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ABSTRACT Genetic variability and population structure of Cercospora sorghi from wild and cultivated sorghum were investigated to gain insight into their potential impact on epidemics of gray leaf spot of sorghum in Africa. Population structure was examined using data derived from amplified fragment length polymorphism (AFLP) of C. sorghi by Nei's test for population differentiation, G(ST), and analysis of molecular variation (AMOVA). Two ecological populations of C. sorghi in Uganda were devoid of population structure (G(ST) = 0.03, small ef, CyrillicF(ST) = 0.01, P = 0.291). AMOVA revealed that genetic variability was due mainly to variations within (99%) rather than between (0.35%) populations, and Nei's genetic distance between the two populations was 0.014. Phenetic analysis based on AFLP data and polymerase chain reaction-restriction fragment length polymorphism analyses of the internal transcribed spacer regions of rDNA and mitochondrial small subunit rDNA separated Cercospora cereal pathogens from dicot pathogens but did not differentiate among C. sorghi isolates from wild and cultivated sorghum. Our results indicate that Ugandan populations of C. sorghi compose one epidemiological unit and suggest that wild sorghum, while not affecting genetic variability of the pathogen population, provides an alternative host for generating additional inoculum.
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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.
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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.
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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.
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summary Leptosphaeria maculans causes blackleg disease, and resistance to this fungal pathogen is an important trait in the breeding of oilseed rape. A better comprehension of the role of the myrosinase-glucosinolate system in this context is of great value. The present study is the first to address effects on multiple components of this complex system, including concentrations of individual glucosinolates, product formation, myrosinase isoform distribution and activity, and levels of myrosinase binding proteins during the infection process. One resistant B. napus cultivar (Maluka) and one susceptible cultivar (Westar) were compared in the investigation. Our results show that the two cultivars had the same histological distribution, isoform expression, and activity of the myrosinase enzymes. The glucosinolate levels were also similar, with the exception of glucobrassicin and neoglucobrassicin, which were significantly lower in the resistant cultivar at 11 days post-infection. Growth of the fungus on the plant tissues did not alter glucosinolate levels, suggesting that L. maculans does not degrade these compounds. When the plants were starved of sulphur, and thereby depleted of glucosinolates, no increased susceptibility was observed. Hence, we suggest that the myrosinase-glucosinolate system does not determine the outcome of the interaction between B. napus and L. maculans.