Melia azedarach is a species of enormous value of pharmaceutical industries. Although the chloroplast genome of M. azedarach has been explored, the information of mitochondrial genome (Mt genome) remains surprisingly limited. In this study, we used a hybrid assembly strategy of BGI short-reads and Nanopore long-reads to assemble the Mt genome of M. azedarach. The Mt genome of M. azedarach is characterized by two circular chromosomes with 350,142 bp and 290,387 bp in length, respectively, which encodes 35 protein-coding genes (PCGs), 23 tRNA genes, and 3 rRNA genes. A pair of direct repeats (R1 and R2) were associated with genome recombination, resulting in two conformations based on the Sanger sequencing and Oxford Nanopore sequencing. Comparative analysis identified 19 homologous fragments between Mt and chloroplast genome, with the longest fragment of 12,142 bp. The phylogenetic analysis based on PCGs were consist with the latest classification of the Angiosperm Phylogeny Group. Notably, a total of 356 potential RNA editing sites were predicted based on 35 PCGs, and the editing events lead to the formation of the stop codon in the rps10 gene and the start codons in the nad4L and atp9 genes, which were verified by PCR amplification and Sanger sequencing. Taken together, the exploration of M. azedarach gap-free Mt genome provides a new insight into the evolution research and complex mitogenome architecture.

Bacterial canker of tomato is an economically important seedborne disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm). Copper‐based bactericides and seed treatment with hydrochloric acid are commonly used for bacterial canker management. Recent studies have shown that some bacteria can enter a viable but nonculturable ( VBNC ) state, and fail to form colonies on microbiological agar media. Bacteria in the VBNC state can recover their culturability when returned to favourable conditions. This study reports the induction of the VBNC state in Cmm by CuSO ₄ and low pH, and resuscitation of VBNC cells on tomato seedlings. Flow cytometry using the nucleic acid dyes SYTO 9 and propidium iodide, combined with agar plating, was used to assess VBNC cell counts. It was demonstrated that CuSO ₄ and low pH induced the VBNC state in Cmm and the rate of induction increased with copper ion concentration and acidity. Pathogenicity tests showed that some of the VBNC cells induced by CuSO ₄ retained their ability to colonize tomato seedlings but failed to produce typical bacterial canker symptoms by 2 months post‐inoculation. This was probably due to low levels of resuscitation of VBNC Cmm cells resulting in low levels of initial inoculum. This study has improved understanding of the VBNC state of Gram‐positive phytopathogenic bacteria. Most importantly, because copper‐based chemicals and low pH conditions are used for disease management, induction of the VBNC state and subsequent resuscitation of Cmm cells on tomato seedlings may limit pathogen detection by culture‐based assays yet present a risk for disease development in the field.

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.

Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-borne pathogen that causes bacterial canker disease of tomato. Cmm is typically detected in tomato seeds using quantitative real-time polymerase chain reaction (qPCR) combined with culture-based isolation. The viable but nonculturable (VBNC) state of Cmm may result in the underestimation or false negative detection of the pathogen. In the present study, propidium monoazide (PMA) and its improved structure PMAxx were used to pretreat Cmm prior to DNA extraction, followed by qPCR. Both PMA and PMAxx could bind to the chromosomal DNA of dead bacterial cells and therefore block DNA amplification by PCR. This effect, however, does not occur in living bacterial cells, as the chemicals cannot penetrate through the undamaged cell membrane. Both viable and dead Cmm cells were treated with PMA and PMAxx at various concentrations. With this treatment, the range of the cell population was determined for effective detection. PMAxx showed a better discrimination effect than PMA on the viable and dead cells of Cmm and was therefore used throughout the present study. VBNC cells of Cmm (10⁸ CFU mL^-1) was induced by 50 μM copper sulfate, which was detected at different sampling times up to a month by using both PMAxx-qPCR and flow cytometry assays. The optimal PMAxx concentration was 20 μM for detecting membrane-intact Cmm cells. High specificity and sensitivity were obtained at Cmm concentrations ranging from 10³ to 10⁷ CFU mL^-1. The accurate and robust results of PMAxx-qPCR were confirmed by flow cytometry method to detect viable Cmm cells. Furthermore, the PMAxx-qPCR assay was successfully used in detecting VBNC Cmm cells in tomato seeds with as few as 10 seeds per set.