Transfer of resistance to clubroot (Plasmodiophora brassicae) to swedes {Brassica napus L. var. napobrassica Peterm) from B. rapa

In 1975, tests with UK populations of Plasmodiophora brassicae not only revealed a lack of effective clubroot resistance in swedes (Brassica napus), but also the outstanding resistance of the European Clubroot Differential (ECD)04 (B. rapa). It was, therefore, decided to transfer the resistance genes from ECD04 to swedes, using the most pathogenic UK population of clubroot (C56) available for screening purposes. An autotetraploid form of ECD04 was crossed with tetraploid kale (B. oleracea) using the latter as female parent. One of the euploid, 2n = 38, hybrids secured by embryo rescue in 1976 was crossed to the swede cultivars Marian and Ruta Øtofte. Three further backcrosses of clubroot resistant plants to lines derived from modern swede cultivars were made over the period 1980 to 1982. Selfing commenced in 1983 to produce F₂ populations. From F₃ to F₅ there was family selection for yield and agronomic characters, as well as single plant selection for clubroot resistance. In 1991, the six most promising F₅ families were multiplied for subsequent evaluation in replicated yield trials in Dundee. The most promising family entered official trials at the beginning of 1993 and, 2 years later, was added to the National List as cv. Invitation and granted Plant Breeders' Rights. The first certified seed was sold in 1996, 20 years after the original synthetic B. napus was produced. The breeding programme provided evidence for only one of the three postulated dominant genes in ECD04 being required for resistance to C56 and also good evidence of differential resistance from tests with other clubroot populations. Hence, whilst the differential resistance in cv. Invitation should prove useful in the UK in the immediate future, it may not be durable in the longer term. It is, therefore, argued that the next and more difficult goal to achieve should be to introduce high levels of non-differential resistance from B. oleracea.     

Peroxidase and chitinase isoenzyme activities during root infection of Chinese cabbage with Plasmodiophora brassicae

Roots of two Chinese cabbage (Brassica campestris L. ssp. pekinensis) varieties, one tolerant and one susceptible, were inoculated with Plasmodiophora brassicae in liquid medium and in soil. Chitinase and peroxidase activities were determined in roots and shoots 1–21 days after inoculation with resting spores of Plasmodiophora and the enzyme activities compared with healthy tissue of the same age. In infected roots of the susceptible variety ‘Granat’ chitinase activity was higher than in the control 10 days after inoculation with spores. In the tolerant variety ‘Parkin’ we detected an increase in chitinase activity at the same time, which was about twice that of ‘Granat’. Chitinase activity in ‘Granat’ was also enhanced on day 13, 14 and 17 after inoculation, whereas chitinase activity in ‘Parkin’ was lower in the infected roots than in the controls during that period. In the shoots no correlation between chitinase activity and infection in the two varieties was observed. Chitinase from Chinese cabbage was further characterized and showed a pH optimum at pH 4.5–5.5 and a temperature optimum at 35–45°C. After isoelectric focusing 7 isoenzymes were discovered, but there were almost no differences between infected and healthy root extracts. Two isoenzymes with pI 8.7 and 8.8 showed cross-reactivity with an antiserum against bean chitinases. The molecular mass of these isoenzymes was determined as 33 kDa. Total peroxidase activity was generally higher in root tissue of both varieties than in the shoots. Peroxidase activity was increased most prominently in infected ‘Granat’ roots on day 13 after inoculation and of both varieties on day 17 compared to the controls. In clubbed tissue of ‘Granat’ a specific peroxidase isoenzyme appeared the first time 21 days after inoculation and was most prominent 28–30 days after inoculation. This isoenzyme had a molecular mass of ca 24 kDa and a pI of ca 8.8. With respect to our results the strategy of the Plasmodiophorales for plant attack is discussed.     

Selection for resistance to clubroot (Plasmodiophora brassicae) in marrowstem kale (Brassica oleracea var. acephala L.)

