Structure and Organization of Effective Peanut and Cowpea Root Nodules Induced by Rhizobial Strain 32H1

A comparison of the structure and organization of nodular tissuesand bacteroids of peanut and cowpea induced by Rhizobium sp.strain 32H1 was madc 4 to 5 weeks after inoculation when nitrogenaseactivity reaches the peak. Observations revealed major differencesthat may have a role in the different rates of nitrogen fixationshown by the two species. All cell types in cowpca nodules werelarger than those of peanut. The inner cortex of cowpea hadan ‘endodermis-like’ layer of cells which was absentin peanut. All cells in the bactcroidai zone of peanut wereinfected but in cowpca many remained free of bactcroids. Thebacteroid containing cells of peanut were isodianietrical anduniform in size with a central vacuole and a nucleus surroundedby tightly arranged bactcroids enclosed singly in peribacteroidalmembrane sacs. Such cells in cowpea were mostly elongated witha nucleus and one or more vacuoles. The bacteroids within cowpeacells were arranged without any particular order with more spacefor host cellular material. They were mostly present singlyin peribacteroidal membrane sacs which sometimes fused to enclosemore than one bactcroid. The hosts seem to play the dominantrole in the differentiation of nodular tissue and the morphogenesisof bacteroids in symbiotic systems induced by the same strainof Rhizobium. Key words: Peanut, Cowpea, nodule structre     

The Structure and Development of Trifolium subterraneum L. Root Nodules: II. IN PLANTS GROWN AT SUB-OPTIMAL ROOT TEMPERATURES

Cold root temperature affected infection thread proliferation,cell invasion, and release of Rhizobium and the subsequent developmentof this infection in Trifotium subterraneum. These events werealso modified by both host cultivar and bacterial strain. At7 °C bacteroid development was only substantial with strainTA1, with either sparsely or abundantly nodulating lines ofthe host. At 11 °C strain SU297 also readily formed effective,bacteroid-filled nodules with both lines. Strain 0403 formeda few bacteroids with the abundant line only at 7 °C andreadily formed bacteroids with the sparse line only at 19 °C.At 15 °C 0403 nodules were effective on abundant lines,but mostly ineffective on sparse lines. The development of Rhizobium rods into bacteroicis and theirsubsequent degeneration wa slower at low temperatures with bothstrains. Low root temperatures favoured the deposition of starchthroughout the nodule. At higher temperatures, when bacteroidswere more active in nitrogen fixation, starch was mostly confinedto a narrow band of the youngest bacteroid filled cells andto the zone of bacteroid degeneration.     

Nodulation of Pole Bean (Phaseolus vulgaris L.) by Rhizobium Species of Two Cross-Inoculation Groups

Physiology and morphology of pole bean (Phaseolus vulgaris L. cv Kentucky Wonder) root nodules induced by two Rhizobium species of different cross-inoculation groups have been compared. Root nodules induced by Rhizobium sp. 127E15, which is a strain of the cowpea group Rhizobium, were pinkish, had irregular shapes, and were only partially effective. Their peak acetylene reduction activity was 4.36 μmol of C₂H₄ formed per g of fresh nodules per h at 30 days after inoculation. The effective nodules induced by Rhizobium phaseoli 127K14, which is a strain of the bean group Rhizobium, were dark red, spherical, and showed peak acetylene reduction activity of 15.95 μmol of C₂H₄ formed per g of fresh nodules per h at 15 days after inoculation. The partial effectiveness of 127E15-induced nodules was associated with fewer infected cells, a delay in the increase of bacteroid population within the host cells, abundance of cytoplasmic vesicles in the host cells, more bacteroids within a membrane envelope (peribacteroid membrane), and the inability of bacteroids to completely fill up the host cytoplasmic space. The 127K14-induced nodules were fully mature, with host cells filled with bacteroids by 12 days after inoculation. In contrast, the 127E15-induced nodules did not reach a similar developmental stage even 30 days after inoculation.     

