The Influence of Root Temperature, Rhizobium Strain and Host Selection on the Structure and Nitrogen-fixing Efficiency of the Root Nodules of Trifolium subterraneum

Low root temperature greatly affected the structure and N2-fixingefficiency of root nodules. More nodule tissue was formed perplant at 11 and 15 °C than at 7 and 19 °C. Low roottemperatures either prevented or slowed bacteroid differentiation;the differentiation zone was 19 per cent of the total noduletissue at 7 °C but only 5 per cent at 19 °C. The amount of bacteroid tissue formed at the different roottemperatures by the two fully effective strains TAi and SU297reflected the environment from which they originated. Both formedthe same amount at 15 and 19 °C but only TAI, which originatedfrom a cold environment formed bacteroids at 7 °C. At 7°C a bacteroid-filled cell did not degenerate until after20 days, cf. less than 10 days at 19 °C. At 7 and 11 °Call strains formed more bacteroids in the abundantly nodulatingthan in the sparse host independently of nodule number. Strain0403 was most sensitive to both temperature and host; it formedbacteroids in nodules on the sparse host at 19 °C only,but formed bacteroids in the abundant host between 7–19°C. The amount of bacteroid tissue formed by TAI and SU297 dependeddirectly on nodule number and was approximately constant between20–40 days only at 19 °C when nodule formation hadalmost stopped. The optimum temperature for maximum fixation of nitrogen wasnot necessarily that for maximum efficiency of fixation, whichfor these experiments was 51 ug N mm-3 bacteroid tissue perday.     

Growth of Trifolium subterraneum L. Selected for Sparse and Abundant Nodulation as Affected by Root Temperature and Rhizobium Strain

Root temperature greatly affected plant growth whether or notplants depended on symbiotic nitrogen fixation. The two plantselections responded differently to the three strains of Rhizobiumand this response was differentially affected by root temperature. Plant yield was significantly decreased by each fall of 4 °Cin temperature from 19 to 7 °C by amounts that dependedboth on the host and Rhizobium strain. Symbiosis with strainTA1, originally isolated from a cold environment, was most tolerantof a root temperature of 11 °C; TA1 produced as much ormore plant material of the abundantly nodulating host in 40days growth at 7 and 11 °C as did the uninoculated plantsgiven KNO³. Root temperature affected the number, rate of formation, anddistribution of nodules on the root system. At 7 °C fewernodules formed than between 11 and 19 °C. At 7 °C nodulesdid not form on secondary roots by 40 days but at 11 °Cthe secondary roots nodulated rapidly between 30 and 40 days.Nodule formation at 19 °C was almost completed at 20 days,when secondary root nodules accounted for 60 per cent of thetotal. Within the range 15 to 19 °C, at which the originalselections for sparse and abundant nodulation were made, plantsnodulated true to selection, but not at 11 °C. At 7 and11 °C plants nodulated with TA1 yielded more with increasingnumber of nodules.     

The Influence of Temperature on Root Hair Infection of Trifolium parviflorum and T. glomeratum by Root Nodule Bacteria: I. THE EFFECTS OF CONSTANT ROOT TEMPERATURE ON INFECTION AND RELATED ASPECTS OF PLANT DEVELOPMENT

