Rape Seedlings Research Articles

Induction of nodule-like structures on oilseed rape by inoculation with rhizobia in the presence of helper bacteria

Abstract Nodule-like structures on the roots of seedlings of oilseed rape (Brassica campestris L. subsp. napus) cv. CO were induced by Bradyrhizobium sp. strain CP283 or A2P5 in the presence of Erwinia carotovora MAFF-301393, a bacterium that produces the cell wall-degrading enzymes cellulase and pectinase. E. carotovora was most effective at an inoculation density of 2.0 × 108 cells plant-1. Lower densities decreased the rate of nodule-like structure formation, while higher densities caused plant death. The induced nodule-like structures were either spherical or elongated. Histological sections of the structures showed a distinct central vascular cylinder and a surrounding swollen region composed entirely of cortical cells. The highest rate of nodule-like structure formation (33.3%) was achieved by combined inoculation of B. sp. strain CP283 and E. carotovora.

Amino acid sequences of three acyl-binding/lipid-transfer proteins from rape seedlings

The complete amino acid sequence of three acyl-binding/lipid-transfer proteins, AB/LTP I, AB/LTP II and AB/LTP III from germinated rape seeds were determined. AB/LTP I and AB/LTP II consist of 93 residues and the M r was determined as 9408 by mass spectrometry and calculated as 9406.8 from the sequence. AB/LTP III consists of 92 residues and the M r was determined as 9424 by mass spectrometry and calculated as 9422.8 from the sequence. The primary structures were determined by automated Edman degradations of the intact proteins and peptides obtained from digestion with trypsin and endoproteinase Asp-N and cyanogen bromide cleavage. Use of 252Cf plasma-desorption mass spectrometry facilitated the identification and verification of peptides.

Effects of methods of cereal straw disposal, seedbed preparation and sowing method on the establishment, yield and oil content of winter oilseed rape (Brassica napus)

SUMMARYIn the first of two series of experiments the effects of barley straw disposal by burning, chopping and spreading or baling and removing on winter oilseed rape were tested after seedbed preparation either by ploughing in the residue or incorporation in the soil by tine cultivation. These treatments were compared in four field experiments on silty clay loam soils at Rothamsted, UK from 1986 to 1989. The winter rape was either sown in late August or mid-September in seedbeds where either none or 50 kg N/ha had been applied.In the first season, August-sown rape was successfully established after tine cultivation but very dry conditions prevented seedbed preparation after ploughing, consequently all ploughed treatments were sown in September. Continuing dry conditions delayed emergence of the September-sown crop, the resultant small rape seedlings suffered substantial winter kill in some treatments during a period of abnormally low temperatures. Yield from the August-sown treatments was large (c. 40 t/ha) and showed no effect of straw disposal treatment or additional seedbed N. The yield of the September-sown crop was influenced by the amount of winter kill sustained; the smallest yields resulted from tine incorporation, and the largest after ploughing, where they approached those of the early sown crop.In the following three seasons more plants emerged from September than from August sowings. The application of seedbed N increased the plant population of the August-sown crop where the straw had been burnt. Plant losses over winter ranged from 15 to 20% and were unaffected by straw disposal treatment. There were significant differences in yield resulting from season and sowing date. Incorporating chopped straw by tine cultivation significantly decreased yield which, coupled with a lower oil content in the September-sown crop, gave a significantly smaller oil yield.In a second series of field experiments from 1987 to 1989, the effects of improving the timeliness of rape establishment after winter wheat by broadcasting rape seed into standing wheat was compared with conventional sowing after preparing a post-harvest seedbed. After cereal harvest, straw was disposed of either by baling and removing or chopping and spreading over the rape seed. The application of 50 kg N/ha to the stubble or seedbed was also tested, as was the effect of increasing the seed rate from 8 to 16 kg/ha in two seasons.There were always fewer plants established from broadcasting than from drilling. Generally there were fewer winter losses from broadcast seed than from drilled. Applying N to seedbed or stubble had no effect on plant population or survival except where 16 kg/ha seed was sown in 1989 and increased yield in two of the three seasons.In spite of a smaller plant population from broadcast seed, yields were often larger from broadcast than drilled treatments. On average broadcasting the seed and baling the straw gave the largest yield although this was significant only in 1989.

