Development and characterisation of Brassica napus-Sinapis arvensis addition lines exhibiting resistance to Leptosphaeria maculans

Blackleg caused by Leptosphaeria maculans is one of the most important diseases affecting oilseed rape worldwide. Sinapis arvensis is valuable for the transfer of blackleg resistance to oilseed rape (Brassica napus) because this species contains high resistance against various aggressive isolates of the blackleg fungus. These include at least one Australian isolate which has been found to overcome resistance originating from species with the Brassica B genome, until now the major source for interspecific transfer of blackleg resistance. Backcross offspring from intergeneric crosses between Brassica napus and S. arvensis were subjected to phytopathological studies and molecular cytogenetic analysis with genomic in situ hybridisation (GISH). The BC₃S progenies included fertile plants exhibiting high seedling (cotyledon) and adult plant resistance associated with the presence of an acrocentric addition chromosome from S. arvensis. In addition, some individuals with adult plant resistance but cotyledon susceptibility were observed to have a normal B. napus karyotype with no visible GISH signals, indicating possible resistant introgression lines. Phytopathological analysis of selfing progenies from 3 different highly resistant BC₃ plants showed that seedling and adult plant resistance are probably conferred by different loci. Received: 20 September 1999 / Accepted: 25 March 2000     

Towards identifying Brassica proteins involved in mediating resistance to Leptosphaeria maculans: a proteomics-based approach

To better understand the pathogen-stress response of Brassica species against the ubiquitous hemi-biotroph fungus Leptosphaeria maculans, we conducted a comparative proteomic analysis between blackleg-susceptible Brassica napus and blackleg-resistant Brassica carinata following pathogen inoculation. We examined temporal changes (6, 12, 24, 48 and 72 h) in protein profiles of both species subjected to pathogen-challenge using two-dimensional gel electrophoresis. A total of 64 proteins were found to be significantly affected by the pathogen in the two species, out of which 51 protein spots were identified using tandem mass spectrometry. The proteins identified included antioxidant enzymes, photosynthetic and metabolic enzymes, and those involved in protein processing and signaling. Specifically, we observed that in the tolerant B. carinata, enzymes involved in the detoxification of free radicals increased in response to the pathogen whereas no such increase was observed in the susceptible B. napus. The expression of genes encoding four selected proteins was validated using quantitative real-time PCR and an additional one by Western blotting. Our findings are discussed with respect to tolerance or susceptibility of these species to the pathogen.     

Identification of QTL involved in field resistance to light leaf spot (Pyrenopeziza brassicae) and blackleg resistance (Leptosphaeria maculans) in winter rapeseed (Brassica napus L.)

 Quantitative trait loci (QTL), involved in the polygenic field resistance of rapeseed (Brassica napus L.) to light leaf spot disease, were mapped using 288 DNA markers on 152 doubled-haploid (DH) lines derived from the cross ‘Darmor-bzh’בYudal’. Over two years (1995 and 1996), the DH population was evaluated for light leaf spot resistance on leaves (L) and stems (S), and for blackleg disease resistance in same field trials. For the L resistance criterion, a total of five and seven QTL were detected in 1995 and in 1996 respectively, accounting for 53% and 57% of the genotypic variation. For the S criterion, three and five QTL were identified in 1995 and in 1996 respectively, explaining 29% and 43% of the genotypic variation. The locations of the QTL detected were quite consistent over the two years (4- and 2-year common QTL for L and S, respectively). Three genomic regions, located on the DY5, DY10 and DY11 groups, were common to the resistance on leaves and stems. In comparison with the QTL for blackleg resistance described by Pilet et al. (1998), two regions on the DY6 and DY10 groups, were associated with the two disease resistances. These ‘multiple disease resistance’ (‘MDR’) QTL may correspond to genes involved in common resistance mechanisms towards the two pathogens or else to clusters of resistance genes. Received: 21 November 1997 / Accepted: 3 March 1998     

Identification of loci contributing to quantitative field resistance to blackleg disease, causal agent Leptosphaeria maculans (Desm.) Ces. et de Not., in Winter rapeseed (Brassica napus L.)

