ERRATA

Delete: J. Ineffective nodule with outer cells filled with ared pigment (x40) Insert: J . Coiled, branched and swollen root hairs in the axilof laterals, with no infection threads (x 100). Delete: C, G Insert: C, E, F. Insert: D. Latus hispidus Delete: D, E. Coiled, branched, and swollen root hairs in theaxils of laterals with no infection threads. Insert: D. Ineffective nodules of L. hispidus with outer cellsfilled with a red pigment (x40). Delete: F. Insert: E Delete: G. Insert: F. Abstract, 4th Line: Delete: 40 ?C Insert: 4 ?C Please.note equation (3) on page 959 should read V₂ = V₁ l?27^{(T₂ - T₁)} and NOT: V₂ = V₁ (l?27)T₁ - T₂ as printed.     

Effectiveness of Lotus Root Nodules: II. RELATIONSHIP BETWEEN ROOT NODULE EFFECTIVENESS AND 'IN VITRO' SENSITIVITY OF FAST-GROWING LOTUS RHIZOBIA TO FLAVOLANS

An extract from the roots of Lotus pedunculatus plants was foundto contain a compound toxic towards fast-growing Lotus rhizobia.This compound was identified as a flavolan, which has a prodeiphinidin:procyanidin ratio of 75:25. A fast-growing strain of Rhizobium(NZP2213) which forms ineffective root nodules on L. pedunculatuswas four times more sensitive to this flavolan (ED₅₀ = 25 ?gml^–1) than another strain (NZP2037, ED₅₀ = 100 ?g ml^–1)which forms effective root nodules on this species. The rootsof another Lotus species, L. tenuis, on which both strains ofRhizobium form effective root nodules, also contained a flavolan( 95% procyanidin) but both strains were relatively insensitiveto this flavolan (EDED₅₀ = 350 to 500 ?g ml^–1) L. pedunculatusplants bearing ineffective root nodules contained two to threetimes more flavolan in their roots (5–7 mg g^–1 fr.wt.)than uninoculated control plants. Experiments with seven otherLotus species and with hybrid plants developed between L. pedunculatusand L. tenuis showed a relationship between the prodeiphinidin:procyanidin ratio of the flavolan in their roots and the effectivenessof root nodules formed on these plants by NZP2213. Quantitativebinding studies of the flavolan from L. pedunculatus to NZP2037and NZP2213 indicated that, while the affinity constants forbinding were similar for both strains, the surface of strainNZP2037 contained four times more binding sites than NZP2213,possibly correlating with this strain's ability to toleratehigher concentrations of this flavolan. It is suggested thatthe differential sensitivity of these two strains of Rhizobiumto flavolans is related to their ability to form effective rootnodules on Lotus species.     

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     

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 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     

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.     

Effect of oxygen availability on nitrogen fixation by two Lotus species under flooded conditions

The pasture legumes Lotus uliginosus (Schk.) and Lotus corniculatus (L.), known to differ in their tolerance to flooding, were inoculated with Rhizobium loti and flooded for 60 d while subjected to two levels of dissolved pO2: 0.241 and 0.094 mol ml^-1. L. uliginosus showed significantly greater growth (shoot and root) and N2 fixation under both pO2s, compared to L. corniculatus, although growth and N2 fixation by L. corniculatus was not affected by the low pO2. Surprisingly, in L. uliginosus., growth, nodulation and N2 fixation were all increased by low pO2 while nodulation of L. corniculatus where low pO2 plants showed a significant increase over that of the higher pO2 plants while L. uliginosus plants showed a decline. Root porosity of L. uliginosus doubled in the low pO2-treatment from a mean of 14.5% in high pO2 roots to 28.5%, whereas that of L. corniculatus was relatively unaffected by pO2, being 7% and 9% for high and low pO2 plants, respectively. The structure of nodules differed little between species and treatments, although nodules/nodulated roots from the L. uliginosus plants had particularly profuse lenticels and aerenchyma. However, L. corniculatus nodules, especially those grown in the lower pO2 showed signs of early senescence with vacuolation of infected cells and green coloration when cut open. Leghaemoglobin (Lb) concentrations in nodules from both species were unaffected by low pO2, although that of L. corniculatus nodules, regardless of pO2, was significantly greater than L. uliginosus. Concentrations of the intercellular glycoprotein recognized by the monoclonal antibody MAC265 were significantly reduced in nodules from the low pO2 treatment in both species. Immunogold labelling showed that the MAC265 antigen was localized primarily within intercellular spaces within nodule cortices from both Lotus species. A marked decrease in deposition of the MAC265 antigen within the cortices of L. uliginosus nodules grown in the lower pO2, is discussed in terms of the relative abilities of the two Lotus spp. to maintain an O2 supply to the N2-fixing bacteroids within submerged nodules.Keywords: Lotus uliginosus, Lotus corniculatus, N2 fixation, flooding, oxygen.      

