Ramularia leaf spot in barley

Ramularia leaf spot is an emerging pathogen across barley growing regions of the world. It's rise from minor to major disease has been rapid over the last twenty years. The causal pathogen, Ramularia collo-cygni is poorly understood but it has been shown to have a complex life cycle and the ability to exist on many hosts in an endophytic state. The rate of development of fungicide resistance in the fungus is also extremely fast and many of the major single site fungicides are no longer effective in many countries. With multisite fungicides having their approval or reconsidered and no consistent varietal resistance available, control of the disease is increasing challenging. This chapter reviews the latest research into Ramularia biology and control and highlights the areas where recent advances have been made.

Fungicide Resistance Evolving in Ramularia Collo-Cygni Population in Estonia

Ramularia leaf spot caused by the fungus Ramularia collo-cygni, has recently become widespread in Estonian barley fields. Currently, disease control in barley fields relies on SDHI and DMI fungicides, which might be threatened by R. collo-cygni isolates that are well-adapted to fungicide pressure. In a two-year study, 353 R. collo-cygni isolates were collected from spring barley fields in Estonia. A total of 153 R. collo-cygni isolates were examined for sensitivity to azoles (DMIs; prothioconazole-desthio, epoxiconazole, mefentrifluconazole) and succinate dehydrogenase inhibitors (SDHIs; boscalid, fluxapyroxad). Epoxiconazole was the least effective and a new fungicide mefentrifluconazole was the most effective DMI. Among SDHIs, fluxapyroxad was more effective than boscalid. Also, single R. collo-cygni isolates with high resistance to tested fungicides occurred, which could affect fungicide control of the pathogen. The entire collection of R. collo-cygni was analysed for mutations in fungicide target proteins. Six mutations were identified in CYP51 gene, the most dominant being I381T, I384T, and S459C. Also, numerous point mutations in the SdhC gene were present. The mutation G143A in strobilurin target protein CytB dominates in over 80% of the R. collo-cygni population, confirming the low efficacy of strobilurin fungicides in barley disease control.

Ramularia leaf spot: PCR-based methods reveal widespread distribution of Ramulariopsis pseudogycines and limited presence of R. gossypii in Brazil

Whilst Brazil is the fourth largest cotton producer globally, incidence of ramularia leaf spot (RLS) has decreased yield across all growing regions. In 2017-18 and 2018-19 growing seasons, ca. 300 fungal samples were collected from seven Brazilian states. Hyphal tip cultures were obtained for DNA extraction and amplification of partial sequences of the RNA polymerase II gene (RPB2), 28S rRNA, the ribosomal DNA internal transcribed spacers (ITS), actin (ACT), elongation factor (EF1-α) and histone H3 (HIS3) gene regions. Genetic diversity of the isolates was evaluated using fourteen molecular markers. Clade assignments based on the concatenated-sequence tree (RPB2, LSU, EF1-α, ITS, ACT, and HIS3) were identical to those in tree clades generated by RPB2-sequences, as well as in an RPB2 haplotype network, in an ISSR (TGTC)4 dendrogram, and based on morphological comparisons. In total, 252 out of 267 isolates were identified as Ramulariopsis pseudoglycines, indicating this species as the most widespread causal agent of cotton RLS in the Brazilian growing regions today. Validation of the ISSR (TGTC)4 primer as a tool to study the diversity and distribution of Ramulariopsis species will make it possible to carry out extensive RLS sampling studies worldwide.

Ramularia leaf spot and Boll rot are affected differently by organic and inorganic nitrogen fertilization in cotton plants

Background: In this work, the interaction among nitrogen fertilization using bovine manure, poultry manure, Jatropha curcas seed cake and urea, and the diseases Ramularia leaf spot (RLS) and Boll rot (BR), caused by Ramulariopsis pseudoglycines and Diplodia gossypina, respectively, on cotton plants (Gossypium hirsutum L.), was studied under field conditions. The intensity (incidence and severity in percentage) of RLS and the incidence (%) of BR were evaluated six times, starting in the reproductive stage B1 (first visible flower bud). A randomized complete block experimental design with a 4x4 factorial arrangement (fertilizers x dose), totaling 16 treatments were used. The disease progress was analyzed with the nonlinear Logistic and Gomperts models, obtaining the initial disease's epidemiological parameters (Y0) and progress rate (r). Results: Cotton plants fertilized with 100 kg N ha-1 of J. curcas seed cake and poultry manure with 100 and 200 kg N ha-1 showed an incidence between 16 and 21% of RLS. In contrast, plants fertilized with bovine manure presented the highest incidence of ramularia leaf spot (33%). Regarding factor B (dose), cotton plants fertilized with 50 kg N ha-1, showed a higher percentage of BR incidence, being different from those fertilized with the other doses. In the analysis of the interaction corresponding to the BR incidence, no response pattern was found in the doses for each fertilizer. No correlation was observed between the three health variables analyzed, finding probabilities between 0.002 and 0.892. In the temporal progress of RLS incidence, it was shown that fertilization with J. curcas seedcake and poultry manure was lower than the rest. In severity, the plants were fertilized by J. curcas seedcake concerning the rest. The progression curve of RLS severity temporarily increased similarly, observing that plants fertilized with higher doses reached a lower final proportion of the disease.Regarding the modeling of epidemics using the nonlinear Logistic and Gompertz models, the first model better represented the RLS, except for factor B, where none of the curves was significant. In the Logistic model, a lower amount of initial disease was found (Y0) of RLS, both in incidence (0.259) as in severity (0.081), in plants fertilized with J. curcas seed cake and poultry manure, compared to the rest. For the case of BR incidence, none of the nonlinear models used could be represented.Conclusions: Plants fertilized with 50 kg N ha-1, presented an incidence twice higher than those obtained with other fertilizers. The Logistic model better fits RLS, but no model was adapted to BR. Only the RLS epidemiological parameters were affected differently in this experiment compared to BR disease.

