Light-dependent oxidative stress determines physiological leaf spot formation in barley

ABSTRACT We reported previously that physiological leaf spot (PLS) formation in winter and spring barley is dependent on genotype-related oxidative stress under field conditions. In the present study, we searched for factors inducing PLS symptoms in the greenhouse similar to those observed in the field and investigated its relationship to reactive oxygen species (ROS) metabolism. We found that in the greenhouse, oxidative stress induced spring barley cv. Extract, which is sensitive to PLS, to express symptoms similar to those observed in the field. Leaves severely affected by PLS showed significantly lower activities of key enzymes in the Halliwell-Asada cycle such as ascorbate peroxidase, glutathione reductase, dehy-droascorbate reductase, and monodehydroascorbate reductase. The sensitive cultivar also showed lower levels of total superoxide dismutase (SOD) and Cu/Zn-SOD activity but a higher level of chloroplast-specific Fe-SOD activity than that of the insensitive cultivar. Thus, an unbalanced ROS metabolism in chloroplasts may trigger PLS incidence in sensitive cultivars, which is in agreement with the fact that light is essential for the induction of PLS expression under both field and greenhouse conditions. Accordingly, under greenhouse conditions, continuous light stress (7 days), but not light shock treatments, induced PLS similar to that of field conditions in sensitive cv. Extract, but not in resistant cv. Scarlett. Light with a high proportion of energy in the blue wavelength spectrum (350 to 560 nm) was significantly more PLS inductive than light with a pronounced red (photosynthetically active radiation) spectrum (580 to 650 nm). Exposure to ozone did not produce PLS-like symptoms. Furthermore, similar to earlier observations in the field, PLS symptom expression was closely correlated with the accumulation of superoxide (O(2) (-)) detected by both biochemical and histochemical assays. Taken together, these data suggest that PLS in barley is genotype-dependent but its expression appears to be induced by certain environmental stress factors, among which photosyn-thetically active radiation plays a major role.     

The development of the leaf rust,Puccinia hordei,population during winter,spring and early summer in 11 winter barley cultivars

The overwintering and the epidemic development in spring of leaf rust was studied in 11 winter barley cultivars at two different sites near Wageningen in 1976/1977. The amount of leaf rust decreased through the winter at both sites. Cultivars differed considerably in the amount of leaf rust in late winter. Both the moment and the rate of increase of leaf rust after the winter varied with cultivars. The ultimate amount of leaf rust in a cultivar was therefore determined by three factors: The amount of overwintering leaf rust, the onset of leaf rust increase and the rate of increase. The latter was determined by the partial resistance of the cultivar. Why the epidemics did not start at the same moment is yet unknown. The differential overwintering could be explained from the amounts of leaf rust and powdery mildew at the start of the winter, the effect of powdery mildew being a negative one. The correlation coefficient between the values observed in March and those predicted from the December leaf rust and powdery mildew readings was 0.93. In a second experiment carried out in 1979/1980 with six winter barley cultivars chosen from the first experiment the powdery mildew was succesfully excluded by treatment with fungicides. There was no decrease in the leaf rust over the winter nor a differential cultivar effect on overwintering.     

Effect of Juglone on Active Oxygen Species and Antioxidant Enzymes in Susceptible and Partially Resistant Banana Cultivars to Black Leaf Streak Disease

The black leaf streak disease (BLSD), caused by Mycosphaerella fijiensis, is the most destructive disease of bananas and plantains around the world. Breeding for resistance is the most promising strategy to fight this disease especially in small farmer plantations. Mycosphaerella fijiensis produces many phytotoxins such as juglone, which can be used, jointly with field and inoculations under controlled conditions, for screening banana cultivars for BLSD-resistance. This non-host specific phytotoxin has been shown to act on chloroplasts and disturbs the proton electrochemical gradient across the plasmalemma membrane. Moreover, an involvement of the oxidative burst during the interaction has been suggested. The present study was carried out using two cultivars that differed for either their juglone-responses or their resistance to BLSD (cv. Grande Naine susceptible to BLSD and juglone and cv. Fougamou partially resistant to BLSD and highly tolerant to juglone). The production of active oxygen species (AOS) and the enhancement of the enzymatic and/or non-enzymatic AOS-scavenging systems were investigated after treatment of the two cultivars with juglone. The time-course of AOS-production and AOS-scavenging was shown to be the key difference between these two tested cultivars after treatment with juglone. Thus, an early release of AOS (O₂⁻ radical and H₂O₂) and a quick stimulation of a preferment anti-oxidant system (superoxide dismutases, catalases, and peroxidases) was observed for cv. Fougamou as compared to cv. Grande Naine for which a late and weak generation of AOS accompanied by a late stimulation of the anti-oxidant systems were detected.     

