Arbuscular mycorrhiza decreases cadmium phytoextraction by transgenic tobacco with inserted metallothionein

The effect of arbuscular mycorrhiza (AM) on the phytoextraction efficiency of transgenic tobacco with increased ability to tolerate and accumulate cadmium (Cd) was tested in a pot experiment. The tobacco plants bearing the yeast metallothionein CUP1 combined with a polyhistidine cluster were compared to non-transgenic tobacco of the same variety at four Cd concentrations in soil, non-inoculated or inoculated with two isolates of the AM fungus Glomus intraradices. Mycorrhizal inoculation improved the growth of both the transgenic and non-transgenic tobacco and decreased Cd concentrations in shoots and root to shoot translocation. Differences were found between the two AM fungal isolates: one isolate supported more efficient phosphorus uptake and plant growth in the soil without Cd addition, while the other isolate alleviated the inhibitory effect of cadmium on plant growth. The resulting effect of inoculation on Cd accumulation was dependent on Cd level in soil and differed between the more Cd tolerant transgenic plants and the less tolerant non-transgenic plants. Mycorrhiza mostly decreased the phytoextraction efficiency of transgenic plants while increased that of non-transgenic plants at Cd levels in soil inhibitory to tobacco growth. Mechanisms of the observed effects of inoculation on growth and Cd uptake are discussed as well as the possible implications of the results for the exploitation of AM in phytoextraction of heavy metals from contaminated soils.     

Limitations to the Potential of Transgenic Trifolium subterraneum L. Plants that Exude Phytase when Grown in Soils with a Range of Organic P Content

Growth and P-nutrition of transgenic Trifolium subterraneum L. which express a chimeric fungal phytase gene (ex::phyA) was compared to azygous and wild-type controls in a range of soils that differed in organic P content. Shoot and root growth by plant lines were measured and effects of reducing the influence of soil microorganisms were investigated by pasteurising the soils. Plants that expressed phyA did not have better P-nutrition than control plants after 56 days growth, except in a soil that contained a large concentration of both total organic P and organic P that was amenable to hydrolysis by a plant-derived phytase. Pasteurisation had little effect on the relative P-nutrition of the various plant lines in any of the soils. Roots of transgenic plants that expressed ex::phyA were shorter than controls up to 21 days growth in a number of soils, which resulted in an initial greater P accumulation efficiency. However, greater P accumulation efficiency was only maintained in the soil where significant growth and P nutrition responses were also observed. Availability of inositol phosphates in soil is a major factor that limits the effectiveness of expressing fungal phytase genes in plants as a means to improve P-nutrition. Reducing the influence of rhizosphere microorganisms appeared to have little effect on the P-nutrition of plant lines, but the longer root system produced by control plants may have initially provided them with greater access to soil P resources. This research highlights the inherent difficulty in improving the P-nutrition of plants by the manipulation of single traits in isolation, but does provide some evidence that such approaches can be successful under certain edaphic conditions.     

Overexpressing <Emphasis Type="Italic">SgNCED1</Emphasis> in Tobacco Increases ABA Level,Antioxidant Enzyme Activities,and Stress Tolerance

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses. 9-cis-epoxycarotenoid dioxygenase (NCED) is the key enzyme of ABA biosynthesis in higher plants. A NCED gene, SgNCED1, was overexpressed in transgenic tobacco plants which resulted in 51–77% more accumulation of ABA in leaves. Transgenic tobacco plants decreased stomatal conductance, transpiration rate, and photosynthetic rate but induced activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate-peroxidase (APX). Hydrogen peroxide (H₂O₂) and nitric oxide (NO) in leaves were also induced in the transgenic plants. Compared to the wild-type control, the transgenic plants improved growth under 0.1 M mannitol-induced drought stress and 0.1 M NaCl-induced salinity stress. It is suggested that the ABA-induced H₂O₂ and NO generation upregulates the stomatal closure and antioxidant enzymes, and therefore increases drought and salinity tolerance in the transgenic plants.     

