Periodic Reporting for period 1 - BIOLACVAC (NEW BIOFERTILIZER IN FUNTIONAL FOODS: EMPLOY OF LACTIC ACID BACTERIA AS VACCINIUM CORYMBOSUM INOCULANT)
Période du rapport: 2020-05-01 au 2022-04-30
In recent years, the commitment of consumers to a balanced diet that allows secondary benefits to be achieved through the consumption of functional foods has been a growing trend. Among these functional foods we find berries such as blueberries, which have a high content of phenolic compounds with a high antioxidant capacity, which together with their high fiber content and low sugar level, have positioned them among one of the foods most demanded functions. In turn, agricultural production is immersed in a process of change and optimization through the incorporation of new techniques that allow increasing its sustainability. In this scenario, the use of bacterial biofertilizer allows reducing the application of inputs to agriculture, improving the use of soil resources and, as has been described in recent years, increasing the concentration of bioactive compounds. However, not all bacteria can be used as biofertilizers and they must meet high biosafety standards to avoid problems for the consumer, especially in fresh foods such as fruits and vegetables. In this project we propose the use of bacterial biofertilizers based on lactic acid bacteria (LAB) with a high level of biosafety to improve blueberry production. This project aims to discover the potential of LAB to be used as blueberry biofertilizers, determining their ability to interact with the crop through the study of their plant growth promotion mechanisms, colonization capacity and their possible compatibility with other isolates. rhizospheric and endophytic. Thus, it intends to study the effect of the inoculation of different bacterial biofertilizers alone or in consortium on the plant transcriptome and the production of phenolic compounds that are decisive for the antioxidant capacity of blueberries.
In this project, the characterization of the autochthonous bacterial populations of wild blueberry (Vaccinium myrtillus) in Portugal has been carried out to determine the presence of lactic acid bacteria in them by metabarcoding them, observing how this plant is capable of selecting endophytic populations. However, lactic acid bacteria (LAB) were not detected in these populations, so it was decided to isolate from cheeses made with raw milk from sheep from the "Serra da Estrela" region. In this way, 6 genera and 8 different LAB species were obtained, of which some strains showed promising capacities to promote plant development (PGP) exhibiting mechanisms in addition to promoting the development in blueberry seedlings and colonizing their rhizosphere. In addition, bacteria from the rhizosphere and endosphere of wild blueberry were selected, which were characterized according to their PGP ability. The next step was to study the compatibility of LAB with blueberry isolates, as well as with two bacterial strains, Phyllobacterium endophyticum PEPV15 and Rhizobium laguerreae PEPV16, which had shown in previous studies a remarkable ability to improve berries production. In this way, the LAB that had shown better PGP qualities only showed compatibility with the PEPV16 strain, the rest of the isolates being either sensitive to the bacteriocins produced by the most promising LAB, or compatible with LAB that had shown negligible potential.
In this way, 4 biofertilization treatments were selected, a treatment with LAB Lactiplantibacillus plantarum QSE79, a treatment with a known strain of biofertilizing potential Rhizobium laguerreae PEPV16, a combined treatment of both and finally, a biofertilization treatment with a native bacterium Paenibacillus sp. VMFR46. The effect of biofertilization at the transcriptomic level was analyzed, observing that the treatments with PEPV16 and VMFR46 generated a response in the plant that presented a large number of overexpressed or repressed genes in common, while in the case of treatment with QSE79, generated a modification in the expression of a smaller number of genes in addition to presenting a greater degree of singularity. This trend was also confirmed when the expression of the genes of the biosynthesis pathway of phenolic compounds in blueberries was specifically analyzed, where the treatments with PEPV16 and VMFR46 generated an advance in the expression of certain genes such as dihydroflavonol reductase and flavonoid-3-O-glucosyltransferase.
