Soyabean Root Research Articles

EFFECT OF INOCULATING Bradyrhizobium liaoningense ON GROWTH, CHLOROPHYLL CONCENTRATION, NODULATION AND YIELD OF SOYA BEANS

Soya bean is important staple food crops in Western Kenya. Legumes play a significant role in agriculture by fixing nitrogen. Currently Kenya is experiencing low yield of soya beans. Approximately 0.8 t/ha is under production and this is due to low soil fertility. This has posed a threat to agricultural productivity. Symbiotic rhizobia inoculation increases crop yields through biological nitrogen fixation. However, the potential of Bradyrhizobium liaoningense in improving yield and productivity of soya beans has not been documented. Rhizobia bacteria are known to improve growth and yield of several other crops but the effect of inoculating soya beans with B. liaoningense on growth, chlorophyll concentration, nodulation and yields has not been determined. This study was conducted at Maseno University under greenhouse conditions. Previously purified local isolates of B. liaoningense (S3) from wild soya beans plants were used to inoculate the soya bean seeds. 4.5 litre plastic pots were filled with 7kg of top soil. Ten seeds of Soya beans coated with Bradyrhizobium liaoningense inoculants were sown in each pot except the control. The seeds were treated with the isolates inoculants as: un-inoculated (control), 1.07x107 cfu/ml, 1.19x107 cfu/ml, 1.31x107 cfu/ml and 2.67x107 cfu/ml inoculation of B. liaoningense. The experiment was laid out as Completely Randomised Design (CRD). The treatments were replicated three times. After two weeks of germination, the seedlings were thinned to three plants per pot. Watering was done daily with 200 ml of water per pot up to the end of the experiment. Data on plant height, number of leaves, leaf area, shoot and root fresh weight and dry weight, chlorophyll concentration, number of nodules and number of pods were determined. Data was subjected to Analysis of Variance (ANOVA). Treatments means were separated using Fisher’s Least Significant Difference at (P = 0.05). Soya bean growth, chlorophyll concentration, number of nodules and number of pods after inoculation treatments were significantly different among the treatments (P ≤ 0.05). Plant height, number of leaves and leaf area were highest in 2.67 x 107 cfu/ml of B. liaoningense inoculation followed by 1.31x 107 cfu/ml inoculation, 1.19x107 cfu/ml, 1.07x107 cfu/ml and lowest in un-inoculated. Inoculation at 2.67 x 107 cfu/ml significantly increased soya bean root and shoot fresh weight and dry weights. Chlorophyll concentration was found to be highest in 2.67 x 107 cfu/ml treatment and lowest in un-inoculated control treatment. Inoculation at 2.67 x 107 cfu/ml increased the number of nodules of soya beans. Number of pod per plant were highest at 2.67 x 107 cfu/ml treatment and lowest in un-inoculated (control treatment). These findings showed that B. liaoningense inoculation was effective in enhancing growth, chlorophyll synthesis, formation of root nodules and pods in soya beans. From the study it can be recommended that B. liaoningense from wild soya bean may be used as a biofertiliser to improve productivity of soya beans by smallholder farmers in Kenya.

Rapid Diagnosis of Soya Bean Root Rot Caused by Fusarium culmorum Using a Loop‐Mediated Isothermal Amplification Assay

AbstractWe report a rapid diagnosis of soya bean (Glycine max L.) root rot caused by Fusarium culmorum, using a loop‐mediated isothermal amplification (LAMP) assay. We used the CYP51C gene sequence to design LAMP assay primers specific for F. culmorum. The LAMP assay amplified the target gene efficiently in 60 min at 63°C. The sensitivity of the assay was 100 pg/μl of genomic DNA. Among the tested soya bean pathogens, a positive colour (sky blue) was only observed in the presence of F. culmorum with the addition of hydroxynaphthol blue (HNB) dye prior to amplification, whereas other species isolates showed no colour change. Suspected diseased soya bean samples collected in the field from Jiangsu, Shandong and Anhui provinces and Beijing were diagnosed successfully using the LAMP assay reported here. This study provides a new and readily available method for rapid diagnosis of soya bean root rot caused by F. culmorum.