The onset and rate of infection in root hairs of T. parviflorumand T. glomeratum inoculated with Rhizobium trifolli strain5 varied much with root temperature. At moderate root temperature(18, 24, and 30 °C) infections were initiated earlier andin larger numbers than at low (6 and 12 °C) or moderatelyhigh (36 °C) temperatures. Both species showed a broad temperatureoptimum between 18 and 30 °C. The site of thread initiation(apically or laterally in a hair) was independent of temperature,as was also the proportion of successful threads penetratingthe root cortex, which increased with seedling age. Threadsgrew more slowly at low temperatures. The size of hair nucleinear infection threads remained unaffected by temperature, butnuclei associated with laterally originating threads were largerthan those associated with apical threads. Infection was non-randomly distributed along the main root atall temperatures. More zones of infection were found at moderatetemperatures than at temperature extremes (6–12 or 36°C). Probit plots of numbers of infections for individualplants were steplike, the linear sloping parts correspondingto normal distributions of infection within zones. Between 18 and 30 °C numbers of infections increased exponentiallyin two phases, the first more rapid phase ending at about thetime nodules appeared. A model devised for the infection processand fitted to the data suggested the existence of two kindsof infections: primary ones occuring randomly at a slow rateand probably not affected by temperature and secondary infectionsthat appeared to increase with rising temperatures in the range12 to 30 °C. Nodule numbers were relatively more sensitive to high and lowtemperatures than infection. The numbers of infections and nodulesand the root lengths of T. parviflorum were twice those of T.glomeratum except at the temperature extremes. Numbers of infectionswere otherwise unrelated to root length or cotyledon or leafareas. The development of lateral organs (primordia, lateralroots, and nodules) was reduced at temperatures below 18 °Cand above 30 °C.     

Viability of Bradyrhizobium japonicum bacteriods

Homogenates from soybean nodules, formed by 12 strains of Bradyrhizobium japonicum, were plated into yeast-extract mannitol agar containing 3 or 37 g mannitol 1^-1. Viable counts ranged from 8.298 to 11.265 log₁₀ cells-gram nodule^-1. When monitored over the life cycle of the symbiosis, the viability of strains USDA 110 and USDA 123 increased with days after planting (DAP), and at 70 DAP was 95% and 81%, respectively. By contrast, the viability of USDA 38 bacteroids decreased with time, and at 70 DAP was only 1.9%. At 49 DAP, nodules induced by USDA 38 had significantly fewer bacteroids per peribacteroid membrane than those formed by USDA 110 or USDA 123, and at 70 DAP, 27% of the USDA 38 bacteroids showed some degree of degeneration. Viable counts of USDA 123 and USDA 110 bacteroids, isolated from the nodules of 12 different cultivars, ranged from 10.963 to 11.463 and from 10.683 to 11.117 log₁₀ viable cells-gram nodule^-1, respectively. Varying the osmolarity of the medium had no predictable effect on bacteroid viability. When surface-sterilized nodules of IPAGO 587 (high bacteroid viability) and USDA 38 (low bacteroid viability) were inoculated into a nonsterile silt loam soil, at rates equivalent to 5.0×10⁸ and 5.0×10⁶ viable bacteroids g^-1 soil, respectively, and then incubated at 28° C for 60 days, 4.3×10⁴ and 1.5×10⁴ surviving cells g^-1 soil, respectively, were recovered. Thus, despite differences due to host and strain variation, bacteroid viability appears to be unrelated to persistence of individual strains following an annual legume crop cycle.Journal paper No. 14930, Agricultural Experiment Station University of Minnesota, St. Paul, MN 55108, USA     

The effect of nitrogen addition on N2-fixation and on glutamate dehydrogenase and glutamate synthase activities. in nodules and roots of soybeans inoculated with various strains of Rhizobium japonicum

The addition of nitrogen in the form of urea decreased the activitiesof glutamate dehydrogenase (GDH) and glutamate synthase (GOGAT)in root nodules of Glycine max, whereas the same addition greatlyenhanced root GDH activity. Division of nodules into a mitochondrialand bacteroid fraction indicated that the addition of nitrogenas urea, ammonia, or nitrate most greatly inhibits GDH activityin the mitochondrial fraction. Studies with plants having floralprimordia indicated that added nitrate inhibits nodular GDHmore than either ammonia or urea, while plants inoculated withan ineffective strain (non-nitrogen fixing) of Rhizobium japonicumshowed an increase in nodular GDH activity with nitrogen addition.GOGAT activity was greatly reduced after floral initiation.GDH, GOGAT, and nitrogenase activities in root nodules appearedto vary with the strain of Rhizobium japonicum used as inoculum.In general, strains which produced nodules with high GDH activityproduced bacteroids with low GOGAT activity and the strain whichproduced nodules with the lowest GDH activity produced bacteroidswith the highest GOGAT activity. (Received May 24, 1976; )     

