Enhancement of cold tolerance and growth of tomato plants using endophytic bacteria isolated from cold-adapted plants

Cold stress is a key abiotic factor that adversely impacts crop production. Climate change is responsible for warm winter periods that allow crop production under partial protection, such as commercial greenhouses and tunnels used for tomato production in Sicily. However, temperature conditions are not constant under commercial greenhouse conditions, and cold stress damage can occur. Microbial communities associated with plants in cold environments can enhance plant growth at low temperatures and boost their tolerance to cold stress. Thus, the use of cold-tolerant endophytic bacteria could be a promising approach to protect crop plants from cold stress. This work aims to investigate the effect of bacterial endophytes isolated from wild cold-adapted alpine plants on the tolerance of tomato seedlings to cold stress. A total of 41 cold tolerant endophytic bacterial isolates collected from the roots of three cold-adapted Rosaceae plants were tested for their growth-promoting ability by root inoculation on tomato plants. Seedlings were grown at 25°C under controlled conditions, and the most promising isolates were selected according to their ability to increase dry and fresh plant biomass. Moreover, the ten best-performing plant growth-promoting isolates were tested for their ability to promote tomato growth at 10°C. In particular, two bacterial isolates, Chryseobacterium sp. GRCS301 (Chryseobacterium sp.) and Pseudomonas sp. GRCS202 (Pseudomonas sp.), were able to improve tomato growth under cold stress conditions, reducing the content of H2O2 and modulating the expression of cold stress-related genes. RNA-Seq was then performed at two different time points, one and 14 days after stress (DAS), and gene ontology analysis was carried out to detect differences in gene modulation related to bacterium inoculation under cold conditions. Genes modulated by cold stress in mock-inoculated and bacterium-inoculated plants showed the regulation of response to acid chemicals at both time points. Moreover, Chryseobacterium sp. inoculation showed the upregulation of gene pathways associated with osmotic and oxidative stress protection at 1 DAS, while Pseudomonas sp. inoculation caused up-regulation of gene pathways associated with osmotic stress and calcium ion transporting, indicating that treatments with cold tolerant bacteria can boosts early and late cold-stress response. Cold tolerant bacteria were also tested under commercial greenhouse conditions (ITAKA Crop Solutions, Ragusa, Italy; partner of the PON project) greenhouse conditions and one bacterial isolate (Pseudomonas sp. GRCS202) confirmed the plant growth promotion activity (enhanced root dry weight compared to mock-inoculated plants) during winter tomato production. Cold tolerant bacteria isolated from Alpine environments represent a valuable resource to enhance plant growth and resilience to cold stress in crop plants.