Infection and Invasion of Roots by Symbiotic, Nitrogen-Fixing Rhizobia during Nodulation of Temperate Legumes

Bacteria belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Azorhizobium (collectively referred to as rhizobia) grow in the soil as free-living organisms but can also live as nitrogen-fixing symbionts inside root nodule cells of legume plants. The interactions between several rhizobial species and their host plants have become models for this type of nitrogen-fixing symbiosis. Temperate legumes such as alfalfa, pea, and vetch form indeterminate nodules that arise from root inner and middle cortical cells and grow out from the root via a persistent meristem. During the formation of functional indeterminate nodules, symbiotic bacteria must gain access to the interior of the host root. To get from the outside to the inside, rhizobia grow and divide in tubules called infection threads, which are composite structures derived from the two symbiotic partners. This review focuses on symbiotic infection and invasion during the formation of indeterminate nodules. It summarizes root hair growth, how root hair growth is influenced by rhizobial signaling molecules, infection of root hairs, infection thread extension down root hairs, infection thread growth into root tissue, and the plant and bacterial contributions necessary for infection thread formation and growth. The review also summarizes recent advances concerning the growth dynamics of rhizobial populations in infection threads.     

Development of selection method for Glycyrrhiza uralensis superior clones with high-glycyrrhizic acid contents using DNA sequence polymorphisms in glycyrrhizic acid biosynthetic genes

Glycyrrhiza plants are important resources for sweeteners and medicines, because underground parts of them contain glycyrrhizic acid (GL), which has sweet taste and various pharmacological activities (ex. anti-inflammatory, antiallergy, antiviral activity, etc.). Although such importance of them, their supply still depends principally on the collection of wild plants. Therefore, it is an important issue to develop stable and efficient production system of Glycyrrhiza plants. To overcome this problem, we established the hydroponic cultivation system of Glycyrrhiza uralensis and selected superior G. uralensis clones with high-GL contents in the containment greenhouse. In this study, we aimed to develop a method of selecting these superior G. uralensis clones by DNA sequence polymorphisms in biosynthetic genes. Among the DNA sequences of GL biosynthetic key enzyme gene (CYP88D6), we found Glycyrrhiza species and clone-specific polymorphisms in intronic regions. By using these polymorphisms, discrimination among Glycyrrhiza species and G. uralensis clones became possible. Furthermore, the appearance frequency of superior clone-specific alleles in cloned CYP88D6 sequences was correlated with GL contents in crude drugs collected from the Japanese market. We also observed the tendency that G. uralensis seedlings having superior clone-specific alleles of CYP88D6 gene showed higher secondary metabolite productivity than those without the alleles. These results indicated that superior clone-specific alleles of CYP88D6 gene could be applied as DNA markers for selecting G. uralensis clones accumulating high secondary metabolites.     

