Curtobacterium flaccumfaciens pv. flaccumfaciens on Soybean in Germany – A Threat for Farming

Plant diseases caused by Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) are distributed in North and South America as well as in South and East Europe and occur mostly on beans (Phaseolus vulgaris). This is the first report of Cff on soybean in Germany. Cff was detected in complex with Pseudomonas syringae pv. glycinea on field‐grown soybeans that were not treated with pesticides. Cff, the causal agent of bacterial tan spot disease, was identified by 16S rDNA sequencing and by artificial infection and re‐isolation from the host plants soybean (Glycine max) and bush bean (Phaseolus vulgaris).     

Enhancing crop legume N2 fixation through selection and breeding

Legume N₂ fixation is variable, but nonetheless is a valuable process in world agriculture. There is great potential to increase the contribution by the crop legumes to the world's supply of soil.N. This will be achieved by (i) increasing the area of legumes sown by farmers; (ii) improved management of the crops in order that the major determinants of productivity, e.g. land area, water availability, are converted to harvested product with maximum efficiency; and (iii) genetic modification of the commonly-grown species to ensure high dependence of the legume crop on N₂ fixation at all levels of productivity. Currently-used methods for measuring N₂ fixation and for assessing heritability and repeatability of N₂ fixation in breeding and selection programs are reviewed. Results from research programs to define genetic variation in N₂ fixation and to enhance N₂ fixation through selection and breeding are presented with particular emphasis on common bean (Phaseolus vulgaris) and soybean (Glycine max).     

Structure and chromosomal arrangement of leghemoglobin genes in kidney bean suggest divergence in soybean leghemoglobin gene loci following tetraploidization

We have determined the structure of one of the leghemoglobin (Lb) genes of Phaseolus vulgaris (kidney bean) and deduced the chromosomal arrangement of leghemoglobin genes by genomic hybridizations with Lb and two other sequences, each specific to the 5' or 3' region of the soybean leghemoglobin loci. By comparing this organization with two other species of legumes, Glycine max (soybean) and G. soja (wild soybean), a phylogeny of leghemoglobin gene loci was traced. The intragenic structure of the kidney bean leghemoglobin gene shows the same intron/exon arrangement as that of soybean leghemoglobin genes and extensive sequence homologies in both coding as well as 5' and 3' non-coding regions. The presence in the kidney bean genome of four leghemoglobin genes suggests that tandem duplications of a single primordial plant globin gene had occurred to generate four leghemoglobin genes in an `Lb-locus' before Glycine and Phaseolus species diverged. Chromosome duplication by tetraploidization in Glycine generated two loci containing four genes each. A large deletion in one of the two four-gene loci in soybean resulted in the generation of the Lbc₂ locus containing two leghemoglobin genes, one functional and another pseudo (LbΨ₂). This pseudogene, unlike that present on the main locus, is represented by only two and a half exons and appears to be truncated. The two other truncated genes (LbT₁ and LbT₂) were probably generated similarly in the genome of Glycine spp. following tetraploidization before the divergence of G. max and G. soja.     

Genome organization in dicots. II. Arabidopsis as a ’bridging species’ to resolve genome evolution events among legumes

Analysis of molecular linkage groups within the soybean (Glycine max L. Merr.) genome reveals many homologous regions, reflecting the ancient polyploidy of soybean. The fragmented arrangement of the duplicated regions suggests that extensive rearrangements, as well as additional duplications, have occurred since the initial polyploidization event. In this study we used comparisons between homoeologous regions in soybean, and the homologous regions in the related diploids Phaseolus vulgaris and Vigna radiata, to elucidate the evolutionary history of the three legume genomes. Our results show that there is not only conservation of large regions of the genomes but that these conserved linkage blocks are also represented twice in the soybean genome. To gain a better understanding of the process of genome evolution in dicots, molecular comparisons have been extended to another well-studied species, Arabidopsis thaliana. Interestingly, the conserved regions we identified in the legume species are also relatively conserved in Arabidopsis. Our results suggest that there is conservation of blocks of DNA between species as distantly related as legumes and brassicas, representing 90 million years of divergence. We also present evidence for an additional, presumably earlier, genome duplication in soybean. These duplicated regions were only recognized by using Arabidopsis as a ’bridging’ species in the genome comparisons. Received: 10 October 2000 / Accepted: 13 January 2001     

New techniques for studying competition by Rhizobia and for assessing nitrogen fixation in the field

