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Extensive efforts are being made to develop oilseed rape cultivars with resistance to Verticillium wilt. Evaluation of these cultivars involves either dipping roots of young plants in a conidial suspension followed by replanting in soil, or sowing the seed directly in artificially infested soil. These methods for screening for resistance take a long time and are expensive. The objective of this research was to develop a rapid and reliable method of screening cultivars for resistance to Verticillium wilt. A box test method in which 4-day-old seedlings were inoculated and grown under sterile conditions in double magenta boxes was developed. The Brassica napus cultivars Accord, Express, Hanna, and Libraska were inoculated with isolates of Verticillium longisporum or V. dahliae. The results from the box test method were compared with root-dip inoculation of seedlings grown in a soil mixture under greenhouse conditions. In addition, plants from the box test were randomly selected and transferred to pots containing sterile soil to permit comparison of plants from the box test at similar developmental stage with plants in the root-dip method. Disease evaluation was based on the number of days after transferring seedlings into the boxes that root browning, root and shoot blackening, and wilting of cotyledons developed. Additional disease evaluation was based on a 0 to 5 disease severity scale based on the percentage of wilted leaves (number) and/or the percentage of stunting (mm) of inoculated plants compared with noninoculated plants. Disease severity was higher on Hanna than on Express, and isolates of V. longisporum were more virulent than isolates of V. dahliae. There was a significant correlation (r = 0.93) of disease rating between the traditional root-dip method and the new box test method. Analysis of variance revealed that the interaction between cultivars and isolates was highly significant (P ≤ 0.01), whereas method of inoculation did not affect the results. In the box test, plants were rated after 25 days, which is at least 3.5 times more rapid than the root-dip method.

Plasmodiophora brassicae causes clubroot on cruciferous plants and causes worldwide huge economical losses on important Brassica crops. P. brassicae infection produces large root galls, the clubroots, which can also affect the upper plant parts by reduced water and nutrient uptake and redirection of assimilates from leaves to roots. P. brassicae is an obligate biotrophic protist in the plasmodiophorids within the eukaryote supergroup of Rhizaria and is unrelated to other known plant pathogens. Plasmodiophorids can be parasites of plants and oomycetes. The recently released genome of P. brassicae is only the third in the poorly studied Rhizaria and the first plant pathogenic genome of this eukaryotic group. The P. brassicae genome was estimated to be 25.5 Mb in size and predicted to contain 9730 gene models. A transcriptome of P. brassicae and Spongospora subterranea, the potato scab pathogen was also presented. Consequently, for the first time large scale data for a eukaryotic plant pathogen group outside the fungi and oomycetes are now available. This review highlights selected characteristics of the P. brassicae genome including molecular events shown or predicted to take place in each phase of its life-cycle, such as manipulation of: 1) host primary metabolism, 2) plant hormone homeostasis, and 3) plant defense. Further, future directions and challenges in the P. brassicae and plasmodiophorid genomic research are discussed.

In order to understand the underlaying causes of new severe turcicum leaf blight outbreaks in East Africa, a survey was undertaken in Uganda to examine the sorghum—Setosphaeria turcica interaction in terms of disease severity and incidence, the overall fungal population structure, and new resistant resources. Highest disease severities were recorded on caudatum accessions, whereas kafir genotypes were most resistant. The disease was more severe in the most humid farmlands compared to moderately dry agro-ecologies. In districts with wide adoption of the Epuripur variety a very high incidence (100%) of turcicum leaf blight was found. The two S. turcica mating type genes MAT1-1 and MAT1-2 assessed on fungal isolates deriving from both sorghum and maize diseased leaves were found in 20 of 23 districts sampled and in equal proportions. Upon cross inoculation on maize differential lines, four S. turcica isolates were identified as race 1, two as race 2, and one isolate corresponded to race 0 and race 3, respectively. The remaining 10 S. turcica isolates did not cause any disease symptoms on the maize lines assessed. Highly resistant accessions originating from a regional collection were found among the five sorghum races (kafir, guinea, caudatum, bicolor and durra), and are now implemented in new sorghum disease resistance programs.

