Siratro Plants Research Articles

First Report of Passiflora virus Y Infecting Macroptilium atropurpureum in Taiwan.

Macroptilium atropurpureum (siratro plants) is a perennial wild legume plant introduced to Taiwan as a forage crop in 1961 (3) and has become a naturalized weed found all over the island. In 2010, siratro plants with virus-like symptoms of mosaic and leaf deformation were observed on the campus of Da-Yeh University in central Taiwan. Flexuous virus-like particles about 750 × 12 nm were observed in the crude sap extracted from symptomatic leaves with a transmission electron microscope. Crude sap was mechanically inoculated to Chenopodium quinoa and local lesions can be observed on inoculated leaves 4 to 5 days after inoculation. Virus was purified from the leaves of inoculated C. quinoa with modified protocols of Gonsalves and Ishii (2). The virus coat protein (CP) consisted of a single major peptide with relative molecular weight of approximately 33 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Viral RNA extracted from the purified virus was used as a template and was primed with several primer sets corresponding to potyviruses and carlaviruses in reverse transcription-PCR to amplify possible corresponding cDNA fragments. After several attempts, a cDNA fragment of about 1,300 bp could be amplified with the degenerated primer set of BCMNV-F (5'CCDTGGACDGTWGGVATGAC3') and BCMNV-R (5'CACCAHACCATRAARCCATTCAT3'), which were designed on the basis of the conserved region of the nuclear inclusion b (NIb) and CP genes of some potyviruses including Bean common mosaic necrosis virus, Soybean mosaic virus, Blackeye cowpea mosaic virus, East Asian passiflora virus, and Passion fruit woodiness virus. BLAST analyses showed the amplicon was highly homologous to that of Passiflora virus Y (PaVY). Together with oligo dT, a specific primer (5'GATGACACTCAAATGGCTG3') corresponding to PaVY CP was used to amplify the cDNA fragment of the most 3' region of the viral RNA (about 800 bp). The assembled cDNA fragment of 1,958 bp (Accession No. AB679294) contains a partial NIb gene (877 nt), a complete CP gene (819 nt), and the 3' noncoding region (262 nt). The CP gene shared sequence identities of 89.4 to 98.9% and 92.7 to 98.9% in nucleotide and amino acid, respectively, to that of documented PaVY isolates. PaVY has also been found to be infecting Vigna trilobata, Rhynchosia minima, Clitoria ternatea, and Passiflora foetida in Australia (1). Here we present the first report to our knowledge of PaVY and its infection of siratro (M. atropurpureum) in Taiwan. Additional work is needed to investigate the spread of PaVY and its interaction with other legume plants in Taiwan.

Effects of Bacillus thuringiensis CRY1A(c) d-endotoxin on growth, nodulation and productivity of beans [Phaseolus vulgaris (L.) and siratro (Macroptilium atropurpureum DC.)

The recent introduction of Bt maize and Bt cotton transgenic crops into Africa has raised concerns on their potential short and long-term ecological effects on the environment. The effects of Bacillus thuringiensis (Bt) Cry1A(c) d-endotoxin on the growth, nodulation and productivity of two leguminous plants grown in clay soil were evaluated. Bt Cry1A(c) d-endotoxin from a local B. thuringiensis isolate (ICIPE L1-2) active against Chilo partellus (Swinhoe) was used. Beans (Phaseolus vulgaris L.) and Siratro (Macroptilium atropurpureum DC.) seedlings were grown in pots treated with Bt Cry1A(c) d-endotoxin solution (100 µg/ml). Control experiments were treated with water. The plants were maintained in the greenhouse until nodulation (8 weeks) and maturity (14 weeks) stages when sampling was done for measurements of morphological, productivity and nodulation traits. Nodulation was observed in both plants species. Nitrogen content in treatments for both bean and siratro plants, with and without Bt-toxin not were significantly different. Leaf area, plant dry weight, number of pods per plants and number of seeds per pod observed in treatments with and without Bt-toxin for both bean and siratro plants were also not significantly different. This shows that Bt Cry1A(c) delta-endotoxin does not interfere with the host plant growth, nodulation and productivity in clay soil. Findings will provide researchers with data to design more robust experiments and will inform the decisions of diverse stakeholders regarding the safety of transgenic crops. Key words: Bacillus thuringiensis, Bt Cry1A(c) δ-endotoxin, Macroptilium atropurpureum (DC.), nodulation.

