Isolation of Rhizobium leguminosarum biovar viciae Variants with Indole-3-acetamide Hydrolase Activity

The IAA biosynthetic pathway from tryptophan to IAA via IAM(IAM pathway) was investigated in Rhizobium spp. (fast-growingrhizobium). Southern hybridization with the bam gene, a structuralgene for IAM hydrolase (the enzyme that converts IAM to IAA)cloned from Bradyrhizobium japonicum J1063, indicated that homologoussequences exist among wild-type Rhizobium spp. However the IAMpathway has not been detected biochemically in free-living bacteria.When 5-methyltryptophan-resistant strains were screened forRhizobium leguminosarum biovar viciae K5 which has DNA sequenceswith high homology to the bam gene, spontaneous mutants showingIAM hydrolase activity were isolated. The results suggest thepossibility that the activity of IAM hydrolase is suppressedin free-living state in Rhizobium leguminosarum biovar viciaeK5. In addition we detected the peak at the same t_R of IAM byHPLC analysis using two columns when a large amount of L-tryptophanwas added to the suspension of 5-methyltryptophan-resistantvariants. Whether or not tryptophan-2-monooxygenase activity,however, actually works in Rhizobium cells remained to be solved. (Received September 20, 1989; Accepted March 6, 1990)     

The Presence of an Enzyme that Converts IndoIe-3-acetamide into IAA in Wild and Cultivated Rice

The enzymatic conversion of indole-3-acetamide (IAM) to IAA,which is the second step in the IAM pathway (tryptophan IAM IAA) was investigated in calluses derived from various dicotyledonousand monocotyledonous plants. A simple method, using analysisby HPLC to measure the conversion of naphthaleneacetamide (NAM)to naphthaleneacetic acid (NAA) was employed for the detectionof IAM hydrolase activity. Among calluses from 27 plants tested,only callus from a cultivated strain of rice (Oryza sativa C5924)had high conversion activity similar to that of crown gall cells,and very weak activity was found in calluses from lucern andorange. In addition to the presence of the conversion activity,we confirmed that radioactivity from ³H-IAM was incorporatedinto IAA in a cell-free system from O. sativa C5924. An extractof roots of rice seedlings exhibited twice the activity of thatin an extract of shoots. IAM hydrolase activity was observedin calluses from all lines of rice callus examined, irrespectiveof whether they were wild or cultivated lines, with the exceptionof O. grandiglumis W1194 and O. branchyantha W656, while otherspecies of Gramineae exhibited no activity. These results suggestthe possibility that this enzyme may play a specific role inrice. (Received August 23, 1990; Accepted November 29, 1990)     

Natural Occurrence of Indoleacetamide and Amidohydrolase Activity in Etiolated Aseptically-Grown Squash Seedlings

Etiolated seedling tissues of aseptically grown squash (Cucurbitamaxima Duch) contain indole-3-acetamide (IAM) as a natural endogenouscompound, conclusively identified by gas chromatography-massspectrometry (GC-MS). Roots of aseptically raised seedlingsalso contain amide hydrolysing activity, which converts IAMto IAA, indoleacetonitrile (IAN) to IAM and IAA, and 1-naphthaleneacetamideto 1-naphthaleneacetic acid. This activity was enriched 48-foldby fractional precipitation with ammonium sulphate, Sephadexgel nitration and anion exchange chromatography. Being hydrolytic,it works equally well in air and in vacuo, without added cofactors.The partially purified enzyme works optimally between pH 7 and7.5, and a K_m value of 80 µM was calculated with IAM asthe substrate. The product of this reaction was definitivelyidentified as IAA by GC-MS. The temperature optimum of thisamidohydrolase lies around 45°C, and it is stable to freezing.A comparison of its properties with the amidohydrolase of Agrobacteriumor crown gall tissue, shows it to be different. In view of thenatural occurrence of both IAM and the amidohydrolase, it issuggested that the IAM pathway of IAA biogenesis is feasiblein etiolated squash seedlings. ⁴Deceased 2/2-1993.     