Transgenically Enhanced Sorbitol Synthesis Facilitates Phloem Boron Transport and Increases Tolerance of Tobacco to Boron Deficiency

The mobility of elements within plants contributes to a plant species' tolerance of nutrient deficiencies in the soil. The genetic manipulation of within-plant nutrient movement may therefore provide a means to enhance plant growth under conditions of variable soil nutrient availability. In these experiments tobacco (Nicotiana tabacum) was engineered to synthesize sorbitol, and the resultant effect on phloem mobility of boron (B) was determined. In contrast to wild-type tobacco, transgenic tobacco plants containing sorbitol exhibit a marked increase in within-plant B mobility and a resultant increase in plant growth and yield when grown with limited or interrupted soil B supply. Growth of transgenic tobacco could be maintained by reutilization of B present in mature tissues or from B supplied as a foliar application to mature leaves. In contrast, B present in mature leaves of control tobacco lines could not be used to provide the B requirements for new plant growth. ¹⁰B-labeling experiments verified that B is phloem mobile in transgenic tobacco but is immobile in control lines. These results demonstrate that the transgenic enhancement of within-plant nutrient mobility is a viable approach to improve plant tolerance of nutrient stress.     

一株耐盐日本曲霉的筛选及其溶磷促生作用

【目的】从内蒙古种植葵花的盐碱地中筛选高效溶磷真菌,为盐碱地增产节肥开发生物肥料提供溶磷菌种资源。【方法】利用ITS r DNA序列鉴定菌株、固体培养基测定耐盐性,液体摇床培养与盆栽试验结合分析菌株溶磷能力,盆栽和田间试验明确菌株M1促进作物生长和增产作用;LC-MS技术测定菌株M1在液体培养基中分泌有机酸和植物激素含量,明确菌株M1的溶磷和促生机理。【结果】溶磷菌株M1鉴定为日本曲霉(Aspergillus japonicus)。液体培养基接种菌株M1培养6 d,以Ca_3(PO_4)_2为磷源时上清液有效磷达1020.89 mg/L,溶解率为63.30%;以AlPO_4为磷源时有效磷达995.69 mg/L,溶解率为48.59%;以贵州开阳磷矿粉、江苏锦屏磷矿粉、云南晋宁磷矿粉、河北钒山磷矿粉和云南昆阳磷矿粉为磷源接种菌株M1,从晋宁磷矿粉释放的有效磷浓度最高,达到363.64 mg/L。菌株M1可耐受10%NaCl。将M1制备的菌剂分别接种于施用Ca_3(PO_4)_2、AlPO_4和开阳磷矿粉3种磷源的4种盆栽试验土壤包括北京石灰性潮土、安徽黏性潮土、安徽水稻土和山东沿海盐潮土。结果显示,菌株M1对玉米植株促生效果显著,玉米植株鲜重比对照提高2.14%–90.91%、干重增加22.15%–268.28%;土壤有效磷提高21.81–24.27 mg/kg。菌株M1与4种土壤的适配性均高于对照菌株DSM 821。田间小区花生产量结果显示,接种溶磷菌剂M1增产效果最好,花生果实产量达4.46 t/hm~2,比不接种菌剂的对照处理增加0.81 t/hm~2,增产22.19%。菌株M1在含有磷酸三钙、磷酸铝和开阳磷矿粉3种难溶磷培养液中经过6 d培养,均产生7种有机酸,其中草酸和柠檬酸含量最高,分别为616.16 mg/L和413.69 mg/L;培养液均能检测到吲哚乙酸(IAA)和玉米素,IAA含量为15.45–77.58 mg/L,玉米素浓度为0.06–0.11 mg/L。【结论】获得了一株高效溶解多种难溶磷的日本曲霉菌M1,它能显著增加土壤有效磷、促进玉米生长和花生增产,与4种典型土壤适配性好,具有良好的农业应用前景。     

Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola

Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene according to the Brassica codon usage and an endoplasmic reticulum (ER) retention signal KDEL that confers an ER accumulation of the recombinant phytase. Secretion of the phytase to the extracellular fluid was also established by the use of the tobacco PR-S signal peptide. Phytase accumulation in mature seed accounted for 2.6% of the total soluble proteins. The enzyme can be glycosylated in the seeds of transgenic plants and retain a high stability during storage. These results suggest a commercial feasibility of producing a stable recombinant phytase in canola at a high level for animal feed supplement and for reducing phosphorus eutrophication problems.     

