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.

Satureja montana Essential Oil, Zein Nanoparticles and Their Combination as a Biocontrol Strategy to Reduce Bacterial Spot Disease on Tomato Plants

Tomato bacterial spot (Bs), caused by Xanthomonas spp., including X. euvesicatoria (Xeu) remains a major threat for tomato production. The emergence of copper resistance strains of Xeu calls urgently for eco-friendly phytosanitary treatments as sustainable green alternatives for disease control. Satureja spp. essential oil (EO) has antimicrobial activity against xanthomonads and combined with zein nanoparticles (ZNPs), might offer a viable option for field applications. This study aims to evaluate the effects of S. montana EO, of ZNPs, and their combination in a nanoformulation, on Xeu quantity, and how these compounds modulate molecular and physiological changes in the pathosystem. Uninfected and infected tomato plants (var. Oxheart) were treated with EO; ZNPs and nanoformulation (EO + ZNPs). Treatments reduced Xeu amount by a minimum of 1.6-fold (EO) and a maximum of 202-fold (ZNPs) and improved plants’ health. Nanoformulation and ZNPs increased plants’ phenolic content. ZNPs significantly increased GPX activity and reduced CAT activity. Overall treatments upregulated transcripts of the phenylpropanoid pathway in infected plants, while ZNPs and nanoformulation upregulated those transcripts in uninfected plants. Both sod and aao transcripts were downregulated by treatments in infected plants. These findings demonstrate that S. montana EO, ZNPs and their nanoformulation are suitable to integrate tomato bacterial spot management strategies, mainly due to their antimicrobial activity on Xeu, however further field studies clarifying the long-term action of these products are required. These results also support the prophylactic potential of ZNPs on tomato bacterial spot.

A long-amplicon viability-qPCR test for quantifying living pathogens that cause bacterial spot in tomato seed

Bacterial spot is one of the most serious diseases of tomato. It is caused by four species of Xanthomonas: X. euvesicatoria, X. gardneri, X. perforans, and X. vesicatoria. Contaminated and/or infected seed can serve as a major source of inoculum for this disease. The use of certified pathogen-free seed is one of the primary management practices to reduce the inoculum load in commercial production. Current seed testing protocols rely mainly on plating the seed extract and conventional PCR, however, the plating method cannot detect viable but non-culturable cells and the conventional PCR assay has limited capability to differentiate DNA extracted from viable versus dead bacterial cells. To improve the sensitivity and specificity of the tomato seed testing method for the bacterial spot pathogens, a long-amplicon qPCR assay coupled with propidium monoazide (PMA-qPCR) was developed to quantify selectively the four pathogenic Xanthomonas species in tomato seed. The optimized PMA-qPCR procedure was evaluated on pure bacterial suspensions, bacteria-spiked seed extracts, and seed extracts of inoculated and naturally-infected seed. A crude DNA extraction protocol also was developed and PMA-qPCR with crude bacterial DNA extracts resulted in accurate quantification of 104-108 CFU/ml of viable bacteria when mixed with dead cells at concentrations as high as 107 CFU/ml in the seed extracts. With DNA purified from concentrated seed extracts, the PMA-qPCR assay was able to detect DNA of the target pathogens in seed samples spiked with ≥75 CFU/ml (~0.5 CFU/seed) of the viable pathogens. Latent class analysis of the inoculated and naturally-infected seed samples showed that the PMA-qPCR assay had greater sensitivity than plating the seed extracts on the semi-selective MTMB and CKTM media for all four target species. Being much faster and more sensitive than dilution plating, the PMA-qPCR assay has a promising potential to serve as a standalone tool or used in combination with the plating method to improve tomato seed testing and advance the production of clean seed.

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.

Identification of Key Transcription Factors Related to Bacterial Spot Resistance in Pepper through Regulatory Network Analyses

Bacterial spot (BS), caused by Xanthomonas campestris pv. Vesicatoria (Xcv), severely affects the quality and yield of pepper. Thus, breeding new pepper cultivars with enhanced resistance to BS can improve economic benefits for pepper production. Identification of BS resistance genes is an essential step to achieve this goal. However, very few BS resistance genes have been well characterized in pepper so far. In this study, we reanalyzed public multiple time points related to RNA-seq data sets from two pepper cultivars, the Xcv-susceptible cultivar ECW and the Xcv-resistant cultivar VI037601, post Xcv infection. We identified a total of 3568 differentially expressed genes (DEGs) between two cultivars post Xcv infection, which were mainly involved in some biological processes, such as Gene Ontology (GO) terms related to defense response to bacterium, immune system process, and regulation of defense response, etc. Through weighted gene co-expression network analysis (WGCNA), we identified 15 hub (Hub) transcription factor (TF) candidates in response to Xcv infection. We further selected 20 TFs from the gene regulatory network (GRN) potentially involved in Xcv resistance response. Finally, we predicted 4 TFs, C3H (p-coumarate 3-hydroxylase), ERF (ethylene-responsive element binding factor), TALE (three-amino-acid-loop-extension), and HSF (heat shock transcription factor), as key factors responsible for BS disease resistance in pepper. In conclusion, our study provides valuable resources for dissecting the underlying molecular mechanism responsible for Xcv resistance in pepper. Additionally, it also provides valuable references for mining transcriptomic data to identify key candidates for disease resistance in horticulture crops.

Attenuations of bacterial spot disease Xanthomonas euvesicatoria on tomato plants treated with biostimulants

Background The bacterial-spot disease caused by different Xanthomonas species is one of the major tomato diseases that reduce crop production and quality. Pesticides indiscriminate usage has resulted in an increase in resistant bacterial strains as well as contamination of farmers, consumers and the environment. Plant growth-promoting bacteria and humic acids can act as elicitors of plant defence mechanism causing extensive transcriptional and metabolic reprogramming which, in turn, produce a range of plant chemical defences. The purpose of this study was to study how humic acids and plant growth-promoting bacteria, when applied to the substrate, affected the severity of bacterial spot symptoms in tomato leaves. Materials and methods One-month-old Micro-Tom tomato (Solanum lycopersicum L.) were transferred to 3 L pots filled with a sterile mixture of sand and vermiculite (2:1, v:v) and treated or not (control) with 250 mL of 4.5 mmol C. L−1 of humic acids, Herbaspirillum seropedicae (108 CFU. mL−1) and the combination of humic acids plus H. seropedicae. One day after substrate treatment, the leaves were inoculated (or not) with X. euvesicatoria (Xe). The area below the disease progression curve based on severity scores and the number of symptomatic leaflets was used to assess phytopathogen virulence. The concentration of oxalic, citric and succinic acids in leaf extracts were determined using HPLC analysis. Results Sole or combined H. seropedicae (BAC) and humic acids (HA) application promoted shoot and root growth related to control when plants were challenged with Xe pathogen. For plants inoculated with Xe, more significant plant-growth promotion results were obtained for HA + BAC treatment. The first visible symptoms were observed 16 days after inoculation with 2 × 104 CFU. g−1 of Xe cells in leaves of control plants. HA and BAC applied alone or combined reduced disease severity. Only plants treated with HA were able to reduce disease incidence (number of the leaflets with symptoms). Organic acids, such as oxalic, citric and succinic acids, rose in Xe-inoculated leaves. The reduced amount of organic acids in diseased leaves treated with HA + BAC may be linked to a decrease in disease progression. Conclusion Humic acids and H. seropedicae increased growth by modulating the content of organic acids in leaf tissue, attenuating the symptoms of the bacterial spot disease. Graphic abstract