Mechanisms Of Copper Tolerance Research Articles

Comparison of Copper-Tolerance Genes between Different Groups of Acidovorax citrulli.

Acidovorax citrulli populations exhibit genetic and phenotypic variations, particularly in terms of copper tolerance. Group I strains of A. citrulli generally exhibit higher copper tolerance compared to group II strains. This study aims to identify genes involved in copper tolerance to better understand the differences in copper tolerance between group I and group II strains. Representative strains pslb65 (group I) and pslbtw14 (group II) were selected for comparison. Deletion mutants of putative copper-tolerance genes and their corresponding complementary strains were constructed. The copper tolerance of each strain was evaluated using the minimum inhibitory concentration method. The results showed that the copA, copZ, cueR, and cueO genes played major roles in copper tolerance in A. citrulli, while cusC-like, cusA-like, and cusB-like genes had minor effects. The different expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 under copper stress indicated that they had different mechanisms for coping with copper stress. Overall, this study provides insights into the mechanisms of copper tolerance in A. citrulli and highlights the importance of specific genes in copper tolerance.

Systematic investigation of simultaneous copper biosorption and nitrogen removal from wastewater by an aerobic denitrifying bacterium of auto-aggregation

Finding effective methods for simultaneous removal of eutrophic nutrients and heavy metals has attracted increasing concerns for the environmental remediation. Herein, a novel auto-aggregating aerobic denitrifying strain (Aeromonas veronii YL-41) was isolated with capacities for copper tolerance and biosorption. The denitrification efficiency and nitrogen removal pathway of the strain were investigated by nitrogen balance analysis and amplification of key denitrification functional genes. Moreover, the changes in the auto-aggregation properties of the strain caused by extracellular polymeric substances (EPS) production were focused on. The biosorption capacity and mechanisms of copper tolerance during denitrification were further explored by measuring changes in copper tolerance and adsorption indices, as well as by variations in extracellular functional groups. The strain showed extremely strong total nitrogen removal ability, with 67.5%, 82.08% and 78.48% of total nitrogen removal when NH4+-N, NO2−-N, and NO3−-N were used as the only initial nitrogen source, respectively. The successful amplification of napA, nirK, norR, and nosZ genes further demonstrated that the strain accomplished nitrate removal through a complete aerobic denitrification pathway. The production of protein-rich EPS of up to 23.31 mg/g and an auto-aggregation index of up to 76.42% may confer a strong biofilm-forming potential to the strain. Under the stress of 20 mg/L copper ions, the removal of nitrate-nitrogen was still as high as 71.4%. In addition, the strain could achieve an efficient removal of 96.9% of copper ions at an initial concentration of 80 mg/L. Scanning electron microscopy and deconvolution analysis of characteristic peaks confirmed that the strains encapsulate heavy metals by secreting EPS and, meanwhile, form strong hydrogen bonding structures to enhance intermolecular forces to resist copper ion stress. This study provides an innovative and effective biological approach for the synergistic bioaugmentation removal of eutrophic substances and heavy metals from aquatic environments.

Copper Tolerance Mechanism of the Novel Marine Multi-Stress Tolerant Yeast Meyerozyma guilliermondii GXDK6 as Revealed by Integrated Omics Analysis.

Various agricultural products used in food fermentation are polluted by heavy metals, especially copper, which seriously endangers human health. Methods to remove copper with microbial strategies have gained interests. A novel Meyerozyma guilliermondii GXDK6 could survive independently under high stress of copper (1400 ppm). The copper tolerance mechanism of GXDK6 was revealed by integrated omics in this work. Whole-genome analysis showed that nine genes (i.e., CCC2, CTR3, FRE2, GGT, GST, CAT, SOD2, PXMP4, and HSP82) were related to GXDK6 copper tolerance. Copper stress elevated glutathione metabolism-related gene expression, glutathione content, and glutathione sulfur transferase activity, suggesting enhanced copper conjugation and detoxification in cells. The inhibited copper uptake by Ctr3 and enhanced copper efflux by Ccc2 contributed to the decrease in intracellular copper concentration. The improved expression of antioxidant enzyme genes (PXMP4, SOD2, and CAT), accompanied by the enhanced activities of antioxidant enzymes (peroxidase, superoxide dismutase, and catalase), decreased copper-induced reactive oxygen species production, protein carbonylation, lipid peroxidation, and cell death. The metabolite D-mannose against harsh stress conditions was beneficial to improving copper tolerance. This study contributed to understanding the copper tolerance mechanism of M. guilliermondii and its application in removing copper during fermentation.