Expression of the rice cytoplasmic cysteine synthase gene in tobacco reduces ozone-induced damage

Cysteine serves as a precursor for the synthesis of various sulfur-containing metabolites, and the cysteine synthase (CS) gene plays a central role in the sulfur cycle in nature. In the present study, rcs1, a cytosolic CS gene of rice, was introduced into the genome of tobacco (Nicotiana tabacum). The tolerance of wild-type tobacco plants as well as of the resulting transgenic tobacco plants overexpressing the rcs1 gene to toxic levels of ozone (O₃, 0.15 μ mol⁻¹) was measured after various lengths of exposure. Leaf lesions in plants exposed for 2 weeks to O₃ were more prevalent in the leaves of the wild-type plants than in those of the transgenic tobacco plants. Transgenic tobacco plants showed a higher growth rate and a higher chlorophyll content than the wild-type plants. Cysteine synthase activity and cysteine and glutathione contents were higher in transgenic plants than in wild-type plants irrespective of the length of the O₃ treatment. Our results indicate that the CS gene plays a role in the protection of the plant against toxic O₃ gas, probably through the mechanism of an over-accumulation of such sulfur-rich antioxidants as cysteine and glutathione.     

Production and characterization of thermostable alkaline phytase from <Emphasis Type="Italic">Bacillus laevolacticus</Emphasis> isolated from rhizosphere soil

A novel phytase producing thermophilic strain of Bacillus laevolacticus insensitive to inorganic phosphate was isolated from the rhizosphere soil of leguminous plant methi (Medicago falacata). The culture conditions for production of phytase by B. laevolacticus under shake flask culture were optimized to obtain high levels of phytase (2.957 ± 0.002 U/ml). The partially purified phytase from B. laevolacticus strain was optimally active at 70 °C and between pH 7.0 and pH 8.0. The enzyme exhibited thermostability with ∼80% activity at 70 °C and pH 8.0 for up to 3 h in the presence/absence of 5 mM CaCl₂. The phytase from B. laevolacticus showed high specificity for phytate salts of Ca⁺ > Na⁺. The enzyme showed an apparent K _m 0.526 mM and V _max 12.3 μmole/min/mg of activity against sodium phytate.     

Ability of <Emphasis Type="Italic">Emericella rugulosa</Emphasis> to mobilize unavailable P compounds during Pearl millet [<Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.] crop under arid condition

Phosphate solubilizing microorganisms are ubiquitous in soils and could play an important role in supplying P to plants where plant unavailable P content in soil was more. A phosphatase and phytase producing fungus Emericella rugulosa was isolated and tested under field condition (Pearl millet as a test crop) in a loamy sand soil. In the experimental soil 68% organic phosphorous was present as phytin; less than 1% of phosphorous was present in a plant available form. The maximum effect of inoculation on different enzyme activities (acid phosphatase, alkaline phosphatase, phytase, and dehydrogenase) was observed between 5 and 8 weeks of plant age. The depletion of organic P was much higher than mineral and phytin P. The microbial contribution was significantly higher than the plant contribution to the hydrolysis of the different P fractions. A significant improvement in plant biomass, root length, seed and straw yield and P concentration of root and shoot resulted from inoculation. The results suggest that Emericella rugulosa produces phosphatases and phytase, which mobilize P and enhance the production of pearl millet.     

转枯草芽孢杆菌植酸酶基因烟草对不同介质中植酸磷的吸收利用

利用转入枯草芽孢杆菌植酸酶基因的不同烟草株系,分别在无菌培养基、砂培和土培试验中研究了转植酸酶基因烟草对植酸磷的吸收和利用.结果表明,在无菌培养基试验中,所有转植酸酶基因烟草对植酸磷的吸收利用能力均显著高于野生型,其生物量比野生型提高了3.6～10.7倍,总磷吸收量提高了2.2～4.6倍;在沙培和土培中,转植酸酶基因烟草对植酸磷的吸收利用与野生型相比,生物量和总磷吸收量差异不显著.这说明转植酸酶基因在无菌条件下可以提高植物吸收利用植酸磷的能力,但是在自然条件下,由于微生物分解或矿物固定等原因,其作用不稳定,需要进一步研究克服土壤中的限制因素,才能使转基因植物充分发挥作用.     

