H2020 BIOFERTICELLULASER Project: Role of bacterial cellulases in the transition from free-living to root endophytes in rapeseed crops and in the design of efficient biofertilizers
- Type Project
- Status Filled
- Execution 2017 -2019
- Assigned Budget 170.121,6 €
- Scope Europeo
- Main source of financing H2020
- Project website BIOFERTICELLULASER
Isolation of B. napus endophytes with PGP activity and the ability to produce hydrolytic enzymes in plant cell compounds: A collection of rapeseed bacterial endophytes was obtained by combining rich and minimal media to isolate a wide biodiversity of bacterial endophytes. PGP characteristics and hydrolytic enzyme production were evaluated in vitro in all bacterial isolates, and all strains were identified to species level. The ability of the best selected bacterial isolates to promote plant growth was also evaluated in planta.
Identification of genes overexpressed during infection: The bacterium Pseudomonas brassicacearum CDVBN10 was identified as one of the best plant growth promoters. Therefore, it was selected for transcriptomic analysis of gene expression at the root surface. A set of bacterial genes was selected as potentially overexpressed during infection. None corresponded to a bacterial cellulase. Therefore, the initial hypothesis of the project was adapted to evaluate the role of other bacterial enzymes in the interaction with the plant. We focused on an overexpressed gene encoding an N-carbamoyl putrescine amidase, involved in polyamine biosynthesis.
Isolation of mutant strains: We used CRISPR/Cas genome editing to inactivate the gene encoding N-carbamoyl putrescine amidase in P. brassicacearum CDVBN10.
Symbiotic phenotype study of mutant-derived strains: We studied the symbiotic phenotype of the N-carbamoyl putrescine amidase KO mutant in root colonization, root attachment, root hair deformations, and root infection. We have just detected a distinct mutant phenotype in root hair deformations: the WT induces root hair deformations of B. napus, whereas the mutant strain shows levels of root hair deformations comparable to the non-inoculated control. The phenotype is recovered with the addition of putrescine.
Analysis of the role of N-carbamoyl putrescine amidase in PGP efficiency: We compared the ability of WT and mutant strains to promote plant growth and induce stress resistance to biotic (phytopathogen Phoma lingam) and abiotic (salinity) stress conditions, observing a significant impairment of the mutant in these capacities. The phenotypes are recovered with the addition of putrescine.
Analysis of the efficiency of the bacterial endophyte PGP P. brassicacearum CDVBN10 in infecting rapeseeds and increasing crop yields: P. brassicacearum CDVBN10 was tested under field conditions as a biofertilizer for rapeseeds. The genus Pseudomonas was one of the dominant taxa in the inoculated plants, indicating the effectiveness of the bacterium in establishing itself as a root endophyte in the field. Furthermore, plant size and seed production appeared to increase significantly in the inoculated plants, demonstrating its efficiency as a bacterial biofertilizer for these crops.
According to the FAO, by 2050 there will be 2.3 billion more people on the planet, who will need to produce more food while simultaneously combating existing poverty and hunger, using scarce natural resources more efficiently, and adapting to climate change.
Chemical fertilizers increase crop yields, but they have negative effects on human and animal health, as well as the environment. Plant productivity can be improved through the activity of plant growth-promoting bacteria (PGBs), naturally occurring bacteria capable of modulating plant growth through their metabolic activities. However, with the exception of rhizobic strains applied to legume crops, most biofertilizers designed based on in vitro studies fail when applied in the field. This failure could be due to the fact that, once applied to the soil, the in vitro-selected PGP bacteria must compete with a wide variety of soil microorganisms and adapt to the different abiotic conditions of each environment (temperature range, periods of water/dryness, etc.). This fact increases the interest in the PGP potential of bacterial endophytes (those bacteria capable of entering the endorhiza (interior of the root)), since, once inside the plant, they do not need to compete with the dense population of bacteria in the rhizosphere and are protected from extreme abiotic conditions. However, endophytic colonization, apart from the well-studied interactions between rhizobia and legumes, is poorly understood. Shedding more light on the mechanisms by which endophytes actively enter roots will likely allow major advances in the intelligent selection of bacterial strains that can act as efficient biofertilizers in non-leguminous crops.
