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Fungicide tolerant bradyrhizobium japonicum mitigate toxicity and enhance greengram production under hexaconazole stress

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Journal of Environmental Sciences 2019年第4期31卷 92-108页

作者： Mohammad Shahid Mohd.Saghir Khan Department of Agricultural Microbiology Faculty of Agricultural SciencesAligarh Muslim University

Bacterial strain RV9 recovered from greengram nodules tolerated 2400 μg/mL of hexaconazole and was identified by 16 S rDNA sequence analysis as bradyrhizobium japonicum(KY940048). Strain RV9 produced IAA(61.6 μg/mL), ACC deaminase(51.7 mg/(protein·hr)), solubilized TCP(105 μg/mL), secreted 337.6 μg/mL EPS, and produced SA(52.2 μg/mL) and 2,3-DHBA(28.3 μg/mL). Exopolysaccharides produced by strain RV9 was quantified and characterized by SEM, AFM, EDX and FTIR. Beyond tolerance limit,hexaconazole caused cellular impairment and reduced the viability of strain RV9 revealed by SEM and CLSM. Hexaconazole distorted the root tips and altered nodule structure leading thereby to reduction in the performance of greengram. Also, the level of antioxidant enzymes, proline, TBARS, ROS and cell death was increased in hexaconazole treated plants.CLSM images revealed a concentration dependent increase in the characteristic green and blue fluorescence of hexaconazole treated roots. The application of B. japonicum strain RV9 alleviated the fungicide toxicity and improved the measured plant characteristics. Also,rhizobial cells were localized inside tissues as revealed by CLSM. Colonization of B.japonicum strain RV9 decreased the levels of CAT, POD, APX, GPX and TBARS by 80%, 5%,13%, 13% and 19%, respectively over plants grown at 80 μg/(hexaconazole·kg) soil. The ability to detoxify hexaconazole, colonize plant tissues, secrete PGP bioactive molecules even under fungicide pressure and its unique ability to diminish oxidative stress make B.japonicum an attractive choice for remediation of fungicide polluted soils and to concurrently enhance greengram production under stressed environment.

关键词： Hexaconazole toxicity Greengram bradyrhizobium japonicum Cellular damage Nodule ultrastructure Oxidative stress

Dual inoculation of salt tolerant bradyrhizobium and Glomus mosseae for improvement of Vigna radiata L. cultivation in saline areas of West Bengal, India

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Agricultural Sciences 2011年第4期2卷 413-423页

作者： Nicky Singh Nirmalendu Samajpati Amal Kanti Paul 不详

This study is aimed as to evaluate the interaction between salt tolerant bradyrhizobium sp. and Glomus mosseae in the rhizosphere of legume crop Vigna radiata L. under pot culture and field conditions in different saline zones of West Bengal, India. bradyrhizobium sp. when inoculated alone showed marked increase in number of nodules, root and shoot length, total plant biomass, arbuscular mycorrhizal fungal (AMF) colonization and population etc. when compared with plants inoculated only with AMF. However, when used in combination, the in oculants showed marked change in the above mentioned parameters over single inoculation of both salt tolerant AM fungi and bradyrhizobium. These results suggest that AMF along with bradyrhizobium can greatly help in establishment of V. radiata L. cultivation in the saline soils of West Bengal, India. The increased production of the legume crop could also lead to further benefit of the poor farmers by up lifting their socio-economic conditions with the net profit achieved by cultivating this crop in saline stress condition of West Bengal as a second crop during rabi season.

关键词： Vigna radiata L. Arbuscular Mycorrhizal Fungi bradyrhizobium Salinity Glomus mosseae

Soybean Seed Co-Inoculation with bradyrhizobiumspp. and Azospirillum brasilense: A New Biotechnological Tool to Improve Yield and Sustainability

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American Journal of Plant Sciences 2015年第6期6卷 811-817页

作者： Mariangela Hungria Marco Antonio Nogueira Ricardo Silva Araujo Embrapa Soja Londrina Brazil Total Biotecnologia Indústria e Comércio Ltda Curitiba Brazil

Legume nodulation by rhizobia can supply crops with nitrogen and reduce environmental impacts caused by chemical fertilization. The soybean crop in Brazil is an impressive example of how biological N2 fixation can be employed with a plant species of high economic value. However, the development of more productive cultivars, along with the increasing global climatic changes demand agricultural practices to become more productive and yet more environmentally friendly. Plant growth-promoting rhizobacteria (PGPR) are highly beneficial to agriculture worldwide, acting in plant nutrition, protection, and growth stimulation. Azospirillum is, certainly, the most employed PGPR in the world, but little is known about its interaction with rhizobia, when both are applied to legume seeds. We have evaluated the co-inoculation of bradyrhizobia and azospirilla on soybean seeds under different soil and climate conditions in Brazil. Our results demonstrated that co-inoculation is efficient and beneficial to the crop, and promotes yield increases without adding any chemical N fertilizers even in soils where established populations of soybean bradyrhizobia exist. The strategy of co-inoculation thus represents a new biotechnological tool to improve soybean yield without adding any chemical N fertilizers, thus contributing to current practices of sustainability in agriculture.

