Bacterial contamination is a major and constant threat to the establishment and subculture of in vitro plant culture. In this study, we used a slightly modified qualitative disk diffusion method to screen optimal antibiotics to control the growth of bacterial contaminants isolated from explants of cherry rootstock ‘Gisela 6’. Bacterial susceptibility to eight different antibiotics was tested. The results showed that tetracycline was the most effective antibiotic for controlling bacterial growth; cefotaxime, carbenicillin, kanamycin, and streptomycin were less effective, whereas ampicillin, penicillin, and cefazolin did not inhibit growth of the isolated bacteria. Using the quantitative E test, the minimal inhibitory concentration (MIC) of tetracycline was determined to be 1.0 µg mL−1. We also measured the Fv/Fm values, chlorophyll content, and enzymatic activity of superoxide dismutase and peroxidase to explore the effect of different tetracycline concentrations, 0, 0.064, 0.5, 1.0, 16, and 256 µg mL−1, on the growth of bacteria and explants over 30 days. Results indicated that 1.0 µg mL−1 tetracycline was effective in restricting bacterial growth, with non-significant negative effects on explants at low concentrations, but were enhanced negative effects at high concentrations. The application of the disk diffusion method and E test enabled the identification of an antibiotic and its MIC value effective for eliminating bacterial contaminants while causing minimal damage to explants, indicating a high potential of these methods to control bacterial contaminants in in vitro plant culture.

Background Crown gall disease, caused by the pathogenic bacterium Agrobacterium tumefaciens, is responsible for extensive economic losses in orchards. Cherry rootstock ‘CDR-1’ (Prunus mahaleb) shows high resistance but the mechanism remains unclear. Here, we examined the morphology of pathogen-infected root neck surface, determined the activity of 10 defense-related enzymes and the content of salicylic acid (SA) and jasmonic acid (JA), and also applied transcriptome analysis, transient expression and transgenic verification to explore the crown gall resistance genes in ‘CDR-1’ plants. Results In our study, peroxidase increased in the first 10 days, while phenylalanine ammonialyase and lipoxygenase increased in the first 15 days post-infection. Four key enzymes in the AsA-GSH cycle also responded, to a certain extent; although JA content increased significantly after the treatment, the SA content did not. In a follow-up transcriptome analysis, the differentially expressed genes Pm4CL2, PmCYP450, PmHCT1, PmHCT2, and PmCAD were up-regulated. Based on the above results, we focused on the lignin biosynthetic pathway, and further measured lignin content, and found it increased significantly. The Pm4CL2 gene was used to conduct transient expression and transgenic experiments to verify its function in crown gall disease resistance. It showed the relative expression of the treatment group was almost 14-fold that of the control group at 12 h post-treatment. After the infection treatment, clear signs of resistance were found in the transgenic lines; this indicated that under the higher expression level and earlier activation of Pm4CL2, plant resistance was enhanced. Conclusions The crown gall resistance of ‘CDR-1’ is likely related to the lignin biosynthetic pathway, in which Pm4CL2 functions crucially during the plant defense response to the pathogen A. tumefaciens. The results thus offer novel insights into the defense responses and resistance mechanism of cherry rootstock ‘CDR-1’ against crown gall disease.

Understanding herbivore diversity both at the species and genetic levels is a key to effective pest management. We examined moth samples from multiple locations from a major apple growing region in China. For specimen collection, we used a pheromone trap designed to attract Grapholita molesta (Busck) (Lepidoptera: Tortricidae). Surprisingly, we found a second species captured at high proportions. Its external morphology (e.g., male genitalia and forewing coloration) was the same as for Grapholita funebrana Treitschke (Lepidoptera: Tortricidae) specimens from Europe. However, the barcode sequence of the mitochondrial gene cytochrome oxidase I (COI) diverged markedly between specimens from China and Europe, and the genetic distance value between the specimens from the two regions as estimated using the Juke-Cantor (JC) model amounted to 0.067. These morphological and molecular findings together point to a cryptic species in G. funebrana from China. Further molecular analyses based on COI and COII genes revealed its extremely high genetic diversity, indicating that the origin of this species includes the sampling region. Moreover, molecular data suggest that this species passed through a recent population expansion. This is the first report on a cryptic species in G. funebrana, as well as the first report on its genetic diversity.