Efficacy of different copper compounds in the control of Xanthomonas citri subsp. citri pathotypes A and A*

Citrus canker disease is one of the most devastating diseases that attacks citrus, especially limes in the Southern parts of Iran, and is caused by Xanthomonas citri subsp. citri (Xcc). The efficacy of several formulations of copper compounds including Bordeaux mixture, copper oxychloride and copper sulphate in controlling Xcc in Key lime was estimated in vitro and in planta using artificial inoculation. Specific primers were used to detect copper-resistant genes copA, copB and copL in 30 isolates of Xcc. The copA and copL genes were present in all isolates, and copB was detected only in 6 strains. In this study, we observed a very good in vitro growth inhibition activity of copper compounds against Xcc pathotype A. S14 strain (pathotype A^*) was the sole isolate that grew on media amended with 2/4 mM of Bordeaux mixture, copper oxychloride and copper sulphate. All other strains (pathotype A) failed to grow on media amended with this concentration. Bordeaux mixture exhibited high efficacy in controlling Xcc in both conditions. However, there were no significant differences in the efficacy of copper oxychloride and copper sulphate at 1.2 mM concentration in planta. A significantly minimum canker necrotic spot and highest disease control was achieved with Bordeaux mixture and copper oxychloride. There was a significant difference in disease severity of the type strain LMG9322 (pathotype A) and Xcc strain S14 (pathotype A^*). Our experiments showed that Bordeaux mixture exhibited satisfactory efficacy in controlling the causal agent of citrus canker.     

Analysis of the Mutagenic Potential of Copper Compounds and Modification of Their Effect by Silver Iodide

Mutagenic potential of copper compounds and its alteration in case of the interaction with silver compounds were analyzed by use of plant test systems. As test systems,Crepis capillarisL., Tradescantia clone 02, and soybean (Glycine max(L.) Merrill) were used. Mutagenic properties of copper iodide and copper sulfate were not detected. CuI, being not a mutagen by itself, remarkably enhanced mutagenic potential of AgI.     

Assessment of strains of Pseudomonas syringae pv. tomato from Tanzania for resistance to copper and streptomycin

Abstract

Fifty-six strains of Pseudomonas syringae pv. tomato (P.s. pv. tomato) were collected from tomato-producing areas in Tanzania and assessed for resistance to copper and antibiotics. The collection was done from three tomato-producing regions (Morogoro, Arusha and Iringa), representing three different ecological conditions in the country. After isolation and identification, the P. s. pv. tomato strains were grown on King's medium B (KB) amended with 20% copper sulphate (w/v). The strains were also assessed for resistance to antibiotics. Results indicated that there was widespread resistance of the P. s. pv. tomato strains to copper sulphate. The highest level of resistance was recorded from the Arusha region (Northern Tanzania), 83.3% of the P. s. pv. tomato strains from that region showed resistance to copper sulphate. This was followed by Iringa region (Southern Tanzania), from where strains of the pathogen were moderately resistant to copper sulphate, such that 54.0% of them were able to grow on the KB medium amended with 20% (w/v) of the copper compound.

Out of seven strains of P. s. pv. tomato from Morogoro region (Central Tanzania) included in the study, five (71.5%) were resistant to copper sulphate. The only strain of P. s. pv. tomato from the Dodoma region (Central Tanzania, but with a different ecological condition from the Morogoro region) included in the study was unable to grow on the medium containing 20% copper sulphate. None of the P. s. pv. tomato strains in the four regions included in the study were resistant to streptomycin sulphate. These results suggest that in the Arusha and Iringa regions of Tanzania, there might be possibilities of excessive use of copper compounds in tomato production, such that strains of P. s. pv. tomato strains in the areas have become resistant to the compounds.     

The Uniqueness of Superoxide Dismutase (SOD) — Why Cannot Most Copper Compounds Substitute Sod In vivo?

It is shown that the copper zinc superoxide dismutase is unique in its ability to catalyze O₂- dismutation in vivo in contrast to other copper compounds which have this feature only in vitro. The reasons for this difference are discussed in terms of kinetic and thermodynamic parameters.     

