Dual control of avirulence in Leptosphaeria maculans towards a Brassica napus cultivar with 'sylvestris-derived' resistance suggests involvement of two resistance genes

Blackleg disease (phoma stem canker) of Brassica napus (canola, oilseed rape) is caused by the fungus Leptosphaeria maculans . In some regions of Australia, resistance in oilseed rape cultivars derived from B. rapa subs . sylvestris (e.g. cv. Surpass 400) became ineffective within three years of commercial release. The genetic control of avirulence in L. maculans towards cv. Surpass 400 is described. When Australian field isolates were screened on this cultivar, three phenotypic classes were observed; virulent, intermediate and avirulent. Analysis of crosses between fungal isolates varying in their ability to infect cv. Surpass 400 demonstrated the presence of two unlinked avirulence genes, AvrLm1 and AvrLmS . Complementation of isolates (genotype avrLm1 ) with a functional copy of AvrLm1 , and genotyping of field isolates using a molecular marker for AvrLm1 showed that virulence towards Rlm1 is necessary, but not sufficient, for expression of a virulent phenotype on cv. Surpass 400. Taken together, these data strongly suggest that cv. Surpass 400, with ' sylvestris -derived' resistance, contains at least two resistance genes, one of which is Rlm1 .     

Genetic Control and Host Range of Avirulence Toward Brassica napus Cultivars Quinta and Jet Neuf in Leptosphaeria maculans

ABSTRACT Leptosphaeria maculans causes blackleg of oilseed rape. Gene-for-gene interactions between race PG3 and Brassica napus cv. Quinta were related to interaction between the fungal avirulence (Avr) gene AvrLm1 and the corresponding resistance gene Rlm1. AvrLm1 isolates were aviru-lent on cvs. Doublol, Vivol, Columbus, and Capitol, and no recombinant phenotypes were observed in the progeny of two AvrLm1 x avrLm1 crosses, suggesting that all of these cultivars may possess Rlm1 or genes displaying the same recognition spectrum, or that a cluster of Avr genes is present at the Avrlm1 locus. In one cross, segregation distortion was observed at the AvrLm1 locus that could be explained by interaction between AvrLm1 and one unlinked deleterious gene, termed Del1. Incompatibility toward cvs. Jet Neuf and Darmor.bzh was governed by a single gene, unlinked to AvrLm1 or Del1. This avirulence gene was termed AvrLm4. Preliminary plant genetic analysis suggested the occurrence of a corresponding dominant resistance gene, termed Rlm4, present in the Quinta line analyzed and linked to Rlm1.     

The Leptosphaeria maculans – Leptosphaeria biglobosa species complex in the American continent

Stem canker of oilseed rape (canola, Brassica napus ) is associated with a species complex of two closely related fungal species, Leptosphaeria maculans and L. biglobosa . Of these, L. maculans is the most damaging and develops gene-for-gene relationships with the host . Here, a wide scale analysis of the L. maculans - L. biglobosa species complex was performed throughout the American continent (23 locations from Chile to Canada) plus several locations in Western Australia for comparison purposes, based on a collection of 1132 isolates from infected tissues of a susceptible cultivar. Fungal species were discriminated on the basis of morphological, phytopathological and molecular criteria and showed that L. biglobosa was closely associated with L. maculans in most of the locations. Multiple gene phylogeny using sequences of ITS, actin and β-tubulin confirmed the prevalence of the L. biglobosa 'canadensis' sub-clade in Canada, whereas up to three different sub-clades of L. biglobosa were found in Georgia (USA). Race structure of L. maculans was investigated using a combination of pathogenicity tests and PCR amplification of avirulence alleles AvrLm1 , AvrLm4 and AvrLm6 . Three contrasting situations were observed: (i) race structure in Ontario, Chile and Georgia was related to that of European and Western Australian populations, with a low race diversity; (ii) only one race was found in Mexico, and not found outside of this country; (iii) a large diversity of races was observed in central Canada (Manitoba, Alberta and Saskatchewan) with very specific features including maintenance of avirulence alleles absent from Europe, absence of the AvrLm7 allele common in Europe (or eastern Canada) and wide location-to-location variability.     