关键词： Soybean Azospirillum bradyrhizobium Inoculation Yield

Incompatible Nodulation of bradyrhizobium elkaniiStrains BLY3-8 and BLY6-1 with Rj3 Gene-Harboring Soybean Cultivars

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American Journal of Plant Sciences 2017年第2期8卷 178-190页

作者： Aung Zaw Htwe Takeo Yamakawa Laboratory of Plant Nutrition Graduate School of Bioresource and Bioenvironmental Sciences Faculty of Agriculture Kyushu University Fukuoka Japan Department of Agronomy Yezin Agricultural University Yezin Naypyitaw Myanmar

Bradyrhizobia are known symbiotic partners of soybean. However, some soybean cultivars restrict nodulation by some bradyrhizobium bacterial strains. These restrictions are related to compatibility between the Rj genes of soybean cultivars and nodulation types of inoculated bacteria. The objective of this study was to determine nodulation incompatibility of Type B strains with Rj3 soybean cultivars. Newly isolated B. elkanii strains BLY3-8 and BLY6-1 from Myanmar and specific strain bradyrhizobium elkanii USDA33, which are incompatible with Rj3 soybean cultivars, and B. japonicum USDA110 were used as inoculants to check compatibility or incompatibility with Rj3 soybean cultivars. Nitrogen fixation activity was measured by the acetylene reduction method. Ethylene concentration (reduction of acetylene) was determined by flame ionization gas chromatography. According to the inoculation test results, USDA110 was compatible with all soybean cultivars because it formed effective nodules (Figure S1 in Appendix) and possessed nitrogenase activity. Similarly, B. elkanii strains BLY3-8, BLY6-1, and USDA33 were highly compatible with non-Rj and Rj4-gene harboring soybean cultivars because they had the ability to form functional nodules and possessed nitrogenase activity. Inversely, BLY3-8, BLY6-1, and USDA33 were incompatible with Rj3 soybean cultivars because they produced ineffective nodules. Consequently, the ratio of ineffective nodule number to total nodule number was >0.5. Therefore, nodule formation by the newly isolated B. elkanii strains BLY3-8 and BLY6-1 was restricted by the Rj3 soybean cultivars potentially making them useful as specific strains to detect the Rj3 gene in soybean cultivars.

关键词： bradyrhizobium elkanii Nodulation Incompatibility Rj3 Soybean

Phylogenetic Diversity and Evaluation the Effectiveness of Indigenous bradyrhizobiumStrains for Myanmar Black Gram (Vigna mungoL. Hepper) Cultivars

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American Journal of Plant Sciences 2020年第2期11卷 285-306页

作者： Khin Myat Soe Aung Zaw Htwe Kyi Moe Tomomi Abiko Takeo Yamakawa Biofertilizer Laboratory Land Use Division Department of Agriculture Ministry of Agriculture Livestock and Irrigation Yangon Myanmar Department of Agronomy Yezin Agricultural University Yezin Myanmar Laboratory of Agroecology Department of Bioresource Sciences Faculty of Agriculture Kyushu University Harumachi Kasuya-Machi Kasuya-Gun Fukuoka Japan Laboratory of Plant Nutrition Department of Bioresource and Bioenvironmental Sciences Faculty of Agriculture Kyushu University Motooka Nishi-Ku Fukuoka Japan

Black gram (Vigna mungo L. Hepper) is one of the main leguminous crops that provide chief source of food. Several bradyrhizobium species are able to induce effective nodules in black gram cultivars. In the present study, we characterized forty isolates of indigenous black gram bradyrhizobia from Myanmar based on the sequence analysis of the bacterial 16S rRNA gene. The sequence analysis confirmed that all isolates were categorized and identified as the genus bradyrhizobium and they were conspecific with B. elkanii, B. sp., B. liaoningense, B. japonicum and B. yunamingense. Almost all the collected isolates from major black gram growing regions of Nyaunglebin Bago Regio, Chaungzon Mon State, Sittwe Rakhine State, Danubyu Ayeyarwady Region and Launglon Tanintharyi Region were identified as B. liaoningense. At Danubyu Ayeyarwady Region and Pyinmanar Nay Pyi Taw Region, most of the strains were identified as B. japonicum. On the other hand, more or less all the isolates from Launglon Tanintharyi Region and Hpa-an Kayin State were related to B. elkanii. However, all B. sp. strains were found in Salingyi Sagaing Region black gram growing region. This is the first report describing bradyrhizobium strains that were isolated from soil samples of major black gram growing areas in Myanmar. Evaluation of the effectiveness of Myanmar Bradyrhizobim strains isolated from soil samples of major black gram growing areas of Myanmar for plant growth and nitrogen fixation was studied in pot experiments with completely randomized design and three replicates. The nodule dry weight, shoot dry weight and acetylene reduction activity of the plant inoculated with bradyrhizobium elkanii LauBG38 were significantly higher in ARA per plant, nodule and shoot dry weights than the other tested isolates in both Yezin-4 and Yezin-7 black gram varieties. We expect that Myanmar bradyrhizobium elkanii LauBG38 will be able to use as Biofertilizer for black gram cultivars.

