Spread of recombinant DNA by roots and pollen of transgenic potato plants,identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp

Transgenic potato plants with the nptII gene coding for neomycin phosphotransferase (kanamycin resistance) as a selection marker were examined for the spread of recombinant DNA into the environment. We used the recombinant fusion of nptII with the tg4 terminator for a novel biomonitoring technique. This depended on natural transformation of Acinetobacter sp. strain BD413 cells having in their genomes a terminally truncated nptII gene (nptII'; kanamycin sensitivity) followed by the tg4 terminator. Integration of the recombinant fusion DNA by homologous recombination in nptII' and tg4 restored nptII, leading to kanamycin-resistant transformants. DNA of the transgenic potato was detectable with high sensitivity, while no transformants were obtained with the DNA of other transgenic plants harboring nptII in different genetic contexts. The recombinant DNA was frequently found in rhizosphere extracts of transgenic potato plants from field plots. In a series of field plot and greenhouse experiments we identified two sources of this DNA: spread by roots during plant growth and by pollen during flowering. Both sources also contributed to the spread of the transgene into the rhizospheres of nontransgenic plants in the vicinity. The longest persistence of transforming DNA in field soil was observed with soil from a potato field in 1997 sampled in the following year in April and then stored moist at 4 degrees C in the dark for 4 years prior to extract preparation and transformation. In this study natural transformation is used as a reliable laboratory technique to detect recombinant DNA but is not used for monitoring horizontal gene transfer in the environment.     

Spread of Recombinant DNA by Roots and Pollen of Transgenic Potato Plants, Identified by Highly Specific Biomonitoring Using Natural Transformation of an Acinetobacter sp.

Transgenic potato plants with the nptII gene coding for neomycin phosphotransferase (kanamycin resistance) as a selection marker were examined for the spread of recombinant DNA into the environment. We used the recombinant fusion of nptII with the tg4 terminator for a novel biomonitoring technique. This depended on natural transformation of Acinetobacter sp. strain BD413 cells having in their genomes a terminally truncated nptII gene (nptII′; kanamycin sensitivity) followed by the tg4 terminator. Integration of the recombinant fusion DNA by homologous recombination in nptII′ and tg4 restored nptII, leading to kanamycin-resistant transformants. DNA of the transgenic potato was detectable with high sensitivity, while no transformants were obtained with the DNA of other transgenic plants harboring nptII in different genetic contexts. The recombinant DNA was frequently found in rhizosphere extracts of transgenic potato plants from field plots. In a series of field plot and greenhouse experiments we identified two sources of this DNA: spread by roots during plant growth and by pollen during flowering. Both sources also contributed to the spread of the transgene into the rhizospheres of nontransgenic plants in the vicinity. The longest persistence of transforming DNA in field soil was observed with soil from a potato field in 1997 sampled in the following year in April and then stored moist at 4°C in the dark for 4 years prior to extract preparation and transformation. In this study natural transformation is used as a reliable laboratory technique to detect recombinant DNA but is not used for monitoring horizontal gene transfer in the environment.     

Manganese dynamics in the rhizosphere and Mn uptake by different crops evaluated by a mechanistic model