Fungicide strategies and resistance of Ramularia collo-cygni to demethylation and succinate dehydrogenase inhibitors in Austrian winter barley (Hordeum vulgare)

Ramularia collo-cygni B. Sutton and J.M. Waller is a major disease in Austrian barley-growing regions. To date, fungicide application is the most effective method to manage the disease; however, fungicide resistance to demethylation and succinate dehydrogenase inhibitors has developed over the last few years. In the growing seasons 2016/2017 and 2017/2018, field trials were carried out to analyze the efficiency of fungicide strategies based on different fungicide classes. Disease development, growth parameters and monitoring of CYP51 and sdh mutations were determined. Fungicide treatments resulted in higher disease control, green leaf area and grain yield. In Austrian R. collo-cygni field populations, the frequency of the mutations CYP51-I325T and CYP51-I328L was low to moderate. Frequency of mutations sdhC-H146R and sdhC-H153R was low. Frequencies of CYP51-I325T and -I328L were similar and increased following DMI application. Frequency of sdhC-H146R was higher compared to sdhC-H153R. The SDHI benzovindiflupyr showed a higher selection rate for sdh mutations compared to bixafen. These sdh mutations were not selected if chlorothalonil was used as mixing partner, leading to a stable composition of sdh resistance alleles over the last two years. Chlorothalonil was proven to be an effective tool for anti-resistance strategies. Currently, SDHIs and DMIs are the backbone of Ramularia leaf spot control in Austria; however, the level of resistance is likely to increase in absence of suitable anti-resistance strategies and following the ban of chlorothalonil.

Ramularia leaf spot disease of barley is highly host genotype-dependent and suppressed by continuous drought stress in the field

Since the 1980s, Ramularia leaf spot (RLS) is an emerging barley disease worldwide. The control of RLS is increasingly aggravated by a recent decline in fungicide efficacy and a lack of RLS-resistant cultivars. Furthermore, climate change increases drought periods in Europe, enhances variable weather conditions, and thus will have an impact on severity of plant diseases. Hence, identification of RLS-resistant cultivars and understanding of disease progression under abiotic stress are important aims in integrated disease management under climate change. In the present study, we evaluated quantitative RLS resistance of 15 spring barley genotypes under drought, controlled irrigation and field conditions between 2016 and 2019 and monitored microclimatic conditions within the canopy. We identified genotypes that show robust quantitative resistance to RLS in different field environments. Our findings suggest that long-lasting drought periods create unfavourable conditions for the disease and supports that the extent and duration of leaf wetness is a key factor for RLS epidemics.

Ramularia leaf spot disease of barley is highly host genotype-dependent and suppressed by continuous drought stress in the field

Since the 1980s, Ramularia leaf spot (RLS) is an emerging barley disease world-wide. The control of RLS is increasingly aggravated by a recent decline in fungicide efficacy and a lack of RLS-resistant cultivars. Furthermore, climate change increases drought periods in Europe, enhances variable weather conditions and thus will have an impact on severity of plant diseases. Hence, identification of RLS-resistant cultivars and understanding of disease progression under abiotic stress are important aims in integrated disease management under climate change. In the present study, we evaluated quantitative RLS resistance of 15 spring barley genotypes under drought, controlled irrigation and field conditions between 2016 and 2019 and monitored microclimatic conditions within the canopy. We identified genotypes that show robust quantitative resistance to RLS in different field environments. Our findings suggest that long-lasting drought periods create unfavourable conditions for the disease and supports that the extent and duration of leaf wetness is a key factor for RLS epidemics.

The Current Epidemic of the Barley Pathogen Ramularia collo-cygni Derives from a Population Expansion and Shows Global Admixture

Ramularia leaf spot is becoming an ever-increasing problem in main barley-growing regions since the 1980s, causing up to 70% yield loss in extreme cases. Yet, the causal agent Ramularia collo-cygni remains poorly studied. The diversity of the pathogen in the field thus far remains unknown. Furthermore, it is unknown to what extent the pathogen has a sexual reproductive cycle. The teleomorph of R. collo-cygni has not been observed. To study the genetic diversity of R. collo-cygni and get more insights in its biology, we sequenced the genomes of 19 R. collo-cygni isolates from multiple geographic locations and diverse hosts. Nucleotide polymorphism analyses of all isolates shows that R. collo-cygni is genetically diverse worldwide, with little geographic or host specific differentiation. Next, we used two different methods to detect signals of recombination in our sample set. Both methods find putative recombination events, which indicate that sexual reproduction happens or has happened in the global R. collo-cygni population. Lastly, we used these data on recombination to perform historic population size analyses. These suggest that the effective population size of R. collo-cygni decreased during the domestication of barley and subsequently grew with the rise of agriculture. Our findings deepen our understanding of R. collo-cygni biology and can help us to understand the current epidemic. We discuss how our findings support possible global spread through seed transfer, and we highlight how recombination, clonal spreading, and lack of host specificity could amplify global epidemics of this increasingly important disease and suggest specific approaches to combat the pathogen.