Endophytic establishment of Azorhizobium caulinodans through auxin-induced root tumors of rice (Oryza sativa L.)

Rice seedlings developed nodule-like tumors (para-nodules) along primary and secondary roots when treated with the auxin 2,4-dichlor-phenoxy-acetic acid (2,4-D). Histologically, these tumors appeared as cancerous out-grown lateral-root primordes and were thus comparable with stem nodules of the legume Sesbania rostrata. Azorhizobium caulinodans (a diazotroph known as a specific endophyte of Sesbania rostrata) was introduced and became established inside rice para-nodules and in root tissues around tumor bases. The infection with A. caulinodans followed a typical crack-entry invasion at places where paranodule tumors had emerged through the root cortex and epidermis. The bacteria settled with high cell densities in intercellular spaces of the induced tumors and between root cortical cells. Infection of plant cells took place both in the epidermis and in cortical tissue. Intracellularly established A. caulinodans was found inside the cytoplasm, surrounded by membrane-like structures. N₂ fixation by tumor-inhabiting Azorhizobium sp. was increased at low O₂ tensions (1.5–3 kPa) compared with an untreated control. Only a little activity remained at O₂ tensions of 5 kPa and above. The present results confirm that root-tumor induction offers a suitable method of establishing diazotrophs endophytically in the roots of gramineous crops.     

Nitrogenase divarication with histological attributes distinctive for glycosylated aerial stem nodules and nitrosylated root nodules of sesbania

Exceptional symbiotic nitrogen fixation with Sesbania has provided high soil fertility for many past centuries of paddy rice production. Unique stem nodulation results in high nitrogenase activity levels of S. rostrata, Brem, during rapid growth in continuously flooded rice fields that greatly disfavor legume root nodulation and this functional development. The objective of this study was to determine plant nutrient interactions that influence contrasting root and aerial stem nodule histology governing effective nitrogenase activity levels and nitrogen fixation. Top growth, nodulation, and nitrogenase activity levels were significantly increased with increased available soil P. Response to K levels and Ca additions resulted only when soil P was adequate in all treatment combinations. However, there was no significant correlation between fresh nodule weight, nitrogenase activity, and nodules plant^‐1 for both root nodules and aerial stem nodules. Nodule histology was highly contrastive with nodule type and Rhizobium morphology, cytosol composition, and governing enzyme activity levels. Distinctive nonpleomorphic cocci bacteroids of functional aerial stem nodules have tentative designation as Azorhizobium caulinodans gen. nov. sp. nov.     

Genetic diversity and spatial distribution of rhizobial bacteria nodulating on black locust (Robinia pseudoacacia) rhizospheres

Black locust (Robinia pseudoacacia L.) is an economically and ecologically important tree in Japan. The species is widely used for afforestation because of its symbiotic relationship with nitrogen-fixing rhizobia. In this study, genetic diversity of rhizobia isolated from black locust nodules and spatial distribution of their genotypes were examined. From a coastal forest, six black locust saplings including the whole root systems were collected and positions of nodules on the roots were recorded. Bacteria were isolated from each nodule and then genotyped by Polymerase Chain Reaction (PCR)-Restriction Fragment Length Polymorphism (RFLP) and sequencing analysis of the 16S rRNA gene. Dendrogram analysis based on PCR-RFLP and sequencing analysis indicated that Mesorhizobium species dominantly colonized black locust roots in this forest, occupying at least 77% of living nodules. Positions and PCR-RFLP genotypes of the nodules revealed that rhizobia isolated from neighbouring nodules tended to have the same genotype in some cases. Statistical analysis has supported this fact. The clustering distribution of nodules in the same RFLP groups should be considered to contribute to the large genetic diversity of rhizobia.     

