Pathogen lifestyle determines host genetic signature of quantitative disease resistance loci in oilseed rape (Brassica napus).

KEY MESSAGE: Using associative transcriptomics, our study identifies genes conferring resistance to four diverse fungal pathogens in crops, emphasizing key genetic determinants of multi-pathogen resistance. Crops are affected by several pathogens, but these are rarely studied in parallel to identify common and unique genetic factors controlling diseases. Broad-spectrum quantitative disease resistance (QDR) is desirable for crop breeding as it confers resistance to several pathogen species. Here, we use associative transcriptomics (AT) to identify candidate gene loci associated with Brassica napus constitutive QDR to four contrasting fungal pathogens: Alternaria brassicicola, Botrytis cinerea, Pyrenopeziza brassicae, and Verticillium longisporum. We did not identify any shared loci associated with broad-spectrum QDR to fungal pathogens with contrasting lifestyles. Instead, we observed QDR dependent on the lifestyle of the pathogen-hemibiotrophic and necrotrophic pathogens had distinct QDR responses and associated loci, including some loci associated with early immunity. Furthermore, we identify a genomic deletion associated with resistance to V. longisporum and potentially broad-spectrum QDR. This is the first time AT has been used for several pathosystems simultaneously to identify host genetic loci involved in broad-spectrum QDR. We highlight constitutive expressed candidate loci for broad-spectrum QDR with no antagonistic effects on susceptibility to the other pathogens studies as candidates for crop breeding. In conclusion, this study represents an advancement in our understanding of broad-spectrum QDR in B. napus and is a significant resource for the scientific community.

Transcriptomics of temperature-sensitive R gene-mediated resistance identifies a WAKL10 protein interaction network.

Understanding temperature-sensitivity of R gene-mediated resistance against apoplastic pathogens is important for sustainable food production in the face of global warming. Here, we show that resistance of Brassica napus cotyledons against Leptosphaeria maculans was temperature-sensitive in introgression line Topas-Rlm7 but temperature-resilient in Topas-Rlm4. A set of 1,646 host genes was differentially expressed in Topas-Rlm4 and Topas-Rlm7 in response to temperature. Amongst these were three WAKL10 genes, including BnaA07g20220D, representing the temperature-sensitive Rlm7-1 allele and Rlm4. Network analysis identified a WAKL10 protein interaction cluster specifically for Topas-Rlm7 at 25 °C. Diffusion analysis of the Topas-Rlm4 network identified WRKY22 as a putative regulatory target of the ESCRT-III complex-associated protein VPS60.1, which belongs to the WAKL10 protein interaction community. Combined enrichment analysis of gene ontology terms considering gene expression and network data linked vesicle-mediated transport to defence. Thus, dysregulation of effector-triggered defence in Topas-Rlm7 disrupts vesicle-associated resistance against the apoplastic pathogen L. maculans.

Co-inoculation timing affects the interspecific interactions between phoma stem canker pathogens Leptosphaeria maculans and Leptosphaeria biglobosa.

BACKGROUND: Phoma stem canker is an economically important disease of oilseed rape, caused by two co-existing fungal pathogen species, Leptosphaeria maculans (Plenodomus lingam) and Leptosphaeria biglobosa (Plenodomus biglobosus). Leptosphaeria maculans produces a phytotoxin called sirodesmin PL. Our previous work showed that L. biglobosa has an antagonistic effect on the production of sirodesmin PL if it is simultaneously co-inoculated with L. maculans. However, the effects of sequential co-inoculation on interspecific interactions between the two pathogens are not understood. RESULTS: The interactions between L. maculans and L. biglobosa were investigated in liquid culture by inoculation with L. maculans first, followed by L. biglobosa sequentially at 1, 3, 5 or 7 days later and vice versa; the controls were inoculated with L. maculans only, L. biglobosa only, or L. maculans and L. biglobosa simultaneously. The results showed that L. biglobosa inhibited the growth of L. maculans, the production of both sirodesmin PL and its precursors if L. biglobosa was inoculated before, or simultaneously with, L. maculans. However, the antagonistic effects of L. biglobosa were lost if it was co-inoculated 5 or 7 days after L. maculans. CONCLUSION: For the first time, the results of this study provided evidence that the timing when L. maculans and L. biglobosa meet significantly influences the outcome of the interspecific competition between them. Leptosphaeria biglobosa can inhibit the production of sirodesmin PL and the growth of L. maculans if it is inoculated before L. maculans or less than 3 days after L. maculans in liquid culture. There is a need to further investigate the timing of co-inoculation on interactions between L. maculans and L. biglobosa in their host plants for improving the control of phoma stem canker. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Efficient qPCR estimation and discrimination of airborne inoculum of Leptosphaeria maculans and L. biglobosa, the causal organisms of phoma leaf spotting and stem canker of oilseed rape.

