Arboricola Pv Research Articles

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 with wild type, while showing itself to be auxotrophic for arginine invitro. 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 wild type (WT) triggered processes of catabolism and oxidative stress in the host under observed disease symptoms, while most of the host 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 copper-based control methods are losing their effectiveness, and new sustainable methods are urgently needed in orchard environments.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Copper Tolerance in Xanthomonas arboricola pv. pruni in South Carolina Peach Orchards.

Bacterial spot of peach, caused by Xanthomonas arboricola pv. pruni, causes yield loss every year in southeastern U.S. 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 X. arboricola pv. pruni in the United States, adaptation of populations from frequent use is a concern. We collected X. arboricola pv. pruni from shoot cankers, leaves, and fruit of cultivar O'Henry over 2 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. X. arboricola pv. pruni was isolated from four canker types (bud canker, tip canker, nonconcentric canker, and concentric canker) in early spring (bud break), as well as from leaf and fruit tissues later in the season at the phenological stages of pit hardening and final swell. X. arboricola pv. pruni was most frequently isolated from cankers of the organic farm (24% of the cankers) and most isolates (45%) came from bud cankers. X. arboricola pv. pruni 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 X. arboricola pv. pruni 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 the stages of delayed dormancy (high rate; 2,397 μg/ml Cu2+), 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 X. arboricola pv. pruni tolerance to copper in commercial peach orchards in the southeastern United States, and suggests its contribution to bacterial spot development under current management practices.

Complete Genome and Plasmid Sequence Data of Three Strains of Xanthomonas arboricola pv. corylina, the Bacterium Responsible for Bacterial Blight of Hazelnut.

Xanthomonas arboricola pv. corylina is the causal agent of bacterial blight of hazelnut. The bacterium has been listed as an A2 quarantine pathogen in Europe since 1978 and on the regulated non-quarantine pest list since 2019. Three isolates 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.

Oxytetracycline and Streptomycin Resistance Genes in Xanthomonas arboricola pv. pruni, the Causal Agent of Bacterial Spot in Peach.

Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot, a major worldwide disease of Prunus species. Very few chemical management options are available for this disease and frequent applications of oxytetracycline (OTC) in the United States peach orchards have raised concerns about resistance development. During 2017–2020, 430 Xap strains were collected from ten peach orchards in South Carolina. Seven OTC-resistant (OTCR) Xap strains were found in 2017 and 2020 from four orchards about 20–270 km apart. Interestingly, the seven strains were also resistant to streptomycin (STR). Six strains grew on media amended with ≤100 μg/mL OTC, while one strain, R1, grew on ≤250 μg/mL OTC. Genome sequence analysis of four representative OTCR strains revealed a 14–20 kb plasmid carrying tetC, tetR, and strAB in each strain. These three genes were transferable to Xanthomonas perforans via conjugation, and they were PCR confirmed in all seven OTCR Xap strains. When tetC and tetR were cloned and expressed together in a sensitive strain, the transconjugants showed resistance to ≤100 μg/mL OTC. When tetC was cloned and expressed alone in a sensitive strain, the transconjugants showed resistance to ≤250 μg/mL OTC. TetC and tetR expression was inducible by OTC in all six wild-type strains resistant to ≤100 μg/mL OTC. However, in the R1 strain resistant to ≤250 μg/mL OTC, tetR was not expressed, possibly due to the presence of Tn3 in the tetR gene, and in this case tetC was constitutively expressed. These data suggest that tetC confers OTC resistance in Xap strains, and tetR regulates the level of OTC resistance conferred by tetC. To our knowledge, this is the first report of OTC resistance in plant pathogenic xanthomonads.

Xanthomonas arboricola pv. fragariae (bacterial leaf blight of strawberry)

This datasheet on Xanthomonas arboricola pv. fragariae covers Identity, Overview, Distribution, Dispersal, Hosts/Species Affected, Diagnosis, Biology & Ecology, Environmental Requirements, Natural Enemies, Impacts, Uses, Prevention/Control, Further Information.

First Report of Shot-Hole on Flowering Cherry Caused by Burkholderia contaminans and Pseudomonas syringae pv. syringae.