Effects of inoculation with Glomus mosseae, Azorhizobium caulinodans and rock phosphate on the growth of and nitrogen and phosphorus accumulation in Sesbania rostrata

A study was carried out to evaluate the influence of single or dual inoculation with Glomus mosseae and Azorhizobium caulinodans with and without rock phosphate on the growth of and N, P and K accumulation in Sesbania rostrata grown in sterilized soil. Height of plants inoculated with G. mosseae in combination with A. caulinadans and/or rock phosphate was significantly higher than that of uninoculated plants or plants inoculated with A. caulinodans alone before but not after 28 days. However, the oven-dry weight of shoots and roots from all treatments did not vary significantly. Generally, concentration and uptake of N and P but not K in shoots and roots of plants were improved by dual inoculation with G. mosseae and A. caulinodans plus or minus P. Mycorrhizal infection of roots was enhanced significantly by A. caulinodans. Received: 18 March 1996     

A significant proportion of indigenous rhizobia from India associated with soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) distinctly belong to <Emphasis Type="Italic">Bradyrhizobium</Emphasis> and <Emphasis Type="Italic">Ensifer</Emphasis> genera

The diversity among 269 rhizobia isolated from naturally occurring root nodules of soybean collected from two different agro-ecological regions of India, based on RFLP and sequences of the intergenic spacer (IGS) between the 16S and 23S rRNA genes, growth rate, and indole acetic acid production, revealed their significant, site-dependent genomic diversity. Among these bacteria, nine IGS genotypes were identified with two endonucleases. They were distributed into five divergent lineages by sequence analysis of each IGS representative strain, i.e., (1) comprising IGS genotypes I, II, III, and reference Bradyrhizobium yuanmingense; (2) with genotype IV and strains of unclassified bradyrhizobia genomic species; (3) including genotypes V, VI, and Bradyrhizobium liaoningense; (4) with IGS genotype VII and Bradyrhizobium elkanii strains; and (5) comprising IGS genotypes VIII, IX, and different Ensifer genus bacteria. Host-specificity test revealed that all rhizobia-nodulated soybean and cowpea and only part of them formed nodules on Arachis hypogeae and Cajanus cajan. The great diversity of soybean nodulators observed in this study emphasises that Indian soil is an important reservoir of nitrogen-fixing rhizobia.     

Dinitrogen fixation activity of Azorhizobium caulinodans in the rice (Oryza sativa L.) rhizosphere assessed by nitrogen balance and nitrogen-15 dilution methods

 Azorhizobium caulinodans was directly inoculated onto rice plants in three short-term pot trials. Addition of increasing amounts of sucrose (23, 46, 92 kg ha^–1) did not influence the N economy of the A. caulinodans-rice association during the early vegetative growth stage. A. caulinodans inoculation alone and in combination with the highest amount of sucrose had a significantly positive effect on the N balance, with small but significant N gains in the system. Application of 60 kg urea-N ha^–1 had a negative impact on the N economy of the inoculated treatments. N losses increased and the amount of atmospheric N₂ fixed and incorporated decreased significantly as compared to the amounts under the 20 kg urea-N ha^–1 regime. However, N losses were low – a maximum of 8% – at the early vegetative growth stage under the conditions of the experiments. C limitation does not seem to be a limiting factor for the incorporation of fixed N₂ in this bacteria-plant association. Biological N₂ fixation caused by A. caulinodans inoculation was responsible for 14% of the plant N at the vegetative growth stage and under low N conditions. Received: 30 January 2000     

Establishment of Azorhizobium caulinodans in the rhizosphere of wetland rice (Oryza sativa L.)

 Azorhizobium caulinodans strongly colonized the rhizosphere of rice plants after incorporation of Sesbania rostrata in a field trial throughout the growing season and during the fallow period until 19 weeks after incorporation of S. rostrata. A. caulinodans became well established in the rhizosphere (7.17 log cfu g^–1 dry rice root) and colonized subsequent S. rostrata test plants. Three traditional and three improved high-yielding rice varieties were inoculated with A. caulinodans under gnotobiotic conditions. In none of the combinations did acetylene reduction activity significantly increase. Ethylene production on colonized rice roots only started after the growth medium had been supplemented with an extra C source (0.1 to 0.25% Na-lactate). This indicates that the bacterial nitrogenase activity is limited by energy supply. Four possible inoculant-carriers (peat, coir dust, bagasse, rice straw) were compared for long-term survival of the bacterial strain. Independent of the storage temperature (26  °C or 4  °C), the survival of A. caulinodans in peat and coir dust was very high during a 12-month period (>8 log cfu g^–1 dry carrier), whereas the bagasse and rice straw carriers showed a serious decline from 3 months onwards. Received: 6 April 1999     