Effects of NaCl on Root Growth and Cell Wall Composition of Two Soya bean Cultivars with Contrasting Salt Tolerance

AbstractOur objectives were to determine the influence of salinity on root cell wall composition in soya beans and the possible mechanism of salt tolerance. Two soya bean cultivars, Touzan 69 (salt sensitive) and Dare (salt tolerant), were selected as experimental material for comparison. Root growth was clearly inhibited by salinity in both cultivars, but Touzan 69 showed more severe reductions in root length than Dare. In the 0–5 mm root segment (from root tip), the total cell wall sugar content of Touzan 69 decreased considerably due to salinity as were the pectin, hemicellulose and cellulose fractions. In Dare, NaCl treatments only caused a slight decrease in the pectin fraction and no marked change in hemicellulose and cellulose fractions. Without salt treatment, the pectin fraction accounted for about 40 % and cellulose for 30 % of cell wall composition in the 0–5 mm root segment; in the 5–10 segment (from root tip), pectin and cellulose accounted for 27 % and 45 % in Touzan 69, and 34 % and 38 % in Dare. The percentage of pectin decreased and that of cellulose increased in the 5–10 mm root segment compared with the 0–5 mm segment. This indicates that pectin largely regulates cell growth, as the 0–5 mm region is considered the elongation zone of soya bean roots. Salt treatment decreased the percentage of pectin, but increased that of cellulose across root zones of the two cultivars, suggesting that salt presence may increase cell wall rigidity, and thus, inhibits root growth. Dare was able to maintain its main root cell wall substances, an apparent advantage for root cell growth that may overall improve its salt tolerance. Also, the less reduction in cell wall uronic acid was of some benefit in the positive regulation of root cell growth in Dare. The changes in cell wall composition, especially the pectin content had a close relation with the regulation of root growth. The difference in salt tolerance between the two tested cultivars can partly be explained on the basis of these changes in response to salinity. Sugar compounds in each cell wall constituent and their functions in ion transport as well as the relationship between root cell wall and soya bean salt tolerance need to be further investigated.

Plant immunity. Methods and protocols

This volume of the Methods in Molecular Biology series (no. 712) describes newly developed or optimised techniques for understanding the immune response of plants and the ways in which pathogens, including the true fungi, oomycetes, nematodes and bacteria, are able to subvert it. This is a rapidly developing field of investigation precisely because of the adoption of new advances in molecular biology, genomics and genetics. Fifty authors have contributed to this book of 21 chapters. Chapter 1 describes recent protocols for detecting the intracellular localization of NB-LRR proteins during a pathogen attack, whilst Chapter 2 describes a new method, termed ‘fragment complementation’ for studying the intra-molecular interactions of NB-LRR protein domains. Chapters 3 and 4 describe new protocols for purifying low-abundant proteins such as those involved in the immune response. Chapter 5 deals with chromatin immune-precipitation protocols for characterizing protein–DNA interactions, with special reference to WRKY transcription factors, which participate in regulation of the immune response. These techniques could also be applied to the study of intra-nucleus NB-LRR proteins. Chapter 6 describes an inducible transgenic system that can be applied, for example, for the expression of pathogen effectors in planta. Chapter 7 provides a protocol for the detection and quantification of alternatively spliced transcripts, which is important to more fully understand NB-LRR gene regulation. Advances in techniques for the identification, localization and functionality of pathogen effectors are dealt with in Chapters 8–17 and include, for example, the construction of cDNA libraries enriched for effector transcripts, transformation of oomycetes, functional characterization of multiple effectors using high-throughput assays and protocols for subcellular localization of effectors. Chapters 18–20 describe methods for the use of laser micro-dissection of different cell types, in particular nematode feeding cells from soyabean roots and Arabidopsis leaf epidermal cells infected with an obligate biotrophic powdery mildew. Both these examples of laser micro-dissection give explicit instructions on how to prepare tissues for both histology and for RNA extraction. A short introduction giving a background and the principal concepts to be addressed is given for each chapter. The methods are clearly written and detailed enough to be repeated successfully; common pitfalls are noted and instructions given on how to avoid them, for example by including a section on experimental design. Where appropriate, methods are well illustrated with diagrams, photographs of equipment and expected results. In summary, this is a timely addition to this series and will be of immediate interest and use to all those working in this field.

