Detection of Leptosphaeria maculans races on winter oilseed rape in different geographic regions of Germany and efficacy of monogenic resistance genes under varying temperatures

Final Published versio

Conversion of cDNA differential display results (DDRT-PCR) into quantitative transcription profiles

BACKGROUND: Gene expression studies on non-model organisms require open-end strategies for transcription profiling. Gel-based analysis of cDNA fragments allows to detect alterations in gene expression for genes which have neither been sequenced yet nor are available in cDNA libraries. Commonly used protocols for gel-based transcript profiling are cDNA differential display (DDRT-PCR) and cDNA-AFLP. Both methods have been used merely as qualitative gene discovery tools so far. RESULTS: We developed procedures for the conversion of cDNA Differential Display data into quantitative transcription profiles. Amplified cDNA fragments are separated on a DNA sequencer and detector signals are converted into virtual gel images suitable for semi-automatic analysis. Data processing consists of four steps: (i) cDNA bands in lanes corresponding to samples treated with the same primer combination are matched in order to identify fragments originating from the same transcript, (ii) intensity of bands is determined by densitometry, (iii) densitometric values are normalized, and (iv) intensity ratio is calculated for each pair of corresponding bands. Transcription profiles are represented by sets of intensity ratios (control vs. treatment) for cDNA fragments defined by primer combination and DNA mobility. We demonstrated the procedure by analyzing DDRT-PCR data on the effect of secondary metabolites of oilseed rape Brassica napus on the transcriptome of the pathogenic fungus Leptosphaeria maculans. CONCLUSION: We developed a data processing procedure for the quantitative analysis of amplified cDNA fragments separated by electrophoresis. The system utilizes common software and provides an open-end alternative to DNA microarray analysis of the transcriptome. It is expected to work equally well with DDRT-PCR and cDNA-AFLP data and be useful particularly in reseach on organisms for which microarray analysis is not available or economical

Occurrence of Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker of tomato, in Syria

Several surveys were carried out to evaluate the occurrence of bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis (Cmm) in Syria, especially in the North-West provinces Latakiaand Tartous. The surveys revealed typical disease symptoms in greenhouses where the tomato cvs. Dima, Huda and Astona were grown, such as dark brown to black lesions on the leaf margins, wilting of whole plants, stunting, and vascular discoloration. The disease incidence in such greenhouses was 15% in the spring of 2007, and up to 70% by the end of July. Ten isolates obtained from diseased plants at different locations in these two provinces were identified as Clavibacter michiganensis subsp. michiganensis using classical microbiological tests as well as PCR. This is the first detailed proof of the occurrence of bacterial canker of tomato in Syria

Dissection of quantitative blackleg resistance reveals novel variants of resistance gene Rlm9 in elite Brassica napus

Blackleg is one of the major fungal diseases in oilseed rape/canola worldwide. Most commercial cultivars carry R gene-mediated qualitative resistances that confer a high level of race-specific protection against Leptosphaeria maculans, the causal fungus of blackleg disease. However, monogenic resistances of this kind can potentially be rapidly overcome by mutations in the pathogen’s avirulence genes. To counteract pathogen adaptation in this evolutionary arms race, there is a tremendous demand for quantitative background resistance to enhance durability and efficacy of blackleg resistance in oilseed rape. In this study, we characterized genomic regions contributing to quantitative L. maculans resistance by genome-wide association studies in a multiparental mapping population derived from six parental elite varieties exhibiting quantitative resistance, which were all crossed to one common susceptible parental elite variety. Resistance was screened using a fungal isolate with no corresponding avirulence (AvrLm) to major R genes present in the parents of the mapping population. Genome-wide association studies revealed eight significantly associated quantitative trait loci (QTL) on chromosomes A07 and A09, with small effects explaining 3–6% of the phenotypic variance. Unexpectedly, the qualitative blackleg resistance gene Rlm9 was found to be located within a resistance-associated haploblock on chromosome A07. Furthermore, long-range sequence data spanning this haploblock revealed high levels of singlenucleotide and structural variants within the Rlm9 coding sequence among the parents of the mapping population. The results suggest that novel variants of Rlm9 could play a previously unknown role in expression of quantitative disease resistance in oilseed rape

A new set of international Leptosphaeria maculans isolates as a resource for elucidation of the basis and evolution of blackleg disease on Brassica napus

© 2023 The Authors. Plant Pathology published by John Wiley & Sons Ltd on behalf of British Society for Plant Pathology. This is an open access article under the terms of the Creative Commons Attribution-Non Commercial-No Derivs License. https://creativecommons.org/licenses/by-nc-nd/4.0/A collection of isolates of the fungi Leptosphaeria maculans and L. biglobosa, which cause blackleg disease on Brassica napus (canola/oilseed rape) and other Brassicaceae species, was assembled to represent the global diversity of these pathogens and a resource for international research. The collection consists of 226 isolates (205 L. maculans and 21 L. biglobosa) from 11 countries. The genomes of all 205 L. maculans isolates were sequenced, and the distribution and identity of avirulence gene alleles were determined based on genotypic information and phenotypic reactions on B. napus lines that hosted specific resistance genes. Whilst the frequencies of some avirulence alleles were consistent across each of the regions, others differed dramatically, potentially reflecting the canola/oilseed rape cultivars grown in those countries. Analyses of the single-nucleotide polymorphism (SNP) diversity within these L. maculans isolates revealed geographical separation of the populations. This "open access" resource provides a standardized set of isolates that can be used to define the basis for how these fungal pathogens cause disease, and as a tool for discovery of new resistance traits in Brassica species.Peer reviewe