Gene Expression Analysis of Three Putative Copper-Transporting ATPases in Copper-Tolerant Fibroporia radiculosa.

Copper tolerance of brown-rot basidiomycete decay fungi can lessen the efficacy of copper-containing wood preservatives for wood products in-service. The purpose of this study was to evaluate wood mass loss and differential expression of three genes that have putative annotations for copper-transporting ATPase pumps (FIBRA_00974, FIBRA_04716, and FIBRA_01430). Untreated southern pine (SP) and SP treated with three concentrations of ammoniacal copper citrate (CC, 0.6, 1.2, and 2.4%) were exposed to two copper-tolerant Fibroporia radiculosa isolates (FP-90848-T and L-9414-SP) and copper-sensitive Gloeophyllum trabeum isolate (MAD 617) in a 4-week-long standard decay test (AWPA E10-19). Decay of copper-treated wood was inhibited by G. trabeum (p = 0.001); however, there was no inhibition of decay with increasing copper concentrations by both F. radiculosa isolates. Initially, G. trabeum and one F. radiculosa isolate (L-9414-SP) highly upregulated FIBRA_00974 and FIBRA_04716 on copper-treated wood at week 1 (p = 0.005), but subsequent expression was either not detected or was similar to expression on untreated wood (p = 0.471). The other F. radiculosa isolate (FP-90848-T) downregulated FIBRA_00974 (p = 0.301) and FIBRA_04716 (p = 0.004) on copper-treated wood. FIBRA_01430 expression by G. trabeum was not detected, but was upregulated by both F. radiculosa FP-90848-T (p = 0.481) and L-9414-SP (p = 0.392). Results from this study suggest that all three test fungi utilized different mechanisms when decaying copper-treated wood. Additionally, results from this study do not provide support for the involvement of these putative gene annotations for copper-transporting ATPase pumps in the mechanism of copper-tolerance.

Copper stress by nutritional immunity activates the CusS-CusR two-component system that contributes to Vibrio alginolyticus anti-host response but affects virulence-related properties

Copper is indispensable for cellular functions, but high levels of copper will destruct biological macromolecules. Copper accumulation was recently found to appear where the bacterial infection took place. This proved that copper resistance is necessary for bacteria virulence. Almost all bacteria, from isolated environmental bacteria to human pathogenic bacteria, have copper tolerance mechanisms. We found Vibrio alginolyticus, an emerging microbe distributed widely in tropical coastal areas, had the copper resistance phosphate regulon CusS-CusR-CusCFBA. After Cu2+ treatment, the expression of cusS, cusR, cusCFBA, copB and [EC:3.6.3.4] were significantly up-regulated. These genes were related to copper tolerance, among which cusS and cusR played a key role. When cusS and cusR were silenced, the expression levels of cusCFBA were also significantly decreased. Unexpectedly, the adhesion, hemolysis, biofilm formation, motility, and virulence were significantly increased in the silencing strains of cusS and cusR. Meanwhile, promoter region analysis, EMSA, and LacZ reporter gene analysis suggested that CusR played a role in the regulation of cusCFBA and multiple virulence genes. Together, these indicated that although CusSR could confer resistance to the host defense by regulating the cusCFBA to participate in the copper tolerance mechanism, the activation of the CusSR by copper stress from nutritional immunity would reduce the V. alginolyticus pathogenesis.

Copper Tolerance Mediated by FgAceA and FgCrpA in Fusarium graminearum.