Root-colonization ability of antagonistic Streptomyces griseoviridis

Root-colonization ability of Streptomyces griseoviridis was tested on turnip rape (Brassica rapa subsp. oleifera) and carrot (Daucus carota) by the plate test and the sand-tube method. In the plate test, colonized root length of total root length was highly significantly greater for turnip rape roots (72%) from those for carrot roots (1%). In the sand-tube method, root-colonization ability was examined in nonsterile soil, and no water was added after sowing. Seeds were treated with spores of S. griseoviridis or the biofungicide Mycostop. Roots were cut into 2-cm segments, and the root segments and the rhizosphere soil were studied separately. Root-colonization frequencies and population densities of the microbe in the rhizosphere soil indicated that S. griseoviridis successfully colonized turnip rape but weakly colonized carrot. Root-colonization of turnip rape is accounted for as proliferation of S. griseoviridis in the rhizosphere of turnip rape seedlings and is not due to the movement of microbe through the rhizosphere by water infiltration.

Acidification in the Rhizosphere of Rape Seedlings and in Bulk Soil by Nitrification and Ammonium Uptake

AbstractAmmonium salts used as fertilizers may cause soil acidification by two different processes: nitrification in soil and net release of protons from roots. Their influence on soil pH may vary depending on the distance from root surface. The aim of this study was to distinguish between these two processes. For this purpose rape seedlings were grown 10 d in a system which separated roots from soil by a fine‐meshed screen. As a function of distance from the plane root layer formed on the screen, pH, titratable and exchangeable acidity and NO3‐ and NH4‐nitrogen were determined. The soil, a luvisol from loess, was supplied with no N or (NH4)2SO4 either with or without a nitrification inhibitor (DCD). The bulk soil pH remained unaffected when no N or 400 mg NH4N kg−1 soil plus DCD was applied but it decreased from 6.6 to 5.8 without DCD. In contrast, rhizosphere pH decreased in all cases, mainly within a distance of 1 mm from the root plane only, but with gradients extending to between 2 and 4 mm into the soil. The strongest pH decrease, from 6.6 to 4.9, occurred at the root surface of plants treated with both NH4‐N and DCD where most of the mineral N remained as ammonium. In this case Al was solubilized in the rhizosphere as indicated by exchangeable acidity. Total soil acidity produced in the NH4 treatment without DCD was mainly derived from nitrification compared to root released protons. However, acidification of the rhizosphere was diminished by nitrification because nitrate ions taken up by the roots counteracted net proton release. It is concluded that nitrification inhibitors may reduce proton input from ammonium fertilizers but enhance acidification at the soil‐root interface which may cause Al toxicity to plants.

Acyl-binding/lipid-transfer proteins from rape seedlings, a novel category of proteins interacting with lipids

From rape ( Brassica napus) seedlings proteins able to bind fatty acids and their CoA-esters were purified by gel filtration and cation-exchange chromatography. Among the four proteins detected, one of them (peak IV) appeared purified to homogeneity. This protein is a monomer with a molecular mass of about 9 kDa, as estimated by gel filtration and by polyacrylamide gel electrophoresis. The isoelectric point of the rape protein was higher than 10.5 as determined by chromatofocusing. The pure rape protein appeared furthermore to be able to transfer several phospholipids (phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine) between membranes. The rape protein, having a multifunctional property, was thus called acyl-bind-ing/lipid-transfer protein (AB-LTP). In order to compare this protein to plant lipid-transfer proteins (LTPs), its structure was determined. The amino acid analysis of the rape AB-LTP revealed a high amount of alanine, an absence of histidine and tryptophan and the presence of eight cysteine residues. The N-terminal amino acid sequence of the rape protein revealed a high homology to plant LTPs. These observations led us to propose that the rape AB-LTPs belong to a category of plant proteins interacting with lipids and playing a role in the fatty acid dynamics.