 Blackleg, caused by Leptosphaeria maculans, is one of the most important diseases of Brassica napus. Genomic regions controlling blackleg resistance at the adult plant stage were detected using 152 doubled-haploid (DH) lines derived from the F₁‘Darmor-bzh’בYudal’. The rapeseed genetic map used includes 288 DNA markers on 19 linkage groups. Blackleg resistance of each DH line was evaluated in field tests in 1995 and 1996 by measuring the mean disease index (I) and the percentage of lost plants (P). From notations recovered in 1995, ten quantitative trait loci (QTL) were detected: seven QTL for I and six QTL for P, explaining 57% and 41% of the genotypic variation, respectively. Three of them were common to I and P. From data recovered in 1996, seven QTL were identified: five QTL for I and two different QTL for P, accounting for 50% and 23% of the genotypic variation, respectively. One I QTL, located close to a dwarf gene (bzh), was detected with a very strong effect, masking more QTL detection. It was not revealed at the same position and with the same effect in 1995. Four major genomic regions were revealed from 1995 and from 1996 with the same parental contribution. One of them, located on the DY2 group, has a resistance allele from the susceptible parent. Five- and two-year-specific QTL were detected in 1995 and 1996, respectively. Received: 25 April 1997 / Accepted: 5 August 1997     

Pathways of infection of Brassica napus roots by Leptosphaeria maculans

Infection of Brassica napus cotyledons and leaves by germinating ascospores of Leptosphaeria maculans leads to production of leaf lesions followed by stem cankers (blackleg). Leptosphaeria maculans also causes root rot but the pathway of infection has not been described. An L. maculans isolate expressing green fluorescent protein (GFP) was applied to the petiole of B. napus plants. Hyphal growth was followed by fluorescence microscopy and by culturing of sections of plant tissue on growth media. Leptosphaeria maculans grew within stem and hypocotyl tissue during the vegetative stages of plant growth, and proliferated into the roots within xylem vessels at the onset of flowering. Hyphae grew in all tissues in the stem and hypocotyl, but were restricted mainly to xylem tissue in the root. Leptosphaeria maculans also infected intact roots when inoculum was applied directly to them and hyphae entered at sites of lateral root emergence. Hyphal entry may occur at other sites but the mechanism is uncertain as penetration structures were not observed. Infection of B. napus roots by L. maculans can occur via above- and below-ground sources of inoculum, but the relative importance of the infection pathways under field conditions is unknown.     

Enhanced pathogenicity of Leptosphaeria maculans Pycnidiospores from paired co-inoculation of Brassica napus cotyledons with ascospores

BACKGROUND AND AIMS: Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus) worldwide, including Australia. In most cases, the severity of the disease in the field is related to infections caused by airborne ascospores. In contrast, pycnidiospores originating from leaf and stem lesions and stubble are widely assumed to play only a relatively minor role in the epidemiology of blackleg. It is not clear whether, under certain conditions, pycnidiospores can cause severe disease in the field. The aim of the work reported was to determine if the pathogenicity of pycnidiospores is enhanced by paired co-inoculation of B. napus cotyledons with ascospores. METHODS: Three investigations were carried out under controlled-environment conditions using various L. maculans isolates and B. napus cultivars with different levels of host resistance to blackleg. KEY RESULTS: In all three experiments, co-inoculation with ascospores increased the ability of pycnidiospores to cause more disease on B. napus than when inoculations consisted of pycnidiospores alone. This effect was significantly influenced by the host resistance of the cultivar, but overall was independent of the L. maculans isolate used in the different experiments. This effect was also independent of timing of inoculation with the ascospores, with increased disease from pycnidiospores occurring on the cotyledon of the seedling in situations where inoculations with ascospores were carried out 0, 1 or 2 d after pycnidiospore inoculation. This enhanced pathogenicity of pycnidiospores was evident even when low concentrations of pycnidiospores were applied to the other cotyledon of the same seedling. CONCLUSIONS: These results may explain continuing severe blackleg disease cycles throughout the cropping season even when ascospore fallout was low or constrained only to a brief period or phase of the cropping season, and suggest that disease epidemics may be polycyclic rather than monocyclic.     

Rapid identification of the Leptosphaeria maculans avirulence gene AvrLm2 using an intraspecific comparative genomics approach

Five avirulence genes from Leptosphaeria maculans, the causal agent of blackleg of canola (Brassica napus), have been identified previously through map‐based cloning. In this study, a comparative genomic approach was used to clone the previously mapped AvrLm2. Given the lack of a presence–absence gene polymorphism coincident with the AvrLm2 phenotype, 36 L. maculans isolates were resequenced and analysed for single‐nucleotide polymorphisms (SNPs) in predicted small secreted protein‐encoding genes present within the map interval. Three SNPs coincident with the AvrLm2 phenotype were identified within LmCys1, previously identified as a putative effector‐coding gene. Complementation of a virulent isolate with LmCys1, as the candidate AvrLm2 allele, restored the avirulent phenotype on Rlm2‐containing B. napus lines. AvrLm2 encodes a small cysteine‐rich protein with low similarity to other proteins in the public databases. Unlike other avirulence genes, AvrLm2 resides in a small GC island within an AT‐rich isochore of the genome, and was never found to be deleted completely in virulent isolates.     