Heterozygous <Emphasis Type="BoldItalic">tx</Emphasis> mice have an increased sensitivity to copper loading: Implications for Wilson’s disease carriers

Wilson’s disease carriers constitute 1% of the human population. It is unknown whether Wilson’s disease carriers are at increased susceptibility to copper overload when exposed to chronically high levels of ingested copper. This study investigated the effect of chronic excess copper in drinking water on the heterozygous form of the Wilson's disease mouse model – the toxic milk (tx) mouse. Mice were provided with drinking water containing 300 mg/l copper for 4–7, 8–11, 12–15 or 16–20 months. At the completion of the study liver, spleen, kidney and brain tissue were analyzed by atomic absorption spectroscopy to determine copper concentration. Plasma ceruloplasmin oxidase activity and liver histology were also assessed. Chronic copper loading resulted in significantly increased liver copper in both tx heterozygous and tx homozygous mice, while wild type mice were resistant to the effects of copper loading. Copper loading effects were greatest in tx homozygous mice, with increased extrahepatic copper deposition in spleen and kidney – an effect absent in heterozygote and wild type mice. Although liver histology in homozygous mice was markedly abnormal, no histological differences were noted between heterozygous and wild type mice with copper loading. Tx heterozygous mice have a reduced ability to excrete excess copper, indicating that half of the normal liver Atp7b copper transporter activity is insufficient to deal with large copper intakes. Our results suggest that Wilson’s disease carriers in the human population may be at increased risk of copper loading if chronically exposed to elevated copper in food or drinking water.     

ANTIOXIDANT RESPONSES IN SCYTOSIPHON LOMENTARIA (PHAEOPHYCEAE) INHABITING COPPER‐ENRICHED COASTAL ENVIRONMENTS1

Scytosiphon lomentaria (Lingb.) Link. (Phaeophyceae) is one of the two dominant seaweeds in a coastal area of northern Chile affected by copper mine wastes, where the concentration of copper in water and algal tissues remains higher than in nonimpacted sites. Copper‐loaded plants develop oxidative stress, as demonstrated by the increased levels of reactive oxygen species and lipoperoxides. This stress was associated with 1) an enhanced activity of the antioxidant enzymes catalase, glutathione peroxidase, ascorbate peroxidase, monodehydroascorbate reductase, and dehydroascorbate reductase and 2) an inhibition of the glutathione reductase activity. Furthermore, stressed plants showed a decrease in glutathione and phenolic compounds levels and an increase in total ascorbate. Reciprocal transplants revealed that plants rapidly adjusted their antioxidant system in response to the conditions of the receiving site. In individuals transplanted from the copper‐enriched environment to the control site, normal levels of lipoperoxides and antioxidant compounds were restored in 48 h and antioxidant enzymes recovered their basal activities in 96 h. Individuals transplanted from the control site to the copper‐enriched area adjusted their antioxidant compounds and antioxidant enzymes within 48 h and 96 h, respectively, and reached the functional status of the local plants. We conclude that S. lomentaria inhabiting the copper‐enriched area buffered oxidative stress by a simultaneous involvement of antioxidant enzymes and water‐soluble antioxidant compounds. These antioxidant responses were rapid and reversible, suggesting that copper resistance in S. lomentaria is a constitutive trait and that copper enrichment of the area did not result in a locally adapted copper‐tolerant ecotype.     

Bacillus subtilis QST 713 Confers Protection to Tomato Plants Against Pseudomonas syringae pv tomato and Induces Plant Defence‐related Genes

Bacterial speck, caused by Pseudomonas syringae pv. tomato (Pst), is an economically important disease of tomato, resulting in yield loss of marketable fruit. Management of bacterial speck is a challenge in commercial production fields due to the limited efficacy of current disease management strategies, as the pathogen acquires resistance to antibiotics and fixed copper bactericides and host resistance has not proven durable. Therefore, it is essential to develop alternative disease management strategies, like biological control. In this study, the efficacy of the commercially available biocontrol agent Bacillus subtilis QST 713 along with copper hydroxide was tested against Pst under greenhouse conditions. QST 713 reduced significantly disease severity and incidence compared to control and the copper hydroxide treatment; subsequently, the Pst population was lower in the QST 713‐treated plants compared to control. In parallel, QST 713 and copper hydroxide increased plant height compared to control and mock plants. Furthermore, the quantitative PCR analysis of PR1a, PR1b and Pin2 expression suggests a positive role for Pin2 in the plant protective activity of QST 713, as Pin2 expression was significantly higher in the QST 713‐treated plants challenged with Pst compared to the control Pst‐inoculated plants.     

