N fixing bacteria from extreme environments as a remedy for nitrogen deficiency in saline soils

Project Information

NitroFixSal

Grant agreement ID: 101038072

DOI

10.3030/101038072

Project closed

EC signature date 24 March 2021

Start date 1 July 2021

End date 30 June 2023

Funded under

• SPREADING EXCELLENCE AND WIDENING PARTICIPATION

Total cost

€ 137 625,60

EU contribution

€ 137 625,60

Coordinated by

UNIWERSYTET MIKOLAJA KOPERNIKA
Poland

Periodic Reporting for period 1 - NitroFixSal (N fixing bacteria from extreme environments as a remedy for nitrogen deficiency in saline soils)

Reporting period: 2021-07-01 to 2023-06-30

Summary of the context and overall objectives of the project

The current requirement in the agricultural sector is to enhance soil fertility to improve the quality and quantity of food production. To achieve this, significant quantities of inorganic fertilizers are being utilized in agricultural fields to maximize yield. However, the utilization of these chemical fertilizers is detrimental to soil health and is contributing to environmental changes. Therefore, the implementation of biofertilizers emerges as a sustainable solution to mitigate this issue while also ensuring food security for the burgeoning population.

Our project is dedicated to advancing sustainable agricultural practices through a variety of strategies:

1. Developing a membrane-based sensor capable of swiftly and effortlessly detecting nitrogen-fixing bacteria in soil and/or water samples.
2. Isolating halotolerant Plant Growth-Promoting Rhizobacteria (PGPR) from saline alkaline lime and technogenic soils, and assessing the impact of potential isolates on promoting the growth of wheat plants in the presence of salinity-induced stress.
3. Investigating the differentially expressed genes in wheat plants following interactions with PGPR under conditions of salinity stress.

Throughout the course of this project, we have successfully developed a membrane-based sensor designed for the detection of nitrogen-fixing bacteria. This pioneering approach eliminates the necessity for bacterial growth media and significantly reduces detection time in comparison to traditional techniques. Our study encompassed the testing of diazotrophs exhibiting diverse Plant Growth-Promoting (PGP) activities. All these diazotrophs were extracted from technosoils located in Inowrocław, central Poland.

Remarkably, among the tested strains, W4ii and Azo12 demonstrated the most effective growth promotion capabilities. Additionally, they exhibited the ability to alleviate salt-induced stress in wheat plants. To deeply understand the molecular mechanisms governing the interaction between plants and PGPR, we opted to conduct a transcriptome analysis. This analysis involved the sequencing of transcripts to examine the genes that displayed differential expression in wheat plants during their interaction with the W4ii and Azo12 strains, particularly under conditions of salinity stress.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The NitroFixSal project was organized into four interconnected work packages (WP1-WP4) to achieve the main goals of the project.

In WP1, the initial task involved crafting a membrane sensor. Various N-free media with diverse element sources and a dye for the detection of active bacteria were employed. This invention holds promise for biotech firms producing biofertilizers based on nitrogen-fixing bacteria.

Within WP2, the focus shifted to screening N2-fixing bacteria from the rhizospheres of wheat, maize, Aster tripolium, and Salicornia europea grown in technosoils near soda lime repository ponds of CIECH Soda Polska S.A. Over 60 halotolerant strains were obtained from the soil samples, with 11 proving to be nitrogen-fixing bacteria. A comprehensive investigation explored additional growth-promoting properties of these isolates. Notably, strains Azo12 and W4ii significantly facilitated the growth of root and leaf portions of seedlings in salt stress scenarios. Furthermore, Azo12, Azo7, and Azo11 substantially enhanced seedling growth in nitrogen-stressed conditions compared to control seedlings. The results were presented at the 7th Edition of the Global Conference on Plant Science and Molecular Biology (GPMB 2022), Paris, France (September 1-2, 2022) and during the Power of Microbes in Industry and Environment International Conference in Poreč, Croatia (May 15-18, 2023).

WP3 focused on assessing the growth promotion effects and salt stress mitigation potential of the selected PGPR isolates, W4ii and Azo12, when applied to wheat plants under saline conditions. Plant properties (e.g. height, weight, chlorophyll content, lipid peroxidation, total soluble, proline content) were examined in treated and non-treated plants. Both PGPR strains demonstrated the capacity to ameliorate the adverse effects of salt stress on wheat plants. The results were presented during the 8th Central European Congress of Life Sciences EUROBIOTECH, held in Kraków, Poland (June 20-22, 2022), and published in Open Research Europe as an associated article titled "Application of halotolerant Azotobacter chroococcum W4ii isolated from technosoils to mitigate salt stress in the wheat plant. Patent applications grounded in strain Azo12 have been developed and submitted to the Polish Patent Office under reference numbers P.444444 P.444469 and P.444472.

WP4 encompassed transcriptome analysis of wheat plants treated with W4ii and Azo12 isolates under salinity stress. The aim was to scrutinize differentially expressed genes in wheat plants following interaction with PGPR.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The NitroFixSal project has yielded significant outcomes, as outlined below:
a) The sensor effectively identified nifH-positive bacteria, a technological breakthrough with vast potential for biotech companies involved in the production of biofertilizers based on nitrogen-fixing bacteria. Consequently, it is poised to stimulate greater biofertilizer production, thereby enhancing sustainable agricultural practices.
b) The W4ii strain demonstrated inherent resilience in challenging environments, allowing it to mitigate salt stress in the Polish winter wheat variety, Ostroga. Ostroga stands out due to its exceptional grain quality, robust yield potential, and frost resistance. Given its frost resistance, this wheat variety can thrive in reclaimed or salt-affected soils. Its collaboration with the W4ii strain further equips it to adapt effectively to saline conditions.
c) Both the W4ii and Azo12 strains display promising potential for the development of biofertilizers. These advancements have the capacity to offer significant benefits to farmers grappling with yield reductions caused by high salinity levels in their soils.

Color development after the detection of N2 fixing bacteria with the help of membrane-based sensor