豇豆与锈菌互作中的活性氧代谢研究

 本文分析了不同抗性水平的豇豆(Vigna sesquipdalis Wight)品种与锈菌(Uromyces vignae Barcl)互作中活性氧(ac-tive oxygen species,AOS)的发生、防御酶活性及膜脂过氧化水平的变化规律及其与豇豆抗锈病性的关系。结果表明,在接种后,免疫品种(益农)和感病品种(揭上和金迪)的超氧物歧化酶(superoxide dismutase,SOD)比活性均升高,且升高幅度都在12 h出现高峰;抗病品种(金山和特青)开始时降低,至12h时回升,在24 h(高抗)和48 h(中抗)出现高峰;在24 h时,免疫和抗病品种的SOD比活性高于感病品种。在接种后12 h内,免疫和抗病品种的过氧化氢酶(catalase,CAT)比活性都下降;而感病品种均上升,并在12 h出现第1个高峰。受锈菌侵染后,豇豆各品种都产生超氧阴离子(superoxide anion,O₂^·),在多数测试时段中,免疫和抗病品种的O₂^·产率净变化值低于感病品种。丙二醛(malondialdehyde,MDA)含量变化幅度与品种抗性呈负相关,与O₂^·产率的变化趋势基本相吻合。这些结果表明AOS代谢在豇豆与锈菌互作中起重要作用。     

Application of real-time and multiplex polymerase chain reaction assays to study leaf blotch epidemics in barley

ABSTRACT Leaf blotch, caused by Rhynchosporium secalis, was studied in a range of winter barley cultivars using a combination of traditional plant pathological techniques and newly developed multiplex and real-time polymerase chain reaction (PCR) assays. Using PCR, symptomless leaf blotch colonization was shown to occur throughout the growing season in the resistant winter barley cv. Leonie. The dynamics of colonization throughout the growing season were similar in both Leonie and Vertige, a susceptible cultivar. However, pathogen DNA levels were approximately 10-fold higher in the susceptible cultivar, which expressed symptoms throughout the growing season. Visual assessments and PCR also were used to determine levels of R. secalis colonization and infection in samples from a field experiment used to test a range of winter barley cultivars with different levels of leaf blotch resistance. The correlation between the PCR and visual assessment data was better at higher infection levels (R(2) = 0.81 for leaf samples with >0.3% disease). Although resistance ratings did not correlate well with levels of disease for all cultivars tested, low levels of infection were observed in the cultivar with the highest resistance rating and high levels of infection in the cultivar with the lowest resistance rating.     

Proteinaceous Metabolites from Pyrenophora teres Contribute to Symptom Development of Barley Net Blotch

ABSTRACT Pyrenophora teres, the causal agent of net blotch of barley (Hordeum vulgare L.), induces a combination of necrosis and extensive chlorosis in susceptible barley cultivars. Cell-free filtrates from both net and spot forms of P. teres; P. teres f. sp. teres, and P. teres f. sp. maculata were found to contain phytotoxic low molecular weight compounds (LMWCs) and proteinaceous metabolites which appear to be responsible for different components of the symptoms induced by the two forms of the pathogen in a susceptible cultivar of barley (cv. Sloop). Proteins induced only brown necrotic spots or lesions similar to those induced by the pathogens 72 h after inoculation. In contrast, LMWCs induced general chlorosis seen 240 h after inoculation but not the localized necrosis. Neither hydrolyzed or heat- or protease-treated proteinaceous metabolites induced the symptoms. This is the first report of the involvement of proteins produced by P. teres in symptom development during net blotch disease of barley.     