Ninety-six cultivars of Brassica oleracea were screened for clubroot resistance in a seedling test using two populations of Plasmodiophora brassicae. The most resistant cultivars were kales. Sixteen resistant marrowstem kale cultivars of diverse geographical origin were used to start a selection programme for clubroot resistance. Four generations of selection, involving single plants, half-sib and full-sib families, reduced a disease index averaged over six clubroot populations from 41.2 to 12.5. This was lower than the most resistant cultivar in the original population, cv. Mixti 28.8, and as good as a German landrace of cabbage noted for its resistance, Bohmerwaldkohl 10.5. In comparison, the mean of five kale controls, cvs Bittern, Canson, Condor, Kestrel and Merlin, was 61.1 and the value for the most susceptible control, cabbage cv. Septa, was 89.3. In the final assessment, there were no clubroot population x B. oleracea genotype interactions and in the initial assessment of cultivars there were only small interactions which could be removed by an angular transformation of the data. It was concluded that a high level of non-differential resistance had been achieved and that it may prove durable. It was also concluded from a small field trial that this level of resistance would prevent serious yield losses in practice.     

Germination of surface-disinfected resting spores ofPlasmodiophora brassicae and their root hair infection in turnip hairy roots

Germination of surface-disinfected resting spores ofPlasmodiophora brassicae and its infection of turnip hairy root hairs were studied. Surface-disinfected resting spores showed higher germination than non-disinfected resting spores. Root hair infection was most frequent in the section of root formed 1 d before inoculation. Root hair infection began 4 d after inoculation, increased up to 6 d, and continued to increase more slowly until 10 to 12 d after inoculation. Growth ofP. brassicae in the root hair of hairy roots was observed serially. Most primary plasmodia differentiated to mature zoosporangia 8–10 d after inoculation. The secondary zoospores were initially released 6 d after inoculation.     

Dynamics of infection by Leptosphaeria maculans on canola (Brassica napus) as influenced by crop rotation and tillage

Abstract

This study, intending to understand the effects of crop rotation and tillage on blackleg disease, was conducted in a field at Carman, Manitoba, Canada, from 1999 – 2002. Canola, wheat and flax were among the rotated crops. Rotations were performed under conventional or zero-till conditions. The number of infected plants, infected leaves per plant, lesions per plant, and percentage of leaf coverage with lesions decreased when canola was rotated with wheat and flax under zero till. The number of lesions per plant and percentage of leaf coverage with lesions were strongly correlated with stem disease severity, and the number of infected plants with stem disease incidence. Ascospores and pycnidiospores of Leptosphaeria maculans were reduced by crop rotation and tillage. This study suggests that the appropriate combination of rotation and tillage may lower airborne inoculum and reduce infection of canola plants by L. maculans.     

Plasmodiophora brassicae-induced Cell Death and Medium Alkalization in Clubroot-resistant Cultured Roots of Brassica rapa

Plasmodiophora brassicae causes clubroot in the turnip, Brassica rapa L. We used organ cultures of adventitious roots from B. rapa seedlings to investigate the initial response of resistant and susceptible cultivars to P. brassicae infection. Primary plasmodia of P. brassicae were observed in root hairs of both susceptible and resistant cultured roots. On the other hand, secondary plasmodia were able to proliferate only in the susceptible root culture but not in the resistant one. Root cultures from the susceptible cultivar all developed clubroot 4 weeks after treatment with 10⁴, 10⁵ or 10⁶ spores/ml, but roots from the resistant cultivar did not develop clubroot under the same conditions. Cell death, as measured by Evans blue and TTC dye methods, was observed in cultured roots from the resistant cultivar but did not occur in roots from the susceptible cultivar after exposure to P. brassicae spores. Cell death was inhibited almost completely by EGTA and verapamil but not by the calmodulin antagonist W7. These results suggest the involvement of Ca²⁺ in P. brassicae‐induced cell death. Alkalization of the root culture medium of the resistant cultivar was observed 2 days after treatment with P. brassicae spores but was not observed in root culture medium from the susceptible strain. We conclude that our root culture system must be a useful tool for further studies of the molecular mechanism of clubroot resistance.     