箭舌豌豆根瘤液泡中细菌周膜来源的研究

电镜观察结果表明,幼龄箭舌豌豆根瘤侵染细胞的细胞质较少,中央是一些体积较大的液泡。细胞质中侵入线经常可见,由侵入线释放出来的细菌均有细菌周膜。这些细菌只位于细胞质中,不出现在液泡里面。成熟根瘤中的侵染细胞与此不同,它们中有大量的成熟侵染细胞,细胞质丰富,里面充满大量细菌,中央常有一个大液泡。当中央液泡发育到一定程度时,位于其附近的细菌可通过液泡膜内吞、液泡膜与细菌周膜融合及液泡膜破裂3种途径进入液泡,后一种途径常伴有寄主细胞质。液泡中的细菌绝大部分裸露在外,只有个别细菌具有细菌周膜且多位于液泡膜的破损处附近,因此细菌周膜可能是原来就有的。     

The Infection of Trifolium subterraneum Root Hairs by Rhizobium trifolii

Both host cultivar and Rhizobium strain influence the numberof infected root hairs of Trifolium subl-errctneum, seedlings;Yarloop had fewer infections than Cranmore, Mount Barker, orTallarook and Rhizobium trifolii strain 5 infected fewer hairsthan strain TA1. Hybrid lines bred for sparse or abundant nodulationhad similar numbers of infected hairs, but. as in the cultivars,these always greatly exceeded the number of nodules formed.More infection threads aborted early during growth in the roothairs of Cranmore than in other hosts and early abortion wasmore common with strain 5 than strain TA1 In all hosts and with both Rhizobium strains, infection beganon day 3 and was initially restricted to one or two zones alongthe root with later infections extending these zones or initiatingnew ones. The exponential rate of infection (least for Yarloop)slows sharply when nodules appear. Early nodules and lateral roots formed at different places indifferent hosts, and in most cultivars and hybrid lines nodulesand laterals occurred in mutually exclusive zones. Primordiaarising above the first nodule failed to develop.     

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

The structure of effective nodules on Trifolium subterraneumL. grown at a root temperature favourable for nitrogen fixationwas examined by light microscopy. Rhizobium were released intohost cells from vesicles on infection threads which were closelyassociated with the host cell nucleus. Enlargement into bacteroidforms was rapid and synchronous in similarly-aged host cells,but groups of rhizobia close to the nucleus sometimes did notdevelop into bacteroids. Rhizobium changed in shape from smallrods to more elongate and then to irregularly swollen formsbefore finally becoming coccoid. On degeneration clusters ofbacteroids became un evenly stained before coalescing at thecentre of the host cell and finally disintegrating. Small vegetativerods multiplied amongst the degenerating bacteroids and in thegreatly enlarged intercellular spaces; host cell walls becamefolded and distorted and sometimes broke before collapsing ontoeach other.     

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.     

Differential Inhibition of Shootvs.Root Growth at Low Temperature and its Relationship with Carbohydrate Accumulation in Different Wheat Cultivars

Shoot and root growth rate, carbohydrate accumulation (includingfructan), reducing sugar content and dry matter percentage weremeasured in six wheat cultivars, ranging from winter to springtypes, grown at either 5 or 25 °C. At 5 °C (comparedwith 25 °C), the relative growth rate (RGR) of shoots wassimilarly reduced in all cultivars, but the RGR of shoots wasmore affected in winter wheats. This difference resulted insmaller root:shoot ratios than in spring wheats, which alsodeveloped more first-order lateral roots. A direct relationshipbetween carbohydrate accumulation at low temperatures and reductionin root growth was established. These results suggest that differentialshootvs.root growth inhibition at low temperature may play akey role in carbohydrate accumulation at chilling temperatures.This differential response might lead to improvements in survivalat temperatures below 0 °C, regrowth during spring, andwater and nutrient absorption at low temperatures.Copyright1997 Annals of Botany Company Wheat; Triticum aestivum; low temperatures; root growth; root: shoot ratio; sugar accumulation     