Identification and Characterization of a Potyvirus Isolated from Siratro Plants

AbstractThe present work describes the identification and characterization of a potyvirus isolated from siratro (Macroptilium atropurpureum Urb.) in the north‐west region of the State of São Paulo, Brazil. The virus was transmitted by mechanical inoculation. Its host range was restricted mainly to members of the Fabaceae. A cDNA fragment of about 930 bp was amplified by RT/PCR, cloned and sequenced. The fragment, which included the coat protein gene, had amino acid identity percentages between 88 and 98% with isolates of Bean common mosaic virus (BCMV). Phylogenetic analysis grouped the siratro potyvirus and BCMV isolates in 99% of the replicates, including Azuki mosaic virus, Dendrobium mosaic virus, Blackeye cowpea mosaic virus and Peanut stripe virus, which have been classified as BCMV strains. This is the first citation on the presence of BCMV in siratro plants in Brazil.

Characterization of Acid-Aluminium Sensitive Mutants of Soybean Symbiont Bradyrhizobium japonicum Generated by Transposon Mutagenesis

Acid-aluminium sensitive mutants of symbiotic bacterium Bradyrhizobium japonicum BJ11 (designated as AAS11) and KDR15 (designated as AAS15) were constructed by mini-Tn5 transposon mutagenesis to study genes involved in acid-aluminium tolerance (AAT) in B. japonicum. Transposon delivery was carried out through conjugation between B. japonicum strains as recipients and Escherichia coli S17-1 (ë pir) carrying pUTmini- Tn5Km1 as a donor strain. The result showed that frequency of transconjugation was in the range of 6.7 x 10-7 to 7.1 x 10-6 cell per recipients. AAS11 and AAS15 mutants did not grow on Ayanaba media (pH 4.5) containing 50 μM Aluminium. These mutants remained able to form root nodules of Siratro (Macroptilium arthropurpureum) plants revealing genes interrupted by transposon which were responsible for acid-Al tolerance did not correlate with the nodulation genes. Strains tolerant to acid-aluminium and their mutants with a wild type sensitive to acidaluminium were characterized by accumulating phosphate and aluminium absorption. Compared to the wild type acid-aluminium tolerant B. japonicum, there was approximately a three- to eight-times decrease in phosphate accumulation and a five- to seven-times increase in aluminium absorption by these mutants. These results suggest that aluminium and phosphate contents in the bacterial cells may be involved in mechanisms of acid-Al tolerance of B. japonicum grown in acid-aluminium stress conditions.

Rhizobium purine auxotrophs, perturbed in nodulation, have multiple changes in protein synthesis

We investigated the infectivity, growth ability and the overall biochemical status of Rhizobium purine auxotrophs to determine why these strains show varied nodulation phenotypes on their host plants. Strains ANU2861 (purM::Tn5) and ANU2866 (purY::Tn5), both derivatives of ANU280, are unable to nodulate siratro plants. In contrast, strain L1 (purF::Tn5ssgusA40), a derivative of ANU843, is able to induce fully-developed nodules and normal levels of nodulation on several clover hosts. To our knowledge, strain L1 is the only genetically-defined pur– mutant capable of inducing fully-developed nodules on a legume host. None of the pur– mutants are able to grow at normal rates in vitro even in the presence of purine pathway supplements including AICA-riboside. Further, the nodulation ability of the strain ANU280 pur– mutants is unable to be restored by the addition of AICA-riboside. Full growth and nodulation ability can only be restored to the pur– mutants by genetic complementation. We confirmed that supplementation of pur intermediates failed to fully restore wild-type growth by using proteome analysis to examine the overall biochemical status of the mutants. Proteome analysis demonstrated that the purine mutants possess multiple changes in the protein species present. In addition, strain ANU2866 but not strains ANU2861 or L1 could not tolerate the effects of nutrient step-up or step-down transitions, and failed to produce any colonies on laboratory media. Collectively, these data show that purine auxotrophs suffer pleiotropic effects at the level of protein synthesis and their overall metabolism is compromised. Therefore, care should be exercised in concluding that purine intermediates or by-products of this pathway are required per se for nodule development.