BACKGROUND: Detection of the inoculum of phytopathogens greatly assists in the management of diseases, but is difficult for pathogens with airborne fungal propagules. Here, we present experiments to determine the abundance and distribution frequencies of the ascospores of Leptosphaeria (Plenodomus) species that were collected on the tapes of volumetric Hirst-type traps near oilseed rape fields in Poznan, Poland and Harpenden, UK. Fungal detection and species discrimination were achieved using a SYBR-Green quantitative polymerase chain reaction (qPCR) with two different pairs of primers previously reported to differentiate Leptosphaeria maculans (Plenodomus lingam) or L. biglobosa (P. biglobosus). RESULTS: Detection was successful even at fewer than five spores per m3 of air. The primer pairs differed in the correlation coefficients obtained between DNA yields and the daily abundance of ascospores that were quantified by microscopy on duplicate halves of the spore trap tapes. Important differences in the specificity and sensitivity of the published SYBR-Green assays were also found, indicating that the Liu primers did not detect L. biglobosa subclade 'canadensis', whereas the Mahuku primers detected L. biglobosa subclade 'canadensis' and also the closely related Plenodomus dezfulensis. CONCLUSIONS: Comparisons confirmed that application of qPCR assays to spore trap samples can be used for the early detection, discrimination and quantification of aerially dispersed L. maculans and L. biglobosa propagules before leaf spot symptoms are visible in winter oilseed rape fields. The specificity of the primers must be taken into consideration because the final result will greatly depend on the local population of the pathogen. © 2023 Society of Chemical Industry.

Novel gene loci associated with susceptibility or cryptic quantitative resistance to Pyrenopeziza brassicae in Brassica napus.

KEY MESSAGE: Quantitative disease resistance (QDR) controls the association of the light leaf spot pathogen with Brassica napus; four QDR loci that were in linkage disequilibrium and eight gene expression markers were identified. Quantitative disease resistance (QDR) can provide durable control of pathogens in crops in contrast to resistance (R) gene-mediated resistance which can break down due to pathogen evolution. QDR is therefore a desirable trait in crop improvement, but little is known about the causative genes, and so it is difficult to incorporate into breeding programmes. Light leaf spot, caused by Pyrenopeziza brassicae, is an important disease of oilseed rape (canola, Brassica napus). To identify new QDR gene loci, we used a high-throughput screening pathosystem with P. brassicae on 195 lines of B. napus combined with an association transcriptomics platform. We show that all resistance against P. brassicae was associated with QDR and not R gene-mediated. We used genome-wide association analysis with an improved B. napus population structure to reveal four gene loci significantly (P = 0.0001) associated with QDR in regions showing linkage disequilibrium. On chromosome A09, enhanced resistance was associated with heterozygosity for a cytochrome P450 gene co-localising with a previously described locus for seed glucosinolate content. In addition, eight significant gene expression markers with a false discovery rate of 0.001 were associated with QDR against P. brassicae. For seven of these, expression was positively correlated with resistance, whereas for one, a HXXXD-type acyl-transferase, negative correlation indicated a potential susceptibility gene. The study identifies novel QDR loci for susceptibility and resistance, including novel cryptic QDR genes associated with heterozygosity, that will inform future crop improvement.

Leptosphaeria biglobosa inhibits the production of sirodesmin PL by L. maculans.