One of the key factors limiting the proper assessment and use of rhizobial strains in the field is the lack of suitable methodology to screen the success of individual isolates in competing for nodule occupancy with different cultivars of legumes and in different soil and agronomic conditions. The use of marker genes enables individual rhizobial strains to be identified by a simple colour assay, thus enabling a dramatic increase in throughput of strain screening. One such marker system for rhizobial ecology, the GUS system, is already in use to facilitate rapid screening of rhizobial isolates. Other markers, which will allow the competitive behaviour of several strains to be studied at once, are under development.Likewise, breeding of the host legume for a high efficiency of nitrogen fixation is hampered by the difficulty in assessing this property. The method which currently gives the highest throughput of analysis, and has been successfully used in soybean breeding programs, is the ureide technique. However, it remains somewhat laborious for use in routine breeding programs. In this paper we discuss the potential use of reporter genes to provide information on the relative levels of ureides and other nitrogenous compounds in plants growing in the field. This would greatly increase the rate at which this trait could be scored, and would thus enable routine assays for increased symbiotic nitrogen fixation for breeding or management purposes in legume crops such as soybean (Glycine max) and common bean (Phaseolus vulgaris).     

Assembly and annotation of a draft genome sequence for Glycine latifolia,a perennial wild relative of soybean

Glycine latifolia (Benth.) Newell & Hymowitz (2n = 40), one of the 27 wild perennial relatives of soybean, possesses genetic diversity and agronomically favorable traits that are lacking in soybean. Here, we report the 939‐Mb draft genome assembly of G. latifolia (PI 559298) using exclusively linked‐reads sequenced from a single Chromium library. We organized scaffolds into 20 chromosome‐scale pseudomolecules utilizing two genetic maps and the Glycine max (L.) Merr. genome sequence. High copy numbers of putative 91‐bp centromere‐specific tandem repeats were observed in consecutive blocks within predicted pericentromeric regions on several pseudomolecules. No 92‐bp putative centromeric repeats, which are abundant in G. max, were detected in G. latifolia or Glycine tomentella. Annotation of the assembled genome and subsequent filtering yielded a high confidence gene set of 54 475 protein‐coding loci. In comparative analysis with five legume species, genes related to defense responses were significantly overrepresented in Glycine‐specific orthologous gene families. A total of 304 putative nucleotide‐binding site (NBS)‐leucine‐rich‐repeat (LRR) genes were identified in this genome assembly. Different from other legume species, we observed a scarcity of TIR‐NBS‐LRR genes in G. latifolia. The G. latifolia genome was also predicted to contain genes encoding 367 LRR‐receptor‐like kinases, a family of proteins involved in basal defense responses and responses to abiotic stress. The genome sequence and annotation of G. latifolia provides a valuable source of alternative alleles and novel genes to facilitate soybean improvement. This study also highlights the efficacy and cost‐effectiveness of the application of Chromium linked‐reads in diploid plant genome de novo assembly.     

A COMPARATIVE STUDY OF OVULE AND MEGAGAMETOPHYTE DEVELOPMENT IN FIELD-GROWN AND GREENHOUSE-GROWN PLANTS OF GLYCINE MAX AND PHASEOLUS AUREUS (PAPILIONACEAE)

A study of ovule and megagametophyte development in field- and greenhouse-grown plants of Glycine max (L.) Merrill and Phaseolus aureus Roxb. reveals several consistent features for both species. These features include: a multiple archesporium, enlargement of a primary sporogenous cell directly into a megasporocyte, production of unequal dyad cells, a functional chalazal megaspore, Polygonum-type development, and a hypostase. A filiform apparatus was not observed in either species. Several marked differences in development also occur. Phaseolus usually produces one sporogenous cell per ovule; Glycine produces 2–3 sporogenous cells per ovule. Meiosis II is synchronous in Phaseolus but nonsynchronous in Glycine. Linear tetrads are produced in Phaseolus, whereas linear and T-shaped tetrads are found in Glycine. Starch grains accumulate in the mature megagametophyte of Glycine but are absent at that stage in Phaseolus. The usefulness of the modified clearing fluid, benzyl benzoate-4½, for the study of ovule and megagametophyte development in Glycine max and Phaseolus aureus is here demonstrated. In addition, the study indicates for both species that megagametophyte development in plants grown under field conditions is markedly similar to development in plants grown in the more uniform conditions of the greenhouse. Accordingly, these findings suggest generally that embryological data collected from plants grown under greenhouse conditions will reflect those from plants found in nature.     

