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     

Effects of light leaf spot (Pyrenopeziza brassicae) on yield of winter oilseed rape (Brassica napus)

The relationship between development of light leaf spot and yield loss in winter oilseed rape was analysed, initially using data from three experiments at sites near Aberdeen in Scotland in the seasons 1991/92, 1992/93 and 1993/94, respectively. Over the three seasons, single-point models relating yield to light leaf spot incidence (% plants with leaves with light leaf spot) at GS 3.3 (flower buds visible) generally accounted for more of the variance than single-point models at earlier or later growth stages. Only in 1992/93, when a severe light leaf spot epidemic developed on leaves early in the season, did the single-point model for disease severity on leaves at GS 3.5/4.0 account for more of the variance than that for disease incidence at GS 3.3. In 1991/92 and 1992/3, when reasonably severe epidemics developed on stems, the single-point model for light leaf spot incidence (stems) at GS 6.3 accounted for as much of the variance. Two-point (disease severity at GS 3.3 and GS 4.0) and AUDPC models (disease incidence/severity) accounted for more of the variance than the single-point model based on disease incidence at GS 3.3 in 1992/93 but not in the other two seasons. Therefore, a simple model using the light leaf spot incidence at GS 3.3 (x) as the explanatory variable was selected as a predictive model to estimate % yield loss (y_r): y_r= 0.32x– 0.57. This model fitted all three data sets from Scotland, When data sets from Rothamsted, Rosemaund and Thurloxton in England were used to test it, this single-point predictive model generally fitted the data well, except when yield loss was clearly not related to occurrence of light leaf spot. However, the regression lines relating observed yield loss to light leaf spot incidence at GS 3.3 often had smaller slopes than the line produce, by the model based on Scottish data.     

Effects of previous cropping and fungicide timing on the development of light leaf spot (Pyrenopeziza brassicae), seed yield and quality of winter oilseed rape (Brassica napus)

Light leaf spot, caused by Pyrenopeziza brassicae, was assessed regularly on double-low cultivars of winter oilseed rape during field experiments at Rothamsted in 1990-91 and 1991-92. Previous cropping and fungicide applications differed; seed yield and seed quality were measured at harvest. In each season, both the initial incidence of light leaf spot and the rate of disease increase were greater in oilseed rape crops sown after rape than those sown after cereals. The incidence of diseases caused by Phoma lingam or Alternaria spp. was also greater in second oilseed rape crops. In 1991-92 there was 42% less rainfall between September and March than in 1990-91, and much less light leaf spot developed. However, P. lingam and Alternaria spp. were more common. Only fungicide application schedules including an autumn spray decreased the incidence of light leaf spot on leaves, stems and pods, as indicated by decreased areas under the disease progress curves (AUDPC) and slower rates of disease increase. Summer sprays decreased incidence and severity of light leaf spot on pods only. In 1990-91, all fungicide treatments which included an autumn spray increased seed and oil yields of cv. Capricorn but only the treatment which included autumn, spring and summer sprays increased yields of cv. Falcon. No treatment increased the yields of cv. Capricorn or cv. Falcon in 1991-92. Fungicide applications decreased glucosinolate concentrations in the seed from a crop of cv. Cobra severely infected by P. brassicae in 1990-91, but did not increase yield.     

Diagnosis of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) in the UK

Light leaf spot lesions were generally first observed as light green areas on leaves of UK winter oilseed rape crops in January or February and later became brittle and bleached. Elongated lesions, which were brown with indistinct edges, developed on stems in the spring and summer, when lesions were also observed on flower buds, pedicels and pods. Development of diagnostic white pustules (spore masses of Pyrenopeziza brassicae, which erupt through surfaces of infected tissues) for confirmation of light leaf spot infection on symptomless plants or plants with indistinct or ambiguous symptoms in the autumn, winter or spring was enhanced by incubating plants in polyethylene bags. In experiments with artificially inoculated plants, glasshouse-grown plants exposed in infected crops and plants sampled from crops, white pustules developed at all incubation temperatures from 2^oC to 20^oC on infected leaves of different cultivars. The period of incubation required before the appearance of pustules decreased as the time that had already elapsed since the initial infection increased. The longest periods of incubation were required at the lowest temperatures (2^oC or 5^oC) but leaves senesced and abscised from plants most quickly at the highest temperatures (15^oC or 20^oC), suggesting that the optimal incubation temperature was between 10^oC and 15^oC.     

