*3.4. Abundance of ARGs in the Pakchoi Endophytic System*

In total, 23 ARGs corresponding to three antibiotics were detected in antibiotic-treated vegetables using the PCR technique. Among them, only one *tet* gene (*tet*X), one β-lactamase gene (*bla*CTX-M), and two sul genes (*sul*1 and *sul*2) responsible for TC, CPL, and SMX resistance, respectively, were

present in the corresponding antibiotic-treated samples. Thus, further quantification using qPCR was conducted to monitor their responses to different treatment doses. To minimize the differences in background bacterial abundances and DNA extraction efficiency, 16S rRNA gene was also quantified and the absolute numbers of the above four quantified ARGs were normalized to that of the ambient 16S rDNA (Figure 4). In the control plants without antibiotic treatment, the values of the *tet*X gene were under the detection limit but the other three ARGs were detected at ~10−<sup>6</sup> copies/16S rRNA gene copies. For the *tet*X gene, the relative abundance continuously increased as the TC dose increased. Sul and bla genes, on the whole, showed similar changes during the planting period. The four ARGs all reached their highest relative abundances of 10−<sup>5</sup> to 10−<sup>4</sup> copies/16S rRNA gene copies at the MIC exposure levels, which were one to two orders of magnitude greater than those in the control samples. Thus, the variation trends of the *tet*X, *sul*1, *sul*2, and *bla*CTX-M genes during different antibiotic treatments demonstrated great approximations.

**Figure 4.** Abundance of antibiotic resistance genes (ARGs) in the endophytic system of pakchoi under different dosages of antibiotic exposure. (**A**) *tet*X gene in TC-treated plants; (**B**) *bla*CTX-M gene in CPL-treated plants; (**C**) *sul*1 and *sul*2 genes in SMX-treated plants. Values are mean ± SD (*n* = 3). Different letters on the top of the error bars indicate statistical difference among the treatments (*p* < 0.05).