BACKGROUND: Phoma stem canker is caused by two coexisting pathogens, Leptosphaeria maculans and L. biglobosa. They coexist because of their temporal and spatial separations, which are associated with the differences in timing of their ascospore release. L. maculans produces sirodesmin PL, while L. biglobosa does not. However, their interaction/coexistence in terms of secondary metabolite production is not understood. RESULTS: Secondary metabolites were extracted from liquid cultures, L. maculans only (Lm only), L. biglobosa only (Lb only), L. maculans and L. biglobosa simultaneously (Lm&Lb) or sequentially 7 days later (Lm+Lb). Sirodesmin PL or its precursors were identified in extracts from 'Lm only' and 'Lm+Lb', but not from 'Lm&Lb'. Metabolites from 'Lb only', 'Lm&Lb' or 'Lm+Lb' caused significant reductions in L. maculans colony area. However, only the metabolites containing sirodesmin PL caused a significant reduction to L. biglobosa colony area. When oilseed rape cotyledons were inoculated with conidia of 'Lm only', 'Lb only' or 'Lm&Lb', 'Lm only' produced large gray lesions, while 'Lm&Lb' produced small dark lesions similar to lesions caused by 'Lb only'. Sirodesmin PL was found only in the plant extracts from 'Lm only'. These results suggest that L. biglobosa prevents the production of sirodesmin PL and its precursors by L. maculans when they grow simultaneously in vitro or in planta. CONCLUSION: For the first time, L. biglobosa has been shown to inhibit the production of sirodesmin PL by L. maculans when interacting simultaneously with L. maculans either in vitro or in planta. This antagonistic effect of interspecific interaction may affect their coexistence and subsequent disease progression and management. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Azole fungicide sensitivity and molecular mechanisms of reduced sensitivity in Irish Pyrenopeziza brassicae populations.

BACKGROUND: Light leaf spot, caused by Pyrenopeziza brassicae, is amongst the most damaging diseases of winter oilseed rape (Brassica napus), and currently the sterol 14α-demethylase (CYP51) inhibitors (azoles) represent the main class of fungicides used to control light leaf spot development. However, a shift in sensitivity to azole fungicides in P. brassicae populations has been observed in different European countries, including Ireland. RESULTS: To assess the sensitivity status of Irish P. brassicae populations to azole fungicides, three collections of P. brassicae from 2018-2020 were tested in vitro against tebuconazole and prothioconazole-desthio, and the PbCYP51 gene targeted by this class of fungicides was genotyped in different isolates. A change in sensitivity to azole fungicides was observed and differences in sensitivity to tebuconazole between Irish populations were present. There were two substitutions within PbCYP51 (G460S and S508T) and inserts of different sizes in its promoter region. The presence of the G460S/S508T double mutant was reported for the first time, and the diversity in insert size was greater than previously known. Compared to wild type isolates, those carrying G460S or S508T were less sensitive to both fungicides and, where inserts were also identified, they further reduced sensitivity to azole fungicides. CONCLUSIONS: The results of this study suggest that azole fungicides are still very effective in controlling light leaf spot in Ireland. However, using azole fungicides in mixtures of fungicides with different modes of action is recommended. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Distribution and Biodiversity of Seed-Borne Pathogenic and Toxigenic Fungi of Maize in Egypt and Their Correlations with Weather Variables.

Studies of the biodiversity of plant pathogenic and toxigenic fungi are attracting great attention to improve the predictability of their epidemics and the development of their control programs. Two hundred maize grain samples were gathered from 25 maize-growing governorates in Egypt and 189 samples were processed for the isolation and identification of seed-borne fungal microbiome. Twenty-six fungal genera comprising 42 species were identified according to their morphological characteristics and ITS DNA sequence analysis. Occurrence and biodiversity indicators of these fungal species were calculated. Ustilago maydis, Alternaria alternata, Aspergillus flavus, A. niger, Penicillium spp., Cladosporium spp. and Fusarium verticillioides were the highly frequent (>90% for each), recording the highest relative abundance (˃50%). Al-Menia governorate showed the highest species diversity and richness, followed by Sohag, Al-Nobaria and New Valley governorates. Correlations of 18 fungal species with temperature, relative humidity, precipitation, wind speed, and solar radiation were analyzed using canonical correspondence analysis. Results showed that relative humidity, temperature, and wind speed, respectively, were the most impactful weather variables. However, the occurrence and distribution of these fungi were not clearly grouped into the distinctive climatic regions in which maize crops are grown. Monitoring the occurrence and distribution of the fungal pathogens of maize grains in Egypt will play an important role in predicting their outbreaks and developing appropriate future management strategies. The findings in this study may be useful to other maize-growing countries that have similar climatic conditions.