Infection and Root-Nodule Development in Stylosanthes Species by Rhizobium

Root nodules of the tropical forage legume Stylosanthes occurredonly at lateral root junctions and resulted from direct invasionby rhizobia through spaces between epidermal cells. No infectionthreads were present in either the root hairs or nodules. Invasionof the host cortical cells was through structurally alteredcell walls. The bacteria reached the site of nodule initiationin the lateral root cortex by progressive collapse of the initiallyinvaded cells which were compressed by neighbouring cells toform intercellular thread-like infection zones. The bacteriamultiplied in the invaded cells of the nodule initial whichdivided repeatedly to form the nodule. Bacteroids formed onlywhen the host cells ceased to divide. Some abnormal associations occurred in S. capltata and S. hamata40264A. Division of invaded cells was restricted in S. capitataand the bacteria became enlarged and grossly deformed. In S.hamata restricted cell division was immediastely followed bythe brcakdown of the host cells and, although the bacteria multiplied,no bacteroids were formed. Bacteria isolated from these nodulesformed both effective and abnormal nodules when inoculated ontothe same host.     

Infection and Nodule Development in Aotus ericoides (Vent.) G. Don, A Woody Native Australian Legume

Infection and nodule development were studied by light and electronmicroscopy in Aotus ericoides, a woody native Australian legume,inoculated with a slow-growing field isolate of Rhizobium. Rhizobiabound to straight, but not deformed, root hairs, as detectedby immunofluorescence. Neither markedly curled root hairs norroot hairs with infection threads were seen. Nodules were indeterminate(astragaloid), with a peripheral meristematic layer, few vasculartraces and both infected and uninfected cells in the centralinfected zone. Infection threads containing contorted bacteriawere present throughout the nodule. Swollen, rod-shaped bacteriain infected cells were in groups in vesicles bounded by plasmalemma-derivedperibacteroid membranes. Senescence in infected cells was associatedwith accumulation of a fibrillar matrix inside peribacteroidmembranes, distortion of bacteria and destruction of most cytoplasmiccontents of the bacteria and host cells; however, most bacterialand plant membranes and plant cell walls remained intact. Ineffectivenesswas associated with relatively little, short-lived infectedtissue. Events in infection and nodule development were similarto those in most herbaceous legumes but showed characters ofboth determinate and indeterminate nodules. Key words: Bacteroids, Legume, Nitrogen-fixing, Nodule, Rhizobium     

Effects of the plant host on the detergent sensitivity and viability of Rhizobium bacteroids

Bacteroids prepared from different legume species showed large differences in detergent sensitivity as judged by changes in turbidity and the release of cytochrome c oxidase activity after detergent treatments. There was a strong correlation between the detergent sensitivity and non-viability of bacteroids. Differences in the detergent sensitivity of bacteroids were determined by the plant host rather than the Rhizobium strain or the effectiveness of the symbiosis. The most common level of detergent sensitivity observed amongst bacteroids from 34 legume species was intermediate between lupin bacteroids and brothcultured bacteria.     

Development of the bacteroid in the root nodule of barrel medic (Medicago tribuloides Desr.) and subterraneum clover (Trifolium subterraneum L.)

Summary The development of the bacteriod is traced from thin sections of slices of nodules fixed in KMnO₄ and OsO₄. While in the infection thread the Rhizobium cell has the ultrastructure characteristic of gram-negative bacteria, with two unit membranes bounding a granular cytoplasm containing dense bodies, a nucleoid area and inclusion granules. A 10–12 fold increase in size, a loss of inclusion granules and the formation of a membrane envelope around each Rhizobium cell follows the dispersal of the rhizobia through the host cytoplasm. As the bacteriods develop there is a loss of fibrillar material from the nucleoid region and changes occur in the distribution of ribosome-like particles in both host and bacterial cells. When fully differentiated and presumably fixing nitrogen the bacteroids from the red zone of subterraneum clover nodules but not barrel medic have a well developed intra-cytoplasmic membrane system.     