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     

Siderophore production byBradyrhizobium spp. strains nodulating groundnut

EighteenBradyrhizobium spp. strains, fourRhizobium spp. strains and oneAzorhizobium caulinodans strain were grown under Fe limitation and assayed for siderophore production. It was further assessed if Fe accumulation in two groundnut cultivars was influenced by inoculant strain or nitrate fertilisation. Growth ofBradyrhizobium spp. strains nodulating groundnut was slow with mean generation times from 11–24 h. All strains, except MAR 967, showed a reduced growth rate when deprived of Fe; none of the strains showed starvation at 1 M Fe. In the CAS (chrome azurol S)-agar assay, all strains, which formed colonies, produced siderophores as visualised by orange halos around the colonies on blue plates.Bradyrhizobium strains produced much smaller halos than the referenceRhizobium meliloti strain. In the CAS-supernatant assay, all strains, except MAR 967, gave positive responses (measured as absorbance at 630 nm) when supernatants of Fe-depleted cultures were assayed with CAS-indicator complex in comparison with Fe-supplemented cultures. Responses of all fourRhizobium spp. strains were large, while responses of allBradyrhizobium strains, exceptB. japonicum MAR 1491 (USDA 110), were small and mostly insignificant. A small response, i.e. a low Fe-scavenging ability, implies either the production of small quantities of siderophores or the production of low affinity siderophores. Among theBradyrhizobium strains, MAR 1574 and MAR 1587 gave the largest responses taken over the two assays. Fe accumulation in groundnut cultivar Falcon was seven times larger than in cultivar Natal Common. No correlation was found between the quantity of nodule tissue and Fe accumulation, making it unlikely that bacteroids are involved in Fe acquisition by groundnuts. Nitrate-fertilised plants accumulated significantly more Fe, suggesting involvement of nitrate reductase in Fe assimilation in groundnut. The two most successful Fe-scavengingBradyrhizobium spp. strains were also the most effective in nodulating groundnut, the reverse also being true. Strain MAR 967, with the lowest Fe requirement, produced the largest nodule dry weight. These data indicate that improved Fe scavenging properties and/or reduced Fe requirement improve rhizospheric growth and with that nodulation effectiveness.     

The Effect of Mutations in the T-DNA Encoded Auxin Pathway on the Endogenous Phytohormone Content in Cloned Nicotiana tabacum Crown Gall Tissues

We analyzed the endogenous auxin and cytokinin levels of clonedNicotiana tabacum SR 1-lines induced either by the wild-typeAgrobacterium tumefaciens C58 strain or by mutants affectedin the T-DNA-encoded IAA biosynthesis pathway. The wild-typeSR1-C58 line contained up to 20 times more IAA than a nontransformedSRI-callus line. The mutant lines affected in gene 1 (iaaM^–)or gene 2 (iaaH^–) contained intermediate levels of IAA. Analysis of the endogenous levels of indole-3-acetamide (IAM)in the nontransformed SR 1 callus line, the wild-type SR1-C58and the two mutant lines confirmed the T-DNA-induced IAA biosynthesispathway in the transformed tumor cells. Supplementing auxinto the mutant lines resulted in complete suppression of theshoot-forming ability, but no changes in the endogenous IAAlevels. There was no marked difference in the cytokinin level betweenthe nontransformed callus line and the wild type tumor line.The two mutant lines, however, showed a 20- to 30-fold highercytokinin level which was not affected by the addition of NAA.The T-DNA encoded hormone biosynthetic pathways are discussedin relation to pathways of the host plant. (Received July 29, 1986; Accepted February 14, 1987)     

In vitro Hormonal Regulation of Laticifer Differentiation in Calotropis procera

Cultures of Calotropis procera were maintained on MS mediumsupplemented with 4·6 µM FAP and 3 µM NAA.Laticifer initials were observed in 2-week-old cultures whichwere converted into matured, branched, non-articulated laticifersin 4 weeks. It was observed that laticifer differentiation increasedwith the age of cultures, from 2·78% (in the second passage)to 15·11% (in the 12th passage). It has been establishedthat laticifer differentiation in vitro is a cytokinin-dependentprocess and among the cytokinins, FAP was more effective thanBA and 2-iP. But the type of auxin and its concentration alsoplay an important role in modifying the effect of cytokinin.Among the different auxins used IAA was more effective for laticiferdifferentiation than IBA and NAA, while 2,4D was inhibitory.Maximum laticifer differentiation (17·01% was observedon MS medium supplemented with 4·6 µM FAP and 1µM IAA.Copyright 1995, 1999 Academic Press Calotropis procera, callus culture, laticifer, differentiation, hormonal regulation     

In Planta Production of Indole-3-Acetic Acid by Colletotrichum gloeosporioides f. sp. aeschynomene

The plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene utilizes external tryptophan to produce indole-3-acetic acid (IAA) through the intermediate indole-3-acetamide (IAM). We studied the effects of tryptophan, IAA, and IAM on IAA biosynthesis in fungal axenic cultures and on in planta IAA production by the fungus. IAA biosynthesis was strictly dependent on external tryptophan and was enhanced by tryptophan and IAM. The fungus produced IAM and IAA in planta during the biotrophic and necrotrophic phases of infection. The amounts of IAA produced per fungal biomass were highest during the biotrophic phase. IAA production by this plant pathogen might be important during early stages of plant colonization.     

