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.     

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.     

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.     

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.     

Dissemination of Information About Management Strategies and Changes in Farming Practices for the Exploitation of Resistance to Leptosphaeria maculans (Phoma Stem Canker) in Oilseed Rape Cultivars

The management of phoma stem canker (blackleg disease, caused by Leptosphaeria maculans) is an integral component of oilseed rape production. In this paper, we discuss the information about management strategies that is disseminated in Europe and Australia. New cultivars have been introduced with improved resistance to disease, but sometimes this resistance has been overcome as new races of the pathogen have emerged. When cultivars with single major gene resistance have been introduced into areas with high inoculum concentrations, significant economic damage has been caused by new races of L. maculans within 2–3 years. Quantitative or polygenic resistance has also been used successfully against stem canker and offers more durable disease resistance if plant breeders and farmers deploy this resistance more effectively. Strategies to improve the durability of resistance need to be developed and tested in practice. New information on the occurrence of virulence and avirulence genes in populations of Leptosphaeria maculans and modelling of the durability of resistance provide opportunities for plant breeders, specialist technical organisations, cooperatives, advisory services and farmers to collaborate and better exploit cultivar resistance. Changing economic and environmental factors influence cropping practices and, if to be considered successful, management strategies must show clear financial benefits. Technology transfer will need to address all aspects of managing stem canker and other diseases of oilseed rape and using effective written, verbal and electronic methods of communication.     

Methods for Sampling and Assessment in Relation to the Spatial Pattern of Phoma Stem Canker (Leptosphaeria maculans) in Oilseed Rape

A sound assessment of phoma stem canker symptoms is needed to develop epidemiological, agronomical and physiological studies on the pathosystem. A specific analysis was therefore carried out to: (i) compare four methods of crown canker assessment; (ii) test the among and within assessor repeatability of one of the methods compared; (iii) characterise the spatial pattern of the disease; and (iv) define the sample size required to achieve a given level of disease assessment precision. The methods compared examined the symptoms with different procedures and graded the plants observed into six severity classes. A disease index (DI) summarised the severity distribution observed. Examination of crown cross-sections was the most precise method for assessing crown cankers. The method was repeatable, though an 'assessor effect' was apparent. The disease generally had a random pattern although significant spatial correlations were detected for four out of the fifteen plots studied at the scales examined. A relationship between the coefficient of variation of the DI and the sample size was established, evaluated with experimental field data and exemplified for typical severity distributions.     

Improved Resistance Management for Durable Disease Control: A Case Study of Phoma Stem Canker of Oilseed Rape (Brassica napus)

Specific resistance loci in plants are generally very efficient in controlling development of pathogen populations. However, because of the strong selection pressure exerted, these resistances are often not durable. The probability of a resistance breakdown in a pathosystem depends on the evolutionary potential of the pathogen which is affected by: (i) the type of resistance (monogenic and/or polygenic), (ii) the type of reproduction of the pathogen (sexual and/or asexual), (iii) the capacity of the pathogen for dispersal, (iv) the resistance deployment strategy (pyramiding of specific resistances, mixture of cultivars, spatio-temporal alternation), (v) the size of the pathogen population, which is affected by control methods and environmental conditions. We propose the concept of Integrated Avirulence Management (IAM) to enhance the durability of specific resistances. IAM involves a strategy to limit the selection pressure exerted on pathogen populations and, at the same time, reduce the size of pathogen populations by combining cultural, physical, biological or chemical methods of control. Several breakdowns of resistance specific to Leptosphaeria maculans, the causal agent of phoma stem canker have occurred in Europe and in Australia. This review paper examines control methods to limit the size of L. maculans populations and discusses how this limitation of population size can enhance the durability of specific resistances. It proposes pathways for the development of a spatially explicit model to define IAM strategies. Simulation results are presented to demonstrate the potential uses of such a model for the oilseed rape/L. maculans pathosystem.     

