The Effect of Ageing on the Leghemoglobin of Cowpea Nodules

Nodulated cowpea (Vigna unguiculata [L.] Walp. cv Vita 3: Bradyrhizobium strain CB756) and soybean (Glycine max [L.] Merr. cv White Eye: Bradyrhizobium strain CB1809) were grown with their root systems maintained in a flowing gas stream containing a range of pO(2) (1-80%, v/v) in N(2) for up to 28 days after planting. At the extremes of sub- and supra-ambient pO(2), the levels of leghemoglobin (Lb) in nodules were reduced. However, neither the proportional composition of Lb component proteins (eight in soybean, three in cowpea) nor their oxidation state was affected by pO(2). Short-term changes in pO(2) (transferring plants grown with sub- or supra-ambient pO(2) in the rhizosphere to air or vice versa) caused a significant decline in Lb content and, in cowpea but not soybean, where pO(2) was increased, a higher percentage of oxidation of Lb. Combining data on changes in Lb level of cowpea nodules grown in sub-ambient pO(2) with those for their structural adaptation to an under supply of O(2) indicated that, despite the nodules having a lower level of Lb, the amount per infected cell was increased by up to twofold and per bacteroid up to fivefold (in those from 1% O(2)) compared to those grown in air. Progressive decline in pO(2) resulted in a progressive increase on this basis, indicating a close relationship between Lb content and the adaptation of nodule functioning to external O(2) level.

As a control measure against weeds, the use of herbicides is an effective and inexpensive alternative. However, there are no products recommended for the cultivation of cowpea in Brazil, making it necessary to search for alternative solutions. The objective of this study was to evaluate the effect of herbicides applied in the pre- and post-emergence on cowpea nodulation and production under conditions of the Amazonian savannah. Two experiments were carried out in a randomized block design with four replicates, using the cowpea cultivar BRS Aracê subjected to the pre-emergence herbicides: Metribuzin, Sulfentrazone, Smetolachlor, Pendimethalin, Oxadiazon, Alachlor, Metribuzin + Pendimethalin, Metribuzin + Alachlor and Quizalofop-p-ethyl, Bentazon, Fomesafen, Imazethapyr, Imazamox + Bentazon, Quizalofop-p-ethyl + Imazethapyr, Quizalofop-p-ethyl + Imazamox and Quizalofop-p-ethyl + Bentazon, and post-emergence herbicides: Quizalofop-p-ethyl, Bentazon, Fomesafen, Imazethapyr, Imazamox + Bentazon, Quizalofop-p-ethyl + Imazethapyr, Quizalofop-p-ethyl + Imazamox, and Quizalofop-p-ethyl + Bentazon. The number of nodules in each plant, the dry matter of nodules, dry matter of roots and the grain yield were evaluated. According to the results obtained, the management of weeds in pre- or post-emergence according to the herbicide used affects the nodulation and productivity of cowpea under the conditions of the Amazonian savannah. The herbicides Metribuzin in preemergence,and Fomesafen and the mixture of Quizalofop-pethyl + Imazethapyr in post-emergence are not recommended for weed control in cowpea. The application of Oxadiazon, Alachlor, and Pendimethalin in pre-emergence can be considered interesting because they do not inhibit the development of the root system or the nodulation of cowpea which provides agreater grain yield. Regarding weed control strategies at postemergence, the application of the herbicide Imazethapyr and the combination of the herbicides quizalofop-p-ethyl + imazamox, Quizalofop-p-ethyl + Bentazon and Imazamox + Bentazon allow satisfactory levels of grain yield, root system development and nodulation of cowpea.

