Effects and side effects of penicillin injection in huanglongbing affected grapefruit trees Keumchul Shin a, b , Marina S. Ascunce a, b , Hossein A. Narouei-Khandan a, b , Xiaoan Sun c , Debra Jones c , Oluwaseun Olawale Kolawole a, b , Erica M. Goss a, b , Ariena H.C. van Bruggen a, b, * a Emerging Pathogens Institute, University of Florida, Gainesville, FL 32611, USA b Department of Plant Pathology, IFAS, University of Florida, Gainesville, FL 32610, USA c Florida Department of Agriculture & Consumer Services, Division of Plant Industry, Gainesville, FL 32614, USA article info Article history: Received 15 July 2016 Received in revised form 22 August 2016 Accepted 24 August 2016 Keywords: Candidatus Liberibacter asiaticus Colony forming units (CFU) qPCR Penicillin resistance Glyphosate resistance Phytophthora nicotianae abstract Trunk injection with penicillin has been tested to control citrus huanglongbing (HLB), but side effects and environmental safety must be assured before approval of penicillin injection can be considered. We investigated effects of penicillin injection on densities of Candidatus Liberibacter asiaticus (Las) in leaves, as well as culturable bacterial populations in rhizospheres and petioles of grapefruit trees in field and greenhouse experiments. Trees were injected with penicillin G, and leaf and root concentrations were assessed in bioassays with Bacillus subtilis. Las densities were determined by qPCR, and bacteria were isolated on a low carbon medium from roots plus rhizosphere and surface-sterilized petioles at various times after penicillin injection. Selected bacterial isolates were tested for penicillin resistance (20 mg/mL) and glyphosate resistance (7000 mg/mL), because glyphosate is widely used and cross-resistance against antibiotics had been documented. One month after penicillin injection half of the greenhouse trees were inoculated with Phytophthora nicotianae. Cycle threshold (Ct) values of Las in old and young leaves significantly increased 90 days after trunk injection with penicillin. Bacterial populations in petioles and root-rhizospheres initially increased after penicillin injections, probably due to nutrient release, then returned to control levels after one week. Penicillin resistance was common in isolates from penicillin- injected and control trees (30e94%). Significantly more glyphosate resistant than sensitive isolates were penicillin resistant (81% versus 52%). Phytophthora root rot was not increased after penicillin injection. Thus, side effects of penicillin injection tested here were minimal, while Las titers were reduced after three months. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction Huanglongbing (HLB) also known as citrus greening is one of the most destructive diseases for Florida citrus production, which constitutes 71% of U.S. citrus production with a value of $1.4 billion in 2012 (Farnsworth et al., 2014; FDACS, 2013). The putative causal agent is the gram negative and phloem-limited a-Proteobacterium Candidatus Liberibacter asiaticus (Las) which is transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama and grafting (Bov e, 2006; Narouei-Khandan et al., 2016; Shimwela et al., 2016; Zhang et al., 2012). While the psyllids, both nymphs and adults, feed on the phloem of infected trees, they acquire the pathogen that can then be transferred to neighboring healthy trees (Chiyaka et al., 2012). The pathogen spreads systemically in the infected tree but few or no symptoms occur for several months up to years after infection (Shen et al., 2013b). The bacterial pathogen causes root degeneration by unknown mechanisms and phloem plugging in infected trees, interfering with the transport of nutrients and eventually leading to tree decline (Bov e, 2006; Johnson et al., 2014). HLB needs to be controlled as soon as possible to avoid collapse of the citrus industry in Florida. Management for HLB has focused on reducing pathogen inoculum and insect vector populations, and production of healthy trees, which can delay disease development (Boina and Bloomquist, 2015; Bov e, 2006; Shen et al., 2013a). However, these practices have not been fully successful. Foliar application of nutrients and systemic resistance-enhancing com- pounds have reduced titers of Las and symptom development * Corresponding author. Emerging Pathogens Institute, University of Florida, Gainesville, FL 32611, USA. E-mail address: ahcvanbruggen@ufl.edu (A.H.C. van Bruggen). Contents lists available at ScienceDirect Crop Protection journal homepage: www.elsevier.com/locate/cropro http://dx.doi.org/10.1016/j.cropro.2016.08.025 0261-2194/© 2016 Elsevier Ltd. All rights reserved. Crop Protection 90 (2016) 106e116