Ramularia leaf spots of barley

The deuteromycete Ramularia collo-cygni is a fungal pathogen which has gained increasing importance as the inciting agent of a novel leaf spot disease, called Ramularia leaf spots, on winter and spring barley within the last decade. Ramularia collo-cygni was discovered by Cavara 1893 in northern Italy and described as Ophiocladium hordei Cav. Since 20 years symptoms of Ramularia leaf spot have been observed in barley in Europe with increasing tendency, but also in other countries like New Zealand, South America (Sachs, 2002; Sheridan ,1996; Sutton & Waller, 1988) and Canada (Oxley pers. comm.). The pathogen was finally named 1988 by Sutton and Waller R. collo-cygni after the S-shape of the conidiophores looking like a swan’s neck (Figure 1).

The first report of significant yield losses (-10%) caused by massive infection of barley by R. collo-cygni was based on observations carried out in Austria (Huss et al., 1987). Heavy infections were also reported from Scotland and Ireland in 1997 (Millar, 1998; Burke et al., 2000), and from Norway (Salamati, 2002). Showing, that this fungi fulfil the Koch`sche postulate proved that this fungi is a real parasitic fungi (Huss & Sachs 1998). It is still unclear if it belongs to the group of hemi-biotrophic pathogens because of its long endophytic phase in the leaves or if it is a pertotrophic fungi. Despite its increasing importance as a pathogen less is known about the live-cycle of these fungi. One main R. collo-cygni inoculum are conidiophores. Because of their small size it can be assumed that conidiophores spread out with the wind and get transported via long distances by this way. However, till now the source of inoculum is not known but it is supposed from surveys that one source might be wild grasses, contaminated seeds or straw left in the fields. It is also likely that R. collo-cygni is hibernating in winter barley after sawing, spreading out in the spring and is jumping over to spring barley. We established one field experiment with different tillage intensities and different type of crop rotation to evaluate if these factors influence the intensity or spread of the disease and are feasible sources of inoculum. To detect and quantify R. collo-cygni in plant material ELISA technique and a more sensitive molecular method using PCR were established and RCC was detected first in October in winter barley after sawing in this experimental site. Since October RCC was detected monthly in this site. Quantitative PCR will now be established to enable us to make a quantitative determination of R. collo-cygni. Although, till now no teleomorph of R. collo-cygni is known, strong evidences from molecular studies exist that the teleomorph will belong to the Mycosphaerella genus as this fungus was phylogenetic clustered together with Cercospora, Cladosporium and Septoria tritici, all known to belong to the Mycosporella genus (Crous et al., 2001).

For spore germination on the leaf the fungus deserves high humidity and permanent leaf wetness, while radiation and temperature seems to be no key factor (Formayer et al., 2002). After spore germination on the surface of a leaf penetration occurs through the stomata followed by the invasion of the apoplast. Phenotypically the fungus becomes visible after appearance of small necrotic spots on the older senescing (lower) leaves, then spread rapidly on the younger leaves with the further maturation of the plants. Ramularia leaf spot results in the production of small, 1-2 mm long, dark brown spots on leaves, leaf sheaths, stems, and awns of barley (Figure 2,3). These symptoms normally develop only after ear emergence in the crop. The lesions typically are contained by the leaf veins. Their brown colour intensifies toward the centre, while the edges are relatively pale. Later the conidiophores are growing out of the stomata at the under surface of the leave and are visible as small bunches in a line following the stomata (Figure 1).

In field- and greenhouse experiments with a controlled inoculation system we found strong evidences that the epidemic spread and pathogenicity of R. collo-cygni is closely related to the ontogenetic plant development and distinct alterations in the antioxidative protection system of barley leaves. Our results provide evidence that the antioxidative protection system of the leaves plays a key role in the incidence of Ramularia leaf spot as susceptibility increases dramatically with the age-related breakdown of the antioxidative capacity (Schützendübel et al., 2008). For getting a deeper insight into the development of R. collo-cygni on barley and the epidemiology we developed an in vitro system using leaf segments (Figure 4). This system enables us to compare strains of R. collo-cygni collected from different field sites regarding to differences in pathogenicity as well as to test barley for differences in the resistance against R. collo-cygni dependent on age and genotype.