Shot-hole disease (SH) is one of the most common and important diseases affecting flowering cherry (FC; Prunus × yedoensis Matsumura; Somei-yoshino) trees in South Korea every year, resulting in premature defoliation and reduced flowering in the following year. However, pathogens associated with the disease remain unknown, which has rendered disease management challenging. Here, the pathogens associated with SH, their biochemical characteristics, and their host range were elucidated. Detached-leaf and in planta assays revealed that two biofilm-forming bacteria-namely, Burkholderia contaminans and Pseudomonas syringae pv. syringae-caused SH of FC trees. These pathogens were recorded for the first time as the causes of SH of FC trees in South Korea. Additionally, the two pathogens induced similar disease symptoms in several stone fruit belonging to the genus Prunus, including peach (Prunus persica), plum (P. salicina), and apricot (P. mume), with peach being the most susceptible. These results indicate that B. contaminans and P. syringae pv. syringae caused SH on FC trees and presented a broad spectrum of hosts. Furthermore, Xanthomonas arboricola pv. pruni, the causative agent of leaf spot on stone fruit, incited brown spots and shot holes on FC leaves. Therefore, FC trees are susceptible to infections by various pathogenic bacteria, including B. contaminans, P. syringae pv. syringae, and X. arboricola pv. pruni. These findings will be of great importance as a reference for effective management of SH in the face of possible cross-infection between Prunus spp. in the future.

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.

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

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.

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.

PM 7/064 (2) Xanthomonas arboricola pv. pruni

Specific scopeThis Standard describes a diagnostic protocol for Xanthomonas arboricola pv. pruni.1This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.Specific approval and amendmentApproved in 2005–09. Revised in 2021–03.

Introduction to Diagnostics Standards

Introduction to Diagnostics Standards

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.

Antagonism and biocontrol of walnut blight by sweet osmanthus endophytic bacterium OFE17

Walnut blight caused by Xanthomonas arboricola pv. juglandis (Xaj) is an important bacterial disease for walnut production worldwide. The objective of the present study was to characterize one endophytic bacterium, namely OFE17 from Osmanthus fragrans leaves, evaluate its potential biocontrol efficiency against the disease, and identify the underlying probable mechanisms of its function. Based on morphology, biochemical and physiological characteristics, 16S-rDNA and gyrB sequences, and antibiotic production genes, the endophyte OFE17 was tentatively identified as Bacillus sp. A disease control efficiency of up to 68.69% was observed through a biocontrol test on detached immature walnut fruits under controlled conditions. OFE17 can produce protease, cellulase, amylase, siderophores, and demonstrates phosphate dissolving ability. However, the OFE17 is unable to produce extracellular lipase, IAA (indoleacetic acid), and has no nitrogen fixation capability. The active compounds of OFE17 were primarily non-protein compounds, and the optimum organic extraction solvent was chloroform. Through specific PCR detection, it contains the genes ituA and ituD which play a key role in active compound synthesis of iturin A synthetase. This study added a promising biocontrol agent candidate for the disease control and laid a foundation for further exploration.

Determining the tolerance of the Turkish hazelnut cultivars (Coryllus avellana) against the pathovar Xanthomonas arboricola pv. corylina (causing agent of fireblight)

Objective: This study was conducted to define the tolerance of various hazelnut cultivars against Xanthomonas arboricola pv. corylina causing agent of bacterial blight. 
 Material and Methods: The Turkish hazelnut cultivars (Acı, Çakıldak, Foşa, İncekara, Kalınkara, Kan, Kargalak, Kuş, Mincane, Palaz, Sivri, Tombul, Uzunmusa, Yassı Badem and Yuvarlak Badem) were used as plant material. AT8/Z strain of Xanthomonas arboricola pv. corylina isolated from the field was tested. Parameters measured to detect bacterial blight tolerance were infected leaf percentage and leaf lesion rate. 
 Results: İncekara showed the minimum percentage of infected leaves (%17.57) and Yassı Badem reflected the highest (%77.51). Yassı Badem had the highest leaf lesion degree (3.00) and İncekara the lowest (1.34). The results suggested that Yassı Badem cultivar was the most susceptible and İncekara the most resistant to the pathogen. The highest antimicrobial activity was represented by Mincane with leaf diameter of 20 mm.
 Conclusion: In this study, resistance to Xanthomonas arboricola pv. corylina pathogen tolerance of Turkish hezelnut cultivars grown in our country was determined. It is thought that the results obtained from the study will shed light on the breeding studies of resistant varieties against this pathogen in the future.

Xanthomonas arboricola pv. juglandis and pv. corylina: Brothers or distant relatives? Genetic clues, epidemiology, and insights for disease management.