关键词： Black Gram Myanmar 16S rRNA Gene bradyrhizobium Nitrogen Fixation

Low-Density Co-Inoculation of Myanmar bradyrhizobium yuanmingenseMAS34 and Streptomyces griseoflavusP4 to Enhance Symbiosis and Seed Yield in Soybean Varieties

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American Journal of Plant Sciences 2013年第9期4卷 1879-1892页

作者： Khin Myat Soe Takeo Yamakawa Plant Nutrition Laboratory Graduate School of Bioresource and Bioenvironmental Sciences Faculty of Agriculture Kyushu University Fukuoka Japan Plant Nutrition Laboratroy Division of Molecular Biosciences Department of Biosciences & Biotechnology Faculty of Agriculture Kyushu University Fukuoka Japan.

This study examined whether low-density co-inoculation of Myanmar bradyrhizobium yuanmingense strain MAS34 and Streptomyces griseoflavus P4 would enhance nodulation, N2 fixation, and seed yield in two soybean varieties. A field experiment was conducted during the July to November 2012 growing season at Kyushu University Farm, Japan, using a split-plot design with three replications and the following four treatments: T1, an uninoculated treatment with peat moss (uninoculated);T2, a single inoculation with S. griseoflavus P4 (P4);T3, a single inoculation of B. yuanmingense MAS34 (MAS34);and T4, a dual inoculation of P4 with MAS34 (P4 + MAS34). Two varieties of soybean, Yezin-3 (Rj4) and Yezin-6 (non-Rj), were used. The N2 fixation activity of soybean was evaluated by the relative ureide method using xylem solute from root bled sap at the early pod-fill stage (R3.5). Dry matter production, N2 fixation, and seed yield were significantly (P 0.01) different between the inoculated treatments. The effect of variety was also significant (P 0.05) for nodule dry weight at the V6 stage, percentage of N derived from the atmosphere at the R3.5 stage, and seed yield at the maturity stage. The number of nodules on the tap roots was significantly higher in Yezin-3 than in Yezin-6. The single inoculation of P4 did not have a significant effect on dry matter production, N2 fixation, and seed yield in either soybean variety. The dry matter production, relative ureide index, percentage of N derived from the atmosphere, and seed yield were significantly (P 0.01) enhanced by a single inoculation of MAS34 in Yezin-3 and by dual inoculation of P4 + MAS34 in Yezin-6. These results indicate that low inoculum concentrations (105 cells seed-1) increase N2 fixation and seed yield in these soybean varieties under open field conditions. Myanmar B. yuanmingense MAS34 and S. griseoflavus P4 are expected to be useful biofertilizers for soybean production.

关键词： bradyrhizobium yuanmingense N2 Fixation S. Griseoflavus P4 Seed Yield Soybean

野生大豆根瘤菌bradyrhizobium sp. GZL13-3的鉴定研究

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野生大豆根瘤菌Bradyrhizobium sp. GZL13-3的鉴定研究

作者：蒋春玲吉林农业大学

学位级别：硕士

本研究以从野生大豆根瘤中分离、纯化得到的菌株为研究对象,采用表型性状鉴定、16S rDNA全序列分析、持家基因分析、共生固氮基因分析和盆栽回接结瘤试验等方法进行研究。从野生大豆的根瘤里分离得到一株根瘤菌GZL13-3,具有典型的根瘤... 详细信息