Tannery industry releases high amounts of tannery sludge which are currently composted and used in agricultural soils. The consecutive amendment of such composted tannery sludge (CTS) may affect soil microrganisms, such as rhizobia. In this study, we evaluated the effects of 5-year repeated CTS amendment on growth, nodulation, and yield of cowpea (Vigna unguiculata L.) CTS was applied in different amounts (0, 2.5, 5, 10 and 20 Mg/ha) to a sandy soil. Amendment of CTS increased soil pH, electrical conductivity (EC), sodium and chromium content. Plant growth, nodulation, N accumulation, and cowpea yield increased up to 10 Mg/ha; however, above this rate, these variables decreased. After 5 years of CTS amendment, the increase in soil chemical properties, particularly EC and Na content, exerted negative effects on the growth, nodulation, and yield of cowpea.

O objetivo deste trabalho foi avaliar o efeito da associação entre o rizóbio e o Bacillus subtilis sobre a nodulação, a fixação de N2 e o crescimento do feijão-caupi (Vigna unguiculata) e da leucena (Leucaena leucocephala) cultivados em um Latossolo vermelho. Os tratamentos consistiram de: a) testemunha; b) fertilização com NPK; c) inoculação com rizóbio + PK; e d) inoculação com rizóbio + Bacillus subtilis + PK. Houve um aumento na nodulação do feijão-caupi com a coinoculação. A massa da parte aérea seca, o acúmulo de N e a leitura da clorofila em feijão-caupi foram maiores nos tratamentos fertilizados (NPK) e no tratamento coinoculado. No caso da leucena, não houve diferenças entre os tratamentos NPK, inoculado e coinoculado. As massas das raízes secas do feijão-caupi e leucena aumentaram com a coinoculação. A coinoculação rizóbio e Bacillus subtilis favoreceu o aumento na nodulação em feijão-caupi e tem potencial para aumentar a fixação biológica do N2 e o crescimento das plantas.

Cowpea (Vigna unguiculata) is recognized as a promiscuous legume in its symbiotic relationships with rhizobia, capable of forming associations with a wide range of bacterial species. Our study focused on assessing the diversity of bacterial strains present in cowpea nodules when inoculated with soils from six indigenous lands of Mato Grosso do Sul state, Central-Western Brazil, comprising the Cerrado and the Pantanal biomes, which are known for their rich diversity. The DNA profiles (BOX-PCR) of 89 strains indicated great genetic diversity, with 20 groups and 23 strains occupying single positions, and all strains grouped at a final similarity level of only 25%. Further characterization using 16S rRNA gene sequencing revealed a diverse array of bacterial genera associated with the cowpea nodules. The strains (number in parenthesis) were classified into ten genera: Agrobacterium (47), Ancylobacter (2), Burkholderia (12), Ensifer (1), Enterobacter (1), Mesorhizobium (1), Microbacterium (1), Paraburkholderia (1), Rhizobium (22), and Stenotrophomonas (1), split into four different classes. Notably, only Ensifer, Mesorhizobium, Rhizobium, and Paraburkholderia are classified as rhizobia. Phylogenetic analysis was conducted based on the classes of the identified genera and the type strains of the closest species. Our integrated analyses, combining phenotypic, genotypic, and phylogenetic approaches, highlighted the significant promiscuity of cowpea in associating with a diverse array of bacteria within nodules, showcasing the Brazilian soils as a hotspot of bacterial diversity.

The significance of cowpea (Vigna unguiculata L. Walp) as nutritional, economic and a soil fertility promoter had already been elucidated by many researchers. The present work was aimed at isolation and characterization of rhizobial and non-rhizobial endophytes of cowpea nodules recovered from smallholder farms in northern Nigeria. The study soils were analyzed and found to have varying physical and chemical characteristics. Nine (75 %) of the soil samples were sandy loam while the others included sand loam, sand and clay loam each. The pH (water) of the soils ranged from moderately acidic to neutral. The fertility status of the soils in respect to total nitrogen (%), (phosphorus mg kg-1) potassium (Exc K), and organic carbon (%) varied among sites. Each of the 40 nodular endophytes was isolated using yeast mannitol agar and phenotypically characterized using 14 assays (5-growth, 4-sugar fementation, 5-temperature courses). In all, 35 % of the isolates tested positive for the 14 assays. All isolates grew in nutrient agar, King’s B agar; and 30 °C and 40 °C. Hierarchical Cluster Analysis (HCA) indicated diversity and similarities among isolates from same and different localities. Soil origin, its features or conditions did not appear to determine the phenotypes of the bacterial isolates. Growth of some of the isolates on Pseudomonas selective agar suggests that bacteria resident in cowpea nodules included at least rhizobia and pseudomonads flora. Keywords: Legumes, Pseudomonas, Rhizobia, Soil, Sub-saharan Africa