Carbon Loss from the Roots of Forage Rape (Brassica napus L.) Seedlings Following Pulse-labelling with 14CO2

The loss of organic material from the roots of forage rape (Brassica napus L.,) was studied by pulse-labelling 25-d-old non-sterile sand-grown plants with 14CO2. The distribution of 14C within the plant was measured at 0, 6 and 13 d after labelling whilst 14 C accumulating in the root-zone was measured at more frequent intervals. The rates of 14C release into the rhizosphere, and loss of 14CO2 from the rhizosphere were also determined. These data were used to estimate the accumulative loss of 14C from roots and loss respiratory 14CO2 from both roots and associated micro-organisms. Approximately 17-19% of fixed 14CO2 was translocated to the roots over 2 weeks, of which 30-34% was released into the rhizosphere, and 23-24% was respired by the roots as 14 CO2. Of the 14C released into the rhizosphere, between 35-51% was assimilated and respired by rhizosphere micro-organisms.

Identification of Catabolites of Chlorophyll-Porphyrin in Senescent Rape Cotyledons.

Developing shoots of rape seedlings (Brassica napus L.) were excised and fed with 4-[14C]5-aminolevulinic acid to label the pyrroles in chlorophyll (Chl) synthesized during the final phase of expansion and greening of the cotyledons. About 80% of 14C taken up into the cotyledons was incorporated into Chl. The subsequent incubation of labeled shoots in permanent darkness caused the rapid loss of labeled Chl while increasing proportions of 14C appeared in the fraction of water-soluble compounds. Reversed-phase high performance liquid chromatography resolved three nonfluorescent polar catabolites of Chl-porphyrin that were progressively accumulated as senescence advanced. At intermediate stages of senescence, the cotyledons contained a fluorescent radio-active derivative of Chl that was also detectable, together with traces of other putative fluorescent catabolites, in isolated senescent chloroplasts. The nonfluorescent catabolites, identified by means of radiolabeling, were also found to accumulate in attached cotyledons senescing under photoperiod; under these conditions, one of the compounds, NCC-1, was particularly abundant. The catabolites of rape exhibited the same ultraviolet spectra, characterized by a maximum at 320 nm, as a previously reported secoporphinoid catabolite from barley (B. Krautler, B. Jaun, W. Amrein, K. Bortlik, M. Schellenberg, P. Matile [1992] Plant Physiol Biochem 30: 333-346). Different polarities suggest, however, that the structures may be different. A terminology for Chl catabolites is proposed because present knowledge suggests that a large number of different structures results from species-specific processing of breakdown products and may require a suitable nomenclature.

The Effect of Exogenous Sugar Supply and Nitrogen Deficiency on Dry Matter Allocation in Rape Seedlings Grown in vitro

Summary Dry matter allocation is a very good indicator of conditions of both the mineral nutrition of plants and the photosynthetic conditions in vivo. We determined the response of dry matter accumulation and shoot/root ratio of rape seedlings (Brassica napus L.) to different levels of sucrose supply and nitrogen deficiency in vitro. The dry weight of roots and shoots increased significantly with increasing concentration of sugar in the medium from 0 % to 9 % (w/v) but shoot/ root ratio decreased. Nitrogen deficiency caused a further decrease in the shoot/root ratio. The results show that exogenous sugar supply can induce a similar shift in partitioning of dry matter as favourable photosynthetic conditions and that the response to exogenous sugar is influenced by nitrogen deficiency.

Downstream DNA sequences are required to activate a gene expressed in the root cortex of embryos and seedlings.

We showed previously that a gene, designated AX92, which is expressed at an early stage of cortex differentiation in the root apex of oilseed rape seedlings, is also expressed in embryos. To compare AX92 gene regulation during embryo-genesis and postembryonic growth, we constructed a chimeric gene consisting of AX92 5' and 3' untranslated and flanking regions fused with a beta-glucuronidase protein coding region. We showed that the chimeric gene is active in both developing cortex cells in the root apical meristems of transgenic oilseed rape seedlings and in cortex cells at the root end of embryonic axes. To determine whether the AX92 gene is regulated by a common mechanism in embryos and seedlings, we analyzed the expression of modified chimeric genes. We showed that the AX92 chimeric gene is regulated combinatorially and that DNA sequences located 3' of the protein coding region are necessary for its activation in the root cortex of both embryos and seedlings. Our results suggest that common regulatory sequences are required to activate the gene in the embryonic and postembryonic root cortex.