Efficacy of major resistance genes against Leptosphaeria maculans in oilseed rape and influence of temperature on the plant-pathogen interaction

Stem canker caused by Leptosphaeria maculans is an important disease in oilseed rape (Brassica napus L.) and causes significant yield losses. The cultivation of resistant cultivars is a reliable tool for protecting the crop against pathogen damage. In controlled environment experiments, the efficacy of major resistance genes against L. maculans was tested under varying temperatures for cotyledons and stems. Therefore, the resistant cultivars Caiman with Rlm7 resistance and Uluru with LepR3 resistance, as well as Lirabon as susceptible control, were used. For each resistant cultivar an avirulent and a virulent L. maculans isolate were selected. Cotyledon resistance was tested with a spore suspension, whereas adult resistance was tested at the stem base by inoculation with a mycelium plug. The plant‐pathogen interactions were examined at different temperature regimes. Incompatible interactions found on cotyledons of Uluru turned to be compatible, whereas only an increase of L. maculans DNA was found for cotyledons of Caiman at higher temperatures (≥27°C). Major gene resistance could actively reduce disease severity in stem tissue. Especially, Caiman was strongly dependent on its Rlm7 resistance gene, whereas resistance of Uluru relied more on quantitative resistance. High temperature treatment did not change incompatibility into compatibility at stem bases.Non peer reviewe

Role of Salicylic Acid and Components of the Phenylpropanoid Pathway in Basal and Cultivar-Related Resistance of Oilseed Rape (Brassica napus) to Verticillium longisporum

Enhanced resistance is a key strategy of controlling ‘Verticillium stem striping’ in Brassica napus caused by the soil-borne vascular pathogen Verticillium longisporum. The present study analyses the role of a broad range of components in the phenylpropanoid and salicylic acid (SA) pathways in basal and cultivar-related resistance of B. napus towards V. longisporum. A remarkable increase of susceptibility to V. longisporum in SA-deficient transgenic NahG plants indicated an essential role of SA in basal resistance of B. napus to V. longisporum. Accordingly, elevated SA levels were also found in a resistant and not in a susceptible cultivar during early asymptomatic stages of infection (7 dpi), which was associated with increased expression of PR1 and PR2. In later symptomatic stages (14 or 21 dpi), SA responses did not differ anymore between cultivars varying in resistance. In parallel, starting at 7 dpi, an overall increase in phenylpropanoid syntheses developed in the resistant cultivar, including the activity of some key enzymes, phenylalanine ammonium lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POX) and the expression of key genes, PAL4, CCoAMT, CCR, POX. As a consequence, a remarkable increase in the levels of phenolic acids (t-cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid) occurred associated with cultivar resistance. A principal component analysis including all 27 traits studied indicated that component 1 related to SA synthesis (PR1, PR2, POX, level of free SA) and component 2 related to lignin synthesis (level of free ferulic acid, free p-coumaric acid, conjugated t-cinnamic acid) were the strongest factors to determine cultivar-related resistance. This study provides evidence that both SA and phenolic acid synthesis are important in cultivar-related resistance, however, with differential roles during asymptomatic and symptomatic stages of infection

Greenhouse Evaluation of Clubroot Resistant-<i>Brassica napus</i> cv. Mendel and Its Efficacy Concerning Virulence and Soil Inoculum Levels of <i>Plasmodiophora brassicae</i>

Clubroot resistance of oilseed rape (OSR) cultivars frequently relies on a major resistance gene originating from cv. Mendel. The efficacy of this resistance was studied in greenhouse experiments using two Plasmodiophora brassicae isolates, which were either virulent (P1(+)) or avirulent (P1) on Mendel. Seeds of clubroot-susceptible cultivar Visby and clubroot-resistant cultivar Mendel were sown in soil mixtures inoculated with different concentrations of resting spores (101, 103, 105, and 107 resting spores/g soil). Clubroot severity, plant height, shoot and root weight as well as resting spore propagation were assessed for each isolate and cultivar separately at four dates after sowing. The OSR cultivars behaved significantly different in the measured parameters. The threshold of inoculum density to cause disease depended strongly on the virulence of the pathogen and susceptibility of the host plant. In Visby grown in soil infested with P1, clubroot symptoms and increases in root weight and the number of propagated resting spores occurred at inoculum levels of 101 resting spores and higher, whereas Mendel was not affected in soils under the three lowest inoculum densities. In contrast, the P1(+) isolate led to earlier and more severe symptoms, heavier galls, and a significantly higher number of new resting spores in both cultivars