Effects of pH, calcium (Ca), and aluminium (Al) on the growth and nodulation of cowpea (Vigna unguiculata) were studied in large-volume, dilute nutrient solutions. Ca concentrations over the range 0.05 mM to 2.5 mM, and pH over the range 4.5 to 5.5, had no significant effect on the growth of the host plant. However, the combination of low pH and low Ca delayed nodulation and strongly depressed nodule number and nodule dry weight. At the highest Ca concentration, effects of pH on nodule number were small and not significant, and effects on nodule weight, although significant, were much smaller than at the lowest Ca concentration. It was concluded that nodulation was more sensitive to low Ca and low pH than was host plant growth. At pH 4.5 and 0.5 mM Ca, A1 delayed and depressed nodulation. An activity of 12.7 8M Al delayed nodulation by four days, and reduced nodule number and nodule dry weight drastically. At higher Al activities, nodulation was reduced to zero. The critical Al activities (10% reduction) were approx. 0.3 and 0.5 ,8M for nodule number and nodule dry weight. The dry weight of tops and roots showed a roughly linear decrease with increasing Al activity over the range 0 to 8 8M, both in inoculated plants supplied with 'starter nitrogen' and in plants supplied continuously with adequate mineral nitrogen. Critical A1 activities for growth were in the range of 4 to 8 PM. It was concluded that nodulation was much more sensitive to Al than was host plant growth. The results are discussed in relation to recent literature on the acid tolerance of cowpea.

Cowpea is kharif pulse grown mostly under rainfed conditions. It acts as a major protein source with 25 per cent content. Cowpea besides fixing atmospheric nitrogen continues to produce under harsh conditions such as low moisture and nutrient supply, which makes it a suitable candidate for dryland conditions. A field experiment was conducted during kharif 2019 at Research Farm, Department of Agronomy, CSK Himachal Pradesh Krishi Vishwavidyalaya, Palampur to study the effect of organic nutrient sources on yield levels, nodulation, nutrient content and uptake of cowpea. Organic nutrient sources include farmyard manure, vermicompost, vermiwash, Bijamrita, Jiwamrita and Ghanajiwamrita can be prepared using on-farm inputs at a reasonable cost. These nutrient sources supply nutrients at a steady rate and in fewer amounts than inorganic fertilizers. The prime role of such organic sources is to sustain the soil ecosystems for longer functioning. The results of the experiment revealed that T5 [Farm yard manure (10 t/ha) + Ghanajiwamrita at sowing (250 kg/ha)] was most effective and resulted in significantly higher yield level (grain yield -10.71 q/ha, straw yield - 53.14 q/ha and biological yield - 63.84 q/ha), nodulation (number of nodules - 31.7 per plant and weight of nodules - 0.47 g/plant), nutrient content (N- 3.54%, P - 0.41% and K - 1.36%) and uptake (N -141.08 kg/ha, P -19.86 kg/ha and K - 112.34 kg/ha) in cowpea

