Plasmodiophora brassicae: aspects of pathogenesis and resistance inBrassica oleracea

Summary Clubroot is one of the most damaging diseases inBrassica oleracea crops world-wide. The pathogenicity ofPlasmodiophora brassicae is highly variable between as well as within field populations. Several sources of resistance to clubroot have been identified inB. oleracea. Generally, resistance tends to inherit partly as a recessive, partly as an additive trait, and appears to be controlled by few major genes. Progress in the understanding of the inheritance of resistance is being made through the use of single-spore isolates of the pathogen, and the use of molecular markers for resistance genes.Abbreviations cv cultivar - DH doubled haploid - ECD European Clubroot Differential set - RFLP Restriction Fragment Length Polymorphism     

The exploitation of genetic resources of Brassica oleracea in breeding for resistance to clubroot (Plasmodiophora brassicae)

Summary About 1000 Brassica oleracea accessions were evaluated in glasshouse tests for response to Plasmodiophora brassicae (clubroot). Resistance was confirmed in some north and west European kales and cabbage. A new source of resistance in cabbage, from Eire, is reported. Most other accessions were highly susceptible but lower levels of susceptibility were observed in open pollinated Brussels sprouts and forms of south European cabbage, cauliflower and broccoli. Modern breeding (as in the production of hybrid cultivars) appears to have resulted in increased susceptibility in several crop types. The implications of these results for the exploitation of germplasm are discussed.     

Screening of Portuguese cole landraces (Brassica oleracea L.) with Peronospora parasitica and Plasmodiophora brassicae

Summary Forty-four landraces of Portuguese coles (Brassica oleracea L.), representing different morphotypes, were screened at the cotyledon stage for downy mildew resistance, caused by Peronospora parasitica. Two isolates of the pathogen, PHW 630 and PHW 828, were used in the experiment. The mean disease interaction phenotype varied among the landraces. Overall, the Portuguese coles have shown resistance to both isolates, particularly to isolate PHW 828. Some landraces were identified with differential reaction to the two P. parasitica isolates. Some of the Portuguese coles are, therefore, good sources of resistance genes to P. parasitica, and could be used in breeding programs or in the development of a downy mildew host differential set. Among the morphotypes tested, Couve Algarvia (ISA 207), Couve Galega (ISA 243 and 187) and Couve Glória de Portugal (ISA 84) have presented the highest resistance interaction. For the development of host differential sets, the landraces ISA 2 (Penca de Mirandela), ISA 35 (Couve de Valhascos) and ISA 600 (Penca Espanhola) could be further selected for the differentiation of P. parasitica isolates. They are resistant to PHW 828 and susceptible to PHW 643.The same morphotypes and landraces were also screened at the seedling stage for resistance to clubroot, caused by Plasmodiophora brassicae Woron.. Although some variation for resistance could be noticed in some the landraces tested, mainly Galega kale types, none of them presented a sufficient level of resistance as to be directly be useful in breeding for resistance to race 6 of P. brassicae.     

Selection for resistance to Plasmodiophora brassicae Wor. in oriental subspecies of Brassica rapa L.

Summary Selection for resistance to Plasmodiophora brassicae Wor. in oriental groups of Brassica rapa L.Two hundred and sixty-five cultivars of leafy, oriental bassicas were tested for resistance to 18 collections of Plasmodiophora brassicae, the causal agent of clubroot. The tests were conducted in the greenhouse at low and high level inoculum concentrations. Eleven cultivars of B. rapa pe-tsai, five cultivars of B. rapa pak-choy and three cultivars of B. rapa choy-sum consistently segregated for resistance at the lower concentration of inoculum (1000 spores/ml). All 265 cultivars were susceptible at the higher concentration (1 000 000 spores/ml). Three cultivars were used in pedigree and recurrent selection schemes for increased resistance. After three cycles of selfing resistant individuals, significantly more resistant S₃ lines were derived from each cultivar. Lines derived from two cultivars. Chinese White and PI 257236, continued to improve with each cycle of selection and demonstrated increased resistance to higher levels of inoculum (up to 1 000 000 spores/ml) New cultivars based on intercrosses of S₂ resistant individuals also had significantly better resistance than the original cultivar. After two cycles of selection in the third cultivar, PI 419007, resistance did not increase and its S₂ mass did not differ significantly from the original cultivar. Evidence that indicates resistance is pathotype-non-differential and offers an alternative to major gene, pathotype-differential types of resistance currently being introduced to the leafy oriental brassicas from other Brassica rapa groups.     

