Root nodulation studies inAeschynomene aspera

Summary Fifteen isolates of nodule bacteria were isolated from root and stem nodules ofAeschynomene aspera and they were characterized as Rhizobium by well known laboratory tests. All these isolates together with other efficient strains of known rhizobia belonging to different cross-inoculation groups were evaluated for their nodulation abilities onAeschynomene aspera, Cajanus cajan (pigeon pea),Cicer arietinum (chickpea),Pisum sativum (pea),Trifolium repens (clover),Medicago sativa (lucerne),Lens culinaris (lentil),Glycine max (soybean),Vigna sinensis (cowpea),Vigna radiata (mung bean),Vigna mungo (urd bean) andArachis hypogea (peanut). The results demonstrated that Rhizobium fromAeschynomene could form nodules only on its homologous host (Aeschynomene) but not on other legumes tested. Secondly, none of the rhizobia of other cross-inoculation groups could nodulateA. aspera.     

Subgroups of the Cowpea Miscellany: Symbiotic Specificity within Bradyrhizobium spp. for Vigna unguiculata, Phaseolus lunatus, Arachis hypogaea, and Macroptilium atropurpureum

Rhizobia classified as Bradyrhizobium spp. comprise a highly heterogeneous group of bacteria that exhibit differential symbiotic characteristics on hosts in the cowpea miscellany cross-inoculation group. To delineate the degree of specificity exhibited by four legumes in the cowpea miscellany, we tested the symbiotic characteristics of indigenous cowpea bradyrhizobia on cowpea (Vigna unguiculata), siratro (Macroptilium atropurpureum), lima bean (Phaseolus lunatus), and peanut (Arachis hypogaea). The most-probable-number counts of indigenous bradyrhizobia at three sites on Maui, Hawaii, were substantially different on the four hosts: highest on siratro, intermediate on cowpea, and significantly lower on both lima bean and peanut. Bradyrhizobia from single cowpea nodules from the most-probable-number assays were inoculated onto the four hosts. Effectiveness patterns of these rhizobia on cowpea followed a normal distribution but were strikingly different on the other legumes. The effectiveness profiles on siratro and cowpea were similar but not identical. The indigenous cowpea-derived bradyrhizobia were of only moderate effectiveness on siratro and were in all cases lower than the inoculant-quality reference strain. Between 5 and 51% of the bradyrhizobia, depending on site, failed to nodulate peanut, whereas 0 to 32% failed to nodulate lima bean. No significant correlation was observed between the relative effectiveness of the bradyrhizobia on cowpea and their corresponding effectiveness on either lima bean or peanut. At all sites, bradyrhizobia that were ineffective on cowpea but that effectively nodulated lima bean, peanut, or both were found. Eighteen percent or fewer of the bradyrhizobia were as effective on lima bean as the reference inoculant strain; 44% or fewer were as effective on peanut as the reference strain. Only 18% of all cowpea-derived bradyrhizobia tested were able to form N(2)-fixing nodules on both lima bean and peanut. These results indicate the need to measure indigenous bradyrhizobial population characteristics directly with the crop of interest to obtain an accurate assessment of the need to inoculate.     

Host specificity among five species in the cowpea cross-inoculation group

Summary In a cross-inoculation experiment using crushed nodules from cowpea (Vigna unguiculata), groundnut (Arachis hypogea), bambara groundnut (Voandzeia subterranea), lima bean (Phaseolus lunatus) and soybean (Glycine max.), it was found that soybean did not nodulate with Rhizobia from any of the other species whilst its Rhizobia nodulated with all species. Cowpea and lima bean, on the other hand, nodulated with Rhizobia from all species, but their Rhizobia nodulated only with each other. Groundnut and bambara groundnut nodulated with Rhizobia from all species except cowpea and lima bean, and their Rhizobia also nodulated with all species except soybean.     