All organisms must secure essential trace elements (e.g., Cu) for survival and reproduction. However, excess trace element accumulation in cells is highly toxic. The maintenance of copper (Cu) homeostasis has been extensively studied in mammals, bacteria, and yeast but not in plant pathogens. In this study, we investigated the molecular mechanisms of copper tolerance in Fusarium graminearum, the important wheat head scab fungus. RNA-seq revealed induced expression of the P-type ATPase transporter FgCrpA and metallothionein (MT) FgCrdA after excess Cu treatment. Deletion of FgCrpA but not FgCrdA resulted in reduced tolerance to Cu toxicity. The “Cu fist” transcription factor FgAceA was involved in Cu detoxification through activation of FgCrpA. △FgAceA was more sensitive to copper toxicity than △FgCrpA and overexpression of FgCrpA restored copper tolerance in △FgAceA. FgAceA negatively regulated aurofusarin production and its biosynthetic gene expression. △FgCrpA and △FgAceA were reduced in virulence in flowering wheat heads and synthesized decreased amounts of the mycotoxin deoxynivalenol when challenged with excess Cu. Taken together, these results suggest that mediation of Cu tolerance in F. graminearum mainly relies on the Cu efflux pump and that FgAceA governs Cu detoxification through activation of FgCrpA.

Copper-induced concomitant increases in photosynthesis, respiration, and C, N and S assimilation revealed by transcriptomic analyses in Ulva compressa (Chlorophyta)

BackgroundThe marine alga Ulva compressa is the dominant species in copper-polluted coastal areas in northern Chile. It has been shown that the alga tolerates micromolar concentrations of copper and accumulates copper at the intracellular level. Transcriptomic analyses were performed using total RNA of the alga cultivated with 10 μ M copper for 0, 1, 3 and 5 days using RNA-seq in order to identify processes involved in copper tolerance.ResultsThe levels of transcripts encoding proteins belonging to Light Harvesting Complex II (LHCII), photosystem II (PSII), cytochrome b6f, PSI, LHCI, ATP synthase and proteins involved in repair of PSII and protection of PSI were increased in the alga cultivated with copper. In addition, the level of transcripts encoding proteins of mitochondrial electron transport chain, ATP synthase, and enzymes involved in C, N and S assimilation were also enhanced. The higher percentages of increase in the level of transcripts were mainly observed at days 3 and 5. In contrast, transcripts involved protein synthesis and degradation, signal transduction, and replication and DNA repair, were decreased. In addition, net photosynthesis and respiration increased in the alga cultivated with copper, mainly at days 1 to 3. Furthermore, the activities of enzymes involved in C, N and S assimilation, rubisco, glutamine synthase and cysteine synthase, respectively, were also increased, mainly at days 1 and 3.ConclusionsThe marine alga U. compressa tolerates copper excess through a concomitant increase in expression of proteins involved in photosynthesis, respiration, and C, N and S assimilation, which represents an exceptional mechanism of copper tolerance.

Essential Gene Clusters Involved in Copper Tolerance Identified in Acinetobacter baumannii Clinical and Environmental Isolates.

Copper is widely used as antimicrobial in agriculture and medicine. Copper tolerance mechanisms of pathogenic bacteria have been proven to be required for both copper tolerance and survival during bacterial infections. Here, we determined both copper-tolerant phenotype and genotype in A. baumannii originated from clinical and environmental samples. Using copper susceptibility testing, copper-tolerant A. baumannii could be found in both clinical and environmental isolates. Genotypic study revealed that representative copper-related genes of the cluster A (cueR), B (pcoAB), and D (oprC) were detected in all isolates, while copRS of cluster C was detected in only copper-tolerant A. baumannii isolates. Moreover, we found that copper-tolerant phenotype was associated with amikacin resistance, while the presence of copRS was statistically associated with blaNDM-1. We chose the A. baumannii strain AB003 as a representative of copper-tolerant isolate to characterize the effect of copper treatment on external morphology as well as on genes responsible for copper tolerance. The morphological features and survival of A. baumannii AB003 were affected by its exposure to copper, while whole-genome sequencing and analysis showed that it carried fourteen copper-related genes located on four clusters, and cluster C of AB003 was found to be embedded on genomic island G08. Transcriptional analysis of fourteen copper-related genes identified in AB003 revealed that copper treatment induced the expressions of genes of clusters A, B, and D at the micromolar level, while genes of cluster C were over-expressed at the millimolar levels of copper. This study showed that both clinical and environmental A. baumannii isolates have the ability to tolerate copper and carried numerous copper tolerance determinants including intrinsic copper tolerance (clusters A, B, and D) and acquired copper tolerance (cluster C) that could respond to copper toxicity. Our evidence suggests that we need to reconsider the use of copper in hospitals and other medical environments to prevent the selection and spread of copper-tolerant organisms.

