Characterization ofAzospirillum and related diazotrophs associated with roots of plants growing in saline soils

Diazotrophs, especially of genusAzospirillum were readily isolated from roots of many plants using semi-solid nitrogen free malate medium (NFM). These isolates formed fine, white sub-surface pellicle in nitrogen-free malate medium within 24h, which gradually moved to the surface, and exhibited high acetylene reduction rates. Using selected cultural and biochemical tests, most of the isolates were identified asAzospirillum brasilense. Four isolates from Kallar grass root surface and one isolate fromAtriplex root interior formed phenotypically a homogenous group. It shared many characteristics with the species of genusAzospirillum except shape. All the biochemical tests performed, categorized them withA. brasilense. However, the shape and the protein profile on SDS polyacrylamide gel electrophoretograms suggested that the group of these five isolates is clearly distinct and differs widely from all the type strains, belonging to various genera.

---

Résumé It est apparu facile d'isoler des diazotrophes, principalement du genreAzospirllium à partir de racines de nombreuses plantes et utilisant un milieu malate semisolide exempt d'azote (NFM). Ces isolats forment une pellicule fine et blanche sous la surface dans le milieu malate exempt d'azote, endéans les24h. Ces pellicules ae déplacent progressivement vers la surface et exhibent des vitesses élevées de production d'acétylène. L'emploi de tests de culture sélective ou biochimique, a permis d'identifier la plupart des isolats commeAzospirillum brasilense. Quatre isolats à partir de la surface des racines d'herbe Kallar et un isolat à partir de l'intérieur d'une racine d'Atriplex forment an groupe homogène sur le plan phénotypique. Celui-ci partage de nombreuses caractéristiques avec les espèces du genreAzospirillum excepté la forme. Tous les tests biochimiques exécutés les rangent avecA. brasilense. Toutefois, leur forme ainsi que le profil protéique sur un électrophérogramme sur gel de polyacrylamide-SDS suggèrent que le groupe de cinq isolats est nettement distinct et diffère largement de toutes les souches types, appartenant à de nombreux genres.

     

Use ofAzospirillum brasilense as biofertilizer in intensive wheat cropping

Summary Three field experiments were conducted on ten cultivars of winterwheat and four cultivars of springwheat to estimate the growth promoting effect ofAzospirillum brasilense under varying nitrogen doses. Independent of cultivar selection or nitrogen dose a highly significant yield increase could be observed in winterwheat: strains S631 and SpBr14 increased the average grain yield with 9.14% and 14.82% respectively. When the yield components were studied a coinciding increase in ear density could be demonstrated of resp. 10.57% and 13.55%. Less significant results were obtained with springwheat although in one experiment strain SpBr14 significantly increased grain yield. As with winterwheat tillering of the plant was markedly affected by inoculation with both strains. In a companion greenhouse experiment it was found that inoculation with Azospirillum can cause a decrease in the root mass of wheatplants except when strain SpBr14 is used. Therefore it is suggested that the presence of a higher tillering together with an undisturbed nutrient uptake capacity can result in yield increases after inoculation withAzospirillum brasilense.     

Selection and evaluation ofAzospirillum brasilense strains growing at a sub-optimum temperature in rhizocoenosis with wheat

FourteenAzospirillum brasilense strains growing at a sub-optimum temperature were selected based on their ability to grow and carry out plant growth promoting activities at 22°C. The strains were tested for their response to inoculation in wheat (two popular cultivars, HD2285 and WH547, under sterile conditions) crop using sterile and nonsterile rooting medium. Significant increase in plant growth parameters was observed: the overall response to inoculation was better in cultivar HD 2285. Based on their performance under sterile conditions, 4 strains were selected and compared under nonsterile conditions with strain sensitive to a sub-optimum temperature in pots using wheat variety HD2285. The strains capable of growing at the sub-optimum temperature can colonize the wheat endorhizosphere efficiently and improve the plant growth and yield as compared to sensitive strain; a 25–27% increase in grain yield was found on inoculating two selected strains compared to NO ₃ ^- control.     