Frequency of a mutant allele of cytochrome b conferring resistance to Ool fungicides in the Czech population of Ramularia collo-cygni

Journal of Plant Diseases and Protection - Ramularia collo-cygni (RCC) is a fungal pathogen infecting both spring and winter barley and causing Ramularia leaf spot (RLS). Fungicides based on...     

Properties of a hordeivirus from Anthoxanthum odoratum

A virus having tubular particles (124, 142 and 167 nm) was manually transmitted from and to Anthoxanthum odoratum (sweet vernal grass). Other experimental hosts were Chenopodium quinoa, C. amaranticolor, Zea mays, Avena sativa and Hordeum vulgare (barley); winter cultivars of barley were more often infected than spring cultivars. In tests which simulated mechanical injury, the virus was transmitted from infected to healthy A. odoratum but not to healthy barley. The virus particles, which were seen as aggregates in the cytoplasm of A. odoratum leaf cells and in anther walls, reacted strongly in immunosorbent electron microscopy tests with antisera to strains of barley stripe mosaic hordeivirus. Nevertheless, the virus could be distinguished from the Type strain of barley stripe mosaic virus by reciprocal serological tests and by symptoms in infected barley cv. Black Hulless. Using an antiserum produced against the virus, a survey of A. odoratum in the field revealed one of 72 tested plants to be naturally infected. The virus is probably that described by Catherall & Chamberlain (1980) and named anthoxanthum latent blanching virus.     

Effects of fungicides JS399-19, azoxystrobin, tebuconazloe, and carbendazim on the physiological and biochemical indices and grain yield of winter wheat

The impact of four modern fungicides JS399-19 (2-cyano-3-amino-3-phenylancryic acetate) (novel fungicide), azoxystrobin (a strobilurin), tebuconazole (a triazole) and carbendazim (a benzimidazole), applied as foliar spray at the recommended field rate, on the physiology and biochemistry of the senescence process and grain yield was studied in winter wheat (Triticum aestivum L. cv. ‘Nannong No. 9918’) under natural environmental conditions. Fungicide treatments to wheat plants at growth stage [ZGS] 57 (3/4 of head emerged) significantly increased the chlorophyll (CHL) and soluble protein (SP) content and decreased the malondialdehyde (MDA) content and electrolyte leakage. Additionally, activities of the antioxidative enzymes superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in flag leaves of the fungicide-treated plants were also higher than that in untreated plants. These coincided with elevated levels of H₂O₂ and reduced level of in the fungicide-treated plants. The results suggested that the fungicide-induced delay of senescence was due to an enhanced antioxidant enzyme activity protecting the plants from harmful active oxygen species (AOS). Because all fungicides can induce the delay of wheat senescence, fungicide-treated wheat shown higher grain yield than untreated wheat. Of all tested fungicides, JS399-19, azoxystrobin and tebuconazole showed similar effects on delaying senescence of wheat and enhancing the grain yield of wheat, but JS399-19 was more efficient in general.     

Seasonal changes in pathotype complexity in French populations of barley powdery mildew

Airborne conidia of Erysiphe graminis f.sp. hordei were sampled in three regions and a single locality in the northern part of France for 2 years. Sampling was carried out in early spring, in late spring and in autumn, in order to separate the effects of winter barley cultivars, carrying few specific resistance alleles, and of spring barley cultivars, carrying diverse resistance alleles, on the structure of the pathogen population. Although complex pathotypes with three to 10 virulences were selected by spring cultivars, simple pathotypes, including a pathotype with the single unnecessary virulence allele Va22 , which formed a clear majority of the samples, remained dominant in early spring, when winter but not spring cultivars were growing. In early spring, simple pathotypes were more prevalent in the north, where the winter cultivars represented 90% of the barley acreage, than in the east, where winter cultivars represented 65%. In the west, the frequency of simple pathotypes was limited compared to the north, possibly because of the resistance allele Mlg in winter cultivars. The high frequency of simple pathotypes in early spring could be explained by a differential adaptation between simple and complex pathotypes or by delayed epidemics on spring cultivars compared to winter cultivars.     