意大利苍耳与乌拉尔甘草种间竞争能力比较

意大利苍耳已经开始入侵乌拉尔甘草农田,然而对其入侵后果目前知之甚少。通过研究意大利苍耳与乌拉尔甘草的种间竞争关系,以期为意大利苍耳对乌拉尔甘草农田生态系统的入侵能力及入侵后果的评估提供试验依据。模拟了甘草农田的土壤水肥条件,采用取代试验法,设置意大利苍耳与乌拉尔甘草的单种种植和混种种植2种种植模式,待意大利苍耳生育期结束后进行收获,分别比较了该两种植物的个体生长及生物量积累在单种种植与混种种植两种处理间的差异,并比较单种和混种模式下乌拉尔甘草的地下器官中甘草酸含量的差别,通过计算相对产量、相对竞争强度和竞争攻击系数来比较该两种植物种间竞争能力的相对强弱。结果表明：与单种模式相比,混种模式下意大利苍耳个体生长的形态学性状和有性繁殖能力均有显著增加,其株高、冠幅和种子数量较单种处理分别增加了13%、27%和56%。而乌拉尔甘草的个体生长及克隆繁殖能力均显著降低,其根瘤的形成也受到显著的抑制,混种处理的乌拉尔甘草的株高、冠幅、根总长度、根总表面积、根平均直径以及根瘤数量比单种处理分别下降了35%、45%、55%、63%、19%和76%。单种处理下每株甘草根状茎的平均条数为3条,而与意大利苍耳混种后,其根状茎的发育被完全抑制。与单种处理相比,混种处理中的意大利苍耳生物量积累均显著的增加了,其中根、茎、叶、果实及总生物量与单种模式相比分别增加了84%、73%、84%、73%和77%,而混种模式却极显著降低了乌拉尔甘草生物量和地下器官甘草酸含量的积累,使其根、茎、叶、总生物量以及甘草酸含量与单种模式相比分别下降了72%、80%、65%、71%和63%。混种模式中的意大利苍耳相对产量（RY_a）大于1,而乌拉尔甘草相对产量（RY_b）小于1,表明意大利苍耳受到邻株同种其他个体的种内竞争压力大于来自邻株乌拉尔甘草的种间竞争压力,而乌拉尔甘草受到邻株同种其他个体的种内竞争压力则小于来自邻株意大利苍耳的种间竞争压力。混种模式中的意大利苍耳相对竞争强度（RCI_a）小于0,其竞争攻击系数（A_q）大于0,而乌拉尔甘草的相对竞争强度（RCI_b）则在0-1之间,其竞争攻击系数（A_b）小于0,表明在该两种植物的混生群落中,乌拉尔甘草的竞争能力弱于意大利苍耳。总体来看,在二者混生的群落中,意大利苍耳在竞争中占据明显的优势地位,对乌拉尔甘草的产量和品质均造成强烈的负面影响。     

STABILIZING MECHANISMS IN A LEGUME–RHIZOBIUM MUTUALISM

Preferential rewarding of more beneficial partners may stabilize mutualisms against the invasion of less beneficial, that is cheater, genotypes. Recent evidence suggests that both partner choice and sanctioning may play roles in preventing the invasion of less-beneficial rhizobia in legume–rhizobium mutualisms. The importance of these mechanisms in natural communities, however, remains unclear. We grew 12 Medicago truncatula maternal families with a mixture of three rhizobium strains from their native range for three plant generations and estimated the symbiotic benefits (nodule number and size) conferred to each rhizobium strain. In this experiment, the majority of M. truncatula genotypes formed more nodules with more beneficial rhizobium strains, providing evidence for adaptive partner choice. We also found that three generations of symbiosis resulted in an increase in the relative frequency of rhizobium strains that were most beneficial to plants—suggesting that partner choice affects rhizobium fitness. By contrast, we found no evidence that plants differentially rewarded rhizobia postnodulation via sanctioning leading to differences in nodule size. Taken together, our data suggest that plants have evolved to recognize beneficial rhizobial signals during the early stages of symbiosis, and that signaling between plants and rhizobia may be subject to coevolutionary pressures.     

Invasive Lespedeza cuneata and native Lespedeza virginica experience asymmetrical benefits from rhizobial symbionts

Background and Aims

Lespedeza cuneata (Dum. Cours.) G. Don is an invasive legume that displaces populations of native N. American congeners. Our aims are to determine the growth benefits of different rhizobacterial strains for L. cuneata and native Lespedeza virginica (L.) Britton, and to determine if these strains influence competition between these plants.

Methods

Plants were grown under nitrogen-limiting conditions in sterilized soil in pairs consisting of two L. cuneata, two L. virginica, or one of each species, and then plants were inoculated with one of seven rhizobial isolates, or with a no-strain control. After 3 months, plants were harvested for determination of biomass and nodulation rate.

Results

Five of the assayed stains improved L. cuneata biomass over uninoculated controls, but none of the strains benefited L. virginica. L. cuneata plants had more biomass and root nodules when grown in competition with L. virginica than with a conspecific.

Conclusions

Asymmetrical benefits from these symbionts accrued to invasive L. cuneata but not to native L. virginica, and this may provide the invader with a growth advantage in the field. Changes in the availability of effective symbionts in the soils of invaded sites can shape performance of native and invasive plants.     

