Plant Pathogen Interaction

PLANT-PATHOGEN INTERACTIONS Mission: To develop effective, durable, economic and environmentally sound strategies for the control of crop diseases through an improved understanding of the interactions between plants, pathogenic agents and the environment.

During summer 2003, around two thirds of the Division re-located into new laboratory accommodation in the Centenary Building. The soil microbiology group, led by Penny Hirsch, transferred into the Division, integrating research on rhizosphere microbiology with studies on rootinfecting pathogens such as potato cyst nematode. The Rothamsted Centre for Bioimaging, incorporating the JeolGatan Cryomicroscopy Laboratory, was commissioned in autumn 2003. The unit is equipped with three new electron microscopes as well as the confocal microscope facility transferred from Long Ashton.

Head of Division: John Lucas

Research demonstrated that the cereal ear blight pathogen Fusarium graminearum can also infect arabidopsis florets, thus providing a model system to investigate host-pathogen interactions and pathogenicity mechanisms. Analysis of the newly sequenced F. graminearum genome identified a 28kb region that contains many homologues of pathogenicity and virulence genes found in other pathogenic fungi. This is the first report of a possible pathogenicity region in the genome of a plant pathogenic fungus. In barley, the recessive rym 4, 5 and 6 genes for resistance to soilborne barley mosaic viruses were shown to encode a component of the ribosomal complex, revealing a novel evolutionarily conserved class of plant resistance genes found in both monocotyledons and dicotyledons.

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Managing fungicide resistance

Bart Fraaije, Hans Cools, John Lucas

Wheat leaves infected with Septoria tritici.

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For the past 40 years, fungicides have played a key role in the management of disease caused by fungal pathogens in arable crops, especially cereals. This role is under threat due to the development of fungicide resistance, the lack of new active ingredients and the pressure to reduce chemical inputs in agriculture. Research at Rothamsted, aimed at detecting and characterising the mechanisms leading to fungicide resistance, as well as the way resistance develops in pathogen populations, is helping to devise strategies to manage the problem in the field.

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were not stable. It was predicted that more than one genetic change was needed to generate levels of resistance that would threaten field efficacy. However, resistance problems were first encountered within two years of introduction in powdery mildew, Blumeria graminis (Figure 1), of wheat in Germany in 1998.

Figure 1. Scanning electron micrograph of powdery mildew, Blumeria graminis.

Fungicide resistance is not new, but has become a greater problem since the introduction of fungicides that act at a single site in the target organism during growth. Unfortunately, a small change in the target, often caused by a single mutation, may be sufficient to completely abolish activity and confer resistance. Under field conditions, application of the fungicide will have little or no effect on resistant strains which, following repeated cycles of selection, come to predominate in the population. Within a short time, often only two or three seasons, the fungicide no longer controls the disease. Strobilurin fungicides, which act by inhibiting mitochondrial energy production, were first introduced in 1997 and quickly took a large share of the cereal fungicide market, due to their activity against the most important cereal pathogens and additional

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effects boosting yield. The risk of resistance development was initially estimated to be moderate because mutants generated in the laboratory showed low levels of resistance and

Resistance in field isolates was invariably associated with a single mutation, known as G143A, in the mitochondrial cytochrome b target protein. This mutation quickly became common in UK wheat mildew populations (Figure 2). The same mutation was also detected in populations of barley powdery mildew in 2000 and, more worryingly, in Septoria tritici, the causal agent of the most serious foliar disease of wheat in

Figure 2. Increase in strobilurin resistance allele frequency in mildew populations at Rosemaund, Herefordshire.

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Europe, at Rothamsted in summer 2002. Research on fungicide resistance development is mainly in three areas: • Understanding the mechanisms whereby pathogens develop resistance to fungicides • Developing molecular tools to detect and monitor the evolution of resistance in pathogen populations • Evaluating strategies for managing fungicide resistance in commercial crops. The main fungicide groups of interest are benzimidazoles (MBCs), triazoles and strobilurins (QoIs), due to their past and/or current importance in the control of fungal diseases in a wide variety of crops. The main pathogens being studied are S. tritici, B. graminis and Rhynchosporium secalis. Fungicide resistance diagnostics and practical applications To test large numbers of isolates quickly for fungicide sensitivity, highthroughput assays using microtitre plates have been developed. S. tritici metabolic activity, in the presence of a normally inhibitory concentration of fungicide, can be detected rapidly by measuring colour/fluorescence released after conversion of a growth indicator substrate present in the medium (Figure 3). Recent advances in DNA diagnostics also make it possible to detect specific alleles based on single nucleotide polymorphisms (SNPs). By using different probes labelled with ROTHAMSTED RESEARCH • 2003-2004