Early development ofRhizobium-induced root nodules ofParasponia rigida. I. Infection and early nodule initiation

Summary The first of two major steps in the infection process in roots ofParasponia rigida (Ulmaceae) following inoculation byRhizobium strain RP501 involves the invasion ofRhizobium into the intercellular space system of the root cortex. The earliest sign of root nodule initiation is the presence of clumps of multicellular root hairs (MCRH), a response apparently unique amongRhizobium-root associations. At the same time or shortly after MCRH are first visible, cell divisions are initiated in the outer root cortex of the host plant, always subjacent to the MCRH. No infection threads were observed in root hairs or cortical cells in early stages. Rhizobial entry through the epidermis and into the root cortex was shown to occur via intercellular invasion at the bases of MCRH. The second major step in the infection process is the actual infectionper se of host cells by the rhizobia and formation of typical intracellular infection threads with host cell accommodation. This infection step is probably the beginning of the truly symbiotic relationship in these nodules. Rhizobial invasion and infection are accompanied by host cortical cell divisions which result in a callus-like mass of cortical cells. In addition to infection thread formation in some of these host cortical cells, another type of rhizobial proliferation was observed in which large accumulations of rhizobia in intercellular spaces are associated with host cell wall distortion, deposition of electron-dense material in the walls, and occasional deleterious effects on host cell cytoplasm.     

Carbon and nitrogen metabolism in legume root nodules

The literature concerning the metabolism of carbon compounds during the reduction, assimilation and translocation of nitrogen in root nodules of leguminous plants is reviewed. The reduction of dinitrogen requires an energy source (ATP) and a reluctant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid or urcide synthesis during the assimilation of ammonia produced by the bacteria within the nodule tissue. Competition for photosynthates occurs between the bacteroids, nodule tissue and the various vegetative and reproductive sinks in the host plant. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolises between the various sites of utilization are only poorly understood. It is apparent that dinitrogen is reduced to ammonia in the bacteroids. Both fast- and slow-growing strains of Rhizobium possess the Entner-Doudoroff pathway of glucose catabolism, and some, if not all, enzymes of the Emden-Meyerhof pathway. Some bacterial cultures also metabolize carbon through the ketogluconate pathway but only the fast-growing strains of cultured rhizobia possess the key enzyme of the pentose phosphate pathway (6-phosphogluconate dehydrogenase). The host cells are thought to contain the complete Emden-Meyerhof pathway and tricarboxylic acid cycle, which provides the carbon skeletons for assimilation of the ammonia, formed by the bacteroids, into α-amino acids. A pathway of anapleurotic carbon conservation, operative in the host cells, synthesizes oxaloacetic acid through β-carboxylation of phosphoenol pyruvate. This process could be important in the recapture and assimilation of respired CO₂ in the rhizosphere. The main route of assimilation of ammonia produced by the bacteroids would appear to be via the glutamine synthetase-glutamate synthase pathway in the host cells. However, glutamate dehydrogenase may also be involved in ammonia assimilation. These enzymes also occur in in vitro cultures of Rhizobium and in bacteroids where they presumably participate in the synthesis of amino acids for growth of the bacteria or bacteroids. Nitrogen assimilated into glutamine or glutamate is exported from the nodules in a variety of forms, which include asparagine, glutamine, aspartate, homoserine and allantoates, in proportions which depend on the legume species. Studies on regulation of the overall process have focussed on expression of bacteroid genes and on the control of enzyme activity, at the level of nitrogenase and enzymes of nitrogen assimilation in particular. However, due to the wide range of experimental techniques, environmental conditions and plant species which have been used, no clear conclusions can yet be drawn. The pathways of carbon flow in nitrogen metabolism, particularly in relation to the synthesis of ureides and the regulation of carbon metabolism, remain key areas for future research in symbiotic nitrogen fixation.     

Fine structure changes in the development of the nodules of Trifolium subterraneum L. and Medicago tribuloides Desr.

