H. Brun

World-Wide Importance of Phoma Stem Canker (Leptosphaeria maculans and L. biglobosa) on Oilseed Rape (Brassica napus)

Phoma stem canker is an internationally important disease of oilseed rape (Brassica napus, canola, rapeseed), causing serious losses in Europe, Australia and North America. UK losses of €56M per season are estimated using national disease survey data and a yield loss formula. Phoma stem canker pathogen populations comprise two main species, Leptosphaeria maculans, associated with damaging stem base cankers, and Leptosphaeria biglobosa, often associated with less damaging upper stem lesions. Both major gene and quantitative trait loci mediated resistance to L. maculans have been identified in B. napus, but little is known about resistance to L. biglobosa.Leptosphaeria maculans, which has spread into areas in North America and eastern Europe where only L. biglobosa was previously identified, now poses a threat to large areas of oilseed rape production in Asia. Epidemics are initiated by air-borne ascospores; major gene resistance to initial infection by L. maculans operates in the leaf lamina of B. napus. It is not clear whether the quantitative trait loci involved in the resistance to the pathogen that can be assessed only at the end of the season operate in the leaf petioles or stems. In countries where serious phoma stem canker epidemics occur, a minimum standard for resistance to L. maculans is included in national systems for registration of cultivars. This review provides a background to a series of papers on improving strategies for managing B. napus resistance to L. maculans, which is a model system for studying genetic interactions between hemi-biotrophic pathogens and their hosts.

Quantitative resistance increases the durability of qualitative resistance to Leptosphaeria maculans in Brassica napus.

It has frequently been hypothesized that quantitative resistance increases the durability of qualitative (R-gene mediated) resistance but supporting experimental evidence is rare. To test this hypothesis, near-isogenic lines with/without the R-gene Rlm6 introduced into two Brassica napus cultivars differing in quantitative resistance to Leptosphaeria maculans were used in a 5-yr field experiment. Recurrent selection of natural fungal populations was done annually on each of the four plant genotypes, using crop residues from each genotype to inoculate separately the four series of field trials for five consecutive cropping seasons. Severity of phoma stem canker was measured on each genotype and frequencies of avirulence alleles in L. maculans populations were estimated. Recurrent selection of virulent isolates by Rlm6 in a susceptible background rendered the resistance ineffective by the third cropping season. By contrast, the resistance was still effective after 5 yr of selection by the genotype combining this gene with quantitative resistance. No significant variation in the performance of quantitative resistance alone was noted over the course of the experiment. We conclude that quantitative resistance can increase the durability of Rlm6. We recommend combining quantitative resistance with R-gene mediated resistance to enhance disease control and crop production.

Major gene and polygenic resistance to Leptosphaeria maculans in oilseed rape (Brassica napus)

The most common and effective way to control phoma stem canker (blackleg) caused by Leptosphaeria maculans in oilseed rape (Brassica napus) is through the breeding of resistant cultivars. Race specific major genes that mediate resistance from the seedling stage have been identified in B. napus or have been introgressed from related species. Many race specific major genes have been described and some of them are probably identical in B. napus (allotetraploid AACC) and the parental species B. rapa (diploid AA). More work is needed using a set of well-characterised isolates to determine the number of different major resistance genes available. In some B. napus cultivars, there is resistance which is polygenic (mediated by Quantitative Trait Loci) and postulated to be race non-specific. Many of these major genes and Quantitative Trait Loci for resistance to L. maculans have been located on B. napus genetic maps. Genes involved in race specific and polygenic resistance are generally distinct.

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

Abstract 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.

Major gene resistance in Brassica napus (oilseed rape) is overcome by changes in virulence of populations of Leptosphaeria maculans in France and Australia

Resistance of Brassica napus (oilseed rape, canola) conferred by three different major resistance genes has been overcome by changes in virulence of Leptosphaeria maculans populations in France and Australia. In South Australia where B. napus cultivars with major gene resistance derived from Brassica rapa ssp. sylvestris were grown extensively, resistance was rendered ineffective within 3 years of commercial release of the cultivar. Disease severity was higher on cultivars with sylvestris-derived resistance than cultivars with polygenic resistance. This Australian situation is compared to that in France, where resistance conferred by the Rlm1 gene was overcome nation-wide in 5 years under commercial cropping practices, and also where a source of resistance introgressed into B. napus from B. juncea was rendered inefficient in 3 years in experimental field plots near Rennes.

Repeat-induced point mutation (RIP) as an alternative mechanism of evolution toward virulence in Leptosphaeria maculans.

Three avirulence genes, AvrLm1, AvrLm6, and AvrLm4-7, were recently identified in Leptosphaeria maculans and found to be localized as solo genes within large noncoding, heterochromatin-like regions mainly composed of retrotransposons, truncated and degenerated by repeat-induced point mutation (RIP). The Rlm6 resistance gene has been overcome within 3 years in outdoor experiments in France and, here, we investigate the molecular basis of evolution toward virulence at the AvrLm6 locus. A region of 235 kb was sequenced in a virulent isolate and showed the deletion of AvrLm6 and three divergent mosaics of retrotransposons. AvrLm6 was found to be absent from 66% of 70 virulent isolates, with multiple events of deletion. The sequencing of virulent alleles in 24 isolates revealed a few cases of point mutations that had created stop codons in the sequence. The most frequent mutation events, however, were RIP, leading to the modification of 4 to 9% of the bases compared with the avirulent allele and generating 2 to 4 stop codons. Thus, RIP is described for the first time as an efficient mechanism leading to virulence and the multiple patterns of mutation observed suggest that multiple RIP events could occur independently in a single field population during 1 year.