A single nuclear locus phylogeny of soybean based on DNA sequence

Soybean [Glycine max (L.) Merr.] evolution was examined by sequencing portions of the restriction fragment length polymorphism (RFLP) locus A-199a of 21 taxa from the Glycininae and 1 from the Phaseoleae. Four hundred nucleotides were determined in each, aligned, and then compared for these taxa. Within the annual soybean subgenus (Soja), the four accessions differed at as many as 2.2% of the nucleotides. Among 13 perennial soybean species (subgenus Glycine), nucleotide variation ranged from 1.7% to 8.4%. The nucleotide difference between the two soybean subgenera was 3.0–7.0%. Nucleotide variation between the genus Glycine and the related genera of Neonotonia, Amphicarpa, Teramnus, and Phaseolus ranged from 8.2% to 16.4%. In addition to nucleotide substitutions, insertions/deletions (indels) differences were also observed and were consistent with nucleotide-based analysis. Cladistic analysis of the A-199a sequences was performed using Wagner parsimony to construct a soybean phylogeny. Sixteen equally parsimonious trees were produced from these data. The trees were 246 steps in length with a consistency index of 0.78. Indels distribution upon the consensus topology revealed a pattern congruent with the nucleotide-based phylogeny. The current taxonomic status of the soybean subgenera and the related genera of Neonotonia, Amphicarpa, and Teramnus were well-supported and appear monophyletic in this analysis. Homoplasy within the subgenus Glycine led to a lack of resolved topology for many of these 13 taxa. However, the Glycine clade topology was consistent with phylogenies proposed using crossing experiments and cpDNA RFLPs. These genera were arranged from ancestral to derived as: Teramnus, Amphicarpa, Neonotonia, and Glycine when Phaseolus vulgaris was used as an outgroup.     

Activity of antioxidant enzymes in response to cadmium in Crotalaria juncea

The aromatic amine, -phenethylamine, was identified in various field-grown leguminous plants by analyses with HPLC, GC, GC-MS and ¹H-NMR. High concentration of -phenethylamine was generally detected only in mature root nodules, but not in other plant organs such as root, stem, leaf, pod and grain. Occurrence was specific to the root nodules formed by Bradyrhizobium infection. Ten of eleven legume crops including soybean [Glycine max (L.) Merr.], pigeon pea [Cajanus cajan (L.) Millsp.], adzuki bean (Vigna angularis), mung bean [V. radiata (L.) Wilczek] and cowpea (V. unguiculata) contained this aromatic amine, but groundnut (Arachis hypogaea L.) also nodulated by Bradyrhizobium sp. did not. Root nodules collected from garden pea (Pisum sativum L.), broad bean (Vicia fava L.), kidney bean (Phaseolus vulgaris L.) and various other herbaceous legumes nodulated by Rhizobium sp., Mesorhizobium sp., Sinorhizobium sp. or Azorhizobium caulinodans, and root-nodulated, woody non-legumes, nodulated by Frankia spp., contained little -phenethylamine.The amount of -phenethylamine in Bradyrhizobium-infected nodules varied with the legume species and their cultivars, and most significantly, with nodule age. In field-grown soybean plants, nodule -phenethylamine attained maximum concentration at the flowering stage and far exceeded that of the major polyamines of soybean nodules, putrescine and spermidine.     

Complete Chloroplast Genome Sequence of <Emphasis Type="Italic">Glycine max</Emphasis> and Comparative Analyses with other Legume Genomes

Lack of complete chloroplast genome sequences is still one of the major limitations to extending chloroplast genetic engineering technology to useful crops. Therefore, we sequenced the soybean chloroplast genome and compared it to the other completely sequenced legumes, Lotus and Medicago. The chloroplast genome of Glycine is 152,218 basepairs (bp) in length, including a pair of inverted repeats of 25,574 bp of identical sequence separated by a small single copy region of 17,895 bp and a large single copy region of 83,175 bp. The genome contains 111 unique genes, and 19 of these are duplicated in the inverted repeat (IR). Comparisons of Glycine, Lotus and Medicago confirm the organization of legume chloroplast genomes based on previous studies. Gene content of the three legumes is nearly identical. The rpl22 gene is missing from all three legumes, and Medicago is missing rps16 and one copy of the IR. Gene order in Glycine, Lotus, and Medicago differs from the usual gene order for angiosperm chloroplast genomes by the presence of a single, large inversion of 51 kilobases (kb). Detailed analyses of repeated sequences indicate that many of the Glycine repeats that are located in the intergenic spacer regions and introns occur in the same location in the other legumes and in Arabidopsis, suggesting that they may play some functional role. The presence of small repeats of psbA and rbcL in legumes that have lost one copy of the IR indicate that this loss has only occurred once during the evolutionary history of legumes.     