Study of Resistance to Orobanche ramosa in Host (Oilseed Rape and Carrot) and Non-host (Maize) Plants

Orobanche ramosa is a parasitic Angiosperm responsible for severe yield losses in several economical crops. It is a serious threat in oilseed rape in France and Morocco and is appearing in carrot crops in Morocco. In this study, several varieties of oilseed rape and carrot were screened in order to identify resistant cultivars and to characterize the resistance mechanisms involved. All the 15 oilseed rape varieties tested were susceptible. In carrot, the varieties 'Colmar à coeur rouge' and 'Nantaise demi-longue' were susceptible, whereas 'Palaiseau' and 'Buror' were resistant. In the susceptible 'Colmar à coeur rouge' carrot no defence reactions were found and the development of the parasite inhibited carrot tap root formation. In the resistant carrot varieties, the parasite germinated, became attached to the host root but became necrotic before emergence. In 'Buror' carrot, formation of a mechanical barrier was associated with the restriction to the cortex of the parasite. In maize cv. 'Vigni', a non-host of O. ramosa, thickening of xylem vessels, cell divisions in the central cylinder and formation of an encapsulation layer were observed in association with restricted development of Orobanche haustoria.     

Breaching by a new strain of Leptosphaeria maculans of anatomical barriers in cotyledons of Brassica napus cultivar Surpass 400 with resistance based on a single dominant gene

Infection processes were examined to investigate the breach by a strain of Leptosphaeria maculans of anatomical barriers in cv. Surpass 400, a cultivar containing single dominant gene-based resistance (SDGBR). Two strains, UWA 192 and UWA P11, were used to inoculate cvs. Surpass 400 and Westar. The pre-penetration and penetration behaviour of both strains was similar in both cultivars. However, they differed significantly after penetration. When UWA P11 infected cv. Surpass 400 through stomata, guard cells rapidly died within a few hours and the surrounding mesophyll cells became necrotic, constituting a hypersensitive reaction (HR). Hyphal growth continued, albeit slowly, through the intercellular palisade mesophyll and spongy mesophyll spaces, but hyphae rarely spread beyond the HR region, and did not sporulate. Polyphenolic compounds accumulated in the area bordering the HR. However, when UWA 192 infected through stomata, symptoms were not evident until 10–12 days postinoculation (dpi) and were typically characterized by pale tan to grey circular lesions in which abundant pycnidia were produced by 14 dpi. Subsequently, hyphae extensively spread beyond the lesion border, reaching the veins and progressing down the petiole towards the stem. Where the SDGBR remained effective (i.e. against strain UWA P11) death of cells was restricted to a few palisade cells within the HR, even though hyphae were present in the lower tissue layers of the cotyledon. In contrast, where the SDGBR was not present (cv. Westar) or was overcome (cv. Surpass 400 with UWA 192), extensive death of epidermal and upper and lower palisade cells occurred throughout infected areas of the cotyledon, with subsequent abundant production of pycnidia. Polyphenolic compounds, which are also associated with resistance, did not accumulate in this instance. It was evident that the ability of the host to instigate the HR mechanism displayed by cv. Surpass 400 was lost with UWA 192 resulting in a “normal” susceptible response. This is the first study of the specific processes involved in the breaching of the HR in cv. Surpass 400.     

Survey of mutations of a histidine kinase gene BcOS1 in dicarboximide-resistant field isolates of Botrytis cinerea

Previously, we cloned a putative osmosensing histidine kinase gene (BcOS1) and revealed that a single amino acid substitution, isoleucine to serine at codon 365, conferred dicarboximide resistance in field isolates of Botrytis cinerea. This point mutation (type I) occurred within the restriction enzyme TaqI site of the wild-type BcOS1 gene. Thus, a procedure was developed for detecting the type I mutation of the BcOS1 gene using a polymerase chain reaction (PCR) in combination with restriction fragment-length polymorphism (RFLP). Diagnosis by PCR-RFLP was conducted on the 105 isolates isolated from 26 fields in Japan. All dicarboximide-sensitive isolates (49 isolates) had the wild-type BcOS1 gene, and the 43 isolates with the type I mutation were resistant to dicarboximides without exception. These data indicate that dicarboximide-resistant isolates with type I mutation are widespread throughout Japan. However, other types of dicarboximide resistance were detected among isolates from Osaka; among the 24 resistant isolates from Osaka, 12 had the BcOS1 gene without the type I mutation. BcOS1 gene sequencing of these resistant isolates classified them into two groups, type II and type III. The type II isolates have three amino acid substitutions within BcOS1p (³⁶⁸Val to Phe, ³⁶⁹Gln to His, and ⁴⁴⁷Thr to Ser). The type III isolates have two amino acid substitutions within BcOS1p (³⁶⁹Gln to Pro and ³⁷³Asn to Ser). These amino acid changes are located on the amino acid repeat domain in BcOS1p. The three types of resistant isolates were all moderately resistant to dicarboximides without significant osmotic sensitivity, and their pathogenicity on cucumber leaves was also very similar to that of the wild-type isolate.     

