Multilocus Sequence Analysis and Copper Ion Resistance Detection of 60 Xanthomonas arboricola pv. juglandis Isolates...

Walnut bacterial blight caused by Xanthomonas arboricola pv. juglandis has serious repercussions for walnut production around the world. Between 2015 and 2017, disease samples were collected from six counties (Danjiangkou, Baokang, Suizhou, Shennongjia, Zigui, and Xingshan) in Hubei Province, China. Fifty-nine X. arboricola pv. juglandis isolates were identified by morphology and specific PCR primers from 206 isolates. The genetic diversity of 60 X. arboricola pv. juglandis isolates (59 from Hubei plus 1 from Beijing) was evaluated by multilocus sequence analysis, and their resistance to copper ion (Cu²⁺) treatment was determined. A neighbor-joining phylogenetic dendrogram was constructed based on four sequences of housekeeping genes (atpD-dnaK-glnA-gyrB). Two groups of isolates whose clustering was consistent with that of glnA were identified. The minimal inhibitory concentration of Cu²⁺ on representative X. arboricola pv. juglandis strain DW3F3 (the first genome sequenced X. arboricola pv. juglandis from China) was 115 μg/ml. Setting the copper-resistant threshold value to 125 μg/ml, 47 and 13 isolates were considered sensitive and resistant to Cu²⁺, respectively. Furthermore, five isolates showed Cu²⁺ resistance at 270 μg/ml. Compared with the copper resistance gene B (copB) from sensitive isolates, the copB gene in resistant isolates had a 15-bp insertion and eight scattered single-nucleotide polymorphisms. Interestingly, the clustering based on multilocus sequence analysis was distinct between X. arboricola pv. juglandis Cu²⁺-resistant and -sensitive isolates.