Deciphering Molecular Host-Pathogen Interactions During Ramularia Collo-Cygni Infection on Barley.

Ramularia collo-cygni is the causal agent of Ramularia leaf spot disease (RLS) on barley and became, during the recent decades, an increasing threat for farmers across the world. Here, we analyze morphological, transcriptional, and metabolic responses of two barley cultivars having contrasting tolerance to RLS, when infected by an aggressive or mild R. collo-cygni isolate. We found that fungal biomass in leaves of the two cultivars does not correlate with their tolerance to RLS, and both cultivars displayed cell wall reinforcement at the point of contact with the fungal hyphae. Comparative transcriptome analysis identified that the largest transcriptional differences between cultivars are at the early stages of fungal colonization with differential expression of kinases, calmodulins, and defense proteins. Weighted gene co-expression network analysis identified modules of co-expressed genes, and hub genes important for cultivar responses to the two R. collo-cygni isolates. Metabolite analyses of the same leaves identified defense compounds such as p-CHDA and serotonin, correlating with responses observed at transcriptome and morphological level. Together these all-round responses of barley to R. collo-cygni provide molecular tools for further development of genetic and physiological markers that may be tested for improving tolerance of barley to this fungal pathogen.

The genome of the emerging barley pathogen Ramularia collo-cygni.

BACKGROUND: Ramularia collo-cygni is a newly important, foliar fungal pathogen of barley that causes the disease Ramularia leaf spot. The fungus exhibits a prolonged endophytic growth stage before switching life habit to become an aggressive, necrotrophic pathogen that causes significant losses to green leaf area and hence grain yield and quality. RESULTS: The R. collo-cygni genome was sequenced using a combination of Illumina and Roche 454 technologies. The draft assembly of 30.3 Mb contained 11,617 predicted gene models. Our phylogenomic analysis confirmed the classification of this ascomycete fungus within the family Mycosphaerellaceae, order Capnodiales of the class Dothideomycetes. A predicted secretome comprising 1053 proteins included redox-related enzymes and carbohydrate-modifying enzymes and proteases. The relative paucity of plant cell wall degrading enzyme genes may be associated with the stealth pathogenesis characteristic of plant pathogens from the Mycosphaerellaceae. A large number of genes associated with secondary metabolite production, including homologs of toxin biosynthesis genes found in other Dothideomycete plant pathogens, were identified. CONCLUSIONS: The genome sequence of R. collo-cygni provides a framework for understanding the genetic basis of pathogenesis in this important emerging pathogen. The reduced complement of carbohydrate-degrading enzyme genes is likely to reflect a strategy to avoid detection by host defences during its prolonged asymptomatic growth. Of particular interest will be the analysis of R. collo-cygni gene expression during interactions with the host barley, to understand what triggers this fungus to switch from being a benign endophyte to an aggressive necrotroph.

Ramularia collo-cygni--An Emerging Pathogen of Barley Crops.

Ramularia collo-cygni is the biotic factor responsible for the disease Ramularia leaf spot (RLS) of barley (Hordeum vulgare). Despite having been described over 100 years ago and being considered a minor disease in some countries, the fungus is attracting interest in the scientific community as a result of the increasing number of recorded economically damaging disease epidemics. New reports of disease spread and fungal identification using molecular diagnostics have helped redefine RLS as a global disease. This review describes recent developments in our understanding of the biology and epidemiology of the fungus, outlines advances made in the field of the genetics of both the fungus and host, and summarizes the control strategies currently available.

Control of foliar pathogens of spring barley using a combination of resistance elicitors.

The ability of the resistance elicitors acibenzolar-S-methyl (ASM), ß-aminobutyric acid (BABA), cis-jasmone (CJ), and a combination of the three products, to control infection of spring barley by Rhynchosporium commune was examined under glasshouse conditions. Significant control of R. commune was provided by ASM and CJ, but the largest reduction in infection was obtained with the combination of the three elicitors. This elicitor combination was found to up-regulate the expression of PR-1b, which is used as a molecular marker for systemic acquired resistance (SAR). However, the elicitor combination also down-regulated the expression of LOX2, a gene involved in the biosynthesis of jasmonic acid (JA). In field experiments over 3 consecutive years, the effects of the elicitor combination were influenced greatly by crop variety and by year. For example, the elicitor combination applied on its own provided significant control of powdery mildew (Blumeria graminis f.sp. hordei) and R. commune in 2009, whereas no control on either variety was observed in 2007. In contrast, treatments involving both the elicitor combination and fungicides provided disease control and yield increases which were equal to, and in some cases better than that provided by the best fungicide-only treatment. The prospects for the use of elicitor plus fungicide treatments to control foliar pathogens of spring barley in practice are discussed.