Sensitivity of the common bean (Phaseolus vulgaris L.) symbiosis to high soil temperature

Summary Experiments were done to test whether N fixation is more sensitive to high soil temperatures in common bean than in cowpea or soybean. Greenhouse experiments compared nodulation, nitrogenase activity, growth and nitrogen accumulation of several host/strain combinations of common bean with the other grain legumes and with N-fertilization, at various root temperatures. Field experiments compared relative N-accumulation (in symbiotic relative to N-fertilized plants) of common bean with cowpea under different soil thermal regimes. N-fertilized beans were unaffected by the higher temperatures, but nitrogen accumulation by symbiotic beans was always more sensitive to high root temperatures (33°C, 33/28°C, 34/28°C compared with 28°C) than were cowpea and soybean symbiosis. Healthy bean nodules that had developed at low temperatures functioned normally in acetylene reduction tests done at 35°C. High temperatures caused little or no suppression of nodule number. However, bean nodules produced at high temperatures were small and had low specific activity. ForP. vulgaris some tolerance to high temperature was observed among rhizobium strains (e.g., CIAT 899 was tolerant) but not among host cultivars. Heat tolerance ofP. acutifolius andP. lunatus symbioses was similar to that of cowpea and soybean. In the field, high surface soil temperatures did not reduce N accumulation in symbiotic beans more than in cowpea, probably because of compensatory nodulation in the deeper and cooler parts of the soil.     

Recognition of Leguminous Hosts by a Promiscuous Rhizobium Strain

The lima bean (Phaseolus lunatus L.) and the pole bean (Phaseolus vulgaris L.) are nodulated by rhizobia of two different cross-inoculation groups. Rhizobium sp. 127E15, a cowpea-type Rhizobium, can induce effective nodules on the lima bean and partially effective nodules on the pole bean. Rhizobium phaseoli 127K14 can induce effective nodules on the pole bean but does not reciprocally nodulate the lima bean. Root hairs of the lima bean when inoculated with Rhizobium sp. 127E15 showed tip curling and swelling and infection thread formation as observed by light microscopy and scanning electron microscopy. When lima bean root hairs were inoculated with R. phaseoli 127K14, no host-specific responses were observed. Pole bean root hairs that had been inoculated with R. phaseoli 127K14 or Rhizobium sp. 127E15 also showed tip curling and swelling and infection thread formation. Colonization of lima bean root hairs by Rhizobium sp. 127E15 and pole bean root hairs by R. phaseoli 127K14 or Rhizobium sp. 127E15 appeared to involve the elaboration of microfibrils. This study showed that when Rhizobium sp. 127E15 nodulates a host of a different cross-inoculation group, it elicits the same specific host responses as it does from a host of the same cross-inoculation group.     

Growth and nodulation of tropical food legumes in dilute solution culture

Twenty-two tropical food legumes were grown in dilute nutrient solution with or without rhizobium inoculation and supplied with either low or adequate amounts of inorganic N. Growth of legumes supplied with adequate inorganic N was generally satisfactory. However, solution phosphorus (P) concentration (15μM) was excessive for black gram, while the initial solution manganese concentration (1.8μM) was excessive for green gram. Growth responses to inoculation with rhizobium at low inorganic N supply were obtained in only 9 of the 22 legumes studied, and shoot dry matter yields were ≤ 51% of those obtained with adequate N supply. Poor growth by inoculated plants with a low N supply was attributed to failure of the inoculated strain of Bradyrhizobium to infect roots (lima bean and Mexican yam bean), to low nodule numbers (green gram, black gram and navy bean), or to excessive uptake of P (black gram, adzuki bean, pigeonpea, winged bean and cowpea cv. Vita 4) and/or manganese (green gram and black gram). High solution temperatures may have limited N fixation by some of the legumes, particularly chickpea.     

Nodulation of Pole Bean (Phaseolus vulgaris L.) by Rhizobium Species of Two Cross-Inoculation Groups

Physiology and morphology of pole bean (Phaseolus vulgaris L. cv Kentucky Wonder) root nodules induced by two Rhizobium species of different cross-inoculation groups have been compared. Root nodules induced by Rhizobium sp. 127E15, which is a strain of the cowpea group Rhizobium, were pinkish, had irregular shapes, and were only partially effective. Their peak acetylene reduction activity was 4.36 μmol of C₂H₄ formed per g of fresh nodules per h at 30 days after inoculation. The effective nodules induced by Rhizobium phaseoli 127K14, which is a strain of the bean group Rhizobium, were dark red, spherical, and showed peak acetylene reduction activity of 15.95 μmol of C₂H₄ formed per g of fresh nodules per h at 15 days after inoculation. The partial effectiveness of 127E15-induced nodules was associated with fewer infected cells, a delay in the increase of bacteroid population within the host cells, abundance of cytoplasmic vesicles in the host cells, more bacteroids within a membrane envelope (peribacteroid membrane), and the inability of bacteroids to completely fill up the host cytoplasmic space. The 127K14-induced nodules were fully mature, with host cells filled with bacteroids by 12 days after inoculation. In contrast, the 127E15-induced nodules did not reach a similar developmental stage even 30 days after inoculation.     