Copper Nitrate Catalyzed Three-Component One-Pot Synthesis of 3,4-Dihydropyrimidin-2(1H)-Ones

Abstract

An efficient synthesis of 3,4-dihydropyrimidin-2(1H)-ones from aldehydes, 1,3-dicarbonyl compounds, and urea using copper nitrate under refluxing temperature in ethanol was described. Compared with other Lewis copper salts, copper nitrate proved to be the most efficient. The advantages of the new method were good yields (61–93%), short reaction time (0.4–3 h), and inexpensive catalyst.     

Hepatic copper-transporting ATPase ATP7B: function and inactivation at the molecular and cellular level

Copper-transporting ATPase ATP7B (Wilson disease protein) is a member of the P-type ATPase family with characteristic domain structure and distinct ATP-binding site. ATP7B plays a central role in the regulation of copper homeostasis in the liver by delivering copper to the secretory pathway and mediating export of excess copper into the bile. The dual function of ATP7B in hepatocytes is coupled with copper-dependent intracellular relocalization of the transporter. The final destination of ATP7B in hepatocytes during the copper-induced trafficking process is still under debate. We show the results of immunocytochemistry experiments in polarized HepG2 cells that support the model in which elevated copper induces trafficking of ATP7B to sub-apical vesicles, and transiently to the canalicular membrane. In Atp7b ^-/- mice, an animal model of Wilson disease, both copper delivery to the trans-Golgi network and copper export into the bile are disrupted despite large accumulation of copper in the cytosol. We review the biochemical and physiological changes associated with Atp7b inactivation in mouse liver and discuss the pleiotropic consequences of the common Wilson disease mutation, His1069Gln.     

Metal complexes of carboxamidrazone analogs as antitubercular agents: 1. Synthesis, X-ray crystal-structures, spectroscopic properties and antimycobacterial activity against Mycobacterium tuberculosis H37Rv

N¹-Benzylidene-pyridine carboxamidrazones and their metal conjugates have emerged as a new class of potential antimycobacterial agents. Nine such carboxamidrazone analogs (L₁–L₉) along with their Cu(II) (MC₁–MC₉) and Fe(III) (MC₁₀–MC₁₈) complexes were synthesized. Single crystal X-ray structures of copper complexes MC₁ and MC₅ were determined which suggest slightly distorted square planer geometries for copper complexes and octahedral geometries for ferric compounds. All compounds were evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H₃₇Rv. The results show 32–64-fold enhancement in antitubercular activity upon copper complexation.     

ATP6H, a subunit of vacuolar ATPase involved in metal transport: evaluation in canine copper toxicosis

Copper toxicosis (CT), resulting in liver disease, occurs commonly in Bedlington terriers. Canine CT is of particular interest because identification of the causative gene may lead to the discovery of another important gene in the copper transport pathway possibly related to human copper diseases not yet identified. Homologs of the copper transporting ATPase ATP7B, defective in Wilson disease, and the copper chaperone ATOX1 were potential candidates, but both have been excluded. The CT locus in Bedlington terriers has been mapped to canine chromosome region CFA10q26, which has a syntenic human chromosome region, HAS2p13-21. The gene ATP6H, for human vacuolar proton-ATPase subunit M9.2, is associated with copper and iron transport in yeast and has been mapped to HAS2p21 and suggested as a candidate gene for CT. We cloned canine ATP6H, which encodes a predicted protein with 99% amino acid sequence identity to the orthologous human protein. Canine ATP6H shows a conserved potential metal binding site, CSVCC, and a glycosylation site, NET. The canine ATP6H is organized into four exons, with a 246-bp open reading frame. Sequence analysis of the coding regions showed no mutations in ATP6H from genomic DNA of an affected dog. We have also identified two, apparently non-transcribed canine ATP6H pseudogenes. Mapping of the true ATP6H gene and a marker closely linked to the CT locus on a canine radiation hybrid panel indicted lack of close physical association. We have therefore excluded canine ATP6H as a candidate gene for canine copper toxicosis, indicating that some other unidentified gene is responsible for this copper storage disease. Received: 8 February 2001 / Accepted: 12 April 2001     

Evaluation of toxic effects of heavy metals on unicellular algae. V-Influence of extracellular products on toxicity and on type of inhibition

Abstract

Results suggest that the toxicity of mercury and copper to two unicellular algae (Cyanidium caldarium and Chlorella saccharophila) can be decreased either through their subtraction from the culture medium by living or dead cells, or by the extracellular products.