Controlling crop diseases using induced resistance: challenges for the future.

A number of different types of induced resistance have been defined based on differences in signalling pathways and spectra of effectiveness, including systemic acquired resistance and induced systemic resistance. Such resistance can be induced in plants by application of a variety of biotic and abiotic agents. The resulting resistance tends to be broad-spectrum and can be long-lasting, but is rarely complete, with most inducing agents reducing disease by between 20 and 85%. Since induced resistance is a host response, its expression under field conditions is likely to be influenced by a number of factors, including the environment, genotype, crop nutrition and the extent to which plants are already induced. Although research in this area has increased over the last few years, our understanding of the impact of these influences on the expression of induced resistance is still poor. There have also been a number of studies in recent years aimed at understanding of how best to use induced resistance in practical crop protection. However, such studies are relatively rare and further research geared towards incorporating induced resistance into disease management programmes, if appropriate, is required.

Cultivar Effects on the Expression of Induced Resistance in Spring Barley.

The influence of host genotype on the expression of induced resistance was examined in several cultivars of spring barley (Hordeum vulgare). Induced resistance was activated using a combination of elicitors (acibenzolar- S-methyl, ß-aminobutyric acid, and cis-jasmone) shown in previous work to induce resistance effectively in barley. The barley cultivars examined were Cellar, Chalice, Decanter, Oxbridge, Tipple, Troon, and Westminster, which differed in their genetic resistance to two major pathogens of barley, Rhynchosporium secalis and Blumeria graminis f. sp. hordei. Controlled-environment studies showed that, although the elicitor combination reduced levels of R. secalis in all but one cultivar, the magnitude of the reduction differed among cultivars. Similar results were obtained in field experiments in 2007, 2008, and 2009, although there was inconsistency in cultivar effects between years, with the elicitor providing disease control in some cultivars in some years and not others. Use of the elicitor combination produced no significant effect on grain yield compared with untreated plants in most cases, although significant increases in grain yield were obtained with the elicitor treatment in two cultivars in 2007 and one cultivar in 2009. Analysis of the defense-related enzyme cinnamyl alcohol dehydrogenase in leaf samples from the field experiment in 2007 showed that activity of the enzyme was already high prior to elicitor application, although activity was increased further in one cultivar following elicitor treatment. It is possible, therefore, that these plants were already induced. Further work is required to confirm this and to determine whether prior induction has any bearing on the variable disease control obtained from elicitors in spring barley.

Ramularia collo-cygni: the biology of an emerging pathogen of barley.

Ramularia collo-cygni is now recognized as an important pathogen of barley in Northern Europe and New Zealand. It induces necrotic spotting and premature leaf senescence, leading to loss of green leaf area in crops, and can result in substantial yield losses. The fungus produces a number of anthraquinone toxins called rubellins, which act as host nonspecific toxins with photodynamic activity. These toxins induce lipid peroxidation and are possibly the cause of the chlorosis and necrosis observed in leaves infected with R. collo-cygni. The fact that the fungus can remain latent in barley plants until flowering, coupled with its very slow growth in vitro, makes it difficult to detect in crops. As a result, the epidemiology of this pathogen remains poorly understood. However, the recent development of rapid and reliable PCR methods for specific detection of R. collo-cygni offers the prospect of increased understanding of its epidemiology and improved disease control.

Rapid nested PCR-based detection of Ramularia collo-cygni direct from barley.

Ramularia collo-cygni is a barley pathogen of increasing importance in Northern and Central Europe, New Zealand and South America. Accurate visual and microscopic identification of the pathogen from diseased tissue is difficult. A nested PCR-based diagnostic test has been developed as part of an initiative to map the distribution of the pathogen in Scotland. The entire nuclear ribosomal internal transcribed spacer and 5.8S rRNA gene regions from 14 isolates of diverse global origin exhibited complete homology following sequence characterization. Two pairs of species-specific primers, based on inter-specific sequence divergence with closely related species, were designed and empirically evaluated for diagnostic nested PCR. Nested primers Rcc3 and Rcc4 consistently amplified a single product of 256 bp from DNA of 24 R. collo-cygni isolates of diverse global provenance, but not from other Ramularia species, or other fungi commonly encountered in cereal pathosystems, as well as Hordeum or Secale DNA preparations. Using this approach, R. collo-cygni was successfully identified from naturally infected barley leaf, awn and grain samples of diverse geographical provenance, in particular from symptoms that lacked the presence of characteristic conidiophores. It is envisaged that this assay will become established as an important tool in continuing studies into the ecology, aetiology and epidemiology of this poorly understood yet economically damaging plant pathogen.