L. R. Mytton

Expression of insecticidal activity in Rhizobium containing the δ-endotoxin gene cloned from Bacillus thuringiensis subsp. tenebrionis

Bacillus thuringiensis subsp. tenebrionis produces a 65 kilodalton polypeptide toxin which is lethal to various coleopteran insect larvae. The gene encoding this toxin was cloned in E. coli in the broad host range vector pKT230 and subsequently transferred to Rhizobium leguminosarum by conjugation. Western blot analysis showed that the toxin gene was expressed in the free living state of Rhizobium producing two major polypeptides of 73 and 68 kilodalton in size. The level of expression of the toxin gene in Rhizobium varied from strain to strain. Cell extracts from toxin-producing rhizobia were toxic to larvae of Gasterophysa viridula. Bioassays also showed that the δ-endotoxin was toxic to larvae of the clover weevil Sitona lepidus. Furthermore, pea (Pisum sativum) and white clover (Trifolium repens) plants suffered less root and nodule damage by Sitona larvae when they were inoculated with Rhizobium strains containing the toxin gene. This suggests that such rhizobia could be useful in the biological control of this important legume pest.

Developing a breeding strategy to exploit quantitative variation in symbiotic nitrogen fixation

SummaryThis paper examines evidence which quantifies the relative importance of legume and Rhizobium genotypes as determinants of phenotypic variation in symbiotic nitrogen fixation. It demonstrates potentially large and unpredictable effects of the Rhizobium genotype. The likely importance of such effects on crop yield is considered. The information is then used to assess ways in which legume breeding programmes may be altered to encompass the effects of genetic variation in Rhizobium.

Reply to ‘Measurement of nitrogenase activity in legume root nodules: In defense of the acetylene reduction assay’ by J.K. Vessey

This article is in response to that of Vessey (1994) who argues that the traditional, closed acetylene reduction assay can still be a valuable tool for measuring relative differences in nitrogenase activity of legumes. To counter this assertion we consider the practical uses of the traditional assay procedure in relation to real research situations. This requires the use of the assay to be considered separately in the different circumstances of pot-grown and field-grown plants. We conclude that for pot-grown legumes there are a few practical applications where the use of the traditional, closed assay procedure is valid and we accept that these can be extended by the careful use of calibrations against open, flow-through systems. However, we doubt that there are many situations where such a calibration approach would have practical advantages over using the flow-through system to obtain the actual measurements. We cannot recommend any form of the uncalibrated acetylene reduction assay for field-based studies and suggest that researchers consider the merits of simple, alternative measurements such as dry weight, yield and total nitrogen.

The potential for breeding white clover (Trifolium repens L.) with improved nodulation and nitrogen fixation when grown with combined nitrogen

SummarySodium nitrate applications ranging from 0.36 to 22.84 mM N were shown to depress rates of nodule formation and reduce total nitrogen fixation (acetylene reduction) in white clover plants grown in aseptic test tube culture.Low nitrate levels gave an initial depression in symbiotic activity but the reduction was of short duration and these treatments were subsequently associated with enhanced rates of nodule formation and nitrogen fixation. As a result, phenotypic variation appeared to be strongly differentially affected by the amount of nitrate present. A subsequent experiment suggested that much of the variation was a consequence of early enhancement of plant growth rates by low levels of nitrate followed by rapid depletion thus giving a transitory inhibitory effect. This was confirmed in a third experiment in which the range of nitrate concentration was held constant. Differential effects on variability in nodule formation and nitrogen fixation were then greatly reduced but there was still a residual level of plant-to-plant variation. The results have clear implications for selecting genetic variants capable of fixing di-nitrogen in the presence of combined N. The provision of a single limiting dose of combined nitrogen to a population containing individuals with inherently different growth rates can bring about variations in the phenotypic expression of symbiotic characters. These variations are unlikely to be based on genetic factors which have a direct and stable effect on nodule development and nitrogenase activity. The implications of the results for plant breeding are discussed.

The physiology and biochemistry of cultivar-strain interactions in the white clover-Rhizobium symbiosis

Two white clover cultivars were inoculated with two Rhizobium leguminosarum bv. trifolii strains in a factorial series of experiments. Plants were grown in axenic conditions in nitrogen free nutrient solution in a controlled environment room. Variations in nitrogen fixation were dependent partly upon general strain effects, partly upon general cultivar effects but there were also substantial differences attributable to precise interactions between specific combinations. The physiological and biochemical basis of these differences was examined. There were variations in the onset of nodulation and nitrogenase (acetylene reduction) activity. The rate at which nitrogenase activity developed also differed between associations as did the average size and number of nodules but none of these effects correlated well with differences in plant dry matter accumulation. Studies on nodule biochemistry revealed that the major nitrogen fixation enzymes were present in all four associations. Nodule protein content and enzyme activity (on a g nodule fresh weight basis) were substantially greater in associations formed by the more effective strain but cannot explain the interactive effect on dry matter accumulation. The relevance of these data to our understanding of factors regulating variations in nitrogen fixation is discussed.

