Chemical composition of extracellular polysaccharides of cowpea rhizobia

The extracellular polysaccharides (EPS) of six strains of cowpea rhizobia were examined. The strains (MI50A, M6-7B, IRC253) produced polysaccharides containing glucose, galactose and mannose in a molar ratio of 2:1.1:1, 1:1.3:3.1 and 1:1.3:3.5 respectively. Two strains (513-B and Ez-Aesch) produced polysaccharides containing galactose and mannose in a molar ratio of 2:3. Mannose was the only sugar detected in the EPS of strain IRC291. Pyruvate, acetate, glucuronic acid and galacturonic acid were not detected in any strain.Abbreviations EPS Extracellular polysaccharide - YEMA yeast-extract mannitol agar - YEMB yeast extract mannitol broth     

Isolation of unique nucleic acid sequences from rhizobia by genomic subtraction: Applications in microbial ecology and symbiotic gene analysis

A subtraction hybridization and PCR amplification procedure was used to isolate two Rhizobium DNA probes which exhibited high degrees of specificity at different levels of taxonomic organization and which could be used as tools for detection of rhizobia in ecological studies. First, a probe was isolated from Rhizobium leguminosarum bv. trifolii strain P3 by removing those Sau3A restriction fragments from a P3 DNA digest which cross hybridized with pooled DNA from seven other strains of the same biovar. The remaining restriction fragments hybridized to DNA from strain P3 but not to DNA from any of the seven other strains. In a similar experiment another DNA probe, specific for the Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici group, was generated by removing sequences from R. leguminosarum bv phaseoli strain Kim 5s with pooled subtracter DNA from eight other Rhizobium, Bradyrhizobium and Agrobacterium species. The same subtraction hybridization technique was also used to isolate symbiotic genes from a Rhizobium species. Results from a 1:1 subtractive DNA hybridization of the broad host range Rhizobium sp NGR234 against highly homologous S. fredii USDA257, combined with those from competitive RNA hybridizations to cosmid digests of the NGR234 symbiotic plasmid, allowed the identification of several NGR234 loci which were flavonoid-inducible and not present in S. fredii USDA257. One of these, ORF-1, was highly homologous to the leucine responsive regulatory protein of E. coli.     

Intrinsic antibiotic resistance and streptomycin uptake in cowpea rhizobia

Abstract Uptake of [³H]dihydrostreptomycin in sensitive and resistant strains of cowpea rhizobia was examined to explain one of the possible mechanisms of intrinsic antibiotic resistance. The rate of accumulation of labelled streptomycin in resistant strains was drastically reduced when compared to that of sensitive strains. Our results indicated that high levels of intrinsic antibiotic resistance was due to decreased permeability causing failure to accumulate the drug.     

Growth and nutritional characteristics of cowpea rhizobia

Summary Twenty-five slow-growing strains of cowpea rhizobia were examined for growth and nutritional characteristics. Growth and nutritional data of these isolates were surprisingly homogeneous given their proposed genetic diversity. Most strains tested were capable of anaerobic growth in the presence of nitrate and all were found capable of autotrophic growth in a defined atmosphere of CO₂ and H₂ with oxygen or nitrate as terminal electron acceptors. These isolates grew heterotrophically with various carbohydrates and organic acids. Nitrogen utilization was consistent with that of other slow-growing rhizobia. Medium composition strongly affected the final pH of the culture. Cowpea rhizobia generally did not require vitamins; those requiring vitamins exhibited good growth when biotin was supplemented to the medium.     

Diverse genomic species and evidences of symbiotic gene lateral transfer detected among the rhizobia associated with Astragalus species grown in the temperate regions of China

Based on the analyses of ribosomal DNA and housekeeping genes, a total of 118 bacterial isolates obtained from 13 Astragalus species grown in the temperate region of China were identified as 19 genomic species of Mesorhizobium, Rhizobium, Sinorhizobium and Bradyrhizobium, two of them being putatively new species. Phylogenetic comparison of symbiotic genes (nodC and nifH) and housekeeping genes showed that the symbiotic genes of the Astragalus rhizobia were maintained by both vertical and horizontal transfer. The results demonstrated that the Astragalus species were very promiscuous hosts for rhizobia and that their rhizobia had very diverse genomic and symbiotic gene backgrounds.     