Using cultured neonatal ventricular myocytes, we investigated whether nitric oxide (NO) directly influences myocyte growth. Treatment of myocytes with phenylephrine stimulated growth, as indicated by increases in atrial natriuretic factor, brain natriuretic peptide (BNP) mRNA and BNP secretion, activator protein 1 activity (activation of early-response genes), and total cellular protein content. NO was stimulated by treatment of myocytes with interleukin-1 beta (IL-1 beta) or was generated by the NO donor nitroglycerin, and its effects on total protein content and BNP secretion were measured. Treatment of cardiocytes with 3.4 nmol/L IL-1 beta for 24 hours stimulated NO (nitrite) production by threefold, which resulted from an increase in the inducible isoform of NO synthase mRNA. Dexamethasone inhibited IL-1 beta induction of nitrite production, whereas the protein kinase C inhibitor staurosporine had no effect. IL-1 beta had no effect on either basal or phenylephrine-stimulated protein content but inhibited phenylephrine-stimulated BNP secretion. Nitroglycerin (10(-7) to 10(-3) mol/L) dose-dependently increased NO production; however, only the highest dose (10(-3) mol/L) reduced basal and phenylephrine-stimulated total protein content and BNP secretion. cGMP, a second messenger of NO, had no effect on either basal or phenylephrine-stimulated BNP secretion or total protein content. In conclusion, our data indicate that BNP mRNA is stimulated by phenylephrine as shown previously for atrial natriuretic factor. Although both BNP and total protein content are increased by phenylephrine, these effects are not inhibited by NO. However, IL-1 beta inhibits phenylephrine-stimulated BNP secretion but not total protein content, suggesting that regulation of BNP secretion can be dissociated from total protein synthesis during myocyte growth.

ABSTRACTCowpea derives most of its N nutrition from biological nitrogen fixation (BNF) via symbiotic bacteroids in root nodules. In Sub-Saharan Africa, the diversity and biogeographic distribution of bacterial microsymbionts nodulating cowpea and other indigenous legumes are not well understood, though needed for increased legume production. The aim of this study was to describe the distribution and phylogenies of rhizobia at different agroecological regions of Mozambique using PCR of the BOX element (BOX-PCR), restriction fragment length polymorphism of the internal transcribed spacer (ITS-RFLP), and sequence analysis of ribosomal, symbiotic, and housekeeping genes. A total of 122 microsymbionts isolated from two cowpea varieties (IT-1263 and IT-18) grouped into 17 clades within the BOX-PCR dendrogram. The PCR-ITS analysis yielded 17 ITS types for the bacterial isolates, while ITS-RFLP analysis placed all test isolates in six distinct clusters (I to VI). BLASTn sequence analysis of 16S rRNA and four housekeeping genes (glnII, gyrB, recA, and rpoB) showed their alignment with Rhizobium and Bradyrhizobium species. The results revealed a group of highly diverse and adapted cowpea-nodulating microsymbionts which included Bradyrhizobium pachyrhizi, Bradyrhizobium arachidis, Bradyrhizobium yuanmingense, and a novel Bradyrhizobium sp., as well as Rhizobium tropici, Rhizobium pusense, and Neorhizobium galegae in Mozambican soils. Discordances observed in single-gene phylogenies could be attributed to horizontal gene transfer and/or subsequent recombinations of the genes. Natural deletion of 60 bp of the gyrB region was observed in isolate TUTVU7; however, this deletion effect on DNA gyrase function still needs to be confirmed. The inconsistency of nifH with core gene phylogenies suggested differences in the evolutionary history of both chromosomal and symbiotic genes.IMPORTANCE A diverse group of both Bradyrhizobium and Rhizobium species responsible for cowpea nodulation in Mozambique was found in this study. Future studies could prove useful in evaluating these bacterial isolates for symbiotic efficiency and strain competitiveness in Mozambican soils.

Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules on soybean (Glycine max [L.] Merr.) in a cultivar-specific manner. This strain forms nodules on primitive soybean cultivars but fails to nodulate agronomically improved North American cultivars. Soybean cultivar specificity is regulated by the nolXWBTUV locus, which encodes part of a type III secretion system (TTSS). NolX, a soybean cultivar specificity protein, is secreted by TTSS and shows homology to HrpF of the plant pathogen Xanthomonas campestris pv. vesicatoria. It is not known whether NolX functions at the bacterium-plant interface or acts inside the host cell. Antibodies raised against S. fredii USDA257 NolX were used in immunocytochemical studies to investigate the subcellular localization of this protein. Immunostaining of paraffin-embedded sections of developing soybean and cowpea (Vigna unguiculata [L.] Walp) nodules revealed localization of NolX in the infection threads. Protein A-gold immunocytochemical localization studies utilizing affinity-purified NolX antibodies revealed specific deposition of gold particles in the fibrillar material inside infection threads. Similar immunogold localization studies failed to detect NolX in thin sections of mature soybean and cowpea nodules. The results from this study indicate that NolX is expressed in planta only during the early stages of nodule development.