A secondary effect of transformation in Rhizobium leguminosarum transgenic for Bacillus thuringiensis subspecies tenebrionis δ-endotoxin (cryIIIA) genes. Part 2.

Abstract By introducing Bacillus thuringiensis subspecies tenebrionisδ-endotoxin genes (cryIIIA) into Rhizobium leguminosarum we have produced strains for the biological control of Sitona larvae. Comparisons between a transgenic and the parent strain show that transformation has induced changes not associated with the intended function of the transgene. Although growth rates in laboratory cultures are similar for both strains, the ability to compete for nodule occupancy is greater in the transgenic than in the non-transformed parent strain. This result demonstrates the importance of studying ecological and agronomic characters of transgenic micro-organisms that could have a bearing on the safety and success of their release into the environment, even if they are not thought to be connected with the transgenes introduced.

The effect of toxin-producing Rhizobium strains, on larvae of Sitona flavescens feeding on legume roots and nodules

Larvae of the weevil Sitona spp. specifically eat the root nodules formed on legume plants by the soil bacterium Rhizobium. This can adversely affect the nitrogen fixing activity in the root nodules and lead to decreases in yield. Transgenic rhizobia were used in a novel approach to the biological control of Sitona. Two transcriptional fusions were made in which the coding sequence of the insecticidal crystal protein gene (cryIIIA) from Bacillus thuringiensis subsp. tenebrionis was fused to either the promoter of the rhizosphere enhanced rhiA gene from R. leguminosarum biovar viciae, or the promoter of the nodule specific nifH gene from R. leguminosarum bv. trifolii. The two chimeric genes were transferred to R. leguminosarum bv. viciae and R. leguminosarum bv. trifolii, respectively. Bioassay systems which allowed easy inspection of the plant root systems were used to test the transgenic strains for their ability to protect their host plant from damage by larvae of Sitona flavescens. In both white clover and pea plants nodulated by the transgenic rhizobia a slightly smaller proportion of root nodules were damaged compared to the wild type control plants. In some of the bioassays this was accompanied by increased nitrogenase activity, but it was not reflected in increased plant growth, which was similar in both treatments. These results suggest that a toxin with much higher toxicity against Sitona larvae is necessary for this approach to biocontrol to succeed.

The response of white clover (Trifolium repens L.) seedlings to spring root temperatures: The relative roles of the plant and the Rhizobium bacteria

Three experiments are reported which examine the relative roles of host and Rhizobium genotypes as factors limiting clover (Trifolium repens L.) growth at low soil temperatures.In the first experiment un-nodulated clover and perennial ryegrass (Lolium perenne L.) were grown with non-limiting nitrate at root temperatures of 8, 10 and 12°C. The ryegrass had substantially better relative growth rates (RGR) than the clover with the biggest difference occurring at 8°C. Alterations in growth rate with temperature were more marked in clover than in ryegrass but the latter still produced several times more dry matter than clover at each temperature.In the subsequent experiments clover nodulated with different strains of rhizobia was grown with and without non-limiting additions of nitrate at root temperatures of 9, 12 and 15°C. Plants receiving nitrate generally produced more dry matter than those dependent upon Rhizobium for nitrogen but differences in yield between these treatments did not alter with temperature. This suggests that limitations imposed by nitrogen fixation are similar at both high and low temperatures. Indeed, there was some evidence that nitrogen limitations were rather more pronounced at the highest temperature. The first experiment clearly demonstrated that the clover genotype makes particularly poor use of nitrate at low root temperatures when compared to its common companion perennial ryegrass.It can be concluded that improvements in spring growth of clover will rest largely with alterations to the plant genotype and its ability to use combined nitrogen for growth at lower temperatures rather than with changes in rhizobia or any symbiotic characters.

The nitrogen fixing potential ofVicia faba rhizobia (R. leguminosarum) from different agricultural locations

SummaryThe size and symbiotic effectiveness, withVicia faba, ofRhizobium leguminosarum populations from five locations in southern Britain has been estimated. Population numbers varied from 4.54×103 to 1.69×105. Nitrogen fixing potential differed by up to 30%. The implications of the results for improving the productivity of field beans are discussed.

Nitrogen limitations to field bean productivity: A comparison of combined nitrogen applications with Rhizobium inoculation

SummaryNitrogen limitations to the yield of a field crop ofVicia faba have been examined. Application of nitrogen totalling 560 kg/ha increased dry matter yield at flowering by 674 kg/ha (32%) and grain yield at final harvest by 1.6 tonnes/ha (24%). Attempts to reduce nitrogen limitations by replacing the native rhizobia with strains ofRhizobium leguminosarum selected for high rates of nitrogen fixation were unsuccessful but the introduction of poor rhizobia reduced grain yield. The reasons for this and the implications of the results for crop improvement are discussed.