The subtraction of the heavy metals tested manifests itself in each case by a shortening of the lag phase. Further, at least in the case of the combination Chlorella saccharophila-copper, a transition from type III inhibition (increased lag phase) to type II (decreased growth rate) as a results of an increase in the concentration of copper in the presence of extracellular products was observed.

This phenomenon was explained by the different toxic action exerted by copper in its ionic form (type III) as contrasted with copper combined in metalorganic compounds (type II).     

Neurospora tyrosinase: structural,spectroscopic and catalytic properties

Summary Tyrosinase is a copper containing monooxygenase catalyzing the formation of melanin pigments and other polyphenolic compounds from various phenols. This review deals with the recent progress on the molecular structure of the enzyme from Neurospora crassa and the unique features of the binuclear active site copper complex involved in the activation of molecular oxygen and the binding of substrates. The results of the spectroscopic properties of Neurospora tyrosinase will also be discussed in the light of the structural similarity of the copper complex in the oxygen binding hemocyanins.     

Interference of Copper Sulphate in Growth of Erwinia amylovora

To understand the toxicity of copper salts on Erwinia amylovora, which are used in the control of fire blight, bacterial growth and cell metabolism was assayed with copper sulphate in the presence or absence of complex-forming compounds such as various amino acids or citrate. In minimal medium without amino acids copper sulphate strongly interfered with the growth of E. amylovora. A concentration of 15 μm CuSO₄ resulted in about 50% growth inhibition. In contrast to a strong effect of streptomycin, copper ions barely killed the cells when incubated in minimal medium for 1 h. The addition of 4 g asparagine per litre relieved a‘bacteriostatic’effect of copper ions and allowed growth of the bacteria at 2 mm CuSO₄. Other amino acids had a similar effect in the protection of E. amylovora against copper ions. This was in contrast to glycine betain, which was unable to suppress growth inhibition by CuSO₄. Presumably, the free ammonium groups of amino acids participated in the protective effect. The addition of citrate, exceeding the amount of copper-ions, was also protective. Bioluminescence of E. amylovora cells was expressed via a constitutive promoter from the lux-operon of Vibrio fischeri. The light emission is dependent on active cell metabolism. In a novel approach to determine the immediate response of E. amylovora after the addition of copper sulphate, the change of bioluminescence was determined. Addition of copper ions to MM3 medium strongly affected the bioluminescence, but no change in light production was noticed, when citrate or asparagine were present in addition to copper sulphate. A decrease of bioluminescence to 50% was observed for 50 μm CuSO₄ in the absence of amino acids.     

Copper transporting P-type ATPases and human disease

Copper transporting P-type ATPases, designated ATP7A and ATP7B, play an essential role in mammalian copper balance. Impaired intestinal transport of copper, resulting from mutations in the ATP7A gene, lead to Menkes disease in humans. Defects in a similar gene, the copper transporting ATPase ATP7B, result in Wilson disease. This ATP7B transporter has two functions: transport of copper into the plasma protein ceruloplasmin, and elimination of copper through the bile. Variants of ATP7B can be functionally assayed to identify defects in each of these functions. Tissue expression studies of the copper ATPases and their copper chaperone ATOX1 indicate that there is not complete overlap in expression. Other chaperones may be important for the transport of copper into ATP7A and ATP7B.     

Metal binding and antibacterial activity of ciprofloxacin complexes

Four novel cobalt(II), copper(II), nickel(II) and zinc(II) complexes of the fluoroquinolone antibiotic ciprofloxacin have been prepared. The compounds were characterized by IR, UV-Visible, molar conductivity and elemental analyses. In all of the complexes, the drug ligand, ciprofloxacin (CFL) was coordinated through two carbonyl oxygen atoms. Octahedral and square-planar geometries have been proposed for the cobalt(II), nickel(II) and zinc(II), and copper(II) complexes, respectively. In vitro tests of susceptibility to these metal complexes showed stronger activity than that of ciprofloxacin against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Bacillus dysenteriae.     