宁夏荒漠草原优势植物生长及生物量分配对放牧干扰的响应

于2011年植物生长旺季(8月)在围封禁牧(NG)、轻度放牧(LG)、中度放牧(MG)和重度放牧(HG)区分别随机选取荒漠草原优势植物甘草(Glycyrrhiza uralensis)和牛心朴子(Cynanchum komarovii)各15株为研究对象,对比分析其生长特征、各植物构件生物量及生物量资源分配差异对不同放牧强度的响应机制,为退化草原的恢复演替提供依据。结果表明:(1)甘草株高和地径、牛心朴子株高均随放牧强度的增加呈先升高后下降的趋势,而且均在轻度放牧条件下最高,重度放牧时则显著降低。(2)甘草和牛心朴子的总生物量、茎生物量和叶生物量随着放牧强度的增加呈先升高后降低的趋势,且不同放牧强度间差异显著;甘草和牛心朴子根系生物量随放牧强度的加强变化趋势不同。(3)甘草和牛心朴子生物量分配的总体格局为:根叶茎;随着放牧强度的增加,甘草根生物量比呈先升高后降低趋势,茎生物量比呈下降的趋势,叶生物量比呈上升趋势,而牛心朴子根生物量比呈先下降后升高的趋势,茎生物量和叶生物量呈先增加后下降的趋势。研究认为,不同放牧强度下两种植物形态可塑性和生物量分配格局的差异反映出植物生态适应策略的不同。     

Legume growth-promoting rhizobia: An overview on the Mesorhizobium genus

The need for sustainable agricultural practices is revitalizing the interest in biological nitrogen fixation and rhizobia-legumes symbioses, particularly those involving economically important legume crops in terms of food and forage. The genus Mesorhizobium includes species with high geographical dispersion and able to nodulate a wide variety of legumes, including important crop species, like chickpea or biserrula. Some cases of legume-mesorhizobia inoculant introduction represent exceptional opportunities to study the rhizobia genomes evolution and the evolutionary relationships among species. Complete genome sequences revealed that mesorhizobia typically harbour chromosomal symbiosis islands. The phylogenies of symbiosis genes, such as nodC, are not congruent with the phylogenies based on core genes, reflecting rhizobial host range, rather than species affiliation. This agrees with studies showing that Mesorhizobium species are able to exchange symbiosis genes through lateral transfer of chromosomal symbiosis islands, thus acquiring the ability to nodulate new hosts. Phylogenetic analyses of the Mesorhizobium genus based on core and accessory genes reveal complex evolutionary relationships and a high genomic plasticity, rendering the Mesorhizobium genus as a good model to investigate rhizobia genome evolution and adaptation to different host plants. Further investigation of symbiosis genes as well as stress response genes will certainly contribute to understand mesorhizobia-legume symbiosis and to develop more effective mesorhizobia inoculants.     

Growth response of Casuarina equisetifolia Forst. rooted stem cuttings to Frankia in nursery and field conditions

Casuarina equisetifolia Forst. is a tree crop that provides fuel wood, land reclamation, dune stabilization, and scaffolding for construction, shelter belts, and pulp and paper production. C. equisetifolia fixes atmospheric nitrogen through a symbiotic relationship with Frankia, a soil bacterium of the actinobacteria group. The roots of C. equisetifolia produce root nodules where the bacteria fix atmospheric nitrogen, which is an essential nutrient for all plant metabolic activities. However, rooted stem cuttings of elite clones of C. equisetifolia by vegetative propagation is being planted by the farmers of Pondicherry as costeffective method. As the vegetative propagation method uses inert material (vermiculite) for rooting there is no chance for Frankia association. Therefore after planting of these stocks the farmers are applying 150 kg of di-ammonium phosphate (DAP)/acre/year. To overcome this fertilizer usage, the Frankia-inoculated rooted stem cuttings were propagated under nursery conditions and transplanted in the nutrient-deficient soils of Karaikal, Pondicherry (India), in this study. Under nursery experiments the growth and biomass of C. equisetifolia rooted stem cuttings inoculated with Frankia showed 3 times higher growth and biomass than uninoculated control. These stocks were transplanted and monitored for their growth and survival for 1 year in the nutrient-deficient farm land. The results showed that the rooted stem cuttings of C. equisetifolia significantly improved growth in height (8.8 m), stem girth (9.6 cm) and tissue nitrogen content (3.3 mg g^?1) than uninoculated controls. The soil nutrient status was also improved due to inoculation of Frankia.     