Overexpression of phyA and appA Genes Improves Soil Organic Phosphorus Utilisation and Seed Phytase Activity in Brassica napus

Phytate is the major storage form of organic phosphorus in soils and plant seeds, and phosphorus (P) in this form is unavailable to plants or monogastric animals. In the present study, the phytase genes phyA and appA were introduced into Brassica napus cv Westar with a signal peptide sequence and CaMV 35S promoter, respectively. Three independent transgenic lines, P3 and P11 from phyA and a18 from appA, were selected. The three transgenic lines exhibited significantly higher exuded phytase activity when compared to wild-type (WT) controls. A quartz sand culture experiment demonstrated that transgenic Brassica napus had significantly improved P uptake and plant biomass. A soil culture experiment revealed that seed yields of transgenic lines P11 and a18 increased by 20.9% and 59.9%, respectively, when compared to WT. When phytate was used as the sole P source, P accumulation in seeds increased by 20.6% and 46.9% with respect to WT in P11 and a18, respectively. The P3 line accumulated markedly more P in seeds than WT, while no significant difference was observed in seed yields when phytate was used as the sole P source. Phytase activities in transgenic canola seeds ranged from 1,138 to 1,605 U kg^–1 seeds, while no phytase activity was detected in WT seeds. Moreover, phytic acid content in P11 and a18 seeds was significantly lower than in WT. These results introduce an opportunity for improvement of soil and seed phytate-P bioavailability through genetic manipulation of oilseed rape, thereby increasing plant production and P nutrition for monogastric animals.     

Stable transformation and long-term maintenance of transgenic <Emphasis Type="Italic">Taxus</Emphasis> cell suspension cultures

A cell line of Taxus cuspidata has been transformed with wild-type Agrobacterium rhizogenes ATCC strain 15834 containing binary vector pCAMBIA1301 and, separately, with A. tumefaciens strain EHA105 containing binary vector pCAMBIA1305.2. Additionally, a cell line of T. chinensis has been transformed with wild-type A. rhizogenes ATCC strain 25818 containing binary vector pCAMBIA1301. The two transgenic T. cuspidata cell lines have been maintained in culture for more than 20 months, and the transgenic T. chinensis cell line for more than 9 months, with no loss of reporter gene expression or antibiotic resistance. The introduced genes had no discernable effect on growth or Taxol production in the transgenic cell lines when compared to the parent control. The methods for transforming non-embryogenic Taxus suspension cultures are described.     

蔗叶堆肥中一株泡盛曲霉溶磷能力的鉴定及其对辣椒的促生效果

[目的]从甘蔗叶堆肥中分离筛选具有高效溶磷及促生功能的菌株,为微生物肥料制备提供一种可利用的菌种资源.[方法]以Ca3(PO4)2和Zn3(PO4)2为磷源,进行平板溶磷筛选实验;采用形态学特征和ITS rDNA序列分析法进行菌种鉴定;采用液体摇瓶培养测定菌株的溶磷能力;将溶磷菌接种至辣椒幼苗根部分析其促生效应.[结果...     

Two Phosphate- and Potassium-solubilizing Bacteria Isolated from Tianmu Mountain, Zhejiang, China