Gene silencing may have a direct or indirect impact on many biological processes in eukaryotic cells, and is a useful tool for the determination of the roles of specific genes. In this article, we report silencing in Phytophthora infestans, an oomycete pathogen of potato and tomato. Gene silencing is known to occur in P. infestans, but its genetic basis has yet to be determined. Genes encoding the major components of the RNA interference (RNAi) pathway, Dicer-like (Pidcl1), Argonaute (Piago1-5) and RNA-directed RNA polymerase (Pirdr1), were identified in the P. infestans genome by comparative genomics, together with families of other genes potentially involved in gene silencing, such as histone deacetylases, histone methyltransferases, DEAD helicases, chromodomain proteins and a class 1 RNaseIII. Real-time reverse transcription-polymerase chain reaction demonstrated transcript accumulation for all candidate genes throughout the asexual lifecycle and plant infection, but at different levels of mRNA abundance. A functional assay was developed in which silencing of the sporulation-associated Picdc14 gene was released by the treatment of protoplasts with in vitro-synthesized double-stranded RNAs homologous to Pidcl1, Piago1/2 and histone deacetylase Pihda1. These results suggest that the components of gene silencing, namely Dicer-like, Argonaute and histone deacetylase, are functional in P. infestans. Our data demonstrate that this oomycete possesses canonical gene silencing pathways similar to those of other eukaryotes.

A collection of Mycosphaerella fijiensis, in total 71 isolates from 11 countries, of which 54 isolates were derived from the Ugandan Lake Victoria region, were analysed with regard to molecular markers and benomyl resistance to characterize the population structure. Forty-seven alleles from seven restriction fragment length polymorphism (PCR-RFLP) and five microsatellite markers were scored and utilized in the study. Using analysis of molecular variance (AMOVA), no significant genetic differentiation could be found between isolates from different leaves compared to isolates from a single leaf (P = 0.07). Exact tests of the allelic frequencies revealed no differences between isolates from different districts or between single-leaf-derived isolates compared to isolates derived from different leaves (P > 0.05 in all cases except two, which, however, were shown to be of type-I error). Resistance to benomyl, analysed both by PCR and fungicide screening, revealed that all isolates were susceptible. Compiled information from the various datasets implied that the Ugandan M. fijiensis population in the Lake Victoria Basin constitutes a homogenous population, probably as the result of a recent founder event.

Spot blotch, caused by Bipolaris sorokiniana, has become an increasingly important disease on barley in Sweden. In order to enhance our knowledge of this disease, eight Swedish B. sorokiniana isolates were assessed for their virulence pathotype on 30 barley genotypes including eight lines of mutant origin, designated B. sorokiniana tolerant (bst). A leaf screen assay revealed the presence of virulence groups 1 and 7 in Sweden. Several mutant genotypes demonstrated enhanced resistance responses (P = 0.0001), of which bstl:3 had the lowest leaf scoring value to the most virulent isolate, Swe2 (virulence group 7). To evaluate the host root responses, two contrasting isolates Swe2 and Swe4 (virulence group 7 and 1) were used to inoculate a set of eight differential lines, including four promising mutant lines from the leaf screen. Mutant bstl:6 was significantly less susceptible (P = 0.001) compared with the other barley genotypes tested, independent of the two isolates used. Mutants bst1, bst1:3 and bstl:4, which displayed less or no spots, are also considered promising genotypes for further breeding attempts.

Abscisic acid (ABA) is a defense hormone with influence on callose-dependent and -independent resistance against Leptosphaeria maculans acting in the RLM1Col pathway. ABA-deficient and -insensitive mutants in Ler-0 background (aba1-3 and abi1-1) displayed susceptibility to L. maculans, along with a significantly decreased level of callose depositions, whereas abi2-1 and abi3-1 remained resistant, together with the abi5-1 mutant of Ws-0 background. Suppressor mutants of abi1-1 confirmed that the L. maculans-susceptible response was due to the dominant negative nature of the abi1-1 mutant. Highly induced camalexin levels made ABA mutants in Col-0 background (aba2-1, aba3-1, and abi4-1) appear resistant, but displayed enhanced susceptibility as double mutants with pad3-1, impaired in camalexin biosynthesis. β-Aminobutyric acid (BABA) pretreatment of Ler-0 contributed to an elevated level of endogenous ABA after L. maculans inoculation. Comparisons between (RLM1Col)pad3 and rlm1Lerpad3 showed that ABA and BABA enhancement of callose deposition requires induction from RLM1Col·ABI1, but not ABI2, was found to be involved in a feedback mechanism that modulates RLM1Col expression. Genetic analysis showed further that this feedback occurs upstream of ABI4 and that components downstream of ABI4 modulate ABI1 activity. ABA and BABA treatments of the L. maculans-susceptible callose synthase mutant pmr4 showed that ABA also induces a callose-independent resistance. Similar treatments enhanced callose depositions and induced resistance to L. maculans in oilseed rape, and BABA-induced resistance was found to be independent of salicylic acid.