The Development of Primary Root Nodules on Vicia faba L. Grown at Two Temperatures

The development of primary root nodules on the field bean (Viciafaba L.) grown at 10 and 18 °C was examined. The sequenceof anatomical changes observed was the same in both temperatureregimes. Nodules developed much more slowly at 10 °C andthe nodules were much larger when corresponding anatomical changesoccurred. Primary root nodules eventually ceased growth in bothtemperature regimes but ultimate nodule volume was nearly twiceas great as 10 °C as at 18 °C. The larger size did notcompensate for the lower specific nitrogenase activity of cold-grownnodules: nitrogen fixation (acetylene reduction) rates on awhole plant basis were much lower at 10 °C than at 18 °C.There was no difference in the total number of primary rootnodules in the two temperature regimes but their distributionwas biased towards the upper part of the root and the epicotylat 18 °C. Vicia faba L., field bean, nodulation, nitrogen fixation, temperature     

The Formation of Nitrogen-Fixing Bacteroids Is Delayed but Not Abolished in Soybean Infected by an [alpha]-Ketoglutarate Dehydrogenase-Deficient Mutant of Bradyrhizobium japonicum

A mutant strain of Bradyrhizobium japonicum USDA 110 devoid of [alpha]-ketoglutarate dehydrogenase activity (LSG184) was used to test whether this tricarboxylic acid cycle enzyme is necessary to support nitrogen fixation during symbiosis with soybean (Glycine max). LSG184 formed nodules about 5 d later than the wild-type strain, and the nodules, although otherwise normal in structure, contained many fewer infected host cells than is typical. At 19 d after inoculation cells infected with the mutant strain were only partially filled with bacteroids and showed large accumulations of starch, but by 32 d after inoculation the host cells infected with the mutant appeared normal. The onset of nitrogen fixation was delayed about 15 d for plants inoculated with LSG184, and the rate, on a per nodule fresh weight basis, reached only about 20% of normal. However, because nodules formed by LSG184 contained only about 20% of the normal number of bacteroids, it could be inferred that the mutant, on an individual bacteroid basis, was fixing nitrogen at near wild-type rates. Therefore, the loss of [alpha]-ketoglutarate dehydrogenase in B. japonicum does not prevent the formation or the functioning of nitrogen-fixing bacteroids in soybean.     

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     

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.     

Leaf Extension in Zea mays: I. LEAF EXTENSION AND WATER POTENTIAL IN RELATION TO ROOT-ZONE AND AIR TEMPERATURES

The temperature of the roots and shoots of Zea mays plants werevaried independently of each other and the rates of leaf extensionand leaf water potentials were measured. Restrictions of leafextension occurred when root temperatures were lowered from35 to 0 °C, but leaf water potentials were lowered onlyat root temperatures below 5 °C. Similar changes in ratesof leaf extension were measured at air temperatures from 30to 5 °. Between 30 and 35 °C air temperature, in anunsaturated atmosphere, restrictions of leaf extension wereassociated with low leaf water potentials. It was concluded that, at root temperatures 5 to 35 °C,and shoot temperatures 5 to 30 °C, water stress was notthe main factor restricting the extension of Zea mays leaves.     

Biological and Cultural Characteristics of the Effective Frankia Strain HFPCcl3 (Actinomycetales) from Casuarina cunninghamiana (Casuarinaceae)