Relationship between root colonization and initial adsorption ofAzospirillum to plant roots

Four strains ofAzospirillum were ranked according to numbers of cells adsorbed on the roots of seedlings in liquid medium, and the rankings were evaluated for their usefulness in predicting success of colonization of the roots of pot-grown plants. Different rankings were observed on different parts of the roots and on different host plants. Rhizosphere colonization results for rice were similar to those for clover and showed little difference between bacterial strains. The population densities were approximately equal to those of the most dense strains in the wheat rhizosphere, whereas the highest concentrations in the root interior of clover and rice were only about one-tenth of those in wheat. Rankings of initial adsorptive ability on various parts of the roots showed potential for predicting the best strains for colonizing the root interiors of wheat and clover. On wheat, the two strains (Cd and SpBr14) which showed best initial adsorption to the root cap were best at colonizing the endorhizosphere of pot-grown plants. For rice, strains Cd and SpBr14 gave lowest and highest values, respectively, both for adsorption to the terminal 2 cm of roots and for subsequent colonization of the root interior. Data on initial adsorption were of no value in predicting the relative success of strains in colonizing the root surface of any host plants or the interior of clover roots.     

Growth distribution and surface pH patterns along maize roots

The distribution of elongation and surface pH patterns along the primary roots of maize (cv. LG 11), maintained vertically in humid air (darkness, 22°C), have been analysed quantitatively. A new technique employing Sephadex G 25 beads containing a pH indicator dye (bromocresol purple), was used for measuring both the growth gradient of the roots (Sephadex beads as markers) and at the same time, the surface pH changes (referring to a standard scale). The optimal axial growth was located between 2 and 4 mm from the tip. This coincides with the optimal decrease in surface pH.     

Intracellular distribution of enzymes associated with nitrogen assimilation in roots

The cellular distribution of enzymes involved in nitrogen assimilation: nitrate reductase (EC 1.6.6.2), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) has been studied in the roots of five plants: maize (Zea mays L. hybrid W 64A x W 182E), rice (Oryza sativa L. cv. Delta), bean (Phaseolus vulgaris L. cv. Contender), pea (Pisum sativum L. cv. Demi-nain), and barley (Hordeum vulgare L.). Initially, cell organelles were separated from soluble proteins by differential centrifugation. Cell organelles were also subjected to sucrose density gradients. The results obtained by these two methods indicate that nitrite reductase and glutamate synthase are localized in plastids, nitrate reductase and glutamine synthetase are present in the cytosol, and glutamate dehydrogenase is a mitochondrial enzyme.     

Composition ofAzospirillum species associated with wetland rice plant grown in different soils

Total aerobic heterotrophs and N₂-fixing putative azospirilla associated with rice plant grown in long-term unfertilized wetland rice field at 5 sites in the Philippines were enumerated. Several azospirilla isolates were identified based on cellular morphology, biochemical tests and reaction to immunodiffusion. Azospirilla constitute about one percent of the total aerobic heterotrophs. Most (85%) of the Azospirillum isolates belong toA. lipoferum indicating its preferential colonization to the rice plant.     

Aerenchyma formation and associated oxygen movement in seminal and nodal roots of wheat