The responses of Arabidopsis accessions and characterized genotypes were used to explore components in the early defense responses to the soilborne fungus Verticillium longisporum. V. longisporum susceptibility was found to be a complex trait, in which different disease phenotypes, such as stunting, altered flowering time, weight loss, and chlorosis were perceived differently across genotypes. A Bay-0 × Shahdara recombinant inbred line population was used to identify two loci on chromosomes 2 and 3 of Bay-0 origin that caused enhanced chlorosis after V. longisporum challenge. Furthermore, the observation that a mutation in RFO1 in Col-0 resulted in susceptibility whereas the natural rfo1 allele in Ty-0 showed a high degree of resistance to the pathogen supports the hypothesis that several resistance quantitative trait loci reside among Arabidopsis accessions. Analysis of mutants impaired in known pathogen response pathways revealed an enhanced susceptibility in ein2-1, ein4-1, ein6-1, esa1-1, and pad1-1, but not in other jasmonic acid (JA)-, ethylene (ET)-, or camalexin-deficient mutants, suggesting that V. longisporum resistance is regulated via a hitherto unknown JA- and ET-associated pathway. Pretreatments with the ET precursor 1-aminocyclo-propane-1-carboxylic acid (ACC) or methyl jasmonate (MeJA) caused enhanced resistance to V. longisporum. Mutants in the salicylic acid (SA) pathway (eds1-1, NahG, npr1-3, pad4-1, and sid2-1) did not show enhanced susceptibility to V. longisporum. In contrast, the more severe npr1-1 allele displayed enhanced V. longisporum susceptibility and decreased responses to ACC or MeJA pretreatments. This shows that cytosolic NPR1, in addition to SA responses, is required for JA- and ET-mediated V. longisporum resistance. Expression of the SA-dependent PR-1 and PR-2 and the ET-dependent PR-4 were increased 7 days postinoculation with V. longisporum. This indicates increased levels of SA and ET in response to V. longisporum inoculation. The R-gene signaling mutant ndr1-1 was found to be susceptible to V. longisporum, which could be complemented by ACC or MeJA pretreatments, in contrast to the rfo1 T-DNA mutant, which remained susceptible, suggesting that RFO1 (Fusarium oxysporum resistance) and NDR1 (nonrace specific disease resistance 1) activate two distinct signaling pathways for V. longisporum resistance.

Verticillium wilt of oilseed rape (Brassica napus) is caused primarily by Verticillium longisporum and has become a serious problem in northern Europe. In order to evaluate whether V. longisporum and V. dahliae differ in their interaction with oilseed rape, phenotypical and molecular assessments were made. Oilseed rape plants for fungal assessments were inoculated with V. longisporum and V. dahliae via root-dipping and samples were taken from roots, stems, leaves, flowers, pods and seeds during plant development. The infection by V. longisporum was found to start mainly in lateral roots and root-hairs, followed by colonization of the xylem vessels and extensive spread in stems and leaves, whereas V. dahliae infected the main roots and remained in the region below the cotyledon node of the plants. Re-isolation studies, together with PCR analysis of samples taken from early growth stages through to fully ripe plants, showed that the onset of flowering was a critical phase for V. longisporum to colonize plants. No seeds infected with V. longisporum were found. Mycelial growth from V. dahliae but not V. longisporum was significantly reduced on media containing tissue from a low glucosinolate B. napus genotype compared with growth on media containing tissue from a high glucosinolate cultivar. The results of this study suggest that V. longisporum favours B. napus as host and that the transition from the vegetative to the generative phase is of importance for the spread of the fungus in oilseed rape plants.

Verticillium longisporum is a soil-borne fungal pathogen causing vascular wilt of Brassica crops. This study was conducted to enhance our knowledge on the host range of V. longisporum. Seven crop species (barley, oat, oilseed rape, pea, red clover, sugar beet and wheat) and five weed species (barren brome, black-grass, charlock, cleavers and scentless mayweed) all common in southern Sweden were evaluated for infection by response to V. longisporum. Oat, spring wheat, oilseed rape, scentless mayweed and charlock inoculated with V. longisporum in a greenhouse showed stunting to various degrees close to the fully ripe stage. Based on the extent of microsclerotia formation, explants were separated into four groups: for pea and wheat, 80%. The results suggest that plant species outside the Brassicaceae can act as reservoirs of V. longisporum inoculum. Soil inoculum densities in nine fields were monitored over a period of 12 months, which ranged from 1 to 48 cfu g−1 soil. Density of microsclerotia was lowest just after harvest, reaching its maximum six months later. No significant correlation between inoculum density in soil and disease incidence on oilseed rape plants was found. However, the data suggest that a threshold of 1 cfu g−1 soil is needed to cause disease on oilseed rape. Species identification based on microsclerotia morphology and PCR analysis showed that V. longisporum dominated in soil of seven, and V. dahliae in two of the nine fields studied.