Copper ions influence the toxicity of β-amyloid(1–42) in a concentration-dependent manner in a <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> model of Alzheimer’s disease

β-amyloid (Aβ) and copper play important roles in the pathogenesis of Alzheimer’s disease (AD). However, the behavioral correlativity and molecular mechanisms of Aβ and copper toxicity have been investigated less often. In the present study, we investigated the interaction and toxicity of Aβ1–42 and copper in the Aβ1–42 transgenic Caenorhabditis elegans worm model CL2006. Our data show that the paralysis behavior of CL2006 worms significantly deteriorated after exposure to 10⁻³ mol L⁻¹ copper ions. However, the paralysis behavior was dramatically attenuated with exposure to 10⁻⁴ mol L⁻¹ copper ions. The exogenous copper treatment also partially changed the homeostatic balance of zinc, manganese, and iron. Our data suggest that the accumulation of reactive oxygen species (ROS) was responsible for the paralysis induced by Aβ and copper in CL2006. The ROS generation induced by Aβ and copper appear to be through sod-1, prdx-2, skn-1, hsp-60 and hsp-16.2 genes.     

Identification of a copper-responsive promoter and development of a copper biosensor in the soil bacterium <Emphasis Type="Italic">Achromobacter</Emphasis> sp. AO22

A number of human activities result in environmental contamination with copper compounds that can cause severe detrimental effects on the ecosystem as well as human health. The physico-chemical methods of metal detection have limitations such as inability to distinguish between total versus bio-available metals and differences in metal uptake in different organisms. The heavy metal resistance-encoding genetic systems of certain bacteria provide critical tools for development of biosensors for these purposes. This study reports a copper biosensor utilizing the cop operon of the heavy metal resistant bacterial isolate, Achromobacter sp. AO22, isolated from a contaminated site in Australia. A section located between the divergently transcribed putative response regulator gene copR and multicopper oxidase gene copA that included a palindromic cop box was identified as a copper-responsive promoter using a lacZ reporter construct, pCOPRP, in E. coli. The expression was found to be enhanced by inclusion of copR. Another engineered strain, AO22(pCOPRP), showed stronger induction, and the lacZ expression in both backgrounds was enhanced significantly (250–400 fold) by copper but minimally by other metals. The construct in Achromobacter sp. AO22 thus has a high potential as biosensor for detecting copper bioavailability (hence potential toxicity) in a soil bacterial background, while the construct in E. coli is ideal for laboratory-based testing.     

Chronological changes in tissue copper, zinc and iron in the toxic milk mouse and effects of copper loading

The toxic milk (tx) mouse is a rodent model for Wilson disease, an inherited disorder of copper overload. Here we assessed the effect of copper accumulation in the tx mouse on zinc and iron metabolism. Copper, zinc and iron concentrations were determined in the liver, kidney, spleen and brain of control and copper-loaded animals by atomic absorption spectroscopy. Copper concentration increased dramatically in the liver, and was also significantly higher in the spleen, kidney and brain of control tx mice in the first few months of life compared with normal DL mice. Hepatic zinc was increased with age in the tx mouse, but zinc concentrations in the other organs were normal. Liver and kidney iron concentrations were significantly lower at birth in tx mice, but increased quickly to be comparable with control mice by 2 months of age. Iron concentration in the spleen was significantly higher in tx mice, but was lower in 5 day old tx pups. Copper-loading studies showed that normal DL mice ingesting 300 mg/l copper in their diet for 3 months maintained normal liver, kidney and brain copper, zinc and iron levels. Copper-loading of tx mice did not increase the already high liver copper concentrations, but spleen and brain copper concentrations were increased. Despite a significant elevation of copper in the brain of the copper-loaded tx mice no behavioural changes were observed. The livers of copper-loaded tx mice had a lower zinc concentration than control tx mice, whilst the kidney had double the concentration of iron suggesting that there was increased erythrocyte hemolysis in the copper-loaded mutants.