Amplicon sequencing and culture-dependent approaches reveal core bacterial endophytes aiding freezing stress tolerance in alpine Rosaceae plants

Wild plants growing in alpine regions are associated with endophytic microbial communities that may support plant growth and survival under cold conditions. The structure and function of endophytic bacterial communities were characterized in flowers, leaves, and roots of three alpine Rosaceae plants in Alpine areas using a combined amplicon sequencing and culture-dependent approaches to determine the role of core taxa on plant freezing stress tolerance. Amplicon sequencing analysis revealed that plant tissue, collection site, and host plant are the main factors affecting the richness, diversity, and taxonomic structure of endophytic bacterial communities in alpine Rosaceae plants. Core endophytic bacterial taxa were identified as 31 amplicon sequence variants highly prevalent across all plant tissues. Psychrotolerant bacterial endophytes belonging to the core taxa of Duganella, Erwinia, Pseudomonas, and Rhizobium genera mitigated freezing stress in strawberry plants, demonstrating the beneficial role of endophytic bacterial communities and their potential use for cold stress mitigation in agriculture.IMPORTANCEFreezing stress is one of the major abiotic stresses affecting fruit production in Rosaceae crops. Current strategies to reduce freezing damage include physical and chemical methods, which have several limitations in terms of costs, efficacy, feasibility, and environmental impacts. The use or manipulation of plant-associated microbial communities was proposed as a promising sustainable approach to alleviate cold stress in crops, but no information is available on the possible mitigation of freezing stress in Rosaceae plants. A combination of amplicon sequencing, culture-dependent, and plant bioassay approaches revealed the beneficial role of the endophytic bacterial communities in alpine Rosaceae plants. In particular, we showed that culturable psychrotolerant bacterial endophytes belonging to the core taxa of Duganella, Erwinia, Pseudomonas, and Rhizobium genera can mitigate freezing stress on strawberry seedlings. Overall, this study demonstrates the potential use of psychrotolerant bacterial endophytes for the development of biostimulants for cold stress mitigation in agriculture.