Methods for Isolation and Characterization of Nitrogen-Fixing Legume-Nodulating Bacteria

Tak, Nisha; Bissa, Garima; Gehlot, H. S.

doi:10.1007/978-1-4939-9790-9_12

Cited by 5 publications

(8 citation statements)

References 40 publications

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“…seeds were transferred to sterile pots filled with soils collected from various sampling sites and placed in glasshouse maintained at 28 o C under natural light. Plants were watered periodically as required with sterile tap water, and were harvested after 7-8 weeks to assess their nodulation status (Tak et al 2020).…”

Section: Methodsmentioning

confidence: 99%

“…Randomly amplified polymorphic DNA (RAPD) patterns were studied using the single RPO1 primer described by Richardson et al (1995). The PCR reaction mixture was prepared as described in Tak et al (2020) and the PCR cycling conditions used are given in Table S1. On the basis of RPO1 profiling, forty-eight strains were selected for sequencing of their recA (coding for Recombination protein A) and nodA (symbiotic gene encoding N-acyltransferase nodulation protein) genes.…”

Section: Isolation and Purification Of Rhizobiamentioning

confidence: 99%

“…The PCR reaction mixture (25 µl total volume) consisted of 1X Taq buffer, MgCl 2 variable depending on the gene of interest (Table S1), 150 µM each of dNTPs, 60 mM of DMSO, 1 µM of each of forward and reverse primers, 0.75 Units of Taq DNA polymerase, 100-1000 ng µl -1 DNA template, and nuclease-free water to make up the volume. The band sizes (Table S1) of PCR amplicons were ascertained with the help of agarose gel electrophoresis using the BioRad gel documentation system and were pooled for column purification as described in Tak et al (2016Tak et al ( , 2020. Nucleotide sequencing was done with the same set of primers through the Sanger sequencing facilities of AgriGenome Labs Pvt.…”

Section: Isolation and Purification Of Rhizobiamentioning

confidence: 99%

“…Individual and concatenated phylogenetic trees were reconstructed using the Maximum likelihood (ML) method with General Time Reversible (GTR) model and 1000 bootstrap values as described by Tak et al (2020).…”

Section: Analysis Of Nucleotide Sequences and Phylogenetic Studiesmentioning

confidence: 99%

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Evolution of novel strains of Ensifer nodulating the invasive legume Leucaena leucocephala (Lam.) de Wit in different climatic regions of India through lateral gene transfer

Chouhan

Tak

Bissa

et al. 2022

FEMS Microbiology Ecology

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More than 200 root-nodule bacterial strains were isolated from Leucaena leucocephala growing at 42 sampling sites across 12 states and three union territories of India. Genetic diversity was observed among 114 strains from various climatic zones; based on recA these were identified as strains of Ensifer, Mesorhizobium, Rhizobium and Bradyrhizobium. In MLSA strains clustered into several novel clades and lineages. Ensifer were predominant nodulating genotype isolated from majority of alkaline soils, while Mesorhizobium and Rhizobium strains were isolated from a limited sampling in North-Eastern states with acidic soils. Positive nodulation assays of selected Ensifer representing different genetic combinations of housekeeping and sym genes suggested their broad host range within the closely related mimosoid genera Vachellia, Senegalia, Mimosa and Prosopis. Leucaena selected diverse strains of Ensifer and Mesorhizobium as symbionts depending on available soil pH, climatic and other edaphic conditions in India. Lateral gene transfer seems to play a major role in genetic diversification of Ensifer exhibited in terms of Old World vs. Neotropical genetic make-up and mixed populations at several sites. Although Neotropical Ensifer strains were most symbiotically effective on Leucaena the native Ensifer are promiscuous and particularly well-adapted to a wide range of sampling sites with varied climates and edaphic factors.

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Section: Methodsmentioning

confidence: 99%

Section: Isolation and Purification Of Rhizobiamentioning

confidence: 99%

Section: Isolation and Purification Of Rhizobiamentioning

confidence: 99%

Section: Analysis Of Nucleotide Sequences and Phylogenetic Studiesmentioning

confidence: 99%

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Evolution of novel strains of Ensifer nodulating the invasive legume Leucaena leucocephala (Lam.) de Wit in different climatic regions of India through lateral gene transfer

Chouhan

Tak

Bissa

et al. 2022

FEMS Microbiology Ecology

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“…Nitrogenase activity was estimated through acetylene reduction assay (ARA) which is the most commonly used method of indirect quantification of N 2 fixation rate [25]. ARA is based on reduction of acetylene to ethylene by nitrogenase enzyme, instead of reducing N 2 to NH 3 [26]. The nodulated roots were incubated at room temperature in 10 mL air-tight glass vials to which acetylene (10% v/v) was injected.…”

Section: Authentication Of Putative Rhizobia and Assessment Of N 2 Fixationmentioning

confidence: 99%

Phylogenetic diversity analysis reveals Bradyrhizobium yuanmingense and Ensifer aridi as major symbionts of mung bean (Vigna radiata L.) in Pakistan

2020

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The present study was carried out to evaluate the diversity of rhizobia associated with nodules of mung bean in Pakistan, because this information is necessary for inoculum development. Based on sequence analysis of 16S rRNA gene of thirty-one bacteria, 11 were assigned to genus Bradyrhizobium, 17 to Ensifer, and 3 to Rhizobium. Phylogenetic analyses on the basis of 16S-23S ITS region, atpD, recA, nifH, and nodA of representative strains revealed that B. yuanmingense is the predominant species distributed throughout different mung bean-growing areas. Among the fast-growing rhizobia, Ensifer aridi was predominant in Faisalabad, Layyah, and Rawalpindi, while E. meliloti in Thal desert. Sequence variations and phylogeny of nifH and nodA genes suggested that these genes might have been co-evolved with the housekeeping genes and maintained by vertical gene transfer in rhizobia detected in the present study. Host infectivity assay revealed the successful nodulation of host by rhizobia related to genera Bradyrhizobium, Ensifer and Rhizobium. Among all, Bradyrhizobium and Ensifer spp. inoculation exhibited a significantly higher number of nodules (11-34 nodules plant −1 ) and nitrogenase activity (nodule ARA 60-110 μmol g −1 h −1 ). Contrary to the previous studies, our data reveal that B. yuanmingense and E. aridi are predominant species forming effective nodules in mung bean in Pakistan. Furthermore, to the best of our knowledge, this is the first report showing the effective symbiosis of E. aridi, E. meliloti, and Rhizobium pusense with mung bean. The diversity of rhizobia in different habitats revealed in the present study will contribute towards designing site-specific inocula for mung bean.

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The widely distributed legume tree Vachellia (Acacia) nilotica subsp. indica is nodulated by genetically diverse Ensifer strains in India

et al. 2019

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Methods for Isolation and Characterization of Nitrogen-Fixing Legume-Nodulating Bacteria

Cited by 5 publications

References 40 publications

Evolution of novel strains of Ensifer nodulating the invasive legume Leucaena leucocephala (Lam.) de Wit in different climatic regions of India through lateral gene transfer

Evolution of novel strains of Ensifer nodulating the invasive legume Leucaena leucocephala (Lam.) de Wit in different climatic regions of India through lateral gene transfer

Phylogenetic diversity analysis reveals Bradyrhizobium yuanmingense and Ensifer aridi as major symbionts of mung bean (Vigna radiata L.) in Pakistan

The widely distributed legume tree Vachellia (Acacia) nilotica subsp. indica is nodulated by genetically diverse Ensifer strains in India

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