Field trials showed that treatments with PEPV16 and VMFR46 were able to produce an increase in production per plant, as well as the combined treatment of PEPV16 and QSE79. Although all the biofertilization treatments generated an improvement in the ripening index, the treatments with QSE79 and VMFR46 were those that presented improvements in the Brix degree of the fruits. In addition, it was generally observed that the use of biofertilizers produced a modification in the profile of phenolic compounds in the fruits without affecting their mineral composition. However, depending on the field trial, the biofertilized treatments showed different behaviors with respect to the total concentration of phenolic compounds. However, all the biofertilized treatments showed an increase in the antioxidant capacity of the fruits against the synthetic radical DPPH.
In this way, 4 biofertilization treatments were selected, a treatment with LAB Lactiplantibacillus plantarum QSE79, a treatment with a known strain of biofertilizing potential Rhizobium laguerreae PEPV16, a combined treatment of both and finally, a biofertilization treatment with a native bacterium Paenibacillus sp. VMFR46. The effect of biofertilization at the transcriptomic level was analyzed, observing that the treatments with PEPV16 and VMFR46 generated a response in the plant that presented a large number of overexpressed or repressed genes in common, while in the case of treatment with QSE79, generated a modification in the expression of a smaller number of genes in addition to presenting a greater degree of singularity. This trend was also confirmed when the expression of the genes of the biosynthesis pathway of phenolic compounds in blueberries was specifically analyzed, where the treatments with PEPV16 and VMFR46 generated an advance in the expression of certain genes such as dihydroflavonol reductase and flavonoid-3-O-glucosyltransferase.
Field trials showed that treatments with PEPV16 and VMFR46 were able to produce an increase in production per plant, as well as the combined treatment of PEPV16 and QSE79. Although all the biofertilization treatments generated an improvement in the ripening index, the treatments with QSE79 and VMFR46 were those that presented improvements in the Brix degree of the fruits. In addition, it was generally observed that the use of biofertilizers produced a modification in the profile of phenolic compounds in the fruits without affecting their mineral composition. However, depending on the field trial, the biofertilized treatments showed different behaviors with respect to the total concentration of phenolic compounds. However, all the biofertilized treatments showed an increase in the antioxidant capacity of the fruits against the synthetic radical DPPH.
This project has allowed us to discover the capacities of LAB to be used as biofertilizers with a high level of safety, showing that although their use alone may not entail a similar benefit as the use of rhizospheric or endophytic bacteria from edaphic environments, their combination With them, you can generate similar benefits and even exceed them in terms of improvement at a qualitative level. This is the first time that the capacity of different LAB to promote blueberry plant growth has been analyzed and the first time that their colonization capacity has been studied using immunodetection techniques. Thus, the results obtained in the analysis of the populations of LAB in PDO “Serra da Estrela” cheese have shown that the cheeses present a high diversity population with a high biotechnological potential. Regarding the study of wild blueberry populations, they have shown that this plant has an outstanding ability to modulate endophytic populations and that, in turn, the most isolated populations in the interior of Portugal present a relative degree of singularity. In turn, the analysis of the transcriptome of the interaction between the different selected biofertilizer treatments will allow knowing the response of the crops to the inoculation and knowing the molecular mechanisms that govern the interaction. This biofertilization has shown an improvement in production as well as an increase in the concentration of phenolic compounds and a modification in their organic profile, as well as an increase in antioxidant activity.
These results will allow the design of biofertilizers to improve agricultural production and obtain agricultural production capable of increasing its yield while improving the biological activity of a functional food such as blueberries. In turn, the involvement of local producers in carrying out tests with bacterial biofertilizers has made it possible to disseminate and publicize the usefulness of this type of input, showing its possibilities and how it can be a key tool in obtaining a differentiated product. more competitive and with market potential that allows producers from the interior of Portugal to differentiate themselves and improve their projection.
These results will allow the design of biofertilizers to improve agricultural production and obtain agricultural production capable of increasing its yield while improving the biological activity of a functional food such as blueberries. In turn, the involvement of local producers in carrying out tests with bacterial biofertilizers has made it possible to disseminate and publicize the usefulness of this type of input, showing its possibilities and how it can be a key tool in obtaining a differentiated product. more competitive and with market potential that allows producers from the interior of Portugal to differentiate themselves and improve their projection.