Nodulation of Oilseed Rape (Brassica napus) by Rhizobia

Nodules were induced on the non-legume oilseed rape, followingenzyme treatment of seedling roots and inoculation with Rhizobiumlegnminosarum, Bradyrhizobtum 32H1 or a mixture of R. lott withBradyrhizobium 32H1 in the presence of PEG. A Nod– strainof R leguminosarum also induced nodules, but a Nod– strainfailed to elicit this response Nodules induced on oilseed rapewere morphologically similar, when examined by light microscopyand cryo-scanning electron microscopy, to those induced on rootsof white clover by R trifolu. Transmission electron microscopyshowed rhizobia within cells of the nodules These observationsare discussed with respect to the extension of Rhizobium symbiosisto non-legumes. Key words: Brassica napus, Bradyrhizobium, Rhizobium, non-legumes, nodulation, transmission electron microscopy, cryo-scanning electron microscopy, acetylene reduction, cell wall degrading enzymes     

Formation of nodular structures on the non-legumes Brassica napus,B. campestris,B. juncea and Arabidopsis thaliana with Bradyrhizobium and Rhizobium isolated from Parasponia spp. or legumes grown in tropical soils

Strains of Bradyrhizobium formed nodule-like structures on Arabidopsis and species of Brassica in pots with sandvermiculite and in glass tubes on a nitrogen-free mineral salts agar. Broad-host-range Rhizobium strains NGR234 from Lablab purpureus and NGR76 from Phaseolus vulgaris formed similar nodule-like structures on Brassica spp. The size of these structures on plants in pots were large, often reaching 10 mm in diameter.The frequency of inoculated Brassica plants in pots with nodule-like structures was 25–50%, depending on the inoculum strain. The inheritable nature of factors involved in the formation of the nodule-like structures was demonstrated when the structures occurred on 100% of inoculated B. napus seedlings derived from plants with the nodule-like structures.Nodule-like structures occurred without, but not with, the application of a cellulase-pectolyase-PEG treatment to the roots. Attempts to isolate Bradyrhizobium or Rhizobium from the nodule-like structures failed. Internal infection of these structures could not be detected using either the light or electron microscope. The inoculum strains of root-nodule bacteria were detected in high numbers in the rhizosphere of plants 5 months after inoculation. On agar plates bacterial colonies could be seen, with undiminished growth, over the surface of the agar extending to the root surface. However, ground root tissue of Brassica was toxic to Bradyrhizobium strains. This suggested that Bradyrhizobium strains would not survive after infecting the roots of Brassica spp. Nitrogen fixation was associated with high rhizosphere populations of Azospirillum and not with Bradyrhizobium induced nodule structures of Brassica spp.     

The Pigeonpea-Rhizobium Symbiosis as Affected by High Root Temperture: Effect on Nodule Formation

The effect of elevated temperature on root hair formation, adsorptionof rhizobia, and nodulation of pigeonpea was studied. Nodulationwas adversely affected at both 28°C and 37°C, and theeffect was more pronounced during the first 3 d of nodule formation.Temperatures above 32°C resulted in the reduction or evencomplete absence of root hairs. The root hairs formed at elevatedtemperatures were spheroid and stunted in growth. The numberof loosely and firmly adsorbed cells of Bradyrhizobium spp.(Cajanus) strain CC1021 on pigeonpea roots were reduced to 49%and 38%, respectively, at 37°C. Key words: Pigeonpea, Bradyrhizobium spp. (Cajanus), high root temperature, root hairs, adsorption     

Signal molecules in the peanut–bradyrhizobia interaction

Main nodulation signal molecules in the peanut–bradyrhizobia interaction were examined. Flavonoids exuded by Arachis hypogaea L. cultivar Tegua were genistein, daidzein and chrysin, the latest being released in lower quantities. Thin layer chromatography analysis from genistein-induced bacterial cultures of three peanut bradyrhizobia resulted in an identical Nod factor pattern, suggesting low variability in genes involved in the synthesis of these molecules. Structural study of Nod factor by mass spectrometry and NMR analysis revealed that it shares a variety of substituents with the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium spp. Nodulation assays in legumes nodulated by these rhizobia demonstrated differences between them and the three peanut bradyrhizobia. The three isolates were classified as Bradyrhizobium sp. Their fixation gene nifD and the common nodulation genes nodD and nodA were also analyzed. Accession numbers: AY427207, EF202193, EF158295 (16S rRNA gene of strains NLH25, NOD31 and NDEHE, respectively); DQ295199, DQ295200, DQ295201 (Partial nifD gene sequences of strains NLH25, NOD31 and NDEHE, respectively).     