During growth within leaf apoplast the fungus produces a photosensitive toxin which probably plays a major role in the necrotic life style of R. collo-cygni. Heiser et al (2003) reported that R. collo-cygni produce several fractions of this anthraquinone derivate. Because of their strong similarities to the Rubellins produced by Mycosphaerella rubella this toxins are called Rubellins (Figure 5). This group of photoactive molecules produced by several pathogenic fungi like Cercospora and Mycosporella spec. will be activated by light. The potent and broad-spectrum toxicity of these photosensitizers is due to their production of activated oxygen species, which occurs when the photosensitizer is converted, through absorption of light energy, to an electronically excited triplet state. Although the triplet photosensitizer may react directly with bio-molecules such as lipids, most toxicity is attributed to the effects of radical and nonradical species of activated oxygen generated by the reaction of the excited triplet state photosensitizer with molecular oxygen (Daub & Ehrensaft, 2000). These reactions, known as result in the production of reduced and radical forms of oxygen such as superoxide (O2- . ), hydrogen peroxide (H2O2), hydroxyl radical (OH.) and the highly toxic, but non radical, singlet oxygen (1O2).

It can be speculated that the pathogenicity of R. collo-cygni depends on inducing elevated levels of activated oxygen species via its phototoxin requiring a specific senescence state of the host tissue enabling the pathogen to overcome the antioxidative defence of the plant. One main topic of our research is which plant signal or physiological change during the ontogenesis or environmental impact triggers the switch between the systemic and the parasitic lifestyle of R. collo-cygni.

Selected references

  • Burke JI, O'Reilly B, and Thomas TM, (2000):Overcoming the spotting disorder and fungicide use in Spring Barley. Teagasc National Tillage Conference2000 Program pp.7. http://www.teagasc.ie/publications/ntc2000/paper01.htm
  • Cavara F, (1893): Über einige parasitische Pilze auf Getreide.- Zeitschrift für Pflanzenkrankheiten 3, 16-26.
  • Crous PW, Kang JC, and Braun U (2001): A phylogenetic redefinition of anamorph genera in Mycosphaerella based on ITS rDNA sequences and morphology. Mycologia 93, 1081-1101.
  • Daub ME, Ehrenshaft M (2000): The photoactivated Cercospora toxin Cercosporin.: Contributions to plant disease and fundamental biology. Annual Reviews of Phytopathology 38, 461 - 90.
  • Formayer H, Huss H, Eckhardt S, Gerersdorfer T, and Kromp-Kolb H (2002): Die Sprenkelkrankheit auf der Gerste: Untersuchung der meteorologischen Ursachen der Krankheit insbesondere der Entwicklung des Pilzes Ramularia collo-cygni. Endbericht des Forschungsprojektes Nr. 1223 des BMLFUW Austria.
  • Heiser I, Sachs E, Liebermann B (2003): Photodynamic oxygen activation by rubellin D, a phytotoxin produced by Ramularia collo-cygni (Sutton et. Waller) Physiological and Molecular Plant Pathology 62, 29 - 36.
  • Huss H, Mayrhofer H, and Wetschnig W (1987): Ophiocladium hordei CAV. (Fungi imperfecti), ein für Österreich neuer parasitischer Pilz der Gerste.- Der Pflanzenarzt 40,167-169.
  • Huss, H., E. Sachs (1998): Ramularia-Blattflecken- oder Sprenkelkrankheit der Gerste. Der Pflanzenarzt 11–12, 15–18.
  • Miethbauer S, Haase S, Schmidtke K-U, Günther W, Heiser I, Liebermann B (2006): Biosynthesis of photodynamically active rubellins and structure elucidation of new anthraquinone derivatives produced by Ramularia collo-cygni. Phytochemistry 67, 1206 - 1213.
  • Millar D (1998): Ramularia rampage. Crops 17(1), 6.
  • Sachs E (2002): Monitoring zur Verbreitung der Ramularia-Blattfleckenkrankheit an Wintergerste in Deutschland im Jahre 2000. Nachrichtenblatt Deutscher Pflanzenschutzdienst 54, 31 - 5.
  • Salamati S, Reitan L, Flataker KE (2002): Occurrence of Ramularia collo-cygni on spring barley in Norway. In: Yahyaoui AH, Brader L, Tekauz A, Wallwork H, eds. Proceedings of the Second International Workshop on Barley Leaf Blights (http://www.icarda.cgiar.org), 2002. Aleppo, Syria, 355 – 359.
  • Schützendübel A, Stadler M, Wallner D, v. Tiedemann A. (2008): A hypothesis on physiological alterations during plant ontogenesis governing susceptibility of winter barley to ramularia leaf spot. Plant Pathology 57, 518-526.
  • Sheridan JE, (1996): Cereal diseases 1995 - 1996. Mycology and Plant Pathology - Report 33, 47.
  • Sutton B, Waller JM (1988): Taxonomy of Ophiocladium hordei, causing leaf lesions on Triticale and other Gramineae. Transactions of the British Mycological Society 90, 55 - 61.