• Summary description of the project objectives

Plant breeding for resistance genes and chemical treatments are currently the main strategies to control plant diseases. However, withdrawal of agrochemicals under new EU legislation and co-evolution of diseases to overcome crop resistance genes have increased the need to find alternative means to improve crop disease management. Beneficial rhizosphere-colonizing microorganisms, such as arbuscular mycorrhiza fungi (AMF) and plant growth-promoting rhizobacteria (PGPR), can prime the plant immune system against pathogen attack. However, the complexity of the plant root microbiome has hampered wide-ranging exploitation of these biocontrol organisms in current crop protection schemes. The objectives of this work have been evaluating the interaction between two beneficial microorganisms, a mycorrhizal fungus (Glomus intraradices) and a plant growth-promoting rhizobacterial strain (Pseudomonas putida) and investigating the mechanisms by which these microbes synergistically prime wheat (Triticum aestivum) for enhanced defensive responses.

Bacteria-GFP
Pseudomonas putida tagged with green fluorescent protein. The presence of such fluorescent protein allows to differentiate the colonies of these bacteria from others.
Arbuscules 01
Glomus intraradices arbuscula developing inside root cells. The arbuscula present their characteristic branched form. The thicker and longer filaments are the fungus hyphae.

• Description of the work performed since the beginning of the project

During the work, two different wheat cultivars were grown in a rhizotron system (allowing root observation) and inoculated either with the mycorrhizal fungus (G. intraradices), the rhizobacteria (P. putida) or both. Several physiological parameters were measured (chlorophyll content, chlorophyll fluorescence, plant weigh) during the experiments. At two different time points, the colonisation of the roots by the rhizobacteria and the mycorrhizal fungus were evaluated. At the same time points, leaves were collected and infiltrated with chitosan, an elicitor of defensive responses. The leaves defensive response was assessed by staining for callose (a well known marker for such responses) and measuring its depositions in cells under the microscope. Finally, metabolite profiles of leaf tissue for all treatment combinations were determined using a non-targeted approach using broad-spectrum metabolite fingerprinting.

Rhizotron

Wheat growing using the rhizotron system. The roots can be monitored at every moment when needed.

• Description of the main results achieved so far

Our results showed that wheat genotype strongly influences root colonization by the mycorrhizal fungus and rhizobacteria. In addition, the mycorrhizal fungus has a positive effect on the number of rhizobacteria on wheat roots, and also recruiting other rhizobacteria when the selected strain is not present. On the other hand, the presence of rhizobacteria reduces the abundance of mycorrhiza arbuscules in wheat. Root colonization by both beneficial microorganisms has a synergistic effect on the induction of callose deposition (priming) in leaves of young wheat plants. The metabolomic analysis clustered apart the treatments either with only mycorrhizal fungus or with both microorganisms with respect to the non-treated plants or those treated only with rhizobacteria. The search for compounds in the databases showed that several of those identified were linked to lipid metabolism pathways.

Callose
Callose depositions in wheat leaf. The white-bluish dots are callose deposits accumulated inside leaf cells. They work enhancing the cell wall resistance against pathogen intrusion.

• Expected final results and their potential impact and use

These results confirm the hypothesis that wheat genotype can influence the root colonization by beneficial microorganism and that mycorrhizal fungi can help recruiting specific rhizobacteria strains. With such knowledge, breeding programmes can be improved aiming to get cultivars allowing a better establishment of mycorrhizal associations. Such improvement will allow better interaction of the plants with the soil microbiome, leading to the recruitment of other beneficial microorganisms such as plant growth-promoting rhizobacteria. Additionally, field inoculations with mycorrhizal fungi and rhizobacteria are starting to be used nowadays as an alternative method for improving crop production by reducing the amount of fertilizers. If such inoculations also help to prevent diseases in the field by priming plant defences as we have seen in our experiments, it means that a reduction in the amount of pesticides (i.e., fungicides) would be possible too. Both things should contribute to a reduction of the negative impact that the use of agrochemicals (fertilizers, fungicides) has in agriculture.

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