Endophytic Occupation of Root Nodules and Roots of Melilotus dentatus by Agrobacterium tumefaciens

Agrobacterium strains have been frequently isolated from the root nodules of different legumes. Various possible mechanisms have been proposed to explain the existence of these bacteria in nodules, but there is no sufficient experimental evidence to support the estimations. In this work, we proved that the Agrobacterium strain CCBAU 81181, which was originally isolated from the root nodules of Onobrychis viciaefolia, and a symbiotic strain of Sinorhizobium meliloti CCBAU 10062 could coinhabit the root nodules of Melilotus dentatus. Analyses were performed by using a fluorescence marker, reisolation of bacteria from nodules, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of whole cellular proteins, and polymerase chain reaction amplification of symbiotic genes. The inoculation of A. tumefaciens CCBAU 81181 did not affect the growth and nodulation of plants. CCBAU 81181 and 24 other Agrobacterium strains isolated from nodules were incapable of nodulating on their original or alternative host and 22 strains of these strains were endophytes in the roots and stems of their hosts. Also, the tumor-inducing A. tumefaciens strains IAM 13129^T and C58 were found capable of entering the roots of Glycyrrhiza pallidiflora, but did not cause pathogenic symptoms. With these results, we conclude that A. tumefaciens strains could be endophytic bacteria in the roots, stems, and root nodules. This finding partially explains why Agrobacterium strains were frequently isolated from the surface-sterilized nodules.     

Stem Nodulation in Legumes: Diversity,Mechanisms, and Unusual Characteristics

Rhizobia can establish a nitrogen-fixing symbiosis with plants of the Leguminosae family. They elicit on their host plant the formation of new organs, called nodules, which develop on the roots. A few aquatic legumes, however, can form nodules on their stem at dormant root primordia. The stem-nodulating legumes described so far are all members of the genera Aeschynomene, Sesbania, Neptunia, and Discolobium. Their rhizobial symbionts belong to four genera already described: Rhizobium, Bradyrhizobium, Sinorhizobium, and Azorhizobium. This review summarizes our current knowledge on most aspects of stem nodulation in legumes, the infection process and nodule development, the characterization and unusual features of the associated bacteria, and the molecular genetics of nodulation. Potential use as green manure in lowland rice of these stem-nodulating legumes, giving them agronomical importance, is also discussed.     

The presence of nodules on legume root systems can alter phenotypic plasticity in response to internal nitrogen independent of nitrogen fixation

All higher plants show developmental plasticity in response to the availability of nitrogen (N) in the soil. In legumes, N starvation causes the formation of root nodules, where symbiotic rhizobacteria fix atmospheric N₂ for the host in exchange for fixed carbon (C) from the shoot. Here, we tested whether plastic responses to internal [N] of legumes are altered by their symbionts. Glasshouse experiments compared root phenotypes of three legumes, Medicago truncatula, Medicago sativa and Trifolium subterraneum, inoculated with their compatible symbiont partners and grown under four nitrate levels. In addition, six strains of rhizobia, differing in their ability to fix N₂ in M. truncatula, were compared to test if plastic responses to internal [N] were dependent on the rhizobia or N₂‐fixing capability of the nodules. We found that the presence of rhizobia affected phenotypic plasticity of the legumes to internal [N], particularly in root length and root mass ratio (RMR), in a plant species‐dependent way. While root length responses of M. truncatula to internal [N] were dependent on the ability of rhizobial symbionts to fix N₂, RMR response to internal [N] was dependent only on initiation of nodules, irrespective of N₂‐fixing ability of the rhizobia strains.     

人和动物条件致病菌环境菌株侵染植物的研究进展北大核心CSCD

越来越多的研究表明某些在环境中普遍存在的人与动物的病原微生物能够跨界侵染不同生物界的寄主。本文就Serratia marcescens,Enterobacter cloacae,Pseudomonas aeuriginosa,Klebsiella pneumoniae等动物条件病原细菌环境菌株跨界侵染植物的研究现状进行了综述。这些病原菌在自然界中普遍存在,能够利用与感染人类相同或不同的侵染策略跨界侵染植物,以拓宽其寄主范围。其中,肺炎克雷伯氏菌(Klebsiella pneumoniae)能在自然条件下引起玉米发生顶腐病,揭示了环境中的某些植物可作为各种病原细菌的天然储存库,在条件合适的情况下可能会感染人类和动物,以及在食品生产中的潜在危害。对这些跨界病原菌的研究,在人、动物和植物流行病学上具有非常重要意义,也为环境科学提出了新的研究热点。     