The effects of infection by barley yellow mosaic virus on the yield and components of yield of barley

Infection by barley yellow mosaic virus (BaYMV) was greater in a crop sown in October than in November and in cv. Maris Otter than in cv. Sonja. However, a greater proportion of plants of Sonja than of Maris Otter were infected, and more severely, with the soil-borne fungus vector of BaYMV. Polymyxagraminis. Indifferent years the yield of individual plants of Maris Otter was decreased by 15% and of Sonja by 40%. The spring barley cultivars Athos. Porthos. Georgie and Ark Royal were all infected with BaYMV when sown in autumn in infected soil. Infected winter barley cv. Igri lost up to 50% of yield when this was measured across a naturally infected patch of crop.     

A comparative assessment of potential components of partial disease resistance to <Emphasis Type="Italic">Fusarium</Emphasis> head blight using a detached leaf assay of wheat,barley and oats

The relative resistance of 15 winter barley, three winter wheat and three winter oat cultivars on the UK recommended list 2003 and two spring wheat cultivars on the Irish 2003 recommended list were evaluated using Microdochium nivale in detached leaf assays to further understand components of partial disease resistance (PDR) and Fusarium head blight (FHB) resistance across cereal species. Barley cultivars showed incubation periods comparable to, and latent periods longer than the most FHB resistant Irish and UK wheat cultivars evaluated. In addition, lesions on barley differed from those on wheat as they were not visibly chlorotic when placed over a light box until sporulation occurred, in contrast to wheat cultivars where chlorosis of the infected area occurred when lesions first developed. The pattern of delayed chlorosis of the infected leaf tissue and longer latent periods indicate that resistances are expressed in barley after the incubation period is observed, and that these temporarily arrest the development of mycelium and sporulation. Incubation periods were longer for oats compared to barley or wheat cultivars. However, oat cultivars differed from both wheat and barley in that mycelial growth was observed before obvious tissue damage was detected under macroscopic examination, indicating tolerance of infection rather than inhibition of pathogen development, and morphology of sporodochia differed, appearing less well developed and being much less abundant. Longer latent periods have previously been related to greater FHB resistance in wheat. The present results suggest the longer latent periods of barley and oat cultivars, than wheat, are likely to play a role in overall FHB resistance if under the same genetic control as PDR components expressed in the head. However the limited range of incubation and latent periods observed within barley and oat cultivars evaluated was in contrast with wheat where incubation and latent periods were shorter and more variable among genotypes. The significance of the various combinations of PDR components detected in the detached leaf assay as components of FHB resistance in each crop requires further investigation, particularly with regard to the apparent tolerance of infection in oats and necrosis in barley, after the incubation period is observed, associated with retardation of mycelial growth and sporulation.     

Susceptibility of German spring barley cultivars to loose smut populations from different European origins

Forty-two registered spring barley cultivars from the German official list were tested under natural infection conditions for susceptibility to loose smut (Ustilago tritici f.sp. hordei) during two test cycles at two locations. Only cv. Steffi was found to be resistant to the local loose smut population. Cultivar Sigrid showed lowest susceptibility because of flowering inside the leaf sheath. Less than 1% infection at all sites showed up in cvs Auriga, Jacinta and Hendrix. Twenty-one cultivars had an infection rate of less than 2%. Cultivar Danuta displayed the highest susceptibility with an average of 12.6%. Another 23 spring barley accessions with expected loose smut resistance were inoculated artificially with loose smut populations obtained from 11 locations in Germany and neighbouring countries. Only Jet with the resistance Un3/6, CDC Freedom with Un8, CIho9973 with quantitative resistance, as well as Lino and GangTuoQuingKeHao1 remained disease-free. In addition to these, another eight accessions in this test group are recommended to become part of a differential tester set to distinguish origins of loose smut. Statistical analysis showed that for scoring of cultivars more importance has to be given to the number of locations for infestation than to the number of test locations to determine the degree of attack. In view of the existing inspection limits for production of certified seed in European countries, the currently registered German barley cultivars put organic seed producers and breeders at high risk in respect to loose smut infection, if the number of generations for multiplication under organic farming increases.     