Abstract The present paper describes the effects of growth of roots of wheat (Triticum aestivum cv. Gamenya) in hypoxic nutrient solutions on acrenchyma formation and O₂ movement from shoots to roots. Two types of roots were investigated: (1) seminal roots of 4–7-d-old seedlings, and (2) seminal and nodal roots of 10–28-d-old plants. Gas-filled porosity of seminal and nodal roots increased from 3 to 12% and from 5–7 to 11–15%, respectively, when the roots emerged in stagnant or N₂-flushed solutions (0.003 mol m ^?3 O₂) compared with growth in continuously acrated solutions (0.26 mol m ^?3 O₂). However, neither root type increased in porosity when they were longer than 100–200 mm at the start of the exposure to these stagnant or N₂-flushed treatments. A vernier microscope and cylindrical platinum-electrode were used to examine the relationship between root extension and transport of O₂ from shoots to roots via the gas spaces. Measurements were made when the roots were in an anoxic medium and were dependent solely on O₂ supplied from the shoots. For seminal roots of 5–7-d-old seedlings raised in stagnant solutions (90–100 mm), internal O₂ transport was sufficient to support a rate of root elongation in the O₂-free medium of between 0.03 and 0.17 mm h^?1. When the O₂ pressure around the shoots was increased from 20 to 100 kPa O₂, the O₂ concentrations at the walls of the expanding zone (2–7 mm from the tip) of these roots increased from 0.006 mol m^?3 to between 0.04 and 0.26 mol m^?3, and the rate of root extension increased five-fold. Oxygen transport to roots grown continuously in acrated solutions was considerably less than for roots raised in stagnant solutions; this difference was greater for seminal than for nodal roots. When the acrated seminal roots were longer than 100 mm and transferred to an O₂-free root medium, O₂ concentration became zero at the root tip causing elongation to cease. After 24 h of anoxia, none of these roots were able to resume elongation following a return to acrated solutions.     

冬小麦根系各种参数垂直分布实验研究

本文根据田间实测资料研究了冬小麦根系各种参数(长度、重量、活性表面和根径)的垂直分布。结果表明,在所观测地区的土壤条件下,冬小麦根系的长度、重量、表面积和体积随深度呈指数分布,而累积根系的长度、重量和表面积随深度呈双曲线型分布。根据作者的观测资料和国内外其它观测资料分析研究表明,对于不同土壤,根区各层土壤中累计根重及根长的百分比随相对深度的变化都符合下列双曲线函数形式:这一研究结果可为根系生态研究和作物对水分吸收的模拟工作提供参考。     

Salt distribution around roots of wheat under different transpiration rates

Summary Magnitude of Na and Cl accumulation around wheat roots was studied under different transpiration conditions in a loamy sand soil salinized with sodium chloride to an electrical conductivity of 4.1 mmho/cm in the saturation extract. A significant correlation was observed between rate of water loss per unit root length and Na and Cl content of the soil closely adhering to the roots. Under high transpiration condition, maximum ion accumulation occurred in the apparent free space of roots followed by the soil closely adhering to the roots. Results indicate that salt concentration at the root surface is markedly altered under the influence of transpiration rate such that under high transpiration conditions, the plant roots may be exposed to a much higher salt concentration than that anticipated from an analysis of the bulk soil samples.Department of Soil and Water EngineeringDepartment of Soils     

Use of fluorochromes for direct observation of microorganisms associated with wheat roots.

The use of several fluorochromes and staining methods was tried out for observation of microorganisms on the surface of wheat roots sampled from soil. Good contrast between fluorescence of microorganisms and fluorescence of root and soil structures was obtained after successive staining of roots with coriphosphine (1:2000, 15 min), congo red (1:10 000, 10 min), and acridine-orange (1:10 000, 2 min).     

Nitrogen fixation (acetylene reduction) associated with roots of winter wheat and sorghum in Nebraska

Root segments and root-soil cores (6.5-cm diameter) from fields and nurseries of winter wheat and sorghum were tested for N2 fixation by using the acetylene reduction assay. Wheat samples (approximately 1,200) from 109 sites generally had low or no activity (0 to 3.1 nmol of C2H4 produced per h per g [dry weight] of root segments), even after 24 h of incubation. However, a commercial field of Scout 66, located in western Nebraska, exhibited appreciable activity (290 nmol of C2H4 produced per h per g [dry weight] of root segments). Of 400 sorghum lines and crosses, grain sorghums (i.e., CK-60A, Wheatland A, B517, and NP-16) generally exhibited higher nitrogenase activity than forage sorghums or winter wheats. CK-60A, a male sterile grain sorghum, was sampled at four locations and had the most consistent activity of 24 to 1,100 nmol of C2H4 produced per h per core. The maximum rate extrapolated to 2.5 g of N per hectare per day. Numerous N2-fixing bacterial isolates were obtained from wheat and sorghum roots that exhibited high nitrogenase activity. Most isolates were members of the Enterobacteriacae, i.e., Klebsiella pneumoniae, Enterobacter cloacae, and Erwinia herbicola.     

