**3. Biology of the pathogen and epidemiology of the** *L. maculans*

The pathogen can infect several crucifers, including cruciferous weeds. Up to 28 crucifer species have been reported as hosts [14]. During infection, the pathogen grows systemically down towards the tap root of the plant, producing severe disease symptoms at the adult plant stage characterised by stem cankers. *L. maculans* reproduces both asexually and sexually on host species and can complete several disease cycles during a single growing season. The fungus survives as mycelium, pycnidia and pseudothecia on crop residues, mainly on stubble [15, 16] subsisting from one season to the next. Sexual mating occurs on crop residues, resulting in the production of ascospores which can travel up to 8 km [17]. High humidity and moderate temperatures during vegetative growth promote disease development [18].

In Australia and most parts of Europe, *L. maculans* infection generally occurs during the seedling stage from infected seed and wind-dispersed ascospores (sexual spores), released from pseudothecia. In western Canada and Poland, asexual pycnidospores are the primary source of inoculum [19], dispersed largely by rain-splash. Under high humidity conditions, ascospores and pycnidiospores adhere to cotyledons or young leaves and germinate to produce hyphae which penetrate through stomata and wounds [9, 20, 21] and grow into substomatal cavities without forming appressoria [22]. After entering into substomatal cavities, the fungus grows between the epidermis and palisade layer and then into intercellular spaces in the mesophyll of lamina. The fungus then reaches the vascular strands and grows within the plant asymptomatically, until eventually invading and killing cells of the stem cortex and causing the stem canker symptom [22-24]. Variability for virulence in *L. maculans* for the first time was reported in 1927 [25]. Australian populations of *L. maculans* have a high level of genetic variability as compared to European and North American isolates [26], along with a high diversity of avirulence genes [27]. Molecular analyses of populations of *L. maculans* have shown high gene flow within and between populations. Isolates of *L. maculans* are usually classified either on the basis of their aggressiveness or into pathogenicity groups [28].

#### **4. Management of the** *L. maculans*

Various practices such as crop rotation, stubble management, time of sowing, seed dressing and foliar application of fungicide, and deployment of genetic resistance have been employed to control this disease and subsequently reduce yield losses [9, 29]. Deployment of host resistance has been used as the most cost-effective and environmentally sound measure for disease control in various crops including in rapeseed. This strategy has been extensively used to manage blackleg disease especially in Australia, Canada, France, and Germany.

## **5. Evaluation of germplasm for** *L. maculans* **resistance**

**2. Symptoms**

shatter and seed infection.

86 Plant Breeding from Laboratories to Fields

Blackleg disease causes two distinct symptoms; leaf lesions and stem canker. Outbreak of the fungus is characterised by dirty-whitish spots on leaves with small dark fruiting bodies (pycnidia). Black lesions are generally also seen on the leaves and deep brown lesions with a dark margin can be seen on the base of stem [11]. In severe epidemic conditions fungus girdles the stem at the crown, leading to lodging of the plant and possible severance of the stem. Typical lesions of blackleg can also occur on pods. Pod infection may leads to premature pod

The pathogen can infect several crucifers, including cruciferous weeds. Up to 28 crucifer species have been reported as hosts [14]. During infection, the pathogen grows systemically down towards the tap root of the plant, producing severe disease symptoms at the adult plant stage characterised by stem cankers. *L. maculans* reproduces both asexually and sexually on host species and can complete several disease cycles during a single growing season. The fungus survives as mycelium, pycnidia and pseudothecia on crop residues, mainly on stubble [15, 16] subsisting from one season to the next. Sexual mating occurs on crop residues, resulting in the production of ascospores which can travel up to 8 km [17]. High humidity and moderate

In Australia and most parts of Europe, *L. maculans* infection generally occurs during the seedling stage from infected seed and wind-dispersed ascospores (sexual spores), released from pseudothecia. In western Canada and Poland, asexual pycnidospores are the primary source of inoculum [19], dispersed largely by rain-splash. Under high humidity conditions, ascospores and pycnidiospores adhere to cotyledons or young leaves and germinate to produce hyphae which penetrate through stomata and wounds [9, 20, 21] and grow into substomatal cavities without forming appressoria [22]. After entering into substomatal cavities, the fungus grows between the epidermis and palisade layer and then into intercellular spaces in the mesophyll of lamina. The fungus then reaches the vascular strands and grows within the plant asymptomatically, until eventually invading and killing cells of the stem cortex and causing the stem canker symptom [22-24]. Variability for virulence in *L. maculans* for the first time was reported in 1927 [25]. Australian populations of *L. maculans* have a high level of genetic variability as compared to European and North American isolates [26], along with a high diversity of avirulence genes [27]. Molecular analyses of populations of *L. maculans* have shown high gene flow within and between populations. Isolates of *L. maculans* are usually classified either on the basis of their aggressiveness or into pathogenicity groups [28].

Various practices such as crop rotation, stubble management, time of sowing, seed dressing and foliar application of fungicide, and deployment of genetic resistance have been employed

**3. Biology of the pathogen and epidemiology of the** *L. maculans*

temperatures during vegetative growth promote disease development [18].

**4. Management of the** *L. maculans*

An efficient and reliable method for phenotyping resistance to *L. maculans* is required for germplasm evaluation and predictive breeding including molecular mapping and gene cloning research. Various criteria are used to assess disease severity, such as severity of cotyledon or stem canker lesions, which rely principally on scales or estimates of the percent of diseased leaf tissue at either seedling (intact and detached leaf) or at adult plant stages. Symptom expression can vary with the environmental conditions, test locations (glasshouse, environment chamber and field conditions), and the method of inoculations (cotyledon, leaf and stem).