Understanding of the mechanisms of Mn supply from the soil and uptake by the plants can be improved by using simulation models that are based on basic principles. For this, a pot culture experiment was conducted with a sandy clay loam soil to measure Mn uptake by summer wheat (Triticum aestivum L. cv. Planet), maize (Zea mays L. cv. Pirat) and sugar beet (Beta vulgaris L. cv. Orbis) and to simulate Mn dynamics in the rhizosphere by means of a mechanistic model. Seeds of three crops were sown in pots containing 2.9 kg soil in a controlled growth chamber. Root and shoot weight, Mn content of plants, root length and root radius were determined 8 (13 days in case of sugar beet) and 20 days after germination. Soil and plant parameters were determined to run nutrient uptake model calculations. Manganese content of the shoot varied from 25 mg kg^-1 for sugar beet to 34 mg kg^-1 for maize. Sugar beet had the lowest root length/shoot weight ratio but the highest relative shoot growth rate, resulting in the highest shoot demand on the root. This is reflected by the Mn influx which was 0.9 × 10^-7, 1.7 × 10^-7 and 2.5 × 10^-7 nmol cm^-1 s^-1 for wheat, maize and sugar beet, respectively. Nutrient uptake model calculations predicted similar influx values. Initial Mn concentration of 0.2 μM in the soil solution decreased to only 0.16 μM for wheat, 0.13 μM for maize and 0.11 μM for sugar beet at the root surface. This shows that manganese transport to the root was not a limiting step. This was confirmed by the fact that an assumed 20 times increase in maximum influx (I_max) increased the calculated Mn influx by 3.7 times. Sensitivity analysis demonstrated that for controlling Mn uptake the initial soil solution concentration (C _Li), the root radius (r₀), I_max and the Michaelis constant (K _m) were the most sensitive factors in the listed order. This revised version was published online in August 2006 with corrections to the Cover Date.     

Crop-weed interactions in the Beta vulgaris complex at a local scale: allelic diversity and gene flow within sugar beet fields

Crop-wild hybrids and weed beets are the main source of agronomic concern for sugar beet production all over Europe. In order to understand the dynamics of crop-wild interactions and the evolution of weediness in Beta vulgaris, we investigated genetic features of bolting individuals occurring at a local scale, i.e. within two sugar beet fields of the French northern area of sugar beet production. By analysing ploidy level, mitochondrial DNA and microsatellite polymorphism, the genetic diversity and the genetic relationships among three different classes of individuals (variety, in-row and out-row weed-beets) from a given field were examined. Such genetic analyses provide a unique opportunity to obtain evidence for the weeds origin and the evolutionary hypotheses previously stated. All the individuals shared in common the Svulg mitochondrial haplotype, and thus a common maternal origin. Conversely, the large genetic diversity at microsatellite loci highlighted the large diversity of the pollinator plants (cultivated and wild plants) during the-seed production process, as well as during the further evolution of weed beets in the sugar production area. Received: 23 April 2001 / Accepted: 15 June 2001     

Plastid DNA diversity in natural populations of Beta maritima showing additional variation in sexual phenotype and mitochondrial DNA

Summary Plants of two natural populations of Beta maritima, characterized by high percentages of male-sterile plants, have been investigated for organelle DNA polymorphism. We confirm the two classes of mitochondrial DNA variation previously described: (i) mitochondrial DNA (mtDNA) type N is associated with male fertility, whereas mtDNA type S can cause cytoplasmic male sterility (CMS); (ii) the 10.4-kb linear plasmid is observed in both types of mitochondria and is not correlated with the cytoplasmic male sterility occurring in this plant material. A third polymorphism is now described for chloroplast DNA (ctDNA). This polymorphism occurs within single populations of Beta maritima. Three different ctDNA types have been identified by HindIII restriction analysis. Among the plants studied, ctDNA type 1 is associated with N mitochondria and type 2 with S mitochondria. Chloroplast DNA type 3 has been found both in a fertile N plant and in a sterile S plant. This finding suggests that the chloroplast DNA polymorphism reported is not involved in the expression of male sterility. A comparison with Beta vulgaris indicates that ctDNA type 3 of Beta maritima corresponds to the ctDNA of fertile sugar beet maintainer lines. The three types of Beta maritima ctDNA described in this study differ from the ctDNA of male-sterile sugar beet.     

Intraspecific diversity of sugar beet (Beta vulgaris) mitochondrial DNA

Summary Mitochondrial (mt) DNA, isolated from different sugar beet populations, was analyzed using BamHI and EcoRI restriction enzymes. It was shown that plants possessing the new mtDNA types are revealed among O-type fertilizers quite frequently. Among cytoplasmic male sterile (cms) plants, which evolved during cultivation of O-type fertilizers, plants with altered mt genome were found.     

