Understanding the role of microbiome interactions in the freezing stress tolerance of plants

Climate change-associated extreme weather events such as spring frosts have increased in frequency and intensity over the past decades in Europe. Freezing stress is one of the main limiting factors for the production of many fruit crops including apple (Malus × domestica, Rosaceae family). Current methods used by growers to prevent or reduce freezing damage include mainly physical approaches, which have several limitations in terms of cost, efficacy, and environmental impact. Thus, the development of natural approaches for the mitigation of frost damage on crops will provide a sustainable tool for growers with beneficial impacts on society (i.e. high quality and quantity of fruits) and the economy (i.e. sustainable use of natural resources). The use of natural plant-beneficial microorganisms is a promising approach against freezing stress. Plants growing in cold regions, such as Alpine regions, are hypothesized to survive freezing stress thanks to their symbiotic relationship with endophytic microorganisms. However, the structure and function of endophytic microbial communities associated with such plants are poorly understood.
FreezingBioprotector project aims to taxonomically and functionally characterize the endophytic bacterial communities associated with wild alpine Rosaceae plants, to develop new microbial-based products for freezing protection of apple.
The overall objectives of this project that were each addressed by a work package (WP) are (i) characterising the bacterial communities associated with Dryas octopetala anthosphere and identifying potential bacteria for freezing protection (WP1) (ii) identifying novel bacterial strains by a rapid-throughput screening for their ice-active traits and environmental stress tolerance (WP2) and (iii) identifying bacterial strains with freezing protection abilities on strawberry and feasibility checking of industrial production (WP3).

Flower, leaf and root samples were collected from three plant species (D. octopetala, Alchemilla sp., and Geum montanum) from seven sites in one ecoregion (Alpine ecoregion in Trentino-Alto Adige) from two expositions (North and South). Culture-independent analysis of 16S rRNA amplicon gene sequencing was performed, demonstrating that bacterial alpha-diversity indices (richness and diversity) partially decreased from roots to leaves and then to flowers. Alpha- and beta-diversity indices were influenced by the plant tissue, plant genus, and collection site. Core endophytic bacterial taxa of alpine Rosaceae plants accounted for 41% of the total relative abundance and was composed of 31 amplicon sequence variants (ASVs). Co-occurrence network analysis showed that the same ASVs constituting the hub taxa (ASVs that are significantly more connected within the network than other ASVs) were also classified as core taxa in the flower and leaf networks. Culture-dependent analysis of targeted isolation method was implemented to test the functional relevance of bacterial endophytes belonging to the most abundant ASVs. As a result, a large (n = 686 isolates) and phylogenetically diverse (4 phyla and 65 genera) collection of psychrotolerant bacterial endophytes was successfully obtained from alpine Rosaceae plants.
The rapid-throughput screening allowed the selection of 97 bacterial isolates as representative psychrotolerant bacterial endophytes and used for the subsequent functional characterization to assess their freezing protection ability on strawberry seedlings and micropropagated apple plantlets. Results showed that most bacterial endophytes could reduce electrolyte leakage in strawberry seedlings with the best stress protection level from isolates belonging to the core taxa. Moreover, leaf spray inoculation with some isolates reduced freezing damage on potted apple plants.
FreezingBioprotector has generated a review article (https://doi.org/10.1093/femsec/fiab161) and three peer-reviewed publications will be available in the next few months. Results obtained with this project have been disseminated in ten national and/or international conferences and seminars, and communications by the website (https://www.centro3a.UNITN.it/en/1001/freezing-bioprotector) Twitter (https://twitter.com/FBioprotector) LinkedIn (https://www.linkedin.com/in/malek-marian-ph-d-64683b15a/) and ResearchGate (https://www.researchgate.net/lab/Michele-Perazzolli-Lab). Results have also been communicated to the public by a video (https://www.youtube.com/watch?v=uH_SExhNv_Q) and in non-scientific events (e.g. European Researchers' Night).

FreezingBioprotector contributed to the taxonomic and functional characterization of endophytic bacterial communities in wild alpine Rosaceae plants, particularly unravelling the key role of core bacterial taxa in modulating plant tolerance to freezing stress. The outcome of this project fills a gap in the knowledge of the scientific community regarding the role of plant-associated microbiomes in the stress tolerance of host plants growing in natural extreme environments. The project may have an impact on growers by alleviating the impacts of climate changes on fruit tree crops using psychrotolerant bacterial endophytes and on EU economy by contributing to the development of a bacterial-based product for freezing protection of fruit crops for the EU market, thereby increasing the income stability of EU growers.

Periodic Reporting for period 1 - FreezingBioprotector (Understanding the role of microbiome interactions in the freezing stress tolerance of plants)

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