Summary Two phosphate- and potassium-solubilizing strains (KNP413 and KNP414) were isolated from the soil of Tianmu Mountain, Zhejiang Province (China) and they were phenotypically and phylogenetically characterized. Both isolates effectively dissolved mineral phosphate and potassium, while strain KNP414 showed higher dissolution capacity even than Bacillus mucilaginosus AS1.153, the inoculant of potassium fertilizer widely used in China. When grown on Aleksandrov medium, both strains were rod-shaped spore-formers with a large capsule, and they formed slimy and translucent colonies. The DNA G+C contents were 57.7 mol% for strain KNP413 and 56.1 mol% for strain KNP414. Strain KNP413 shared a 16S rRNA gene sequence similarity of more than 99.1% with strain KNP414 and Bacillus mucilaginosus strains HSCC 1605 and YNUC0001, and a 94.6% similarity with Bacillus mucilaginosus VKM B-1480D, the type strain of Bacillus mucilaginosus. Strains KNP413 and KNP414 together with other Bacillus mucilaginosus were clustered with Paenibacillus strains in a group. The use of a specific PCR primer PAEN515F designed for differentiating the genus Paenibacillus from other members of the Bacillaceae showed that strains KNP413 and KNP414 had the same amplified 16S rRNA gene fragment (0.9-kb) as members of the genus Paenibacillus. In conclusion, phosphate- and potassium-solubilizing strains KNP413 and KNP414 should be integrated into the same species different from strain VKM B-1480D and they might be transferred to the genus of Paenibacillus, i.e. Paenibacillus mucilaginosus.The GenBank accession numbers of the 16S rRNA gene sequences are AY646227 for KNP413 and AY646228 for KNP414.     

Some peculiarities of steroid metabolism in transgenic <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> plants bearing the <Emphasis Type="Italic">CYP11A1</Emphasis> cDNA of cytochrome P450<Subscript>SCC</Subscript> from the bovine adrenal cortex

In the mitochondria of animal steroidogenic tissues, cytochrome P450_SCC encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolone—the general precursor of all steroid hormones. In this work we study the steroid metabolism in transgenic tobacco plants carrying the CYP11A1 cDNA encoding cytochrome P450_SCC from the bovine adrenal cortex. The transgenic plants under investigation markedly surpass the control wild-type plants by size and are characterized by a shortened period of vegetative growth (by rapid flowering); their leaves contain pregnenolone—the product of a reaction catalyzed by cytochrome P450_SCC. The level of progesterone in transgenic tobacco leaves is higher than in the control plants of the wild type. The seeds of the transgenic plants contain less (24R)-brassinosteroids than the wild-type tobacco plants. The results obtained indicate that the synthesis of an active P450_SCC cytochrome in transgenic Nicotiana tabacum plants has a profound effect on steroid metabolism and is responsible for the specific phenotypic features of transgenic plants bearing CYP11A1 cDNA.     

Utilization of phosphorus by pasture plants supplied with myo-inositol hexaphosphate is enhanced by the presence of soil micro-organisms

A range of pasture grass (Danthonia richardsonii and Phalaris aquatica) and legume (Medicago polymorpha, M. sativa, Trifolium repens and T. subterraneum) species showed limited capacity to obtain phosphorus (P) from inositol hexaphosphate (IHP), when grown in either sterile agar (pH 5.0 or 5.5) or sand-vermiculite media (pH 5.0). The total P content of shoots from IHP-supplied plants grown in agar was between 20% and 34% of that for seedlings supplied with an equivalent amount of P as inorganic phosphate (P_i), while in sand-vermiculite, the total P content of IHP-grown plants was between 5 and 10% of control plants. The poor ability of plants to utilize P from IHP resulted in significantly lower tissue P concentrations and, in general, reduced plant dry weight accumulation. In contrast, the P nutrition of plants supplied with IHP was significantly improved by inoculating media with either a cultured population of total soil micro-organisms or with a specific isolate of Pseudomonas sp., selected for its ability to release phosphate from IHP (strain CCAR59; Richardson and Hadobas, 1997 Can. J. Micro. 43, 509-516). In agar and sand-vermiculite media, respectively, the P content of IHP-grown plants increased with inoculation by up to 3.9- and 6.8-fold, such that the dry weight and P content of the plant material were equivalent to those observed for control plants supplied with P_i. However, the response to inoculation was dependent on the growth medium and the source of micro-organisms used. In sand-vermiculite, the cultured population of soil micro-organisms was effective when IHP was supplied at an equivalent level of P_i required for maximum plant growth. By comparison, inoculation of plants with the Pseudomonas strain was only effective at very high levels of IHP supply (×36), whereas in agar a response to inoculation occurred at all levels of IHP. The ability of pasture plants to acquire P from phytate was, therefore, influenced by the availability of IHP substrate, which was further affected by the presence of soil micro-organisms. Our results show that in addition to having an effect on the sorption characteristics of the growth media, soil micro-organisms also provided a source of phytase for the dephosphorylation of phytate for subsequent utilization of P_i by plants.     