Analysis of Leptosphaeria maculans Race Structure in a Worldwide Collection of Isolates

ABSTRACT Leptosphaeria maculans, the causal agent of stem canker of oilseed rape, develops gene-for-gene interactions with its hosts. To date, eight L. maculans avirulence (Avr) genes, AvrLm1 to AvrLm8, have been genetically characterized. An additional Avr gene, AvrLm9, that interacts with the resistance gene Rlm9, was genetically characterized here following in vitro crosses of the pathogen. A worldwide collection of 63 isolates, including the International Blackleg of Crucifers Network collection, was genotyped at these nine Avr loci. In a first step, isolates were classified into pathogenicity groups (PGs) using two published differential sets. This analysis revealed geographical disparities as regards the proportion of each PG. Genotyping of isolates at all Avr loci confirmed the disparities between continents, in terms of Avr allele frequencies, particularly for AvrLm2, AvrLm3, AvrLm7, AvrLm8, and AvrLm9, or in terms of race structure, diversity, and complexity. Twenty-six distinct races were identified in the collection. A larger number of races (n = 18) was found in Australia than in Europe (n = 8). Mean number of virulence alleles per isolate was also higher in Australia (5.11 virulence alleles) than in Europe (4.33) and Canada (3.46). Due to the diversity of populations of L. maculans evidenced here at the race level, a new, open terminology is proposed for L. maculans race designation, indicating all Avr loci for which the isolate is avirulent.     

A Cluster of Major Specific Resistance Genes to Leptosphaeria maculans in Brassica napus

ABSTRACT Two types of genetic resistance to Leptosphaeria maculans usually are distinguished in Brassica napus: qualitative, total resistance expressed at the seedling stage and quantitative, partial resistance expressed at the adult plant stage. The latter is under the control of many genetic factors that have been mapped through quantitative trait loci (QTL) studies using 'Darmor' resistance. The former usually is ascribed to race-specific resistance controlled by single resistance to L. maculans (Rlm) genes. Three B. napus-originating specific Rlm genes (Rlm1, Rlm2, and Rlm4) previously were characterized. Here, we report on the genetic identification of two novel resistance genes, Rlm3 and Rlm7, corresponding to the avirulence genes AvrLm3 and AvrLm7. The identification of a novel L. maculans- B. napus specific interaction allowed the detection of another putative new specific resistance gene, Rlm9. The resistance genes were mapped in two genomic regions on LG10 and LG16 linkage groups. A cluster of five resistance genes (Rlm1, Rlm3, Rlm4, Rlm7, and Rlm9) was strongly suggested on LG10. The relation between all these specific resistance genes and their potential role in adult-plant field resistance is discussed. These two Rlm-carrying regions do not correspond to major QTL for Darmor quantitative resistance.     

Frequency of Avirulence Alleles in Field Populations of Leptosphaeria maculans in Europe

This paper describes the first large-scale Europe-wide survey of avirulence alleles and races of Leptosphaeria maculans. Isolates were collected from the spring rape cultivar Drakkar, with no known genes for resistance against L. maculans, at six experimental sites across the main oilseed rape growing regions of Europe, including the UK, Germany, Sweden and Poland. Additionally in Poland isolates were collected from cv. Darmor, which has resistance gene, Rlm9. In total, 603 isolates were collected during autumn in 2002 (287 isolates from Germany and the UK) and 2003 (316 isolates from Poland and Sweden). The identity of alleles at eight avirulence loci was determined for these isolates. No isolates had the virulence allele avrLm6 and three virulence alleles (avrLm2, avrLm3 and avrLm9) were present in all isolates. The isolates were polymorphic for AvrLm1, AvrLm4, AvrLm5 and AvrLm7 alleles, with virulence alleles at AvrLm1 and AvrLm4 loci and avirulence alleles at AvrLm7 and AvrLm5 loci predominant in populations. Virulent avrLm7 isolates were found at only one site in Sweden. Approximately 90% of all isolates belonged to one of two races (combinations of avirulence alleles), Av5-6-7 (77% of isolates) or Av6-7 (12%). Eight races were identified, with four races at frequencies less than 1%. The study suggested that Rlm6 and Rlm7 are still effective sources of resistance against L. maculans in oilseed rape in Europe. The results are comparable to those of a similar survey done in France in autumn 2000 and 2001.     