Influence of Elevated Temperatures on Resistance Against Phoma Stem Canker in Oilseed Rape.

Cultivar resistance is an important tool in controlling pathogen-related diseases in agricultural crops. As temperatures increase due to global warming, temperature-resilient disease resistance will play an important role in crop protection. However, the mechanisms behind the temperature-sensitivity of the disease resistance response are poorly understood in crop species and little is known about the effect of elevated temperatures on quantitative disease resistance. Here, we investigated the effect of temperature increase on the quantitative resistance of Brassica napus against Leptosphaeria maculans. Field experiments and controlled environment inoculation assays were done to determine the influence of temperature on R gene-mediated and quantitative resistance against L. maculans; of specific interest was the impact of high summer temperatures on the severity of phoma stem canker. Field experiments were run for three consecutive growing seasons at various sites in England and France using twelve winter oilseed rape breeding lines or cultivars with or without R genes and/or quantitative resistance. Stem inoculation assays were done under controlled environment conditions with four cultivars/breeding lines, using avirulent and virulent L. maculans isolates, to determine if an increase in ambient temperature reduces the efficacy of the resistance. High maximum June temperature was found to be related to phoma stem canker severity. No temperature effect on stem canker severity was found for the cultivar ES Astrid (with only quantitative resistance with no known R genes). However, in the controlled environmental conditions, the cultivar ES Astrid had significantly smaller amounts of necrotic tissue at 20°C than at 25°C. This suggests that, under a sustained temperature of 25°C, the efficacy of quantitative resistance is reduced. Findings from this study show that temperature-resilient quantitative resistance is currently available in some oilseed cultivars and that efficacy of quantitative resistance is maintained at increased temperature but not when these elevated temperatures are sustained for a long period.

Quantitative Trait Locus Mapping for Resistance Against Pyrenopeziza brassicae Derived From a Brassica napus Secondary Gene Pool.

Use of host resistance is the most economical and environmentally safe way to control light leaf spot disease of oilseed rape (Brassica napus). The causal organism of light leaf spot, Pyrenopeziza brassicae, is one of the most economically damaging pathogens of oilseed rape in the United Kingdom and it is considered to have a high potential to evolve due to its mixed reproduction system and airborne ascospores. This necessitates diverse sources of host resistance, which are inadequate at present to minimize yield losses caused by this disease. To address this, we screened a doubled haploid (DH) population of oilseed rape, derived from a secondary gene pool (ancestral genomes) of B. napus for the introgression of resistance against P. brassicae. DH lines were phenotyped using controlled-environment and glasshouse experiments with P. brassicae populations obtained from three different geographic locations in the United Kingdom. Selected DH lines with different levels of resistance were further studied in a controlled-environment experiment using both visual (scanning electron microscope - SEM) and molecular (quantitative PCR) assessment methods to understand the mode/s of host resistance. There was a clear phenotypic variation for resistance against P. brassicae in this DH population. Quantitative trait locus (QTL) analysis identified four QTLs with moderate to large effects, which were located on linkage groups C1, C6, and C9. Of these, the QTL on the linkage group C1 appeared to have a major effect on limiting P. brassicae asexual sporulation. Study of the sub-cuticular growth phase of P. brassicae using qPCR and SEM showed that the pathogen was able to infect and colonise both resistant and susceptible Q DH lines and control B. napus cultivars. However, the rate of increase of pathogen biomass was significantly smaller in resistant lines, suggesting that the resistance segregating in this DH population limits colonisation/sporulation by the pathogen rather than eliminating the pathogen. Resistance QTLs identified in this study provide a useful resource for breeding cultivar resistance for effective control of light leaf spot and form a starting point for functional identification of the genes controlling resistance against P. brassicae that can contribute to our knowledge on mechanisms of partial resistance of crops against pathogens.