Host specificity of Rhizobium strains isolated from nitrogen-fixing trees and nitrogenase activities of strain GRH2 in symbiosis with Prosopis chilensis

Host plant specificity was examined in symbiosis between Rhizobium strains isolated from legume-tree root nodules and herbaceous or woody legumes from which they were isolated. Strain GRH2 isolated from Acacia cyanophylla formed effective nodules on Acacia, Prosopis and Medicago sativa as well. Nitrogenase activity, measured as acetylene reduction, of strain GRH2 in symbiosis with Prosopis chilensis was the highest (P 0.05) among the tropical legumes studied and was similar to those found for other associations involving herbaceous legumes. Relative efficiency of nitrogenase varied from 0.3 to near 1 during the light time of the photoperiod. However no hydrogen uptake activity was detected by the amperometric method used. Rhizobium strains GRH3, GRH5 and GRH9 isolated from A. melanoxylon, P. chilensis and Sophora microphylla, respectively, also showed a very low host-range specificity. All isolates were infective and effective on at least one of the herbaceous legumes tested. These data demonstrate the lack of specificity of Rhizobium strains isolated from nitrogen-fixing tree root nodules and that these strains can form effective nodules on herbaceous legumes.     

Effect of plant root symbionts on performance of native woody species in competition with an invasive grass in multispecies microcosms

The majority of terrestrial plants form mutualistic associations with arbuscular mycorrhizal fungi (AMF) and rhizobia (i.e., nitrogen‐fixing bacteria). Understanding these associations has important implications for ecological theory and for restoration practice. Here, we tested whether the presence of AMF and rhizobia influences the performance of native woody plants invaded by a non‐native grass in experimental microcosms. We planted eight plant species (i.e., Acacia acuminata, A. microbotrya, Eucalyptus loxophleba subsp. loxophleba, E. astringens, Calothamnus quadrifidus, Callistemon phoeniceus, Hakea lissocarpha and H. prostrata) in microcosms of field‐conditioned soil with and without addition of AMF and rhizobia in a fully factorial experimental design. After seedling establishment, we seeded half the microcosms with an invasive grass Bromus diandrus. We measured shoot and root biomass of native plants and Bromus, and on roots, the percentage colonization by AMF, number of rhizobia‐forming nodules and number of proteaceous root clusters. We found no effect of plant root symbionts or Bromus addition on performance of myrtaceous, and as predicted, proteaceous species as they rely little or not at all on AMF and rhizobia. Soil treatments with AMF and rhizobia had a strong positive effect (i.e., larger biomass) on native legumes (A. microbotrya and A. acuminata). However, the beneficial effect of root symbionts on legumes became negative (i.e., lower biomass and less nodules) if Bromus was present, especially for one legume, i.e., A. acuminata, suggesting a disruptive effect of the invader on the mutualism. We also found a stimulating effect of Bromus on root nodule production in A. microbotrya and AMF colonization in A. acuminata which could be indicative of legumes’ increased resource acquisition requirement, i.e., for nitrogen and phosphorus, respectively, in response to the Bromus addition. We have demonstrated the importance of measuring belowground effects because the aboveground effects gave limited indication of the effects occurring belowground.     

Characterization of bacteria associated with nodules of two endemic legumes of Algeria,Hedysarum naudinianum and H. perrauderianum

The root nodules of two wild legume species endemic to Algeria, Hedysarum naudinianum and He. perrauderianum, were investigated with regard to their anatomy and histology, and the identity of the associated bacteria. Both plants were found to form root nodules with regular features and well infected by rod-shaped bacteria. The culturable fraction of bacteria that could be obtained from surface-sterilized nodules included a prevailing presence of Enterobacteriaceae having 100 % 16S rDNA sequence identity with both Enterobacter cloacae and E. ludwigii. In H. perrauderianum, this taxon was the sole cultured isolate, while from H. naudinianum we also found Bacillus, Lactobacillus, Staphylococcus, Rothia, and isolates that were 100 % identical to Corynebacterium pseudodiphthericum, which is known to be an agent of respiratory and cardiac infections in humans. Whereas no culturable rhizobia and alike could be obtained on plates, PCR-based culture-independent approaches revealed in both plants the presence of a Mesorhizobium sp., which in H. perrauderianum was identical to isolates nodulating other legumes from Africa, European Mediterranean countries, and Asia, while in H. naudinianum it bore a single nucleotide polymorphism which is so far unique for any observed mesorhizobia. Data from the microsymbionts appear to suggest interesting clues to interpret the evolutionary ecology of their host plants.     