Grey leaf spot incited by Cercospora zeae-maydis is a new devastating foliar disease of maize in East Africa. For effective control, elucidation of the most critical elements of the grey leaf spot disease pyramid is important. This study investigated the role of mineral nutrition, pathogen variability and host resistance in the epidemic. Trials were conducted under field and controlled environments. The 28 isolates used in the controlled environment varied significantly (P ≤ 0.05) in parasitic fitness measured indirectly as disease efficiency, but no infection pattern could be attributed to known C. zeae-maydis pathotypes. Data from field trials showed that host resistance and mineral nutrition significantly (P ≤ 0.05) affected disease efficiency, with highest disease development occurring in nitrogen-augmented plots. Exclusive phosphorus application had no clear effect on grey leaf spot epidemics but combined application with nitrogen significantly (P ≤ 0.05) reduced the predisposition effects of nitrogen to the disease. Overall, treated plots had less disease than unfertilised plots. Fertiliser application had no effect on sporulation capacity, while cultivars significantly affected it. Geographic differences in amount of disease were observed, suggesting environment influences on grey leaf spot incidence. The results suggest that the current grey leaf spot epidemics in East Africa are due to favourable cultivars, poor mineral nutrition and environmental interactions.

Using a polymerase chain reaction-based strategy, we have isolated a cDNA sequence from Brassica nigra, denoted Lm1, which significantly improves blackleg resistance when expressed in transgenic oilseed rape, B. napus. Lm1 was shown to map to locations on linkage groups 3 and 8 in the Brassica B-genome previously linked to both cotyledon, adult leaf, and stem resistance. B. napus plants transformed with Lm1 under the control of a constitutive promoter exhibited broad spectrum resistance to all L. maculans isolates tested, but enhanced resistance to Alternaria brassicae and Verticillium longisporum was not observed. A transcript corresponding to the cDNA size was induced in B. nigra 12 h after fungal challenge from a level of weak constitutive expression previous to inoculation. The Lm1 sequence bears no resemblance to previously characterized plant resistance genes but has two predicted transmembrane motifs. Several sequences with high homology to Lm1 were found in the databases. Lm1 appears to be a member of a larger group of related genes present in a variety of plant species. Most of them have unknown functions, but homology between Lm1 and the nodule inception gene of Lotus japonicus suggests an intriguing link between defense-related and symbiotic pathways.

Two hundred and ninety-nine accessions representing 11 cultivar groups of Brassica oleracea and eight additional accessions of the wild species B. cretica, B. incana, B. insularis and B. villosa were screened for resistance to Verticillium wilt. A disease index (DI) was calculated for each accession, and a correction of the DI was carried out to compensate for a fluctuating infection level between 11 independent trials. A total of 235, or 77% of the accessions tested, had a DIcorr less or equal to the oilseed rape cv. ‘Express’ (DIcorr= 2.81), the reference cultivar. Only one accession of the wild species, B. incana, showed an enhanced level of resistance (DIcorr= 2.01). Twenty-four accessions, distributed over eight cultivar groups of B. oleracea were selected for subsequent crosses involving B. rapa. Hybrid plants with 14 accessions were resultant and seed was obtained from crosses where the cultivar groups acephala, alboglabra, botrytis, capitata, gemmifera, italica and sabellica were used as female parents. When progeny of the produced resynthesized rapeseed lines were evaluated for Verticillium wilt resistance, three lines showed a significantly lower disease index (P ≥ 0.01) compared with the cv. ‘Express’. This source of resistance is now being crossed to advanced breeding material of oilseed rape.

Amplified fragment length polymorphism (AFLP) analysis was used to study genetic variation among 76 isolates of Verticillium. A dendrogram based on the AFLP data revealed three main groups. One group consisted of 35 European isolates derived from Brassica napus together with five Californian isolates taken from B. oleracea. This group displayed a high degree of genetic similarity and included three isolates earlier classified as Verticillium longisporum, indicating that all isolates in this group probably should be regarded as members of V. longisporum. V. dahliae isolates constituted the second group while the third group contained four V. albo-atrum isolates. In addition to these three groups, a cluster of six V. nigrescens isolates was observed. However, the genetic distances between the isolates of V. nigrescens were much higher than those between members in the other groups and the bootstrap value for the V. nigrescens cluster was subsequently low. Four isolates classified as V. tricorpus were highly diverse and did not cluster together. Analysis of molecular variance revealed that the isolates of V. longisporum were separated into four subgroups, based on geographic origin. The study furthermore shows that AFLP is a suitable method for studying population structure in Verticillium.