Effect of low light intensity on growth and accumulation of secondary metabolites in roots of Glycyrrhiza uralensis Fisch

Effects of low light intensity on growth and accumulation of secondary metabolites of a medicinal plant Glycyrrhiza uralensis Fisch. were investigated. Hydroponic-cultivated one year-old rhizome seedlings were grown under three low irradiances, 200, 100, and 50 μmol m⁻² s⁻¹ for 135 days. Control plants were cultured under natural light conditions. Low light intensity stress decreased leaf thickness, photosynthesis and biomass, but increased leaf area and chlorophyll concentrations. Low light intensity also significantly increased accumulation of glycyrrhizic acid and liquiritin in the root, while the maximum values of both secondary metabolites were obtained under an irradiance of 100 μmol m⁻² s⁻¹. Concentrations of both secondary metabolites were negatively correlated with root biomass. The results suggested that G. uralensis could endure an environment with low light intensity and suitable light control might increase the secondary metabolite contents within agroforestry systems.     

Endoplasmic reticulum-targeted GFP reveals ER remodeling in Mesorhizobium-treated Lotus japonicus root hairs during root hair curling and infection thread formation

The endoplasmic reticulum (ER) of the model legume Lotus japonicus was visualized using green fluorescent protein (GFP) fused with the KDEL sequence to investigate the changes in the root hair cortical ER in the presence or absence of Mesorhizobium loti using live fluorescence imaging. Uninoculated root hairs displayed dynamic forms of ER, ranging from a highly condensed form to an open reticulum. In the presence of M. loti, a highly dynamic condensed form of the ER linked with the nucleus was found in deformed, curled, and infected root hairs, similar to that in uninoculated and inoculated growing zone I and II root hairs. An open reticulum was primarily found in mature inoculated zone III root hairs, similar to that found in inactive deformed/curled root hairs and infected root hairs with aborted infection threads. Co-imaging of GFP-labeled ER with light transmission demonstrated a correlation between the mobility of the ER and other organelles and the directionality of the cytoplasmic streaming in root hairs in the early stages of infection thread formation and growth. ER remodeling in root hair cells is discussed in terms of possible biological significance during root hair growth, deformation/curling, and infection in the Mesorhizobium–L. japonicus symbiosis.     

Charcoal and shrubs modify soil processes in ponderosa pine forests of western Montana

In split-root systems of alfalfa (Medicago sativa L.), already existing nodules or arbuscular mycorrhizal roots suppress further establishment of symbiosis in other root parts, a phenomenon named autoregulation. Roots treated with rhizobial nodulation signals (Nod factors) induce a similar systemic suppression of symbiosis.In order to test the hypothesis that flavonoids play a role in this systemic suppression, split-root systems of alfalfa plants were inoculated on one side of the split-root system with Sinorhizobium meliloti or Glomus mosseae or were treated with Nod factor. HPLC-analysis of alfalfa root extracts from both sides of the split-root system revealed a persistent local and systemic accumulation pattern of some flavonoids associated with the different treatments. The two flavonoids, formononetin and ononin, could be identified to be similarily altered after rhizobial or mycorrhizal inoculation or when treated with Nod factor.Exogenous application of formononetin and ononin partially restored nodulation and mycorrhization pointing towards the involvement of these two secondary compounds in the autoregulation of both symbioses.     

Genotypic variation of nodules’ enzymatic activities in symbiotic nitrogen fixation among common bean (Phaseolus vulgaris L.) genotypes grown under salinity constraint

The effect of salt stress, under glasshouse conditions, was studied on plant biomass, nodulation, and activities of acid phosphatases (APase, EC 3.1.3.2) and trehalose 6-phosphate phosphatase (TPP, EC 3.1.3.12) in the symbiosis common bean (Phaseolus vulgaris L.)-rhizobia nodules. Four common bean recombinant inbred lines (147, 115, 104 and 83) were separately inoculated, with CIAT 899 or RhM11 strains and grown in hydroaeroponic culture. Two NaCl levels (0 and 25 mM NaCl plant^?1 week^?1 corresponding, respectively, to the control and the salt treatment) were applied and the culture was assessed during 42 days after their transplantation. The results showed that the nodulation of these lines was not affected by salinity except for the line 83 inoculated with CIAT 899, whose nodule dry weight decreased by 48.24 % compared with the corresponding controls. For the other symbiotic combinations, shoot and root biomasses were not significantly affected by salt constraint. Salinity stress generally reduced acid phosphatise and trehalose phosphate phosphatase activities in nodules that were less affected in plants inoculated with RhM11. Based on our data, it appears that nodule phosphatase activity may be involved in salinity tolerance in common beans and the levels of salt tolerance depend principally on specific combination of the rhizobial strain and the host cultivar.     