BackgroundThe species Xanthomonas arboricola comprises up to nine pathovars, two of which affect nut crops: pv. juglandis, the causal agent of walnut bacterial blight, brown apical necrosis, and the vertical oozing canker of Persian (English) walnut; and pv. corylina, the causal agent of the bacterial blight of hazelnut. Both pathovars share a complex population structure, represented by different clusters and several clades. Here we describe our current understanding of symptomatology, population dynamics, epidemiology, and disease control.Taxonomic statusBacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Lysobacterales (earlier synonym of Xanthomonadales); Family Lysobacteraceae (earlier synonym of Xanthomonadaceae); Genus Xanthomonas; Species X. arboricola; Pathovars: pv. juglandis and pv. corylina.Host range and symptomsThe host range of each pathovar is not limited to a single species, but each infects mainly one plant species: Juglans regia (X. arboricola pv. juglandis) and Corylus avellana (X. arboricola. pv. corylina). Walnut bacterial blight is characterized by lesions on leaves and fruits, and cankers on twigs, branches, and trunks; brown apical necrosis symptoms consist of apical necrosis originating at the stigmatic end of the fruit. A peculiar symptom, the vertical oozing canker developing along the trunk, is elicited by a particular genetic lineage of the bacterium. Symptoms of hazelnut bacterial blight are visible on leaves and fruits as necrotic lesions, and on woody parts as cankers. A remarkable difference is that affected walnuts drop abundantly, whereas hazelnuts with symptoms do not.DistributionBacterial blight of walnut has a worldwide distribution, wherever Persian (English) walnut is cultivated; the bacterial blight of hazelnut has a more limited distribution, although disease outbreaks are currently more frequently reported. X. arboricola pv. juglandis is regulated almost nowhere, whereas X. arboricola pv. corylina is regulated in most European and Mediterranean Plant Protection Organization (EPPO) countries.Epidemiology and controlFor both pathogens infected nursery material is the main pathway for their introduction and spread into newly cultivated areas; additionally, infected nursery material is the source of primary inoculum. X. arboricola pv. juglandis is also disseminated through pollen. Disease control is achieved through the phytosanitary certification of nursery material (hazelnut), although approved certification schemes are not currently available. Once the disease is present in walnut/hazelnut groves, copper compounds are widely used, mostly in association with dithiocarbamates; where allowed, antibiotics (preferably kasugamycin) are sprayed. The emergence of strains highly resistant to copper currently represents the major threat for effective management of the bacterial blight of walnut.Useful websites https://gd.eppo.int/taxon/XANTJU, https://gd.eppo.int/taxon/XANTCY, https://www.euroxanth.eu, http://www.xanthomonas.org

Complete Genome of Xanthomonas arboricola pv. corylina Strain A7 Isolated from Southern Chile.

The complete genome sequence of Xanthomonas arboricola pv. corylina A7 was obtained by a hybrid approach combining PacBio and Illumina HiSeq sequence data. A single circular chromosome of 5.1 mb with 65.47% G + C content was obtained, including 4,344 coding sequences identified as well as some genes involved in copper resistance. The information obtained corresponds to the first report of a high-quality whole genome of X. arboricola pv. corylina, isolated from infected hazelnut trees in southern Chile.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A comparative genomic analysis of Xanthomonas arboricola pv. juglandis strains reveal hallmarks of mobile genetic elements in the adaptation and accelerated evolution of virulence

Xanthomonas arboricola pv. juglandis (Xaj) is the most significant aboveground walnut bacterial pathogen. Disease management uses copper-based pesticides which induce pathogen resistance. We examined the genetic repertoire associated with adaptation and virulence evolution in Xaj. Comparative genomics of 32 Xaj strains reveal the possible acquisition and propagation of virulence factors via insertion sequences (IS). Fine-scale annotation revealed a Tn3 transposon (TnXaj417) encoding copper resistance genes acquired by horizontal gene transfer and associated with adaptation and tolerance to metal-based pesticides commonly used to manage pathogens in orchard ecosystems. Phylogenomic analysis reveals IS involvement in acquisition and diversification of type III effector proteins ranging from two to eight in non-pathogenic strains, 16 to 20 in pathogenic strains, besides six other putative effectors with a reduced identity degree found mostly among pathogenic strains. Yersiniabactin, xopK, xopAI, and antibiotic resistance genes are also located near ISs or inside genomic islands and structures resembling composite transposons.