Biotic factors affecting the expression of partial resistance in pea to ascochyta blight in a detached stipule assay

The expression of partial resistance in pea to ascochyta blight (caused by Mycosphaerella pinodes) was studied in a detached stipule assay by quantifying two resistance components (fleck coalescence and lesion expansion) using the method of point inoculation of stipules. Factors determining optimal conditions for the observation of partial resistance are spore concentration, the age of the fungal culture prior to spore harvest and the pathogenicity of the isolate used for testing. Partial resistance was not expressed when spore concentration was high or when the selected isolate was aggressive. Furthermore, assessments of components of partial resistance were highly correlated with disease severity in a seedling test. A screening protocol was developed based on inoculations of detached stipules to study partial resistance in pea. To simplify the rating process, a more comprehensive disease rating scale which took into account fleck coalescence and lesion expansion was tested by screening a large number of genotypes.     

Identification of a CAPS marker tightly linked to the Tomato yellow leaf curl disease resistance gene <Emphasis Type="Italic">Ty-1</Emphasis> in tomato

During the process of breeding programmes, several resistance genes have been introgressed into tomato (Solanum lycopersicum) cultivars from different wild tomato relatives. A number of these resistance genes have been mapped to chromosome 6. Among them, Ty-1 and Mi, which confer resistance to Tomato yellow leaf curl disease and to Meloidogyne spp., respectively, are in most cases incorporated in commercial hybrids. Several molecular markers tightly linked to Mi have been identified. This study was conducted in order to find an informative molecular marker linked to Ty-1. Six markers mapped in the same region as Ty-1 were analysed in plant material carrying different combinations of Ty-1 and Mi alleles. Three of the six markers revealed polymorphism among the assayed accessions. One allele of JB-1 marker showed association with Ty-1. Furthermore, the presence of Mi did not interfere with the results. The analysis of several accessions of wild tomato relatives with the three polymorphic markers allowed the establishment of the origin of the alleles found in cultivated plant material, showing that introgressions from S. lycopersicum, S. pimpinellifolium and S. habrochaites will not interfere with the results of this marker which tags Ty-1. Furthermore this analysis enabled the location of CT21, the RFLP marker from which JB-1 was designed.     

Systemic resistance to bacterial leaf speck pathogen in Arabidopsis thaliana induced by the culture filtrate of a plant growth-promoting fungus (PGPF) Phoma sp. GS8-1

The culture filtrate (CF) from the plant growth-promoting fungus Phoma sp. GS8-1 was found to induce systemic resistance in Arabidopsis thaliana against the bacterial leaf speck pathogen Pseudomonas syringae pv. tomato DC3000 (Pst), and the underlying mechanism was studied. Roots of A. thaliana were treated with CF from GS8-1, and plants expressed a clear resistance to subsequent Pst infection; disease severity was reduced, and proliferation of pathogen was suppressed. Various mutants of A. thaliana were used to test whether the CF induced resistance through one of the known signaling pathways: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). The CF was fully protective against Pst in Arabidopsis mutants jar1 and ein2 similar to wild-type plants. However, its efficacy was reduced in plants containing transgene NahG. Examination of systemic gene expression revealed that CF modulates the expression of SA-inducible PR-1, PR-2 and PR-5 genes, the JA/ET-inducible ChitB gene, and the ET-inducible Hel gene. Moreover, the expression of these genes was further enhanced upon subsequent stimulation after attack by Pst. Our data suggest that in addition to a partial requirement for SA, the signals JA and ET may also play a role in defense signaling in Arabidopsis.