The influence of colonizing methylobacteria on morphogenesis and resistance of sugar beet and white cabbage plants to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

The influence of colonization of sugar beet (Beta vulgaris var. saccharifera (Alef) Krass) and white cabbage (Brassica oleracea var. capitata L.) plants by methylotrophic bacteria Methylovorus mays on the growth, rooting, and plant resistance to phytopathogen bacteria Erwinia carotovora was investigated. The colonization by methylobacteria led to their steady association with the plants which had increased growth speed, root formation and photosynthetic activity. The colonized plants had increased resistance to Erwinia carotovora phytopathogen and were better adapted to greenhouse conditions. The obtained results showed the perspectives for the practical implementation of methylobacteria in the ecologically clean microbiology substances used as the plant growth stimulators and for the plant protection from pathogens.     

Life table parameters of large white butterfly Pieris brassicae (Lepidoptera: Pieridae) on different cabbage varieties

Effect of four different cole crops (Brassica oleracea var. botrytis, Brassica oleracea var. capitata, Brassica oleracea var. italica and Brassica oleracea var. viridis) on biological parameters of the large white butterfly Pieris brassicae was evaluated at temperature 26 ± 1 °C, 60 ± 5% R. H. and a photoperiod of 16: 8 (L:D) h. The shortest larval and pupal period stages were recorded on B. oleracea var. botrytis (22.18 ± 0.20 days) and (13.32 ± 0.17 days), respectively. The life span was longest on B. oleracea var. viridis (60.43 ± 2.34 days) and shortest on B. oleracea var. botrytis (50.19 ± 0.51 days). The highest percentage of larval and pupal mortality was observed on B. oleracea var. viridis (74%), and (53%), respectively. We found that P. brassicae prefers B. oleracea var. botrytis and B. oleracea var. capitata among cole crops; it is due to the lowest percentage of larval and pupal mortality and the highest rate of life table parameters, including survival rate (lx) and life expectancy (ex), which makes them to be susceptible varieties to this pest. Contrary, a longer developmental time on B. oleracea var. viridis may be attributed to poor nutritional status and reduced survival of the cohort, resulting in high rates of mortality, which was partial resistance to pest. Knowledge of the biology and life table parameters of P. brassicae on different cole crops could be effective in detecting and monitoring the pest infestation, variety selection and crop breeding.     

Identification of quantitative trait loci controlling developmental characteristics of Brassica oleracea L

A segregating population of F₁-derived doubled haploid (DH) lines of Brassica oleracea was used to detect and locate QTLs controlling 27 morphological and developmental traits, including leaf, flowering, axillary bud and stem characters. The population resulted from a cross between two very different B. oleracea crop types, an annual cauliflower and a biennial Brussels sprout. A principal component analysis (PCA), based on line means, allowed all the traits to be grouped into distinct categories according to the first five Principal Components. These were: leaf traits (PC1), flowering traits (PC2), axillary bud traits (PC3 and 5) and stem traits (PC4). Between zero and four putative QTL were located per trait, which individually explained between 6% and 43% of the additive genetic variation, using the multiple-marker regression approach to QTL mapping. For lamina width, bare petiole length and stem length two QTL with opposite effects were detected on the same linkage groups. Intra- and inter-specific comparative mapping using RFLP markers identified a QTL on linkage group O8 accounting for variation in vernalisation, which is probably synonymous with a QTL detected on linkage group N19 of Brassica napus. In addition, a QTL for petiole length detected on O3 of this study appeared to be homologous to a QTL detected on another B. oleracea genetic map (Camargo et al. 1995). Received: 28 March 2001 / Accepted: 25 June 2001     

During photosynthetic induction,biochemical and stomatal limitations differ between Brassica crops

Interventions to increase crop radiation use efficiency rely on understanding of how biochemical and stomatal limitations affect photosynthesis. When leaves transition from shade to high light, slow increases in maximum Rubisco carboxylation rate and stomatal conductance limit net CO₂ assimilation for several minutes. However, as stomata open intercellular [CO₂] increases, so electron transport rate could also become limiting. Photosynthetic limitations were evaluated in three important Brassica crops: Brassica rapa, Brassica oleracea and Brassica napus. Measurements of induction after a period of shade showed that net CO₂ assimilation by B. rapa and B. napus saturated by 10 min. A new method of analyzing limitations to induction by varying intercellular [CO₂] showed this was due to co-limitation by Rubisco and electron transport. By contrast, in B. oleracea persistent Rubisco limitation meant that CO₂ assimilation was still recovering 15 min after induction. Correspondingly, B. oleracea had the lowest Rubisco total activity. The methodology developed, and its application here, shows a means to identify the basis of variation in photosynthetic efficiency in fluctuating light, which could be exploited in breeding and bioengineering to improve crop productivity.     