Stem canker (blackleg) caused by Leptosphaeria maculans is a widespread disease of Brassica napus. In contrast, most Arabidopsis thaliana accessions are highly resistant. Hence, novel material derived from symmetric and asymmetric somatic hybrids between B. napus and A. thaliana was utilised in a screen for L. maculans resistance. Initially, both cotyledon and adult-leaf resistance traits were transferred from A. thaliana to B. napus. In later generations the two traits segregated and cotyledon resistance was lost. The adult-leaf resistance was investigated with respect to genome localisation and protein expression. Analyses of remaining A. thaliana DNA in resistant plants showed co-segregation between adult-leaf resistance and chromosome-3 molecular markers. Resistant offspring from asymmetric hybrid plants that contained fragments of chromosome 3 were studied in more detail. Two regions at positions 9.8-10.4 Mbp and 18-19.5 Mbp, where several defence-related genes are located, were identified. A proteomic approach was taken to further investigate genes involved in the defence interaction. Forty eight hours after inoculation with L. maculans, only a few proteins, such as glycolate oxidase, were identified as differentially expressed in the resistant line compared to B. napus, despite the presence of additional A. thaliana chromosomes. The plant materials described in the present study constitute a new genetic source of L. maculans resistance and are currently being incorporated into B. napus breeding programmes.

Somatic hybrids between Brassica napus (oilseed rape) and its wild relative Sinapis arvensis (Xinjiang wild mustard) from northwestern China were produced by fusing mesophyll protoplasts. Fifty-four plants were identified as symmetric hybrids and four as asymmetric hybrids by random amplified polymorphic DNA analysis and nuclear DNA content. The morphology of investigated 58 hybrid plants resembled characters from both parental species. Highly fertile hybrids were recovered where the fertility was associated with the choice of B. napus genotype. Enhanced disease resistance to Leptosphaeria maculans was found in S. arvensis and in the hybrid offspring. This plant material has great potentials not only for use as a bridge for the introduction of a number of valuable traits from the wild species to Brassica crops but also for breeding new varieties with improved blackleg resistance.

Leptosphaeria maculans isolates have been assigned to one of two groups, A or B, on the basis of differences in their characteristics. Group A can further be divided into pathogenicity groups (PG) 2, 3 and 4 and group B into PG1. To determine if isolates belonging to the aggressive canker forming group A are present in Sweden, physiological and genetic characterisation of 120 isolates collected in the year 2000 were performed. Thirty-seven isolates were classified as belonging to pathogenicity group PG3 and 63 isolates as PG4, based on a cotyledon assay. Twenty isolates did not cause any symptoms at all, and were classified as PG1. When comparing two geographical regions, Skane and Ostergotland, equal numbers of PG3 and PG4 isolates were found. By analysing the isolates by PCR, the collection was further classified into 100 group A isolates and 20 group B isolates. A corresponding classification of the isolates was observed when the ability to produce pigments in Czapek Dox broth was examined. The results showed a clear predominance of group A. This was also the case for the isolate collection from 2001. In a detailed survey of disease development in a L. maculans infected winter oilseed rape field in southern Sweden (Skane), basal stem canker was not observed until early June Although the disease index value increased from 8.4 in June to 18.0 in July, few severely damaged plants were observed before harvest in mid-July, despite infection with group A isolates.

Eight isolates of Verticillium longisporum and seven of Verticillium dahliae were examined for their pathogenicity to Arabidopsis thaliana. Two-week-old seedlings of two A. thaliana ecotypes were inoculated with conidial suspensions (1.6 × 10 4 conidia/ml) and maintained in sterile conditions on microtitre plates for 25 days. The seedlings were assessed daily for flowering time, height at flowering and mortality. From this data the proportion of plants flowering was calculated and the susceptibility of each ecotype to individual isolate was quantified. The A. thaliana ecotypes were inoculated by several isolates of V. dahliae and V. longisporum with the disease symptoms observed being similar to those characteristic of Verticillium-infected Brassica napus. Responses to Verticillium isolates varied and for both ecotypes significant differences (P < 0.05) were recorded in days to flowering between the V. longisporum isolates VD6 and VD12 and the V. dahliae isolate INRA617 and all other isolates. There were no other significant effects correlated with isolate origin or Verticillium species.