Bodenbewohnende Schädlinge und Seitenwurzelfäule der Zuckerrübe in Abhängigkeit von der Rotation – Ergebnisse eines Fruchtfolgeversuches

Soil inhabiting pests and rot of feeding roots of sugar beet depending on rotation - Results of a long-term trial Over a period of 17 years a trial was carried out with sugar beet, cereals and oilseed rape in different crop rotations on a field near Göttingen (Lower Saxony). The frequency of sugar beet in the rotation was 17, 25, 33 and 67 %. In absence of beet nematodes, root and sugar yield of the beet decreased after repeated growing of sugar beet in short rotations compared to variants with long rotations. Sugar content and beet quality were only slightly influenced. By applying a bioassay (BW-Test) with young beet plants in the greenhouse it was shown that increasing infections on the tips of rootlets of the beet plants were the cause for decreasing beet yield in close rotations. Mortality of young beet plants and progress of infection in the test indicated roughly the quantity of pathogenic fungi in the soil. In the roots of the bait platits the parasitic fungus Aphanomyces cochlioides predominated. Rate of infection and yield reduction in the field were decisively influenced by weather conditions. Differences in yield between sugar beet grown in a three-year and a four-year rotation, however, were not significant. An occurrence of beet pests depending on crop rotation was stated only for Atomaria linearis and this only in a few years.     

Helicotylenchus vulgaris and its association with damage to sugar beet

Severe and irregular stunting of sugar beet has been associated with Helicotylenchus vulgaris but little is known of the pathogenicity of this nematode. A survey was made at 10 sites in East Anglia on calcareous soils (Wantage series) on which the original damage was reported. Numbers were usually maintained under a variety of crops, despite fluctuations during the period. In field trials, aldicarb treatment did not improve the yield of either sugar beet or winter wheat on land infested with H. vulgaris. In pot tests growth of sugar beet was stimulated by low nematode inocula; this effect diminished as numbers inoculated increased. Sugar beet grown at 10 °C and 15 °C with H. vulgaris grew markedly better in sterilised soil than in untreated field soil, especially at the higher temperature early in the experiment. It is concluded that though H. vulgaris may damage sugar beet it requires an atypical combination of external conditions to cause significant yield loss.     

Control of the larvae ofConorhynchus mendicus (Col.: curculionidae) bySteinernema carpocapsae andSteinernema feltiae (nematoda,steinernematidae) in the gharb area (Morocco)

Two entomopathogenic nematodes,Steirnernema carpocapsae andS. feltiae were evaluated in the laboratory and under field conditions against larvae ofConorhynchus mendicus, a pest of sugar beet in the Gharb area of Morocco. Nematodes were applied in an artificially infested field using a knapsack sprayer either during the day or in the evening after sunset. The number of insect cadavers recovered from both soil and sugar beet plants was greater in the treated plots compared with the untreated ones. The percent reduction in live larvae was significantly greater in theS. carpocapsae treated plots (89.5% and 59.5% for night an day application respectively) compared with those treated withS. feltiae (68.5% and 28% respectively). The efficacy of entomopathogenic nematodes was therefore increased by application at night particularly in the case ofS. carpocapsae where control was maintained for 45 days.     

Genotypic Variation for Drought Tolerance in Beta vulgaris

Insufficient soil moisture during summer months is now the majorcause of sugar beet yield losses in the UK. However, selectionfor increased drought tolerance has not been a breeding priorityuntil recently. Genetic variation for drought tolerance is anessential prerequisite for the development of more stress-tolerantvarieties, but commercial sugar beet varieties seem to havesimilar yield responses to drought. The objective of this studywas to assess the degree of genotypic variation for droughttolerance within a wide range of sugar beet germplasm and genebankaccessions within Beta. Thirty sugar beet genotypes were screenedunder field drought conditions, and putative drought tolerantand sensitive lines (in terms of yield reduction in polythene-coveredvs. irrigated plots) were identified. Significant genotype xwater treatment interactions were found for dry matter yieldand relative leaf expansion rate. Genotypic differences fordrought susceptibility index were also significant. Differentialsensitivity of seedling shoot growth to water deficit was examinedby comparing 350 genebank accessions in a simple growth chamberscreen. Methods of data management were devised to highlightlines for entry into subsequent field tests. The results ofthe field and seedling screens indicate that there is variationfor tolerance to water deficits within sugar beet and relatedtypes, and that there are lines that show greater drought tolerancethan selected commercial varieties. Divergent lines showingcontrasting behaviour should aid in the identification of keymorpho-physiological traits that confer drought tolerance.     

