Psychrotolerant bacteria isolated from the leaf apoplast of cold-adapted wild plants improve the cold resistance of bean (Phaseolus vulgaris L.) under low temperature

We have isolated psychrotolerant bacteria from the leaf apoplast of cold-adapted wild plants and aimed to investigate their effect on the cold resistance of bean (Phaseolus vulgaris L.). Based on the findings of 16S rRNA gene sequence analysis, 20 isolates belonging to 5 bacteria species (Pseudomonas fragi, P. chloropaphis, P. fluorescens, P. proteolytica and Brevibacterium frigoritolerans) were identified in the leaf apoplastic fluid of Draba nemorosa, Galanthus gracilis, Colchicum speciousum, Scilla siberica, Erodium cicutarium, respectively. We have determined that 6 of the 20 isolates have exhibited ACC (1-aminocyclopropane-1-carboxylate) deaminase activity and secreted different extracellular proteins under cold condition (+4 degrees C) compared to normal growth condition (28 degrees C). The six isolates were then inoculated independently of each other to the leaves of 10-day-old bean seedlings growing under normal conditions (25/22 degrees C, 16/8 h photoperiod), and the inoculated and un-inoculated (control) seedlings were transferred to cold (9/5 degrees C, 16/8 h photoperiod) for 3 days. The bacterial inoculations have decreased freezing injury, ice nucleating activity and lipid peroxidation content in parallel with the decrease of reactive oxygen species level such as O-2(center dot-) and H2O2 in the inoculated seedlings compared to the control. In addition, the inoculations of the isolates have stimulated the activity of apoplastic antioxidant enzymes including superoxide dismutase, catalase, peroxidase, and glutathione reductase. The results show that the inoculations improve the cold resistance of bean seedlings and the psychrotolerant bacterial isolates can be evaluated within the group of plant growth promoting bacteria (PGPB) which can increase tolerance of cold sensitive crops.