Biochemical characterisation of an allantoate-degrading enzyme from French bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>): the requirement of phenylhydrazine

In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the atmospheric nitrogen fixed in nodules is used for synthesis of the ureides allantoin and allantoic acid, the major long distance transport forms of organic nitrogen in these species. The purpose of this investigation was to characterise the allantoate degradation step in Phaseolus vulgaris. The degradation of allantoin, allantoate and ureidoglycolate was determined “in vivo” using small pieces of chopped seedlings. With allantoate and ureidoglycolate as substrates, the determination of the reaction products required the addition of phenylhydrazine to the assay mixture. The protein associated with the allantoate degradation has been partially purified 22-fold by ultracentrifugation and batch separation with DEAE-Sephacel. This enzyme was specific for allantoate and could not use ureidoglycolate as substrate. The activity was completely dependent on phenylhydrazine, which acts as an activator at low concentrations and decreases the affinity of the enzyme for the substrate at higher concentrations. The optimal pH for the activity of the purified protein was 7.0 and the optimal temperature was 37°C. The activity was completely inhibited by EDTA and only manganese partially restored the activity. The level of activity was lower in extracts obtained from leaves and fruits of French bean grown with nitrate than in plants actively fixing nitrogen and, therefore, relying on ureides as nitrogen supply. This is the first time that an allantoate-degrading activity has been partially purified and characterised from a plant extract. The allosteric regulation of the enzyme suggests a critical role in the regulation of ureide degradation.     

The Occurrence of Vascular Cavities and Specialized Parenchyma Cells in the Roots of Cool-season Legumes

The seeds of 20 legume species were grown in the greenhouse or in growth chambers at different temperatures. Under warm temperature conditions (above 15 °C), six species, pea (Pisum sativum), broadbean (Vicia faba), chickpea (Cicer arietinum), lentil (Lens culinaris), wild lupine (Lupinus latifolius), and soybean (Glycine max), formed cavities in the vascular cylinder of their primary roots, which in turn became filled by the ingrowth of specialized parenchyma cells (SP cells). When these species were grown at low temperature (below 15 °C), however, a “normal” vascular cylinder formed in the primary roots with late-maturing metaxylem vessel members differentiating in the center. These species were all cool-season legumes except soybean, a warm-season legume, which sometimes also formed cavities and SP cell ingrowths. The occurrence of cavities and SP cells therefore was restricted to the cool-season legumes (except soybean) when they were grown under warm temperature conditions. The position and size of cavities varied among these species. Pea and broadbean usually formed large, axially elongated cavities in the central vascular cylinder, or in the xylem poles. Others formed smaller cavities of various lengths.     

Induction of Glutamine Synthetase Activity in Nonnodulated Roots of Glycine max, Phaseolus vulgaris, and Pisum sativum

Nitrate or ammonium fertilization significantly increased glutamine synthetase (GS) activity in nonnodulated roots of French bean (Phaseolus vulgaris), soybean (Glycine max), and pea (Pisum sativum). Western analysis revealed substantial GS antibody-positive protein in root extracts that had minimal GS activity, indicating that an inactive form of GS may be present in nonfertilized plants.     

Nodulation and nitrogen fixation by fast- and slow-growing rhizobia strains of soybean on several temperate and tropical legumes