Biodiversity of Pathogenic and Toxigenic Seed-Borne Mycoflora of Wheat in Egypt and Their Correlations with Weather Variables.

Surveillance investigations for pathogenic and toxigenic fungi are important to refine our understanding of their epidemiology and help in predicting their outbreaks. During 2019, 198 samples of wheat grains were collected from 25 wheat-growing governorates in Egypt to detect and identify seed-borne mycoflora in vitro. Forty-four fungal species belonging to 20 genera were identified. Molecular data for these fungi were analyzed to construct a phylogenetic tree. Occurrence and biodiversity indicators were calculated. Two prevalent pathogens (average incidence > 40%) were Alternaria alternata and Cladosporium spp. Ustilago tritici was present in only seven of the 25 governorates, and less abundant than Tilletia tritici, the causal agent of stinking smut. Sinai governorate recorded the greatest species diversity, while the greatest species richness was in Qena and Sohag governorates. Canonical correspondence analysis of data for 20 fungal genera with temperature, relative humidity, precipitation, wind speed or solar radiation revealed that relative humidity was the most influential weather variable. It showed that occurrence and distribution of the 20 genera corresponded well with three out of four Egyptian climatic regions: Mediterranean, semi-arid, and arid. Knowing pathogen occurrence and distribution in Egypt is the first step to developing future disease management strategies to limit yield losses and improve food security. Despite this study being conducted on the wheat-growing areas in Egypt, our findings are useful for other wheat-growing countries that share the same climatic conditions. The correlation between a given fungus and the climatic variables can be useful in other ecosystems.

Mycovirus-Induced Hypervirulence of Leptosphaeria biglobosa Enhances Systemic Acquired Resistance to Leptosphaeria maculans in Brassica napus.

Phoma stem canker (blackleg) is one of the most important diseases of winter oilseed rape (Brassica napus) worldwide and is caused by a complex that comprises at least two species: Leptosphaeria maculans and L. biglobosa. Screening a panel of field Leptosphaeria isolates from B. napus for the presence of mycoviruses revealed the presence of a novel double-stranded RNA quadrivirus in L. biglobosa and no viruses in L. maculans. Following elimination of the mycovirus, virus-infected and virus-free isogenic lines of L. biglobosa were created. A direct comparison of the growth and virulence of these isogenic lines illustrated that virus infection caused hypervirulence and resulted in induced systemic resistance toward L. maculans in B. napus following lower leaf preinoculation with the virus-infected isolate. Analysis of the plant transcriptome suggests that the presence of the virus leads to subtle alterations in metabolism and plant defenses. For instance, transcripts involved in carbohydrate and amino acid metabolism are enriched in plants treated with the virus-infected isolate, while pathogenesis-related proteins, chitinases and WRKY transcription factors are differentially expressed. These results illustrate the potential for deliberate inoculation of plants with hypervirulent L. biglobosa to decrease the severity of Phoma stem canker later in the growing season.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Oilseed rape (Brassica napus) resistance to growth of Leptosphaeria maculans in leaves of young plants contributes to quantitative resistance in stems of adult plants.

