Defoliation of Brassica napus increases severity of blackleg caused by Leptosphaeria maculans: implications for dual-purpose cropping

Canola (Brassica napus) crops for grazing and grain (dual-purpose) production provide an economic break-crop alternative for dual-purpose cereals in Australian mixed farming systems. Infection by Leptosphaeria maculans is the most prevalent disease in Australian canola crops with airborne inoculum released throughout the autumn and winter when crops are grazed. Glasshouse and field experiments were conducted to investigate the effect of mechanical defoliation (simulated grazing) on disease severity at plant maturity. In glasshouse experiments, stem canker severity increased from 4% to 24% in severely defoliated plants, but light defoliation had no effect compared with undefoliated control plants. Disease severity was increased with defoliation in all field experiments. Defoliation increased crown canker severity from 22.6% to 39.3% at Wagga Wagga and from 3.0% to 7.1% at Canberra and lodging from 9.6% to 11.9% at Naracoorte in the same set of cultivars assessed at each site. The increase in disease severity with defoliation was less in canola lines with moderate to high levels of stem canker resistance. Plants defoliated before stem elongation tended to develop less disease than those defoliated during the reproductive phase of plant growth. These findings suggest that the impact of grazing on L. maculans infection of canola crops can be minimised by sowing cultivars with a high level of stem canker resistance and grazing during the vegetative stage of plant growth prior to stem elongation. Further research is required to determine whether these management strategies are applicable in canola crops defoliated by grazing animals.     

Identification and mapping of a novel blackleg resistance locus LepR4 in the progenies from Brassica napus × B. rapa subsp. sylvestris

Blackleg, caused by Leptosphaeria maculans, is one of the most economically important diseases of Brassica napus worldwide. Two blackleg-resistant lines, 16S and 61446, were developed through interspecific hybridization between B. napus and B. rapa subsp. sylvestris and backcrossing to B. napus. Classical genetic analysis demonstrated that a single recessive gene in both lines conferred resistance to L. maculans and that the resistance alleles were allelic. Using BC₁ progeny derived from each resistant plant, this locus was mapped to B. napus linkage group N6 and was flanked by microsatellite markers sN2189b and sORH72a in an interval of about 10 cM, in a region equivalent to about 6 Mb of B. rapa DNA sequence. This new resistance gene locus was designated as LepR4. The two lines were evaluated for resistance to a wide range of L. maculans isolates using cotyledon inoculation tests under controlled environment conditions, and for stem canker resistance in blackleg field nurseries. Results indicated that line 16S, carrying LepR4a, was highly resistant to all isolates tested on cotyledons and had a high level of stem canker resistance under field conditions. Line 61446, carrying LepR4b, was only resistant to some of the isolates tested on cotyledons and was weakly resistant to stem canker under field conditions.     

Analysis of leaf appearance,leaf death and phoma leaf spot,caused by Leptosphaeria maculans,on oilseed rape (Brassica napus) cultivars

Development of phoma leaf spot (caused by Leptosphaeria maculans) on winter oilseed rape (canola, Brassica napus) was assessed in two experiments at Rothamsted in successive years (2003–04 and 2004–05 growing seasons). Both experiments compared oilseed rape cultivars Eurol, Darmor, Canberra and Lipton, which differ in their resistance to L. maculans. Data were analysed to describe disease development in terms of increasing numbers of leaves affected over thermal time from sowing. The cultivars showed similar patterns of leaf spot development in the 2003–04 experiment when inoculum concentration was relatively low (up to 133 ascospores m⁻³ air), Darmor developing 5.3 diseased leaves per plant by 5 May 2004, Canberra 6.6, Eurol 6.8 and Lipton 7.5. Inoculum concentration was up to sevenfold greater in 2004–05, with Eurol and Darmor developing 2.4 diseased leaves per plant by 16 February 2005, whereas Lipton and Canberra developed 2.8 and 3.0 diseased leaves, respectively. Based on three defined periods of crop development, a piece-wise linear statistical model was applied to the progress of the leaf spot disease (cumulative diseased leaves) in relation to appearance (‘birth’) and death of leaves for individual plants of each cultivar. Estimates of the thermal time from sowing until appearance of the first leaf or death of the first leaf, the rate of increase in number of diseased leaves and the area under the disease progress line (AUDPL) for the first time period were made. In 2004–05, Canberra (1025 leaves ×°C days) and Lipton (879) had greater AUDPL values than Eurol (427) and Darmor (598). For Darmor and Lipton, the severity of leaf spotting could be related to the severity of stem canker at harvest. Eurol had less leaf spotting but severe stem canker, whereas Canberra had more leaf spotting but less severe canker.     