Identification and characterization of a novel Bradyrhizobium japonicum gene involved in host-specific nitrogen fixation

To understand the genetic mechanism of host specificity in the interaction between rhizobia and their hosts, it is important to identify genes that influence both early and late steps in symbiotic development. This paper focuses on the little-understood genetics of host-specific nitrogen fixation. A deletion mutant of Bradyrhizobium japonicum, strain NAD163, was found to induce effective, nitrogen-fixing nodules on soybean and siratro plants but produced ineffective nodules on cowpea plants. Additional transposon and deletion mutants defined a small region that conferred this phenotype, and this region was sequenced to identify two putative open reading frames (ORFs). Data indicate that only one of these ORFs is detectable in bacteroids. This ORF was termed hsfA, with a predicted protein product of 11 kDa. The transcriptional start site of hsfA was determined and found to coincide with a predicted RpoN-dependent promoter. Microscopic studies of nodules induced by the wild type and hsfA mutants on cowpea and soybean plants indicate that the cowpea mutant nodules are slow to develop. The data indicate that hsfA appears to play a crucial role in bacteroid development on cowpea but does not appear to be essential for nitrogen fixation on the other hosts tested.

Histochemical Localization of Acetylcholinesterase in Leguminous Plant, Siratro (Macroptilium atropurpureum).

Acetylcholine (ACh) and acetylcholinesterase (AChE) have previously been demonstrated to occur in leguminous plant, Siratro (Macroptilium atropurpureum). In the present work, the localizatiion of acetylcholinesterase (AChE) within selected organs of Siratro is described. Histochemical techniques have been employed to show the presence of acetylcholinesterase activity in petiole, stem, root and the primary and secondary pulvini. No histochemical evidence was observed in petiole, stem and root segments. The enzyme was only present in the primary and secondary pulvini. The AChE in tissues of the primary and secondary pulvini appeared to be in the endodermal cells around the vascular system. Particularly, after the cytochemical reaction dark spots of copper sulfide strongly stained the endodermal cells that circled around the vascular system in the primary pulvinus. The results confirmed a high ACh content and a hige enzyme activity was present in the primary and secondary pulvini of Siratro. In addition, it is demonstrated that the primary pulvinus is associated with ACh function in Siratro plants.

Tissue-Specific Expression of the Alternative Oxidase in Soybean and Siratro

Alternative oxidase activity (cyanide-insensitive respiration) was measured in mitochondria from the shoots, roots, and nodules of soybean (Glycine max L.) and siratro (Macroptilium atropurpureum) plants. Activity was highest in the shoots and lowest in the nodules. Alternative oxidase activity was associated with one (roots) or two (shoots) proteins between 30 and 35 kilodaltons that were detected by western blotting with a monoclonal antibody against Sauromatum guttatum alternative oxidase. No such protein was detected in nodule mitochondria. Measurements of oxygen uptake by isolated soybean root and nodule cells in the presence of cyanide and salicylhydroxamic acid indicated that alternative oxidase activity was confined to the uninfected cortex cells of the nodule. Immunoprecipitation of translation products of mRNA isolated from soybean shoots revealed a major band at 43 kilodaltons that is assumed to be the precursor of an alternative oxidase protein. This band was not seen when mRNA from nodules was treated in the same fashion. The results indicate that tissue-specific expression of the alternative oxidase occurs in soybean and siratro.

The effect of high temperature on Vigna radiata nodulation and growth with different bradyrhizobial strains

A study was conducted to examine the effect of constant high temperatures and diurnally administered temperature regimes (day/night temperature was maintained at 30°C and increasing 2 hr temperature shocks of 36, 42 and 48°C were applied daily) on growth, nodulation and nitrogen fixation of mungbean ( Vigna radiata L. Wilczek) plants and growth responses of five different cowpea bradyrhizobial strains. Mungbean genotype and bradyrhizobial strains responded differently to high temperatures. Mungbean plants survived at (1) diurnal regimens of high temperature and (2) constant root temperatures of 42 and 48°C (day and night), but germination at 48°C was reduced to 38%. The bradyrhizobial strains survived, grew and remained infective and effective after incubation at constant temperatures up to 42°C and diurnal regimen high temperatures. Two strains, Vml and Vrl16, survived and multiplied at a constant temperature of 48°C. These strains were also effective on mungbean and siratro ( Macroptilium alropurpureum) plants after incubation at 48°C for 5–10 days. Elevated temperatures (36°C) depressed nodulation and nitrogen fixation. Constant high temperatures (42 and 48°C) at the seedling stage markedly affected nitrogenase activity (ARA).

Induction of pathogenic-like responses in the legume Macroptilium atropurpureum by a transposon-induced mutant of the fast-growing, broad-host-range Rhizobium strain NGR234.