Three slow-growingBradyrhizobium japonicum (G₃, USDA-110 and KUL-150) of diverse origins and two fast-growing strains ofRhizobium fredii (USDA-192 and USDA-193) were tested with a cropped soybean (Glycine max L. Merrill) cultivar, two cowpeas (Vigna unguiculata), one mung-bean (Phaseolus radiata), one winged-bean (Psophocarpus tetragonolobus) and one field bean (Phaseolus vulgaris) varieties.TheR. fredii strains nodulated and fixed Nitrogen as effectively as the strains ofB. japonicum in a modern european soybean cultivar, namely Fiskeby V. The other western bred soybeans tested were not nodulated by theseR. fredii strains. All of the soybean rhizobia produced nodules in both cowpeas and in mung-bean; theR. fredii strains showed effective N₂-fixation in the cowpeas, particularly USDA-193, yielding shoot dry weights greater than those from theB. japonicum. The symbiotic performance of theR. fredii strains with soybean and other legumes indicated that they should be placed in an intermediate group between the slow-growingB. japonicum and cowpearhizobium sp.The hydrogen uptake activites suggested a possible host effect on the expression of such genes in one out of theB. japonicum strains tested. Furthermore, the slow-growing rhizobia showed significantly higher nitrate-reduction than theR. fredii in the nodules.     

The content of N-acylphosphatidylethanolamines in the seeds of cultivated plants and grain products

The content of the minor class of phospholipids, N-acylphosphatidylethanolamines (NAPEs) in mature seeds of cultivated plants: kidney bean (Phaseolus vulgaris L.), soybean (Glycine max (L.) Merr.), soft spring wheat (Triticum aestivum L. emend. Fiori et Paol), barley (Hordeum vulgare L.), and oat (Avena sativa L.), and the products of technological processing of grain cultures (floor, bran) was studied. Reliable NAPE identification was performed by a comparison of their chromatographic mobility with the marker NAPE sample from wheat flour and synthetic phosphatidylmethanol, and also using specific reagents. Kidney bean cv. Shchedraya seeds contained the highest amount of NAPEs. In legumes, the content of NAPEs varied not only in different species but also in different cultivars of a single species. In cereals, the highest NAPE content was detected in the barley seeds and best quality wheat flour. The content of NAPEs in cereal seeds was less variable than in legumes. NAPE quantification showed that accumulation of this phospholipid class occurred in parallel with accumulation of total phospholipids in seeds. The relation between NAPE content and the processes of seed development and their lipid composition is discussed. The seeds and products best suitable for NAPE isolation are recommended.     

Nitrogen nutrition of seedling grain legumes: some taxonomic, morphological and physiological constraints

Abstract The growth of young plants of the epigeal species Phaseolus vulgaris and Glycine max is compared with that of the hypogeal species Pisum sativum and Vicia faba, with particular reference to synchronization between the exhuastion of seed reserves of N and the availability of fixed N. It is argued that the N stress symptoms which occur when these two processes are not synchronized are more common and obvious in Phaseolus or Glycine than in Pisum or Vicia. This is primarily because in these species (a) the first fixed N is used for nodule growth rather than being exported to the shoot system and (b) the first foliage leaves have a much greater area and contain a larger proportion of N reserves from the seed. It is further suggested that Phaseolus and Glycine may show the greater response to nitrogen fertilizer applied at sowing since (a) most of the applied nitrate is passed directly to the shoots (rather than being reduced in the roots as in Pisum or Vicia) and (b) in addition to being used for growth (following reduction), it may also be used prior to reduction as part of the osmotic force driving cell expansion.     

Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus,Medicago, and Phaseolus

Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution.     

Isoflavone Content in Subterranean Clover Germplasm from Sardinia

Subterranean clover (Trifolium subterraneum) is an important pasture legume, and Sardinia is known as a major centre of diversification of this species. As other legumes, this clover produces biologically active flavonoids including the subclass of isoflavones that are natural phytoestrogens with positive health effects. Present sources of isoflavones for medical/nutraceutical treatments are red clover (Trifolium pratense) and soybean (Glycine max). This study assessed the content and composition of flavonoids in 14 subterranean clover genotypes from Sardinia, grown ex‐situ in comparison with two red clover ecotypes, to acquire information on the potential of the species as an alternative source of isoflavones for possible exploitation. Twenty compounds were tentatively identified across the two clovers after HPLC and LC/ESI‐MS analyses, including clovamide, four flavonols, and 15 isoflavones. Most compounds were present as glucosides or glucosyl malonates. Subterranean clover extracts mainly comprised of derivatives of the isoflavones genistein, biochanin A, and formononetin. Compared to red clover, subterranean clover had higher content of total isoflavones and lower concentration of total flavonols. The isoflavone concentration in subterranean clover was higher than literature data for soybean or red clover. The existing genotypic variation warrants the possibility of selecting varieties with high isoflavone concentration for nutraceutical or pharmaceutical purposes.