Studies on Ramularia Leaf Spots on Barley - Resistance Phenotyping, Epidemiology and Pathogenicity

Abstract

The deuteromycete, Ramularia collo-cygni (Rcc) has gained increasing importance as the causal agent of a novel leaf spot disease, Ramularia leaf spot (RLS), on barley. The disease occurs conspicuously late in the growing season. When the crop has passed the flowering stage, the disease severity in the field increases dramatically causing complete browning and leaf death within 12 days. Studies on Ramularia collo-cygni are fairly recent and classical and molecular knowledge on the disease epidemiology and control are continuously improved. Recently, molecular bioanalytical tools based on PCR have been developed and are now available for the detection of Rcc in plant tissues. This study aimed to improve knowledge on the epidemiology, pathogenicity, sources of inoculum and spread of Rcc, and to develop reliable phenotyping assays for resistance evaluation in different barley genotypes under field and greenhouse conditions. Initially, a SYBR Green-based quantitative polymerase chain reaction (qPCR) assay for quantification of fungal DNA in plant material was developed. Rcc was successfully detected and identified down to 0.1 pg in culture, in host plant tissues and in seeds as well as in rain and snow. Qualitative PCR analyses revealed the transmission of the fungus from seeds into emerging plants and confirmed the spread of the fungus in adult plants in a symptom-less manner. Under greenhouse conditions, the symptomless stage of RLS persisted throughout the first generation of plants emerging from infested seeds until maturity and grain formation. Hot water treatment of seeds did not eliminate Rcc or impair the transfer of the pathogen into the developing plant. Further studies on effects of seed dressings and consecutive applications of foliar fungicides during plant development were conducted in order to produce pathogen-free plants. The efficacy of the seed dressing fungicide Zardex G (cyproconazole and imazalil) and the systemic foliar fungicide Proline (prothioconazole) on fungal systemic spread was assessed during different growth stages by means of real time PCR. Results show that both fungicides were not effective in controlling fungal development inside the plants when used separately. However, using Zardex G before sowing the seeds and applying Proline at early growth stages (39-41) had the strongest inhibitory effect on fungal development. In a parallel study, the mobility and spread of Rcc inoculum through the air and over large distances was investigated using spore traps placed either close to a barley field or in a place at long distance from any fields. Rcc conidia were detectable during late autumn and winter months at larger distance from fields and in higher elevation above ground. This suggests that Rcc inoculum is widespread also in the cooler season and may spread over large distances via the atmosphere and in rain water or snow. In the screening assay, different spring barley genotypes were evaluated for resistance to RLS. Evaluations were conducted in replicated experiments in a growth chamber (with leaf segments) and under greenhouse and field conditions (with whole plants) at mature growth stages (73-75). Genotypes displayed significant differences in their response to Rcc infection in the field, greenhouse, and the growth chamber experiments. Upon naturally infection in the field, the cultivar IPZ 24727 was significantly more resistant to Rcc compared to the other cultivars. A significant correlation has been shown between greenhouse experiment (whole plant inoculation) and field experiment 2009 (p=0.005, rs=0.483) and field experiment 2010 (p=0.03, rs=0.384). A significant correlation was found between leaf segment assay and severity of leaf symptoms in the greenhouse experiments (p=0.0002, rs=0.592) and leaf segment assay between field experiment 2009 (p=0.0005, rs=0.576) and field experiment 2010 (p=0.002, rs=0.513). A significant correlation has been also observed between field experiments in two different years (p=0.04, rs=0.419). By using qPCR, DNA of Rcc was detected in all barley genotypes. This suggests that none of the spring barley cultivars was completely resistant to Rcc. The results indicate that the PCR was able to detect the presence of the pathogen before appearance of the symptoms at early growth stages. Quantitative real time PCR analyses demonstrated a strong correlation (p=0.00179, rs=0.851) between the visual disease symptoms and fungal DNA concentration in leaf F-1, suggesting that quantitative real-time PCR can be used for the selection of resistant barley. Furthermore, a novel detection method for the Rcc phytotoxin, rubellin, was developed by using HPLC with fluorescence detector. With this method, the presence of the toxin was detected before appearance of the symptoms at early growth stages. Levels of fungal toxins in infected leaf tissue correlated strongly (p=0.00005, rs=0.966) with the visual disease symptoms. These results demonstrate the potential for screening barley cultivars for Rcc resistance under controlled conditions.