Ecology and genetics of tropical rhizobia species

Biological nitrogen fixation (BNF) technology with special reference to Rhizobium-legume symbiosis is growing very rapidly with the hope of combatting world hunger by producing cheaper protein for animal and human consumption in the Third World. One can see rapid progress made in the biochemistry and molecular biology of symbiotic nitrogen fixation in general; however, less progress has been made on the ecological aspects despite the fact that an enormous amount of literature is available on inoculation problems and on agronomic aspects of symbiotic nitrogen fixation. So far most information on Rhizobium concerns fast-growing rhizobia and their host legume. Although it is essential that food production using BNF technology should be maximized in the Third World, the least work has been done on slow-growing rhizobia, which are generally found in tropical and sub-tropical soils. The majority of the developing countries are in tropical and sub-tropical regions. Except for R. japonicum, a microsymbiont partner of soybean (Glycine max), the majority of the slow-growing rhizobia belong to the cowpea group, and we refer to cowpea rhizobia as tropical rhizobia species. In this review we have tried to consolidate the recent progress made on ecology and genetics of tropical rhizobia. By using recombinant DNA technology techniques it is expected that super strains of rhizobia with desirable characteristics can be produced. One must evaluate the efficiency and effectiveness of these genetically manipulated laboratory strains under field conditions. In conclusion, if one aims at combatting hunger in the Third World using BNF technology, an intensive research programme on fundamental and applied aspects of tropical rhizobia species is suggested. This involves close cooperation between molecular biologists and microbial ecologists.     

Diversity of ectomycorrhizal fungi in Britain: a test of the species–area relationship, and the role of host specificity

The host range of ectomycorrhizal (ECM) fungi in Britain was examined by compilation of a data matrix from published literature sources, based primarily on accounts of sporocarp associations with particular host genera. Information was gathered for 577 species of ECM fungi belonging to 51 genera, and 25 genera of host trees, representing the majority of ECM fungal species and host genera recorded in Britain.
Pronounced variation was recorded in the number of ECM fungal species associated with different host genera, with over 200 species recorded with Betula , Fagus , Pinus and Quercus . There was a positive linear relationship ( r ²=0·47, P =0·007) between the number of species of ECM fungi associated with different host genera and the total area occupied by each tree genus in Britain (both values log-transformed). There was also variation in the number of species of ECM fungi which were apparently specific to particular host genera, values ranging from zero (in 15 genera) to >40 in the case of Betula and Fagus . In total, 233 fungal species appeared to be specific to a single host genus (i.e. 40% of those surveyed). Comparison of the ECM mycota associated with different host genera by PCA accounted for 17% of the total variation, with genera belonging to the Fagaceae ( Quercus , Fagus and Castanea ) tending to cluster together, indicating a degree of overlap in their ECM associates. Exotic conifer species, which displayed a lower ECM diversity than would be expected from their distributional areas, were characterized by a high degree of overlap with the ECM associates of Pinus and Betula .
These results indicate that the abundance of different genera of host trees and variation in host specificity could provide a basis for understanding patterns of diversity in ECM fungi within Britain.     

Properties of streptomycin dependent non-nodulating mutants of cowpea rhizobia and Bradyrhizobium japonicum

We have isolated and characterized mutants from cowpea rhizobia strains JRW3 and IRC256 and Bradyrhizobium japonicum USDA110, which show dependence on streptomycin (Sm) for growth. In the presence of Sm, the majority of the Sm^D (streptomycin dependent) mutants showed cross-resistance to other aminoglycoside antibiotics and some showed no growth at 37°C and 40°C. When nodulation abilities of Sm^D mutants (derived from all three strains) were examined, most of them (> 91%) showed non-nodulating phenotypes to their respective hosts. Preliminary biochemical and genetic characterization indicated that drug-uptake function was altered in Sm^D mutant, and the wild type strain JRW3 could be transformed to streptomycin dependent by Sm^D DNA.     

Genetic characterization of fast-growing rhizobia able to nodulate Prosopis alba in North Spain

Prosopis is a Mimosaceae legume tree indigenous to South America and not naturalized in Europe. In this work 18 rhizobial strains nodulating Prosopis alba roots were isolated from a soil in North Spain that belong to eight different randomly amplified polymorphic DNA groups phylogenetically related to Sinorhizobium medicae, Sinorhizobium meliloti and Rhizobium giardinii according to their intergenic spacer and 16S rRNA gene sequences. The nodC genes of isolates close to S. medicae and S. meliloti were identical to those of S. medicae USDA 1,037(T) and S. meliloti LMG 6,133(T) and accordingly all these strains were able to nodulate both alfalfa and Prosopis. These nodC genes were phylogenetically divergent from those of the isolates close to R. giardinii that were identical to that of R. giardinii H152(T) and therefore all these strains formed nodules in common beans and Prosopis. The nodC genes of the strains isolated in Spain were phylogenetically divergent from that carried by Mesorhizobium chacoense Pr-5(T) and Sinorhizobium arboris LMG 1,4919(T) nodulating Prosopis in America and Africa, respectively. Therefore, Prosopis is a promiscuous host which can establish symbiosis with strains carrying very divergent nodC genes and this promiscuity may be an important advantage for this legume tree to be used in reforestation.     

