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

Abstract

Walnut bacterial blight caused by Xanthomonas arboricola pv. juglandis (Xaj) 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 Xaj strains were identified by morphology and specific PCR primers from 206 isolates. The genetic diversity of 60 Xaj strains (59 from Hubei plus one from Beijing) was evaluated by Multilocus Sequence Analysis (MLST), 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 strains were identified whose clustering was consistent with that of glnA. The minimal inhibitory concentration of copper ion on representative Xaj strain DW3F3 (the first genome sequenced Xaj from China) was 115 μg/ml. Setting the copper resistant threshold value to 125 μg/ml, 47 and 13 strains were considered sensitive and resistant to Cu2+, respectively. Furthermore, five strains showed Cu2+ resistance at 270 μg/ml. Compared to the copB from sensitive strains, the copB gene in resistant strains had a 15-bp insertion and eight scattered single nucleotide polymorphisms. Interestingly, the clustering based on MLSA was distinct between Xaj copper ion resistant and sensitive strains.

Short Communication: Genetic diversity of walnut blight-associated bacteria from China using rep-PCR

“…Picking up those colonies that have characteristics consistent with typical Xaj morphology, purifying every single representative colony (Xaj suspected) from the independent sample several times. Sixty isolates identified as Xaj (Fu et al 2021) were cultured on YPGA medium at 26℃ for 3 days, and then stored at -80°C within 30% glycerol for future use.…”

Multilocus Sequence Analysis and Detection of Copper Ion Resistance of Xanthomonas phaseoli pv. manihotis Causing Bacterial Blight in Cassava

“…Using a Cu 2+ concentration of 125 µg/mL (0.5 mM) as the threshold dose, Fu et al (2021) categorized X. arboricola pv. juglandis isolates into resistant and sensitive groups [33]. Previous studies have reported that only some of the studied strains of plant pathogenic bacteria were copper-resistant.…”

“…The gyrB sequences were highly conserved, with the same sequence from 60 X. arboricola pv. juglandis isolates from China [33]. The genetic diversity analysis of 59 X. arboricola pv.…”

“…juglandis was common, the high ratio of copper-resistant isolates was also associated with the heavy use of copper-based bactericides. Two isolates from Danjiangkou, where copper compounds had been frequently applied for more than 10 years, were extremely highly resistant to Cu 2+ (270 µg/mL) [33].…”

Development of Real-time PCR Method for Detection of Xanthomonas campestris pv. juglandis Causing Walnut Bacterial Blight

“…The growing demand for walnut has led to the growing area of walnut cultivation, and walnut pests and diseases are becoming more frequent. Among them, walnut bacterial blight occurs continuously in many places which is widely distributed in walnut producing areas of many cities in China, and is also found in most walnut producing regions in the world (Fu et al 2021;Kim et al 2021;Sagawa et al 2021). This disease is severely harmful to walnut, affecting the healthy development of the walnut industry.…”