Nitrogen fixation (acetylene reduction) associated with roots of winter wheat and sorghum in Nebraska.

Root segments and root-soil cores (6.5-cm diameter) from fields and nurseries of winter wheat and sorghum were tested for N2 fixation by using the acetylene reduction assay. Wheat samples (approximately 1,200) from 109 sites generally had low or no activity (0 to 3.1 nmol of C2H4 produced per h per g [dry weight] of root segments), even after 24 h of incubation. However, a commercial field of Scout 66, located in western Nebraska, exhibited appreciable activity (290 nmol of C2H4 produced per h per g [dry weight] of root segments). Of 400 sorghum lines and crosses, grain sorghums (i.e., CK-60A, Wheatland A, B517, and NP-16) generally exhibited higher nitrogenase activity than forage sorghums or winter wheats. CK-60A, a male sterile grain sorghum, was sampled at four locations and had the most consistent activity of 24 to 1,100 nmol of C2H4 produced per h per core. The maximum rate extrapolated to 2.5 g of N per hectare per day. Numerous N2-fixing bacterial isolates were obtained from wheat and sorghum roots that exhibited high nitrogenase activity. Most isolates were members of the Enterobacteriacae, i.e., Klebsiella pneumoniae, Enterobacter cloacae, and Erwinia herbicola.     

不同抗旱性冬小麦根系时空分布与产量的关系

为明确不同抗旱性冬小麦品种(Triticum aestivum L.)根系时空分布及其与产量的关系,以抗旱性品种长武134、长旱58和干旱敏感性品种小偃22、西农979为材料,采用根箱试验研究干旱胁迫和充分供水条件下4个品种在拔节期、开花期和成熟期根系总生物量、总根长密度、根系在表层(0—20 cm)和深层(20 cm以下)土壤中的垂直分布、动态变化及其对产量的影响。结果表明,干旱胁迫下抗旱性品种产量显著高于干旱敏感性品种,其中长旱58产量最高,西农979最低;充分供水条件下,西农979产量最高,长武134最低,长旱58与小偃22之间没有差异。相关分析表明,产量与各生育时期根系性状均有显著关系。多元逐步回归分析的结果显示,干旱胁迫和充分供水条件下,拔节期深层根生物量对产量有正效应,而成熟期总根长密度对产量表现为负效应。通径分析表明,干旱胁迫下,根系性状对产量的直接贡献大小为开花期总根长密度(|0.54|)拔节期深层根生物量(|0.36|)成熟期总根长密度(|-0.31|);充分供水时,成熟期总根长密度(|-1.56|)拔节期深层根生物量(|0.83|)。研究表明,减少成熟期总根长密度,增加拔节期深层根生物量对抗旱性及干旱敏感性冬小麦品种产量均有显著的正效应,增加开花期根长密度有利于提高抗旱性冬小麦产量。     

Nodulation in clover roots: Correlation with vacuolar pH

Abstract Nodulation patterns of three clover cultivars inoculated with three strains of Rhizobium leguminosarum bv. trifolii were established. Profiles of the frequency of nodule formation in the root-hair zone and no-root-hair zone of Trifolium subterraneum (cvs. Clare and Mount Barker) indicated a more frequent development of nodules in the no-root-hair zone. A different nodulation pattern was observed in Trifolium fragiferum cv. Palestine in which the number of nodules per centimeter did not change along the root.
In order to assess the mechanisms responsible for the different capacity of root tissues to initiate meristematic activity for nodule formation, intracellular pH of the different root zones was measured using ³¹P-NMR spectroscopy. In the two clovers with distinct zones of nodule formation, the vacuolar pH of the no-root-hair zone was 0.3 units higher than that of the root-hair zone, with a lower pH being associated with a lower nodulation frequency.     