Cross-inoculation specificity ofPsophocarpus tetragonolobus (L.) DC

Summary Only legumes of the cowpea cross-inoculation group, including the winged bean (Psophocarpus tetragonolobus) were found to form nodules in a temperate zone soil with no previous history of legume cropping. Isolates from root nodules from uninoculated winged beans grown in the field only nodulated legumes in the cowpea cross-inoculation group.Rhizobium japonicum formed ineffective nodules with the winged bean. Contribution No.5852, Scientific Article No.A2802 of the Maryland Agricultural Experiment Station, Department of Botany.     

An Isotopic Study of the Fixation of Nitrogen associated with Nodulated Plants of Alnus, Myrica, and Hippophae

Nodulated plants of Alnus glutinosa, Myrica gale, and Hippophaërhamnoides, the root systems of which had been exposed to excessfree ¹⁵N, showed substantial enrichment in fixed ¹⁵N contentin all parts of the plant, but particularly in the root nodules.The data resemble closely those obtained by the present andprevious authors with legumes under comparable conditions, andsupport the conclusion already drawn from experiments of traditionaltype that the nodules of these non-legumes are similar to thoseof legumes in their function and relation to the rest of theplant. Nodulated plants of Alnus and Myrica continued to fixfree nitrogen concurrently with the uptake of combined nitrogenwhen the latter was supplied in the rooting medium in amountsunlikely to be exceeded in the field. Isotopic tests on detachednodules of one of the species (Alnus glutinosa) showed thatfixation continued, and though much reduced as compared withthat shown by attached nodules it considerably exceeded thatexperienced by the present and previous authors with detachedlegume nodules, on the basis of fixation per unit of total nodulenitrogen. It is probable that detached Alnus nodules presentconvenient material for the further study of various aspectsof the fixation process.     

Long-distance control of nodulation: Molecules and models

Legume plants develop root nodules to recruit nitrogen-fixing bacteria called rhizobia. This symbiotic relationship allows the host plants to grow even under nitrogen limiting environment. Since nodule development is an energetically expensive process, the number of nodules should be tightly controlled by the host plants. For this purpose, legume plants utilize a long-distance signaling known as autoregulation of nodulation (AON). AON signaling in legumes has been extensively studied over decades but the underlying molecular mechanism had been largely unclear until recently. With the advent of the model legumes, L. japonicus and M. truncatula, we have been seeing a great progress including isolation of the AON-associated receptor kinase. Here, we summarize recent studies on AON and discuss an updated view of the long-distance control of nodulation.     

Mixture of endophytic Agrobacterium and Sinorhizobium meliloti strains could induce nonspecific nodulation on some woody legumes

Agrobacterium sp. II CCBAU 21244 isolated from root nodules of Wisteria sinensis was verified as an endophytic bacterium by inoculation and reisolation tests. However, inoculation with a mixture of this strain and a Sinorhizobium meliloti strain could induce root nodules on W. sinensis and two other woody legumes, which do not form a symbiosis with S. meliloti alone. Rod-shaped and irregular nodules were found on the inoculated plants, in which the S. meliloti strain was detected in all of the nodules; while the Agrobacterium strain was inside of the rod-shaped nodules, or occupied only the nodule surface of the irregular globe-shaped nodules. These findings revealed novel interactions among the symbiotic bacteria, endophytic bacteria and the legume plants, although the mechanisms are still unknown.     