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.     

Expression of resistance to Leptosphaeria maculans in Brassica napus double haploid lines in France and Australia is influenced by location

Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus), worldwide, including Australia and France. The aims of these studies were first, to determine if higher levels of resistance to L. maculans could be generated in double haploid (DH) lines derived from spring‐type B. napus cv. Grouse, which has a good level of field resistance to blackleg; and second, to determine whether the resistance to blackleg disease of individual DH lines responds differentially to different L. maculans field populations within and between the two countries. DH lines were extracted from cv. Grouse and tested in field experiments carried out in both France and Australia against natural L. maculans populations. Extracting and screening DH lines were an effective means to select individual lines with greatly improved expression of resistance to blackleg crown canker disease in comparison with the original parental population. However, relative disease resistance rankings for DH lines were not always consistent between sites. The higher level of resistance in France was shown to be because of a high expression level of quantitative resistance in the French growing conditions. Big differences were observed for some DH lines between the 2004 and the 2005 field sites in Australia where the L. maculans populations differed by their virulence on single dominant gene‐based resistant lines derived from Brassica rapa ssp. sylvestris. This differential behaviour could not be clearly explained by the specific resistance genes until now identified in these DH lines. This investigation highlights the potential to derive DH lines with superior levels of resistance to L. maculans compared with parental populations. However, in locations with particularly high pathogen diversity, such as in southern Australia, multiyear and multisite evaluations should be performed to screen for the most efficient material in different situations.     

Selection of stable Brassica napus-B. juncea recombinant lines resistant to blackleg (Leptosphaeria maculans). 1. Identification of molecular markers, chromosomal and genomic origin of the introgression

 A scheme of selection combining selfing and backcross was applied to a B. napus line with the blackleg resistance from B. juncea in order to transfer this resistance to a winter oilseed rape variety. Cytogenetic analyses combined with cotyledon blackleg resistance tests at each generation allowed us to obtain a recombinant line showing regular meiotic behavior. The resistance is monogenic and is highly efficient under field conditions. Four-hundred RAPD primers were tested on two segregating populations by bulk segregant analysis. Three markers totally linked to the introgression were identified. The analysis of these markers on both sets of B. napus-B. nigra and B. oleracea-B. nigra addition lines revealed that they are not located on the B4 chromosome of B. nigra, which has already been shown to carry a blackleg resistance gene, but rather on the B8 chromosome. We confirmed that the resistance gene is carried by the B genome of B. juncea. Based on these data, two hypotheses, one involving chromosome rearrangements between the two B genomes of B. nigra and B. juncea, and the other based on a more probable digenic control of the resistance within B. juncea, are discussed. Received: 30 May 1997 / Accepted: 23 June 1997     

STS markers linked to Phoma resistance genes of the Brassica B-genome revealed sequence homology between Brassica nigra and Brassica napus

The RFLP and AFLP techniques are laborious and expensive and therefore of limited use for marker-assisted selection, demanding a high throughput of samples in a short time. But marker-assisted selection is most useful for traits which are hard to score on single plants and influenced by environmental factors. Four RFLP and three AFLP markers have been found to be linked to genes of the B-genome of Brassica mediating resistance against Phoma lingam in oilseed rape. One RFLP and one AFLP marker were converted into three PCR-based STS markers: one of dominant, as well as one of codominant inheritance separated in a standard agarose gel and a third one of codominant inheritance to be separated in a polyacrylamide gel on an automated sequencer. As expected, the STS markers mapped at the same position as the original RFLP and AFLP markers. The STS markers are efficient in marker-assisted backcross programs of the resistant B-genome/Brassica napus recombinant lines with most of the tested oilseed rape varieties and breeding lines. More than 90% of the tested oilseed rape varieties and breeding lines exhibited no resistance marker alleles. The mapping results obtained with the markers, as well as comparative sequencing of the marker alleles, indicate synteny and homology between the B-genome resistance gene donors and B. napus in the region of the resistance genes. The location of the resistance genes in the B-genome/B. napus recombinant lines is most likely on the A genome. Thus the transfer of the B-genome resistance genes into Brassica campestris is also possible. Received: 9 December 1999 / Accepted: 21 June 2000     

Recurring challenges from a necrotrophic fungal plant pathogen: a case study with Leptosphaeria maculans (causal agent of blackleg disease in brassicas) in Western Australia

