Isolation and molecular identification of endophytic diazotrophs from seeds and stems of three cereal crops.

Huawei Liu,Junbiao Zhang,Lei Zhang,Li Qiu,Baohong Zhang,Shushen Yang,Miaomiao Xie,Dylan Chase Faulk,Aihua Meng,Yaohong Qin

doi:10.1371/journal.pone.0187383

Huawei Liu, Junbiao Zhang + Show 8 more

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https://doi.org/10.1371/journal.pone.0187383

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Abstract

Ten strains of endophytic diazotroph were isolated and identified from the plants collected from three different agricultural crop species, wheat, rice and maize, using the nitrogen-free selective isolation conditions. The nitrogen-fixing ability of endophytic diazotroph was verified by the nifH-PCR assay that showed positive nitrogen fixation ability. These identified strains were classified by 879F-RAPD and 16S rRNA sequence analysis. RAPD analyses revealed that the 10 strains were clustered into seven 879F-RAPD groups, suggesting a clonal origin. 16S rRNA sequencing analyses allowed the assignment of the 10 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Paenibacillus, Enterobacter, Klebsiella and Pantoea. These representative genus are not endophytic diazotrophs in the conventional sense. They may have obtained nitrogen fixation ability through lateral gene transfer, however, the evolutionary forces of lateral gene transfer are not well known. Molecular identification results from 16S rRNA analyses were also confirmed by morphological and biochemical data. The test strains SH6A and MZB showed positive effect on the growth of plants.

Highlights

Nitrogen is important for the growth of plants and animals [1]
Ten bacteria were isolated from wheat seeds (SH6A, SH6B and ZY1A), maize stems (KHSA), maize seeds (B73A and KHB), and rice seeds (MZA, MZB, GYA and GYB) by using the selective medium yeast manitol agar (YMA) and yeast extract mannitol (YEM)
Endophytic diazotrophs have tremendous potential applications in agricultural practices because their ability to fix nitrogen can be put to good use [6]

Summary

Introduction

Nitrogen is important for the growth of plants and animals [1]. Some micro-organisms are able to fix atmosphere nitrogen and provide it to host plants in a process known as biological nitrogen fixation [4]. These micro-organisms are called nitrogen fixing bacteria and they can convert nitrogen into ammonia [5]. Numerous nitrogen-fixing bacteria were isolated and identified from gramineous plants such as maize, rice, and wheat [6]. In the last two decades, new nitrogen-fixing bacteria were isolated from plant tissues, they were called

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