A Phytophthora conserved transposon‐like DNA element as a potential target for soyabean root rot disease diagnosis

A transposon‐like element, A3aPro, with multiple copies in the Phytophthora sojae genome, was identified as a suitable detection target for this devastating soyabean root rot pathogen. The PCR primers TrapF1/TrapR1 were designed based on unique sequences derived from the transposon‐like sequence. A 267‐bp DNA fragment was amplified using this primer pair, the specificity of which was evaluated against 118 isolates of P. sojae, 72 isolates of 25 other Phytophthora spp., isolates of Pythium spp. and isolates of true fungi. In tests with P. sojae genomic DNA, detection sensitivities of 10 pg and 10 fg DNA were achieved in standard PCR (TrapF1/TrapR1) and nested PCR (TrapF1/TrapR1 and TrapF2/TrapR2), respectively. Meanwhile, PCR with TrapF1/TrapR1 primers detected the pathogen at the level of a single oospore, and even one zoospore. These primers also proved to be efficient in detecting pathogens from diseased soyabean tissues, residues and soils. In addition, real‐time quantitative PCR (qPCR) assays coupled with the TrapF1/TrapR1 primers were developed to detect and quantify the pathogen. The results demonstrated that the TrapF1/TrapR1 and TrapF2/TrapR2 primer‐based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in plants and in production fields.

Plant Species, Host Age and Host Genotype Effects on Meloidogyne incognita Biocontrol by Pseudomonas fluorescens Strain CHA0 and its Genetically‐Modified Derivatives

Abstract Pseudomonas fluorescens strain CHA0 and its antibiotic overproducing derivative CHA0/pME3424 repeatedly reduced Meloidogyne incognita galling on tomato, brinjal, mungbean and soya bean roots but not in chilli. An antibiotic‐deficient derivative, CHA89, did not reduce nematode invasion in any of the plant species tested. When plant species were compared, bacterial inoculants afforded better protection to tomato, mungbean and soya bean roots against root‐knot nematodes than to brinjal and chilli. Antibiotic overproducing strain CHA0/pME3424 markedly reduced fresh shoot weights of chilli and mungbean while antibiotic‐deficient strain CHA89 enhanced fresh shoot weights of mungbean. While strains CHA0 had no significant impact on fresh root weights of any of the plant species, strain CHA0/pME3424 consistently reduced fresh root weights of brinjal and mungbean. In none of the plant species the bacterial strains had an influence on protein contents of the leaves. Regardless of the plant species, the three bacterial strains did not differ markedly in their rhizosphere colonization pattern. However, colonization was highest in brinjal rhizosphere and lowest in the mungbean rhizosphere. A slight host genotype effect on the biocontrol performance of the bacterial inoculants was also detected at cultivar level. When five soya bean cultivars were compared, biocontrol bacteria exhibited best suppression of the root‐knot nematode in cv. Ajmeri. Antibiotic overproducing strain CHA0/pME3424 substantially reduced fresh shoot weights of the soya bean cultivars Centuray 84 and NARC‐I while strain CHA89 enhanced shoot weights of the cultivars Ajmeri, William‐82 and NARC‐II. Wild type strain CHA0 had no significant impact on fresh shoot weights of any of the soya bean cultivars. Strain CHA0/pME3424 reduced fresh weights of root of Century 84, NARC‐I and NARC‐II while strain CHA89 increased root weights. Bacterial rhizosphere colonization was highest in variety NARC‐I and lowest in variety Ajmeri. Plant age had a significant impact on the biocontrol performance of bacterial inoculants against nematodes. The biocontrol effect of all bacterial strains was more prominent during early growth stage (7 days after nematode inoculation). A strong negative correlation between bacterial rhizosphere colonization and nematode invasion in soya bean roots was observed.