VuNIP1 (NOD26-like) and VuHSP17.7 gene expression are regulated in response to heat stress in cowpea nodule

Xylem sap composition was examined in nodulated and nonnodulated cowpea (Vigna unguiculata [L.] Walp.) plants receiving a range of levels of NO(3) and in eight other ureide-forming legumes utilizing NO(3) or N(2) as sole source of nitrogen. A (15)N dilution technique determined the proportions of plant nitrogen derived from N(2) in the nodulated cowpeas fed NO(3). Xylem sap composition of NO(3)-fed, nodulated cowpea varied predictably with the relative extents to which N(2) and NO(3) were being utilized. The ratios of asparagine to glutamine (N/N) and of NO(3) to ureide (N/N) in xylem sap increased with increasing dependence on NO(3) whereas per cent of xylem nitrogen as ureide and the ratio of ureide plus glutamine to asparagine plus NO(3) (N/N) in xylem sap increased with increasing dependence on N(2) fixation. The amounts of NO(3) and ureides stored in leaflets, stems plus petioles, and roots of cowpea varied in a complex manner with level of NO(3) and the presence or absence of N(2) fixation. All species showed higher proportions of organic nitrogen as ureide and several-fold lower ratios of asparagine to glutamine in their xylem sap when relying on N(2) than when utilizing NO(3). In nodulated (minus nitrate) cowpea and mung bean (Vigna radiata [L.] Wilczek) the percentage of xylem nitrogen as ureide remained constant during growth but the ratio of asparagine to glutamine varied considerably. The biochemical significance of the above differences in xylem sap composition was discussed.

Kacang tolo (Vigna unguiculata) have potency as protein source instead soybean with appropriate processing such as fermentation of tempe. The objective of research was to study the nutrient changing especially total protein and digestible protein content during fermentation of tempefrom kacang tolo. The research method was the experimental method with complete block design. Watercontent was analyzed by thermogravimetry, total protein by micro Kjieldahl, and digestible protein by in vitro method. Data were analyzed by one way anova at significant level 5 %. If there are significant different, then is followed with Duncan Multiple Range Test (DMRT). Research result concluded that water content, total protein content, and digestible protein were increasing during tempefermentation.

Leaf extracts and mulch from 14 MPTs were used to study their effects on germination, initial growth and nodulation of cowpea. Aqueous leaf extracts of , , and full concentrations were added to cowpea seeds placed in Petri dishes. In the pot experiment, cowpea was planted in plastic pots filled with 1200 g of soil, and mulch was either surface‐applied or incorporated into the substrate at rates equivalent to 5, 10 and 20 tons · ha−1. Leaf extracts from Gliricidia sepium, Senna siamea, Milletia thoningii, Grewia pubescens and Tetrapleura tetraptera reduced cowpea germination significantly at concentrations above 25 % of the full concentration 7 days of incubation. Root length was also strongly reduced by extracts from Gmelina arborea, Senna siamea, Alchornea coordifolia, and Tetrapleura tetraptera. These inhibiting effects were also observed in the pots. Mulch incorporation significantly reduced growth and nodulation of cowpea in the pots. These reductions were more pronounced with the incorporation of mulch from fast decomposing trees such as Leucaena leococephala, Gliricidia sepium and Grewia pubescens. Although Leucaena leucocephala and Gliricidia sepium released much nitrogen, they strongly reduced cowpea growth when incorporated. This may suggest that other reasons than N immobilization caused growth reduction when the plant residues were applied to cowpea.