Techniques for observing phosphorus mobilization in the rhizosphere

The techniques described here were developed to visualize the dissolution of sparingly soluble calcium phosphate and the presence of root-borne phosphatase in the rhizosphere. Newly formed calcium phosphate precipitate was suspended in agar containing other essential nutrients. The agar was poured into Petri dishes and acrylglass boxes and was used as a growth medium for seedlings of wheat, rape, buckwheat, and rice. With NH 4 + applied as the N source, the precipitate dissolved in the root vicinity and this was attributed to acidification. No dissolution occurred with NO 3 − as the N source. The release of a neutral phosphatase from roots was verified by embedding the roots of young seedlings in agar at pH 7 containing phenolphthalein phosphate. After pH was raised to the alkaline range by adding sodium hydroxide, the agar around the roots turned purple, especially around the roots of P-deprived plants. The most intensive phosphatase activity was found in apical root regions.

Frost-seeding of summer turnip rape in the subarctic

Previous research has indicated that spring-planted summer turnip rape (Brassica rapa L.) can produce good seed yields with high oil content in subarctic Alaska. However, short growing seasons often prevent seeds from reaching full maturity, resulting in high proportions of green seeds and lowered market quality. In an attempt to allow summer turnip rape seedlings to begin growth earlier in the growing season, we planted the seeds in frozen soil in late fall and early spring. Fall seeding of summer turnip rape always resulted in low plant populations, low seed yields, and high percentages of green seeds. Percentages of green seeds were highly variable and early spring seedings did not result in significantly lower percentages of green seeds than conventional (May) seedings. The effects of early spring frost-seeding on plant populations and seed yields were variable but only rarely did early spring seeding produce significantly higher seed yields than conventional seeding. Planting into barley (Hordeum vulgare L.) stubble resulted in poor stands which produced low seed yields. This study did not indicate any advantage from fall planting of summer turnip rape. Results from spring frost-seeding were not consistent; therefore, this practice cannot be recommended until these inconsistencies are better understood.Key words: Frost-seeding, summer turnip rape, subarctic, Alaska

Alternaria alternata from oilseed rape: mycotoxin production, and toxicity toArtemia salina larvae and rape seedlings

A survey, carried out in Southern Italy on fungi colonizing oilseed rape (Brassicae napua L subspoleifera DC) in the field, showedAlternaria alternata (Fries) Keissler as one of the predominant species. 11 strains ofAlternaria alternata isolated from oilseed rape were cultured on rice to test the ability to produce mycotoxins. All strains produced mycotoxins, including tenuazonic acid (up to 12,000mg/kg), alternariol (up to 200mg/kg), alternariol mortomethyl ether (up to 200mg/kg), altertoxin-l (2 to 250mg/kg), and altertoxin-ll (2 to 70mg/kg). Culture extracts containing tenuazonic acid were toxic toArtemia salina and inhibited seedling growth of oilseed rape. Little or no activity with the same bioassays was shown by culture extracts containing the remaining mycotoxins. No mycotoxins were detected in 16 samples of oilseed rape from processing factories.

Induction by chromium ions of chitinases and polyamines in barley ( Hordeum vulgare L.) and rape ( Brassica napus L. ssp. oleifera)

Barley and rape seedlings were grown in hydroponic culture with increasing concentrations of CrO 3 (Cr(VI)) of CrCl 3 (Cr(III)). The chitinase activity and the concentrations of putrescine, spermidine and spermine were determined in the third leaf of barley seedlings and in the second leaf of rape seedlings. With increasing concentrations of Cr(VI) (30–100 μg/l) a 5–10-fold increase in chitinase activity and 4–5-fold increase in putrescine concentrations were found in barley and rape seedlings after 4 days. Levels of spermidine and spermine were not consistently influenced by exposure to Cr(VI). Cr(III) at concentrations of 10–50 μg/ml did not significantly alter the concentrations of polyamines nor the chitinase activities in either species, however, at 100 μg/ml in barley Cr(III) induced an increase in putrescine concentration after 6 days of exposure. The induction of chitinases and the increases in the putrescine level caused by Cr(VI) but not by Cr(III) (10–50 μg/ml) exposure is similar in the two species suggesting an analogous defense system in both mono- and di-cotyledonous plants. The anionic form of Cr(VI) seems to be more potent than the cationic form of Cr(III). Grain of barley plants grown in enriched peat and watered with increasing concentrations of Cr(VI) (250, 1250, 6250 or 10 000 μg/ml) for 4 days from 10 days post anthesis (dpa) showed increasing contents of chitinases.