The effect of phytase on the availability of P from myo-inositol hexaphosphate (phytate) for maize roots

The effect of adding phytase to the root medium of maize plants on the P-availability of added myo-inositol hexaphosphate (phytin) has been studied in pot experiments. When 40 mM phytin-P in nutrient solution was incubated in quartz-sand for 15 days in the absence of plants, 80% of it could be recovered from the solution as soluble organic P. Maize plants growing on this mixture assimilated P from phytin at rates comparable to those from inorganic phosphate (Pi). At a lower addition rate (2 mM phytin-P) only 10% was recovered in the soil solution, and plant growth was severely limited by P. At this low phytin level, the addition of phytase (10 enzyme units per kg sand) increased the plants' dry weight yield by 32%. The relative increases of the Pi concentration in the solution and of the amount of P in the plants were even higher, indicating that the observed growth stimulation was due to an increased rate of phytin hydrolysis. The enzyme-induced growth stimulation was also observed with plants growing in pots filled with soil low in P, when phytin was added. However, on three different soils the addition rates of phytin and phytase necessary for obtaining a significant phytase effect were both about 10 times higher than those required in quartzsand. It is concluded that the P-availability from organic sources can be limited by the rate of their hydrolytic cleavage.Abbreviation Pi inorganic phosphate     

Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene <Emphasis Type="Italic">BADH</Emphasis> and a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene <Emphasis Type="Italic">SeNHX1</Emphasis>

Three types of transgenic tobacco plants were acquired by separate transformation or co-transformation of a vacuolar Na⁺/H⁺ antiporter gene, SeNHX1, and a betaine synthesis gene, BADH. When exposed to 200 mM NaCl, the dual gene-transformed plants displayed greater accumulation of betaine and Na⁺ than their wild-type counterparts. Photosynthetic rate and photosystem II activity in the transgenic plants were less affected by salt stress than wild-type plants. Transgenic plants exhibited a greater increase in osmotic pressure than wild-type plants when exposed to NaCl. More importantly, the dual gene transformed plants accumulated higher biomass than either of the single transgenic plants under salt stress. Taken together, these findings indicate that simultaneous transformation of BADH and SeNHX1 genes into tobacco plants can enable plants to accumulate betaine and Na⁺, thus conferring them more tolerance to salinity than either of the single gene transformed plants or wild-type tobacco plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Response of Leucaena/Rhizobium symbiosis to mineral nutrients in Southwestern Nigeria

Pot and field experiments carried out at the International Institute of Tropical Agriculture (IITA) and at Fashola, Southwestern Nigeria, examined the effect of inoculation and N, P and micronutrients on nodulation and growth ofLeucaena leucocephala (Lam.) de Wit. In pot studies all parameters measured, except the percentage of nitrogen in shoots, were improved by inoculation, nitrogen and phosphorus. Micronutrients increased only nitrogen and allantoin contents. Interactions between inoculation and P, N and micronutrients on nodulation and growth of leucaena were observed. The effect of inoculation and fertilization with phosphorus or micronutrients was further investigated in field experiments. Establishment of uninoculated and unfertilized leucaena was poor at both locations due to low soil fertility and the presence of only a few native leucaena rhizobia. At one site, only inoculated plants were nodulated, while at the other, all plants produced nodules. Shoot dry weight, total nitrogen and phosphorus of inoculated plants were statistically equal to nitrogen-fertilized plants. Uninoculated plants were stunted. Generally, micronutrients did not influence nodulation, total nitrogen or growth of leucaena. They had only a positive effect on nitrogenase activity. Phosphorus increased total nitrogen and phosphorus uptake and plant growth. A 75% increase in shoot dry weight was obtained when 80 kg P ha^–1 was applied to inoculated leucaena with Rhizobium strain IRc 1045. Inoculated plants contained more allantoins than uninoculated ones but no significant correlation was found between these compounds and other parameters of N fixation.