Gram-positive bacteria are prominent members of plant-associated microbial communities. Although many are hypothesized to be beneficial, some are causative agents of economically important diseases of crop plants. Because the features of Gram-positive bacteria are fundamentally different relative to those of Gram-negative bacteria, the evolution and ecology as well as the mechanisms used to colonize and infect plants also differ. Here, we discuss recent advances in our understanding of Gram-positive, plant-associated bacteria and provide a framework for future research directions on these important plant symbionts.

Clavibacter michiganensis, the causal agent of bacterial canker of tomato, is a Gram‐positive bacterium and a model for studying plant diseases. The real‐time quantitative reverse transcription PCR (real‐time qRT‐PCR) assay is widely used to quantify gene expression in plant pathogenic bacteria. However, accurate quantification of gene expression requires stably expressed reference genes that are consistently expressed during the experimental conditions of interest. The use of inappropriate reference genes leads to a misinterpretation of gene expression data and false conclusions. In current study, we empirically assessed the expression stability of six housekeeping genes (gyrB, rpoB, tufA, bipA, gapA, and pbpA) of C. michiganensis under five experimental conditions using two algorithms, geNorm and NormFinder. C. michiganensis expressed gyrB, bipA, and gapA stably when growing in nutrient‐rich broth (TBY broth and modified M9 broth). We concluded that pbpA, tufA, and gyrB were suitable reference genes in C. michiganensis—tomato interaction studies. We also recommended bipA and rpoB to be used to study bacterial gene expression under nutrient‐poor conditions. Finally, gyrB, pbpA, and rpoB can be used to normalize the quantification of C. michiganensis gene expression while the bacterium is in the viable but nonculturable (VBNC) state. This study identified the most suitable reference genes depending on the experimental conditions for calibrating real‐time qRT‐PCR analyses of C. michiganensis and will be useful in studies that seek to understand the molecular interactions between C. michiganensis and tomato.

Previous research has shown that penicillin-binding proteins (PBPs), enzymes involved in peptidoglycan (PG) assembly, could play an important role during the induction of viable but non-culturable (VBNC) state, which allows non-spore forming bacteria to survive adverse environmental conditions. The current study found that C. michiganensis has a total of seven PBP proteins. Mutant analysis indicated that deletion of either of the class B PBPs was lethal, and that the class A PBP, PBPC, had an important role in PG synthesis, with the ΔpbpC mutant having an altered cellular morphology that resulted in longer cells that were swollen at one end, and had thinner cell walls. The ΔpbpC mutant was also found to produce mucoid colonies in solid culture and a lower final cell titer in liquid medium, as well as having increased sensitivity to osmotic stress and lysozyme treatment, and surprisingly increased pathogenicity. The double mutant, ΔdacB/ΔpbpE also had a slightly altered phenotype resulting in longer cells. Further analysis revealed that both mutants had increased sensitivity to copper, which resulted in quicker induction into the VBNC state. However, only the ΔpbpC mutant had significantly reduced survivorship in the VBNC state. The study also confirmed the VBNC state significantly improved the survivorship of wild-type C. michiganensis cells in response to environmental stresses, and systemically demonstrated the protective role of the VBNC state in C. michiganensis, which is an important finding regarding its epidemiology, and has serious implications for disease management.

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 Cu 2+ 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 Cu 2+ 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.

Xanthomonas campestris pv. campestris (Xcc) is a typical seedborne phytopathogenic bacterium that causes large economic losses worldwide, and this is the first original research article to investigate the role of ppGpp in this important species. Here, we revealed the function of RelA and SpoT in ppGpp production, physiology, pathogenicity, and stress resistance in Xcc.

Clavibacter michiganensis is the causal agent of bacterial canker of tomato, which causes significant economic losses because of the lack of resistant tomato varieties. Chemical control with streptomycin or cupric bactericides is the last defensive line in canker disease management. Streptomycin is an aminoglycoside antibiotic that inhibits protein synthesis and targets the 30S ribosomal protein RpsL. Streptomycin has been used to control multiple plant bacterial diseases. However, identification and characterization of streptomycin resistance in C. michiganensis have remained unexplored. In this study, a naturally occurring C. michiganensis strain TX-0702 exhibiting spontaneous streptomycin resistance was identified, with a minimum inhibitory concentration of 128 μg/ml. Additionally, an induced streptomycin-resistant strain BT-0505-R was generated by experimental evolution of the sensitive C. michiganensis strain BT-0505. Genome sequencing and functional analyses were used to identify the genes conferring resistance. A point mutation at the 128th nucleotide in the rpsL gene of strain BT-0505-R is responsible for conferring streptomycin resistance. However, in TX-0702, resistance is not attributed to mutation of rpsL, streptomycin inactivation enzymes, or multidrug efflux pumps. The mechanism of resistance in TX-0702 is independent of previously reported bacterial loci. Taken together, these data highlight diverse mechanisms used by a Gram-positive plant pathogenic bacterium to confer antibiotic resistance.

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