Variation in host range, systemic infection and epidemiology of Leptosphaeria maculans

The A and B groups (aggressive and non-aggressive) of Leptosphaeria maculans were compared in studies of host range, infection phenotypes and epidemiology. Isolates of both groups infected a wide range of cruciferous hosts including Brassica napus, B. rapa, B. oleracea, B. juncea, B. carinata. B. nigra, Thlaspi arvense and Raphanus sativus. On cotyledons, B-group isolates were generally more aggressive than A-group isolates, causing local lesions and subsequent systemic invasion of the majority of test species. On susceptible stems, A-group isolates caused cortical lesions; B-group isolates, if they reached the stem, usually caused pith lesions with no external symptoms. In a susceptible line of B. napus , CrGC5, systemic infections of the leaf and petiole were similar with both groups, but the B-group rarely entered the stem to form cortical lesions or cankers. At two different field locations, natural epidemics caused by the two groups on oilseed rape leaves were similar in overall pattern, B-group lesions occurred slightly later but the incidence increased more rapidly and reached a maximum slightly earlier than that of the A group. The two groups differed markedly in stem infection patterns. Infection of the cortex near the base of the stem and the development of a typical stem canker was caused entirely by the A group. However the B group often caused considerable damage to the pith. Superficial chlorotic lesions on stems and inflorescences were mainly attributable to the B group. In view of the stem pith infection by the B group without external symptoms, its importance on oilseed rape may have been underestimated previously.     

Strategies to prevent spread of Leptosphaeria maculans (phoma stem canker) onto oilseed rape crops in China; costs and benefits

Field experiments in Europe have shown that Chinese cultivars of winter oilseed rape ( Brassica napus ) are very susceptible to the pathogen Leptosphaeria maculans (cause of phoma stem canker). Climatic and agronomic conditions in China are suitable for L. maculans since the closely related but less damaging pathogen L. biglobosa occurs on the winter and spring oilseed rape crops there. Major gene resistance to L. maculans is not durable; when introduced into commercial oilseed rape cultivars it is rapidly rendered ineffective by changes in the pathogen population. The threat to Chinese oilseed rape production from L. maculans is illustrated by the way in which L. maculans has spread into other areas of the world where previously only L. biglobosa was present, such as Canada and Poland. Models were developed to describe the spread (in space and time) of L. maculans across Alberta province, Canada, based on survey data collected over a 15-year period. These models were used to estimate the potential spread of L. maculans across the Yangtze river oilseed rape growing areas of China and its associated costs. Short-term strategies to prevent occurrence of severe phoma stem canker epidemics in China include training of extension workers to recognise symptoms of the disease and use of PCR-based diagnostics to detect the pathogen on imported seed. Long-term strategies include the introduction of durable resistance to L. maculans into Chinese oilseed rape cultivars as a component of an integrated disease management programme. The costs of such strategies in relation to costs of a phoma stem canker epidemic are discussed.     