Phytohormonal changes in greening and senescing intact cotyledons of oilseed rape and pumpkin: influence of the growth retardant BAS 111 W

The triazole growth retardant BAS 111‥W delayed senescence in cotyledons of pumpkin (Cucurbita maxima L. cv. Gelbe genetzte Riesenmelone) and stimulated chlorophyll synthesis in greening cotyledons of oilseed rape (Brassica napus L. cv. Petranova) seedlings. In both cases, changes of phytohormone-like substances in the cotyledons were analyzed on a fresh weight basis by immunoassay. After soil treatment with increasing retardant concentrations, a close correlation was observed in senescing cotyledons of pumpkin between a reduced loss in total chlorophyll and increasing levels of dihydrozeatin riboside (DZR) and trans-zeatin riboside (ZR)-type cytokinins. In contrast, the levels of isopentenyladenosine (IPA)-type cytokinins, 3-indoleacetic acid (IAA) and gibberellin (GA) did not change significantly. The levels of abscisic acid (ABA) were slightly elevated at low retardant concentrations but dropped considerably below those of controls at higher doses. Consequently, the molar ratio of total cytokinin to ABA content changed from approximately 1:40 in controls (50% of initial chlorophyll) to 1:3 in cotyledons treated with 3 mg BAS 111‥W plant−1 (85% of initial chlorophyll). These changes, together with the known reduction of ethylene production by plants treated with nitrogen-heterocyclic retardants, can explain the delayed senescence in pumpkin cotyledons. Likewise, when etiolated, BAS 111‥W-treated seedlings of oilseed rape were exposed to light, the stimulation of chlorophyll synthesis in the cotyledons was accompanied by an accumulation of DZR- and, particularly, ZR-type cytokinins and IAA. In contrast, GA and ABA contents decreased slightly. We conclude that the influence of BAS 111‥W on cytokinin levels might be involved in the stimulation of greening.

Scanning electron microscopy of hyphal interaction between Streptomyces griseoviridis and some plant pathogenic fungi

The interaction between Streptomyces griseoviridis and the pathogens Alternaria brassicicola, Botrytis cinerea, Fusarium oxysporum, Mycocentrospora acerina, Rhizoctonia solani and Sclerotinia sclerotiorum was studied by SEM both on autoclaved seeds and living seedlings of turnip rape and carrot and the fungi Phomopsis sclerotioides and Pythium ultimum on cucumber seedlings. The samples were prepared by the standard method for examination by scanning electron microscope. The hyperparasitism of S. griseoviridis was clearly shown. S. griseoviridis tightly wound around Alternaria conidia and Sclerotinia hyphae, eventually disintegrating them. It grew along the hyphae of B. cinerea, P. sclerotioides and M. acerina, dissolving them. The hypha of F. oxysporum seemed to be slightly affected, and its conidia not at all. The hyperparasite grew only loosely on the hypha of R. solani and on the mycelium and oogonia of Pythium which seemed not to sustain much injury.

