The role of seeds and airborne inoculum in the initiation of leaf blotch (Rhynchosporium secalis) epidemics in winter barley

Both airborne spores of Rhynchosporium secalis and seed infection have been implied as major sources of primary inoculum for barley leaf blotch (scald) epidemics in fields without previous history of barley cropping. However, little is known about their relative importance in the onset of disease. Results from both quantitative real‐time PCR and visual assessments indicated that seed infection was the main source of inoculum in the field trial conducted in this study. Glasshouse studies established that the pathogen can be transmitted from infected seeds into roots, shoots and leaves without causing symptoms. Plants in the field trial remained symptomless for approximately four months before symptoms were observed in the crop. Covering the crop during part of the growing season was shown to prevent pathogen growth, despite the use of infected seed, indicating that changes in the physiological condition of the plant and/or environmental conditions may trigger disease development. However, once the disease appeared in the field it quickly became uniform throughout the cropping area. Only small amounts of R. secalis DNA were measured in 24 h spore‐trap tape samples using PCR. Inoculum levels equivalent to spore concentrations between 30 and 60 spores per m³ of air were only detected on three occasions during the growing season. The temporal pattern and level of detection of R. secalis DNA in spore tape samples indicated that airborne inoculum was limited and most likely represented rain‐splashed conidia rather than putative ascospores.     

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.     

Detection of Ramularia collo-cygni DMI- and SDHI-resistant field populations in Austria and the effect of fungicides on the population and genetic diversity

European Journal of Plant Pathology - Winter barley (Hordeum vulgare L.) is the third most cultivated crop after corn and wheat in Austria but one of the most challenging for disease control. The...     

Role of Ascospores in Further Spread of QoI-Resistant Cytochrome b Alleles (G143A) in Field Populations of Mycosphaerella graminicola

ABSTRACT Strobilurin fungicides or quinone outside inhibitors (QoIs) have been used successfully to control Septoria leaf blotch in the United Kingdom since 1997. However, QoI-resistant isolates of Mycosphaerella graminicola were reported for the first time at Rothamsted during the summer of 2002. Sequence analysis of the cytochrome b gene revealed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Extensive monitoring using real-time polymerase chain reaction (PCR) testing revealed that fungicide treatments based on QoIs rapidly selected for isolates carrying resistant A143 (R) alleles within field populations. This selection is driven mainly by polycyclic dispersal of abundantly produced asexual conidia over short distances. In order to investigate the role of sexually produced airborne ascospores in the further spread of R alleles, a method integrating spore trapping with real-time PCR assays was developed. This method enabled us to both quantify the number of M. graminicola ascospores in air samples as well as estimate the frequency of R alleles in ascospore populations. As expected, most ascospores were produced at the end of the growing season during senescence of the wheat crop. However, a rapid increase in R-allele frequency, from 35 to 80%, was measured immediately in airborne ascospore populations sampled in a wheat plot after the first QoI application at growth stage 32. After the second QoI application, most R-allele frequencies measured for M. graminicola populations present in leaves and aerosols sampled from the treated plot exceeded 90%. Spatial sampling and testing of M. graminicola flag leaf populations derived from ascospores in the surrounding crop showed that ascospores carrying R alleles can spread readily within the crop at distances of up to 85 m. After harvest, fewer ascospores were detected in air samples and the R-allele frequencies measured were influenced by ascospores originating from nearby wheat fields.     

Infection strategy of Ramularia collo‐cygni and development of ramularia leaf spot on barley and alternative graminaceous hosts

Ramularia leaf spot (RLS) is a newly important disease of barley across temperate regions worldwide. Despite this recent change in importance, the infection biology of the causal agent Ramularia collo‐cygni (Rcc) remains poorly understood. Confocal microscopy of the infection process of two transgenic Rcc isolates, expressing either GFP or DsRed reporter markers, was combined with light microscopy during field infection to track the progression of Rcc in planta. Infection of stomata, including the development of a previously unreported stomatopodium structure, results in symptomless development and intercellular colonization of the mesophyll tissue. Transition to necrotrophy is associated with breakdown of host chloroplasts and the formation of aggregates of conidiophores. In addition to barley, Rcc forms a compatible interaction with winter wheat and a number of perennial grass species. An incompatible reaction was observed with two dicotyledonous species. These results provide further insights into the host interactions of this fungus and suggest that RLS could be a potential threat to other agriculturally important crops.     

