Induction and Resuscitation of the Viable but Non-culturable (VBNC) State in Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch of Cucurbitaceous Crops.

Yumin Kan,Ronald Walcott,Jianqiang Li,Saul Burdman,Xin Xu,Laixin Luo,Vinoj Gopalakrishnan,Na Jiang,Qingyang Lyu

doi:10.3389/fmicb.2019.01081

Abstract

Acidovorax citrulli is a gram-negative bacterium that infects a wide range of cucurbits causing bacterial fruit blotch (BFB) disease. Copper-based compounds are the most widely-used chemicals for managing BFB and other bacterial diseases in the field. Many bacteria can enter a viable but non-culturable (VBNC) state in response to stress, including exposure to copper, and recover the culturability when favorable conditions return. The present study demonstrates that A. citrulli strain AAC00-1 is able to enter into the VBNC state by treatment with different concentrations of copper sulfate. It took 3 h, 5 and 15 days for all viable cells to lose culturability upon exposure to copper sulfate concentrations of 50, 10, and 5 μM, respectively. The VBNC A. citrulli cells regained culturability when the Cu2+ ions were removed by chelation with EDTA or by transfer of cells to LB broth, a cell-free supernatant from a suspension of AAC00-1, oligotrophic media amended with casein hydrolysate or watermelon seedling juice. We also found that the VBNC cells induced by Cu2+ were unable to colonize or infect watermelon seedlings directly, but the resuscitated cells recovered full virulence equivalent to untreated bacterial cells in the log phase. To the best of our knowledge, this is the first report on the VBNC state in A. citrulli and the factors that facilitate resuscitation and restoration of pathogenicity.

Highlights

Bacterial fruit blotch (BFB) is a devastating disease of cucurbits that has caused significant economic losses worldwide since the late 1980s (Somodi et al, 1991; Bahar et al, 2009; Burdman and Walcott, 2012)
Six of the eight species of plant–pathogenic bacteria investigated in this topic have been shown to enter the viable but non-culturable (VBNC) state in response to copper, and four of them can resuscitate under appropriate conditions (Alexander et al, 1999; Ghezzi and Steck, 1999; Grey and Steck, 2001; Ordax et al, 2006; Campo et al, 2009; Jiang et al, 2016)
Our study shows for the first time that the gram-negative bacterium A. citrulli can enter the VBNC state in response to exposure to copper sulfate

Summary

Introduction

Bacterial fruit blotch (BFB) is a devastating disease of cucurbits that has caused significant economic losses worldwide since the late 1980s (Somodi et al, 1991; Bahar et al, 2009; Burdman and Walcott, 2012). The causal agent of BFB is Acidovorax citrulli, a seed borne gram-negative bacterium that can infect most cucurbitaceous crops, but is damaging to watermelon and melon (Bahar and Burdman, 2010; Burdman and Walcott, 2012). To date, there are no resistant varieties of cucurbits resistant to A. citrulli (Zhao and Walcott, 2018). Applications of copper-based bactericides, agricultural antibiotics and other protective chemicals can reduce

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