本研究以从野生大豆根瘤中分离、纯化得到的菌株为研究对象,采用表型性状鉴定、16S rDNA全序列分析、持家基因分析、共生固氮基因分析和盆栽回接结瘤试验等方法进行研究。从野生大豆的根瘤里分离得到一株根瘤菌GZL13-3,具有典型的根瘤菌菌落特征和生理生化特性。生长代时18h, BTB试验产碱,说明是慢生型根瘤菌;能耐受0.50%的NaCl浓度,在pH5.0-8.0都能生长;能抗0.1%的刚果红、溴酚蓝;不能利用乳糖、柠檬酸盐、丙二酸盐;对不同的抗生素敏感性差异很大等。用热变性温度法测得GZL13-3的G+C mol%为61.6%。16S rDNA基因序列测定分析结果显示出供试菌株GZL13-3属于bradyrhizobium属,但与该属的其他已知种的16S rDNA有很大的差异,与B.yuanmingense的序列相似性最高(95.3%)。持家基因(recA、atpD)序列的研究印证了16S rDNA基因序列测定分析的结果,在以recA、atpD为基础构建的进化树中,供试菌株GZL13-3同样属于bradyrhizobium属。以共生固氮基因(nodC、nifH)序列构建的系统发育树与保守基因发育树存在差异。在nodC及其同源序列为基础构建的系统发育树中,GZL13-3与M. loti比bradyrhizobium属显现出更为接近的遗传距离,推测nodC共生基因片段可能来源于Mesorhizobium属的水平转移。以nifH基因为基础构建的系统树中,GZL13-3与bradyrhizobium属新种B.jicamae组成一个系统发育分支,而与bradyrhizobium属其他的菌株亲缘关系较远,是一类特殊的nifH基因。回接实验表明,能与寄主植物大豆(Glycine max)、野生大豆(Glycine soja)形成根瘤。结瘤再分离菌株通过SDS-PAGE全蛋白分析与供试菌株为同一菌株,证明了GZL13-3的结瘤能力。综上所述,根瘤菌GZL13-3属于bradyrhizobium属,但是由于保守基因同源性与已知菌株较低,该菌株也可能是一个潜在的新种。其明确的系统分类地位还需进一步的试验来证明。

关键词：根瘤菌 bradyrhizobium 16s rDNA 固氮基因系统发育

Effects of Long-Term Fertilization Strategies on Soil Productivity and Soybean Rhizobial Diversity in a Chinese Mollisol

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Pedosphere 2019年第6期29卷 784-793页

作者： YAN Jun HAN Xiaozeng CHEN Xu LU Xinchun CHEN Wenfeng WANG Entao ZOU Wenxiu ZHANG Zhiming Northeast Institute of Geography and Agroecology Chinese Academy of SciencesHarbin 150081(China State Key Laboratory of Agrobiotechnology College of Biological Sciences and Rhizobia Research CenterChina Agricultural UniversityBeijing 100193(China Department of Microbiology National College of Life SciencesNational Institute of TechnologyMéxico 11340(México

Rhizobial diversity is affected by interactions between soil features, fertilization strategy, and cropping system. However, interactions among the rhizobial community, chemical-organic manure fertilization, and plant production have not been well documented in Mollisols from long-term experiments. Aimed at maintaining and recovering the productivity of Chinese Mollisols, a long-term fertilization experiment had been carried out for 29 years under a wheat-maize-soybean rotation system, involving the application of recycled organic manure (ROM), chemical fertilizers (N, P, and/or K), or ROM plus N, P, and/or K. In the present study, the effects of different treatments were evaluated by determining soil physicochemical features, soybean production, and soybean rhizobial diversity. The results showed that application of ROM plus NPK maintained or increased soil fertility, which was accompanied by higher production and higher diversity of rhizobia, as compared with the other treatments. The negative association of bradyrhizobium japonicum with N fertilizer, positive association of B. diazoefficiens with soil pH, and alleviation of N-inhibition on the diversity of bradyrhizobium by the addition of ROM were recorded as new findings. Therefore, application of ROM or ROM plus NPK could be a feasible strategy for maintaining and recovering the fertility of Chinese Mollisols, whereas rhizobial diversity could be an indicator of soil fertility.

关键词： bradyrhizobium Chemical fertilizer Housekeeping gene Multilocus sequence analysis Organic manure Rhizobial community Soil fertility

Characterization of Avirulent TnphoA Mutants in Agrobacterium tumefaciensto Enhance Transformation Efficiency

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Advances in Microbiology 2014年第9期4卷 579-593页

作者： Dilip K. Das Eugene W. Nester Department of Microbiology School of Medicine University of Washington Seattle USA P.G. Department of Biotechnology Tilka Manjhi Bhagalpur University Bhagalpur India

Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane protein. Agrobacterium strain A6007 was mutagenized with E. coli strain mm294A plasmid pRK609 having TnphoA to obtain mutants defective in virulence. Because alkaline phosphatase activity is only detected when the PhoA gene product from the transposon is secreted out of the protoplasm, the virulence mutants are located in genes that code for transmembrane or periplasmic proteins. Attempts were made to obtain the sequences adjacent to the TnphoA inserts through several different approaches including Inverse PCR, Cloning, and Tail PCR. Transposon-adjacent sequence was obtained from one membrane anchor subunit in bradyrhizobium japonicum i.e. succinate dehydrogenase which has enhanced transformation efficiency.

关键词： Transposon Mutagenesis Agrobacterium tumefaciense bradyrhizobium japonicum