Association mapping of quantitative resistance for <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Stem canker caused by the fungus Leptosphaeria maculans is a major disease of Brassica napus. Quantitative resistance factors appear to be important components for effective and durable control of this pathogen. Quantitative trait loci (QTL) for stem canker resistance have previously been identified in the Darmor variety. However, before these QTL can be used in marker-assisted selection (MAS) to breed resistant varieties, they must be validated in a wide range of genetic backgrounds. We used an association mapping approach to confirm the markers located within the QTL previously identified in Darmor and establish their usefulness in MAS. For this, we characterized the molecular diversity of an oilseed rape collection of 128 lines showing a large spectrum of responses to infection by L. maculans, using 72 pairs of primers for simple sequence repeat and other markers. We used different association mapping models which either do or do not take into account the population structure and/or family relatedness. In all, 61 marker alleles were found to be associated with resistance to stem canker. Some of these markers were associated with previously identified QTL, which confirms their usefulness in MAS. Markers located in regions not harbouring previously identified QTL were also associated with resistance, suggesting that new QTL or allelic variants are present in the collection. All of these markers associated with stem canker resistance will help identify accessions carrying desirable alleles and facilitate QTL introgression.     

Transgenic canola lines expressing pea defense gene DRR206 have resistance to aggressive blackleg isolates and to Rhizoctonia solani

We have previously demonstrated that transgenic Brassica napus plants expressing pea DRR206 constitutively are resistant to the hemibiotrophic blackleg fungus, Leptosphaeria maculans isolate PG2. The present work seeks to determine whether DRR206 is effective against a wider range of fungi. Transgenic plants expressing DRR206 exhibit decreased severity of stem canker in adult plants inoculated with aggressive L. maculans isolates PG3 and PG4. Decreased seedling mortality with the biotrophic root pathogen Rhizoctonia solani is also seen. Finally, leaves of DRR206 transgenic plants inoculated with the necrotroph Sclerotinia sclerotiorum show smaller lesions at 48 h after inoculation, leading to a delay, but not a prevention, of disease development. These results demonstrate the effectiveness of DRR206 against several fungal species with three distinct modes of pathogenicity. Although its precise function remains to be determined, a recent report shows that pea DRR206 shares strong amino acid sequence similarity with `dirigent proteins' which couple monolignol radicals to form the lignan (+) pinoresinol.     

Molecular and phenotypic characterization of near isogenic lines at QTL for quantitative resistance to Leptosphaeria maculans in oilseed rape (Brassica napus L.)

The most common and effective way to control phoma stem canker (blackleg) caused by Leptosphaeria maculans in oilseed rape (Brassica napus) is by breeding resistant cultivars. Specific resistance genes have been identified in B. napus and related species but in some B. napus cultivars resistance is polygenic [mediated by quantitative trait loci (QTL)], postulated to be race non-specific and durable. The genetic basis of quantitative resistance in the French winter oilseed rape ‘Darmor’, which was derived from ‘Jet Neuf’, was previously examined in two genetic backgrounds. Stable QTL involved in blackleg resistance across year and genetic backgrounds were identified. In this study, near isogenic lines (NILs) were produced in the susceptible background ‘Yudal’ for four of these QTL using marker-assisted selection. Various strategies were used to develop new molecular markers, which were mapped in these QTL regions. These were used to characterize the length and homozygosity of the ‘Darmor-bzh’ introgressed segment in the NILs. Individuals from each NIL were evaluated in blackleg disease field trials and assessed for their level of stem canker in comparison to the recurrent line ‘Yudal’. The effect of QTL LmA2 was clearly validated and to a lesser extent, QTL LmA9 also showed an effect on the disease level. This work provides valuable material that can be used to study the mode of action of genetic factors involved in L. maculans quantitative resistance.     