A Field Method for Evaluating the Potential Durability of New Resistance Sources: Application to the Leptosphaeria maculans-Brassica napus Pathosystem.

ABSTRACT To increase the longevity of new resistance genes by avoiding a rapid change in pathogen populations, we established a new field method to determine, before the release of a resistant cultivar, whether and how rapidly the pathogen population is capable of responding to the selective pressure we impose. This method was applied to the Leptosphaeria maculans-Brassica napus pathosystem. The potential durability of two new major resistance genes introgressed into B. napus from the Brassica B genome was tested separately for each gene under field conditions for 4 years. Successive inoculations with residues of the resistant lines mixed with susceptible contaminated plant material recovered at harvest the previous year were performed in autumn. The Jlm1 resistance gene originating from B. juncea conferred complete resistance on the B. napus-B. juncea recombinant lines MX and MXS to inoculation of the cotyledons with a large diversity of L. maculans isolates. It also gave a high level of stem canker resistance in the field against natural populations of the pathogen. A similar level of resistance was obtained in the B. napus-B. nigra addition line LA4+, containing B. nigra chromosome 4 in a B. napus background. In the second year of the field experiment (i.e., the first in which residues from the resistant lines were included in the inoculation material), both MX and LA4+ maintained a high level of resistance. In the third and fourth years of the field experiment, the resistance of MX and MXS exposed to inoculum produced from their own residues broke down, but against fungal populations from susceptible B. napus or resistant B. nigra material remained effective. In contrast, LA4+ remained highly resistant to all sources of inoculum for the 4-year experiment.

Detection, introgression and localization of genes conferring specific resistance to Leptosphaeria maculans from Brassica rapa into B. napus.

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.

Leptosphaeria maculans and cryptogein induce similar vascular responses in tissues undergoing the hypersensitive reaction in Brassica napus

Cotyledons of Brassica napus cultivars displayed an hypersensitive reaction (HR) after either inoculation with an avirulent isolate of Leptosphaeria maculans (L. maculans), the fungus responsible for blackleg of crucifers, or infiltration with an elicitin, cryptogein. In the first case, ultrastructural observations revealed that the lumen of vessels located in the areas displaying a hypersensitive response was occluded by a fibrillar-like material. This material was labeled with anti-pectin antibodies, but not with antibodies specific for cellulose, callose, hemicellulose, or hydroxyproline-rich glycoprotein. Xylem and phloem parenchyma cells displayed ultrastructural features including morphological changes of nuclei and coagulation of the cytoplasm, reminiscent of cell death. None of these reactions were observed in plants infected with a virulent isolate of L. maculans or in controls. The host responses following infiltration with cryptogein appear similar to these following inoculation with an avirulent isolate of L. maculans. The data show that responses of vascular parenchyma cells are associated with the HR induced by an avirulent isolate of L. maculans and that cryptogein can mimic effects of an avirulent isolate of L. maculans at the vascular level.

Frequency of avirulence genes in Leptosphaeria maculans in western Canada

Abstract Changes in pathogenicity of populations of Leptosphaeria maculans, the cause of blackleg disease of canola and other Brassica spp., have been observed in western Canada. These changes in the population are believed to have resulted from the use of specific resistance (Rlm) genes in Brassica spp., many of which have only recently been identified. We determined the frequency of 10 avirulence alleles for 96 isolates of L. maculans collected between 1997 and 2005 from Alberta, Saskatchewan and Manitoba, Canada from infested canola stubble in field plots or farmers’ fields. Cotyledon interaction phenotypes were scored after inoculation of each isolate on a differential set of Brassica varieties or lines carrying the known resistance genes LepR3 and Rlm1 to Rlm10, except for Rlm8. The avirulence (AvrLm) alleles present in each isolate were inferred from the data. Due to the presence of confounding resistance genes in the host differentials, AvrLm1 and AvrLm2 could not be determined in nine of the isolates, AvrLm6 in two isolates and AvrLepR3 in 49. Based on the number of isolates in which the avirulence genes were confirmed, AvrLm1 was detected in 46.0% and AvrLm9 in 60.4% of the isolates in this collection. A high proportion of isolates carried AvrLm2 (96.6%) and AvrLepR3 (97.9%), and all isolates carried AvrLm6 and AvrLm10. The proportion of L. maculans isolates carrying AvrLm3, AvrLm4, AvrLm5 and AvrLm7 was much lower and varied between 10.4 and 29.2%. In total, 16 races of L. maculans were identified based on 10 avirulence alleles, with seven races accounting for 89.7% of the isolates. Information on the avirulence frequency in the pathogen population will be useful for development of resistance management strategies to control blackleg disease in B. napus oilseed rape in western Canada.