The complete chloroplast genome of copper-tolerance plant Elsholtzia splendens

Elsholtzia splendens is a copper-tolerance plant colonized in copper mines in southern China. In this study, we sequenced and de novo assembled the complete chloroplast genome of E. splendens. The complete chloroplast genome is 150,761 bp (37.8% of GC) in length and contains 87 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Phylogenetic analysis revealed that among the 11 Lamiaceae species, Perilla citriodor is the closest relative of E. splendens. The complete chloroplast genome of E. splendens provides a valuable resource for comparative and evolutionary analysis among Lamiaceae species and may be helpful in understanding the molecular mechanism of copper tolerance in E. splendens.

GENE EXPRESSION ANALYSIS OF WOOD DECAY FUNGUS FIBROPORIA RADICULOSA GROWN IN ACQ-TREATED WOOD

Copper-tolerant brown-rot fungi are able to degrade wood treated with copper or copper-based wood preservatives. This research used quantitative reverse transcriptase polymerase chain reaction to explore what genes of the brown-rot fungus, Fibroporia radiculosa, were expressed when the fungus was overcoming the wood preservatives and decaying the wood. Aryl alcohol oxidase, catalase, oxalate decarboxylase 2, and copper resistance P-type ATPase pump had higher expression on alkaline copper quat type D treated wood compared with week 1. In addition, two genes had high expression at week 5; glycoside hydrolase 5 and glycoside hydrolase 10 when wood strength loss was around 50%. Glyoxylate dehydrogenase had high expression until week 8. This gene might be involved in the production of oxalate. Laccase, oxalate decarboxylase 1 and isocitrate lyase were not differentially expressed, suggesting that these genes were not involved in the decay process of alkaline copper quat-treated wood. These results are important to understand the genes that are involved in the mechanism of copper tolerance and wood decay in F. radiculosa.

Fungal–copper interactions in wood examined with large field of view synchrotron-based X-ray fluorescence microscopy

ABSTRACTThe goal of this study was to demonstrate how synchrotron-based X-ray fluorescence microscopy (XFM) can be used to better understand the mechanisms of copper tolerance in wood decay fungi. Copper is a major component in commercial wood preservatives as it is toxic to many wood decay fungi. However, certain fungi are copper tolerant and can attack preservative-treated wood, resulting in structural damage to treated wood members. Here we used large-field XFM to visualize six different elements (K, Ca, Mn, Fe, Cu, and Zn) in the mycelia and wood inoculated with four different species of brown rot wood decay fungi. Wood blocks were partially dipped into a solution of copper sulfate, exposed to fungi in malt extract agar petri dish assays for nine weeks, and then imaged and compared to blocks that were partially dipped in water. The blocks were imaged immediately adjacent to an end-matched control that was placed in malt extract agar petri dish assays for 9 weeks, but not exposed to the fungi so that the differences in the elemental distributions could be directly compared. The colonized wood and mycelia were rich in K, Ca, Mn, and Fe; however, the elements and the spatial distribution in the mycelia and wood differed across fungal species. The most interesting results were the maps showing the copper distribution. While three of the four fungi grew on the copper-rich region of the wood, only one species, Fibroporia radiculosa, dramatically reduced the copper concentration in the region of fungal growth.