Association of RFLP markers with trait loci affecting clubroot resistance and morphological characters in Brassica oleracea L.

Summary Resistance to Plasmodiophora brassicae Wor. race 7, the causal agent of the disease clubroot, was examined in an F₂ population of a cross between a clubroot resistant broccoli (Brassica oleracea var. italica) and a susceptible cauliflower (B. oleracea var. botrytis). A genetic linkage map was constructed in the same population based on the segregation of 58 dispersed restriction fragment length polymorphism (RFLP) markers. Associations between the inheritance of RFLP marker genotypes and segregation for disease resistance, morphological and maturity characteristics were examined. For each triat examined, several chromosomal regions marked by RFLP probes appeared to contain trait loci, suggesting that each trait was under polygenic control. RFLP marker linkage to a major factor imparting dominance for clubroot resistance from the broccoli parent was observed in this population. Additionally, RFLP marker linkage to an independently segregating factor contributing clubroot resistance from the cauliflower parent was observed, indicating that it should be possible to use RFLP markers to facilitate selection of transgressive segregants having the combined resistance from both parental sources. In some instances, RFLP markers from the same or closely linked chromosomal regions were associated with both clubroot resistance and morphological traits. Analysis of RFLP marker genotypes at linked loci should facilitate the selection of desired disease resistant morphotypes.     

Transfer of resistance to race 2 of Plasmodiophora brassicae from Brassica napus to cabbage (B. oleracea ssp. capitata). III. First backcross and F2 progenies from interspecific hybrids between B. napus and B. oleracea ssp. capitata

Summary The first backcross and F₂ progenies from triploid F₁ and tetraploid F₁ hybrids between B. napus and 2x and 4x B. oleracea ssp. capitata (cabbage) were studied for their general morphology, resistance to race 2 of the clubroot pathogen, chromosome number and meiotic chromosome behavior. No linkage was apparent between resistance and the major morphological characters. Unreduced gametes played a large part in the successful formation of seed of the B₁ and F₂ progeny. B₁ plants with low chromosome numbers were selected for use in recurrent backcrosses. The potential use of anther culture to extract gametic progenies from resistant B₁ and F₂ plants with higher chromosome numbers was suggested. The presence of homoeologous pairing observed in all the plants is considered advantageous for selecting suitable progeny in later generations.     

Transfer of resistance to race 2 of Plasmodiophora Brassicae from Brassica napus to cabbage (B. oleracea var. Capitata). I. Interspecific hybridization between B. napus and B. oleracea var. Capitata

Summary Interspecific hybridization between Brassica napus L. (2n=38, a₁a₁c₁c₁) and B. oleracea var. capitata L. (2x- and 4x-cabbage; 2n=2x=18, cc and 2n=4x=36, cccc) was carried out for the purpose of transferring clubroot disease resistance from the amphidiploid species to cabbage. Nineteen hybrids with three different chromosome levels (2n=28, a₁c₁c; 2n=37, a₁c₁cc and 2n=55, a₁c₁cccc) were obtained. The F₁ plants were mostly intermediate between the two parents but as the number of c genomes in the hybrids increased, the more closely the hybrids resembled the cabbage parent. All F₁ hybrids were resistant when tested against race 2 of Plasmodiophora brassicae wor. The complete dominance of resistance over susceptibility suggested that the gene(s) controlling resistance to this particular race of the clubroot pathogen is probably located on a chromosome of the a genome in Brassica.Contribution No. J654.     

Transfer of resistance to race 2 of Plasmodiophora brassicae from Brassica napus to cabbage (B. oleracea var. capitata). II. Meiosis in the interspecific hybrids between B. napus and 2x and 4x cabbage

Summary Meiosis in 14 interspecific F₁ hybrids with three chromosomal levels (triploid, tetraploid, hexaploid; 2n=28, 37 and 55) between Brassica napus L. and 2x and 4x cabbage (B. oleracea var. capitata L.) was studied. The oleracea genome from B. napus maintained close homology with the c genome of cabbage while the campestris genome of B. napus showed partial homology with the c genome contained in the hybrids. Genotypic influence on chromosome pairing was indicated. Structural chromosome differences and spontaneous chromosome breakage and reunion were suggested as causes for the abnormalities which related to the unbalance of the genotypes. The divergence of the genomes of B. napus and B. oleracea and the need for the qualification of the term secondary association were discussed.Contribution No. J. 673, Research Station, Agriculture Canada, St. Jean, Québec.     