Organic Carbon Inputs by Soyabean in the West African Moist Savanna

ABSTRACT This study was carried out to assess the carbon returned in soyabean crop residues as a function of soyabean maturity class and P fertilizer. Three levels of P were applied to four soyabean varieties in three replications at four sites. Late varieties gave greater root + litter C input than early and medium varieties, and their responses to P were larger. P application increased root + litter C by 44–49%, and total C, comprising C in soyabean root, litter and stover, by 45–46%. Root + litter C was 60% of total C input by soyabean. While soyabean was not able to maintain soil organic C (SOC) at an annual C input decomposition rate of 70%, the application of P provided 105–107% of the C input required to maintain SOC at 5.1 g kg−1 at C input decomposition rate of 50%. For the low SOC levels of 5.1–8.7 g kg−1 at the sites, it was estimated that total C inputs of 12.2–14.2 Mg ha−1 are required to increase organic C by 1 g kg−1. Depending on decomposition rate, however, total C input by the varieties was 7–22% of total C required to increase organic C by the same amount. Although the late varieties responded positively to P, the total C required to increase organic C by 1 g kg−1 was reduced by only 4–10% with the application of P. Organic C input by soyabean in a cereal-based cropping system remains minimal despite increased soyabean duration and the application of P.

The Flux of14C-Labelled Photosynthate through Soyabean Root Nodules during N2Fixation

Experiments are described which examine the flux of photosynthetic assimilates from leaves to nodules of soyabean during N2 fixation. The first part, where the respiratory efflux of 14C02 by nodules was used as a means of assessing the import of labelled photosynthate from leaves, shows that most 14C02 loss from nodulated roots is due to the metabolic activity of nodules. Much less photosynthate was imported by nodules if the metabolic activity associated with N2 fixation was inhibited by low 02 concentration. The second part describes the chemical fate of current photosynthate as it is utilized by nodules. Labelled material was detected in nodules within c.15 min of supplying 14C02 to the leaf. This rose to a maximum at c.70 min before declining by 85% within the following 4 h. Most (80%) 14carbon imported by nodules was either lost as respiratory 14C02 or re-exported as products of N2 fixation. Ten per cent of imported carbon was found as structural material and 10% as starch. Of the 14C soluble in ethanol, most was found in the neutral fraction (80% declining to 50% as sucrose) with smaller amounts as amino acids, organic acids (each category rising from 10% to 20%) and phosphate esters ( < 5%). Comparison of the distribution of 14C among amino acids, amides and ureides in the nodules with that of xylem exudates indicated that selected compounds were exported from nodules. The 14C data indicate that c.80% of the nitrogen exported from nodules was in the form of ureides (mainly allantoic acid) and only 10-12% as amides.

Water uptake by soya-bean roots as affected by their depth and by soil water content

SUMMARYA field experiment was made to determine water uptake patterns of soya beans and water use and to compare relative effectiveness of roots at various depths in the profile.Depth of water extraction by the root systems increased with rooting depth. Water uptake rates decreased with soil water content at all soil depths and the soil water content at which roots extracted almost no water increased with depth. The maximum rate of uptake of the deep roots was greater, per unit of length, than shallow roots.

Aflatoxin B1 influence on excised soya-bean root growth, 14C-leucine uptake and incorporation.

The present work reports a portion of our continuing effort to determine the mechanism(s) whereby aflatoxins cause toxic responses in in vitro cultured plant tissues. Few investigations have dealt with the mode of action of aflatoxin B1 (AFB1) in excised plant tissues. Here is detailed AFB1 influence on growth, uptake and incorporation of 14C-leucine by excised, incubated soya-bean roots. Pure AFB1 was added to culture medium prior to autoclaving. One gram fresh weight portions of roots from three-day old soya-bean seedlings were excised and incubated for 4, 8, 12 and 24 hours. Growth was assayed by following changes in root dry weight. Aflatoxin B1 inhibited root dry weight at both 20 and 30 micrograms/ml. Uptake of 14C-leucine was checked by following its depletion from the medium. Reduced 14C-leucine uptake by roots exposed to 20 micrograms/ml AFB1 suggests that the toxin may alter the plasmalemma. A possible role for AFB1 in modification of membrane-associated amino acid transport mechanisms is discussed. Incorporation of 14C-leucine into trichloroacetic acid-precipitable cytoplasm was assayed. Inhibition of this incorporation at 20 micrograms/ml AFB1 was most apparent at 12 hours. Thus, AFB1 may also impair the ability of excised soya-bean roots to carry out protein synthesis.