Phloem translocation of gibberellins in three species of higher plants

Phloem sap was collected from white lupin (Lupinus albus L.), cowpea (Vigna unguiculata L.) and castor bean (Ricinus communis L.) and analysed for gibberellins (GAs) using gas chromatography-mass spectrometry (GC-MS). A large number of GAs were found in the phloem exudate of all three species, particularly where the sap was collected from pods (white lupin and cowpea) and in these legumes GAs representing both the early C-13-hydroxylation and non-hydroxylation pathways of biosynthesis were identified. In the sap collected from the vegetative tissues of castor bean the number of GAs identified was fewer than that in the other species, representing mainly the non-hydroxylation pathway. Data from sap collected from the pedicel and stylar ends of pods and by making feeds of radiolabelled GAs to seeds in situ in white lupin indicate that the GAs present in the phloem are derived mainly from the vegetative tissues of the plant. No evidence for metabolism of GAs in the phloem could be found.     

Effects of environment and host on vector biology and incidence of two whitefly-spread diseases of legumes in Nigeria

Seasonal periodicity in the incidence of cowpea golden mosaic (CGM) and lima bean golden mosaic (LBGM), two whitefly-spread virus-like diseases, corresponded closely with fluctuations in population density of their vector, Bemisia tabaci, at two contrasting sites in southern Nigeria. Peak catches of the vector followed the onset of rains after very high temperatures during the dry season; populations declined abruptly with continuous, heavy rainfall. At Onne, B. tabaci infested legumes at emergence and remained and reproduced on them but at Ibadan infestation was delayed and there was no evidence of reproduction on legumes. B. tabaci preferred LBGM-susceptible lima beans (Phaseolus lunatus) to resistant ones and more pupae of B. tabaci occurred on CGM-susceptible cowpea (Vigna unguiculata) cultivars than on resistant ones but these observations did not relate to the yellow-sensitivity of whiteflies. Resistance to CGM in cowpea, and to LBGM in lima bean, was identified amongst germplasm exposed to natural infection. Resistance was, in each case, associated with lower disease incidence, slower spread and milder expression of symptoms relative to susceptible cultivars. Vector non-preference for resistant cultivars may have contributed to reduced secondary spread.     

Survival and life table parameters of soybean pod borer Maruca vitrata (Geyer) (Lepidoptera: Crambidae) on leguminous crop cultivars

The soybean pod borer, Maruca vitrata is one of the key insect pests of tropical legumes. It damages tender leaf axils, flower buds, flowers and pods by webbing and boring clusters of flowers and pods. In this study, we investigated the survival and life table parameters of M. vitrata on several leguminous crops; soybean (cvs. Daewon, Poongsannamool and Socheongja), azuki bean (cv. Hongeon), mung bean (cv. Sanpo), and cowpea (cv. Jangchae), compared to artificial diet to assess the antibiosis resistance to M. vitrata. The life‐variables of M. vitrata were significantly affected by the tested legume cultivars. None of the larvae fed cowpea cultivar Jangchae survived. The azuki bean cultivar Hongeon and mung bean cultivar Sanpo were found susceptible to M. vitrata, whereas cowpea cultivar Jangchae and soybean cultivar Daewon showed antibiosis resistance to M. vitrata. Further studies should examine the chemicals associated with leguminous crop cultivars and its mechanism to develop a control method against M. vitrata.     

Rhizobial-induced increase in internode length and identification of endogenous GAs of cowpea (Vigna unguiculata [L.] Walp) stems and nodules

Inoculation with Bradyrhizobium sp. strain 127E14 has been shown to cause a dramatic increase in the internode length of lima bean (Phaseolus lunatus L.), when compared to control plants inoculated with strain 127E15. This rhizobial-induced growth also occurs in cowpea (Vigna unguiculata [L.] Walp), an alternate host for the symbiont. Cowpea plants inoculated with strain 127E14 were 23% taller than those inoculated with strain 127E14 after 6 weeks of growth. Petiole length was found to be significantly greater in plants inoculated with strain 127E14. Cowpea plants treated at the apex with exogenous GA₃ or GA_4/7 responded by increasing internode length when compared to controls. As in lima beans, the rhizobial-induced growth response observed in cowpeas may be in response to an imbalance in the levels of GA-like substances within the plants. Gibberellins A₁, A₃, A₈, A₁₉, A₂₀, A₂₉, and A₄₄have been identified by GC-MS analysis in stems of cowpea, whereas the gibberellins A₁, A₁₉, A₂₀, A₂₉, and A₄₄ were found to be present in nodule tissue formed by strain 127E14. The presence of these GAs indicates that the early 13-hydroxylation biosynthetic pathway is operative in cowpea. GAs identified in cowpea nodules are similar to those found in lima bean nodules formed by the same rhizobia. The finding that rhizobial strain 127E14 induces GA-dependent growth responses in two host legumes further supports the hypothesis that the presence of this bacteria alters the GA balance within the plant.     