Transfer of resistance to race 2 of Plasmodiophora brassicae from Brassica napus to cabbage (B. Oleracea spp. Capitata). IV. A resistant 18-chromosome B1 plant and its B2 progenies

Summary The somatic karyotype and meiotic chromosome behavior were studied in an 18-chromosome B₁ plant derived from backcrossing a triploid (Brassica napus x B. oleracea ssp. capitata) F₁ hybrid to cabbage. It is considered that cabbage chromosomes no. 1 and no. 7 were substituted by two shorter B. napus chromosomes. Meiotic disturbances were more apparent during the late stages of second division. Seed fertility of this plant was largely restored in the second backcrosses with both cabbage and broccoli. 18-chromosome B₂ plants resistant to race 2 of Plasmodiophora brassicae were recovered among the progenies.Contribution no. J. 725 from the Research Station, Research Branch, Agriculture Canada, St-Jean, Québec J3B 6Z8.     

Identificaiton of a clubroot resistance locus conferring resistance to a Plasmodiophora brassicae classified into pathotype group 3 in Chinese cabbage (Brassica rapa L.)

In Chinese cabbage (Brassica rapa), the clubroot resistance (CR) genes Crr1 and Crr2 are effective against the mild Plasmodiophora brassicae isolate Ano-01 and the more virulent isolate Wakayama-01, but not against isolate No. 14, classified into pathotype group 3. ‘Akiriso’, a clubroot-resistant F₁ cultivar, showed resistance to isolate No. 14. To increase the durability of resistance, we attempted to identify the CR locus in ‘Akiriso’. CR in ‘Akiriso’ segregated as a single dominant gene and was linked to several molecular markers that were also linked to CRb, a CR locus from cultivar ‘CR Shinki’. We developed additional markers around CRb and constructed partial genetic maps of this region in ‘Akiriso’ and ‘CR Shinki’. The positions and order of markers in the genetic maps of the two cultivars were very similar. The segregation ratios for resistance to isolate No. 14 in F₂ populations derived from each of the two cultivars were also very similar. These results suggest that the CR locus in ‘Akiriso’ is CRb or a tightly linked locus. The newly developed markers in this study were more closely linked to CRb than previously reported markers and will be useful for marker-assisted selection of CRb in Chinese cabbage breeding.     

Genetic analysis of resistance to clubroot (Plasmodiophora brassicae Woron) in two Brassica oleracea groups (ssp. acephala and ssp. botrytis) through diallel analysis

Clubroot, caused by Plasmodiophora brassicae Woron, is a major disease of cruciferous crops and few sources of resistance have been detected and genetically studied in the Brassica oleracea species. In this study, two diallel mating systems using resistant kale lines and susceptible cauliflower lines were performed. Resistance was assessed from a disease index evaluated on young plants artificially inoculated with local isolates of the parasite. Both general and specific combining ability effects (GCA and SCA) and partly reciprocal effects were demonstrated. Resistance inheritance was, however, characterized by a predominance of additive genetic effects (GCA). Three groups of parent lines could be separated; kale lines with very high resistance, kale lines with intermediate resistance and susceptible cauliflower lines. In the two kale groups, two genetically different resistance types were suggested and various recurrent selection procedures are proposed following the diallel results.     

Disease response of resynthesized Brassica napus L. lines carrying different combinations of resistance to Plasmodiophora brassicae Wor.

Resistance responses of resynthesized Brassica napus lines to infection with Plasmodiophora brassicae were investigated. Lines that were derived from interspecific crosses between clubroot-resistant B. rapa and resistant B. oleracea exhibited very broad and effective resistance in both greenhouse and field tests. When clubroot resistance was introduced into resynthesized lines from the B. oleracea parent only, the plants were mainly susceptible. Interspecific hybrids from the most resistant parental genotypes, i.e. B. campestris ECD-04 and the B. oleracea cultivars ECD-15 or ‘Bohmerwaldkohf’, were used to initiate a B. napus resistance-breeding programme. These artificial rapeseed lines were resistant to isolates that were virulent on all B. napus differential lines and/or parental lines. Preliminary segregation analysis suggests that their resistance is due to at least two dominant and unlinked genes. In some cases progenies from selfed resynthesized plants exhibited resistance reactions that differed from those of the parental hybrid plant; this may have been the result of cytological instability.     