Production and analysis of intergeneric somatic hybrids between <Emphasis Type="Italic">Brassica oleracea</Emphasis> and<Emphasis Type="Italic"> Matthiola incana</Emphasis>

The gene pool of Brassica oleracea was enriched via intergeneric somatic hybridization between B. oleracea (2n = 18) and Matthiola incana (2n = 14). One hundred and eighteen plants were obtained from 96 calli. Only four plants (H1, H2, H3 and H4) showed an intermediate phenotype from the parents; among these, H1 and H3 arose from the same callus. Random amplified polymorphic DNA (RAPD), sequence-related amplified polymorphism (SRAP), and cytological analyses confirmed that H1 and H3 were hybrids. The nuclear DNA content of the regenerated plants was determined by flow cytometry. More than half of the plants contained a nuclear DNA content of 1.3 to 3.9 pg/cell, which was higher than the content of B. oleracea but lower than that of M. incana. H1 contained 4.89 ± 0.02 pg of DNA per cell, while H3 nuclear DNA content was estimated at 4.87 ± 0.06 pg/cell. Cytological study of the root-tip cells revealed that the majority of the H1 and H3 hybrid cells contained 28 chromosomes.     

Pythium root rot of crucifers

A destructive root disease of cabbage (Brassica oleracea L. var. capitata) and cauliflower (Brassica oleracea L. var. botrytis) incited by a species of Pythium Pringsheim is described as occurring in Varanasi, U.P. The pathogen was isolated on potato dextrose and corn meal agar. Pathogenicity and host range of the disease were studied. Cultural characters, morphology and developmental stages and life cycle of the fungus indicated its identity with Pythium middletonii Sparrow.     

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.     

Molecular systematics of Brassica and allied genera (Subtribe Brassicinae,Brassiceae) —chloroplast genome and cytodeme congruence

Summary Chloroplast DNA restriction sites for 20 endonucleases were mapped using cpDNA probes from Brassica juncea and site variation was surveyed in 33 diploid taxa of the Subtribe Brassicinae. A total of 419 mutations was observed, including both site (i.e., gain/ loss) and fragment length (i.e., insertions or deletions); 221 (53%) mutations showed variation at the interspecific level. Phylogenetic analysis indicated a clear division of the subtribe into two ancient evolutionary lineages. These were (I) the Nigra lineage: Brassica nigra, B. fruticulosa, B. tournefortii, Sinapis pubescens, S. alba, S. flexuosa, S. arvensis, Coincya cheiranthos, Erucastrum canariense, and Hirschfeldia incana, and (II) the Rapa/ Oleracea lineage: Brassica rapa, B. oleracea ssp. oleracea and ssp. alboglabra, B. rupestris-villosa complex (B. rupestris, B. drepanensis, B. macrocarpa, B. villosa), B. barrelieri, B. deflexa, B. oxyrrhina, B. gravinae, Diplotaxis erucoides, D. tenuifolia, Eruca sativa, Raphanus raphanistrum, R. sativus, and Sinapis aucheri. In the Nigra lineage, Brassica nigra was most closely related to the annual Sinapis species, S. arvensis and S. alba. In the Rapa/Oleracea lineage, the Brassica rapa and B. oleracea genomes formed a distinct group whose closest relatives were the wild species of the B. oleracea (n=9) complex (i.e., B. rupestris-villosa complex). Species with n=7 chromosomes exist in both lineages. Hirschfeldia incana (n=7), in the Nigra lineage, was most closely related to Sinapis pubescens. In the Rapa/Oleracea lineage three taxa with n=7 — B. deflexa, D. erucoides, and S. aucheri — were closely related, advanced in the lineage, and were the closest apparent relatives (particularly D. erucoides) to B. rapa, B. oleracea, and its wild relatives. Levels of genetic divergence suggested by the cpDNA data were consistent with cytodeme recognition in the subtribe, but provided evidence for inconsistencies in the current generic delimitations based on morphology. Very low levels of genetic divergence were evident among taxa/accessions within a cytodeme. Raphanus was closely related to the Brassica rapa and B. oleracea genomes and clearly belongs in Subtribe Brassicinae. Several cytoplasmic genetic markers of potential use in plant breeding programs were identified for each of the cytodemes.     