Figure 3. Bioassays showing the growth of Septoria tritici in liquid medium amended with azoxystrobin. Alamar Blue was used as a growth indicator. Pink indicates growth, blue indicates no growth measured.

contrasting fluorescent dyes in the polymerase chain reaction (PCR), sensitive and resistant alleles can be quantified both individually and simultaneously in a single reaction (Figure. 4). Provided that the phenotype correlates well with the genotype, these real-time PCR assays can be used to analyse the resistance status of a given sample, and provide estimates of Rallele frequencies. Using these techniques on archived wheat samples from the Broadbalk Experiment, resistance to MBC and QoI fungicides in populations of S. tritici was first detected in samples from1985 and 2002, respectively (Figure 5). As part of a Sustainable Arable LINK

programme, ‘Providing a scientific basis for the avoidance of fungicide resistance in plant pathogens’, both assays are being used to test the three factors likely to influence the evolution of resistance and be amenable to manipulation in an anti-resistance strategy – fungicide dose, number of sprays and alternating or mixing fungicides with different modes of action. The evolution of resistance Fungicide resistance is not always associated with a simple genetic change in the pathogen. The sudden and unexpected emergence of resistance to strobilurins has put renewed pressure on alternative

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Figure 4. Multiplex PCR targeting strobilurin-resistant (R) alleles and/or –sensitive (S) alleles with three different fluorogenic probes and a reference dye. The blue signal represents cleavage of a probe binding to both R- and S-alleles, green shows detection of R-alleles, grey detection of S-alleles and red is the signal of the reference dye. In this leaf sample, the frequency of R-alleles in the Septoria tritici population was estimated to be approximately 70%.

Figure 5. The development of MBC and QoI resistance in populations of Septoria tritici in the Broadbalk experiment using b-tubulin (E198A) and cytochrome b alleles as markers. For some years, data are lacking due to the absence of S. tritici DNA in the archived wheat samples.

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fungicides, especially the triazoles which are important not only in crop protection but also in the clinical management of candidiasis and aspergillosis. Although the failure of triazoles to control plant disease has not yet occurred, it seems that the efficacy of some triazoles has decreased. Three different resistance mechanisms have been reported so far: (i) over-expression of the target sterol 14a-demethylase encoded by the CYP51 gene, (ii) mutational changes in the target gene CYP51 gene and (iii) enhanced active efflux, mediated by the up-regulation of ATP-binding cassette transporters. The last two mechanisms have been detected in UK isolates of S. tritici. Using real-time PCR we have shown constitutive and induced overexpression of known and novel genes encoding ABC-transporters in the least triazole-sensitive isolates tested (Figure. 6). However, increased transcript levels were not found exclusively in less-sensitive isolates. This suggests that reduced triazole sensitivity in S. tritici is conferred by a combination of mechanisms, including mutations in the CYP51 gene and up-regulation of ABC transporters. Further studies are in progress to determine the precise contribution of individual mechanisms to the less sensitive phenotype. The way forward. The success of fungicides in the recent past has tended to reduce the importance of breeding for disease ROTHAMSTED RESEARCH • 2003-2004

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resistance in cereals and alternative disease management practices. Because of the development of fungicide resistance and the lack of new active compounds, disease control could eventually be compromised. Future sustainable disease control requires an integrated approach that exploits diversity in host plant resistance genes, chemicals and alternative agents such as plant defence activators and biological control agents.

Figure 6. Real-time PCR analysis of the expression of Septoria tritici ABC transporter encoding gene ATR3. Triazole sensitive (S) and resistant (R) isolates shown in the absence (-) and presence (+) of the triazole epoxiconazole. Transcript abundance (dR) is shown relative to the calibrator sensitive strain.

The Broadbalk experiment is a source of archived wheat samples.

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