Resistance to Brevicoryne brassicae in horticultural brassicas

Summary A wide range of cultivated brassica accessions including broccoli, Brussels sprouts, Chinese cabbage, cauliflower, collard, kale and swede material was tested against the cabbage aphid, Brevicoryne brassicae, at HRI, Wellesbourne in the field and laboratory in both 1992 and 1993. In the field, B. brassicae attack was assessed as the proportion of infested plants and the numbers of aphid colonies present. In the laboratory, aphid performance was measured in terms of the pre-reproductive and reproductive period, population increase, and insect mortality. Interpretation of the data was facilitated by plotting sorted accession means against normal order statistics. This statistical approach indicated the spread of variation amongst the accessions and permitted identification of extremes. Partial levels of antixenosis resistance were discovered in red brassicas. Glossy accessions of cabbage and cauliflower possessed antixenosis and antibiosis resistance that lasted throughout the season of crop growth in the field. Other accessions were shown to withstand aphid attack and therefore possessed tolerance. The laboratory studies provided information on mechanisms of antibiosis resistance. The potential value of the different sources of resistance is discussed.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. I. Production of F1 hybrids through embryo rescue

Summary Triazine resistant Brassica napus ssp. oleifera and ssp. rapifera were hybridized to cultivars of B. oleracea ssp. italica, ssp. botrytis, ssp. capitata and ssp. fimbriata. The interspecific embryos did not survive in vivo but could be rescued in vitro using a culture medium developed by Monnier (1973). The embryos did not grow directly into normal plants but were successfully regenerated using the protocol developed by Keller (1984). Hybridization efficiency ranged from 0 to 2.64 hybrids per pollination. Interspecific embryo abortion may be related to abnormal endosperm development.     

Identification of S-alleles in Brassica oleracea

Summary S-alleles of Brassica oleracea were identified using a method which is based on the amplification of S-sequences from genomic DNA, followed by digestion of the PCR products with selected restriction enzymes (PCR-RFLP). A study was made in which the same S-allele was present in the homozygous state in a range of different crop types. This showed that, with minor exceptions, characteristic restriction patterns were obtained, and therefore that it was possible to identify the S-allele. To test whether the method was also suitable for the identification of both the S-alleles present in heterozygotes, a number of S-heterozygotes together with an F₂ population were screened. The results showed that the standard method was not very reliable for the identification of both of the S-alleles. This is because firstly, one of the S-alleles may be amplified preferentially, and secondly, the restriction patterns are not unique to a particular combination of S-alleles. Finally, although it is not possible to identify unequivocally both S-alleles of heterozygotes using a standard technique, the procedure can be modified for particular combinations of alleles to enable the identification to be made.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. II. Morphology,fertility and cytology of the F1 hybrid

Summary F₁ hybrids of triazine resistant Brassica napus and triazine susceptible B. oleracea were morphologically intermediate to the parent species. Of 49 hybrids examined, 44 had 28 chromosomes, two had 37, one had 38 and two had 56. The 38-chromosome plant was thought to be a matromorph, the others, A₁C₁C (28), A₁C₁CC (37) or A₁A₁C₁C₁CC (56) type hybrids. Pollen stainability averaged 9.0% in the sesquidiploid, 32.0% in the tetraploids and 89.5% in the hexaploids. All the interspecific hybrids were resistant to 1.0×10^-4 mol L^-1 atrazine. The sesquidiploid hybrids produced gametes with chromosome numbers ranging from 9 to 17 and the tetraploid hybrid gametes had chromosome numbers from 15 to 22. Most hybrids produced self-seed. The partial fertility of these hybrids may permit their backcrossing to one or both parents.     