Occurrence of a new subclade of Leptosphaeria biglobosa in Western Australia

Stem canker of crucifers is caused by an ascomycete species complex comprising of two main species, Leptosphaeria maculans and L. biglobosa. These are composed of at least seven distinct subclades based on biochemical data or on sequences of internal transcribed spacer (ITS), the mating type MAT1-2 or fragments of actin or beta-tubulin genes. In the course of a wide-scale characterization of the race structure of L. maculans from Western Australia, a few isolates from two locations failed to amplify specific sequences of L. maculans, i.e., the mating-type or minisatellite alleles. Based on both pathogenicity tests and ITS size, these isolates were classified as belonging to the L. biglobosa species. Parsimony and distance analyses performed on ITS, actin and beta-tubulin sequences revealed that these isolates formed a new L. biglobosa subclade, more related to the Canadian L. biglobosa 'canadensis' subclade than to the L. biglobosa 'australensis' isolates previously described in Australia (Victoria). They are termed here as L. biglobosa 'occiaustralensis'. These isolates were mainly recovered from resistant oilseed rape cultivars that included the Brassica rapa sp. sylvestris-derived resistance source, but not from the susceptible cv. Westar. The pathogenicity of L. biglobosa 'occiaustralensis' to cotyledons of most oilseed rape genotypes was higher than that of L. biglobosa 'canadensis' or L. biglobosa 'australensis' isolates.     

Epidemiology and management of Leptosphaeria maculans (phoma stem canker) on oilseed rape in Australia, Canada and Europe

Phoma stem canker (blackleg), caused by Leptosphaeria maculans , is an important disease on oilseed rape (canola, rapeseed, Brassica napus , Brassica juncea , Brassica rapa ) causing seedling death, lodging or early senescence in Australia, Canada and Europe, but not in China. The two forms of L. maculans (A group and B group) that occur on oilseed rape are now considered to be separate species. The epidemiology and severity of phoma stem canker differs between continents due to differences in the pathogen population structure, oilseed rape species and cultivars grown, climate and agricultural practices. Epidemics are most severe in Australia, where only the A group occurs, and can be damaging in Canada and western Europe, where both A and B groups occur, although their proportions vary within regions and throughout the year. Epidemics are slight in China, where the A group has not been found. Dry climates (Australia, western Canada) lengthen the persistence of infected debris and may synchronize the release of airborne ascospores (after rain) with seedling emergence. L. maculans spreads from cotyledon and leaf infections down petioles to reach the stem, with infections on cotyledons and leaves early in the season producing the most damaging stem cankers at the stem base (crown). Development of both crown cankers and phoma stem lesions higher up stems is most rapid in regions with high temperatures from flowering to harvest, such as Australia and Canada. Breeding for resistance (genetic, disease escape or tolerance), stubble management, crop rotation and fungicide seed treatments are important strategies for control of phoma stem canker in all areas. Fungicide spray treatments are justified only in regions such as western Europe where high yields are obtained, and accurate forecasts of epidemic severity are needed to optimize their use.     

Pathogenicity of Leptosphaeria maculans Isolates on a Brassica napus-B. juncea Recombinant Line

ABSTRACT The Brassica napus-B. juncea recombinant line (MX), resistant to Leptosphaeria maculans, was produced by interspecific crosses and bears one gene (Jlm1) from the B. juncea B genome. We investigated whether this new resistance was race specific by characterizing protection against a large sample of L. maculans isolates. The pathogenicity of 119 isolates of L. maculans comprising 105 A-group isolates and 14 B-group isolates was studied at the cotyledon stage under controlled conditions using the MX line, the susceptible B. napus cultivar Westar, and the resistant B. juncea cultivar Picra. All but one of the isolates were pathogenic on 'Westar'. Only 3 of the 105 A-group isolates caused very mild symptoms on 'Picra'. Two of these strains were isolated from the MX line and the other from Sinapis arvensis. The other 102 strains caused hypersensitive-type responses. Most B-group isolates were pathogenic on 'Picra'. There were differences in pathogenicity among A-group isolates tested on the MX line, whereas all B-group isolates were pathogenic on this line. A-group isolates obtained from the MX line were more frequently pathogenic on the MX line than those obtained from B. napus cultivars. One isolate from S. arvensis infected the MX line. These results suggest that the resistance of the MX line is unlikely to be durable. Thus, the new resistance gene Jlm1 should probably be used in association with other sources of resistance, in plant breeding schemes, to prevent the breakdown of this resistance.     

