De novo arginine synthesis is required for full virulence of Xanthomonas arboricola pv. juglandis during Walnut Bacterial Blight Disease

Walnut blight (WB) disease caused by Xanthomonas arboricola pv. juglandis (Xaj) threatens orchards worldwide. Nitrogen metabolism in this bacterial pathogen is dependent on arginine, a nitrogen enriched amino acid that can either be synthesized or provided by the plant host. The arginine biosynthetic pathway uses argininosuccinate synthase (argG), associated with increased bacterial virulence. We examined the effects of bacterial arginine and nitrogen metabolism on the plant response during WB by proteomic analysis of the mutant strain Xaj argG-. Phenotypically, the mutant strain produced 42% fewer symptoms and survived in the plant tissue with 2.5-fold reduced growth compared to wild-type (WT) while auxotrophic for arginine in vitro. Proteomic analysis of infected tissue enabled the profiling of 676 Xaj proteins and 3,296 walnut proteins using isobaric labeling in a data-dependent acquisition approach. Comparative analysis of differentially expressed proteins revealed distinct plant responses. Xaj WT triggered processes of catabolism and oxidative stress in the host under observed disease symptoms, while most host’s biosynthetic processes triggered by Xaj WT were inhibited during Xaj argG- infection. Overall, the Xaj proteins revealed a drastic shift in carbon and energy management induced by disruption of nitrogen metabolism while the top differentially expressed proteins included a Fis transcriptional regulator and a peptidyl-prolyl isomerase. Our results show the critical role of de novo arginine biosynthesis to sustain virulence and minimal growth during WB. This study is timely and critical as current copper-based control methods are losing their effectiveness, and new sustainable methods are urgently needed in orchard environments.

Copper tolerance in Xanthomonas arboricola pv. pruni in South Carolina peach orchards

Bacterial spot of peach, caused by Xanthomonas arboricola pv. pruni (Xap), causes yield loss every year in southeastern United States peach orchards. Management is mainly driven by season-long applications of copper-based products, site location, and choice of cultivar. Although tolerance to copper has not been reported in Xap in the United States, adaptation of populations due to frequent use is a concern. We collected Xap from shoot cankers, leaves, and fruit of cv. O'Henry over two years from three conventional farms and one organic farm in South Carolina, one orchard per farm. The four farms had been using copper extensively for years to control bacterial spot. Xap was isolated from four canker types (bud canker, tip canker, non-concentric canker, and concentric canker) in early spring (‘bud break’), as well as from leaf and fruit tissues later in the season at phenological stages ‘pit hardening’ and ‘final swell’. Xap was most frequently isolated from cankers of the organic farm (24% of the cankers) and most isolates (45%) came from bud cankers. Xap isolates were assessed for sensitivity to copper using minimal glucose yeast agar and nutrient agar amended with 38 µg/ml or 51 µg/ml of Cu2+. Two phenotypes of copper-tolerance in Xap were discovered: low copper tolerance (LCT: growth up to 38 µg/ml Cu2+) and high copper tolerance (HCT: growth up to 51 µg/ml Cu2+). A total of 26 (23 LCT and 3 HCT) out of 165 isolates in 2018 and 32 (20 LCT and 12 HCT) out of 133 isolates in 2019 were tolerant to copper. Peach leaves on potted trees were sprayed with copper rates typically applied at ‘delayed dormancy’ (high rate; 2,397 µg/ml Cu2+), at ‘shuck split’ (medium rate; 599 µg/ml Cu2+), and during ‘summer cover sprays’ (low rate; 120 µg/ml Cu2+) and subsequently inoculated with sensitive, LCT and HCT strains. Results indicated that the low and medium rates of copper reduced bacterial spot incidence caused by the sensitive strain but not by the LCT and HCT strains. This study confirms existence of Xap tolerance to copper in commercial peach orchards in the southeastern United States and suggests its contribution to bacterial spot development under current management practices.

Susceptibility of Some Corylus avellana L. Cultivars to Xanthomonas arboricola pv. corylina

Bacterial blight of hazelnut (Corylus avellana L.) is caused by Xanthomonas arboricola pv. corylina (Xac). In the past, bacterial blight has been a key disease impacting the Oregon hazelnut industry where 99% of the United States hazelnut crop is grown. The disease is re-emerging in young orchards, as acreage of newly released hazelnut cultivars rapidly increases. This increase in hazelnut acreage is accompanied by renewed interest in developing control strategies for bacterial blight. Information on susceptibility of hazelnut cultivars to Xac is limited, partially due to lack of verified methods to quantify hazelnut cultivar response to artificial inoculation. In this research, Xac inoculation protocols were adapted to two hazelnut growing environments to evaluate cultivar susceptibility: in vitro tissue culture under sterile and controlled conditions, and in vivo potted tree conditions. Five hazelnut cultivars were evaluated using the in vitro inoculation protocol and seven hazelnut cultivars were evaluated using the in vivo inoculation protocol. Under in vitro conditions, there were severe bacterial blight symptoms on each cultivar consistent with those seen in the field, but no significant differences in the susceptibility of the newly released cultivars were observed compared to known Xac-susceptible cultivar (“Barcelona”). Under in vivo conditions, the proportion of necrotic buds were significantly higher in “Jefferson” and “Dorris” compared to all of the other tested cultivars, including “Barcelona.” The symptom progression seen in vivo mirrored the timing and symptom progression of bacterial blight reported from field observations. The in vitro conditions significantly reduced the amount of time required to measure the inoculation efficiency compared to the in vivo environment and allowed for greater replication. Further studies on the effects of Xac can use the results of these experiments to establish a dose–response model for bacterial blight, a wider range of germplasm can be tested under in vitro conditions, and management strategies that can be evaluated on large populations of new cultivars using the in vivo methods.

