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.

Evaluation of Streptomyces species from historically sewage sludge contaminated soils for tolerance to copper(II) toxicity

Pollution of the environment by copper(II) exerts numerous adverse impacts on soils, microorganisms, plants, animals and humans. Assessing these impacts on microorganisms is essential as they are potential agents of bioremediation to purify the ecosystem. The aim of this study was to investigate the tolerance of Streptomyces species obtained from historically contaminated soils to copper (II) toxicity. Soil samples were collected from farms around sewage sludge dump sites (tests) and where there were no dumps (control). Standard microbiological methods were used to isolate, characterize and identify the three best copper tolerant species. Also, the impact on Streptomyces growth characteristics such as radial growth (R2), index of growth (GI) and percentage inhibition of radial growth (PIRG) by copper (II) was examined. Results obtained indicated that; the minimum inhibitory concentration of copper (II) for some of the Streptomyces isolates was 600 mg/l. However, three isolates; SW2B, SW7A and SW7B had MIC of 3000 mg/l and were selected for further investigations. Increasing metal concentration from 100 mg/l to 2000 mg/l reduced R2 from 40.2 to 4.0 and GI from 0.77 to 0.08, corresponding to 69.48 % increase in PIRG for SW2B. For isolate SW7A, R2 and GI decreased from 33.7 to 3.8 and from 0.67 to 0.08 respectively as copper (II) concentration increased from 100 mg/l to 2000 mg/l, giving 59.33 % increase in PIRG. The highest increase in PIRG (88.04 %) was obtained for SW7B, corresponding to a decrease of R2 and GI from 39.2 to 5.2 and 0.77 to 0.10 respectively. Molecular method was used to identify the isolates as Streptomyces chartreusis (SW2B), Streptomyces aureoverticillatus (SW7A) and Streptomyces subrutilus (SW7B).

Local adaptation of Legionella pneumophila within a hospital hot water system increases tolerance to copper

In large-building water systems, Legionella pneumophila is exposed to common environmental stressors such as copper. The aim of this study was to evaluate the susceptibility to copper of L. pneumophila isolates recovered from various sites: two clinical and seven environmental from hot water systems biofilm & water, and from cooling tower water. After one-week acclimation in simulated drinking water, strains were exposed to various copper concentrations (0.8 to 5 mg/L) for over 672 hours. Complete loss of culturability was observed for three isolates, following copper exposure to 5 mg/L for 672h. Two ST1427-like isolates were highly sensitive to copper, while the other two, isolated from biofilm samples, maintained higher culturability. The expression of the copper resistance gene copA evaluated by RT-qPCR was significantly higher for the biofilm isolates. All four ST1427-like isolates were recovered from the same water system during an outbreak. Whole genome sequencing results confirmed that the four isolates are very close phylogenetically, differing by only 29 single nucleotide polymorphisms, suggesting in situ adaptation to microenvironmental conditions, possibly due to epigenetic regulation. These results indicate that the immediate environment within a building water distribution system influences the tolerance of L. pneumophila to copper. Increased contact of L. pneumophila biofilm strains with copper piping or copper alloys in the heat exchanger might lead to local adaptation. The phenotypic differences observed between water and biofilm isolates from the hot water system of a healthcare facility warrants further investigation to assess the relevance of evaluating disinfection performances based on water sampling alone. Importance Legionella pneumophila is a pathogen indigenous to natural and large building water systems in the bulk and the biofilm phases. The immediate environment within a system can impact the tolerance of L. pneumophila to environmental stressors, including copper. In healthcare facilities, copper levels in water can vary, depending on water quality, plumbing materials and age. This study evaluated the impact of the isolation site (water vs biofilm, hot water system vs cooling tower) within building water systems. Closely related strains isolated from a healthcare facility hot water system exhibited variable tolerance to copper stress shown by differential expression of copA, with biofilm isolates displaying highest expression and tolerance. Relying on the detection of L. pneumophila in water samples following exposure to environmental stressor such as copper may underestimate the prevalence of L. pneumophila, leading to inappropriate risk management strategies and increasing the risk of exposure for vulnerable patients.

Subnuclear gene positioning through lamina association affects copper tolerance

The nuclear lamina plays an important role in the regulation of chromatin organization and gene positioning in animals. CROWDED NUCLEI (CRWN) is a strong candidate for the plant nuclear lamina protein in Arabidopsis thaliana but its biological function was largely unknown. Here, we show that CRWNs localize at the nuclear lamina and build the meshwork structure. Fluorescence in situ hybridization and RNA-seq analyses revealed that CRWNs regulate chromatin distribution and gene expression. More than 2000 differentially expressed genes were identified in the crwn1crwn4 double mutant. Copper-associated (CA) genes that form a gene cluster on chromosome 5 were among the downregulated genes in the double mutant exhibiting low tolerance to excess copper. Our analyses showed this low tolerance to copper was associated with the suppression of CA gene expression and that CRWN1 interacts with the CA gene locus, enabling the locus to localize at the nuclear lamina under excess copper conditions.