Soil and plant water relations in a crested wheatgrass pasture: response to spring grazing by cattle

Summary Few field studies have attempted to relate effects of actual livestock grazing on soil and plant water status. The present study was initiated to determine the effects of periodic defoliations by cattle during spring on soil moisture and plant water status in a crested wheatgrass (Agropyron cristatum (L.) Gaertn. and A. desertorum (Fisch. ex Link) Schult.) pasture in central Utah. Soil moisture in the top 130 cm of the soil profile was depleted more rapidly in ungrazed plots than in grazed plots during spring and early summer. Soil moisture depletion was more rapid in grazed plots in one paddock after 1 July due to differential regrowth, but there was no difference in soil water depletion between plots in another paddock during the same period. This difference in soil water depletion between paddocks was related to a difference in date of grazing. Although more water had been extracted from the 60 cm to 130 cm depths in ungrazed plots by late September, cumulative soil moisture depletion over the entire 193 cm profile was similar in grazed and ungrazed plots. Prior to 1 July, grazing had no effect on predawn leaf water potentials as estimated by a pressure chamber technique; however, after 1 July, predawn leaf water potentials were lower for ungrazed plants. Midday leaf water potentials were lower for grazed plants before 1 July, but did not differ between grazed and ungrazed plants after 1 July. A 4- to 8-day difference in date of defoliation did not affect either predawn or midday leaf water potentials. The observed differences in water use patterns during spring and early-summer may be important in influencing growth and competitive interactions in crested wheatgrass communities that are subject to grazing by domestic livestock.     

Populations of Rhizoctonia solani in soil under crops in rotation with sugar beet

Using a soil debris isolation method, populations of Rhizoctonia solani were monitored over a 4 -yr period in four fields which were initially cropped to sugar beet and in which four areas of Rhizoctonia crown rot diseased beets (DA) and four areas of apparently healthy beets (AH) had been selected and precisely located. Soil from these areas was assayed during the subsequent crops, which included sugar beet, tomato, cucumber, maize and soybean. No significant differences in colony counts were found between the soils in DA and AH on any of 30 sampling dates. R. solani population counts were, in general, quite low, except under sugar beet and following tomato harvest. Areas of diseased beet and high R. solani soil populations that developed in subsequent sugar beet crops did not necessarily coincide with the previously selected diseased areas. High R. solani populations developed from parasitic activity on sugar beet or saprophytically on tomato crop residues. Of the other crops, both maize and soybean may have slightly increased the low R. solani residual populations in soil. The monitoring of R. solani populations in the season prior to, and during the early season of sugar beet cropping did not provide a basis for forecasting disease in fields or sites within fields. The initiation of disease patches in these sugar beet fields was therefore governed by factors other than inoculum density.     

Growth of crop roots in relation to soil moisture extraction

Growth of the roots of sugar beet, potato and barley in the field was observed through glass panels and related to changes in soil moisture measured by a neutron probe during 1969–71. The depth of observed root growth was generally related to, but 10–15 cm deeper than, the maximum depth of soil-moisture extraction. On average of three years, sugar beet, potato and barley used water from the top 23, 33 and 45 cm soil respectively by the beginning of June, and from the top 70, 68 and > 100 cm soil by the end of June. Maximum soil drying in each horizon gave an in situ measure of available water capacity, and showed that sugar beet and barley eventually extracted similar amounts of water from each horizon, but potatoes extracted less, especially from below 60 cm. Between 30 and 100 cm deep, the in situ available water capacity (per 10 cm soil) progressively decreased from 16 to 10, 15 to 5 and 16 to 8 mm under sugar beet, potato and barley respectively. The calculated soil-moisture deficit (potential evapotranspiration minus rainfall) and measured soil moisture deficit were not related early in the growing period before the crops established much leaf cover.     