Different strategies for salt tolerance in determined and indeterminate nodules of Lotus japonicus and Medicago truncatula

In this study, we examine how indeterminate and determined nitrogen-fixing root nodules of model legumes Lotus japonicus and Medicago truncatula adapt their non-structural carbohydrate pool during salt stress, with particular emphasis on trehalose, a compatible solute abundant in nodules of some legumes. M. truncatula and L. japonicus plants were inoculated with Sinorhizobium meliloti and Mesorhizobium loti, respectively, and the effect of 50 mM sodium chloride (NaCl) added to the nutrient solution was studied in a time-course experiment. Sucrose and pinitol were the predominant carbohydrates in nodules of both legumes, contributing to osmoprotection in nodules of L. japonicus under salt stress. Trehalose concentration increased under salt stress in L. japonicus nodules; however, compared with sucrose and pinitol, its concentration was too low to contribute efficiently to osmoregulation. By contrast, proline showed a dramatic increase in nodules and leaves of M. truncatula under salt stress, contributing to osmotic adjustment in this species. Results found in this study showed different mechanisms for salt tolerance in determined and indeterminate nodules of model legumes L. japonicus and M. truncatula that might be a general feature in the mentioned different types of nodules.     

Nodule-specific expression of Rhizobium meliloti symbiotic promoters P1 and P2 in chickpea-Rhizobium sp. symbiosis

Developmentally specific expression of Rhizobium spp. genes involved in symbiotic N₂ fixation is known to operate through cascade regulation of various nif and fix operons. Fusion constructs of lacZ under symbiotic promoters P1 (for nifHDK operon) and P2 (for fixABCX operon) of Rhizobium meliloti were mobilized into Rhizobium spp. (Cicer) strains Rcd301 and RCR13. The assays for -galactosidase activity to monitor the expression of lacZ under these promoters was performed in host backgrounds of Escherichia coli, R. meliloti, and Rhizobium spp. (Cicer). The enzyme assays indicated significant levels of expression from P1 and P2 promoters in chickpea rhizobia, specifically in symbiotic cells from nodules. However, as in R. meliloti, these promoters did not induce strong expression in free-living cells of Rhizobium spp. (Cicer). This indicates functional homology of R. meliloti promoters in rhizobium spp. (Cicer). Functional cross-reactivity of trans regulatory factors like NtrA, NtrC, and NifA between these rhizobia seems evident from the nodule-specific expression of P1 and P2 cis elements.     

Maize growth and yield responses to seed‐inoculated N2‐fixing bacteria under Dryland production conditions

Maize (Zea Mays L.) seeds were inoculated with the N₂‐fixing bacteria, Azospirillum brasilense and Azorhizobium caulinodans. Shoot growth, shoot nitrogen (N) concentration, and grain yield was determined under dryland production conditions in a silt loam. Fertilizer N was applied according to soil test recommendations at either 0, 50, 75, or 100% of the recommended N requirements for a 7,500 kg ha^‐1 yield goal. Both A. brasilense and A. caulinodans increased shoot dry matter production, shoot N concentration, and grain yield somewhat at the lower N recommended rates. There was no agronomic benefit with either A. brasilense and A. caulinodans inoculations under dryland conditions for high N fertility soils under dryland production conditions in a subhumid or semi‐arid moisture regime.     