Optimierte Strategien zur Krankheitsbekämpfung in der Gerste unter Berücksichtigung neuer Erkenntnisse zur Epidemiologie des Blattfleckenkomplexes

The leaf spot complex constitutes an increasing challenge for integrated pest control in barley. The complex consists of biotic and abiotic factors causing early ripening after a rapid loss of green leaf area, in particular in upper, exposed leaves. Severe symptoms appear late in the growing season after heading. The experience from recent years shows that the control of the leaf spot complex improves yield. Best control is achieved by new, effective fungicides. Efficiency is improved by late fungicide application and is dose-dependent. In contrast, late treatments provide poor control of early appearing diseases. For a successful disease control in barley good efficiency in the control of the leaf spot complex appears based on a sufficient control of the other diseases. For optimization of disease control, we require improved knowledge of the epidemics of the leaf spot complex and of the contribution of individual biotic and abiotic factors. Besides the weather, the fungal pathogen Ramularia collo-cygni and its photodynamic toxins play a mayor role in symptom development. By combination of conventional and molecular diagnostics we aim at a better understanding of the complex as a basis for early and reliable prediction of epidemics. Because non fungicidal factors like the physiological plant age play a significant role in the occurrence of the leaf spot complex, they should be taken into account for integrated control.     

Aetiology of leaf spot of garlic and onion caused by Stemphylium vesicarium in Spain

Surveys between 1989 and 1993 in the major garlic production areas of Spain identified a new leaf spot disease, characterized by white and purple lesions followed by extensive necrosis. Isolation and pathogenicity tests with fungal isolates taken from these spots indicated that Stemphylium vesicarium was the causal agent. Pseudothecia of the teleomorph stage, Pleospora sp., were found on leaf debris from affected plants. Inoculation of garlic and onion plants with residues carrying mature pseudothecia, or with ascospore suspensions obtained from the pseudothecia, resulted in the development of white and purple leaf spots. Wetness periods longer than 24 h were required for symptom development under controlled conditions. Isolates of S. vesicarium from garlic, onion and asparagus caused disease in all three hosts. In garlic, cv. Blanco de Vallelado was most susceptible, while lines B4P17 and B6P1, and cvs Iberose and Golourose were less susceptible to the disease.     

Differential effects of spot blotch on photosynthesis and grain yield in two barley cultivars

Barley spot blotch (SB), caused by Cochliobolus sativus, is an important barley disease which causes extensive grain yield losses. These losses may not always correlate directly with the amount of diseased leaf area. Two barley cultivars, Quebracho (susceptible to SB) and Carumbé (with intermediate susceptibility to SB), were compared in field experiments in 2003, 2004 and 2006. Plots of each cultivar were either inoculated with C. sativus or protected with fungicide under field conditions to generate contrasting treatments: i) diseased, and ii) free of disease, respectively. SB severity over the growing season, photosynthetic rate on leaves with no visible symptoms and grain yield were assessed for each treatment and year. There was no treatment effect on cv. Carumbé, while cv. Quebracho showed a significant yield reduction, even though SB severity during the grain filling period was <10 %. This yield reduction was associated with a reduced photosynthetic rate at the beginning of the grain filling period in cv. Quebracho. A similar experiment was conducted under greenhouse conditions, adding a treatment without inoculum or fungicide. There were no differences in photosynthetic rate or grain yield per plant among treatments. These results suggest a distinct physiological response to SB infection among cultivars affecting leaf photosynthetic rate, and SB severity may not be the best estimator of yield losses caused by SB.     

Photodynamic oxygen activation by rubellin D,a phytotoxin produced by Ramularia collo-cygni (Sutton et Waller)

Ramularia collo-cygni, a pathogenic deuteromycete, is the causal agent of Ramularia leaf spot disease on barley. It produces a number of coloured metabolites of which one was identified as rubellin D, a phytotoxin only described for Mycosphaerella rubella until now. Rubellin D induces light- and concentration-dependent necrosis on barley leaves. In a biochemical model system we could demonstrate that rubellin D exhibits photodynamic activity triggering the light-dependent production of reactive oxygen species (ROS). The use of inhibitors of ROS production confirmed mainly the production of singlet oxygen and to a lesser extent superoxide. In addition, light-dependent peroxidation of α-linolenic acid by rubellin D was shown. Based on our results we present a model for ROS-mediated symptom induction on barley leaves by Ramularia collo-cygni.     