Summary Electron microscope observations of thin sections of nodules of subterranean clover and barrel medic, after fixation in KMnO₄ or OsO₄, show that following infection there is a marked increase in the amount of endoplasmic reticulum, in the number of ribosomes, Golgi bodies, mitochondria and proplastids in the host cells.As the infection thread approaches the nucleus, large gaps appear in the nuclear membrane. During the formation of the membrane envelopes around the rhizobia, after their release from the infection thread, the reticulum changes from a predominantly plate-like to a vesicular form. As the bacteroids develop the plastids of the host cells become filled with starch, and become aligned, with the mitochondria, against the cell walls of the host cells. Plastids in noninvaded cells also become starch-filled. Bacteroids and host cells enlarge further and finally the bacterioids occupy most of the cytoplasm of the host cell, except for the nuclear region and vacuole. With OsO₄ fixation the nucleoplasm, predominantly fibrillar before infection, with a dense staining nucleolus, becomes packed with dense ribosome-like (150 A° diameter) granules. No such changes occur in the nuclei of non-infected cells. In the proplastids and plastids many small, electron dense particles (60 A° diameter) (phytoferritin?) are observed.     

Nitrogenase (C2H2) Activities of Isolated Peanut and Cowpea Bacteroids at Optimal Oxygen Availability and Comparison with Whole Nodule Activities

Bacteroids, formed by the same strain of Rhizobium, were isolatedanaerobically from peanut and cowpea root nodules and theirC₂H₂ reduction activities were measured. Measurements were startedin a pure N₂ atmosphere followed by stepwise addition of smallamounts of O₂. The procedures may have general application andare described in detail. With increasing O₂ level, a gradualincrease in nitrogenase activity was observed which reacheda peak, presumably at the optimum availability of O₂ to bacteroids,and then declined. The maximum activity attained by isolatedbacteroids of cowpea was much higher than that obtained frommeasurement of activities of intact nodules and their bacteroidcontent, whereas for peanut the two were nearly equal. The resultsindicated that intranodular conditions are probably responsiblefor the difference in nitrogenase activities of peanut and cowpeanodules rather than the unique morphological modification ofpeanut bacteroids. Key words: Root nodules, Peanut, Cowpea, Bacteroids, Nitrogenase activity     

Primary metabolites of Phaseolus vulgaris nodules

More ethanol soluble material (carbohydrate and amino nitrogen) was found in both host cell and bacteroid components of Phaseolus vulgaris nodules from plants grown at 28 W/m² than from plants grown at 7 W/m². The range of compounds identified was similar at the two irradiances. On feeding ¹⁴CO₂ to the plant tops at either irradiance the labelling patterns of carbohydrates and organic acids in the nodule host cells and bacteroids suggested that any or all of the following substances could be donated by the host to the bacteroids for general metabolism: sucrose, fructose, glucose, an unidentified carbohydrate, malic acid and an organic acid co-chromatographing with 6-phosphogluconate. Distribution and labelling patterns of nodule amino compounds were consistent with the hypothesis that ammonia is the primary product of nitrogen fixation within bacteroids, and that this ammonia is transported to host cells for assimilation, initially into glutamine and glutamate.     

Rhizobial lipo-oligosaccharide nodulation factors: multidimensional chromatographic analysis of symbiotic signals involved in the development of legume root nodules

Nod factors are a group of biologically active oligosaccharidesignals that are secreted by symbiotically competent bacteriaof the family Rhizobiaceae. Their biosynthesis is determinedby rhizobial nodulation (nod) genes, and is specifically inducedin response to flavonoids secreted from the roots of host leguminousplants. The biological activity of Nod factors on these hostlegumes dramatically mimics the early developmental symptomsof the Rhizobium-legame symbiosis including, amongst other effects,root hair deformations and nodule initiation. Structurally,all Nod factors are short oligomers of ß-1,4-linkedN-acetylglucos-amine residues [usually degree of polymerization(dp) 4 or 5] that are N-acylated on the distal glucosarnine.This common ‘core’ structure may be modified bya number of species-specific substituents on the distal or reducingsugars. These modifications are governed by rhizobial host specificitynod genes. The biological activity of purified Nod factors mirrorsthis host specificity, indicating that the symbiotic host rangeof individual Rhizobium species is, at least partially, determinedby the variety of Nod factors they are able to produce. Herewe describe techniques that are universally applicable to theextraction, chromatographic separation and identification ofNod factors. We have applied these techniques to Nod factorsfrom the broad-host-range species Rhizobium fredii USDA257 andRhizobium spp. NGR234, and the more narrow-host-range Bradyrhizobiumjaponicum USDA110, and have identified a group of novel, relativelyhydrophilic Nod factors from the NGR234 species that may haveimplications for Nod factor biosynthesis. lipo-oligosaccharide Nod factor rhibozobia singals TLC     