pH control in nitrate uptake studies with excised roots

Summary In the investigation of NO ₃ ^– uptake by excised roots, pH of salt solutions can be effectively controlled by periodic additions of a cation exchange resin, Amberlite IR-120, in the H⁺ form. This method of pH control provides for the addition of H⁺ to systems without an accompanying anion which might influence the absorption of NO ₃ ^– . The resin appeared to provide a favorable buffering capacity during an extended period and the method was especially useful for NO ₃ ^– uptake studies conducted at high root-to-solution ratios.Contribution from the Plant Nutrition Laboratory, Department of Botany, The University of Michigan, paper number 39. This work was supported by research grant GM-07339-02 from the National Institutes of Health, U. S. Public Health Service.     

Ultrastructural localization and identification ofAzospirillum brasilense Cd on and within wheat root by immuno-gold labeling

Azospirillum brasilense Cd localization in wheat roots was studied by light microscopy, by scanning, and by transmission electron microscopy.A. brasilense Cd cells were specifically identified immunocytochemically around and within root tissues.A. brasilense Cd cells found both outside and inside inoculated roots were intensively labeled with colloidal gold. In non-axenic cultures other bacterial strains or plant tissue were not labeled, thereby providing a non-interfering background. The roots of axenic grown wheat plants were colonized both externally and internally byA. brasilense Cd after inoculation, whereas non-axenic cultures were colonized by other bacterial strains as well.A. brasilense Cd cells were located on the root surface along the following zones: the root tip, the elongation, and the root-hair zone. However, bacteria were located within the cortex only in the latter two zones. In a number of observations, an electron dense material mediated the binding of bacterial cells to outer surfaces of epidermal cells, or between adjacent bacterial cells.A. brasilense Cd were found in root cortical intercellular spaces, but were not detected in either the endodermal layer or in the vascular system. This study proposes that in addition to root surface colonization,A. brasilense Cd forms intercellular associations within wheat roots.     

A mycorrhiza-responsive protein in wheat roots

A small protein, designated Myk15, was found to be strongly induced in wheat ( Triticum aestivum) roots colonized by the arbuscular mycorrhizal fungus Glomus intraradices. This protein, which is most abundant in root fractions characterized by strong mycorrhizal colonization, has been characterized using two-dimensional polyacrylamide gel electrophoresis and microsequencing. It has an apparent molecular mass of 15 kDa and an isoelectric point of 4.5. The N-terminal sequence has high similarity to a peptide sequence deduced from an expressed sequence tag (EST) clone derived from Medicago truncatula roots colonized by G. intraradices. This EST clone is predicted to code for a protein with a similar size and isoelectric point as Myk15. The N-terminus of the deduced M. truncatula protein contains a highly hydrophobic stretch of 24 amino acid residues preceding the region with high similarity to the Myk15 N-terminus. This hydrophobic stretch is predicted to form a transmembrane alpha-helix and may correspond to a cleavable targeting domain.     

Soil pH changes associated with lupin and wheat plant materials incorporated in a red–brown earth soil

Both alkalization and acidification of soil occurred when shoot and root materials from lupin and wheat were incubated in a red–brown earth soil, but with three different starting pH values, during a 70-day period. The response of soil pH change to the addition of organic matter depended on the type of plant materials and starting pH. The net effect of addition of lupin and wheat shoots to acid soils (pH<5) caused soil pH to increase, the addition of lupin roots to soils caused soil pH to decrease slightly, whilst with a higher pH soil (6.5) the wheat straw and lupin shoots raised pH and pH was unchanged for soil with addition of lupin roots. The ash alkalinity of plant materials and the mineralization of organic N are major reasons for the soil pH increase, and the nitrification of mineralized N results in soil pH to decrease. Whilst the data given here would suggest the likelihood of soil acidification occurring, particularly on poorly buffered soil given the inevitable influence of legume root materials, the overall directions of soil pH change in a cropping system that is legume-based will be very much influenced by the balance of many factors associated with the soil and plant system.