The variation obtained in storage fatty acids induced by the procedures of tissue culture and transformation with Agrobacterium tumefaciens was investigated and compared in rapeseed, Brassica napus, cv. Hanna. An increased variation in the fatty acid profiles was noted after tissue culture and transformation compared with plants derived directly from seeds. In the second generation of rapeseed transformants, T2, the content of oleic acid ranged from 39–72%, 12–31% for linoleic acid and 7–16% for linolenic acid. This could be compared with the oleic acid content in the T2 generation of tissue culture-derived plants which ranged between 47–76% and in seed-derived material where oleic acid ranged between 55–69%.In the T3 generation the ranges in transgenic seeds were decreased but still larger than in the seed derived plants. The range in transgenic plants was 49–64% for oleic acid, 20–28% for linoleic acid and 9–18% for linolenic acid. The most extreme individuals, both highest and lowest in the common fatty acids, were found in the group of transformed plants independent of generation. The total lipid content was also affected by the two treatments and seeds with the lowest and highest lipid content were both found among the transformed plants. In conclusion, care should be taken to use proper controls when performing transformation experiments in order to distinguish variation in the fatty acid profiles induced by the transformation procedure and tissue culture treatments from the changes due to transgenic expression.

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.

Offspring of somatic hybrids between the zero-erucic acid rapeseed cv Hanna and Lesquerella fendleri were analysed regarding their fatty acid profiles. In the first back-cross generation one plant was found that produced a seed containing up to 16.5% erucic acid and 15% eicosaenoic acid (Line 1), as well as a seed having 4.3% ricinoleic acid (Line 2). This was interpreted as due to a contribution of elongase and hydroxylase genes from the L. fendleri genome since these two fatty acids are not produced in the recipient rapeseed cultivar Hanna. Crosses between Line 1 and cv Hanna resulted in the production of seeds with 35% erucic acid (F2). Furthermore, crosses between the F2 plants and the rapeseed cultivar Gulle, producing 35% erucic acid in the seeds, resulted in F3 seeds with 48% erucic acid. The highest amount of erucic acid, 61.5%, was found in the F6 generation after crossing Line 1 with a high erucic acid rapeseed line, HEAR, followed by self-fertilisation for two generations. When performing Southern-blot analysis on the F6 plants, seven of the nine analysed plants hybridised with the L. fendleri species-specific repetitive probe. The presence of the hydroxylase gene was also observed in the F6 generation of Line 1 according to Southern-blot analysis. Hybridisation with a hydroxylase probe was seen although no hydroxy fatty acids could be detected in any of the F6 plants. In parallel, Line 2 was crossed with HEAR cv Gulle and self fertilised. No hydroxy fatty acids were detected in the F2 generation of Line 2 and no specific hybridisation patterns could be found in the Southern-blot analysis.

The inheritance of resistance to Leptosphaeria maculans was studied in near-isogenic lines derived from asymmetric somatic hybrids between Brassica napus+Brassica nigra and Brassica napus+Brassica juncea, respectively. The hybrids had been backcrossed to B. napus for seven generations before the genetic segregation of the blackleg resistance was determined. The results of the inheritance studies suggested that one single dominant allele controls the resistance in the Brassica napojuncea line, whereas two independent dominant loci were found in the Brassica naponigra line. Total leaf DNA from the near-isogenic lines was isolated and 89 loci were detected by hybridization to 66 restriction fragment length polymorphism (RFLP) markers previously mapped in the B. nigra genome. Out of the 89 loci, eight loci were detected in the B. naponigra line and six were found in the B. napojuncea line. RFLP markers co-segregating with blackleg resistance in adult leaves were also found. Two markers associated with linkage group 5 and 8, respectively, of the B genome were found in the B. naponigra line and one marker was associated with linkage group 2 in the B. napojuncea line.

Chromosome counts and RFLP markers mapped to Arabidopsis thaliana were used to determine the proportion of eliminated chromosomes and retained A. thaliana DNA in the back-crossed (BC) progeny derived from symmetric and asymmetric somatic hybrids between Brassica napus and A. thaliana. All plants were analysed for the presence of two RFLP markers per chromosome, preferably with one located on each chromosome arm. A reduction in both A. thaliana RFLP markers and chromosome numbers was found in the BC1 and BC2 generations of the symmetric hybrids as well as in the BC1 generation of the asymmetric hybrids. In the symmetric hybrids, two back-crosses to B. napus were required to reduce the frequency of retained A. thaliana loci to 42.4% and mean chromosome number to 39.4. In comparison, the BC1 progeny of the asymmetric hybrids had 16% of the analysed A. thaliana loci present and an average of 38.4 chromosomes maintained. When the frequency of A. thaliana chromosomes with both analysed loci maintained was compared with the frequency of chromosomes with one locus lost and one kept, a reduction in the number of complete chromosomes between BC1 and BC2 derived from the symmetric hybrids was observed. Among the BC1 plants in the asymmetric group the situation was different, with higher amounts of incomplete donor chromosomes compared to whole chromosomes. The results indicate that A. thaliana chromosome fragments are more often found in the progeny of irradiated hybrids, while back-crossed symmetric hybrids have more complete chromosomes.