Enterococci and Bacilli from surface water: assessment of their resistance to copper and antibiotics

Heavy metal-resistant bacteria can be efficient bioremediators of metals and might provide an alternative method for metal removal in contaminated environments. The present study aims to isolate bacteria from the aquatic environment and evaluate their potential tolerance to copper metal, aiming at bioremediation processes. Also, compare co-resistance to heavy metal and antibiotics. The morphology of isolates was observed, and sequence analysis (16S ribosomal DNA) revealed that isolated strains were closely related to species belonging to the genera Enterococcus and Bacillus. Bacterial isolates were resistant to CuSO4, with a minimum inhibitory concentration of 0.78 mg ml-1. Enterococcus lactis was resistant to a combination of copper and tetracycline. The other tested isolates were sensitive to the tested antimicrobials. The metal removal ability of these isolates was assayed using atomic absorption spectroscopy, and the strains 27, 23, and E. lactis were best at removing heavy metals, at 87.7%. Enterococcus casseliflavus EC55 was 62%, followed by Bacillus aerius (18.4%), E. casseliflavus EC70 (10%) and Bacillus licheniformis (10%). Based on our findings, Enterococcus sp and Bacillus sp. have potential applications in enhanced remediation of contaminated environments.

Local adaptation of Legionella pneumophila within a hospital hot water system increases tolerance to copper

In large-building water systems, Legionella pneumophila is exposed to common environmental stressors such as copper. The aim of this study was to evaluate the susceptibility to copper of L. pneumophila isolates recovered from various sites: two clinical and seven environmental from hot water systems biofilm & water, and from cooling tower water. After one-week acclimation in simulated drinking water, strains were exposed to various copper concentrations (0.8 to 5 mg/L) for over 672 hours. Complete loss of culturability was observed for three isolates, following copper exposure to 5 mg/L for 672h. Two ST1427-like isolates were highly sensitive to copper, while the other two, isolated from biofilm samples, were resistant. The expression of the copper resistance gene copA evaluated by RT-qPCR was significantly higher for the biofilm isolates. All four ST1427-like isolates were recovered from the same water system during an outbreak. Whole genome sequencing results confirmed that the four isolates are very close phylogenetically, differing by only 29 single nucleotide polymorphisms, suggesting in situ adaptation to microenvironmental conditions, possibly due to epigenetic regulation. These results indicate that the immediate environment within a building water distribution system influences the tolerance of L. pneumophila to copper. Increased contact of L. pneumophila biofilm strains with copper piping or copper alloys in the heat exchanger might lead to local adaptation. The phenotypic differences observed between water and biofilm isolates from the hot water system of a healthcare facility warrants further investigation to assess the relevance of evaluating disinfection performances based on water sampling alone.ImportanceLegionella pneumophila is a pathogen indigenous to natural and large building water systems in the bulk and the biofilm phases. The immediate environment within a system can impact the tolerance of L. pneumophila to environmental stressors, including copper. In healthcare facilities, copper levels in water can vary, depending on water quality, plumbing materials and age. This study evaluated the impact of the isolation site (water vs biofilm, hot water system vs cooling tower) within building water systems. Closely related strains isolated from a healthcare facility hot water system exhibited variable tolerance to copper stress shown by differential expression of copA, with biofilm isolates displaying highest expression and tolerance. Relying on the detection of L. pneumophila in water samples following exposure to environmental stressor such as copper may underestimate the prevalence of L. pneumophila, leading to inappropriate risk management strategies and increasing the risk of exposure for vulnerable patients.

Reduced Virulence of an Introduced Forest Pathogen over 50 Years

Pathogen incursions are a major impediment for global forest health. How pathogens and forest trees coexist over time, without pathogens simply killing their long-lived hosts, is a critical but unanswered question. The Dothistroma Needle Blight pathogen Dothistroma septosporum was introduced into New Zealand in the 1960s and remains a low-diversity, asexual population, providing a unique opportunity to analyze the evolution of a forest pathogen. Isolates of D. septosporum collected from commercial pine forests over 50 years were compared at whole-genome and phenotype levels. Limited genome diversity and increased diversification among recent isolates support the premise of a single introduction event. Isolates from the 1960s show significantly elevated virulence against Pinus radiata seedlings and produce higher levels of the virulence factor dothistromin compared to isolates collected in the 1990s and 2000s. However, later isolates have no increased tolerance to copper, used in fungicide treatments of infested forests and traditionally assumed to be a strong selection pressure. The isolated New Zealand population of this forest pathogen therefore appears to have become less virulent over time, likely in part to maintain the viability of its long-lived host. This finding has broad implications for forest health and highlights the benefits of long-term pathogen surveys.

Bacteria Associated with Onion Foliage in Michigan and Their Copper Sensitivity

Bacterial stalk and leaf necrosis of onion, Allium cepa L., is a concern of Michigan producers. Plants with symptoms of bacterial stalk and leaf necrosis were collected in 2013 and 2014 from 17 fields in six Michigan counties. Bacterial isolates were identified using Biolog and confirmed by sequencing the 16s rDNA gene. From a total of 414 isolates, 10 bacterial species were identified. Pantoea agglomerans (42.5%), Pantoea ananatis (17.4%), and Enterobacter cowanii (7.5%) were most prevalent and were tested for pathogenicity on onion foliage and bulbs. More P. ananatis isolates (92%) were pathogenic on seedlings than P. agglomerans or E. cowanii isolates (approximately 50%). When 197 bacterial isolates were tested for sensitivity to copper hydroxide (200 µg/ml), 41% of P. agglomerans isolates were found to be tolerant to copper hydroxide, whereas 19 and 22% of P. ananatis and E. cowanii isolates were tolerant, respectively. Identifying the bacterial species associated with foliar disease symptoms on onions in Michigan, their pathogenicity and tolerance to copper is an important step in developing improved disease management strategies.