Inoculum of Endophytic Bacillus spp. Stimulates Growth of Ex Vitro Acclimatised Apple Plantlets

In vitro shoot culture and cryopreservation (CP) are techniques essential for the ex situ preservation of genetic resources and the production of plant propagation material of clonally propagated horticultural crops. Changes in plant-associated microbiota diversity and composition induced by in vitro cultivation and CP treatment could have a negative effect on the growth and ex vitro adaptation of the in vitro propagated shoots. Therefore, the aim of the present study was to assess changes in endophytic bacteria diversity in domestic apple tissues induced by in vitro cultivation and CP treatment and to investigate the potential of the bacterial inoculum to improve the rooting and ex vitro acclimatisation of the propagated shoots. Metataxonomic analysis revealed a variation in the endophytic bacteria diversity and taxonomic composition between the field-grown tree dormant bud and the in vitro propagated or CP-treated shoot samples of apple cv. Gala. Whereas Sphingobacteriaceae, Sphingomonadaceae, Pseudomonadaceae, and Beijerinckiaceae families were the most prevalent families in the bud samples, Enterobacteriaceae, Bacillaceae, and Lactobacillaceae were dominant in the in vitro shoots. The bacterial inoculum effect on rooting and ex vitro acclimatisation was assessed using four isolates selected by screening the endophytic isolate collection. Bacillus sp. L3.4, B. toyonensis Nt18, or a combined inoculum resulted in a 21%, 36%, and 59% increase in cumulative root length and a 41%, 46%, and 35% increase in the biomass accumulation of ex vitro acclimatised plantlets, respectively. Root zone microbiota functional diversity analysis implied that growth stimulation was not related to improved nutrient uptake but could involve a pathogen-suppressing effect. The results demonstrate that the application of plant growth-promoting bacteria can potentially improve the performance of the in vitro propagated germplasm.