A Secreted Chorismate Mutase from Xanthomonas arboricola pv. juglandis Attenuates Virulence and Walnut Blight Symptoms

Walnut blight is a significant above-ground disease of walnuts caused by Xanthomonas arboricola pv. juglandis (Xaj). The secreted form of chorismate mutase (CM), a key enzyme of the shikimate pathway regulating plant immunity, is highly conserved between plant-associated beta and gamma proteobacteria including phytopathogens belonging to the Xanthomonadaceae family. To define its role in walnut blight disease, a dysfunctional mutant of chorismate mutase was created in a copper resistant strain Xaj417 (XajCM). Infections of immature walnut Juglans regia (Jr) fruit with XajCM were hypervirulent compared with infections with the wildtype Xaj417 strain. The in vitro growth rate, size and cellular morphology were similar between the wild-type and XajCM mutant strains, however the quantification of bacterial cells by dPCR within walnut hull tissues showed a 27% increase in XajCM seven days post-infection. To define the mechanism of hypervirulence, proteome analysis was conducted to compare walnut hull tissues inoculated with the wild type to those inoculated with the XajCM mutant strain. Proteome analysis revealed 3296 Jr proteins (five decreased and ten increased with FDR ≤ 0.05) and 676 Xaj417 proteins (235 increased in XajCM with FDR ≤ 0.05). Interestingly, the most abundant protein in Xaj was a polygalacturonase, while in Jr it was a polygalacturonase inhibitor. These results suggest that this secreted chorismate mutase may be an important virulence suppressor gene that regulates Xaj417 virulence response, allowing for improved bacterial survival in the plant tissues.

Screening of Antagonistic Bacteria from Endophytes against Walnut Blight Pathogen Xanthomonas arboricola pv. juglandis

Walnut blight caused by Xanthomonas arboricola pv. juglandis (Xaj) is the most important bacterial disease in walnut production worldwide. To seek biocontrol agents against Xaj, we screened 152 endophytic bacteria isolated from 87 plants. Through dual-culture method screening, we obtained four antagonistic bacteria, ATE17, BME17, CIE17, and OFE17 which were isolated from Amaranthus tricolor, Bambusa multiplex, Canna indica, and Osmanthus fragrans plants respectively. The inhibition ratios of ATE18, BME17, CIE18, and OFE17 against Xaj on plates were 1.5, 1.6, 1.3, and 1.6, respectively. These indicated they have good biocontrol potential for walnut bacterial blight. Subsequently, the four endophytic bacteria were identified by morphology, Gram staining, Microbial Identification System (fatty acid methyl ester analysis), as well as 16S rDNA and gyrB sequencing. It turns out that all four strains were identified as Bacillus sp. Furthermore, the two strains BME17 and OFE17 can suppress multiple plant fungal pathogens and bacterial pathogens on plates.