Key message: One QTL for resistance against Leptosphaeria maculans growth in leaves of young plants in controlled environments overlapped with one QTL detected in adult plants in field experiments. The fungal pathogen Leptosphaeria maculans initially infects leaves of oilseed rape (Brassica napus) in autumn in Europe and then grows systemically from leaf lesions along the leaf petiole to the stem, where it causes damaging phoma stem canker (blackleg) in summer before harvest. Due to the difficulties of investigating resistance to L. maculans growth in leaves and petioles under field conditions, identification of quantitative resistance typically relies on end of season stem canker assessment on adult plants. To investigate whether quantitative resistance can be detected in young plants, we first selected nine representative DH (doubled haploid) lines from an oilseed rape DY ('Darmor-bzh' × 'Yudal') mapping population segregating for quantitative resistance against L. maculans for controlled environment experiment (CE). We observed a significant correlation between distance grown by L. maculans along the leaf petiole towards the stem (r = 0.91) in CE experiments and the severity of phoma stem canker in field experiments. To further investigate quantitative trait loci (QTL) related to resistance against growth of L. maculans in leaves of young plants in CE experiments, we selected 190 DH lines and compared the QTL detected in CE experiments with QTL related to stem canker severity in stems of adult plants in field experiments. Five QTL for resistance to L. maculans growth along the leaf petiole were detected; collectively they explained 35% of the variance. Two of these were also detected in leaf lesion area assessments and each explained 10-12% of the variance. One QTL on A02 co-localized with a QTL detected in stems of adult plants in field experiments. This suggests that resistance to the growth of L. maculans from leaves along the petioles towards the stems contributes to the quantitative resistance assessed in stems of adult plants in field experiments at the end of the growing season.

Identification, Molecular Characterization, and Biology of a Novel Quadrivirus Infecting the Phytopathogenic Fungus Leptosphaeria biglobosa.

Here we report the molecular characterisation of a novel dsRNA virus isolated from the filamentous, plant pathogenic fungus Leptosphaeria biglobosa and known to cause significant alterations to fungal pigmentation and growth and to result in hypervirulence, as illustrated by comparisons between virus-infected and -cured isogenic fungal strains. The virus forms isometric particles approximately 40⁻45 nm in diameter and has a quadripartite dsRNA genome structure with size ranges of 4.9 to 4 kbp, each possessing a single ORF. Sequence analysis of the putative proteins encoded by dsRNAs 1⁻4, termed P1⁻P4, respectively, revealed modest similarities to the amino acid sequences of equivalent proteins predicted from the nucleotide sequences of known and suspected members of the family Quadriviridae and for that reason the virus was nominated Leptosphaeria biglobosa quadrivirus-1 (LbQV-1). Sequence and phylogenetic analysis using the P3 sequence, which encodes an RdRP, revealed that LbQV-1 was most closely related to known and suspected quadriviruses and monopartite totiviruses rather than other quadripartite mycoviruses including chrysoviruses and alternaviruses. Of the remaining encoded proteins, LbQV-1 P2 and P4 are structural proteins but the function of P1 is unknown. We propose that LbQV-1 is a novel member of the family Quadriviridae.

Genomic evidence for genes encoding leucine-rich repeat receptors linked to resistance against the eukaryotic extra- and intracellular Brassica napus pathogens Leptosphaeria maculans and Plasmodiophora brassicae.

Genes coding for nucleotide-binding leucine-rich repeat (LRR) receptors (NLRs) control resistance against intracellular (cell-penetrating) pathogens. However, evidence for a role of genes coding for proteins with LRR domains in resistance against extracellular (apoplastic) fungal pathogens is limited. Here, the distribution of genes coding for proteins with eLRR domains but lacking kinase domains was determined for the Brassica napus genome. Predictions of signal peptide and transmembrane regions divided these genes into 184 coding for receptor-like proteins (RLPs) and 121 coding for secreted proteins (SPs). Together with previously annotated NLRs, a total of 720 LRR genes were found. Leptosphaeria maculans-induced expression during a compatible interaction with cultivar Topas differed between RLP, SP and NLR gene families; NLR genes were induced relatively late, during the necrotrophic phase of pathogen colonization. Seven RLP, one SP and two NLR genes were found in Rlm1 and Rlm3/Rlm4/Rlm7/Rlm9 loci for resistance against L. maculans on chromosome A07 of B. napus. One NLR gene at the Rlm9 locus was positively selected, as was the RLP gene on chromosome A10 with LepR3 and Rlm2 alleles conferring resistance against L. maculans races with corresponding effectors AvrLm1 and AvrLm2, respectively. Known loci for resistance against L. maculans (extracellular hemi-biotrophic fungus), Sclerotinia sclerotiorum (necrotrophic fungus) and Plasmodiophora brassicae (intracellular, obligate biotrophic protist) were examined for presence of RLPs, SPs and NLRs in these regions. Whereas loci for resistance against P. brassicae were enriched for NLRs, no such signature was observed for the other pathogens. These findings demonstrate involvement of (i) NLR genes in resistance against the intracellular pathogen P. brassicae and a putative NLR gene in Rlm9-mediated resistance against the extracellular pathogen L. maculans.