On the Relationship Between Soil Moisture and Osmotic Potential in Maize and Sugar Beet Plants

Investigations on the relationship between soil moisture and maize and sugar beet plant osmotic potential were carried out during three years under field trial conditions. It was established that between soil moisture and osmotic potential there is an inverse ratio dependence, expressed in significant correlation coefficients (r =?0.84** for maize and r =?0.88** for sugar beet on an average). The correlation appears more striking in the lower third of the active moisture range, where slight variations in soil moisture determine important changes in osmotic potential. The possibility of using the osmotic potential as an index in estimating plant water requirement is asserted and the limit values indicating a good water supply in maize and sugar beet plants are established according to growth stages.     

Curly Top of Cultivated Plants and Weeds and Report of a Unique Curtovirus from Iran

The incidence of curly top disease on cultivated plants and weeds was investigated in Kerman Province (southeastern Iran) from October 2003 to November 2004. A total of 1186 samples were collected in fields of sugar beet and other crops as well as within commercial plastic houses. Curtovirus infection of four field crops, three vegetables and 11 weeds was verified by indirect enzyme‐linked immunosorbent assay (ELISA) using a polyclonal antibody. An undescribed curtovirus, tentatively designated Iranian beet curly top virus (IBCTV), was isolated from three symptomatic beet samples collected randomly in widely separated regions of south‐eastern, southern and central Iran and used for molecular studies. A 672 bp segment of the coat protein (CP) gene of each isolate was amplified by PCR and sequenced. The results showed that the three isolates shared 98.5–98.7% nucleotide homology with each other but only 72.1–76.5% with other members of the genus Curtovirus. IBCTV was also detected by PCR using specific primers in other samples of sugar beet, tomato, spinach, turnip and several weed species collected in different parts of Iran. These results indicated that IBCTV is the dominant curtovirus in Iran.     

Comparison of chloroplast and mitochondrial DNA from five morphologically distinct Beta vulgaris cultivars: sugar beet,fodder beet,beet root,foliage beet,and Swiss chard

Summary Two cytoplasms, N and S, are used in the breeding of sugar beet, Beta vulgaris var. altissima. These cytoplasms can be distinguished by their mitochondrial DNA. In an attempt to detect new cytoplasms, we compared the restriction profiles of chloroplast and mitochondrial DNA from five different cultivars of Beta vulgaris. All restriction patterns of chloroplast DNA were identical. With the exception of sugar beet with S-cytoplasm, all cultivars studied showed the same restriction profile of mitochondrial DNA, indicating that these cultivars all contain the N-cytoplasm. These results are discussed with regard to the large morphological differences of the cultivars and the cytoplasmic variability found in natural populations of the wild beet, Beta maritima.     

The effect of soil fumigation with D-D on the yields of sugar beet and other crops

An experiment in a field where sugar beet in 1965 had suffered from Docking disorder caused by Longidorus attenuatus tested the effect of fumigating the soil with 3741/ha D-D and two amounts of nitrogen fertilizer on different crop sequences between 1966 and 1969. Although severe Docking disorder did not recur in sugar beet, fumigation increased yield in each of the three following years. Yield of barley was increased for 4 yr and of wheat, potatoes and ryegrass for 1 or 2 yr after treatment. All plant parasitic nematodes were controlled by the first fumigation and the numbers of those in unfumigated plots 3 yr after treatment. Fumigation also largely prevented infection of sugar beet by the fungus Helicobasidium purpureum.     

Mapping of a chloroplast RFLP marker associated with the CMS cytoplasm of sugar beet (Beta vulgaris)

The Owen cytoplasm of male-sterile sugar beet is associated with several alterations of mitochondrial DNA and one additional HindIII site of chloroplast DNA. The region of this HindIII site has been cloned and sequenced. The site maps in a small reading frame (orf32) close to the ycf7 (orf31) gene in the petG-psbE region of chloroplast DNA. Possible functional implications of the results are discussed. The chloroplast RFLP marker described could be useful for studies on chloroplast-mitochondrial interactions, CMS of sugar beet, and the origin of the Owen cytoplasm.