A comparison of the effect of straw incorporation and CO2 enrichment on the growth, nitrogen fixation and yield of soya beans

SUMMARYIn a glasshouse pot culture experiment, the effect of adding straw at 3 and 6 t/ha with and without CO2 enrichment treatments at 1000 mg/1 from flowering to the pod-filling stage in open top chambers was evaluated on the growth and yield of soya beans in relation to nitrogen fixation. N2-ase activity of the soya-bean root nodules as determined by the acetylene reduction technique indicated that (1) straw on average gave significantly 34 and 43% higher N2-ase activity at 3 and 6 t/ha respectively than the controls; (2) CO2 treatments on average increased the activity by 34% compared with the no CO2 treatments; and (3) the mean N2-ase activity nearly doubled from 9·7 μg/h/plant in the control to 18·7 and 19·7 μg/h/plant with straw incorporation in conjunction with CO2 enrichment. High correlations were observed between weight of nodules and dry weight of leaves, between dry weight of nodules and grain yield and between dry weight of leaves and grain yield. Incorporation of straw was found to be beneficial in increasing CO2 content of soil air and in improving the growth and development of the plants. This study lends support to a hypothesis that straw can be considered to provide a partial substitute for the expensive CO2 enrichment treatment for improving N2(C2H2) fixation capacity and thereby the general growth and yield of crops.

The purification and properties of the glutamine synthetase from the cytosol of Soya-bean root nodules.

The major portion of glutamine synthetase activity in root nodules of soya-bean plants is associated with the cytosol rather than with Rhizobium japonicum bacteroids. Glutamine synthetase accounts for about 2% of the total soluble protein in nodule cytosol. Glutamine synthetase from nodule cytosol has been purified by a procedure involving fractionation with protamine sulphate, ammonium sulphate and polypropylene glycol, chromatography on DEAE-Bio-Gel A and Bio-Gel A-5m and affinity chromatography on glutamate-agarose columns. The purified preparation appeared to be homogeneous in the analytical ultracentrifuge. From sedimentation-equilibrium experiments a mol. wt. of about 376000 was determined for the native enzyme and 47300 for the enzyme in guanidinium chloride. From these data and measurements of electron micrographs, we have concluded that glutamine synthetase from nodule cytosol consists of eight subunits arranged in two sets of planar tetramers which form a cubical configuration with dimensions of about 10 nm (100 A) across each side. Glutamine synthetase from nodule cytosol has a higher glycine and proline content and a lower content of phenylalanine than the glutamine synthetase that has been prepared from pea seed. The cytosol enzyme contains four half-cystine molecules per subunit, which is in contrast with two reported for the enzyme from pea seed. Enzyme activity is striking influenced by the relative proportion of Mg2+ and Mn2+ in the assay medium. Activity is inhibited by feedback inhibitors and is influenced by energy charge.

Characterization of the extracellular haemoglobin of Haemopsis sanguisuga (L.)

The haemoglobin from the blood of the horseleech, Haemopsis sanguisuga (L.), had a sedimentation coefficient, SO20, w, of 59.11 +/- 0.55 S, and a molecular weight as determined by sedimentation equilibrium of 3.71 X 10(6)+/-9904 X 10(6). In the electron microscope the molecule appeared to be made up of two hexagonal plates, as is found with other worm haemoglobins, with dimensions 24.4+/-2.0 nm (across the hexagon) and 15.2+/-1.4 nm (height). The amino acid composition and spectrum were closely similar to those of the haemoglobins of other annelids (e.g. Lumbricus). The alpha-helical content, calculated from circular-dichroism measurements in the far-u.v. region, was 56-63%. The haem content was 2.49%, corresponding to a minimum molecular weight per haem group of 24 800, but detergent-gel electrophoresis indicated the presence of polypeptide chains of mol.wts. 12 600, 14 800, 15 500 and 25 100. The pH-induced dissociation of the native molecule yielded compotosol of Soya-bean root nodules.