Effectiveness of Lotus Root Nodules: I. MORPHOLOGY AND FLAVOLAN CONTENT OF NODULES FORMED ON LOTUS PEDUNCULATUS BY FAST-GROWING LOTUS RHIZOBIA

The morphology of root nodules formed on Lotus pedunculatusby two fast-growing strains of Rhizobium, NZP2037 which formseffective (nitrogen-fixing) nodules and NZP2213 which formsineffective (non-nitrogen-fixing) nodules, has been studied.The nodules formed by NZP2037 contained a central zone of bacteroid-filledplant cells surrounded by a cortex. In contrast the nodulesformed by NZP2213 contained no Rhizoblum-infected plant cells,but rhizobia were found in localized areas on the nodule surfaceand between the outer two or three cell layers of the nodule.Electron-dense osmiophilic deposits identified as flavolans(condensed tannins) were present in the vacuoles of many uninfectedplant cells in the nodules formed by both Rhizobium strains.This is the first time that flavolans have been positively identifiedin legume root nodules. In the NZP2037 nodule flavolans werepresent in the outer cortical and epidermal cells. In the ineffecitveNZP2213 nodule fiavolans were present in many of the centralnodule cells. The concentration of flavolan in the NZP2213 nodulewas 12 times higher than in the NZP2037 nodule.     

Hydrogenase in legume root nodule bacteroids: Occurrence and properties

Summary The hydrogenase found in Rhizobium bacteroids is compared with that found in Azotobacter and found, in all respects examined, to be similar. When three host species were inoculated with Rhizobium, strain 311, different amounts of hydrogenase activity were found in Pisum sativum and Vicia bengalensis while the enzyme was absent from nodules of Vicia faba. Of four different strains of Rhizobium examined only two strains possessed the hydrogenase when present in pea root nodules. The role of the hydrogenase in nitrogen fixation is discussed and it is tentatively concluded that the overall efficiency of the nitrogen fixation process is increased by its presence.     

Differential accumulation of hydroxyproline-rich glycoproteins in bean root nodule cells infected with a wild-type strain or a C4-dicarboxylic acid mutant of Rhizobium leguminosarum bv. phaseoli

An antiserum raised against deglycosylated hydroxyproline-rich glycoproteins (HPGPs) from melon (Cucumis melo L.) was used to study the relationship between Rhizobium infection and induction of HRGPs in bean (Phaseolus vulgaris L.) root nodule cells infected with either the wild-type or a C₄-dicarboxylic acid mutant strain of Rhizobium leguminosarum bv. phaseoli. In effective nodules, where fixation of atmospheric dinitrogen is taking place, HRGPs were found to accumulate mainly in the walls of infected cells and in peribacteroid membranes surrounding groups of bacteroids. Internal ramifications of the peribacteroid membrane were also enriched in HRGPs whereas the peribacteroid space as well as the bacteroids themselves were free of these glycoproteins. In mutant-induced root nodules, HRGPs were specifically associated with the electron-dense, laminated structures formed in plastids as a reaction to infection by this mutant. The presence of HRGPs was also detected in the host cytoplasm. The aberrant distribution of HRGPs in infected cells of mutant-induced nodules likely reflects one aspect of the altered host metabolism in relation to peribacteroid-membrane breakdown. The possibility that the antiserum used for HRGP localization may have cross-reacted with ENOD 2 gene products is discussed in relation to amino-acid sequences and sites of accumulation.     

