The leafhopper, Yamatotettix flavovittatus Matsumura is a phytoplasma vector that causes white-leaf disease in sugarcane. The prevalence of Wolbachia infections and their impact on vector biology have been reported; however, their effect on phytoplasma in leafhoppers remains unknown. This research aims to examine the phytoplasma acquisition and retention abilities of Wolbachia-positive and -negative leafhoppers. Phytoplasma infection and titers were evaluated in the vectors after acquisition access periods of 6, 12, 24, and 48 h and latent periods of 7, 14, 21, and 28 days. A significant difference was observed in phytoplasma infection frequency, with a low infection rate in Wolbachia-positive leafhoppers from 6- and 12-h acquisition. Moreover, the phytoplasma titers were reduced in Wolbachia-positive leafhoppers from all acquisition time points (< 7.00 x 10(6) copies), whereas it increased for Wolbachia-negative leafhoppers (ranged from 6.50 to 26.86 x 10(6) copies). At the latent periods, the proportion of phytoplasma infection was 47.06-100% and 83.33-100% for Wolbachia-positive and -negative leafhoppers, respectively. The Wolbachia-positive leafhoppers retained a low phytoplasma titer (< 5 x 10(6) copies) throughout the latent periods, with the titers of Wolbachia and phytoplasma being inversely correlated. By contrast, increased levels, ranging from 12.19 to 30.87 x 10(6) copies were observed in Wolbachia-negative leafhoppers. These results indicate that Wolbachia prevalence negatively affects the phytoplasma acquisition and retention ability of the leafhopper vector. These findings provide useful information that may be utilized in developing new control strategies for vector and phytoplasma diseases.