Microsatellite markers for genome analysis in Brassica. II. Assignment of rapeseed microsatellites to the A and C genomes and genetic mapping in Brassica oleracea L.

Microsatellites are highly polymorphic and efficient markers for the analysis of plant genomes. Primer specificity, however, may restrict the applicability of these markers even between closely related species for comparative mapping studies. We have demonstrated that the majority of microsatellites identified in oilseed rape (Brassica napus L; AC genome) correspond to loci which can be easily assigned to the A and C progenitor genomes. A study with 63 primer pairs has shown that 54% detect two loci, one from each genome, while 25% and 21%, respectively, are either A or C genome-specific. The distribution of rapeseed microsatellites in the C genome was investigated by genetic mapping in Brassica oleracea L. Ninety two dinucleotide microsatellites were screened for polymorphism in an F₂ population derived from a cross between collard and cauliflower, for which an RFLP map has been constructed previously. Thirty three primer pairs (35.7%) have yielded either unspecific or no PCR products whereas the remaining primer pairs amplified one or more distinct loci. The level of polymorphism found in the mapping population was 49.2%. A total of 29 primer pairs disclosed 34 loci of which 31 are evenly distributed on 8 of the 9 B. oleracea linkage groups. For the remaining three markers linkage could not be established. Our results showed that microsatellite markers from the composite genome of B. napus can serve as a useful marker system in genetic studies and for plant-breeding objectives in B. oleracea. Received: 14 April 2000 / Accepted: 3 July 2000     

Chemical-based resistance of Brassica oleracea and Rorippa dubia in response to Spodoptera litura attack

A plant's chemical-based resistance against insects can be characterized by an accumulation of phytochemicals that can severely harm or deter the generalist and non-adapted specialist insects. In the absence of an herbivore threat, plants maintain a basic concentration of defensive compounds as a constitutive resistance mechanism. On the other hand, plants are capable of sharply increasing their production of defensive compounds as induced defence in response to herbivore attacks. In this study, we highlight the role of chemical-based defence by comparing the constitutive and herbivore-induced resistances of cauliflower (Brassica oleracea var. botrytis L.) and yellow cress (Rorippa dubia (Persoon)) with respect to time-dependent changes of phytochemicals that directly influence the performance of the generalist herbivore insect, Spodoptera litura (Fabricius ) (Lepidoptera: Noctuidae). The results of the study showed that both plant species were able to switch, albeit different degrees, between constitutive and induced resistances to combat herbivore attack. Brassica oleracea amassed lesser defensive compounds of constitutive resistance (trypsin inhibitor, polyphenol oxidase and sinigrin) than R. dubia. This led to a higher relative growth rate (RGR) of the S. litura larvae fed on cauliflower than in those fed on yellow cress. Brassica oleracea also demonstrated a lower induced response capability than R. dubia in its production of allelochemicals in herbivore-induced resistances. This was shown by the decrease of the RGR of larvae fed on induced foliage of the cauliflower compared to those fed on yellow cress. These results of the study proved and added to the belief that modern crop variety is not only lower in constitutive resistance but also weaker in induced resistance capability against herbivores, which is likely to be an effect of domestication even Rorippa and Brassica are relatively far from each other in Brassicaceae phylogeny.     

人工合成甘蓝型油菜早期世代及其亲本对盐胁迫的生理响应

以白菜‘矮抗青’(基因组AA)和‘中花芥蓝’(基因组CC)及其人工合成异源四倍体甘蓝型油菜(AACC)的早期世代(F1~F4)为实验材料,采用水培方法分别比较它们在100、200mmol/L NaCl处理下的生理指标差异。结果表明:(1)在盐胁迫条件下,‘中花芥蓝’植株的生物量、脯氨酸和叶绿素含量、抗氧化酶(SOD、POD、CAT)活性均最低,而相对电导率、MDA含量则最高。在100mmol/L NaCl处理下,F2代植株的生物量、叶绿素含量、SOD活性最大,MDA含量最低;在200mmol/L NaCl处理下,F4代的生物量、叶绿素含量、POD活性最大,MDA含量最低。研究发现,亲本‘矮抗青’的耐盐特性高于亲本‘中花芥蓝’,它们的杂种后代(异源四倍体)遗传了AA基因组的耐盐特性,从而比二倍体亲本具有更强的抵御盐胁迫的能力。     