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

Journal of Plant Diseases and Protection - 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...     

Control of foliar diseases in barley: towards an integrated approach

Barley is one of the world??s most important crops providing food and related products for millions of people. Diseases continue to pose a serious threat to barley production, despite the use of fungicides and resistant varieties, highlighting the impact of fungicide resistance and the breakdown of host plant resistance on the efficacy of control measures. This paper reviews progress towards an integrated approach for disease management in barley in which new methods may be combined with existing measures to improve the efficacy of control in the long-term. Advances have been made in genetic mapping of resistance (R) genes and in identifying novel sources of genes in wild barley populations and land races. Marker assisted selection techniques are being used to pyramid R genes to increase the durability of resistance. Elicitors to induce host resistance used in combination with fungicides can provide effective disease control in the field and could delay the evolution of fungicide insensitivity. Traits that may contribute to disease tolerance and escape have been identified and the extent of genetic variation within barley germplasm is being determined. Tools are being developed to integrate the above methods via an assessment of the risk of economic injury occurring from disease to guide decisions on the requirement for fungicide treatment. Barriers exist to the adoption of integrated management approaches from growers and end-users further down the supply chain (e.g. acceptance of variety mixtures) and policy incentives from government may be required for it to be taken up in practice.     

Characterisation of QoI‐resistant field isolates of Botrytis cinerea from citrus and strawberry

BACKGROUND: In 2004, field isolates of Botrytis cinerea Pers. ex Fr., resistant to strobilurin fungicides (QoIs), were first found in commercial citrus orchards in Wakayama Prefecture, Japan. Subsequently, QoI‐resistant isolates of this fungus were also detected in plastic strawberry greenhouses in Saga, Ibaraki and Chiba prefectures, Japan. Biological and molecular characterisation of resistant isolates was conducted in this study. RESULTS: QoI‐resistant isolates of B. cinerea grew well on PDA plates containing kresoxim‐methyl or azoxystrobin at 1 mg L^?1, supplemented with 1 mM of n‐propyl gallate, an inhibitor of alternative oxidase, whereas the growth of sensitive isolates was strongly suppressed. Results from this in vitro test were in good agreement with those of fungus inoculation tests in vivo. In resistant isolates, the mutation at amino acid position 143 of the cytochrome b gene, known to be the cause of high QoI resistance in various fungal pathogens, was found, but only occasionally. The heteroplasmy of cytochrome b gene was confirmed, and the wild‐type sequence often present in the majority of resistant isolates, indicating that the proportion of mutated cytochrome b gene was very low. CONCLUSION: The conventional RFLP and sequence analyses of PCR‐amplified cytochrome b gene are insufficient for molecular identification of QoI resistance in B. cinerea. Copyright © 2009 Society of Chemical Industry     

Distribution and molecular characterization of Corynespora cassiicola isolates resistant to boscalid

A total of 618 isolates of corynespora leaf spot fungus (Corynespora cassiicola) collected from 24 commercial cucumber greenhouses in 12 cities in Ibaraki Prefecture, Japan, were tested for their sensitivity to boscalid. Boscalid‐resistant isolates were detected in 17 out of 19 greenhouses with a history of use of this fungicide and detection frequencies of the resistant isolates exceeded 47% in nine greenhouses. Frequencies of very highly resistant (VHR) isolates with 50% effective concentration (EC₅₀) values of boscalid exceeding 30 μg mL^?1 were higher than those of moderately resistant (MR) isolates with EC₅₀ ranging from 2·0 to 5·9 μg mL^?1 in 11 greenhouses. Additionally, highly resistant (HR) isolates with EC₅₀ from 8·9 to 10·7 μg mL^?1 were first detected. Furthermore, molecular characterization of genes encoding succinate dehydrogenase (SDH) subunits (SdhA, SdhB, SdhC and SdhD) was carried out to elucidate the amino acid substitution responsible for the resistance to boscalid. All 23 VHR isolates had the same mutation from CAC to TAC in the SdhB gene leading to the substitution of histidine with tyrosine at amino acid position 278 (B‐H278Y). At the same position, the substitution to arginine conferred by a mutation to CGC (B‐H278R) was detected in all four HR isolates. Some MR isolates showed a substitution from serine to proline at position 73 in SdhC (C‐S73P), from serine to proline or from glycine to valine at position 89 (D‐S89P) and 109 (D‐G109V), respectively, in SdhD. There was no common mutation in SDH genes of all MR isolates.