Ri-mediated Transformation of <Emphasis Type="Italic">Glycyrrhiza uralensis</Emphasis> with a Squalene Synthase Gene (GuSQS1) for Production of Glycyrrhizin

Root of Glycyrrhiza uralensis, one of the most important medicinal plants, containing bioactive triterpene saponins (glycyrrhizin). Squalene synthase (SQS) plays a regulatory role in the biosynthesis of triterpene saponins. In the present investigation, SQS coding sequence from G. uralensis was cloned by polymerase chain reaction (PCR) and a transgenic system was developed for G. uralensis through Agrobacterium rhizogenes-mediated transformation. The SQS gene placed under a CaMV 35S promoter was transferred into G. uralensis using A. rhizogenes strain ACCC10060. The transformed hairy roots were selected on Murashige and Skoog (1962)-containing phosphinothricin (PPT) and root lines were established. The integration of SQS gene was confirmed by PCR and Southern blot. Three transgenic root lines UP1, UP24, UP31 were obtained and their growth rates were detected. The result showed that transgenic root lines but UP1 line grew faster than control hairy roots; high-performance liquid chromatography (HPLC) analysis demonstrated the highest glycyrrhizin content of transgenic roots was 2.5 mg/g dry weight and was about 2.6 times higher than control hairy roots. The nucleotide sequences GuSQS1 and GUSQS2 reported in this paper appear in the EMBL nucleotide sequence database with the accession number AM182329 and AM182330, respectively.     

Characterization of bacterial endophytes of sweet potato plants

Endophytic bacteria associated with sweet potato plants (Ipomoea batatas (L.) Lam.) were isolated, identified and tested for their ability to fix nitrogen, produce indole acetic acid (IAA), and exhibit stress tolerance. Eleven different strains belonging to the genera, Enterobacter, Rahnella, Rhodanobacter, Pseudomonas, Stenotrophomonas, Xanthomonas and Phyllobacterium, were identified. Four strains were shown to produce IAA (a plant growth hormone) and one strain showed the ability to grow in nitrogen free medium and had the nitrogenase subunit gene, nifH. To determine if IAA production by the endophytes had any role in protecting the cells against adverse conditions, different stress tests were conducted. The IAA producer grew well in the presence of some antibiotics, UV and cold treatments but the response to pH, osmotic shock, thermal and oxidative treatments was the same for both the IAA producer and the no IAA producer. To determine if IAA produced by the strains was biologically relevant to plants, cuttings of poplar were inoculated with the highest IAA producing strain. The inoculated cuttings produced roots sooner and grew more rapidly than uninoculated cuttings. These studies indicate that endophytes of sweet potato plants are beneficial to plant growth.     

Symbiosis-stimulated chitinase isoenzymes of soybean (Glycine mas (L.) Merr.)

Isoforms of endochitinase in soybean were studied in relation to root symbiosis. Five selected cultivars differing in their nodulation potential were inoculated with two strains of Bradyrhizobium japonicum, the broad host-range Rhizobium sp. NGR234, and with the mycorrhizal fungus Glomus mosseae. Total chitinase activity in nodules was up to 7-fold higher than in uninoculated roots and in mycorrhizal roots. The chitinase activity in nodules varied depending on the strain-cultivar combination. On semi-native polyacrylamide gels, four acidic isoforms were identified. Two isoforms (CH 2 and CH 4) were constitutively present in al analysed tissues. The other two isoforms (CH 1 and CH 3) were strongly induced in nodules and were simulated in mycorrhizal roots as compared to uninoculated roots. The induction of CH 1 varied in nodules depending on the soybean cultivar. This isoform was also stimulated in uninfected roots when they were treated with tri-iodobenzoic acid, rhizobial lipochitooloigosaccharides (Nod factors) and chitotetraose. CH 3 was not affected by these stimuli indicating that this isoform could represent a marker for enzymes induced in later stages of the symbiotic interactions.Key words: (Brady)rhizobium, chitinase isoenzymes, mycorrhiza, (restricted) nodulation, Nod factors      

