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.     

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.     

Temperature and leaf wetness duration affect phenotypic expression of Rlm6-mediated resistance to Leptosphaeria maculans in Brassica napus

Near-isogenic Brassica napus lines carrying/lacking resistance gene Rlm6 were used to investigate the effects of temperature and leaf wetness duration on phenotypic expression of Rlm6-mediated resistance. Leaves were inoculated with ascospores or conidia of Leptosphaeria maculans carrying the effector gene AvrLm6. Incubation period to the onset of lesion development, number of lesions and lesion diameter were assessed. Symptomless growth of L. maculans from leaf lesions to stems was investigated using a green fluorescent protein (GFP) expressing isolate carrying AvrLm6. L. maculans produced large grey lesions on Darmor (lacking Rlm6) at 5-25 degrees C and DarmorMX (carrying Rlm6) at 25 degrees C, but small dark spots and 'green islands' on DarmorMX at 5-20 degrees C. With increasing temperature/wetness duration, numbers of lesions/spots generally increased. GFP-expressing L. maculans grew from leaf lesions down leaf petioles to stems on DarmorMX at 25 degrees C but not at 15 degrees C. We conclude that temperature and leaf wetness duration affect the phenotypic expression of Rlm6-mediated resistance in leaves and subsequent L. maculans spread down petioles to produce stem cankers.     

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.     

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.     

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.     

Leptosphaeria maculans AvrLm9: a new player in the game of hide and seek with AvrLm4‐7

Blackleg disease of Brassica napus caused by Leptosphaeria maculans (Lm) is largely controlled by the deployment of race‐specific resistance (R) genes. However, selection pressure exerted by R genes causes Lm to adapt and give rise to new virulent strains through mutation and deletion of effector genes. Therefore, a knowledge of effector gene function is necessary for the effective management of the disease. Here, we report the cloning of Lm effector AvrLm9 which is recognized by the resistance gene Rlm9 in B. napus cultivar Goéland. AvrLm9 was mapped to scaffold 7 of the Lm genome, co‐segregating with the previously reported AvrLm5 (previously known as AvrLmJ1). Comparison of AvrLm5 alleles amongst the 37 re‐sequenced Lm isolates and transgenic complementation identified a single point mutation correlating with the AvrLm9 phenotype. Therefore, we renamed this gene as AvrLm5‐9 to reflect the dual specificity of this locus. Avrlm5‐9 transgenic isolates were avirulent when inoculated on the B. napus cultivar Goéland. The expression of AvrLm5‐9 during infection was monitored by RNA sequencing. The recognition of AvrLm5‐9 by Rlm9 is masked in the presence of AvrLm4‐7, another Lm effector. AvrLm5‐9 and AvrLm4‐7 do not interact, and AvrLm5‐9 is expressed in the presence of AvrLm4‐7. AvrLm5‐9 is the second Lm effector for which host recognition is masked by AvrLm4‐7. An understanding of this complex interaction will provide new opportunities for the engineering of broad‐spectrum recognition.     

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.     

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     

Epidemiology of Leptosphaeria maculans in relation to forecasting stem canker severity on winter oilseed rape in the UK

In the UK, ascospores of Leptosphaeria maculans first infect leaves of oilseed rape in the autumn to cause phoma leaf spots, from which the fungus can grow to cause stem cankers in the spring. Yield losses due to early senescence and lodging result if the stem cankers become severe before harvest. The risk of severe stem canker epidemics needs to be forecast in the autumn when the pathogen is still in the leaves, since early infections cause the greatest yield losses and fungicides have limited curative activity. Currently, the most effective way to forecast severe stem canker is to monitor the onset of phoma leaf spotting in winter oilseed rape crops, although this does not allow much time in which to apply a fungicide. Early warnings of risks of severe stem canker epidemics could be provided at the beginning of the season through regional forecasts based on disease survey and weather data, with options for input of crop-specific information and for updating forecasts during the winter. The accuracy of such forecasts could be improved by including factors relating to the maturation of ascospores in pseudothecia, the release of ascospores and the occurrence of infection conditions, as they affect the onset, intensity and duration of the phoma leaf spotting phase. Accurate forecasting of severe stem canker epidemics can improve disease control and optimise fungicide use.     