Combining R gene and quantitative resistance increases effectiveness of cultivar resistance against Leptosphaeria maculans in Brassica napus in different environments.

Using cultivar resistance against pathogens is one of the most economical and environmentally friendly methods for control of crop diseases. However, cultivar resistance can be easily rendered ineffective due to changes in pathogen populations or environments. To test the hypothesis that combining R gene-mediated resistance and quantitative resistance (QR) in one cultivar can provide more effective resistance than use of either type of resistance on its own, effectiveness of resistance in eight oilseed rape (Brassica napus) cultivars with different R genes and/or QR against Leptosphaeria maculans (phoma stem canker) was investigated in 13 different environments/sites over three growing seasons (2010/2011, 2011/2012 and 2012/2013). Cultivar Drakkar with no R genes and no QR was used as susceptible control and for sampling L. maculans populations. Isolates of L. maculans were obtained from the 13 sites in 2010/2011 to assess frequencies of avirulent alleles of different effector genes (AvrLm1, AvrLm4 or AvrLm7) corresponding to the resistance genes (Rlm1, Rlm4 or Rlm7) used in the field experiments. Results of field experiments showed that cultivars DK Cabernet (Rlm1 + QR) and Adriana (Rlm4 + QR) had significantly less severe phoma stem canker than cultivars Capitol (Rlm1) and Bilbao (Rlm4), respectively. Results of controlled environment experiments confirmed the presence of Rlm genes and/or QR in these four cultivars. Analysis of L. maculans populations from different sites showed that the mean frequencies of AvrLm1 (10%) and AvrLm4 (41%) were less than that of AvrLm7 (100%), suggesting that Rlm1 and Rlm4 gene-mediated resistances were partially rendered ineffective while Rlm7 resistance was still effective. Cultivar Excel (Rlm7 + QR) had less severe canker than cultivar Roxet (Rlm7), but the difference between them was not significant due to influence of the effective resistance gene Rlm7. For the two cultivars with only QR, Es-Astrid (QR) had less severe stem canker than NK Grandia (QR). Analysis of the relationship between severity of stem canker and weather data among the 13 sites in the three growing seasons showed that increased severity of stem canker was associated with increased rainfall during the phoma leaf spot development stage and increased temperature during the stem canker development stage. Further analysis of cultivar response to environmental factors showed that cultivars with both an Rlm gene and QR (e.g. DK Cabernet, Adriana and Excel) were less sensitive to a change in environment than cultivars with only Rlm genes (e.g. Capitol, Bilbao) or only QR (e.g. DK Grandia). These results suggest that combining R gene and QR can provide effective, stable control of phoma stem canker in different environments.

Correction to: Incorporating pleiotropic quantitative trait loci in dissection of complex traits: seed yield in rapeseed as an example.

The article "Incorporating pleiotropic quantitative trait loci in dissection of complex traits: seed yield in rapeseed as an example", written by J. Zou et al, was originally published Online First without open access.

Azole sensitivity in Leptosphaeria pathogens of oilseed rape: the role of lanosterol 14α-demethylase.

Lanosterol 14-α demethylase is a key enzyme intermediating the biosynthesis of ergosterol in fungi, and the target of azole fungicides. Studies have suggested that Leptosphaeria maculans and L. biglobosa, the causal agents of phoma stem canker on oilseed rape, differ in their sensitivity to some azoles, which could be driving pathogen frequency change in crops. Here we used CYP51 protein modelling and heterologous expression to determine whether there are interspecific differences at the target-site level. Moreover, we provide an example of intrinsic sensitivity differences exhibited by both Leptosphaeria spp. in vitro and in planta. Comparison of homologous protein models identified highly conserved residues, particularly at the azole binding site, and heterologous expression of LmCYP51B and LbCYP51B, with fungicide sensitivity testing of the transformants, suggests that both proteins are similarly sensitive to azole fungicides flusilazole, prothioconazole-desthio and tebuconazole. Fungicide sensitivity testing on isolates shows that they sometimes have a minor difference in sensitivity in vitro and in planta. These results suggest that azole fungicides remain a useful component of integrated phoma stem canker control in the UK due to their effectiveness on both Leptosphaeria spp. Other factors, such as varietal resistance or climate, may be driving observed frequency changes between species.