Novel properties of antimicrobial peptide anoplin

Antimicrobial decapeptide anoplin was tested for its antifungal activity against plant pathogen Leptosphaeria maculans and protection of Brassica napus plants from disease. To reveal the mode of action of the peptide, a natural form of anoplin amidated on C-terminus (ANP-NH₂), and its carboxylated analog (ANP-OH) were used in the study. We demonstrated strong antifungal activity of anoplin in vitro regardless C-terminus modification. In addition we show that both ANP-NH₂ and ANP-OH induce expression of defence genes in B. napus and protects plants from L. maculans infection. The results indicate that the amidation of anoplin is not essential for its antifungal and plant defence stimulating activities.     

Arabidopsis thaliana-derived resistance against Leptosphaeria maculans in a Brassica napus genomic background

Stem canker (blackleg) caused by Leptosphaeria maculans is a widespread disease of Brassica napus. In contrast, most Arabidopsis thaliana accessions are highly resistant. Hence, novel material derived from symmetric and asymmetric somatic hybrids between B. napus and A. thaliana was utilised in a screen for L. maculans resistance. Initially, both cotyledon and adult-leaf resistance traits were transferred from A. thaliana to B. napus. In later generations the two traits segregated and cotyledon resistance was lost. The adult-leaf resistance was investigated with respect to genome localisation and protein expression. Analyses of remaining A. thaliana DNA in resistant plants showed co-segregation between adult-leaf resistance and chromosome-3 molecular markers. Resistant offspring from asymmetric hybrid plants that contained fragments of chromosome 3 were studied in more detail. Two regions at positions 9.8-10.4 Mbp and 18-19.5 Mbp, where several defence-related genes are located, were identified. A proteomic approach was taken to further investigate genes involved in the defence interaction. Forty eight hours after inoculation with L. maculans, only a few proteins, such as glycolate oxidase, were identified as differentially expressed in the resistant line compared to B. napus, despite the presence of additional A. thaliana chromosomes. The plant materials described in the present study constitute a new genetic source of L. maculans resistance and are currently being incorporated into B. napus breeding programmes.     

Impact of a resistance gene against a fungal pathogen on the plant host residue microbiome: The case of the Leptosphaeria maculans–Brassica napus pathosystem

Oilseed rape residues are a crucial determinant of stem canker epidemiology as they support the sexual reproduction of the fungal pathogen Leptosphaeria maculans. The aim of this study was to characterize the impact of a resistance gene against L. maculans infection on residue microbial communities and to identify microorganisms interacting with this pathogen during residue degradation. We used near-isogenic lines to obtain healthy and infected host plants. The microbiome associated with the two types of plant residues was characterized by metabarcoding. A combination of linear discriminant analysis and ecological network analysis was used to compare the microbial communities and to identify microorganisms interacting with L. maculans. Fungal community structure differed between the two lines at harvest, but not subsequently, suggesting that the presence/absence of the resistance gene influences the microbiome at the base of the stem whilst the plant is alive, but that this does not necessarily lead to differential colonization of the residues by fungi. Direct interactions with other members of the community involved many fungal and bacterial amplicon sequence variants (ASVs). L. maculans appeared to play a minor role in networks, whereas one ASV affiliated to Plenodomus biglobosus (synonym Leptosphaeria biglobosa) from the Leptosphaeria species complex may be considered a keystone taxon in the networks at harvest. This approach could be used to identify and promote microorganisms with beneficial effects against residue-borne pathogens and, more broadly, to decipher the complex interactions between multispecies pathosystems and other microbial components in crop residues.     