Conversion of SO2−4‐S and Changes in Glucosinolate Content in Leaves of Rape Seedlings Transplanted

AbstractThe conversion of SO2‐4 ‐S and changes in content of S in various constituents in leaves of rape seedlings transplanted were investigated by using (NH4)235SO4 as a tracer to exploit formation and accumulation of glucosinolates in oilseed rape. Seedlings grown under sandy culture absorbed 35SO2‐4 which was added to the cultural solution and incorporated into amino acids, glucosinolates and proteins rapidly. Distribution of extractable 35S with 70 % methanol in glucosinolates in leaves declined with time from labelling, while those in amino acids rised correspondingly. Per cents of 35S incorporated into bound form in total 35S increased linearly and those of 35S into glucosinolates and amino acids decreased with time within five days from labelling. After that the relative amounts of 3SS in three constituents was basically constant. Content on dry weight basis of labelled glucosinolates and amino acids expressed as μmol S/g.d.w. increased linearly with time from labelling with absorption of 35SO2‐4from soil by the seedlings under soil culture. Compared with seedlings grown under sandy culture, more 35S was incorporated into glucosinolates in leaves of seedlings grown under soil culture.

Comparative Analysis of Two Rearing Procedures for Diamondback Moth (Lepidoptera: Plutellidae)

Rape seedlings, Brassica napus L., and a wheat germ-based artificial diet were compared as media for rearing diamondback moth (DBM), Plutella xylostella (L.), for six generations. Mean pupal weight and total number of eggs laid per female were always greater when larvae were reared on artificial diet; however, percentage of eggs hatching was usually greater and development time usually shorter when larvae were reared on rape seedlings. High larval survivorship (>70%) could be obtained on either media. Larvae which were reared on artificial diet were consistently more susceptible to the insecticides methomyl and permethrin, indicating potential problems in using artificial diet for insecticide studies. When larvae were reared on either medium for six generations and then transferred to cabbage, larval survivorship was nearly equal, indicating that either method could be used for artificially incoculating plants for host plant resistance studies. Although it was easier and cheaper to rear DBM on artificial diet, recommendations for using one rearing method over the other must be based on the ultimate use of the colony.

Regulation of endogenous abscisic acid levels and transpiration in oilseed rape by plant growth retardants

Plant growth retardants of the norbornanodiazetine (e.g. tetcyclacis) and triazole (e.g. BAS 111. . W, LAB 150 978) type considerably increased endogenous abscisic acid (ABA) levels in cell suspension cultures, detached leaves and hydroponically grown seedlings of oilseed rape ( Brassica napus ). In detached leaves treated via the vascular system, ABA accumulated in proportion to retardant concentration and was closely correlated with a reduction in transpiration. Thus, the growth retardants appear to influence transpiration by regulating ABA metabolism. In suspension cells and shoots of seedlings, maximal ABA content was achieved 4 and 5 days after treatment, respectively. ABA accumulation may be due to a retardant-caused inhibition of the metabolism of ABA, presumably to phaseic acid, as suggested by [ 14 C] ABA feeding experiments with cell cultures. The initial rise in ABA levels was followed by a sharp decline, an effect that might be explained by a stimulated ABA catabolism and/or by an inhibition of its biosynthesis. In contrast, foliar application of BAS 111. . W to plants at the 4th leaf stage did not change either ABA levels in shoots nor water consumption per shoot fresh or dry weight analysed during 10 days after treatment. In leaves of soil treated plants at the 5th leaf stage ABA levels also remained unaffected or dropped below those of controls, especially at higher BAS 111. . W concentration. It is suggested that the growth retardants regulate ABA metabolism in oilseed rape, dependent on the mode of application and intensity, and duration of action.

Enzymatic fractionation of fatty acids: Enrichment of γ‐linolenic acid and docosahexaenoic acid by selective esterification catalyzed by lipases

Immobilized lipase preparations from seedlings of rape (Brassica napus L.) andMucor miehei (lipozyme) used as biocatalysts in esterification and hydrolysis reactions discriminate strongly against γ‐linolenic and docosahexaenoic acids/acyl moieties. Utilizing this property, γ‐linolenic acid contained in fatty acids of evening primrose oil has been enriched seven to nine‐fold, from 9.5 to almost 85% by selective esterification of the other fatty acids with butanol. Similarly, docosahexaenoic acid of cod liver oil has been enriched four to five‐fold, from 9.4 to 45% by selective esterification of fatty acids (other than docosahexaenoic acid) with butanol. As long as the reaction is stopped before reaching equilibrium, very little of either γ‐linolenic acid or docosahexaenoic acid are converted to butyl esters, which results in high yields of these acids in the unesterified fatty acid fraction.