The Effect of Moisture Stress on Infection of Trifolium subterraneum L. by Rhizobium trifolii Dang

Moisture stress and method of inoculation greatly affected thenumber and distribution of infected root hairs and nodules ofyoung seedlings of Trifolium subterraneum. A reduction of soilmoisture from 5·5 to 3·5% (–0·36to –3·6 x 10⁵ Pa) significantly decreased the numberof infection threads and completely inhibited nodulation, althoughthe number of rhizobia in the rhizosphere was unaffected. Atlow soil moisture levels the root hairs were abnormally shortand swollen. Infection and nodulation were little affected between5·5 and 9·5% moisture (–0·36 to –0·089x 10⁵ Pa). Distribution of infected root hairs depended on the initialplacement of the inoculum; with the inoculum mixed evenly throughthe soil, infection threads occurred at discrete foci alongthe root. With seedlings inoculated at planting, infection threadswere restricted to the top 1–2 cm of root, even at thehighest soil moisture tested. Watering increased the number of infections in plants grownat 3·5% moisture; nodules were formed at a rate equivalentto non-stressed plants. Watering also enabled movement of theseedling-borne inocula; new infections were formed along theroot surface bearing mature root hairs.     

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.     

Competitiveness of a native Rhizobium leguminosarum biovar trifolii strain for nodule occupancy is manifested during infection

The stages in the nodulation process that determined the competitiveness of R. leguminosarum bv. trifolii (Rlt) strain 20–15, which proved to be highly competitive for nodulation in Iceland fields tests over several years, is analysed. White clover (Trifolium repens L.) roots were inoculated with inoculum mixtures containing three strains (Rlt 20-15, Rlt 8-9 and Rlt 32-28) in different proportions and cell densities. Competitiveness in root colonization, formation of infection threads and nodule development was assessed for Rlt 20-15 and its weakest competitor, Rlt 32-28. ERIC-polymerase chain reaction (PCR) DNA fingerprinting was used to identify inoculated strains recovered from root surfaces and individual nodules. GFP or DsRed tagged strains were used to determine identity in root hairs and nodules. Both strains colonized the root equally at all inoculum ratios tested. But, Rlt 20-15 initiated significantly more infection threads and formed more nodules than Rlt 32-28. These results show that Rlt 20-15 expresses its nodulation competitiveness during infection, either at infection thread initiation or during successive growth in the infection threads. The data presented support earlier observations that this strain competed well in the field in spite of its inferior ability to survive in the soil.     

Rhizobium infection and nodule development in soybean are affected by exposure of the cotyledons to light

The initiation of Rhizobium infections and the development of nodules on the primary root of soybean Glycine max L. Merr cv Williams seedlings are strongly affected by exposure of the cotyledons/hypocotyls to light. Seedlings in plastic growth pouches were inoculated with R. japonicum in dim light and the position of the root tip of each seedling was marked on the face of the pouch. The pouches were covered and kept in the dark for various times before exposing the upper portions of the plants (cotyledons and hypocotyls) to light. Maximum nodulation occurred if the plants were kept in the dark until 1 day after inoculation. The exposure of plants to light 2 days before inoculation reduced the number of nodules by 50% while the number of nodules was reduced by 70% if the plants were kept in the dark until 7 days after inoculation. Anatomical studies revealed that exposure to light prior to inoculation reduced both the number of infection centers with visible infection threads and the number of infections which developed nodule meristems. Plants kept in the dark for 7 days after inoculation formed a normal number of infection threads above the root tip mark, but very few of these infections developed a nodule meristem. It appears that light stimulates soybean to produce substances which can both inhibit the formation of infection threads and enhance the development of nodules from established infection threads. The effects of light on nodulation appear to be expressed independently of the Rhizobium-induced suppression of nodule formation in younger regions of the root.     