Effects of salinity and gibberellin on water content,growth and mineral composition of cowpea,calabrese and red radish plants

Salinity had generally little influence on the water content of different parts of cowpea(Vigna sinensis L.), calabrese(Brassica oleracea L. var.botrylis) and red radish(Raphanus salivus L.) plants. Salinity showed a promotive effect on the growth of cowpea, while in calabrese the effect was either promotive or depressive depending upon the concentration of the NaCl, and in red radish plants salinity progressively suppressed growth. Total nitrogen, phosphorus, potassium and sodium contents of cowpea leaves were not affected by salinity treatments, while in calabrese and red radish leaves the contents of N, P and K were generally decreased as the salinity level increased. Gibberellin (GA₃) applied to salt-treated plants had either a stimulatory or inhibitory effect on the growth, water content and contents of N, P, K and Na in the leaves depending upon the plant type, the concentration of GA₃ and level of salinity.     

Identification of QTLs that control clubroot resistance in Brassica oleracea and comparative analysis of clubroot resistance genes between B. rapa and B. oleracea

To perform comparative studies of CR (clubroot resistance) loci in Brassica oleracea and Brassica rapa and to develop marker-assisted selection in B. oleracea, we constructed a B. oleracea map, including specific markers linked to CR genes of B. rapa. We also analyzed CR-QTLs using the mean phenotypes of F₃ progenies from the cross of a resistant double-haploid line (Anju) with a susceptible double-haploid line (GC). In the nine linkage groups obtained (O1-O9), the major QTL, pb-Bo(Anju)1, was derived from Anju with a maximum LOD score (13.7) in O2. The QTL (LOD 5.1) located in O5, pb-Bo(GC)1, was derived from the susceptible GC. Other QTLs with smaller effects were found in O2, O3, and O7. Based on common markers, it was possible to compare our finding CR-QTLs with the B. oleracea CR loci reported by previous authors; pb-Bo(GC)1 may be identical to the CR-QTL reported previously or a different member contained in the same CR gene cluster. In total, the markers linked to seven B. rapa CR genes were mapped on the B. oleracea map. Based on the mapping position and markers of the CR genes, informative comparative studies of CR loci between B. oleracea and B. rapa were performed. Our map discloses specific primer sequences linked to CR genes and includes public SSR markers that will promote pyramiding CR genes in intra- and inter-specific crosses in Brassica crops. Five genes involved in glucosinolates biosynthesis were also mapped, and GSL-BoELONG and GSL-BoPro were found to be linked to the pb-Bo(Anju)1 and Bo(GC)1 loci, respectively. The linkage drag associated with the CR-QTLs is briefly discussed.     

Flow cytometric evidence for endopolyploidy in seedlings of some Brassica species

Flow cytometric analysis of the nuclear DNA contents of somatic tissues from seedlings of Brassica rapa L. and B. oleracea L. revealed extensive endoreduplication, resulting in tissues that contain cells with multiple ploidy levels (also called ’endopolyploidy’). Multiples of the haploid nuclear genome complement (1C) corresponding to 2C, 4C, 8C, 16C, 32C and 64C were observed in Brassica rapa, while B. oleracea exhibited a mixture of cells with five ploidy levels, 2C, 4C, 8C, 16C and 32C. The distribution of cells with the different ploidy levels was tissue-specific and characteristic of the stage of development. Multiploidy was not found in the embryos of dry seeds. Rapid endoreduplication occurred during seedling development. It is most probable that multiploidy is, if not a general feature, at least very common in Brassica species. The physiological and genetic implications of this original feature are discussed. Received: 17 March 2000 / Accepted: 17 April 2000     

Quantification and Localization of Bacteria in Plant Tissues Using Quantitative Real-Time PCR and Online Emission Fingerprinting