Radish introduction affects soil biota and has a positive impact on the growth of a native plant

Introduced plants may out-compete natives by belowground allelopathic effects on soil communities including the symbionts of native plants. We tested for an allelopathic effect of an introduced crucifer, Raphanus sativus, on a common neighboring legume, Lupinus nanus, on the legume’s rhizobium affiliates, and on the broader soil community. In both field observations and a greenhouse experiment, we found that R. sativus decreased the density of nodules on L. nanus roots. However, in the greenhouse experiment, R. sativus soils only decreased the density of small, likely non-beneficial rhizobium nodules. In the same experiment, R. sativus soils decreased fungivorous nematode abundance, though there was no effect of R. sativus introduction on fungal density. In the greenhouse experiment, R. sativus soils had a net positive effect on L. nanus biomass. One explanation of this effect is that R. sativus introduction might alter the mutualistic/parasitic relationship between L. nanus and its rhizobial associates with a net benefit to L. nanus. Our results suggest that introduced brassicas can quickly alter belowground communities, but that the net effect of this on neighboring plants is not necessarily negative.     

Mineralization and herbage recovery of animal manure nitrogen after application to various soil types

Aims

The study aims (1) to evaluate the effect of Mesorhizobium tianshanense on plant proline and polyamine levels of Lotus tenuis and its modulatory effect during plant response to short-term salt stress and (2) to compare these effects with those caused by mycorrhizal symbiosis.

Methods

Experiments consisted of a randomized factorial design of two factors: salinity (two levels, 0 and 150 mM NaCl) and symbiosis (three levels, uninoculated, Glomus intraradices, and M. tianshanense).

Results

Salinization led to increased proline levels regardless of plant organ and symbiotic status, excepting mycorrhizal L. tenuis roots. Salinity diminished the total polyamine level of control and rhizobial plants but not in mycorrhizal ones. Variations in the pattern response of the three individual polyamines (putrescine, spermidine, and spermine) differed in accordance with the symbiotic status of the plant, highlighting a divergence on proline and polyamine metabolisms between rhizobial and mycorrhizal symbiosis.

Conclusions

Spermidine and spermine contributed the most with the salt-induced root polyamine increment observed upon salinization in roots of nodulated plants, suggesting that these polyamines might mediate an adaptive role of the plant–M. tianshanense symbiosis in L. tenuis plants growing in a saline environment.     

Phenotypic plasticity with respect to salt stress response by Lotus glaber: the role of its AM fungal and rhizobial symbionts

Our hypothesis is that Lotus glaber (a glycophytic species, highly tolerant to saline-alkaline soils) displays a plastic root phenotypic response to soil salinity that may be influenced by mycorrhizal and rhizobial microorganisms. Uninoculated plants and plants colonised by Glomus intraradices or Mesorhizobium loti were exposed to either 150 or 0 mM NaCl. General plant growth and root architectural parameters (morphology and topology) were measured and phenotypic plasticity determined at the end of the salt treatment period. Two genotypes differing in their salt tolerance capacity were used in this study. G. intraradices and M. loti reduced the total biomass of non-salinised, sensitive plants, but they did not affect that of corresponding tolerant ones. Root morphology of sensitive plants was greatly affected by salinity, whereas mycorrhiza establishment counteracted salinity effects. Under both saline conditions, the external link length and the internal link length of mycorrhizal salt-sensitive plants were higher than those of uninoculated control and rhizobial treatments. The topological trend (TT) was strongly influenced by genotype x symbiosis interaction. Under non-saline conditions, nodulated root systems of the sensitive plant genotype had a more herringbone architecture than corresponding uninoculated ones. At 150 mM NaCl, nodulated root systems of tolerant plants were more dichotomous and those of the corresponding sensitive genotype more herringbone in architecture. Notwithstanding the absence of a link between TTs and variations in plant growth, it is possible to predict a dissimilar adaptation of plants with different TTs. Root colonisation by either symbiotic microorganisms reduced the level of root phenotypic plasticity in the sensitive plant genotype. We conclude that root plasticity could be part of the general mechanism of L. glaber salt tolerance only in the case of non-symbiotic plants.