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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.

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.

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.

Quantitative resistance against Leptosphaeria maculans in Brassica napus is difficult to assess in young plants due to the long period of symptomless growth of the pathogen from the appearance of leaf lesions to the appearance of canker symptoms on the stem. By using doubled haploid (DH) lines A30 (susceptible) and C119 (with quantitative resistance), quantitative resistance against L. maculans was assessed in young plants in controlled environments at two stages: stage 1, growth of the pathogen along leaf veins/petioles towards the stem by leaf lamina inoculation; stage 2, growth in stem tissues to produce stem canker symptoms by leaf petiole inoculation. Two types of inoculum (ascospores; conidia) and three assessment methods (extent of visible necrosis; symptomless pathogen growth visualised using the GFP reporter gene; amount of pathogen DNA quantified by PCR) were used. In stage 1 assessments, significant differences were observed between lines A30 and C119 in area of leaf lesions, distance grown along veins/petioles assessed by visible necrosis or by viewing GFP and amount of L. maculans DNA in leaf petioles. In stage 2 assessments, significant differences were observed between lines A30 and C119 in severity of stem canker and amount of L. maculans DNA in stem tissues. GFP-labelled L. maculans spread more quickly from the stem cortex to the stem pith in A30 than in C119. Stem canker symptoms were produced more rapidly by using ascospore inoculum than by using conidial inoculum. These results suggest that quantitative resistance against L. maculans in B. napus can be assessed in young plants in controlled conditions. Development of methods to phenotype quantitative resistance against plant pathogens in young plants in controlled environments will help identification of stable quantitative resistance for control of crop diseases.

The fungal genus Rhynchosporium (causative agent of leaf blotch) contains several host-specialised species, including R. commune (colonising barley and brome-grass), R. agropyri (couch-grass), R. secalis (rye and triticale) and the more distantly related R. orthosporum (cocksfoot). This study used molecular fingerprinting, multilocus DNA sequence data, conidial morphology, host range tests and scanning electron microscopy to investigate the relationship between Rhynchosporium species on ryegrasses, both economically important forage grasses and common wild grasses in many cereal growing areas, and other plant species. Two different types of Rhynchosporium were found on ryegrasses in the UK. Firstly, there were isolates of R. commune that were pathogenic to both barley and Italian ryegrass. Secondly, there were isolates of a new species, here named R. lolii, that were pathogenic only to ryegrass species. R. lolii was most closely related to R. orthosporum, but exhibited clear molecular, morphological and host range differences. The species was estimated to have diverged from R. orthosporum ca. 5735 years before the present. The colonisation strategy of all of the different Rhynchosporium species involved extensive hyphal growth in the sub-cuticular regions of the leaves. Finally, new species-specific PCR diagnostic tests were developed that could distinguish between these five closely related Rhynchosporium species.

Abstract 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.

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.

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.

Abstract Experiments over five growing seasons at Rothamsted (1998/992002/03), four seasons at Boxworth (1998/99, 1999/2000, 2001/02, 2002/03) in England (Leptosphaeria maculans) and three seasons (1998/992000/01) at Poznan in Poland (Leptosphaeria biglobosa) suggest that differences in the development of phoma stem canker epidemics between England and Poland relate to differences in weather patterns between the two countries. The duration of ascospore release was longer in England, where winter weather is mild and wet, than in Poland, where winters are cold and often with snow cover, but there was little difference between two sites in England (Rothamsted and Boxworth). Wetness provided by rainfall was essential for release of ascospores of both L. maculans in England and L. biglobosa in Poland. Temperature did not affect release of ascospores over the range 520 C. Diurnal periodicity in release of ascospores of L. maculans in England and L. biglobosa in Poland was similar. The timing (date) of first release of ascospores of L. maculans or L. biglobosa in autumn was related to rainfall in August and September; with increasing rainfall the date was earlier. The incubation periods from first release of ascospores to first appearance of phoma leaf spots for both L. maculans in England and L. biglobosa in Poland, and from first leaf spots to first stem base canker in England, were described using a thermal time (degree-day) approximation. [ABSTRACT FROM AUTHOR]

In Egypt, wheat is the most consumed cereal grain, and its availability and affordability are important for social stability. Irrigation plays a vital role in wheat cultivation, despite intense competition for water resources from the River Nile across various societal sectors. To explore how grain and above-ground biomass yields respond to total seasonal water input from sowing to maturity in six bread wheat cultivars, eight field irrigation experiments were performed at four locations representative of three agro-climatic zones in two consecutive cropping seasons. A three-replicate strip-plot design was used with cultivars nested within the main plots featuring five irrigation treatments, ranging from six to two applications. Overall, irrigation treatment significantly affected nine agronomic traits. Compared with the six irrigation applications treatment (T1), the two irrigation applications treatment (T5) decreased the times to heading and maturity by 6.6 (7.3%) and 8.6 (6.3%) days, respectively. Similarly, T5 reduced the plant height by 14.9 cm (14.3%), flag leaf area by 12.0 cm2 (27.2%), number of spikes per square metre by 77.7 (20.1%), number of kernels per spike by 13.9 (25.2%) and thousand grain weight by 10.0 g (19.6%). T5 also decreased the overall mean grain yield and above-ground biomass yield by 2834.9 (32.0%) and 7910.4 (32.86%) kg/ha, respectively. The grain yield and above-ground biomass production were consistently greater for all six cultivars at Al Mataenah and Sids than at Nubaria and Ismailia in the two cropping seasons. All six cultivars showed significantly greater responses to total seasonal water input for the grain yield and above-ground biomass at Al Mataenah and Ismailia. These results emphasise the necessity for choosing regions with favourable soil and climatic conditions to grow wheat cultivars that respond better to irrigation to enhance the large-scale production of wheat in Egypt. The grain and above-ground biomass yields were mostly linearly and positively associated with the total seasonal water input for all six cultivars at all four locations. This suggests that maintaining the current irrigation schedule of six irrigations is valid and should be practised to maximise productivity, particularly in areas similar to the three representative agro-climatic zones in Egypt.

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.[Graphic: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

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.