Accumulation of an Aromatic Amine, β-Phenethylamine, in Root Nodules of Adzuki Bean Vigna angularis

Neuroactive aromatic amines acting on the central nervous system are widespread in the plant kingdom. We have previously found β-phenethylamine (β-PHA), one of the aromatic alkaloids, in root nodules of various annual legume crops. The present study was undertaken to determine the site of β-PHA accumulation within root nodules of the adzuki bean Vigna angularis. High concentrations of β-PHA were always detected in the alkaloid fraction of adzuki bean root nodules. Related aromatic amines such as tyramine, dopamine, and other β-PHA derivatives, which are found in various medicinal plants, were not detected in adzuki bean root nodules. The amounts of β-PHA in root nodules varied not only with the growth stage of the host plant, but also with nodule age; β-PHA levels increased with nodule development, but declined with nodule senescence. Adzuki bean nodules, after crushing with a grinding medium, were separated into bacteroids and a nodule cytosol fraction. A large amount of β-PHA was detected in the bacteroids, while a very small amount was prsent in the nodule cytosol fraction derived from plant cells. The bacteroids in the mature nodules contained considerably higher amounts of β-PHA than did those in immature or senescent nodules. The formation of β-PHA in root-nodule bacteria was then tested using eight strains of Rhizobiaceae (Rhizobium, Bradyrhizobium and Sinorhizobium), including a strain isolated from root nodules of field-grown adzuki bean plant. None of the cultured cells produced β-PHA in liquid media in the presence or absence of phenylalanine, a putative precursor of β-PHA. Nitrogen-fixing bacteroids within nodules are the cells uniquely differentiated from root-nodule bacteria. The present results suggest that β-PHA is formed associated with the differentiation of vegetative bradyrhizobia into nitrogen-fixing bacteroids with the plant host cells.     

棉铃虫幼虫感染棉铃虫微孢子虫后的组织病理变化

1997年田间调查时发现一种寄生于棉铃虫Helicoverpa armigera（Hübner）的微孢子虫Nosema sp.,它对棉铃虫有较强的致病力并可经卵垂直传播。利用透射电镜对棉铃虫幼虫感染该微孢子虫后的组织病理变化进行了初步观察。结果表明：该微孢子虫可侵染棉铃虫的中肠、马氏管、脂肪体、神经等组织;侵染后可导致寄主中肠的微绒毛脱落,线粒体内脊排列方向发生变化,线粒体整体发生变形并最终瓦解;内质网发生断裂;细胞核体积变小并变形,但该微孢子虫并不入侵细胞核;马氏管膨大,边缘向外突出隆起;神经细胞的细胞核变成长条形,细胞界线模糊;在神经细胞内也发现了微孢子虫孢子,证明该微孢子虫也入侵寄主神经细胞。     

The nodulation of micro-propagated plants of Parasponia andersonii by tropical legume rhizobia

A simple clonal micro-propagation system for Parasponia andersoniiwas employed to study the nodulation response of this non-legumeto inoculation by the broad host range Rhizobium sp. NGR234,isolated from Lablab purpureus, and also to tropical legumerhizobia isolated from Aeschynomene species. Partially effectivenodules, assayed by acetylene reduction and ¹⁵N dilution procedures,were induced with strain NGR234 and its spontaneous streptomycinresistantmutant ANU240. Effective nodules were produced by one of theAeschynomene strains (ORS302) tested, with rates of acetylenereduction comparable to those of root nodules produced by Bradyrhizobiumstrain CP279, originally isolated from P. andersonii. Lightand transmission electron microscopy showed that there was acorrelation between the nitrogen fixing capability of the symbiosisbetween NGR234 and Parasponia and the number of persistent infection(fixation) threads within the nodule cells. Key words: Parasponia, Bradyrhizobium, Rhizobium, Aeschynomene, micro-propagation, root nodules, nitrogen fixation