Sesbania rostrata as a green manure for lowland rice: Growth,N2 fixation,Azorhizobium sp. inoculation,and effects on succeeding crop yields and nitrogen balance

Summary Root and stem nodulation, nitrogen fixation (acetylene-reducing activity), growth and N accumulation bySesbania rostrata as affected by season and inoculation were studied in a pot experiment. The effects ofS. rostrata as a green manure on succeeding wet-season and dry-season rice yields and total N balance were also studied.S. rostrata grown during the wet season showed better growth, nodulation, and greater acetylene-reducing activity than that grown during the dry season. Inoculation withAzorhizobium caulinodans ORS 571 StrSpc® (resistant to streptomycin and spectinomycin) on the stem alone or on both root and stem significantly increased N₂ fixation by the plants. Soil and seed inoculation yielded active root nodules under flooded conditions. Plants that were not inoculated on the stem did not develop stem nodules. The nitrogenase activity of the root nodules was greater than that of the stem nodules in about 50-day-oldS. rostrata. S. rostrata incorporation, irrespective of inoculation, significantly increased the grain yield and N uptake of the succeeding wet season and dry season rice crops. The inoculated treatments produced a significantly greater N gain (873 mg N pot^–1) than the noinoculation (712 mg N pot^–1) treatment. About 80% of the N gained was transferred to the succeeding rice crops and about 20% remained in the soil. The soil N in the flooded fallow-rice treatment significantly declined (–140 mg N pot^–1) but significantly increased in bothS. rostrata-rice treatments (159 and 151 mg N pot^–1 in uninoculated and inoculated treatments respectively). The N-balance data gave extrapolated values of N₂ fixed per hectare at about 303 kg N ha^–1 per two crops forS. rostrata (uninoculated)-rice and 383 forS. rostrata (inoculated)-rice.     

The flavonoid naringenin enhances intercellular colonization of rice roots by<Emphasis Type="Italic"> Azorhizobium caulinodans</Emphasis>

The intercellular colonization of rice roots by Azorhizobium caulinodans and other diazotrophic bacteria has been studied using strains marked with the lacZ reporter gene. A. caulinodans were able to enter the roots of rice at emerging lateral roots (lateral root cracks) by crack entry and this was observed by light microscopy. After colonization of lateral roots, bacteria moved into intercellular space within the cortical cell layer of roots. Naringenin at 1×10^-5 and 5×10^-5 M concentration significantly enhanced root colonization. The role of nodABC and regulatory nodD genes was also studied; lateral root crack (LRC) colonization of rice was shown to be Nod factor and NodD independent. Lateral root crack colonization of rice was also observed with similar frequency following inoculation with Azospirillum brasilense and the colonization by A. brasilense was stimulated by naringenin and other flavonoid molecules.     

Nitrogen-fixing stem nodules on Aeschynomene afraspera

Summary Aeschynomene afraspera is a wild annual legume growing in periodically waterlogged soils in western Africa. This legume is characterized by a profuse stem nodulation. Nodules are formed on the stem at the emergence of lateral root primordia, called nodulation sites. These sites are irregularly distributed on vertical rows all along the stem and branches. Stem nodules are hemispherically shaped. Their outside is dark green and they contain a red-pigmented central zone. Stem nodules exhibit a high nitrogen-fixing potential. Acetylene reduction assays result in stem nodule activity of 309 mol C₂H₄ g^–1 dry nodule h^–1. Field-grown stem nodulated Aeschynomene accumulated more N (51 g N m^–2 in 10 weeks) than the root nodulated one. Because of this nitrogenfixing potential and its ability to grow in waterlogged conditions, A. afraspera could probably be introduced into tropical rice cropping systems.     