Infection of Chinese cabbage by Plasmodiophora brassicae leads to a stimulation of plant growth: impacts on cell wall metabolism and hormone balance

The importance of plant hormones in clubroot infection has long been recognized. The morphological changes, such as cell division and cell elongation leading to gall formation are triggered in the early stages of infection. We analysed cell expansion by localizing Xyloglucan endoTransglucosylase/Hydrolase (XTH)-action and screened the endogenous concentrations of several classes of phytohormones by mass spectrometry in the early stages of Plasmodiophora brassicae infection in Chinese cabbage (Brassica rapa spp. pekinensis). Infected plants showed a general transient growth promotion early in infection. Furthermore a clear XTH action was visible in the epidermal layer of infected roots. Complex changes in the endogenous phytohormone profile were observed. Initially infection resulted in an increased total auxin pool. The auxin increase, together with an increased XTH action, results in wall loosening and consequently cell expansion. When the first secondary plasmodia are formed, thirteen days after infection (DAI), can be considered a switch point in phytohormone metabolism. Twenty-one DAI the plasmodia might act as a plant hormone sink resulting in a reduction in the active cytokinin pool and a lower indole-3-acetic acid content in the infected plants.     

甘蓝型油菜根系突变体lrn1、prl1和野生型根系显微结构的差异

油菜外源细胞分裂素不敏感突变体lrn1和prl1表现为磷高效。营养液培养0.2μmol/L细胞分裂素(6-BA)处理,与甘蓝型油菜野生型‘宁油7号’(WT)相比,突变体lrn1侧根较多,prl1主根较长。本研究利用体式显微技术、非切片压片法以及石蜡切片等技术,对3个基因型在ddH2O和0.2μmol/L 6-BA处理下的根毛、根表皮细胞分化及根尖解剖结构的差异进行了观察,结果表明:ddH2O处理,种子发芽后第1、3、6、9 d,lrn1、prl1和WT根尖成熟区根毛较少。0.2μmol/L 6-BA处理,种子发芽后第3 d,lrn1、prl1和WT根尖形成大量根毛,其中WT根毛最多、密度最大;prl1根毛最少,密度也最小;lrn1处于两者之间。种子发芽后第6 d,lrn1、prl1和WT分生区和伸长区明显缩短,lrn1和prl1分生区面积无显著差异,但两者均显著大于WT;lrn1和prl1根冠细胞结构较正常,而WT根冠细胞结构畸形;lrn1皮层原细胞之间排列较WT和prl1紧密。种子发芽后第9 d,lrn1已有4条侧根,但prl1与WT无侧根形成。6-BA处理,prl1主根较长,与其根尖分生区面积较大密切相关;lrn1侧根较多,可能与中柱原细胞排列密度较高密切相关。     

Detection and analysis of QTLs based on RAPD markers for polygenic resistance to Plasmodiophora brassicae Woron in Brassica oleracea L.

Resistance to Plasmodiophora brassicae Woron, the causal fungus of clubroot, was examined in an F₂ population of a cross between a clubroot-resistant kale (Brassica oleracea L. var. acephala) and a susceptible cauliflower (Brassica oleracea L. var. botrytis). QTL detection was performed with RAPD markers. Two resistance notations, carried out at different times after inoculation, were used. Three markers were associated with these two notations and three were specifically linked to only one notation. QTL analysis suggests the existence of at least two genetic mechanisms implicated in the resistance phenomenon.     

Identification of QTL involved in field resistance to light leaf spot (Pyrenopeziza brassicae) and blackleg resistance (Leptosphaeria maculans) in winter rapeseed (Brassica napus L.)

 Quantitative trait loci (QTL), involved in the polygenic field resistance of rapeseed (Brassica napus L.) to light leaf spot disease, were mapped using 288 DNA markers on 152 doubled-haploid (DH) lines derived from the cross ‘Darmor-bzh’בYudal’. Over two years (1995 and 1996), the DH population was evaluated for light leaf spot resistance on leaves (L) and stems (S), and for blackleg disease resistance in same field trials. For the L resistance criterion, a total of five and seven QTL were detected in 1995 and in 1996 respectively, accounting for 53% and 57% of the genotypic variation. For the S criterion, three and five QTL were identified in 1995 and in 1996 respectively, explaining 29% and 43% of the genotypic variation. The locations of the QTL detected were quite consistent over the two years (4- and 2-year common QTL for L and S, respectively). Three genomic regions, located on the DY5, DY10 and DY11 groups, were common to the resistance on leaves and stems. In comparison with the QTL for blackleg resistance described by Pilet et al. (1998), two regions on the DY6 and DY10 groups, were associated with the two disease resistances. These ‘multiple disease resistance’ (‘MDR’) QTL may correspond to genes involved in common resistance mechanisms towards the two pathogens or else to clusters of resistance genes. Received: 21 November 1997 / Accepted: 3 March 1998     

