rot of tomato. A genetic transformation system based on a split-marker approach was developed and tested to knock out a P. lycopersici gene encoding for a lytic polysaccharide monooxygenase (Plegl1) induced during the disease development. The regions flanking Plegl1 gene were fused with the overlapping parts of hygromycin marker gene, to favour homologous recombination. We were able to obtain four mutants not expressing the Plegl1 gene though, when tested on a susceptible tomato cultivar, Plegl1 mutants showed unaltered virulence, compared with the wild-type strain. The strategy illustrated in the present work demonstrated for the first time that homologous recombination occurs in P. lycopersici. Moreover, a transformation system mediated by Agrobacterium tumefaciens was established and stable genetic transformants have been obtained. The transformation systems developed represent important tools for investigating both the role of genes putatively involved in P. lycopersici interaction with host plant and the function of other physiological traits which emerged to be genetically expanded from the recent genome sequencing of this fungus.

IDENTIFICATION OF TOMATO GENES DIFFERENTIALLY EXPRESSED DURING COMPATIBLE INTERACTION WITH PYRENOCHAETA LYCOPERSICI

Breeding for resistance is the most effective tool for controlling the corky root disease of tomato caused by the fungus Pyrenochaeta lycopersici. However, little is known about the molecular bases of tomato-P. lycopersici interaction. In order to identify genes involved in the basal defence response activated in a susceptible cultivar and in disease symptom development, a set of cDNA-AFLP fragments derived from a profiling experiment was analysed. A total of 247 differentially expressed TDFs (transcript-derived fragments), identified as putative tomato genes, were characterized by similarity searches, and classified into 11 broad functional classes. Timings ranging between the early [48, 72 and 96 h post-infection (hpi)] and the late infection stages (20 and 27 dpi) were used. The changes of tomato root transcriptional profiles showed large differences in quantity and quality between the early and late stages of infection. Mechanisms of basal defence were most likely activated at early stages, when a gene coding for a receptor- like serine-threonine protein kinase and other genes of the signalling class were upregulated. At 20 dpi some of the mechanisms involved in defence were still activated, while at 27 dpi a general repression of gene expression was observed.

Identification of Resistance to Barley Leaf Stripe Using a Pyrenophora graminea Transformant Expressing β-glucuronidase

A Pyrenophora graminea strain expressing the β-glucuronidase gene (GUS) was obtained via genetic transformation, and used to follow the penetration of the pathogen inside barley germinating seeds and the colonization of host tissues. Significant differences between resistant and susceptible barley cultivars were observed in the colonization of artificially-infected embryos by the fungus. These results suggest that the GUS transgenic strain of P. graminea will be useful for the early screening of barley cultivars for resistance to leaf stripe disease.

The genome assembly of the fungal pathogen Pyrenochaeta lycopersici from Single-Molecule Real-Time sequencing sheds new light on its biological complexity

The first draft genome sequencing of the non-model fungal pathogen Pyrenochaeta lycopersici showed an expansion of gene families associated with heterokaryon incompatibility and lacking of mating-type genes, providing insights into the genetic basis of this “imperfect” fungus which lost the ability to produce the sexual stage. However, due to the Illumina short-read technology, the draft genome was too fragmented to allow a comprehensive characterization of the genome, especially of the repetitive sequence fraction. In this work, the sequencing of another P. lycopersici isolate using long-read Single Molecule Real-Time sequencing technology was performed with the aim of obtaining a gapless genome. Indeed, a gapless genome assembly of 62.7 Mb was obtained, with a fraction of repetitive sequences representing 30% of the total bases. The gene content of the two P. lycopersici isolates was very similar, and the large difference in genome size (about 8 Mb) might be attributable to the high fraction of repetitive sequences detected for the new sequenced isolate. The role of repetitive elements, including transposable elements, in modulating virulence effectors is well established in fungal plant pathogens. Moreover, transposable elements are of fundamental importance in creating and re-modelling genes, especially in imperfect fungi. Their abundance in P. lycopersici, together with the large expansion of heterokaryon incompatibility genes in both sequenced isolates, suggest the presence of possible mechanisms alternative to gene re-assorting mediated by sexual recombination. A quite large fraction (~9%) of repetitive elements in P. lycopersici, has no homology with known classes, strengthening this hypothesis. The availability of a gapless genome of P. lycopersici allowed the in-depth analysis of its genome content, by annotating functional genes and TEs. This goal will be an important resource for shedding light on the evolution of the reproductive and pathogenic behaviour of this soilborne pathogen and the onset of a possible speciation within this species.