Mutant strain ANU2861, a transposon Tn5 mutant of the fast-growing, broad-host-range Rhizobium strain ANU280 (NGR234 Smr Rfr) overproduces polysaccharide, is an ade auxotroph, and induces poorly developed nodules on Leucaena leucocephala and Lablab purpureus (H.C. Chen, M. Batley, J.W. Redmond, and B.G. Rolfe, J. Plant Physiol. 120:331-349, 1985). Strain ANU2861 cannot form nodules on Macroptilium atropurpureum Urb. (siratro) or on Desmodium intortum and D. uncinatum and the nonlegume Parasponia. The parent strain, ANU280, effectively nodulates all these legume species except Parasponia, on which it forms ineffective nodules. Ultrastructural examination of infection sites on the legume siratro showed that mutant strain ANU2861 caused root hair curling (Hac+ phenotype), some cortical cell division (Noi+), but no infection threads (Inf-). Localized cellular responses, known to occur in phytopathological interactions, were observed in electron micrographs of the epidermal tissue at or near the infection zone after inoculation with strain ANU2861 but not the wild-type parental strain. These include (i) the rapid (within 20 h) accumulation of osmiophilic droplets attached to membranes at potential sites of strain ANU2861 penetration and (after 48 h) in the epidermal cells in the immediate region of the curled root hairs, and (ii) localized cell death of the epidermal cells. In addition, strain ANU2861 can initiate a systemic response in split-root siratro plants which prevents the successful nodulation of strain ANU280. A 6.3-kilobase fragment of wild-type genomic DNA, which includes the site of Tn5 insertion in strain ANU2861, was cloned and introduced to strain ANU2861. All the phenotypic defects of the mutant strain were corrected by the introduction of this DNA fragment. This indicates that the original Tn5 insertion is responsible for the phenotype.

Nitrogen fixation ability of exopolysaccharide synthesis mutants of Rhizobium sp. strain NGR234 and Rhizobium trifolii is restored by the addition of homologous exopolysaccharides.

Several transposon Tn5-induced mutants of the broad-host-range Rhizobium sp. strain NGR234 produce little or no detectable acidic exopolysaccharide (EPS) and are unable to induce nitrogen-fixing nodules on Leucaena leucocephala var. Peru or siratro plants. The ability of these Exo- mutants to induce functioning nodules on Leucaena plants was restored by coinoculation with a Sym plasmid-cured (Nod- Exo+) derivative of parent strain NGR234, purified EPS from the parent strain, or the oligosaccharide from the EPS. Coinoculation with EPS or related oligosaccharide also resulted in formation of nitrogen-fixing nodules on siratro plants. In addition, an Exo- mutant (ANU437) of Rhizobium trifolii ANU794 was able to form nitrogen-fixing nodules on white clover in the presence of added EPS or related oligosaccharide from R. trifolii ANU843. These results demonstrate that the absence of Rhizobium EPSs can result in failure of effective symbiosis with both temperate and subtropical legumes.

Macroptilium atropurpureum (siratro) host specificity genes are linked to a nodD-like gene in the broad host range Rhizobium strain NGR234

A 6.7 kb HindIII fragment from the Sym-plasmid of strain NGR234 was found to code a nodD-like gene flanked by two loci which were required for siratro host range. Transfer of the 6.7 kb fragment from NGR234 to R. trifolii strain ANU843 conferred extended host range ability to this strain on siratro plants but not to other plants normally nodulated by strain NGR234. Tn5 mutagenesis of the 6.7 kb fragment showed that insertions located into loci flanking the nodD-like gene abolished the extended host range phenotype. A hybridization probe spanning one of the host specificity loci was shown to hybridize to three specific bands in the NGR234 genome. Complementation and DNA hybridization data showed that the nodD-like gene of strain NGR234 was functionally similar to that in R. trifolii. The introduction to R. trifolii of the 6.7 kb HindIII fragment containing Tn5 insertions located in the nodD-like gene did not abolish the ability to extend the host range of R. trifolii to siratro plants. However, transfer of the 6.7 kb HindIII to R. trifolii derivatives containing Tn5 insertions into either nodA, B or C or other R. trifolii nod genes failed to confer siratro nodulation to these recipients. Reconstruction experiments showed that the 6.7 kb fragment from strain NGR234 and the 14 kb nodulation region of R. trifolii could induce the nodulation of siratro plants when introduced together into Sym-plasmid-cured Rhizobium strains.