To evaluate UV-irradiation as an alternative method for chromosome fragmentation in the production of asymmetric somatic hybrids, UV-irradiated protoplasts of Arabidopsis thaliana were fused with protoplasts of Brassica napus . The A. thaliana protoplasts were irradiated with doses between 780 and 28 080 J/m 2 ( λ =254 nm) and hybrid plants were obtained from experiments using doses of 780, 2340 and 4680 J/m 2 . In addition, a control fusion experiment without irradiation was performed. In total, 312 shoots were regenerated and of those 58 plants were analysed for presence of A. thaliana DNA, using 13 mapped A. thaliana RFLP markers (two to four per chromosome). Increased UV-irradiation dose resulted in a higher frequency of asymmetric hybrids and a higher dose was also associated with the generation of hybrids with a higher degree of asymmetry. RFLP data further indicated that irradiation primarily resulted in loss of chromosome fragments rather than whole chromosomes from A. thaliana . Fertility was studied in 119 hybrid plants by selfing as well as pollination by B. napus . The fertility after selfing increased with increasing UV-irradiation dose, while the results of back-crossing were not affected by the irradiation.

Brassica oilseed crops have been grown by Swedish farmers since the early 1940s. Years of high market prices for vegetable oils resulted in intensive cultivation of Brassica oilseeds in various regions, which led to problems with soilborne pathogens including Plasmodiophora brassicae. This pathogen most likely was present in Swedish soils prior to the Brassica oilseed boom after World War II. Currently, reports of clubroot disease outbreaks in Sweden are frequent, with a trend of increasing incidence. Since 2012, DNA-based soil analyses for the presence of P. brassicae have been offered to farmers in order to improve their crop rotation planning. Other means of limiting the damage caused by P. brassicae also are presently under study, such as the effects of boron or different sources of nitrogen. The distribution and identification of different pathotypes/races of P. brassicae in Sweden could so far not be verified. To aid in race diagnostics, resistance breeding efforts, and in understanding the biology of this Plasmodiophorid, the sequencing of the P. brassicae genome has been initiated in Sweden.

Somatic hybrids were produced between hypocotyl protoplasts of Brassica napus (rapeseed) and mesophyll protoplasts of B. tournefortii using protoplast fusion. Fusion products were selected by flow cytometry and cell sorting. Twenty-three plants regenerated from individual calli originating from 4 fusion experiments were successfully transferred to the greenhouse. Nineteen of these were characterized as hybrids according to analysis of morphological characteristics, isoenzyme and nuclear RFLP patterns. Analysis of nuclear DNA content revealed a variation in ploidy level among the hybrids, even though a large number (53%) had a DNA content corresponding to the sum of the parental nuclear DNA. Most of the hybrids were intermediate in morphology between B. napus and B. tournefortii. The flowers displayed a range of variation: from normal flowers to abnormal flowers with reduced number of anthers and petals. Fertility investigation showed that both fertile and sterile hybrids were obtained. Five were self fertile, while 10 were male sterile and 4 were completely sterile. Phoma lingam resistance was transferred from B. tournefortii to the hybrid plants. Out of the 16 hybrids examined 12 showed resistance to P. lingam. Chloroplast DNA (cpDNA) patterns of the hybrids were identified by hybridizing a cpDNA probe to total genomic DNA. Of 18 hybrids analysed, 11 had B. tournefortii and 7 B. napus cpDNA pattern. No recombinant cpDNA banding pattern was found. In contrast to the cpDNA, mitochondrial DNA (mtDNA) rearrangements were detected in the hybrids. Out of 18 analysed hybrids, 7 showed evidence of mitochondrial recombination, while 11 had mtDNA identical to either of the parental types.

The aim of this work was to identify plant resistance genes to the sorghum anthracnose fungus Colletotrichum sublineolum. cDNA-AFLP transcript profiling on two contrasting sorghum genotypes inoculated with C. sublineolum generated about 3,000 informative fragments. In a final set of 126 sequenced genes, 15 were identified as biotic stress related. Seven of the plant-derived genes were selected for functional analysis using a Brome mosaic virus-based virus-induced gene silencing (VIGS) system followed by fungal inoculation and quantitative real-time PCR analysis. The candidate set comprised genes encoding resistance proteins (Cs1A, Cs2A), a lipid transfer protein (SbLTP1), a zinc finger-like transcription factor (SbZnTF1), a rice defensin-like homolog (SbDEFL1), a cell death related protein (SbCDL1), and an unknown gene harboring a casein kinase 2-like domain (SbCK2). Our results demonstrate that down-regulation of Cs1A, Cs2A, SbLTP1, SbZnF1 and SbCD1 via VIGS, significantly compromised the resistance response while milder effects were observed with SbDEFL1 and SbCK2. Expanded genome analysis revealed that Cs1A and Cs2A genes are located in two different loci on chromosome 9 closely linked with duplicated genes Cs1B and Cs2B, respectively. The nucleotide binding-leucine rich repeat (NB-LRR) encoding Cs gene sequence information is presently employed in regional breeding programs.