Novel resistances to four potyviruses in tuber-bearing potato species, and temperature-sensitive expression of hypersensitive resistance to potato virus Y

Novel potyvirus resistance specificities were found in eight tested wild potato species (clones): hypersensitive resistance (HR) to potato Y potyvirus (PVY) strain groups PVY^O in Solanum megistacrolobum and S. polyadenium and PVY^N in S. stoloniferum; HR to potato V potyvirus (PW) in S. maglia, S. polyadenium, S. stoloniferum, S. sparsipilum and S. sucrense, HR to potato A potyvirus (PVA) strain group 1 in S. sucrense, and extreme resistance (ER) to PVA in S. polyadenium. S. commersonii and S. stoloniferum expressed HR to tobacco etch potyvirus (TEV) which has not been reported previously in potato species. The studied clone of S. stoloniferum expressed HR to all potyviruses and potyvirus strains tested. The clone of S. stoloniferum (2n = 48; nuclear DNA content (2C) = 3.6 pg) and S. chacoense (2n = 24; 2C=1.9 pg) were crossed and one hybrid (2n = 36; 2C = 2.9 pg) was obtained. The hybrid expressed HR to all tested potyviruses except PVA, which indicated that HR to PVA was controlled by a gene which is different from the genes (or gene) controlling HR to PVY^O, PVY^N, PVV and TEV in S. stoloniferum. On the other hand, S. chacoense and the hybrid expressed ER to cucumber mosaic cucumovirus (CMV), whereas S. stoloniferum was susceptible to CMV. All tested wild species and the six tested potato cultivars (S. tuberosum subsp. tuberosum) expressed HR to PVV. Expression of HR following infection with PVY^N induced systemic acquired resistance (SAR) in S. chacoense. HR to PVY^N in S. sparsipilum and S. sucrense and to PVY^O in potato cv. Pito was efficiently expressed at lower temperatures (16/18°C) indicated by the development of distinct necrotic lesions and/or vein necrosis in inoculated leaves, whereas the HR was rendered less effective at higher temperatures (19/24°C) which was indicated by the development of systemic infection with leaf-drop and mosaic symptoms.     

Relationships between turnip root fly (Delia floralis) larval development and the sugar content of swede (Brassica napus ssp. rapifera) roots

Eight genotypes of swede (Brassica napus L. ssp. rapifera [Metz.] Sinsk.) at the 8–10 true leaf stage were inoculated with five, 10 or 20 eggs of the turnip root fly Delia floralis (Fall). The roots were sampled, with control roots, after 6 weeks of larval development. D. floralis root damage, as measured by reduction in root weight, was found to be linked to inoculation level. Neither D. floralis egg numbers nor swede genotype had a significant effect on the percentage of larvae developing to pupation. Mean pupal weight varied by a factor of ×1.4 and consistently decreased with increasing egg inoculation level. Changes in the root concentrations of glucose, sucrose and fructose were measured. All swede genotypes showed a similar response in their sugar concentrations after root damage. Glucose and fructose concentrations were reduced whilst sucrose concentration remained unaffected. The concentrations of glucose and fructose were highly correlated. Pupal weight, used as a measure of larval development, was significantly correlated with the concentrations of individual and total sugars in the roots. The implications of sugar responses to damage in brassicas, and the correlation between sugar concentrations in the roots and D. floralis pupal weights are discussed.     