Brassica napus L. (rapeseed) is an important crop not only for human and animal food but also for biodiesel production. In Europe, B. napus seeds are the main source of oil for biodiesel production. However, rapeseed cultivation requires significant amounts of chemical fertilizers, and therefore, alternatives that reduce chemical fertilization for more sustainable cultivation are highly desirable. This involves the use of biofertilizers, which include PGP endophytic bacteria.
Therefore, the objectives of this project were:
- Isolation of PGP endophytes from B. napus
- Identification of genes overexpressed during the infection process.
- Isolation of mutant strains in cellulase-coding genes.
- Study of symbiotic phenotypes of derived mutant strains.
- Analysis of the role of bacterial cellulases in PGP efficiency.
- Analysis of the efficiency of selected bacteria to infect rapeseed and increase crop yield.
One of humanity's main challenges in the coming decades will be to increase food production by exploiting scarce resources and protecting the environment. This is why this is one of the priorities of the European Horizon 2020 Programme. Plant productivity can be improved through the activity of plant growth-promoting bacteria (PGBs), applied to agricultural fields as biofertilizers or probiotics, representing an environmentally friendly way to increase crop yields. Biofertilizers have been used in agriculture for decades, but in many cases, bacteria that showed great potential as PGPs under laboratory conditions fail when applied to natural soils, probably because they are outcompeted by native soil microbial populations or because they cannot adapt to the new environmental conditions.
Based on the Rhizobium-clover model, bacterial cellulases are known to be crucial for bacterial root entry. However, the involvement of these enzymes in the active entry of bacterial endophytes into non-leguminous crops has not yet been studied. This project aims to investigate, through a transcriptomic approach and the isolation of endophyte mutant strains, the role of cellulases in the ability of endophytes to penetrate the roots of non-leguminous plants, using rapeseed (B. napus) as a model plant. If genes encoding cellulases allow active root infection, with an advantage over passive mechanisms, selecting bacterial strains not only based on their PGP capacity, but also on their ability to penetrate the plant—where they have fewer competitors and are protected from abiotic stress—will allow the design of more efficient bacterial biofertilizers. The ultimate goal of this project is to lay the groundwork for the development of microbial-based biological fertilizers that will reduce or even eliminate the use of chemical fertilizers (which are dangerous to human health and the environment, and contribute to climate change), while maintaining or increasing crop production.
In this project, we demonstrate that the rapeseed bacterial endophyte Pseudomonas brassicacearum CDVBN10 is an efficient bacterial fertilizer for rapeseed under laboratory and field conditions and, in addition, induces plant resistance to biotic and abiotic stress conditions.
Furthermore, based on transcriptomics of bacteria on the root surface, we found that the gene encoding an N-carbamoyl putrescine amidase, an enzyme involved in polyamine biosynthesis, was significantly overexpressed, indicating a possible relevant role in the interaction with the plant. The construction of a derived knockout mutant strain showed how the gene plays a relevant role in the bacteria's ability to promote the growth of B. napus and induce its resistance to abiotic stress conditions (saline) and to the attack of phytopathogenic fungi (Phoma lingam). To our knowledge, this is the first report on the effect of deletion of a gene involved in polyamine synthesis in a plant growth-promoting bacterium on its ability to promote plant growth and induce stress resistance.
Therefore, the project builds on existing knowledge about the interactions between non-leguminous plants and PGP bacteria, which is necessary for more efficient bacterial fertilizer design. Furthermore, the project demonstrates the great potential of the Pseudomonas brassicacearum CDVBN10 strain as a biofertilizer for rapeseed crops, which would reduce the use of chemical fertilizers in this crop, with a significant positive impact on the environment and consumer health.
- UNIVERSIDAD DE SALAMANCA (USAL)
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