Besides stem canker severity,oilseed rape host genotype matters for the production of Leptosphaeria maculans fruit bodies

For fungal cyclic epidemics on annual crops, the pathogen carry-over is an important but poorly documented step. Plant resistance affects the pathogen development within the epidemics but we lack data on the inter-annual transmission of inoculum. For Leptosphaeria maculans on 15 oilseed rape genotypes in field during 4 growing seasons, stem canker severity was visually scored at harvest. The number of fruit bodies produced on incubated stubble was quantified using an automated image analysis framework. Our results confirm that fruit body production increases with disease severity and is significantly affected by host genotype and nitrogen supply. Tracking individual stems through incubation, we confirm for the first time that the oilseed rape genotype has a direct effect on inoculum production, not only disease severity. This major effect of genotype on inoculum carry-over should be taken into account in models of varietal deployment strategies.     

Role of hydrogen peroxide and antioxidant enzymes in the interaction between a hemibiotrophic fungal pathogen, Leptosphaeria maculans, and oilseed rape

Reactive oxygen species play a dual role in host-pathogen interaction. They impede the spread of biotrophic pathogens via stimulating cell death and hypersensitive response (HR), and, on the other hand, they provide access to nutrients for necrotrophic pathogens feeding on dead tissues and facilitate their colonizing the host. The participation of ROS in defending plants from pathogens with a combined lifestyle (hemibiotrophs) is not yet understood, and it varies in its dependence on the particular host-pathogen combination. In the present study, we inoculated rapeseed plants (Brassica napus) with a hemibiotrophic fungus, Leptosphaeria maculans, and manipulated the H₂O₂ content in cotyledons by infiltrating catalase and/or H₂O₂ into tissues. The action of catalase resulted in a significant decrease in lesions development, but when H₂O₂ was applied instead, lesion formation was only moderately stimulated compared to the untreated control. When H₂O₂ toxicity to L. maculans was tested in vitro, concentrations above 5 mM and 10 mM H₂O₂ were lethal for germinating conidia and growing mycelia of L. maculans, respectively. We can assume that L. maculans behaves as a necrotroph during this early stage of infection even though its resistance to H₂O₂ does not exceed standard concentrations. To investigate antioxidant mechanisms implicated in the response of B. napus to L. maculans, the cotyledons were both inoculated with conidia and treated with L. maculans elicitor. Increased activities of guaiacol peroxidase, ascorbate peroxidase, glutathione reductase and superoxide dismutase were recorded both in L. maculans-infected and elicitor-treated cotyledons. The results indicate the importance of these enzymes for ROS scavenging in B. napus-L. maculans interaction.     

Dynamics of infection by Leptosphaeria maculans on canola (Brassica napus) as influenced by crop rotation and tillage

Abstract

This study, intending to understand the effects of crop rotation and tillage on blackleg disease, was conducted in a field at Carman, Manitoba, Canada, from 1999 – 2002. Canola, wheat and flax were among the rotated crops. Rotations were performed under conventional or zero-till conditions. The number of infected plants, infected leaves per plant, lesions per plant, and percentage of leaf coverage with lesions decreased when canola was rotated with wheat and flax under zero till. The number of lesions per plant and percentage of leaf coverage with lesions were strongly correlated with stem disease severity, and the number of infected plants with stem disease incidence. Ascospores and pycnidiospores of Leptosphaeria maculans were reduced by crop rotation and tillage. This study suggests that the appropriate combination of rotation and tillage may lower airborne inoculum and reduce infection of canola plants by L. maculans.     

A Comparison of the Disease Reactions of Stems and Detached Leaves of Soil and in vitro Grown Plants and Regenerants of Oilseed Rape to Leptosphaeria maculans and Protocols for Selection for Novel Disease Resistance