Resistance of *B. napus* germplasm to *L. maculans* is tested on the basis of disease reaction under glasshouse and/or field conditions. Cotyledon inoculations, performed under controlled conditions in either a growth chamber or glasshouse, allow for large scale and efficient screening of germplasm. Various environmental conditions such as temperature, light intensity and humidity can be reliably controlled, expediting the development of suitable resistant cultivars [30] as selections can be performed at early stages of plant development. This method also overcomes some of the uncertainties inherent in field testing with its dependence upon growing environment and further reduce the genotype by environment (G x E) interactions. Growth conditions are typically maintained with at 18° C to and 22° C. For uniform infection, a spore suspension is used to inoculate wounded cotyledons of 7 to 15 dayold seedlings [31-33]. Alternatively, seedlings can be sprayed with a spore suspension at up to the third leaf stage and kept at 100 % humidity for 48-72 hr. Spore suspensions of *L. maculans* are generally raised from single-spore isolate cultures grown on different media such as V8-agar, malt-agar and rapeseed leaf extract-agar [21, 22, 34]. Published studies used spore concentrations in the range from 4 x 106 to 1x108 spores per ml [31 - 33].

Doubled haploid (DH) populations were screened for resistance to *L. maculans* in the glass‐ house at three plant growth stages: cotyledon, true leaf and adult plant, as well as under field conditions and reported a high correlation (r≥0.82) for disease severity between glasshouse and field grown lines [33]. Similar observations were also made by McNabb et al [35]. High correlation coefficient values suggest that the resistance to *L. maculans* can be evaluated at all three stages [33]. However among three stages, cotyledon stage was the most promising as inoculum-droplets can be kept at the inoculation site as compared to true leaves.

Assessment of adult plants for resistance to *L. maculans* populations under field conditions is considered very important for the selection of resistant germplasm by the rapeseed breeders. Inoculum is provided by either spreading infected stubble in a disease nursery or spraying plants with fungal spore suspension. Two measures; disease severity and disease incidence are commonly used for evaluating resistance to *L. maculans*. However, disease severity is much more difficult to estimate than disease incidence, due to the G x E interactions and unreliable and inconsistent estimation of canker lesions, even within the same genotype, particularly when infection is not uniform. The use of increased sample size (25 to 50 plants/genotype) and reliable and congenial growing conditions for the disease development will allow better estimation of canker lesions.

Assessment of blackleg resistance under field conditions is usually performed by exposing the plants to a mixed population of *L. maculans* races, which can make the detection of racespecific *R*-genes difficult. No relationship between the degree of cotyledon-lesion develop‐ ment at the seedling stage and crown canker development in mature plants was observed in the intercross population derived from Maluka/Niklas [36]. This study concluded the limited value of the cotyledon test in screening for adult plant blackleg resistance. Similarly a lack of correlationbetweencotyledon(seedling)resistance andstem(adultplant)resistance in*B. napus* andBgenome sourceshas alsobeenreported[37].Recently, apoor correlationbetweenseedling and field reactions was reported in the DH from Skipton/Ag-Spectrum which could have been due to the prevalence of different pathotypes under field conditions as contrary to cotyledon test, where often a specific isolate is used for phenotyping [32]. In order to mimic field condi‐ tions and increase reliability of disease development, an ascospore shower test [38] has been usedforgermplasmevaluationandvarietalrelease inAustralia.Inthis test, stubblewithmature pseudothecia is sprayed with distilled water until run-off, producing 'ascospore shower'. The infected plants can then be assessed for resistance at both the cotyledon and adult plant stages. Thismethodhas shownahighcorrelationwithcankerlesions scoredunderfieldconditions [39].

#### **6. Natural genetic variation for resistance to** *L. maculans*

The introgression of blackleg resistance (*R*) genes into *B. napus* germplasm for blackleg disease management is one of the major objectives of breeding programs aiming to release cultivars in disease-prone areas. Genetic variation for resistance to *L. maculans* exists within *B. napus* germplasm [39, 40, 41]. Some other Brassica species such as *B. rapa*, *B. juncea*, *B. nigra* (black mustard; 2n = 2x = 16, genome BB) and *B. carinata* (Abyssinian or Ethiopian mustard; 2n = 34, genome BBCC), as well as other crucifers such as *Sinapis arvensis* have been reported to carry resistance [42-53]. Some of these sources were utilised in transferring resistance into *B. napus* breeding lines and cultivars. A continuous variation for blackleg resistance in a world-wide collection of *B. rapa* genotypes was reported [54]. None of genotypes were completely suscep‐ tible or completely resistant to either *L. maculans* pathotypes used. However, some *B. rapa* accessions that were either highly resistant or completely susceptible were identified (Raman et al., unpublished) in a set of differential cultivars currently being used in Australia [39].

It has been reported that all B genome Brassica species; *B. nigra, B. carinata* and *B. juncea* carry complete resistance to *L. maculans* which remains effective throughout the life of the plant [40], however susceptible *B. juncea* cultivars have also been identified [55] demonstrating that complete resistance is not a feature of all B genomes. Some B genome resistance genes have been introgressed into *B. napus* lines. [47, 56-59]. Earlier studies have shown that C genome species of the *Brassica* are susceptible to blackleg [50, 53, 60]. However, a recent study [61] evaluated three accessions of *B. oleracea* var. *virids*, collected from the USDA germplasm collection and found that the accession NSL6146 was moderately resistant to *L. maculans*.

Genetic resources for adult plant resistance are very limited and most of them are derived from the French cultivar Jet Neuf [62]. Efforts are currently being made to identify both qualitative and quantitative resistance in the Australian Brassica Germplasm Improvement Programs.
