Role of bacterial cellulases in the transition from free living to root endophytes in rapeseed crops and in the design of efficient biofertilizers

Periodic Reporting for period 1 - BIOFERTICELLULASER (Role of bacterial cellulases in the transition from free living to root endophytes in rapeseed crops and in the design of efficient biofertilizers)

Période du rapport: 2017-06-01 au 2019-05-31

According to FAO’s, in 2050 there will be an additional 2.3 billion people in the Planet, who will require producing more food, while at the same time combating existing poverty and hunger, using scarce natural resources more efficiently and adapting to climate change.

Chemical fertilizers increase crops yields, but they have negative effects for human and animals health and the environment. Plant’s productivity can be enhanced by the activity of plant growth-promoting (PGP) bacteria, which are naturally-occurring bacteria able to modulate plant growth as a result of their metabolic activities. Nevertheless, apart from rhizobial strains applied to legume crops, most of the biofertilizers designed based on in vitro studies fail when applied in the fields. This failure could be due to the fact that, once applied in the soils, in vitro selected PGP bacteria must compete with a wide variety of microorganisms present in the soil and get adapted to the different abiotic conditions of each environment (temperature range, water/desiccation periods, etc.). This fact rises the interest of the PGP potential of bacterial endophytes -those bacteria with the ability to enter the endorhiza (root inside) –since, once inside the plant, they do not need to compete with the dense population of bacteria in the rhizosphere and they are protected from extreme abiotic conditions. Nevertheless, endophytic colonization, apart from the well-studied interactions between rhizobia and legumes, is not well understood. To shed further light in the mechanisms by which endophytes actively enter roots will likely allow great progresses in the wise selection of bacterial strains which can act as efficient biofertilizers in non-legume crops.

Brassica napus L. (rapeseed) is an important crop due to its cultivation not only as food resource (human and animal), but also for biodiesel production. In Europe, the seeds of B. napus are the primary source of oil for biodiesel production. However, rapeseed cultivation requires important amounts of chemical fertilizers and therefore, alternatives that enable the reduction of chemical fertilization for a more sustainable crop are very desirable. This implies the use of biofertilizers, which include endophytic PGP bacteria.

Thus, the aims of this project were:
Isolation of B. napus PGP endophytes
Identification of genes up-regulated during the infection process.
Isolation of mutant strains in cellulase encoding genes.
Study of symbiotic phenotypes of the mutant derivative strains.
Analysis of the role of bacterial cellulases in PGP efficiency.
Analysis of selected bacteria efficiency in infecting rapeseeds and increasing crop yields.

Isolation of B. napus endophytes with PGP activities and capability to produce plant cell compounds hydrolytic enzymes: Using a combination of rich and minimal media to target the isolation of a wider biodiversity of bacterial endophytes, we obtained a colection of rapseed bacterial endophytes. In vitro PGP traits and production of hydrolytic enzymes were tested in all the bacterial isolates and all strains were identified at species level. The capability of the best bacterial isolates selected based on the in vitro test to promote plant growth was also tested in planta.

Identification of genes up-regulated during the infection process: The bacterium Pseudomonas brassicacearum CDVBN10, was identified as one of the best plant growth promoters. Thus, it was selected for a transcriptomic analysis of gene expression over the root surface. A set of bacterial genes was selected as potentially upregulated during the infection process. None of them corresponded to a bacterial cellulase. Thus, the initial hypothesis of the project was adapted to the evaluation of role of other bacterial enzymes in the interaction with the plant. We focused on an up-regulated gene encoding a N-carbamoyl putrescine amidase, implicated in the biosynthesis of polyamines.

Isolation of mutant strains: We used CRISPR/Cas genome editing to knock-out the gene encoding the N-carbamoyl putrescine amidase in P. brassicacearum CDVBN10.

Study of symbiotic phenotypes of the mutant derivative 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 just detected a different mutant phenotype in root hairs deformations -the WT induces the deformation of B. napus root hairs while the mutant strain shows levels of root hair deformations comparable to the uninoculated control. The phenotype is recovered with the addition of putrescine.

Analysis of the role of N-carbamoyl putrescine amidase in the PGP efficiency: We compared the capability of the WT and the mutant strains to promote plant growth and induce plant stress resistance to biotic (phytopathogen Phoma lingam) and abiotic (salinity) stress conditions, observen a significan impairment of the mutant in this capabilities. The phenotypes are recovered with the addition of putrescine.

Analysis of the efficiency of the PGP bacterial endophyte P. brassicacearum CDVBN10 in infecting rapeseeds and increasing crop yields: P. brassicacearum CDVBN10 was assayed in field conditions as rapeseed biofertilizer. The genus Pseudomonas was one of the dominant taxa in inoculated plants, which indicates the effectiveness of the bacterium to be established as a root endophyte in the field. Moreover, the plant size and seeds production appeared significantly increased in the inoculated plants, showing its efficiency as bacterial biofertilizer for this crops.

In this project we show that the rapeseed bacterial endophyte Pseudomonas brassicacearum CDVBN10 is an efficient rapeseed bacterial fertilizer under laboratory and field conditions and moreover, it induces plant resistance to biotic and abiotic stress conditions.
Moreover, based on a transcriptomic of bacteria over the root surface, we found that the gene encoding for a N-carbamoyl putrescine amidase, enzyme implicated in the biosynthesis of polyamines, was significantly overexpressed, indicating a potential relevant role in the interaction with the plant. The construction of a knockout mutant derivative strain showed how the gene plays a relevant role in the capability of the bacterium to promote B. napus growth and induce its resistance to abiotic stress conditions (saline) and the attack of phytopathogenic fungi (Phoma lingam).
To the best of our knowledge, this is the first report on the effect of the deletion of a gene implicated in the synthesis of polyamines in a plant growth promoting bacterium on its capability to promote plant growth and induce stress resistance.
Thus, the project builds upon the existing knowledge on the non-legume plants/PGP bacterial interactions, needed for a more efficient design of bacterial fertilizers.
Moreover, the project shows the good potential of the strain Pseudomonas brassicacearum CDVBN10 as a biofertilizer for rapeseed crops, which would allow the reduction of chemical fertilizers in this plant cropping, with important positive impact on the environment and the consumers' health.

Pseudomonas brassicacearum CDVBN10 colonizing rapeseed roots

Plants from a field experiment to test the efficiency of strain CDVBN10 as PGP biofertilizer

Periodic Reporting for period 1 - BIOFERTICELLULASER (Role of bacterial cellulases in the transition from free living to root endophytes in rapeseed crops and in the design of efficient biofertilizers)

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