Genetic Analysis and Identification of Amplified Fragment Length Polymorphism Markers Linked to the alm1 Avirulence Gene of Leptosphaeria maculans

ABSTRACT A gene-for-gene interaction was previously suggested by mapping of a single major locus (LEM 1) controlling cotyledon resistance to Leptosphaeria maculans isolate PHW1245 in Brassica napus cv. Major. In this study, we obtained further evidence of a gene-for-gene interaction by studying the inheritance of the corresponding avirulence gene in L. maculans isolate PHW1245. The analysis of segregating F(1) progenies and 14 test crosses suggested that a single major gene is involved in the interaction. This putative avirulence gene was designated alm1 after the resistance locus identified in B. napus. Amplified fragment length polymorphism (AFLP) markers were used to generate a rudimentary genetic linkage map of the L. maculans genome and to locate markers linked to the putative avirulence locus. Two flanking AFLP markers, AC/TCC-1 and AC/CAG-5, were linked to alm1 at 3.1 and 8.1 cM, respectively. Identification of markers linked to the avirulence gene indicated that the differential interaction is controlled by a single gene difference between parental isolates and provides further support for the gene-for-gene relationship in the Leptosphaeria-Brassica system.     

Broadening the Genetic Basis of Verticillium longisporum Resistance in Brassica napus by Interspecific Hybridization

ABSTRACT Verticillium wilt caused by the vascular fungal pathogen Verticillium longisporum is one of the most important pathogens of oilseed rape (Brassica napus sp. oleifera) in northern Europe. Because production of this major oilseed crop is expanding rapidly and no approved fungicides are available for V. longisporum, long-term control of the disease can only be achieved with cultivars carrying effective quantitative resistance. However, very little resistance to V. longisporum is available within the gene pool of oilseed rape, meaning that interspecific gene transfer from related species is the only possibility for broadening levels of resistance in current varieties. The amphidiploid species B. napus can be resynthesized by crossing the two progenitor species Brassica oleracea and Brassica rapa, hence resistant accessions of these two diploid species can be used as resistance donors. In this study a total of 43 potential B. rapa and B. oleracea resistance donors were tested with regard to their reaction to a mixture of two aggressive V. longisporum isolates, and resistances from diverse lines were combined by embryo rescue-assisted interspecific hybridization in resynthesized rapeseed lines. Progenies from crosses of the two B. rapa gene bank accessions 13444 and 56515 to the B. oleracea gene bank accessions BRA1008, CGN14044, 8207, BRA1398, and 7518 showed a broad spectrum of resistance in pathogenicity tests. Of 45 tested resynthesized lines, 41 lines exhibited a significantly higher level of resistance than the moderately V. longisporum-tolerant oilseed rape cultivar Express. These lines represent a promising basis for the combination of different resistance resources in new varieties.     

The incidence of Alternaria spp. and Leptosphaeria maculans in commercial brassica seed in the United Kingdom

Tests on samples of oilseed rape seed ( Brassica napus ) sown in the UK between 1981 and 1984 indicated that on average 25% of samples were infected with Alternaria brassicae and 61% with Leptosphaeria maculans , with maximum incidences of infection of 19 and 4.2% respectively. Much infection by Alternaria spp. occurred on vegetable and forage brassica seed produced in the UK between 1979 and 1983. In B. oleracea types A. brassicicola occurred most frequently, affecting 88% of samples and up to 55% of seeds. A. brassicae was detected in 44% of B. oleracea samples and in up to 13% of seeds. Little Alternaria infection occurred in swede or forage rape samples (B. napus ), but A. brassicae affected up to 8–5% of seeds in turnip samples ( B. campestris ). L. maculans occurred in 44% of samples of vegetable and forage brassica seed produced in the UK, with a maximum of 4–6% infected seeds. A. brassicicola was present in 73% of samples of imported B. oleracea seed, affecting up to 25.5% of seeds. A. brassicae was absent from these samples and little L. maculans was detected. Pathogenicity tests on isolates of L. maculans from infected seeds indicated that virulent pathotypes were present in 16 rape seed samples but in only one sample (swede) of vegetable or forage brassica seed. The high incidence of seed infection by these pathogens emphasizes the importance of applying fungicide treatments to all types of brassica seed.     

