Identifying the Physiological Traits of Host-Dependent Endophytes in Grapevines, Using Callus as the Host Material

In vitro-cultured plant calli are colonized by diverse endophytes. As these endophytes are inherited from the maternal plant and appear to be highly dependent on the eco-niche of the host cells, they have been termed host-dependent endophytes (HDEs). HDEs occupy the most intimate microbial environment of plant cells. Nevertheless, our understanding of HDEs and their microenvironmental effects on host plants remains limited due to their cultivation-recalcitrant nature. In this study, grapevine (<i>Vitis vinifera</i> L. × <i>V. labrusca</i> L.) callus was subjected to long-term cultivation in media containing different antibiotics (Q: penicillin; L: streptomycin; Z: nystatin) with the intention of creating grapevine calli with different HDEs. The treated calli were then transferred to an antibiotic-free medium for continuous cultivation. After three cycles of subculture over a total period of 45 days, the endophytic microbiota of the grapevine calli were profiled and their physiological parameters were analyzed. Our results revealed that antibiotic treatments can effectively shape HDEs and create distinct bacterial and fungal HDE microbiota in grapevine calli. Compared to treatment without antibiotics (CK), the Q-treated callus contained more Gram-positive bacterial HDEs but fewer Gram-negative and stress-resistant bacterial HDEs, whereas the Z-treated callus had fewer Gram-positive bacterial HDEs and more Gram-negative, stress-resistant and potentially pathogenic bacterial HDEs. More importantly, grapevine calli with different HDE communities showed varying physiological traits such as respiratory rate, peroxidase activity and total sugar content. Correlative analyses further revealed the functional associations between HDE taxa and callus traits. This work provides an example for studying and utilizing plant HDEs.