Screening of Portuguese cole landraces (Brassica oleracea L.) with Leptosphaeria maculans and Xanthomonas campestris pv. campestris

Summary Fifty-six landraces of Portuguese coles (Brassica oleracea l.), representing fourteen morphotypes, were screened for resistance to Leptosphaeria maculans at the cotyledon stage. Three isolates of the pathogen belonging to three pathogenicity groups were used in the screening. The mean disease interaction phenotype of the B. oleracea landraces with the L. maculans isolates confirmed their differences in pathogenicity. When compared to pathogenicity grouping using a B. napus differential set, a different classification was observed on B. oleracea for the three L. maculans isolates, suggesting possible differences in the genetic interaction between the two plant species and the pathogen. Most of the Portuguese cole landraces were found to be susceptible to all three isolates of L. maculans, although a few have shown intermediate interaction phenotype. Some accessions, on the other hand, elicit a differential reaction with the three isolates tested, and can be further selected to be used in host differential sets or in breeding for resistance to black leg.Fifty-four landraces of Portuguese coles, representing eleven morphotypes, were also screened in the field for resistance to Xanthomonas campestris pv. campestris. A good level of resistance to the bacterium was found in several of the morphotypes.Accessions of the Penca type, particularly, were identified as highly resistant. Landraces belonging to the same Portuguese cole group presented a similar range of mean interaction phenotype with the bacterium. Some Portuguese landraces of B. oleracea are potential sources for use in breeding programs for black rot resistance.     

Brassica coenospecies: a rich reservoir for genetic resistance to leaf spot caused by Alternaria brassicae

Development of leaf spot resistant mustard cultivars is a relevant objective in view of heavy crop losses caused by this pathogen. Thirty-eight species belonging to 9 genera, including cultivated and wild allies, of the genus Brassica were evaluated under epiphytotic conditions for two years. Inoculations were done on whole plants (in vivo) and on detached leaves (in vitro). Data on incubation period, number of lesions per leaf, lesion size and leaf area covered by lesions were recorded. Species which never produced disease symptoms throughout the growing period in pots and until 72 hours after inoculation in detached leaf assays during both years were treated as resistant, while those that produced symptoms were classified as moderately resistant, susceptible or highly susceptible depending upon incubation period, size of lesions and leaf area covered by disease symptoms. Eight species (Brassica desnottesii, Camelina sativa, Coincya pseuderucastrum, Diplotaxis berthautii, D. catholica, D. cretacea, D. erucoides, and Erucastrum gallicum) were found completely resistant, whereas others were classified as moderately resistant (12), susceptible (11) or highly susceptible (9). Since resistance is unavailable within the cultivated species, these 8 resistant wild species could be used as donor parents for introgressing resistance to leaf spot disease in Indian mustard. This revised version was published online in August 2006 with corrections to the Cover Date.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. III. First backcross to parental species

Summary Atrazine resistant Brassica napus × B. oleracea F₁ hybrids were backcrossed to both parental species. The backcrosses to B. napus produced seeds in both directions but results were much better when the F₁ hybrid was the pollen parent. Backcrosses to B. oleracea failed completely but BC₁s were rescued by embryo culture both from a tetraploid hybrid (2n = 4x = 37; A₁C₁CC) and sesquidiploid hybrids (2n = 3x = 8; A₁C₁C). Progeny of crosses between the tetraploid hybrid and B. oleracea had between 25 and 28 chromosomes. That of crosses between the sesquidiploid hybrid and B. oleracea had between 21 and 27. A few plants that had chromosome counts outside the expected range may have originated from either diploid parthenogenesis, unreduced gametes or spontaneous chromosome doubling during in vitro culture. Pollen stainability of the BC₁s ranged from 0% to 91.5%. All the BC₁s to B. oleracea were resistant to atrazine.     

Plant traits associated with resistance to Thrips tabaci in cabbage (Brassica oleracea var capitata)

Thrips tabaci is a major problem in the cultivation of cabbage for storage, as this pest causes symptoms that necessitate the removal of affected leaves from the product. Between cabbage varieties large differences in susceptibility occur. This study aimed to identify plant traits associated with these differences, in field experiments with natural infestation in 2005 and 2006. One factor affecting the amount of thrips damage was the timing of the development of the head. In an experiment with different planting dates especially the early maturing, more susceptible varieties were shown to benefit from later planting. In comparisons of multiple varieties in both years, regression studies showed that more advanced plant development in August and early September increased thrips damage at the final harvest. However, no single plant trait explained more than 25% (2005, Brix) or 48% (2006, compactness) of the variation in thrips damage. Optimal regression models, explaining up to 75% of the variation in thrips damage included Brix and leaf surface wax late in the season, as well as an indicator of plant development earlier in the season, and in 2005 also leaf thickness. The possible role of these plant traits in relation to thrips is discussed.