紫花苜蓿内生和非内生根瘤菌多样性及共生差异

【背景】对根瘤菌多样性的研究有助于推进根瘤菌种质资源的利用。【目的】研究紫花苜蓿内生和非内生根瘤菌的表型和遗传多样性,比较菌株在5个苜蓿品种上的共生效应,验证根瘤菌群体共生效应由苜蓿品种决定的假设。【方法】从甘肃省白银会宁旱作区、兰州安宁灌区、武威凉州灌区3个栽培区域的陇中、清水、WL168HQ、甘农3号、甘农9号等紫花苜蓿品种中分离内生(植株种子、花、叶、茎、根表皮、根中柱和根瘤)和非内生(根际土壤和田间土壤)根瘤菌菌株,通过表型数值分类、 16SrRNA基因限制性片段长度多态性(Restrictionfragmentlengthpolymorphism,RFLP)、16S rRNA基因测序、持家基因多位点序列分型(Multilocus sequence typing,MLST),以及结瘤基因nodC和固氮基因nifH片段序列测定,研究紫花苜蓿根瘤菌的表型和遗传多样性,并采用主成分分析研究根瘤菌菌株在5个紫花苜蓿品种上的共生效应差异。【结果】共分离得到43株内生根瘤菌和10株非内生根瘤菌,叶片和花中没有分离到根瘤菌菌株。53株根瘤菌以及对照菌株R.GN5和S.12531表型特征数值分类聚为8个群,菌株表型多样性丰富。经16SrRNA-RFLP分析共形成22种RFLP分型组合,基因型Ⅰ分布最广泛(24),其次为基因型Ⅻ(5)、ⅩⅤ(5)和ⅩⅨ(3),其余16株菌各代表1种基因型,菌株遗传多样性丰富。16S rRNA基因测序和MLST分析将所有菌株划分为Rhizobiumradiobacter、R.rosettiformans和Ensifermeliloti。仅从7株E.meliloti代表菌株和对照菌株S.12531中扩增到nodC和nifH基因,说明E.meliloti菌株均能结瘤固氮。E.meliloti菌株G3L3接种甘农3号,LP3、LL1和LL2接种陇中,QL2接种清水,LL1、LL2和WLP2接种WL169HQ苜蓿均能显著促进植株的单株结瘤数、地上干重和粗蛋白含量。E.meliloti菌株接种甘农3号、甘农9号和清水苜蓿品种后所有参数值在PC1轴上分别聚在-1-1之间,在PC3轴上聚在-1.5-1.5之间;接种陇中和WL168HQ苜蓿的参数值较分散,PC1轴上分散在-1.5-4之间,PC3轴上分散在-3-4之间。【结论】紫花苜蓿内生和非内生根瘤菌菌株多样性丰富,表型和遗传多样性与其来源没有直接关系。菌株G3L3与甘农3号,LP3、LL1、LL2与陇中,QL2与清水、LL1、LL2、WLP2与WL169HQ苜蓿品种共生匹配和适应能力强。在甘农3号、甘农9号和清水紫花苜蓿品种上群体共生效应相似,在陇中和WL168HQ紫花苜蓿上共生效应差异明显。本研究内生和非内生根瘤菌菌株的群体共生效应根据苜蓿品种而定,根瘤菌菌株与苜蓿品种间的信号识别程度存在差异。     

互惠共生微生物多样性研究概况

所谓互惠共生微生物(mutualistic symbiotic microbes,MSM)是指能定殖其他生物构建互惠共生体系的微生物,主要包括互惠共生细菌、互惠共生放线菌和互惠共生真菌等。MSM种类繁多、分布广泛、物种多样性丰富,涉及原核生物界和真菌界等。MSM定殖人体、动物、植物、藻类或其他真菌,可构建各自相应的互惠共生体系,进而形成范围更加巨大的共生网络,发挥不可替代的生理生态功能。本文在介绍MSM概念的基础上,重点总结了MSM多样性研究进展,指出了目前研究中尚存在的问题,探讨了今后应该开展的工作,MSM多样性研究成果可望为研发MSM应用技术提供依据和材料。     