Transmission of Leptosphaeria maculans from a cropping season to the following one

Current modelling of inoculum transmission from a cropping season to the following one relies on the extrapolation of kernels estimated on data at short distances from punctual sources, because data collected at larger distances are scarce. We estimated the dispersal kernel of Leptosphaeria maculans ascospores from stubble left after harvest in the summer previous to newly sown oilseed rape fields, using phoma stem canker autumn disease severity. We built a dispersal model to analyse the data. Source strengths are described in the spatial domain covered by source fields by a log‐Gaussian spatial process. Infection potentials in the following season are described in the space consisting of the target fields, by a convolution of sources and a power‐exponential dispersal kernel. Data were collected on farmers' fields considered as sources in 2009 and 2011 (72 and 39 observation points) and as targets in 2010 and 2012 (172 and 200 points). We applied the Bayesian approach for model selection and parameter estimation. We obtained fat tail kernels for both data sets. This estimation is the first from data acquired over distances of 0 to 1000 m, using several non‐punctual inoculum sources. It opens the prospect of refining the existing simulators, or developing disease risk maps.     

The Lmgpi15 gene, encoding a component of the glycosylphosphatidylinositol anchor biosynthesis pathway, is required for morphogenesis and pathogenicity in Leptosphaeria maculans

Random insertional mutagenesis was used to investigate pathogenicity determinants in Leptosphaeria maculans. One tagged nonpathogenic mutant, termed m20, was analysed in detail here. The mutant phenotype was investigated by microscopic analyses of infected plant tissues and in vitro growth assays. Complementation and silencing experiments were used to identify the altered gene. Its function was determined by bioinformatics analyses, cell biology experiments and functional studies. The mutant was blocked at the invasive growth phase after an unaffected initial penetration stage, and displayed a reduced growth rate and an aberrant hyphal morphology in vitro. The T-DNA insertion occurred in the intergenic region between two head-to-tail genes, leading to a complex deregulation of their expression. The unique gene accounting for the mutant phenotype was suggested to be the orthologue of the poorly conserved Saccharomyces cerevisiae gpi15, which encodes for one component of the glycosylphosphatidylinositol (GPI) anchor biosynthesis pathway. Consistent with this predicted function, a functional translational fusion with the green fluorescent protein (GFP) was targeted to the endoplasmic reticulum. Moreover, the mutant exhibited an altered cell wall and addition of glucosamine relieved growth defects. It is concluded that the GPI anchor biosynthetic pathway is required for morphogenesis, cell wall integrity and pathogenicity in Leptosphaeria maculans.     

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.     

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     

Factors affecting development of phoma canker (Leptosphaeria maculans) on stems of winter oilseed rape (Brassica napus) in southern England

In winter oilseed rape experiments at Rothamsted in 1997/98 (cvs Lipton and Capitol), 1998/99 (cv. Apex) and 1999/2000 (cvs Apex, Lipton and Capitol), development of crown canker and phoma stem lesions in spring was related to development of phoma leaf spot in the previous autumn/winter. There were differences in thermal time (degree‐days) from the first appearance of phoma leaf spot (autumn) to the first appearance of crown canker (spring) between cultivars (cvs Lipton and Capitol, 1220–1240; cv. Apex, 1120–1140 degree‐days) but not between growing seasons. In 1998/99 and 1999/2000, fungicide (November) treatment delayed the start of crown canker development in the spring but did not affect the rate of increase in severity. In 1997/98, fungicide treatments did not delay the appearance of crown canker but decreased the rate of increase in crown canker severity. In all three seasons, fungicide treatments generally decreased the proportions of plants at harvest with crown canker severity scores 3 or 4 and increased the proportions with scores 0 or 1. There were differences between seasons in the distributions of crown canker severity scores at harvest. The severity of both crown canker and phoma stem lesions increased linearly with accumulated degree‐days in plots with or without fungicide treatment in 1997/98 (cv. Lipton), 1998/99 (cv. Apex) and 1999/2000 (cv. Apex). Regressions showed that severity of crown canker at harvest in July was related to severity in the spring in 1997/98 (early June, cv. Lipton), 1998/99 and 1999/2000 (April, cv. Apex).     