Reaction of Brassica juncea (Indian Mustard) Lines to Australian Isolates of Leptosphaeria maculans under Glasshouse and Field Conditions

Brassica juncea (Indian mustard) lines from diverse geographical locations around the world and from Australian breeding programs were screened for resistance to the blackleg fungus, Leptosphaeria maculans, in both glasshouse and field trials. The five Australian L. maculans isolates used in glasshouse trials could be classified into two groups; those that attacked all B. juncea lines, and those that attacked none. All these isolates caused lesions on cotyledons of B. napus cultivars including Westar, Glacier and Quinta, suggesting that they are in Pathogenicity Group 4 as described by Koch et al. (1991). The two isolates that attacked B. juncea also attacked B. napus lines to a similar extent, but did not attack the two B. carinata lines tested. Brassica lines were sown in a blackleg disease nursery at Lake Bolac, Victoria, Australia, and five indicators of blackleg disease were measured (survival rate, disease rating, disease incidence, external and internal lesion length). All 92 B. juncea lines developed blackleg symptoms. Although they displayed a high disease incidence in the field, almost all of the B. juncea lines were more blackleg-resistant than a B. napus cultivar, Dunkeld, which is amongst the most resistant cultivars in commercial production in Australia. Four B. carinata lines were more resistant than any of the B. juncea lines, suggesting that this species may be a useful source of blackleg resistance in B. napus breeding programs.     

Fitness Cost Associated with Loss of the AvrLm4 Avirulence Function in Leptosphaeria maculans (Phoma Stem Canker of Oilseed Rape)

Near-isogenic isolates of Leptosphaeria maculans differing at the AvrLm4 avirulence locus (AvrLm4 or avrLm4) were produced in vitro. Methods for inoculation of leaves of oilseed rape with ascospores or conidia were compared. The ‘ascospore shower’ inoculation was the most efficient method for use when inoculum is limited (e.g. ascospores produced in vitro). It was used in controlled environments to compare fitness of AvrLm4 and avrLm4 isolates at 5, 10, 15, 20 or 25 °C on leaves of oilseed rape cultivars Eurol and Darmor lacking the resistance gene Rlm4, which corresponds to AvrLm4. At all temperatures tested, AvrLm4 ascospores produced more lesions than avrLm4 ascospores. The diameters of lesions produced by AvrLm4 ascospores were greater than those of lesions produced by avrLm4 ascospores. At 15–20 °C, more lesions initiated by AvrLm4 ascospores produced pycnidia than did lesions initiated by avrLm4 ascospores. However, there were no differences between AvrLm4 and avrLm4 isolates in incubation period (from inoculation to appearance of lesions) or rate of mycelial growth in leaves from lesions towards the stems. In field experiments with winter oilseed rape cultivars lacking Rlm4, the frequency of AvrLm4 isolates increased from 5.7% at the phoma leaf lesion stage (autumn) to 20.5% at the stem canker stage (summer) during 2002/2003 and from 7.9 to 11.5% during 2003/2004 growing seasons. Results of controlled environment and field experiments indicate that avrLm4 isolates have a fitness cost compared to AvrLm4 isolates.     

Differential host-parasite interactions between oilseed rape and Leptosphaeria macufans, the causal fungus of stem canker

Seedlings of 13 cultivars of oilseed rape ( Brassica napus ) were inoculated with 10 isolates of Leptosphaeria maculans. Three virulent isolates were identified which gave a resistant reaction on some cultivars normally considered susceptible. Further experiments to compare these with standard isolates confirmed the existence of a differential host-pathogen interaction. The breeding line 78/271, plus the cultivars Quinta, Garant, Norli and possibly Elvira differentiated the isolates tested while Jet Neuf, Rafal, Primor, Doral, Hercules, Rapora, Liraglu and Erra did not. The relevance of these findings to breeding for canker resistance in oilseed rape is discussed.     