From homogenates prepared from surface-sterilized nodules ofseedlings of Casuarina cunninghamiana grown aeroponically, astrain of Frankia designated HFPCc13 was isolated and has beengrown in pure filamentous culture in a defined synthetic nutrientmedium. Vesicle and sporangium formation can be induced by removalof combined nitrogen from the medium.Frankia strain HFPCc13nodulates young seedlings of C. cunninghamiana and C. equisetifoliawithin three weeks of inoculation with an optimum root mediumpH of 6–7 for nodulation and optimum temperature of 30–35^°C. The presence of combined nitrogen in the root mediuminhibits nodulation with NH₄⁺ more inhibitory than NO₃^–.Frankia HFPCc13 does not nodulate Allocasuarina species withinthe same family nor several other possible actinorhizal plantstested. Thus this strain is quite precise in its host specificity.The rate of acetylene reduction was greater in C. cunninghamianathan the closely related species C. equisetifolia. In neitherof these host species were vesicles observed to occur withinthe infected root nodules which had been demonstrated to beactively fixing dinitrogen. Root nodules were shown to be activein acetylene reduction over a range of O₂ concentration in thegaseous environment with an optimum at about 20 per cent O₂,the ambient P_O2 of the air. The mechanism(s) for oxygen protectionof nitrogenase within the filamentous form of Frankia withinthese nodules remains to be explained. Casuarina, Frankia, nodulation, nitrogen fixation     

Hydrogen Effect on Nitrogenase (C2H2 Reduction) Response to Oxygen in Bradyrhizobium japonicum-Phaseoleae Symbioses

The influence of hydrogenase in Bradyrizobum-Phaseoleae symbioseswas studied ex-planta and in-planra in soybean (Glycine max)and cowpea (Vigna unguiculata). The hydrogenase was activatedby the addition of hydrogen in the incubation gas phase whichmodified the response of nitrogenase activity of Hup⁺ (hydrogenuptake positive) symbiosis to the external oxygen partial pressure.For bacteroids the hydrogenase expression increased nitrogenaseactivity at supraoptimal pO₂, acting possibly as a respiratoryprotection of nitrogenase. However, at suboptimal pO₂, nitrogenaseactivity of Hup⁺ bacteroids decreased with hydrogen, a phenomenonattributed to the lower efficiency of ATP synthesis from hydrogenthan from carbon substrates oxidation. For undisturbed nodules,the hydrogenase expression in soybean increased the optimalpO₂ for ARA (COP), from 35.3 to 40.3 kPa O₂, and the ARA atsupraoptimal pO₂; at suboptimal PO₂ there was a negative effectof hydrogenase on ARA, although this inhibition was less thanon bacteroids and was not detected if plants were grown at 15°C rather than 20 °C root temperature. No H₂ effectwas detected on cowpea nodules. The results on soybean nodulesare consistent with the concept that symbiotic nitrogen fixationis oxygen-limited and that hydrogenase activity has no beneficialeffect on nitrogen fixation in O₂ limitation. Key words: Glycine max, hydrogenase, nitrogenase, nitrogen fixation, nodules, Vigna unguiculata     

Polypeptide synthesis by Rhizobium bacteroids and bacteria.

When Rhizobium bacteroids (strain NZP 2257) from lupin nodules were isolated and incubated aerobically at high osmolarity, they incorporated [35S]-methionine into a characteristic set of polypeptides; many of these polypeptides coelectrophoresed on SDS-polyacrylamide gels with the bacteroid polypeptide bands stained by Coomassie blue. The labelled polypeptides were stable for several hours in pulse-chase experiments. Changes in the concentration of H+, K+ and Mg2+ in the incubation mixture affected overall incorporation of label, but not the relative incorporation into different polypeptides. A similar set of bacteroid polypeptides was labelled in situ when detached nodules were fed [35S]methionine. Distinctive labelling patterns were observed with bacteroid suspensions from mature and immature nodules, with a transitional pattern at the time when nitrogenase activity appeared. Two of the major labelled components in mature bacteroids had estimated molecular weights of 60- and 34-kilodaltons similar to values reported by others for the constituent polypeptides of nitrogenase. Bacteroids of the same Rhizobium strain grown in different plant hosts gave similar polypeptide labelling patterns in purified suspensions, but bacteroids of different Rhizobium strains gave different patterns. The polypeptide labelling patterns obtained using broth-cultured Rhizobium bacteria from various growth stages and growth media differed from those obtained using bacteroids of the same strain.