BACKGROUND: Blackleg disease of Brassica napus, caused by the necrotrophic fungus Leptosphaeria maculans, causes severe yield losses in Australia, Europe and Canada. In Western Australia, it nearly destroyed the oilseed rape industry in 1972 when host genotypes and conducive environmental conditions favoured severe epidemics. The introduction of cultivars with polygenic resistance and the adoption of sound cultural practices two decades later helped to manage the disease. These were abandoned by many farmers in recent years in favour of the effective but ephemeral resistance conferred by the single dominant gene-based resistance derived from B. rapa ssp. sylvestris. Recently, several cultivars carrying this gene have collapsed widely within a period of 3 years after their commercial release. An environment conducive to the disease and the association of the pathogen with susceptible hosts in Western Australia for over 80 years together have led to the proliferation of L. maculans races, amounting to half of all races delineated to date from Europe, including the United Kingdom, Canada and Australia. SCOPE: This review demonstrates the problems that emerge when traditional cultural practices employed, along with cultivars containing polygenic resistance to a serious necrotrophic pathogen, are discarded in preference to the exclusive deployment of effective but ephemeral single dominant gene-based resistance to the disease across Southern Australia. CONCLUSIONS: Single dominant gene-based resistance currently available, on its own, will not confer durable resistance to blackleg disease in oilseed rape. Return to earlier management practices, including reliance upon polygenic resistance and induced resistance, may be the best currently available options to maintain production in regions across Southern Australia predisposed to severe epidemics.     

The genetics of adult-plant blackleg (Leptosphaeria maculans) resistance from Brassica juncea in B. napus

The genetic control of adult-plant blackleg (Leptosphaeria maculans) resistance in a Brassica napus line (579NO48-109-DG-1589), designated R13 possessing Brassica juncea-like resistance (JR), was elucidated by the analysis of segregation ratios in F₂ and F₃ populations from a cross between R13 and the highly blackleg-susceptible B. napus cultivar Tower. The F₂ segregration ratios were bimodal, demonstrating that blackleg resistance in R13 was controlled by major genes. Analysis of the segregation ratios for 13 F₃ families indicated that blackleg resistance in these families was controlled by three nuclear genes, which exhibited a complex interaction. Randomly sampled plants of F₃ progeny all had the normal diploid somatic chromosome number for B. napus. The similarities between the action of the three genes found in this study with those controlling blackleg resistance in B. juncea is discussed.     

Vital staining of plant cell suspension cultures: evaluation of the phytotoxic activity of the phytotoxins phomalide and destruxin B

 A cell suspension culture assay to determine the phytotoxicity of the fungal toxins phomalide, a host-selective toxin produced by the fungus Phoma lingam (Tode ex Fr.) Desm., perfect stage Leptosphaeria maculans (Desm.) Ces. et de Not., and destruxin B, the major host-selective toxin produced by the fungus Alternaria brassicae (Berk.) Sacc., was carried out with three Brassica spp. It was established that phomalide was significantly less phytotoxic to Cutlass (Brassica juncea), the cultivar resistant to L. maculans, than to Westar (B. napus), the cultivar susceptible to L. maculans, at concentrations ≤2×10^–5  M. Similar to phomalide, destruxin B, at concentrations ≤5×10^–5  M, decreased the viability of cells of the cultivar resistant to A. brassicae (Ochre, Sinapis alba) less than the viability of cells of the susceptible cultivar (Westar, B. napus). Considering the high selectivity of phomalide and its direct correlation with plant disease resistance, phomalide may have great potential application in breeding programs screening/selecting for blackleg resistance in brassicas. Received: 23 November 1999 / Revision received: 11 April 2000 / Accepted: 8 May 2000     

Comparison of the genetic maps of Brassica napus and Brassica oleracea

 The genus Brassica consists of several hundreds of diploid and amphidiploid species. Most of the diploid species have eight, nine or ten pairs of chromosomes, known respectively as the B, C, and A genomes. Genetic maps were constructed for both B. napus and B. oleracea using mostly RFLP and RAPD markers. For the B. napus linkage map, 274 RFLPs, 66 RAPDs, and two STS loci were arranged in 19 major linkage groups and ten smaller unassigned segments, covering a genetic distance of 2125 cM. A genetic map of B. oleracea was constructed using the same set of RFLP probes and RAPD primers. The B. oleracea map consisted of 270 RFLPs, 31 RAPDs, one STS, three SCARs, one phenotypic and four isozyme marker loci, arranged into nine major linkage groups and four smaller unassigned segments, covering a genetic distance of 1606 cM. Comparison of the B. napus and B. oleracea linkage maps showed that eight out of nine B. oleracea linkage groups were conserved in the B. napus map. There were also regions in the B. oleracea map showing homoeologies with more than one linkage group in the B. napus map. These results provided molecular evidence for B. oleracea, or a closely related 2n=18 Brassica species, as the C-genome progenitor, and also reflected on the homoeology between the A and C genomes in B. napus. Received: 14 June 1996 / Accepted: 11 October 1996