Choice of hydrogen uptake (Hup) status in legume‐rhizobia symbioses

The H₂ is an obligate by-product of N-fixation. Recycling of H₂ through uptake hydrogenase (Hup) inside the root nodules of leguminous plants is often considered an advantage for plants. However, many of the rhizobium-legume symbioses found in nature, especially those used in agriculture are shown to be Hup⁻, with the plants releasing H₂ produced by nitrogenase activity from root nodules into the surrounding rhizosphere. Recent studies have suggested that, H₂ induces plant-growth-promoting rhizobacteria, which may explain the widespread of Hup⁻ symbioses in spite of the low energy efficiency of such associations. Wild legumes grown in Nova Scotia, Canada, were surveyed to determine if any plant-growth characteristics could give an indication of Hup choice in leguminous plants. Out of the plants sampled, two legumes, Securigera varia and Vicia cracca, showed Hup⁺ associations. Securigera varia exhibited robust root structure as compared with the other plants surveyed. Data from the literature and the results from this study suggested that plants with established root systems are more likely to form the energy-efficient Hup⁺ symbiotic relationships with rhizobia. Conversely, Hup⁻ associations could be beneficial to leguminous plants due to H₂-oxidizing plant-growth-promoting rhizobacteria that allow plants to compete successfully, early in the growing season. However, some nodules from V. cracca tested Hup⁺, while others were Hup⁻. This was similar to that observed in Glycine max and Pisum sativum, giving reason to believe that Hup choice might be affected by various internal and environmental factors.     

Compensatory response of a legume root-nodule system to nodule herbivory by Sitona hispidulus

A laboratory study was conducted to examine the hypothesis that herbivory of nitrogen-fixing root nodules on legumes causes an exact compensatory response in nodule growth. Plants of Medicago sativa (L.) were grown hydroponically in clear plastic growth pouches so that the number and biomass of root nodules could be estimated nondestructively before, and 10 and 18 days after, partial denodulation. For treatments, plants were subjected to 23% denodulation by first-instar larvae of Sitona hispidulus (F.) (a common herbivore of Medicago and Trifolium) or 50% nodule pruning; additional plants were left untreated. Results indicated that nodule herbivory and nodule pruning caused an overcompensatory response in number of nodules. This was also true for number of nodule units (an indirect measure of nodule biomass) per plant at 10 days after denodulation but had changed to an exact compensatory response by day 18. An inverse relationship between change in number of nodule units and initial number of nodules indicated that compensatory nodulation was regulated by a feedback mechanism. Shoot and root biomasses were not affected by denodulation in this study.     

Prosopis glandulosa and the nitrogen balance of rangelands: extent and occurrence of nodulation

Summary We report the recovery of root nodules from P. glandulosa var. glandulosa in the eastern portion of its range, where the species reaches its greatest vegetational development. Single cores 4.7 cm in diameter and up to 250 cm deep yielded from 0 to over 250 nodules. Nodules were found at all depths below 10 cm, with the highest concentration often around 100 cm. Detailed studies of three trees revealed relatively small volume densities of about 0.02 nodules cm^–3, high surface area densities of 2–4 nodules cm^–2, and high nodule biomass of 8–23 g m^–2, when compared to cultivated legumes. Nodules are small, weakly attached to roots that are seldom over 0.5 mm in diameter, and not easily observed under field conditions. No nodules were recovered from cores from the more arid western portion of P.glandulosa's range, although seedlings nodulated readily in these soils in the glasshouse as well as in most unamended soils from throughout mesquite's geographical range. Local differences in nodulating potential of soils included a negative association with mesquite canopies and a positive association with depth. These results suggest a significant role for biological fixation in the nitrogen regime and vegetation dynamics of Prosopis-dominated ecosystems.     

Nod factors of Rhizobium are a key to the legume door

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lopo-oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis-specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR-factors to signal-production-deficient mutants of the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium japorticum strain USDA110. Between 10 ⁻⁷ M and 10⁻⁶ M NodNGR factors permitted these NodABC mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a non-host. Detailed cytological investigations of V. unguiculata showed that the NodABC mutant UGR AnodABC, in the presence of NodNGR factors, entered roots in the same way as the wild-type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.     

Signaling and Gene Expression for Water-Tolerant Legume Nodulation

In the symbiotic interaction with rhizobia, legumes develop nodules in which nitrogen fixation takes place. Upon submersion, most temperate legumes are incapable of nodulation, but tropical legumes that grow in waterlogged soils have acquired water stress tolerance for growth and nodulation. One well-studied model plant, the tropical, semi-aquatic Sesbania rostrata, develops stem-located adventitious root primordia that grow out into adventitious roots upon submergence and develop into stem nodules after inoculation with the microsymbiont, Azorhizobium caulinodans. Sesbania rostrata also has a nodulated underground root system. On well-aerated roots, nodules form via root hair curling infection in the zone, just above the root tip, where root hairs develop; on hydroponic roots, an alternative process is used, recruiting a cortical intercellular invasion program at the lateral root bases that skips the epidermal responses. This intercellular cortical invasion entails infection pocket formation, a process that involves cell death features and reactive oxygen species. The plant hormones ethylene and gibberellin are the major signals that act downstream from the bacterial nodulation factors in the nodulation and invasion program. Both hormones block root hair curling infection, but cooperate to stimulate lateral root base invasion and play a role in infection thread formation, meristem establishment, and differentiation of meristem descendants.     