Prunus persica plant endogenous peptides PpPep1 and PpPep2 cause PTI-like transcriptome reprogramming in peach and enhance resistance to Xanthomonas arboricola pv. pruni

BackgroundRosaceae species are economically highly relevant crops. Their cultivation systems are constrained by phytopathogens causing severe losses. Plants respond to invading pathogens through signaling mechanisms, a component of which are of them being plant elicitor peptides (Peps). Exogenous application of Peps activates defense mechanisms and reduces the symptoms of pathogen infection in various pathosystems. We have previously identified the Rosaceae Peps and showed, in an ex vivo system, that their topical application efficiently enhanced resistance to the bacterial pathogen Xanthomonas arboricola pv. pruni (Xap).ResultsHere we demonstrate the effectiveness of Prunus persica peptides PpPep1 and PpPep2 in protecting peach plants in vivo at nanomolar doses, with 40% reduction of the symptoms following Xap massive infection. We used deep sequencing to characterize the transcriptomic response of peach plants to preventive treatment with PpPep1 and PpPep2. The two peptides induced highly similar massive transcriptomic reprogramming in the plant. One hour, 1 day and 2 days after peptide application there were changes in expression in up to 8% of peach genes. We visualized the transcriptomics dynamics in a background knowledge network and detected the minor variations between plant responses to PpPep1 and PpPep2, which might explain their slightly different protective effects. By designing a P. persica Pep background knowledge network, comparison of our data and previously published immune response datasets was possible.ConclusionsTopical application of P. persica Peps mimics the PTI natural response and protects plants against massive Xap infection. This makes them good candidates for deployment of natural, targeted and environmental-friendly strategies to enhance resistance in Prunus species and prevent important biotic diseases.

Identification and Characterization of Xanthomonas arboricola pv. juglandis Causing Bacterial Blight of Walnuts in Korea.

The present study describes the bacterial blight of walnut, caused by Xanthomonas arboricola pv. juglandis (Xaj) in the northern Gyeongbuk province, Korea. Disease symptoms that appear very similar to anthracnose symptoms were observed in walnut trees in June 2016. Pathogens were isolated from disease infected leaves, fruits, shoots, bud, flower bud of walnut, and cultured onto yeast dextrose carbonate agar plates. Isolated bacteria with bacterial blight symptoms were characterized for their nutrient utilization profiles using Biolog GN2 and Vitek 2. In addition, isolates were subjected to physiological, biochemical, and morphological characterizations. Furthermore, isolates were identified using 16S rDNA sequence analysis, and multi-locus sequence analysis using atpD, dnaK, efp, and rpoD. To confirm pathogenicity, leaves, fruits, and stems of 3-year-old walnut plants were inoculated with bacterial pathogen suspensions as a foliar spray. One week after inoculation, the gray spots on leaves and yellow halos around the spots were developed. Fruits and stems showed browning symptoms. The pathogen Xaj was re-isolated from all symptomatic tissues to fulfill Koch’s postulates, while symptoms were not appeared on control plants. On the other hand, the symptoms were very similar to the symptoms of anthracnose caused by Colletotrichum gloeosporioides. When walnut plants were inoculated with combined pathogens of Xaj and C. gloeosporioides, disease symptoms were greater in comparison with when inoculated alone. Xaj population size was more in the month of April than March due to their dormancy in March, and sensitive to antibiotics such as oxytetracycline and streptomycin, while resistant to copper sulfate.

Selection and validation of reference genes for gene expression studies in Xanthomonas arboricola pv. juglandis subjected to abiotic stress

AbstractThe selection and validation of reference genes for reverse‐transcription quantitative PCR (RT‐qPCR) is key in the normalization of data to evaluate the expression levels of target genes. In this study, the expression levels of nine candidate reference genes in Xanthomonas arboricola pv. juglandis (Xaj) undergoing three abiotic stresses, including thermal stress at 15 ± 1°C and 35 ± 1°C and UV light, were evaluated by RT‐qPCR. A ranked list of reference genes based on expression stability was assessed using BestKeeper, NormFinder, GeNorm, and ΔCt methods. In Xaj undergoing all tested abiotic stress conditions, two reference genes (RGs) were sufficient for effective normalization of RT‐qPCR data. ATP synthase subunit δ (atpD) and RNA polymerase δ factor (rpoD) were the most stably expressed RGs, while gyrase subunit A (gyrA) and gyrase subunit B (gyrB) were the least stable. A combination of the most and least stable RGs was used for the normalization of two isoforms of three target genes, catalase (cat1 and cat2), superoxide dismutase (sod1 and sod2), and glutathione peroxidase (gpx1 and gpx2), showing that the validation of RGs was needed in each experimental condition to avoid misinterpretation of the expression levels of target genes. These results can be useful in studies that seek to understand the biochemical and molecular complex pathways involved in stress tolerance mechanisms in Xaj and to evaluate the effects of stress conditions on plant–pathogen interactions.