Nodulation and biological nitrogen fixation of 80 soybean cultivars in symbiosis with indigenous rhizobia

Eighty soybean cultivars were assessed for their potential for nodulation and nitrogen fixation with indigenous rhizobia in a Nigerian soil. Seventy-six days after planting (DAP) 87%, 3% and 10% of the soybean cultivars had from 0 to 30, 31 to 60 and over 61 nodules/plant, respectively. Only 8% had a nodule dry weight of 600 to 1100 mg/plant. At 84 DAP the proportion of nitrogen derived from the atmosphere (Ndfa) ranged from 0 to 65% 16% of the cultivars derived 51 to 65% of their N₂ from the atmosphere. The diversity of soybean germplasm and the variation in nodulation and N₂ fixation permitted the selection of the five best cultivars in terms of their compatibility with indigenous rhizobia, % Ndfa and the amount of N₂ which they fixed.     

Rapid preliminary characterisation of host specificity of leaf-beetles (Coleoptera: Chrysomelidae)

Rapid characterisation of host specificity is important both in biological control of weeds and studies in ecology and evolution. A means for doing this was developed and tested on four species of leaf beetles of interest for biological control of the weed Mimosa pigra (Mimosaceae). We identified the most promising for the more detailed tests necessary to obtain release approval. The impact of time-dependent effects and effects of experience were also investigated as part of this study but were not detected. Short-term host specificity tests on adult feeding accurately predicted the results of longer term trials. The long-term trial showed that survival on a plant species depended on feeding on it. Hence short-term feeding trials can predict the longer term survival of adults on other plant species. Different feeding results were obtained in cut foliage versus entire plants but no consistent pattern was shown: of the two insect species tested, one species ate more of the cut material while the opposite was demonstrated for the second species. Species in order of priority for further consideration as biocontrol agents were Syphrea bibiana, Genaphthona sp., Syphrea sp. and Paria sp. The latter three species were probably not sufficiently specific for release. The tests done here allow the identification of the most likely species upon which to conduct the more laborious and difficult larval developmental tests, saving considerable resources.     

刺槐根瘤菌的表型多样性研究

从杨陵地区采集豆科树种刺槐的根瘤,经分离、纯化,获得40株未知菌株,并选取35株参比菌株,进行唯一碳源、氮源利用、对抗生素和染料的抗性、耐盐性、初始pH生长、生长温度范围及石蕊牛奶反应等共105项生理生化测定。结果表明：供试的刺槐根瘤菌在碳、氮源利用、抗生素敏感性、对染料的抗性程度等方面存在着差异。部分菌株具有较强的耐盐碱能力,其中42．5％的菌株能耐受3.0％的NaCl,17．5％的菌株可在初始pH12的YMA培养基上生长。从数值分类树状图可见,在86％的相似水平上未知菌株构成了3个新的类群,其中第1、2类群各有10株菌,中心菌株分别为NWYC113和NWYCl29,第3类群有7株菌,中心菌株为NWYC147。其分类地位需进一步研究和确定。     

Ticks (Acari: Ixodoidea) in China: Geographical distribution,host diversity,and specificity

Ticks are obligate blood‐sucking ectoparasites, which not only directly damage through bites but also transmit many pathogens. China has a high diversity of tick species, 125 species have been reported, including 111 hard tick and 14 soft tick species. Many of the ticks are important vectors of pathogens, resulting in zoonoses. The dynamics of ticks are affected by both the host and habitat environment. However, systematic studies on the geographical distribution, host diversity, and specificity of ticks are limited in China. To achieve this goal, the relevant available data were summarized and analyzed in this study. Ticks are distributed in all parts of China and Xinjiang has the most records of ticks. The distribution of ticks in adjacent areas is similar, indicating that the habitat environment affects their distribution. Most ticks are widely distributed, whereas some species are endemic to their distributed regions. Ticks are parasitic on mammals, birds, and reptiles, of which mammals are the main host species. Overall, most ticks parasitize different hosts, only a few ticks have strict host specificity, such as ticks that are specifically parasitic on reptiles and bats. In addition, environmental changes and control efforts also influence the dynamics of ticks. These results can better reveal tick biological traits and are valuable for tick control.     