Cold winters and geographic isolation have hitherto protected the Nordic countries from many plant pathogens and insect pests, leading to a comparatively low input of pesticides. The changing climate is projected to lead to a greater rise in temperature in this region, compared to the global mean. In Scandinavia, a milder and more humid climate implies extended growing seasons and possibilities to introduce new crops, but also opportunities for crop pests and pathogens to thrive in the absence of long cold periods. Increased temperatures, changed precipitation patterns and new cultivation practices may lead to a dramatic change in crop health. Examples of diseases and insect pest problems predicted to increase in incidence and severity due to global warming are discussed.

SUMMARY The Bipolaris sorokiniana tolerant 1 (bst1) barley mutant is derived from fast neutron-irradiated seeds of wild-type Bowman(Rph3). The induced mutation was genetically localized to a position on chromosome 5HL distal to the centromere using amplified fragment length polymorphism markers. In addition, the defence responses and related gene expression in the bst1 mutant after fungal challenge were compared with those occurring in wild-type plants. Hydrogen peroxide generation, determined by 3,3-diaminobenzidine staining, revealed a clearly reduced level of bst1, compared with the wild-type, during the entire experimental time: 8-120 h post-inoculation (hpi). At 48 hpi, the wild-type samples displayed twice as much fungal mass and three times greater H(2)O(2) production than bst1. At the same time, staining of B. sorokiniana showed less fungal growth in the spontaneous lesions of bst1 compared with the wild-type. Monitoring of defence-related genes at 48 hpi demonstrated strong expression of PR-1a, PR-2, PR-5 and PR-10 in bst1. A gene coding for a unique oxidoreductase enzyme, designated as HCP1, was expressed at much higher levels in inoculated leaves of the bst1 mutant than in those of the wild-type plant. Taken together, the results suggest that the defence to B. sorokiniana largely relies on salicylic acid-responsive pathogenesis-related (PR) genes, as well as selected reactive oxygen species and unknown HCP1-associated factors.

Out of 400 reciprocal crosses made between Brassica napus and Arabidopsis thaliana, 200 maternal B. napus flowers produced 31 seeds and 10 seeds developed when A. thaliana was used as the female parent. Of the latter, three seeds successfully germinated but did not develop into mature plants. In contrast, plants from all 31 seeds generated from B. napus were established. DNA from these 31 plants was analysed by 18 probes mapped to the A. thaliana genome, two selectable marker genes and one A. thaliana species-specific repetitive probe. It was found that one plant had identical restriction fragments to A. thaliana with two of the 21 probes used. The gene flow within Brassicaceae is discussed in light of this result.

Protoplast fusions between Brassica napus and Orychophragmus violaceus for transfer of valuable traits to oilseed rape resulted in 257 somatic hybrid plants. Hybridity was confirmed by morphological, cytological and molecular means. Symmetric fusions gave rise to 131 plants. Fifty eight of these plants had an intermediate morphology and contained nuclear DNA corresponding to the sum of the parental species. All 131 plants were sterile with no pollen grains observed upon flowering. Another 126 plants were derived from asymmetric fusions in which protoplasts of the donor parent O. violaceus were irradiated by 100 or 200-Gy X-rays prior to fusion. Morphologically these plants showed a larger variation compared to the plants regenerated from symmetric fusion experiments. In contrast to plants obtained from symmetric fusions, fertile hybrids were recovered among regenerants from the asymmetric fusions. Twenty four of these plants released viable pollen grains and 14 of the determined 17 plants set seeds after either selfing or backcrossing to B. napus. Fourteen male-sterile plants were identified with female fertility. This observed male sterility most-likely originated from alloplasmic recombination and would be of great potential for the development of a new cytoplasmic male sterility system. The fatty acid composition of the fertile hybrids and their progenies showed a biased distribution towards the B. napus parent, which has a high erucic acid-content type. However, increased levels of palmitic and linoleic acids compared to B. napus were found in subsequent generations, as well as a reduced level of erucic acid.