A Small GTPase of the Rab Family Is Required for Root Hair Formation and Preinfection Stages of the Common Bean–Rhizobium Symbiotic Association

Legume plants are able to establish a symbiotic relationship with soil bacteria from the genus Rhizobium, leading to the formation of nitrogen-fixing root nodules. Successful nodulation requires both the formation of infection threads (ITs) in the root epidermis and the activation of cell division in the cortex to form the nodule primordium. This study describes the characterization of RabA2, a common bean (Phaseolus vulgaris) cDNA previously isolated as differentially expressed in root hairs infected with Rhizobium etli, which encodes a protein highly similar to small GTPases of the RabA2 subfamily. This gene is expressed in roots, particularly in root hairs, where the protein was found to be associated with vesicles that move along the cell. The role of this gene during nodulation has been studied in common bean transgenic roots using a reverse genetic approach. Examination of root morphology in RabA2 RNA interference (RNAi) plants revealed that the number and length of the root hairs were severely reduced in these plants. Upon inoculation with R. etli, nodulation was completely impaired and no induction of early nodulation genes (ENODs), such as ERN1, ENOD40, and Hap5, was detected in silenced hairy roots. Moreover, RabA2 RNAi plants failed to induce root hair deformation and to initiate ITs, indicating that morphological changes that precede bacterial infection are compromised in these plants. We propose that RabA2 acts in polar growth of root hairs and is required for reorientation of the root hair growth axis during bacterial infection.     

Infection threads in the root hairs of soybean (Glycine max) plants inoculated withRhizobium japonicum

Summary The mode of infection leading to nodulation was studied in soybean (Glycine max) plants inoculated withRhizobium japonicum strains 61A76, 3I1b83, 3Ilb142, and 3Ilb143 and a commercial inoculum. Infection threads were noticed in the root hairs of plants grown in small field plots, Leonard bottle-jar assemblies and on agar slants. Two infection threads per root hair were commonly observed. Root hairs with infection threads were persistent on the nodules. The maximum number of infection threads per plant was observed in Leonard bottle-jar assemblies.Contribution No. 603 from Charles F. Kettering Research Laboratory.     

Interaction of Azospirillum and Rhizobium Strains Leading to Inhibition of Nodulation

Rhizobium-Azospirillum interactions during establishment of Rhizobium-clover symbiosis were studied. When mixed cultures of Azospirillum and Rhizobium trifolii strains were simultaneously inoculated onto clover plants, no nodulation by R. trifolii was observed. R. trifolii ANU1030, which nodulated clover plants without attacking root hairs, i.e., does not cause root hair curling (Hac⁻), did not show inhibition of nodulation when inoculated together with Azospirillum strains. Isolation of bacteria from surface-sterilized roots showed that azospirilla could be isolated both from within root segments and from nodules. Inhibition of nodulation could be mimicked by the addition of auxins to the plant growth medium.     

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.     

The phenotypic,phylogenetic and symbiotic characterization of rhizobia nodulating Lotus sp. in Tunisian arid soils

Fifteen bacterial isolates, representatives of different 16S rRNA-RFLP genomogroups which were isolated from root nodules of Lotus creticus and L. pusillus growing in the arid areas of Tunisia were characterized by phenotypic features and 16S rDNA sequences. Phenotypically, all isolates are fast growers with the ability to grow at a pH between 5.5 and 9. Most of the tested isolates tolerate NaCl concentrations from 1.39 to 3.48 %. Phylogenetically, the studied isolates are affiliated into the genera: Sinorhizobium (5 strains), Rhizobium (2 strains), and Mesorhizobium (4 strains). The 16S rDNA sequences of Tunisian Lotus sp. nodule isolates: LAC7511, LAC733, and Mesorhizobium alhagi (Alhagi sparsifolia symbiont) shared 100 % identical nucleotides similar to the 16S rDNA sequences of LAC831, LAC814 and Mesorhizobium temperatum CCNWSX0012-2 (Astragalus adsurgens symbiont). Non-nodulating bacteria, considered as endophytes of Lotus sp. nodules, were also found in our studies and they were classified into the genera: Phyllobacterium (2 strains), Starkeya (1 strain) and Pseudomonas (1 strain). Except for these four endophytic Lotus sp. bacteria, all other strains under investigation induce nodules on Lotus sp., but they differ in the number of induced root nodules and the effectiveness of atmospheric nitrogen fixation. The Sinorhizobium sp., Mesohizobium sp. and Lotus sp. nodule isolates, forming the most effective symbiosis with the plant host, are potential candidates for inoculants in revegetation programs.     