Genetic and molecular analyses in crosses of race 2 and race 7 of albugo Candida

ABSTRACT The inheritance of avirulence and polymorphic molecular markers in Albugo candida, the cause of white rust of crucifers, was studied in crosses of race 2 (Ac2), using isolates MiAc2-B1 or MiAc2-B5 (metalaxyl-insensitive and virulent to Brassica juncea cv. Burgonde) with race 7 (Ac7), using isolate MsAc7-A1 (metalaxyl-sensitive and virulent to B. rapa cv. Torch). Hybrids were obtained via co-inoculation onto a common susceptible host. Putative F(1) progeny were selfed to produce F(2) progeny. The parents and F(1) progeny were examined for virulence on the differential cultivars B. juncea cv. Burgonde and B. rapa cv. Torch. Segregation of avirulence or virulence of F(2) populations was analyzed on cv. Torch. Putative F(1) hybrids were confirmed by random amplified polymorphic DNA markers specific for each parent. Avirulence or virulence of F (2) progeny to B. rapa cv. Torch suggested 3:1 in each of three populations, supporting the hypothesis of a single dominant avirulence gene. Amplified fragment length polymorphism markers also segregated in regular Mendelian fashion among F(2) progeny derived from two F(1) hybrids (Cr2-5 and Cr2-7) of Cross-2. This first putative avirulence gene in A. candida was designated AvrAc1. These results suggest that a single dominant gene controls avirulence in race Ac2 to B. rapa cv. Torch and provides further evidence for the gene-for-gene relationship in the Albugo-Brassica pathosystem.     

Survival of Leptosphaeria maculans in soil on residues of Brassica napus in South Australia

The survival of Leptosphaeria maculans , which causes phoma stem canker (blackleg), on oilseed rape residues ( Brassica napus ) in South Australia was investigated. Using a quantitative polymerase chain reaction (PCR) assay for L. maculans DNA, the pathogen was mainly detected in the upper 5 cm of the soil profile, including residues on the soil surface. As the size of organic matter particles in the soil decreased, so did the quantity of L. maculans detected in them. To obtain representative data for a field, at least 30 subsamples needed to be collected over the 0·81 ha area studied. In a survey of 49 commercial fields in South Australia, most L. maculans was detected in fields 1 year after oilseed rape had been grown, with less detected after 2 years and negligible amounts 3 years or more after cropping. The diagnostic DNA-based assay for L. maculans reduced the time and cost of studying L. maculans survival in soil and increased the sensitivity and accuracy of results compared with estimates of propagule number of colony-forming units on a semiselective medium.     

A Large-Scale Survey of Races of Leptosphaeria maculans Occurring on Oilseed Rape in France

Nine avirulence genes (AvrLm1–AvrLm9) were identified in Leptosphaeria maculans, the causal agent of stem canker of oilseed rape (OSR), combinations of which could theoretically generate up to 512 different races of the fungus. L. maculans displays a high evolutionary potential to adapt to novel resistance genes as illustrated by the Rlm1 breakdown in France, where virulent populations became prevalent within three growing seasons. An improved knowledge of the race structure of the fungal population is therefore needed to ensure a better use of available major resistance genes. The objective of this study was to characterise the L. maculans population structure in France using a large-scale, rationalised sample of isolates. Experimental fields, planted with “trap plants” harbouring no major resistance gene, were sown at 20 locations. Single-pycnidium isolates were collected from leaf lesions that developed in early autumn and 1797 isolates were genotyped at Avr loci. The frequency of AvrLm6 and AvrLm7 was higher than 99%, whereas avrLm2 and avrLm9 alleles were fixed in the population. AvrLm1, AvrLm4, AvrLm5 and AvrLm8 were polymorphic. AvrLm3 isolates were detected at a very low frequency (less than 1%). Only 11 races were identified in France, with one race prevalent, namely Av5-6-7-(8) (i.e. virulent on Rlm1, Rlm2, Rlm3, Rlm4 and Rlm9), representing around 65% of the population. Disparities between the locations sampled were evident at all scales analysed. Some virulent races, such as those harbouring avrLm5, were present before the introduction of the corresponding resistance gene in the commercial OSR crop.