Extracellular polysaccharides of cowpea rhizobia: compositional and functional studies

Polysaccharides excreted by cowpea Rhizobium strains JLn(c) and RA-1 were mixtures of complex acidic exopolysaccharides and low molecular weight neutral glucans. These polymers were fractionated using gel filtration chromatography. Purified fractions of the acidic heteropolymer reacted with peanut agglutinin to give precipitin bands when subjected to Ouchterlony gel diffusion. The acidic exopolysaccharide was found to contain mainly glucose, galactose, glucuronic acid, mannose and fucose. The non-carbohydrate substituents of the acidic heteropolymer were pyruvate, acetate and uronate which were identified by infrared and proton nuclear magnetic resonance spectroscopy as well as by chemical analysis.Abbreviations EPS Extracellular polysaccharide - YEM yeast extract mannitol - PNA peanut agglutination - ¹H-NMR proton nuclear magnetic resonance     

Genetic diversity of rhizobia associated with Desmodium species grown in China

AIMS: Desmodia are leguminous plants used as important forage and herbal medicine in China. Little information is available about the nodule bacteria of Desmodium species. To understand the genetic diversity of rhizobia associated with Desmodium species grown in China, isolates from temperate and subtropical regions were obtained and analysed. METHODS AND RESULTS: A total of 39 rhizobial strains isolated from 9 Desmodium species grown in China were characterized by PCR-based 16S rDNA gene and 16S-23S rDNA intergenic spacer gene restriction fragment length polymorphism (RFLP) and 16S rRNA gene sequencing. The results showed high diversity among rhizobia symbiotic with Desmodium species. Most microsymbionts of Desmodium species belonged to Bradyrhizobium closely related to Bradyrhizobium elkanii, Bradyrhizobium japonicum and Bradyrhizobium yuanmingense. Several small groups or single strain were related to Rhizobium, Sinorhizobium or Mesorhizobium. CONCLUSIONS: Desmodium species formed nodules with diverse rhizobia in Chinese soils. SIGNIFICANCE AND IMPACT OF THE STUDY: These results offered the first systematic information about the microsymbionts of desmodia grown in the temperate and subtropical regions of China.     

Bradyrhizobium elkanii, Bradyrhizobium yuanmingense and Bradyrhizobium japonicum are the main rhizobia associated with Vigna unguiculata and Vigna radiata in the subtropical region of China

Cowpea (Vigna unguiculata) and mung bean (Vigna radiata) are important legume crops yet their rhizobia have not been well characterized. In the present study, 62 rhizobial strains isolated from the root nodules of these plants grown in the subtropical region of China were analyzed via a polyphasic approach. The results showed that 90% of the analyzed strains belonged to or were related to Bradyrhizobium japonicum, Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense and Bradyrhizobium elkanii, while the remaining represented Rhizobium leguminosarum, Rhizobium etli and Sinorhizobium fredii. Diverse nifH and nodC genes were found in these strains and their symbiotic genes were mainly coevolved with the housekeeping genes, indicating that the symbiotic genes were mainly maintained by vertical transfer in the studied rhizobial populations.     

Gluconate catabolism in cowpea rhizobia: evidence for a ketogluconate pathway

Enzymatic and radiorespirometric analysis of several strains of cowpea rhizobia revealed the presence of key enzymes of the Entner-Doudoroff (ED) pathway with the operation of the hexose cycle for the dissimilation of gluconate. These bacteria lack the oxidative pentose phosphate (PP) pathway when grown on gluconate. Gluconate-grown cells possessed an operational tricarboxylic acid (TAC) cycle. Enzymes of an ancillary pathway, the ketogluconate (KG) pathway for gluconate catabolism were detected. The presence of this pathway was confirmed by techniques of thin-layer chromatography and radiorespirometry.Abbreviations ED Entner Doudoroff - PP pentose phosphate - EMP Embden-Meyerhof-Parnas - KG ketogluconate - TCA tricarboxylic acid - DKG diketogluconate - PFK phosphofructokinase     

Parasitism of cowpea by Striga gesnerioides: variation in virulence and discovery of a new source of host resistance

In glasshouse experiments in the United Kingdom, Striga gesnerioides-resistant cowpea varieties, Suvita-2 and 58–57, were assessed alongside a susceptible variety for their responses to seventeen samples of S. gesnerioides from four West African countries. The susceptible variety was attacked by all samples, while Suvita-2 was resistant to samples from Burkina Faso and Mali, and 58–57 was resistant only to samples from Burkina Faso.
At least three physiological groups of the parasite can be distinguished: samples from Burkina Faso attacking the smallest range of cowpea varieties; those from Mali being intermediate; those from Niger and Nigeria attacking all the cowpea varieties. A sample from Cameroon behaved slightly differently and may represent a fourth type.
A cowpea line from Botswana, B.301, selected for its field resistance to Alectra vogelii proved resistant to all Striga populations to which it was exposed, including those from Niger and Cameroon.