  Citation: Kashyap, A.S.; Manzar, N.; Nebapure, S.M.; Rajawat, M.V.S.; Deo, M.M.; Singh, J.P.; Kesharwani, A.K.; Singh, R.P.; Dubey, S.C.; Singh, D. Unraveling Microbial Volatile Elicitors Using a Transparent Methodology for Induction of Systemic Resistance and Regulation of Antioxidant Genes at Expression Levels in Chili against Bacterial Wilt Disease. Antioxidants 2022, 11, 404. https://doi.org/10.3390/ antiox11020404 Academic Editor: Vittoria Locato Received: 12 January 2022 Accepted: 9 February 2022 Published: 16 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/). antioxidants Article Unraveling Microbial Volatile Elicitors Using a Transparent Methodology for Induction of Systemic Resistance and Regulation of Antioxidant Genes at Expression Levels in Chili against Bacterial Wilt Disease Abhijeet Shankar Kashyap 1,2, * , Nazia Manzar 2 , Suresh M. Nebapure 3 , Mahendra Vikram Singh Rajawat 2 , Man Mohan Deo 4 , Jyoti Prakash Singh 2,5 , Amit Kumar Kesharwani 1 , Ravinder Pal Singh 1 , S. C. Dubey 6,7 and Dinesh Singh 1, * 1 Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi 110012, India; amitmicro1@gmail.com (A.K.K.); ravinder.20033@gmail.com (R.P.S.) 2 Plant Pathology Laboratory, ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan 275103, India; naziamanzar786@gmail.com (N.M.); rajawat.mvs@gmail.com (M.V.S.R.); jyotipsingh58@outlook.com (J.P.S.) 3 Division of Entomology, ICAR-IARI, New Delhi 110012, India; nebapure.mahadev@icar.gov.in 4 Farm Machinery and Power, ICAR-Indian Institute of Pulses Research, Kanpur 208024, India; mm.deo@icar.org.in 5 Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi 110012, India 6 Division of Plant Quarantine, ICAR-NBPGR, New Delhi 110012, India; scdube2002@yahoo.co.in 7 Krishi Bhawan, Indian Council of Agricultural Research, New Delhi 110001, India * Correspondence: abhijeet4497@gmail.com (A.S.K.); dinesh_iari@rediffmail.com (D.S.) Abstract: Microbial volatiles benefit the agricultural ecological system by promoting plant growth and systemic resistance against diseases without harming the environment. To explore the plant growth- promoting efficiency of VOCs produced by Pseudomonas fluorescens PDS1 and Bacillus subtilis KA9 in terms of chili plant growth and its biocontrol efficiency against Ralstonia solanacearum, experiments were conducted both in vitro and in vivo. A closure assembly was designed using a half-inverted plastic bottle to demonstrate plant–microbial interactions via volatile compounds. The most common volatile organic compounds were identified and reported; they promoted plant development and induced systemic resistance (ISR) against wilt pathogen R. solanacearum. The PDS1 and KA9 VOCs significantly increased defensive enzyme activity and overexpressed the antioxidant genes PAL, POD, SOD, WRKYa, PAL1, DEF-1, CAT-2, WRKY40, HSFC1, LOX2, and NPR1 related to plant defense. The overall gene expression was greater in root tissue as compared to leaf tissue in chili plant. Our findings shed light on the relationship among rhizobacteria, pathogen, and host plants, resulting in plant growth promotion, disease suppression, systemic resistance-inducing potential, and antioxidant response with related gene expression in the leaf and root tissue of chili. Keywords: B. subtilis KA9; volatile compounds; GC–MS spectroscopy; systemic resistance; P. fluorescens PDS1; real-time PCR; transmission electron microscopy; Ralstonia solanacearum 1. Introduction Bacterial wilt is a catastrophic soil-borne disease that affects nearly 450 crop species, primarily those belonging to the Solanaceae family [1,2]. Bacterial wilt is found all over the world in tropical and subtropical climates [3]. In the management of soil-borne plant diseases, the biological control approach is an environmentally benign, cost-effective, and simple-to-implement strategy. Plant growth-promoting bacteria are considered the greatest biocontrol agents, with the ability to inhibit the population of pathogenic microbes and induce systemic resistance in plants against disease [4]. Antimicrobial compound produc- tion, nitrogen fixation, phytohormone production, and mineral solubilization are some Antioxidants 2022, 11, 404. https://doi.org/10.3390/antiox11020404 https://www.mdpi.com/journal/antioxidants