东北黑土区大豆根际促生菌群落组成研究

为明确东北黑土区大豆根际促生菌的群落组成,选择内蒙古自治区鄂温克族自治旗、黑龙江省海伦市、黑龙江省克山县和黑龙江省农垦红兴隆农场4个采样点,分析了大豆根际自生固氮菌、解磷菌、溶磷菌和硅酸盐细菌的群落组成,解析了促生菌种与地域之间的对应关系。结果表明:东北黑土区的大豆根际土壤中存在大量促生菌,自生固氮菌达到104cfu.g 1,溶磷菌和解磷菌达到105cfu.g 1,硅酸盐细菌达到103cfu.g 1;分离得到具有自生固氮能力的菌株5株,溶磷菌6株,解磷菌7株,硅酸盐细菌4株;自生固氮菌多样性指数在0.94~1.60之间,溶磷菌多样性指数在0.83~1.52之间,解磷菌的多样性指数在1.07~1.67之间,硅酸盐细菌多样性指数在0.52~0.96之间,4个取样点大豆根际促生菌的多样性指数均大于2。采用对应分析确定了不同地区的典型促生菌,内蒙古鄂温克族自治旗的特征种为自生固氮菌LLN8(Azotobacter beijerinckia indica),黑龙江省海伦市的特征种为溶磷菌DHS13(Micrococcus),黑龙江省克山县的特征种为溶磷菌DHS19(Pseudomo-nas),黑龙江省红兴隆农场的特征种为自生固氮菌LLN1(A.chrooco-ccum)和溶磷菌DHS5(Azotobacter)。同时明确了LLN2(A.azomonas)、LLN6(Bacillus mucilaginosus)、DHS9(Arthrobacter)、DHSO2(Pseudomonas)、DHSO14(Erwinia)、DHSO17(Corynebacterium)和LSJ21(Bacillus)在东北黑土区大豆根际分布较为广泛,这些菌株为研发中国东北黑土区大豆专用型复合生物肥料提供了基础条件。     

Nitrogen fixation in soybeans as influenced by cultivar and Rhizobium strain differences

Summary The effect of soybean variety and Rhizobium strain, as well as the level of nitrogen fertilizer, were tested in a field experiment in Greece. Three nodulating varieties were used and one non-nodulating variety as control, with two Rhizobium strains, one commercially available and one isolated from the soil of the experimental site. Nitrogen fertilizer labeled with ¹⁵N enabled the nitrogen derived from the atmosphere (Ndfa), from the soil (Ndfs), and from the fertilizer (Ndff) to be estimated. Statistical analyses showed that the combined variety-strain effect was responsible for most of the variation observed in all parameters, either measured directly or calculated, while the nitrogen level had no effect. The locally isolated strain generally performed equally as well as the commercial one. With the highest yielding variety in particular, the nitrogen-fixing efficiency of soybean nodules, expressed as plant total nitrogen in excess of that in the non-nodulated control in relation to nodule dry weight, was even superior. Taking into account the low fertilizer recovery by plants, it is suggested that adapted cultivars properly inoculated can fix most of the nitrogen they need for high yields. This is of great economic importance for crops with high nitrogen-fixing efficiency, like the soybeans used in this work, which can fix more than 200 kg N/ha, as ¹⁵N dilution and nitrogen difference evaluations have confirmed. The local cultivars in this connection were superior to the United States cultivar, while the difference between cultivars in symbiotic performance were not due to the ability to nodulate, but rather to a much higher nodule-specific efficiency. The commercially available strain was found to produce generally more nodules and the locally isolated strain produced less but larger and more efficient nodules. Finally, the grains from the nodulated plants contained significantly smaller quantities of Fe, Ni, Cu, Zn, and especially Mo compared with non-nodulated plants, as determined by X-ray fluorescence techniques.     

沙棘根瘤共生固氮的研究

研究了生长季节、坡向、植株年龄、植株性别诸因子对沙棘根瘤共生固氮作用的影响,结果证明:季节变化是造成固氮酶活性变化的主要因子;阴坡沙棘根瘤的共生固氮作用与株龄密切相关,而阳坡则不明显;坡向、树龄和沙棘性别均未构成对沙棘根瘤固氮酶活性的显著差异.     