Ppe.XapF: High Throughput KASP Assays to Identify Fruit Response to Xanthomonas arboricola pv. pruni (Xap) in Peach

Bacterial spot, caused by Xanthomonas arboricola pv. pruni (Xap), is a serious peach disease with symptoms that traverse severe defoliation and black surface pitting, cracking or blemishes on peach fruit with global economic impacts. A management option for control and meeting consumer demand for chemical-free, environmentally friendly fruit production is the development of resistant or tolerant cultivars. We developed simple, accurate, and efficient DNA assays (Ppe.XapF) based on SNP genotyping with KASP technology to quickly test for bacterial spot resistance alleles in peach fruit that allows breeders to cull seedlings at the greenhouse stage. The objective of this research was to validate newly developed DNA tests that target the two major QTLs for fruit resistance in peach with diagnostic utility in predicting fruit response to bacterial spot infection. Our study confirms that only two Ppe.XapF DNA tests, Ppe.XapF1-1 and Ppe.XapF6-2, are needed to distinguish between susceptible and resistant alleles. Use of these efficient and accurate Ppe.XapF KASP tests resulted in 44% reduction in seedling planting rate in the Clemson University peach breeding program.

Recombinase Polymerase Amplification/Cas12a-Based Identification of Xanthomonas arboricola pv. pruni on Peach

Peach bacterial spot caused by Xanthomonas arboricola pv. pruni (Xap) is a devastating disease worldwide and frequently causes massive economic losses. In recent years, it has become a pandemic outbreak in most peach production areas of China, especially on precocious peaches in the middle reach of the Yangtze River. Rapid, user-friendly detection is extremely important to make the correct diagnosis and develop suitable control strategies. In this study, we described a recombinase polymerase amplification (RPA)/Cas12a-based system that combines RPA and CRISPR/Cas12a for Xap identification. A total of three crRNAs were designed to target a highly conserved ABC transporter ATP-binding protein-encoding gene ftsX to make specific detection of Xap. Results showed that crRNA 2 and crRNA 3 could get consistent detection for Xap. To realize the visualization of detection results, we additionally introduced FQ-reporter and FB-reporter. The developed method was highly sensitive and could detect as low as 10–18 M Xap gDNA with a mini-UV torch, corresponding to 1.63 copies/μl or 8.855 fg/μl gDNA of Xap, while with lateral flow strips, the sensitivity was 10–17 M. In addition, this method could specifically detect Xap from other closely related bacteria or pathogens associated with peach diseases. Furthermore, this method could make correct identification for Xap with crude DNA using NaOH-based extraction (3 min) directly from diseased peach samples. Considering that the developed method could get results within 2 h and could be performed at 37°C (body temperature), it is promising to be applied for Xap diagnosis and monitoring in fields.

Complete genome and plasmid sequence data of three Xanthomonas arboricola pv. corylina strains, the bacterium responsible for bacterial blight of hazelnut

Xanthomonas arboricola pv. corylina is the causal agent of bacterial blight of hazelnut. The bacterium is listed as A2 quarantine pathogen in Europe since 1978 and on the Regulated Non-Quarantine Pest (RNQP) list since 2020. Three strains from various geographic regions and isolated at different times were sequenced using a hybrid approach with short- and long-read technologies to generate closed genome and plasmid sequences in order to better understand the biology of this pathogen.

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.

Selection of target genes for pcr diagnostics of Xanthomonas arboricola virulent for cereals and brassicas

Plant pathogenic xanthomonads virulent to wheat, rye, barley, tomato, sunflower, and brassicas were isolated in Russia in 2001–2008. Physiological tests and multilocus sequence typing analysis confirmed their position within the Xanthomonas arboricola species. The obtained draft genome sequence of representative strain 3004 from barley plants, which is also virulent to sunflower, brassicas, and chestnut, demonstrated an absence of the Type 3 Secretion System T3SS and an evidence for the lateral gene transfer of some other virulence genes from distantly related bacteria. It was concluded that T4SS genes can be used as the target for group-specific PCR analysis of the emerging pathogen. It was proposed to use virD4, virB3, virB4, and virB9 genes to design a detection system. After preliminary experiments with classic PCR for the chosen genes, primers and TaqMan(R) probe were designed to specifically amplify a 121 bp fragment of the VirD4 gene. Amplification products were obtained for all target Xanthomonas arboricola strains and were not detected in other Xanthomonas species, or in other pathogenic or epiphytic bacteria occurring on these host plants. The assay readily detected Xanthomonas arboricola infection in diseased plants and from bacterial colonies isolated on semi-selective media, and was more sensitive and specific than traditional plating methods.