Protocols for selecting plant tissues of winter oilseed rape (Brassica napus subsp. oleifera) with resistance to Leptosphaeria maculans by either stem or leaf inoculation of both soil and in vitro grown plant material are described. The stem inoculation procedure gave good correlation (r = 0. 92) between the 50 day stem disease scores of eight out of nine cultivars of soil grown winter oilseed rape inoculated with isolate 41A4 of L. maculans and the N. A. B. esistance ratings or resistance data from field trials. The exception was the cultivar Liradonna. Inoculation of stems of five cultivars with isolates 41A4, 433 and 478 indicated a range of isolate virulence 478 > 41A4 > 433. This was the inverse of that observed in leaf inoculations. Application of the stem inoculation procedure to in vitro shoot cultures allowed differentiation of resistant and susceptible cultivars, including the cultivar Liradonna, after 20 days incubation at 20°C. The protocol was also applicable to plantlets regenerated from thin cell layer explants grown in vitro. Inoculations with isolate 433 allowed the differentiation of resistant, intermediately resistant and susceptible leaf material of soil grown plants, when leaf discs from young leaves were incubated on water agar supplemented with BAP (1 × 10^?5 M) at 25°C for 10 days. Intermediately resistant leaves were resistant after 10 days and susceptible after 15 days of incubation. Leaves of shoot cultures grown in vitro were more susceptible than the corresponding soil grown material. However, inoculation of old leaves with isolate 41A4 (an isolate of less virulence on leaves than 433) distinguished the cultivars after 15 days of incubation. These protocols allow the accurate assessment of resistance to L. maculans at the stem or leaf level and are of use in traditional as well as in vitro selection programmes.     

A new avirulence gene of Leptosphaeria maculans,AvrLm14, identifies a resistance source in American broccoli (Brassica oleracea) genotypes

In many cultivated crops, sources of resistance to diseases are sparse and rely on introgression from wild relatives. Agricultural crops often are allopolyploids resulting from interspecific crosses between related species, which are sources of diversity for resistance genes. This is the case for Brassica napus (oilseed rape, canola), an interspecific hybrid between Brassica rapa (turnip) and Brassica oleracea (cabbage). B. napus has a narrow genetic basis and few effective resistance genes against stem canker (blackleg) disease, caused by the fungus Leptosphaeria maculans, are currently available. B. rapa diversity has proven to be a valuable source of resistance (Rlm, LepR) genes, while B. oleracea genotypes were mostly considered susceptible. Here we identified a new resistance source in B. oleracea genotypes from America, potentially effective against French L. maculans isolates under both controlled and field conditions. Genetic analysis of fungal avirulence and subsequent cloning and validation identified a new avirulence gene termed AvrLm14 and suggested a typical gene-for-gene interaction between AvrLm14 and the postulated Rlm14 gene. AvrLm14 shares all the usual characteristics of L. maculans avirulence genes: it is hosted in a genomic region enriched in transposable elements and heterochromatin marks H3K9me3, its expression is repressed during vegetative growth but shows a strong overexpression 5–9 days following cotyledon infection, and it encodes a small secreted protein enriched in cysteine residues with few matches in databases. Similar to the previously cloned AvrLm10-A, AvrLm14 contributes to reduce lesion size on susceptible cotyledons, pointing to a complex interplay between effectors promoting or reducing lesion development.     

Detection,introgression and localization of genes conferring specific resistance to <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> from <Emphasis Type="Italic">Brassica rapa</Emphasis> into <Emphasis Type="Italic">B. napus</Emphasis>

Blackleg (stem canker) caused by the fungus Leptosphaeria maculans is one of the most damaging diseases of oilseed rape (Brassica napus). Crop relatives represent a valuable source of “new” resistance genes that could be used to diversify cultivar resistance. B. rapa, one of the progenitors of B. napus, is a potential source of new resistance genes. However, most of the accessions are heterozygous so it is impossible to directly detect the plant genes conferring specific resistance due to the complex patterns of avirulence genes in L. maculans isolates. We developed a strategy to simultaneously characterize and introgress resistance genes from B. rapa, by homologous recombination, into B. napus. One B. rapa plant resistant to one L. maculans isolate was used to produce B. rapa backcross progeny and a resynthesized B. napus plant from which a population of doubled haploid lines was derived after crossing with natural B. napus. We then used molecular analyses and resistance tests on these populations to identify and map the resistance genes and to characterize their introgression from B. rapa into B. napus. Three specific genes conferring resistance to L. maculans (Rlm1, Rlm2 and Rlm7) were identified in B. rapa. Comparisons of genetic maps showed that two of these genes were located on the R7 linkage group, in a region homologous to the region on linkage group N7 in B. napus, where these genes have been reported previously. The results of our study offer new perspectives for gene introgression and cloning in Brassicas.     