Crop canopy development and structure influence weed suppression

Weed suppression characteristics of different winter cereal species and cultivars were studied in two field experiments in two successive years. Two cultivars each of barley, oats and wheat were studied using the weed Galium aparine L. at a range of densities. Significant differences were found in the suppressive abilities of the crop species: oats being the most suppressive, followed by barley and then wheat. Percentage yield loss was related to G. aparine density using a rectangular hyperbolic model. In 1995–96, significant differences between the two wheat cultivars were found, with cv. Avalon being able to suppress G. aparine growth much more effectively than cv. Spark. However, in 1996–97, high rainfall in May, June and July enabled G. aparine to escape suppression by the cultivars. Cultivar competitive ability was associated with high overall leaf area, resistance to loss of tillers under competitive pressure, greater height, canopy structure and development. The oat cultivars were more competitive at early growth stages than could be accounted for by their canopy structure; the possible contribution of allelopathic exudates to their suppressive ability is discussed.     

Resistance to Leptosphaeria maculans (phoma stem canker) in Brassica napus (oilseed rape) induced by L. biglobosa and chemical defence activators in field and controlled environments

Effects of pretreatment of Brassica napus leaves with ascospores of Leptosphaeria biglobosa or chemical defence activators [acibenzolar- S -methyl (ASM) or menadione sodium bisulphite (MSB)] on infection by ascospores of Leptosphaeria maculans (phoma stem canker) and development of disease were studied in controlled-environment (phoma leaf spot) and field (phoma leaf spot and stem canker) experiments. In controlled-environment experiments, pretreatment of oilseed rape leaves (cv. Madrigal) with L. biglobosa , ASM or MSB delayed the appearance of L. maculans phoma leaf spot lesions. These pretreatments also decreased the phoma leaf spot lesion area in both pretreated leaves (local effect) and untreated leaves (systemic effect). In winter oilseed rape field experiments in the 2002/03 and 2003/04 growing seasons, pretreatment with L. biglobosa or ASM in October/November decreased not only the number of phoma leaf spot lesions per leaf caused by L. maculans in autumn/winter, but also the severity of phoma stem canker in the subsequent spring/summer. Effects were greater in 2002/03 (when natural L. maculans ascospore release began in September 2002) than in 2003/04 (when ascospore release began in December following a period of dry weather in August/September 2003). These results suggest that pretreatment with biological or chemical defence activators can induce local and systemic resistance to L. maculans , with both short-term effects on the development of phoma leaf spotting and long-term effects on the development of stem canker 8 months later.     

Predicting light leaf spot (Pyrenopeziza brassicae) risk on winter oilseed rape (Brassica napus) in England and Wales, using survey, weather and crop information

Data from surveys of winter oilseed rape crops in England and Wales in growing seasons with harvests in 1987–99 were used to construct statistical models to predict, in autumn (October), the incidence of light leaf spot caused by Pyrenopeziza brassicae on winter oilseed rape crops the following spring (March/April), at both regional and individual crop scales. Regions (groups of counties) with similar seasonal patterns of incidence (percentage of plants affected) of light leaf spot were defined by using principal coordinates analysis on the survey data. At the regional scale, explanatory variables for the statistical models were regional weather (mean summer temperature and mean monthly winter rainfall) and survey data for regional light leaf spot incidence (percentage of plants with affected pods) in July of the previous season. At the crop scale, further explanatory variables were crop cultivar (light leaf spot resistance rating), sowing date (number of weeks before/after 1 September), autumn fungicide use and light leaf spot incidence in autumn. Risk of severe light leaf spot (> 25% plants affected) in a crop in spring was also predicted, and uncertainty in predictions was assessed. The models were validated using data from spring surveys of winter oilseed rape crops in England and Wales from 2000 to 2003, and reasons for uncertainty in predictions for individual crops are discussed.