Structural identification of the iipo-chitin oligosaccharide nodulation signals of Rhizobium loti

Rhizobium loti is a fast-growing Rhizobium species that has been described as a microsymbiont of plants of the genus Lotus. Nodulation studies show that Lotus plants are nodulated by R loti, but not by most other Rhizobium strains, indicating that R. loti produces specific lipo-chitin oligosaccharides (LCOs) which are necessary for the nodulation of Lotus plants. The LCOs produced by five different Rhizobium ioti strains have been purified and were shown to be N-acetylglucosamine pentasaccharides of which the non-reducing residue is N-methylated and N-acylated with c/s-vaccenic acid (C18:1) or stearic acid (C18:O) and carries a carbamoyl group. In one R. loti strain, NZP2037, an additional carbamoyl group is present on the non-reducing terminal residue. The major class of LCO molecules is substituted on the reducing terminal residue with 4-O-acetylfucose. Addition of LCOs to the roots of Lotus plants results in abundant distortion, swelling and branching of the root hairs, whereas spot inoculation leads to the formation of nodule primordia.     

Selecting improved Lotus nodulating rhizobia to expedite the development of new forage species

Aims

In the past decades the increasing focus by Australian pasture development programs on the genus Lotus has seen the evaluation of many species previously untested in Australia. In field trials, nodulation failure was commonplace. This work was undertaken to select effective symbionts for Lotus to ensure further agronomic evaluation of the genus was not compromised. The symbiotic needs of Lotus ornithopodioides were a particular focus of the studies.

Methods

Glasshouse experiments were undertaken to evaluate symbiotic relationships between 15 Lotus spp and 23 strains of nodulating Mesorhizobium loti. This was followed by evaluation of elite rhizobial strains for their ability to persist and form nodules under field conditions.

Results

Complex symbiotic interactions were recorded between strains of lotus rhizobia and the different species of Lotus. Notably, the rhizobia that are currently provided commercially in Australia for the inoculation of Lotus corniculatus (strain SU343) and Lotus uliginosus (strain CC829) did not form effective symbioses with the promising species L. ornithopodioides and L. maroccanus. No strain we evaluated was compatible with all the Lotus species, however several strains with a broad host range were identified. WSM1293 and WSM1348 were the most effective strains on L. ornithopodioides and L. peregrinus.These strains were also moderately effective on L. corniculatus (79 and 52% of SU343), less effective on L. maroccanus (26 and 49% of SRDI110) but were ineffective on L. uliginosus. The latter species overall had very specific rhizobial needs. Both WSM1293 and WSM1348 produced adequate levels of nodulation when inoculated on L. ornithopodioides, over two seasons at three field sites.

Conclusions

Effective and persistent strains are now available that should allow the un-compromised evaluation of many of the contemporary Lotus species in the field. Selecting a strain for use in commercial inoculants will be more problematic, given the very large host-strain interactions for nitrogen fixation. Here, the balance of Lotus species which are adopted by farmers will have a strong bearing on which rhizobial strains are progressed to commerce.     

A Light and Scanning Electron Microscope Study of Stem Nodules in Vicia faba L

Stem nodules were observed on plants of Vicia faba L. cv. ThrowsMS grown in a variety of environmental conditions. Observationsby light and scanning electron microscopy indicated that theseorgans were morphologically similar to root nodules on the sameplants. Nodules arose following infection of stem hairs andsubsequent growth of infection threads into the stem cortex.They developed to a mature, nitrogen-fixing state.     

Phenylpropanoid defence responses in transgenic Lotus corniculatus: II. Modelling plant defence responses in transgenic root cultures using thiol and carbohydrate elicitors

Transformed root cultures of Lotus corniculatus L. cv. Leo weretreated with a range of thiol and carbohydrate elicitors. Boththiol reagents and fungal carbohydrate preparations resultedin an increase in the activity of phenylalanine ammonia lyase(PAL) in a concentration-dependent manner. One representativethiol elicitor, glutathione (GSH), and one fungal elicitor,derived from Rhynchosporium orthosporum autoclaved cell walls(Ro), were analysed in more detail. Both elicitors induced thetransient accumulation of vestitol, an isoflavan phytoalexin,in tissue and in culture medium. Treatment of Lotus root cultureswith the Ro elicitor resulted in a more rapid initial accumulationof this end product when compared with GSH, however, sativan(the 2–methoxy ester of vestitol) previously reportedto co-accumulate in Lotus leaves was only detected followingelicitation with high concentrations of GSH. Ro and GSH elicitorsalso induced the accumulation of a number of other phenylpropanoidcompounds putatively identified as chalcones. The addition ofthiol and carbohydrate elicitors to Lotus root cultures alsoresulted in characteristic changes in root morphology. Glutathione,in particular, resulted in the inhibition of root growth dueto differential damage of meristem cells. Key words: Lotus corniculatus, hairy roots, elicitors, phytoalexins.