A comparison of nitrogen-fixing ability of peanut, cowpea and siratro plants nodulated by different strains of Rhizobium

Acetylene reduction activity and nitrogen accumulation in the plant top per unit nodule mass were compared among peanut, cowpea and siratro plants nodulated by six different strains of Rhizobium. Peanut was found to have several fold higher values than cowpea and siratro for both parameters for all strains of Rhizobium which nodulated it effectively, but the bacteroid content of the peanut nodules was similar to those of cowpea and siratro.

Concentrations of nutrient elements in parts of siratro as affected by phosphorus supply and plant age

Abstract The distribution of some nutrient elements in various parts of Siratro plants of different age and phosphorus status was examined in order to assess which plant parts were best to sample for chemical analysis of nutrient status and to estimate the relative mobility of these elements within the shoot. With increasing physiological age of leaves, concentrations of nitrogen, phosphorus, sulphur and potassium decreased, concentrations of calcium, aluminium, manganese and boron increased and concentrations of magnesium, iron, zinc and copper were little affected. Concentrations of nitrogen, phosphorus, sulphur, calcium, aluminium, boron, zinc and copper in plant parts between the tip and the third youngest, expanded leaf changed little with plant age, suggesting that analysis of this portion of the shoot would be a suitable guide to the sufficiency, or otherwise, of these elements in Siratro plants. By contrast, potassium concentration in all plant parts was markedly depressed by increasing plant age, suggesting that a standardized sampling time would be necessary for interpretation of plant potassium status. Concentrations of magnesium, iron and manganese in all plant parts also decreased with plant age, but to a lesser extent than potassium. Concentrations of all elements, except nitrogen and phosphorus, decreased slightly with increasing phosphorus supply. This decrease could best be attributed to a dilution of these elements with increased plant growth, induced by phosphorus. The present results suggested that potassium, and possibly iron and magnesium, were redistributed in Siratro shoots with time whereas other elements were relatively immobile.

Uptake of potassium by Setaria anceps and Macroptilium atropurpureum from the same standard solution culture

The influence of potassium supply on growth and potassium uptake by Setavia anceps cv. Nandi and Macroptilium atropurpureum cv. Siratro grown in association in standard solution culture was studied. Different degrees of competition were induced by introducing Siratro plants of three different ages (6, 12, 18 days) to the experimental solutions at the commencement of the experiment. Setaria seedlings were 12 days old at this point. Competition between the two species was compared at a high and at a low level of potassium supply. Throughout the duration of the experiment, Setaria plants absorbed potassium at a much greater rate than did the Siratro plants grown in association. Although the potassium absorption rates of Setaria declined more rapidly as the solution supply was exhausted, they generally remained above those for Siratro. The higher potassium concentrations obtained in Setaria than in Siratro plants grown in association ensure a superior ability for the grass to continue growth following exhaustion of the potassium supply. Rapid early growth of Siratro plants in some situations may provide some advantage in competing for the available potassium supply simply through a greater root mass.

Isotopic studies on the uptake of nitrogen by pasture plants. IV. Uptake of nitrogen from labelled plant material by Rhodes grass and Siratro

Rhodes grass (Chloris gayana Kunth.) and Siratro (Phaseolus atropurpureus D.C. var. Siratro) plants were grown separately and together in pots of soil containing 15N-labelled ground Rhodes grass plant material (carbon/nitrogen ratio 44). The added plant material immobilized nitrogen during the first 6 weeks of the experiment, and nitrogen uptake by Rhodes grass was still less than that of the control at 15 weeks. Both the grass and the nodulated legume took up 15N throughout the experiment, despite the reduction in availability of unlabelled soil nitrogen. Siratro grown alone took up as much 15N as Rhodes grass grown alone. When the plants were grown together about one-third of the 15N uptake went into the Siratro and two-thirds into the Rhodes grass. The Siratro roots had a higher 15N enrichment than the tops, indicating that relatively more of the unlabelled nitrogen fixed from the atmosphere went into the Siratro tops, compared with the labelled nitrogen taken up from the soil. With Rhodes grass the tops had a higher enrichment than the roots. The difference between the extractable mineral nitrogen in the pots of soil under grass and in the bare pots accounted for uptake by the grass at 3 weeks but underestimated it at 15 weeks. In addition, there was a significant difference in enrichment between the total extractable mineral nitrogen in the soil and the nitrogen taken up by the Rhodes grass. From the uptake and partition of 15N it was calculated that 43–50% of the nitrogen in the Siratro plants at 3 weeks was taken up from the soil (the rest was attributed to symbiotic fixation); at 15 weeks only 2–4% of the nitrogen in the Siratro was from the soil. Very little nitrogen was transferred from the Siratro to the associated Rhodes grass.