Chitinase isozymes induced by TYMV and <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> during compatible and incompatible interaction with <Emphasis Type="Italic">Brassica napus</Emphasis>

Accumulation of extracellular chitinases in Brassica napus plants infected with Turnip yellow mosaic virus (TYMV) and fungal pathogen Leptosphaeria maculans was studied in both compatible and incompatible interaction. Analysis of apoplast fluid by means of non-denaturing anodic and cathodic PAGE followed by in-gel detection of chitinase activity revealed a number of chitinase isozymes. TYMV induced 8 acidic and 4 basic isozymes in a systemic way. Except for one acidic and one basic isozyme, all other chitinases were also constitutively present in low amounts in mock inoculated control. In TYMV systemically infected plants, chitinases were detected in leaves expressing symptoms as well as in symptomless ones. Both virulent and avirulent L. maculans isolates induced production of chitinase isozymes in cotyledons in a time dependent manner. Some of them were present in plants constitutively and their content increased after inoculation. Three of five acidic and two of three basic isozymes responded to L. maculans infection. Chitinases started to accumulate before symptom appearance. First two acidic isozymes were detected 24 h after inoculation. The difference between compatible and incompatibe interaction reflected two basic isozymes.     

Quantitative PCR analysis of abundance of airborne propagules of Leptosphaeria species in air samples from different regions of Poland

When airborne propagules of Leptosphaeria maculans and L. biglobosa were collected in Poland at three ecologically different sites from 1 September to 30 November in 2004 to 2008, using a Hirst-type seven-day volumetric spore trap, there were fluctuations in timing of ascospore release and diverse ratios between airborne propagules of both species depending on season, field location and weather conditions. The detection was done using the microscope as well as quantitative PCR with species-specific primers targeted against fragments of β-tubulin genes and quantified with a dual-labelled fluorescent probe approach. This detection chemistry is described for the first time for L. maculans and L. biglobosa. Its advantage over the previous ITS-based SYBR-Green chemistry resides in improved sensitivity and the virtual absence of false positives in the detection of these fungi. There were significant, positive correlations between data obtained using visual assessment of ascospore numbers and DNA concentrations that were measured by qPCR. Climatic differences between the oilseed rape growing regions could have significantly affected the biological processes of pseudothecial maturation and ascospore development of the pathogens. The data suggest that regular rain events of intermediate intensity recorded in the Maritime region favoured the maturation of the pathogen more than the drier weather recorded in the Silesia or Pomerania regions. It was observed that the number of rainy days was of greater importance than the cumulative rainfall to obtain the generative sporulation of the pathogen. Accurate detection of airborne inoculum of pathogenic Leptosphaeria spp. facilitates improved targeting of disease management decisions for oilseed rape protection against phoma stem canker and stem necrosis diseases.     

Genetic Variability and Distribution of Mating Type Alleles in Field Populations of Leptosphaeria maculans from France

Leptosphaeria maculans is the most ubiquitous fungal pathogen of Brassica crops and causes the devastating stem canker disease of oilseed rape worldwide. We used minisatellite markers to determine the genetic structure of L. maculans in four field populations from France. Isolates were collected at three different spatial scales (leaf, 2-m² field plot, and field) enabling the evaluation of spatial distribution of the mating type alleles and of genetic variability within and among field populations. Within each field population, no gametic disequilibrium between the minisatellite loci was detected and the mating type alleles were present at equal frequencies. Both sexual and asexual reproduction occur in the field, but the genetic structure of these populations is consistent with annual cycles of randomly mating sexual reproduction. All L. maculans field populations had a high level of gene diversity (H = 0.68 to 0.75) and genotypic diversity. Within each field population, the number of genotypes often was very close to the number of isolates. Analysis of molecular variance indicated that >99.5% of the total genetic variability was distributed at a small spatial scale, i.e., within 2-m² field plots. Population differentiation among the four field populations was low (G_ST < 0.02), suggesting a high degree of gene exchange between these populations. The high gene flow evidenced here in French populations of L. maculans suggests a rapid countrywide diffusion of novel virulence alleles whenever novel resistance sources are used.