Relationships between phoma leaf spot and development of stem canker (Leptosphaeria maculans) on winter oilseed rape (Brassica napus) in southern England

Models were constructed to describe the relationships between incidence of phoma leaf spot at different growth stages in autumn/winter or early spring and incidence of stem canker (basal canker or stem lesions) in summer on winter oilseed rape in southern England. Model 1, describing the phoma leaf spot/basal canker relationship, was y₁=β₀+β₁x₁+β₂(x₂ ‐ x₁) if x₂ > x₁, and y₁=β₀+β₁x₁ if x₂≤x₁, in which y₁ was the incidence (% plants affected) of basal canker at harvest, x₁ was the maximum incidence of phoma leaf spot during the period from sowing to growth stage (G.S.) 1,6‐1,7 (about 100 days after sowing) and x₂ was the maximum incidence of phoma leaf spot between G.S. 1,7 and G.S. 2,0 (start of stem extension). Model 2, describing the phoma leaf spot/stem lesion relationship, was y₂=α₀+α₁x₃+α₂x₄, in which y₂ was the incidence of stem lesions at harvest, x₃ was the incidence of phoma leaf spot at G.S. 3,3–3,5 (flower buds visible) and x₄ was the incidence of phoma leaf spot at G.S. 4,5–5,5 (flower buds opening). Data from field experiments with four winter oilseed rape cultivars at Boxworth or Rothamsted in the 1992/93, 1993/94, 1996/97, 1997/98 or 1998/99 seasons were used to test the models. The values of R² for the regression equations testing model 1 for the phoma leaf spot/basal canker relationship were 0.75, 0.93, 0.91 and 0.89 for cvs Apex, Bristol, Capitol and Envol, respectively. The values of R² for the regression equations testing model 2 for the phoma leaf spot/stem lesion relationship were 0.58, 0.57, 0.54 and 0.71 for cvs Apex, Bristol, Capitol and Envol, respectively. The phoma leaf spot/basal canker relationship (model 1) could also be fitted to the combined data set for all four cultivars (R²= 0.65), whereas the phoma leaf spot/stem lesion relationship (model 2) could not to be fitted to the combined data set for the four cultivars. The relationships between incidence and severity of stem canker were examined and the values of R² for the regressions of severity on incidence were 0.91 for basal canker and 0.89 for stem lesions.     

Resistance to Leptosphaeria maculans is conserved in a specific region of the Brassica B genome

 Offspring from asymmetric hybrids between Brassica napus and the three B-genome species Brassica nigra, Brassica juncea and Brassica carinata were analysed for the presence of B-genome markers and resistance to the fungus Leptosphaeria maculans, the causal agent of blackleg disease. Twenty five plants from each species combination were analysed in the first backcross (BC₁) generation, 30 plants in BC₂ and 60 plants in BC₃. The plants were analysed by 46 RFLP markers detecting 85 loci dispersed throughout the B. nigra genome. The plants with additional B. carinata DNA had a decrease in the presence of RFLP markers ranging from 59% in BC₁ to 36% in BC₂ and down to 11% in BC₃. Similar results were obtained in the lines with additional DNA from B. juncea where the 60% presence of RFLP markers in BC₁ was reduced to 33% in BC₂ and to 10% in BC₃. However presence of the markers were significantly lower in the B. nigra-derived material where BC₁ had 46%, BC₂ 25% and BC₃ 8%. Since at least two loci could be detected on each end of the eight linkage groups of the B genome, the degree of symmetry was estimated. After one back-cross between 0.5 and 1.25% intact chromosomes were retained, whereas in BC₂ this frequency was 0.21% for all three B-genome donor species. The maintenance of half-chromosomes ranged from 2.63% to 5.38% in BC₁ and between 0.73% and 1.15% in BC₂. No chromosome arms were found in any of the BC₃ plants. In total, four co-segregating markers for cotyledon and adult-leaf resistance to L. maculans were found which detected six loci located on linkage groups 2, 5 and 8. When the results from the three donor species were compared, one triplicate region in the B genome had preserved the resistance loci in all three species. Received: 19 January 1999 / Accepted: 30 January 1999     

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.