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.     

Methods for assessment of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) in the UK

Light leaf spot (Pyrenopeziza brassicae) was assessed as % plants with light leaf spot, % leaves with light leaf spot or % leaf area with light leaf spot in winter oilseed rape field experiments done at different sites (Rothamsted, Hertfordshire; Boxworth, Cambridgeshire; near Aberdeen, Scotland), with different cultivars (e.g. Bristol and Capitol), different fungicide treatments, on plants sampled at different dates. Regression analyses on data from these experiments showed that there were consistently good relationships between % leaves with light leaf spot and % plants with light leaf spot for plants sampled during the autumn and winter, until the % plants with light leaf spot approached 100%. The slopes and positions of regression lines were sometimes affected by cultivar, fungicide treatment or sampling date, but not by site. The relationship between % leaf area with light leaf spot (square root-transformed) and % leaves with light leaf spot was less consistent than that between % leaves with light leaf spot and % plants with light leaf spot and was sometimes affected by cultivar, fungicide treatment or sampling date but not by site. The relationship between % leaf area with light leaf spot (square root-transformed) and % plants with light leaf spot was also inconsistent and was sometimes affected by cultivar, fungicide treatment, sampling date and site.     

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     

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.     

Comparative studies of light leafspot (Pyrenopeziza brassicae) epidemics on the growth and yield of winter oilseed rape

Comparisons of epidemics of light leafspot of differing duration and time of initiation were made in two experiments using a single cultivar of Brassica napus. Fungicide was applied before introduction of disease to prevent infection or some time after inoculation to stop further disease development. In the first experiment, substantial reductions in green leaf area and total plant dry-matter were found at flowering when disease was introduced in the autumn or in January. Plant dry weight at maturity was also greatly reduced in these treatments. The detrimental effect of an epidemic initiated in the autumn was avoided to a large extent if fungicide application began in February. Epidemics initiated in March had only small effects on final dry-matter yield. Seed yield was negatively correlated with the length of the epidemic. In a second experiment, early epidemics initiated in the autumn were halted after different time intervals. Commencing fungicide application even as early as December failed to prevent some loss of dry weight at flowering. At maturity, however, dry weight and seed yield were reduced significantly when fungicide application was delayed until February. Failure to control the disease resulted in a 46% loss of seed yield.     

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 blackleg [Leptosphaeria maculans (Desm.) Ces. et De Not.] resistance in rapeseed (Brassica napus L.)

The genetic control of adult-plant blackleg [Leptosphaeria maculans (Desm.) Ces. et De Not.] resistance in rapeseed (Brassica napus L.) was studied in the F₂ and first-backcross populations of the cross Maluka (blackleg-resistant) x Niklas (highly susceptible). A L. maculans isolate possessing high levels of host specificity (MB2) was used in all inoculations. Resistance/susceptibility was evaluated using three separate measures of crown-canker size, i.e. the percentage of crown girdled (%G), external lesion length (E) and internal lesion area (%II). Disease severity scores for the F₂ and first-backcross populations based on E and %II gave discontinuous distributions, indicating major-gene control for these measures of resistance; but those for %G were continuous, indicating quantitative genetic control for this measure. Chi-square tests performed on the (poorly-defined) resistance classes, based on E, in the F₂ and first-backcross populations indicated the likelihood for resistance being governed by a single, incompletely dominant major gene. Although the distributions of the F₂ and first-backcross populations, based on%II, were clearly discontinuous, the observed segregation ratios for resistance and susceptibility did not fit any of the numerous Mendelian ratios which were considered. Differences in inheritance of resistance according to the assessment method and blackleg isolate used, were discussed.     

Mapping of one major gene and of QTLs involved in resistance to clubroot in Brassica napus

Clubroot, caused by Plasmodiophora brassicae, is a damaging disease of Brassica napus. Genetic control and mapping of loci involved in high and partial quantitative resistance expressed against two single spore isolates (Pb137–522 and K92–16) were studied in the F₁ and DH progenies of the cross Darmor-bzh (resistant) x Yudal (susceptible). The high level of resistance expressed by Darmor-bzh to isolate Pb137–522 was found to be mainly due to a major gene, which we have named Pb-Bn1, located on linkage group (LG) DY4. Partial quantitative resistance showed by Darmor-bzh to the K92–16 isolate arose from the association of at least two additive QTLs detected on LGs DY4 and DY15; the QTL on DY4, explaining 19% of the variance, was mapped at the same position as the major gene Pb-Bn1. Epistatic interactions between nine regions with or without additive effects were detected. The total phenotypic variation accounted for by additive and epistatic QTLs ranged from 62% to 81% depending on the isolate. For one isolate, the relative effect due to additivity was similar to that due to epistasis. Received: 10 November 1999 / Accepted:18 February 2000     

Genetics of virulence in Leptosphaeria maculans (Desm.) Ces. et De Not., the cause of blackleg in rapeseed (Brassica napus L.)