Cross-pathogenicity of Rhizoctonia solani strains on pasture legumes in pasture-crop rotations

In Australia, in the past, pasture legumes were rotated mainly with cereals, but increasingly these rotations now involve pasture legumes with a wider range of crops, including legumes. This increasing frequency of the leguminous host in the rotation system may be associated with increased root rots in legumes in the current pasture-crop rotations. The primary aim of this study was to see whether the pathogenicity on pasture legumes of strains of Rhizoctonia solani sourced from lupins and cereals (common crops in rotation with pastures) is associated with increased incidence of root rots in pasture legumes in the disease conducive sandy soils of the Mediterranean regions of southern Australia. The second aim was to determine sources of resistance among newly introduced pasture legumes to R. solani strains originating from rotational crops as this would reduce the impact of disease in the pasture phase. Fifteen pasture legume genotypes were assessed for their resistance/susceptibility to five different zymogram groups (ZG) of the root rot pathogen R. solani under glasshouse conditions. Of the R. solani groups tested, ZG1–5 and ZG1–4 (both known to be pathogenic on cereals and legumes) overall, caused the most severe root disease across the genotypes tested, significantly more than ZG6 (known to be pathogenic on legumes), in turn significantly >ZG4 (known to be pathogenic on legumes) which in turn was >ZG11 (known to be pathogenic on legumes including tropical species). Overall, Ornithopus sativus Brot. cvs Cadiz and Margurita, Trifolium michelianum Savi. cvs Paradana and Frontier and T. purpureum Loisel. cv. Electro showed a significant level of resistance to root rot caused by R. solani ZG11 (root disease scores ≤1.2 on a 1–3 scale where 3 = maximum disease severity) while O. sativus cvs Cadiz and Erica showed a significant level of resistance to root rot caused by R. solani ZG4 (scores ≤1.2). O. compressus L. cvs Charano and Frontier, O. sativus cv. Erica, and T. purpureum cv. Electro showed some useful resistance to root rot caused by R. solani ZG6 (scores ≤1.8). This is the first time that cvs Cadiz, Electro, Frontier, Margurita and Paradana have been recognised for their levels of resistance to root rot caused by R. solani ZG11; and similarly for cvs Cadiz and Erica against ZG4; and for cvs Charano, Erica, and Electro against ZG6. These genotypes with resistance may also serve as useful sources of resistance in pasture legume breeding programs and also could potentially be exploited directly into areas where other rotation crops are affected by these R. solani strains. None of the tested genotypes showed useful resistance to R. solani ZG1–4 (scores ≥2.0) or ZG1–5 (scores ≥2.5). This study demonstrates the relative potential of the various R. solani ZG strains, and particularly ZG1–4, ZG1–5, ZG4 and ZG6 to attack legume pastures and pose a significant threat to non-pasture crop species susceptible to these strains grown in rotation with these pasture legumes. Significantly, the cross-pathogenicity of these strains could result in the continuous build-up of inoculum of these strains that may seriously affect the productivity eventually of legumes in all rotations. In particular, when choosing pasture legumes as rotation crops, caution needs to be exercised so that the cultivars deployed are those with the best resistance to the R. solani ZGs most likely to be prevalent at the location.     

The role of Rhizobium conserved and host specific nodulation genes in the infection of the non-legume Parasponia andersonii

Summary Rhizobium and Bradyrhizobium bacteria gain intercellular entry into roots of the non-legume Parasponia andersonii by stimulating localized sites of cell division which disrupt the epidermis. Infection threads are then initiated from intercellular colonies within the cortex. Infection via the information of infection threads within curled root hairs, which commonly occurs in legumes, was not observed in Parasponia. The conserved nodulation genes nodABC, necded for the curling of legume root hairs, were not essential for the initiation of infection, however, these genes were required for Parasponia prenodule development. In contrast, the nodD gene of Rhizobium strain NGR234 was essential for the initiation of infection. In addition, successful infection required not only nodD but a region of the NGR234 symbiotic plasmid which is not needed for the nodulation of legumes. Agrobacterium tumefaciens carrying this Parasponia specific region, as well as legume nod genes, was able to form nodules on Parasponia which reached an advanced stage of development.