The Influence of Different Cytokinins and Auxins on Flower Neoformation in Thin Cell Layers of Nicotiana tabacum L.

Neoformation of flower buds was studied in thin cell layersderived from floral branches of Nicotiana tabacum L. Organogenesiswas found to be expressed in different ways according to thekind of auxin and cytokinin present in the culture medium. Inthe presence of synthetic auxins (NAA, CTA), more flower budswere generated than with the natural auxins (IAA, IBA), if theculture medium was also supplemented with a cytokinin. Theseconditions also stimulated the development of the buds to flowersor inflorescences. The most active cytokinin was BA. Naturalcytokinins (zeatin, IPA) appeared much less active but led toremarkable development of roots together with flower buds. Anorganogenetic effect of 2,4-D was observed in this experimentalsystem, leading to the conclusion that organogenetic activityof cytokinins and auxins in thin cell layer cultures stronglydepends on the molecular structure of the growth regulators. (Received September 30, 1987; Accepted March 23, 1988)     

Recent developments inRhizobium taxonomy

Recent developments inRhizobium taxonomy are presented from a molecular and evolutionary point of view. Analyses of ribosomal RNA gene sequences provide a solid basis to infer phylogenies in the Rhizobiaceae family. These studies confirmed thatRhizobium andBradyrhizobium are only distantly related and showed thatRhizobium andBradyrhizobium are related to other groups of bacteria that are not plant symbionts.Rhizobium andAgrobacterium species are intermixed. Differences in plasmid content may explain to a good extent the different behavior ofRhizobium andAgrobacterium as symbionts or pathogens. Other approaches to identify and classify bacteria such as DNA-DNA hybridization, fatty acid analysis, RFLP and RPD-PCR techniques and phylogenies derived from other genes are in general agreement to the groupings derived by ribosomal sequences. Only a small proportion of nodulated legumes have been sampled for their symbionts and more knowledge is required on the systematics and taxonomy ofRhizobium andBradyrhizobium species.     

Root nodules of Genista germanica harbor Bradyrhizobium and Rhizobium bacteria exchanging nodC and nodZ genes

A collection of 18 previously unstudied strains isolated from root nodules of Genista germanica (German greenweed) grown in southeast Poland was evaluated for the level of genetic diversity using the BOX-PCR technique and the phylogenetic relationship based on both core (16S rRNA, dnaK, ftsA, glnII, gyrB, recA, rpoB) and nodulation (nodC and nodZ) gene sequences. Each of the 18 G. germanica root nodule isolates displayed unique BOX-PCR patterns, indicating their high level of genomic heterogeneity. Based on the comparative 16S rDNA sequence analysis, 12 isolates were affiliated to the Bradyrhizobium genus and the other strains were most similar to Rhizobium species. Phylogenetic analysis of the core gene sequences indicated that the studied Bradyrhizobium bacteria were most closely related to Bradyrhizobium japonicum, whereas Rhizobium isolates were most closely related to Rhizobium lusitanum and R. leguminosarum. The phylogenies of nodC and nodZ for the Rhizobium strains were incongruent with each other and with the phylogenies inferred from the core gene sequences. All Rhizobium nodZ gene sequences acquired in this study were grouped with the sequences of Bradyrhizobium strains. Some of the studied Rhizobium isolates were placed in the nodC phylogenetic tree together with reference Rhizobium species, while the others were closely related to Bradyrhizobium bacteria. The results provided evidence for horizontal transfer of nodulation genes between Bradyrhizobium and Rhizobium. However, the horizontal transfer of nod genes was not sufficient for Rhizobium strains to form nodules on G. germanica roots, suggesting that symbiotic genes have to be adapted to the bacterial genome.     