In order to quantify and localize specific bacterial target genes in plant tissue, this project has generated relevant new insights in the combined application of quantitative real-time PCR in parallel with the in situ PCR + probe-hybridization and online emission fingerprinting using LSM 510 META. After designing an Enterobacter radicincitans species-specific probe, introduced bacterial cells were monitored in growing plant parts and their colonization behaviour was examined in relation to the native bacterial community. For this purpose, the plant growth-promoting rhizobacterial (PGPR) strain Enterobacter radicincitans was applied to Brassica oleracea plants in increasing inoculum concentrations 10⁷, 10⁸ and 10⁹ cells per plant. Inoculation of 10⁹ E. radicincitans cells per plant to Brassica oleracea leaves and roots resulted in significant increases of root, leaf and tuber growth. Total bacterial cell numbers were estimated using quantitative real-time PCR to be between 10⁷ and 10⁹ cells g⁻¹ fresh leaf weight and about 10⁸ cells g⁻¹ fresh root weight of Brassica oleracea plants. Using quantitative real-time PCR, a significant colonization of Brassica oleracea leaves and roots with E. radicincitans cells was measured. Roots were colonized with a density of 10⁷ cells g⁻¹ fresh root weight up to at least 14 days after inoculation. That is equivalent to a proportion of E. radicincitans 16S rDNA-gene copy numbers compared to the total bacterial communities of about 10–16%. Online emission fingerprinting established that the introduced bacteria proliferated on and inside the root and that they colonized the intercellular spaces of the root cortex layer. Hence, E. radicincitans was able to successfully compete with the native bacterial population.     

Analysis of self-incompatibility interactions in 30 resynthesized Brassica napus lines. I. Fluorescence microscopic studies

Thirty Brassica napus lines have been developed through interspecific hybridization of B. oleracea and B. campestris lines with defined S-allele constitutions. These lines, which represent 29 different S-allele combinations, were tested in a diallel of test-pollinations to determine the activity of the introgressed S-alleles and intergenomic dominance relationships. Some consistent trends were observed: B. oleracea S-alleles high in the dominance series (e.g. S₈, S₁₄, S₂₉) were always active in the resynthesized B. napus lines, whereas recessive S-alleles (S₂, S₁₅) lost their activity in some test combinations. The B. campestris S-alleles were active in most cases, although 2 alleles were partially inactivated by the recessive B. oleracea allele S₁₅.     

‘Alperujo’ compost amendment of contaminated calcareous and acidic soils: Effects on growth and trace element uptake by five Brassica species

The effects of ‘alperujo’ compost on trace element availability and on microbial activity of two contaminated soils, a calcareous soil (S1) with high contents of Pb and Zn, and an acidic soil (S2) with a substantial amount of Al, As, Pb and Zn, were assessed. Additionally, the growth and capacity for contaminant phytoextraction of five Brassica species were studied. Compost amendment did not affect S1, but in S2 it increased soil pH, thus reducing Al and Zn bioavailability and toxicity. Compost application also increased microbial population and bioactivity in both soils. Brassica plants did not survive in S2, yet they thrived in S1. When compost was applied to S2, Brassica carinata, Brassica napus and Brassica oleracea grew adequately. Considering both the capacity to accumulate trace elements in the shoot and the ability to grow in the contaminated soils tested, the most efficient phytoextractors were Brassica juncea in S1 (particularly for Zn) and Brassica oleracea in S2 (for Al, As, Pb and Zn).     

Characterisation of disease resistance gene-like sequences in Brassica oleracea L.

Several cloned disease resistance genes from a wide range of plant species are known to share conserved regions with similar structural motifs. Degenerate primers based on conserved sequences of the nucleotide binding site of the genes RPS2, N and L6 were used for polymerase chain reaction (PCR) amplification from genomic DNA of two doubled haploid lines of Brassica oleracea. Sequences of amplified products were highly variable, but most of them showed similarity to known disease resistance genes, including RPS5, RPS2 and N, and to disease resistance gene-like sequences (RGLs) from different species. Primers based on B. oleracea sequences amplified five groups of RGLs. Products were mapped through cleaved amplified polymorphic sequence assays onto four different linkage groups of B. oleracea. PCR amplification from cDNA and allele analysis indicated that four locus-specific RGL fragments are expressed in cauliflower. Screening of a B. oleracea bacterial artificial chromosome library (BAC) with four B. oleracea RGL probes identified a small number of clones, suggesting that the four RGLs may not be highly copied. Screening of a BAC library of A. thaliana with the same probes identified clones that mapped onto four different chromosomes. These map positions correspond to known disease resistance loci of A. thaliana. Received: 12 November 1999 / Accepted: 19 June 2000