The genetic basis of virulence of 24 isolates of L. maculans collected from various sites throughout south-eastern and south-western Australia were studied using five clone-lines of B. napus. The experimental design allowed the estimation of the environmental and genetic components of variance using a standard analysis of variance. Virulence of these isolates (as measured by the percentage of stem girdling, %G) on the clonelines NCII and Tap was found to be most likely controlled by a small number of genes; the broad-sense heritabilities were 79.7% and 67.5% for virulence on NCII and Tap, respectively. The significance of these results in relation to the potential of L. maculans in adapting to new resistant B. napus cultivars is discussed.     

Conservation of S-locus for self-incompatibility in Brassica napus (L.) and Brassica oleracea (L.)

 Self-incompatibility (SI) in Brassica is a sporophytic system, genetically determined by alleles at the S-locus, which prevents self-fertilization and encourages outbreeding. This system occurs naturally in diploid Brassica species but is introduced into amphidiploid Brassica species by interspecific breeding, so that in both cases there is a potential for yield increase due to heterosis and the combination of desirable characteristics from both parental lines. Using a polymerase chain reaction (PCR) based analysis specific for the alleles of the SLG (S-locus glycoprotein gene) located on the S-locus, we genetically mapped the S-locus of B. oleracea for SI using a F₂ population from a cross between a rapid-cycling B. oleracea line (CrGC-85) and a cabbage line (86-16-5). The linkage map contained both RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) markers. Similarly, the S-loci were mapped in B. napus using two different crosses (91-SN-5263×87-DHS-002; 90-DHW-1855-4×87-DHS-002) where the common male parent was self-compatible, while the S-alleles introgressed in the two different SI female parents had not been characterized. The linkage group with the S-locus in B. oleracea showed remarkable homology to the corresponding linkage group in B. napus except that in the latter there was an additional locus present, which might have been introgressed from B. rapa. The S-allele in the rapid-cycling Brassica was identified as the S₂₉ allele, the S-allele of the cabbage was the S ₅ allele. These same alleles were present in our two B. napus SI lines, but there was evidence that it might not be the active or major SI allele that caused self-incompatibility in these two B. napus crosses. Received: 7 June 1996/Accepted: 6 September 1996     

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.     

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.     

RFLP mapping of resistance to the blackleg disease [causal agent,Leptosphaeria maculans (Desm.) Ces. et de Not.] in canola (Brassica napus L.)

We report the tagging of genes involved in blackleg resistance, present in the French cultivar Crésor of B. napus, with RFLP markers. A total of 218 cDNA probes were tested on the parental cultivars Crésor (resistant) and Westar (susceptible), and 141 polymorphic markers were used in a segregating population composed of 98 doubled-haploid lines (DH). A genetic map from this cross was constructed with 175 RFLP markers and allowed us to scan for specific chromosomal associations between response to blackleg infection and RFLP markers. Canola residues infested with virulent strains of Leptosphaeria maculans were used as inoculum and a suspension of pycnidiospores from cultures of L. maculans, including the highly virulent isolate Leroy, was sprayed to increase disease pressure. QTL mapping suggested that a single chromosomal region was responsible for resistance in each of the four environments tested. This QTL accounted for a high proportion of the variation of blackleg reaction in each of the assays. A second QTL, responsible for a small proportion of the variation of blackleg reaction, was present in one of four year-site assays. A Mendelian approach, using blackleg disease ratings for classifying DH lines as resistant or susceptible, also allowed us to map resistance in the region of the highly significant LOD scores observed in each environment by interval mapping. Results strongly support the presence of a single major gene, named LmFr ₁ controlling adult plant resistance to blackleg in spring oil-seed rape cultivar Crésor. Several RFLP markers were found associated with LmFr ₁.