Studies of the Inhibition of Stomatal Opening by Naphth-1-ylacetic Acid and Abscisic Acid

Evidence is presented which confirms the inhibitory action ofthe synthetic auxin, NAA, on stomatal opening. In contrast tothe natural auxin IAA, NAA exerts effects which, in some respects,resemble those of ABA. The mode of action of NAA on the guardcells is, however, thought to be different from that of ABA.When NAA and ABA are applied together, their combined actionresults in a greater reduction in stomatal aperture than wouldbe predicted from their separate effects. Key words: NAA, ABA, IAA Stomata, Commelina     

The Abundance and Diversity of Legume-Nodulating Rhizobia in 28-Year-Old Plantations of Tropical, Subtropical, and Exotic Tree Species: a Case Study from the Forest Reserve of Bandia, Senegal

Several fast-growing and multipurpose tree species have been widely used in West Africa to both reverse the tendency of land degradation and restore soil productivity. Although beneficial effects have been reported on soil stabilization, there still remains a lack of information about their impact on soil microorganisms. Our investigation has been carried out in exotic and native tree plantations of 28 years and aimed to survey and compare the abundance and genetic diversity of natural legume-nodulating rhizobia (LNR). The study of LNR is supported by the phylogenetic analysis which clustered the isolates into three genera: Bradyrhizobium, Mesorhizobium, and Sinorhizobium. The results showed close positive correlations between the sizes of LNR populations estimated both in the dry and rainy seasons and the presence of legume tree hosts. There were significant increases in Rhizobium spp. population densities in response to planting with Acacia spp., and high genetic diversities and richness of genotypes were fittest in these tree plantations. This suggests that enrichment of soil Rhizobium spp. populations is host specific. The results indicated also that species of genera Mesorhizobium and Sinorhizobium were lacking in plantations of non-host species. By contrast, there was a widespread distribution of Bradyrhizobium spp. strains across the tree plantations, with no evident specialization in regard to plantation type. Finally, the study provides information about the LNR communities associated with a range of old tree plantations and some aspects of their relationships to soil factors, which may facilitate the management of man-made forest systems that target ecosystem rehabilitation and preservation of soil biota.     

Identification of Rhizobium strains on antibiotic concentration gradients

Antibiotic concentration gradients were used to characterise several cultures of R. phaseoli and Rhizobium spp. (isolated from Cicer arietinum) by differences in intrinsic antibiotic resistance (IAR). Differentiation between cultures was facilitated by use of cluster analyses. The method permitted 15/16 cultures of R. phaseoli to be distinguished on 14 antibiotics. Two cultures which exhibited similar IAR patterns were shown to be the same strain obtained from different collections. The validity of the technique for strain identification was demonstrated by fluorescent antibody tests which gave corresponding identity for 50 nodule isolates from plants inoculated with a mixture of three strains of R. phaseoli. The method was less suitable for characterising cultures of the slow-growing Rhizobium spp. because several antibiotics produced growth lacking a clearly defined boundary between resistance and susceptibility. Although 15/16 cultures of Rhizobium spp. could be differentiated, several isolates were distinguishable only by a difference on a single antibiotic. Similarity between stock cultures and derivative nodule isolates indicated that IAR on gradient plates was a stable property unaffected by plant passage.     

Presence of a Pathway for the Biosynthesis of Auxin via Indole-3-Acetamide in Trifoliata Orange

The auxin-biosynthetic pathway from L-tryptophan to indole-3-aceticadd via indole-3-acetamide (IAM), found in plant-pathogenicbacteria such as Agrobacterium tumefaciens and Pseudomonas savastanoi,has not been found in plants. We attempted to detect the enzymaticactivities for this pathway in cell-free systems from varioustissues of trifoliata orange (Poncirus trifoliata Rafin.). Ahigh level of activity of LAM hydrolase, which catalyzes theconversion of IAM to indole-3-acetic acid, was observed in acrude extract prepared from young fruits one week after fullbloom. Using -naphthaleneacetamide as a competitor of IAM hydrolase,a simple assay system was developed for the detection of theconversion of L-tryptophan to IAM (tryptophan monooxygenaseactivity). When this system was used to assay cell-free extractsof young fruit of P. trifoliata, the conversion of L-tryptophanto IAM was clearly demonstrated by the presence of IAM amongreaction products, as demonstrated by GC/MS analysis and theincorporation of ¹⁴C-labeled L-tryptophan into an IAM fraction.This is the first report indicating the presence of an auxin-biosyntheticpathway via IAM in P. trifoliata. Furthermore, it is shown thatboth enzyme activities in auxin biosynthesis increased transientlyduring fruit development. (Received October 9, 1992; Accepted November 2, 1992)     

Effects of auxin and gibberellin on conidial germination in Neurospora crassa

Auxins, IAA, 2,4-D, and NAA, and gibberellin, GA, significantlyenhanced the conidial germination rate in the wild-type Neurosporacrassa. The inhibitory effect of an antiauxin, 2,4,6-T on conidialgermination was overcome by IAA. The present results suggestthat auxin and gibberellin may act as regulators of conidialgermination in Neurospora. (Received November 21, 1977; )