Role of rhizobial exopolysaccharides in crack entry/intercellular infection of peanut

The early stages of the symbiosis in legumes, when rhizobia enter the plant root, penetrate into the root cortex and spread inside nodules, vary among legume species. The most studied infection mechanism involves infection thread formation while the most simple occur by crack entry/intercellular spreading mode as in Arachis hypogaea L. (peanut). Using a peanut nodulating rhizobia strain impaired in the exopolysaccharide production we have examined the nature of its symbiotic deficiency. Our observations indicate that the inoculation of Arachis hypogaea L. with this exopolysaccharide deficient mutant results in the formation of nodule-like structures and very few nitrogen-fixing nodules. These data contribute to our understanding of exopolysaccharides role in the plant-rhizobia interactions leading to the formation of determinate nodules, particularly relevant for legumes that are infected without infection thread formation.     

Multiple occupancy of nodules by nodulating rhizobia on field-grown soybeans with attendance of Sinorhizobium spp.

Abstract

This study analyzed the phenotypic and genotypic characters of nodulating rhizobia isolated from two soybean cultivars, Kyushu 151 and Sachiyutaka, in the same field of the Yamaguchi Prefectural Technology Center of Agriculture and Forestry in Japan. The isolates were classified into groups using phenotypic characteristics, such as growth rate, color change on Bromothymol blue-containing yeast extract-mannitol agar (YMA) plates and colony morphology on YMA plates, and by genotypic characteristics, such as polymerase chain reaction–restriction fragment length polymorphism patterns of the 16S ribosomal RNA genes (16S rDNA) and the internal transcribed sequence (ITS) regions. In Kyushu 151, single phenotypic and genotypic groups were isolated from every nodule examined. In Sachiyutaka, plural strains belonging to distinct groups were obtained frequently from single nodules, indicating that multiple occupancy was established at high frequency. No fixed combination of the groups was found in the composition of multiple occupancy. An increase in the relative abundance of isolates belonging to Sinorhizobium fredii (Ensifer fredii) occurred concomitantly with the increase in the proportion of nodules with multiple occupancy. Nearly 60% of the isolates from Sachiyutaka belonged to S. fredii; 75% of them were obtained from nodules with multiple occupancy.     

滩涂土壤固氮菌群落与环境因子的典范对应分析

选择连续4年进行水稻–紫云英轮作的田块,在0~10、10~20、20~40和40~80 cm剖面深度分别采集土样,利用PCR-DGGE技术分析水稻–紫云英轮作对土壤不同剖面深度固氮菌nif H基因丰度与群落多样性的影响,并对固氮菌群落与8个环境因子进行了典范对应分析(CCA)。试验结果表明,水稻–紫云英轮作能提高滩涂土壤固氮菌的丰度和多样性。水稻-紫云英轮作处理10~20 cm土壤剖面中固氮菌丰度最大,水稻–休闲处理40~80 cm土壤剖面中的最小,其Shannon-Weaver多样性指数分别为3.18和1.51。滩涂土壤环境因子中,总氮与NH_4~+-N、EC与有效磷、pH与速效钾,两两之间的相关性最高;CCA排序图表明土壤有机质、NH_4~+-N和有效磷是影响滩涂土壤固氮菌群落的主要因子。     

Sulfur deficiency of rice plants in the Lower Volta area,Ghana

Abstract

It is well known that stem nodules are formed on the aerial parts of Aeschynomene spp. and Sesbania rostrata grown in the field (Yatazawa and Yoshida 1979; Dreyfus and Dommergues 1981; Yoshida et al. 1985). We have reported that stem nodules were successfully formed by inoculation of Rhizobium isolates derived from both stem and root nodules of A. indica (Yoshida et al. 1985; Sasakawa et al. 1986). The specific activity of nitrogen fixation in stem nodules is comparable to that of root nodules (Sasakawa et al. 1986; Sasakawa 1990). A red pigment, which suggests the presence of leghemoglobin, was detected in stem nodules as well as in root nodules (Yatazawa and Yoshida 1979; Yatazawa and Susilo 1980; Sasakawa et al. 1986).