Incorporating pleiotropic quantitative trait loci in dissection of complex traits: seed yield in rapeseed as an example.

KEY MESSAGE: A comprehensive linkage atlas for seed yield in rapeseed. Most agronomic traits of interest for crop improvement (including seed yield) are highly complex quantitative traits controlled by numerous genetic loci, which brings challenges for comprehensively capturing associated markers/genes. We propose that multiple trait interactions underlie complex traits such as seed yield, and that considering these component traits and their interactions can dissect individual quantitative trait loci (QTL) effects more effectively and improve yield predictions. Using a segregating rapeseed (Brassica napus) population, we analyzed a large set of trait data generated in 19 independent experiments to investigate correlations between seed yield and other complex traits, and further identified QTL in this population with a SNP-based genetic bin map. A total of 1904 consensus QTL accounting for 22 traits, including 80 QTL directly affecting seed yield, were anchored to the B. napus reference sequence. Through trait association analysis and QTL meta-analysis, we identified a total of 525 indivisible QTL that either directly or indirectly contributed to seed yield, of which 295 QTL were detected across multiple environments. A majority (81.5%) of the 525 QTL were pleiotropic. By considering associations between traits, we identified 25 yield-related QTL previously ignored due to contrasting genetic effects, as well as 31 QTL with minor complementary effects. Implementation of the 525 QTL in genomic prediction models improved seed yield prediction accuracy. Dissecting the genetic and phenotypic interrelationships underlying complex quantitative traits using this method will provide valuable insights for genomics-based crop improvement.

Comparative genomics to explore phylogenetic relationship, cryptic sexual potential and host specificity of Rhynchosporium species on grasses.

BACKGROUND: The Rhynchosporium species complex consists of hemibiotrophic fungal pathogens specialized to different sweet grass species including the cereal crops barley and rye. A sexual stage has not been described, but several lines of evidence suggest the occurrence of sexual reproduction. Therefore, a comparative genomics approach was carried out to disclose the evolutionary relationship of the species and to identify genes demonstrating the potential for a sexual cycle. Furthermore, due to the evolutionary very young age of the five species currently known, this genus appears to be well-suited to address the question at the molecular level of how pathogenic fungi adapt to their hosts. RESULTS: The genomes of the different Rhynchosporium species were sequenced, assembled and annotated using ab initio gene predictors trained on several fungal genomes as well as on Rhynchosporium expressed sequence tags. Structures of the rDNA regions and genome-wide single nucleotide polymorphisms provided a hypothesis for intra-genus evolution. Homology screening detected core meiotic genes along with most genes crucial for sexual recombination in ascomycete fungi. In addition, a large number of cell wall-degrading enzymes that is characteristic for hemibiotrophic and necrotrophic fungi infecting monocotyledonous hosts were found. Furthermore, the Rhynchosporium genomes carry a repertoire of genes coding for polyketide synthases and non-ribosomal peptide synthetases. Several of these genes are missing from the genome of the closest sequenced relative, the poplar pathogen Marssonina brunnea, and are possibly involved in adaptation to the grass hosts. Most importantly, six species-specific genes coding for protein effectors were identified in R. commune. Their deletion yielded mutants that grew more vigorously in planta than the wild type. CONCLUSION: Both cryptic sexuality and secondary metabolites may have contributed to host adaptation. Most importantly, however, the growth-retarding activity of the species-specific effectors suggests that host adaptation of R. commune aims at extending the biotrophic stage at the expense of the necrotrophic stage of pathogenesis. Like other apoplastic fungi Rhynchosporium colonizes the intercellular matrix of host leaves relatively slowly without causing symptoms, reminiscent of the development of endophytic fungi. Rhynchosporium may therefore become an object for studying the mutualism-parasitism transition.