  Citation: Srinivasan, J.; Khadka, J.; Novoplansky, N.; Gillor, O.; Grafi, G. Endophytic Bacteria Colonizing the Petiole of the Desert Plant Zygophyllum dumosum Boiss: Possible Role in Mitigating Stress. Plants 2022, 11, 484. https://doi.org/10.3390/ plants11040484 Academic Editor: Bertrand Hirel Received: 7 January 2022 Accepted: 9 February 2022 Published: 11 February 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article Endophytic Bacteria Colonizing the Petiole of the Desert Plant Zygophyllum dumosum Boiss: Possible Role in Mitigating Stress Jansirani Srinivasan 1,2 , Janardan Khadka 1 , Nurit Novoplansky 1 , Osnat Gillor 2 and Gideon Grafi 1, * 1 French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion 84990, Israel; jansirani085@gmail.com (J.S.); janardankhadka@gmail.com (J.K.); nuritnov@bgu.ac.il (N.N.) 2 Zuckerberg Institute for Water Research, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion 84990, Israel; gilloro@bgu.ac.il * Correspondence: ggrafi@bgu.ac.il Abstract: Zygophyllum dumosum is a dominant shrub in the Negev Desert whose survival is accom- plished by multiple mechanisms including abscission of leaflets to reduce whole plant transpiration while leaving the fleshy, wax-covered petioles alive but dormant during the dry season. Petioles that can survive for two full growing seasons maintain cell component integrity and resume metabolic activity at the beginning of the winter. This remarkable survival prompted us to investigate endo- phytic bacteria colonizing the internal tissues of the petiole and assess their role in stress tolerance. Twenty-one distinct endophytes were isolated by culturing from surface-sterile petioles and identified by sequencing of the 16S rDNA. Sequence alignments and the phylogenetic tree clustered the isolated endophytes into two phyla, Firmicutes and Actinobacteria. Most isolated endophytes displayed a relatively slow growth on nutrient agar, which was accelerated by adding petiole extracts. Metabolic analysis of selected endophytes showed several common metabolites whose level is affected by petiole extract in a species-dependent manner including phosphoric acid, pyroglutamic acid, and glutamic acid. Other metabolites appear to be endophyte-specific metabolites, such as proline and trehalose, which were implicated in stress tolerance. These results demonstrate the existence of multiple endophytic bacteria colonizing Z. dumosum petioles with the potential role in maintaining cell integrity and functionality via synthesis of multiple beneficial metabolites that mitigate stress and contribute to stress tolerance. Keywords: desert plants; phyllosphere endophytic bacteria; Zygophyllum dumosum; Actinobacteria; Firmicutes; 16S rRNA genes; endophyte metabolites; stress-related metabolites 1. Introduction Plants are sessile organisms that are constantly exposed to biotic and abiotic stresses. Consequently, plant have evolved a plethora of mechanisms including morphological, chemical, biochemical, molecular, and developmental mechanisms to enable them to cope with their ever-changing environment. This is well demonstrated in desert plants, such as Zygophyllum dumosum Boiss, that rely on multiple mechanisms to survive the vulnerable desert environment [1]. It is a dominant shrub on the rocky limestone southeast-facing slopes of the Negev Desert and is well adapted for growing in the harsh desert environment and can tolerate drought and the high salt conditions that commonly prevail in the desert ecosystem [2,3]. The plant is highly active during the winter producing new leaves and flowers (Figure 1A). The leaf is compound and consist of a pair of fleshy leaflets, which are carried on a thick, fleshy, and wax-covered petiole (Figure 1B). It is semi-deciduous during the dry summer (Figure 1C); that is, it sheds its leaflets, while leaving the thick, fleshy petiole alive but dormant during the dry season (Figure 1D) [4]. At the beginning of the winter, Plants 2022, 11, 484. https://doi.org/10.3390/plants11040484 https://www.mdpi.com/journal/plants