SCAR and CAPS mapping of CRb, a gene conferring resistance to Plasmodiophora brassicae in Chinese cabbage ( Brassica rapa ssp. pekinensis)

Clubroot disease, caused by Plasmodiophora brassicae Wor., is highly damaging for Chinese cabbage. The CR (clubroot resistant) Shinki DH (doubled haploid) line of Chinese cabbage carries a single dominant gene, CRb, which confers resistance to the P. brassicae races 2, 4, and 8. An F₂ population derived from a cross between the CR Shinki DH line and a susceptible line, 94SK, was used to map the CRb gene. Inoculation of F₃ families with SSI (single-spore isolate) resulted in a 1:2:1 segregation ratio. Use of the AFLP technique combined with bulked segregant analysis allowed five co-dominant AFLP markers, and four and seven dominant AFLP markers linked in coupling and repulsion, respectively, to be identified. Six of the 16 AFLP markers showing low frequencies of recombination with the CRb locus among 138 F₂ lines were cloned. A reliable conversion procedure allowed five AFLP markers to be successfully converted into CAPS and SCAR markers. An F₂ population (143 plants) was analyzed with these markers and a previously identified SCAR marker, and a genetic map around CRb covering a total distance of 6.75 cM was constructed. One dominant marker, TCR09, was located 0.78 cM from CRb. The remaining markers (TCR05, TCR01, TCR10, TCR08, and TCR03) were located on the other side of CRb, and the nearest of these was TCR05, at a distance of 1.92 cM.Communicated by R. Hagemann     

Inheritance of resistance derived from the B-genome of Brassica against Phoma lingam in rapeseed and the development of molecular markers

 Genes of the B genome of Brassica conditioning Phoma resistance at the epicotyle were transferred into Brassica napus by interspecific hybridization. The recombinant lines expressed high resistance similar to that of the donor parents. Unlike the oligo- or poly-genically inherited resistance of B. napus known so far, the B-genome resistance genes of the recombinant lines behaved monogenically dominant. No significant differences in the level of resistance or in the phenotype of the resistance mechanisms were observed among homozygous resistant plants when the different B-genome origins investigated, i.e. B. nigra, B. juncea and B. carinata, were compared. Therefore it was assumed that the resistance genes of each B-genome species and the resistance mechanisms of the species are identical. Temperature increased the expression of internal lesions caused by Phoma lingam. High summer temperatures in the greenhouse led to faster development of tissue damage at the epicotyle of plants, resulting in significant deviations in segregation ratios, when fixed scores were used for disease classification. Independent of origin, the three B-genome resistance genes were introgressed at the same location of the rapeseed genome. The arrangement and distances of closely linked RFLP markers on linkage-groups were similar to those of the same markers on linkage group six of the rapeseed map. It is concluded that the B-genome resistance genes were introgressed by homoeologous recombination after allosyndetical pairing of B-genome chromosomes with the A- or C-genome chromosomes. Received: 3 April 1998/Accepted: 22 April 1998     

Molecular cloning of a sulfotransferase in Arabidopsis thaliana and regulation during development and in response to infection with pathogenic bacteria

A cDNA clone (RaRO47) encoding a sulfotransferase (ST) has been isolated from Arabidopsis cell suspensions. The deduced polypeptide of 302 amino acids is highly related to plant flavonol sulfotrans-ferases (FSTs), characterized for the first time in Flaveria, and also to STs from animal tissue. The expression of the Arabidopsis ST gene(s) corresponding to RaR047 was examined during different developmental stages. It was found that, at the level of steady-state mRNA, expression of gene(s) encoding this ST was rapidly induced in the aerial parts of young seedlings, and during growth of Arabidopsis cell cultures. No expression could be detected in roots. Treatment of Arabidopsis seedlings with hormonal or stress-related compounds, showed that RaR047 mRNA accumulation was more particularly induced in response to salicylic acid and methyl jasmonate. Furthermore, in the leaves of mature plants or in cell suspensions, accumulation of RaR047 mRNA was observed upon infection with bacterial pathogens. This expression was observed preferentially in response to avirulent pathogens causing an hyper-sensitive reaction, as compared to virulent pathogens, which lead to disease.     

抗生素抗性基因在环境中的传播扩散及抗性研究方法

抗生素在医药、畜牧和水产养殖业的大量使用造成了环境中抗性耐药菌和抗性基因日益增加,抗生素抗性基因作为一种新型环境污染物引起人们的广泛关注.本文综述了近年来国内外有关抗生素抗性基因的研究进展,其在水、土壤、空气等环境介质中和动,植物体内的传播扩散,以及开展环境中抗生素抗性基因研究的必要性,重点介绍了有关抗生素抗性（包括抗性细菌和抗性基因）的研究方法,指出抗性基因研究中存在的问题,并对未来的相关研究进行了展望.     