Nodule formation and haemoglobin content in some species of Papilionaceae

Summary Characteristics of nodule formation in eleven genera of the Leguminosae, belonging to the tribes Galegeae, Genisteae, Hedysareae and Phaseoleae of the sub-family Papilionaceae are described. Variation existed in the type, size and weight of nodules formed on the legumes when inoculated with effective cowpea rhizobia, in field conditions of plant growth. Among the legumes, the haemoglobin content of nodules indicated their possible effectiveness. Dolichos lablab L., had a higher haemoglobin content per unit nodule volume than other legumes. This host may have a greater potential than the other species in symbiotic activity with legume bacteria.     

Compatibility of Rhizobial Genotypes within Natural Populations of Rhizobium leguminosarum Biovar viciae for Nodulation of Host Legumes

Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.     

Scab disease of cowpea (Vigna unguiculata) caused by Sphaceloma a species of the fungus

Scab disease of cowpea (Vigna unguiculuta) was shown to be caused by Sphaceloma sp. It affects all above ground parts of the plant. The first symptom of the disease, appearing within 3 to 6 days of inoculation, is puckering of the lamina. Spots on mature leaves are white with or without brown margins. Typical scab lesions on petiole, stem, peduncle and pod are white turning dark brown when chlamydospores are formed and are oblong-elongate except for pod lesions that are ovoid. The most destructive phase is scab of the flowering axis which causes flower and, or, pod abortion or completely prevents flower formation. Inoculation of asparagus pea (V. sesquipedalis) with a cowpea isolate of Sphaceloma sp. produced symptoms similar to those on cowpea. Inoculated hyacinth bean (Lablab niger) produced atypical mild lesions. The following legumes were not affected when artificially inoculated with the fungus: black gram (Phaseolus mungo), green gram (P, aureus), French bean (P. vulgaris), Lima bean (P. lunatus), groundnut (Arachis hypogaea), and soyabean (Glycine max). The characteristics of the fungus on potato dextrose agar are described.     

Diversity among Bradyrhizobium isolates nodulating yardlong bean and sunnhemp in Guam

AIMS: To isolate and characterize bradyrhizobia that nodulate yardlong bean and sunnhemp in Guam. METHODS AND RESULTS: Bradyrhizobia populations that nodulate yardlong bean and sunnhemp in Guam were examined for genetic diversity and their relatedness to Bradyrhizobium japonicum and B. elkanii reference strains. Genomic DNA of 58 isolates of Bradyrhizobium spp. was hybridized with B. japonicum nodY and B. elkanii nodK genes. Based on the hybridization patterns, the isolates were classified into three nodY-nodK hybridizing groups. Group I comprised the majority of the isolates and hybridized with nodY whereas group II isolates hybridized with nodK. The group III isolates, that did not hybridize with either nodY or nodK, formed nitrogen-fixing nodules on cowpea but did not nodulate soybean. DNA sequence analysis of a 280-bp fragment of the variable region of the 16S rRNA gene of a few group III isolates showed that these isolates were more similar to Bradyrhizobium spp. than to B. japonicum or B. elkanii. CONCLUSIONS: The majority of the isolates nodulating yardlong bean and sunnhemp in Guam are similar to B. japonicum, although some isolates are similar to Bradyrhizobium spp. that nodulate a miscellaneous group of legumes including cowpea. SIGNIFICANCE AND IMPACT OF THE STUDY: Since both yardlong bean and sunnhemp are nodulated by a range of bradyrhizobia, selection of superior strains may be based on nodulation effectiveness on both legumes.     