Global and Comparative Proteome Analysis of Nitrogen-Stress Responsive Proteins in the Root,Stem and Leaf of <i>Brassica napus</i>

Nitrogen (N) is one of the basic nutrients and signals for plant development and deficiency of it would always limit the productions of crops in the field. Quantitative research on expression of N-stress responsive proteins on a proteome level remains elusive. In order to gain a deep insight into the proteins responding to nitrogen stress in rapeseed (Brassica napus L.), comparative proteomic analysis was performed to investigate changes of protein expression profiles from the root, stem and leaf under different N concentrations, respectively. More than 200 differential abundance proteins (DAPs) were detected and categorized into groups according to annotations, including “binding and catalytic activity”, “involved in primary metabolism and cellular processes”, “stress-response” and so on. Variation in chlorophyll (Chl) content and antioxidant activities further revealed that oxidative stress raised with the increase of N concentration. Bioinformatics analysis based on the expression level of total proteins suggested these DAPs might play important roles in adaptation to N-stress conditions. Generally, these results provides a new aspect into N-stress responding proteins in Brassica plants.     

Temperature and leaf wetness duration affect phenotypic expression of Rlm6-mediated resistance to Leptosphaeria maculans in Brassica napus

Near-isogenic Brassica napus lines carrying/lacking resistance gene Rlm6 were used to investigate the effects of temperature and leaf wetness duration on phenotypic expression of Rlm6-mediated resistance. Leaves were inoculated with ascospores or conidia of Leptosphaeria maculans carrying the effector gene AvrLm6. Incubation period to the onset of lesion development, number of lesions and lesion diameter were assessed. Symptomless growth of L. maculans from leaf lesions to stems was investigated using a green fluorescent protein (GFP) expressing isolate carrying AvrLm6. L. maculans produced large grey lesions on Darmor (lacking Rlm6) at 5-25 degrees C and DarmorMX (carrying Rlm6) at 25 degrees C, but small dark spots and 'green islands' on DarmorMX at 5-20 degrees C. With increasing temperature/wetness duration, numbers of lesions/spots generally increased. GFP-expressing L. maculans grew from leaf lesions down leaf petioles to stems on DarmorMX at 25 degrees C but not at 15 degrees C. We conclude that temperature and leaf wetness duration affect the phenotypic expression of Rlm6-mediated resistance in leaves and subsequent L. maculans spread down petioles to produce stem cankers.     

Functional disruption of the pentatricopeptide protein SLG1 affects mitochondrial RNA editing, plant development, and responses to abiotic stresses in Arabidopsis

Seed oil content is an important agronomic trait in rapeseed. However, our understanding of the regulatory processes controlling oil accumulation is still limited. Using two rapeseed lines (zy036 and 51070) with contrasting oil content, we found that maternal genotype greatly affects seed oil content. Genetic and physiological evidence indicated that difference in the local and tissue-specific photosynthetic activity in the silique wall (a maternal tissue) was responsible for the different seed oil contents. This effect was mimicked by in planta manipulation of silique wall photosynthesis. Furthermore, the starch content and expression of the important lipid synthesis regulatory gene WRINKLED1 in developing seeds were linked with silique wall photosynthetic activity. 454 pyrosequencing was performed to explore the possible molecular mechanism for the difference in silique wall photosynthesis between zy036 and 51070. Interestingly, the results suggested that photosynthesis-related genes were over-represented in both total silique wall expressed genes and genes that were differentially expressed between genotypes. A potential regulatory mechanism for elevated photosynthesis in the zy036 silique wall is proposed on the basis of knowledge from Arabidopsis. Differentially expressed ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-related genes were used for further investigations. Oil content correlated closely with BnRBCS1A expression levels and Rubisco activities in the silique wall, but not in the leaf. Taken together, our results highlight an important role of silique wall photosynthesis in the regulation of seed oil content in terms of maternal effects.