Transgenic cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>) seeds expressing a bean α-amylase inhibitor 1 confer resistance to storage pests,bruchid beetles

Cowpea is one of the important grain legumes. Storage pests, Callosobruchus maculatus and C. chinensis cause severe damage to the cowpea seeds during storage. We employ a highly efficient Agrobacterium-mediated cowpea transformation method for introduction of the bean (Phaseolus vulgaris) α-amylase inhibitor-1 (αAI-1) gene into a commercially important Indian cowpea cultivar, Pusa Komal and generated fertile transgenic plants. The use of constitutive expression of additional vir genes in resident pSB1 vector in Agrobacterium strain LBA4404, thiol compounds during cocultivation and a geneticin based selection system resulted in twofold increase in stable transformation frequency. Expression of αAI-1 gene under bean phytohemagglutinin promoter results in accumulation of αAI-1 in transgenic seeds. The transgenic protein was active as an inhibitor of porcine α-amylase in vitro. Transgenic cowpeas expressing αAI-1 strongly inhibited the development of C. maculatus and C. chinensis in insect bioassays.     

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.     

Microbial Degradation of α-Picolinic Acid, 2-Pyridinecarboxylic Acid

The biological effects of raw winged bean seeds were investigated with feeding experiments on rats, and the effects of lectin (phytohemagglutinin) present in the seeds are discussed. Administration of a 30% raw winged bean diet caused strong growth depression in young rats, and led to death within 10 ~ 20 days, inducing severe damage to the small intestine of the rats. Significant morphological changes of the intestinal mucosa were observed with a microscopic investigation. As the lethal effect was eliminated by autoclaving but not removed with supplementation of 0.5% l-methionine to the raw winged bean diet, the lectin was assumed to be closely related to the deleterious effects of raw winged bean. In vitro and in vivo digestion tests of the lectin revealed that the winged bean lectin had resistance to peptic, pancreatic and membrane digestions. The hemagglutinating activity was also detected in the intestinal mucosa and faeces from rats ingesting the raw winged bean or its purified lectin. The binding action of lection to mucosal epitheliums of the gastrointestinal tract is suggested to be the initial step of the deleterious effects induced by the winged bean lectin.     

Recent changes to the classification of symbiotic,nitrogen-fixing,legume-associating bacteria: a review

The Rhizobia are collectively comprised of gram negative soil bacteria that have the ability to form symbiotic nitrogen-fixing root and/or stem nodules in association with leguminous plants. The taxonomy of these bacteria is continually in a state of flux, in large part due to rapid development of refined molecular biology techniques. The isolation and characterization of new, and often different, legumes-nodulating bacteria on a variety of plant hosts has resulted in the naming of many new rhizobial species. Here we update the taxonomy of the legume-nodulating bacteria and describe newly identified rhizobia capable of nodulating edible legumes and legume trees. In 1990, there was only one bacterial species that was known to nodulate common bean worldwide (Rhizobium leguminosarum sv. phaseoli), one species that nodulated faba bean (Rhizobium leguminosarum sv. viciae), and two species that nodulated soybean (Bradyrhizobium japonicum and Rhizobium fredii). Today, nearly 14, 11, 6, 5, 5, 4, 3 and 2 species have been defined that are capable of nodulating common bean, soybean, cowpea, chickpea, peanut, lentils, faba bean and pea, respectively. The recent use of whole genome based taxonomy (genomotaxonomy) will surely change how we define this important group of bacteria. The identification of several rhizobial species that are able to nodulate and fix nitrogen with edible legumes may enhance the production of these crops and can compensate for worldwide deficiencies in human nutritional needs in the future.     

Phylogeny of nodulation genes and symbiotic properties of Genista tinctoria bradyrhizobia

Pairwise comparisons of Genista tinctoria (dyer’s weed) rhizobium nodA, nodC, and nodZ gene sequences to those available in databanks revealed their highest sequence identities to nodulation loci of Bradyrhizobium sp. (Lupinus) strains and rhizobia from other genistoid legumes. On phylogenetic trees, genistoid microsymbionts were grouped together in monophyletic clusters, which suggested that their nodulation genes evolved from a common ancestor. G. tinctoria nodulators formed symbioses not only with the native host, but also with other plants of Genisteae tribe such as: Lupinus luteus, Sarothamnus scoparius, and Chamaecytisus ratisbonensis, and they were classified as the genistoid cross-inoculation group. The dyer’s weed root nodules were designated as indeterminate with apical meristem consisting of infected and uninfected cells.The GenBank accession numbers for the sequences reported in this paper are as follows: nodC, DQ139776–DQ139781; nodA, DQ135897, Q135898; nodZ, DQ135899–DQ135903.