Copper excess-induced large reversible and small irreversible adaptations in a population of Chlamydomonas reinhardtii CW15 (Chlorophyta)

<p>Two <em>Chlamydomonas reinhardtii</em> CW15 populations modified by an excess of copper in growth medium were obtained: a “Cu” population that was continuously grown under the selection pressure of 5 µM Cu<sup>2+</sup> (for at least 48 weeks) and the “Re” population, where a relatively short (9 week) exposure to elevated copper, necessary for acquiring tolerance, was followed by a prolonged period (at least 39 weeks) of cultivation at a normal (0.25 µM) copper concentration.</p><p>Cells of the Cu population were able to multiply at a Cu<sup>2+</sup> concentration 16 times higher than that of the control population at a normal light intensity and at a Cu<sup>2+</sup> concentration 64 times higher when cultivated in dim light. The potential quantum yield of photosystem II (F<sub>V</sub>/F<sub>M</sub> ratio) under copper stress was also significantly higher for the Cu population than for Re and control populations.</p><p>The Re population showed only residual tolerance towards the elevated concentration of copper, which is revealed by an F<sub>V</sub>/F<sub>M</sub> ratio slightly higher than in the control population under Cu<sup>2+</sup> stress in dim light or in darkness.</p><p>We postulate that in the <em>Chlamydomonas populations</em> studied in this paper, at least two mechanisms of copper tolerance operate. The first mechanism is maintained during cultivation at a standard copper concentration and seems to be connected with photosynthetic apparatus. This mechanism, however, has only low adaptive value under excess of copper. The other mechanism, with a much higher adaptive value, is probably connected with Cu<sup>2+</sup> homeostasis at the cellular level, but is lost during cultivation at a normal copper concentration.</p>

Copper-Induced Modulation of Biomass Growth, Physiological Parameters, Bioactive Centellosides, and Expression of Biosynthetic Pathway Genes in an Important Medicinal Herb, Centella asiatica

Hydroponically established Centella asiatica plants were studied under different concentrations of copper (0, 0.32,1.6, and 3.2 µM) for their morphological, physiological, and biochemical characteristics. The plants grown in the medium without any exogenous supply of copper showed improved biomass accumulation with maximum fresh weight (FW = 24.7 g plant−1) and dry weight (DW = 2.35 g plant−1). The total chlorophyll content, leaf number, and the leaf area were higher (0.27 mg g−1 DW, 20 and 100.54 cm2) in the plants grown under copper-deficient conditions. The toxic effects of increased levels of copper were evident by significant inhibition in growth and other morphological parameters. Copper treatment showed an increase in malondialdehyde (MDA) content and SOD activity. Bioactive phytochemical profiling using HPLC analysis revealed that higher levels of copper (1.6 and 3.2 µM) inhibit the accumulation of total centelloside content. The differential accumulation of centelloside content was further validated by the consistent pattern of expression of key pathway genes related to centelloside production in copper-treated C. asiatica plants. The study unveiled some of the important facts associated with the mechanism of copper tolerance in this important medicinal herb C. asiatica. The findings of the present study can be further used to provide better production of high value, in demand centellosides.

Insights into the mechanism of copper-tolerance in Fibroporia radiculosa: The biosynthesis of oxalate

Copper is currently used as the key component in wood preservatives despite the known tolerance of many brown-rot Basidiomycetes. Copper-tolerant fungi, like Fibroporia radiculosa, produce and accumulate high levels of oxalate when exposed to copper. To gain insight into the mechanism of oxalate production, four F. radiculosa isolates decaying untreated and 1.2% ammoniacal copper citrate-treated wood were evaluated for the differential expression of citrate synthase, isocitrate lyase, glyoxylate dehydrogenase, a succinate/fumarate antiporter, and a copper resistance-associated ATPase pump. Samples were analyzed at 2, 4, 6, and 8 weeks for oxalate production and gene expression. ATPase pump expression increased in the presence of copper when initial oxalate concentrations were low, suggesting it functions in helping the fungus adapt to the copper-rich environment by pumping toxic copper ions out of the cell. A connection in expression levels between citrate synthase, the succinate/fumarate antiporter isocitrate lyase, and glyoxylate dehydrogenase for the four isolates was found suggesting the production of oxalate originates in the mitochondrial TCA cycle via citrate synthase, shunts to the glyoxysomal glyoxylate cycle via the succinate/fumarate antiporter, moves through a portion of the glyoxylate cycle (isocitrate lyase), and ultimately is made in the cytoplasm (gyloxylate dehydrogenase).

Copper induced oxidative stresses, antioxidant responses and phytoremediation potential of Moso bamboo (Phyllostachys pubescens).

Moso bamboo is recognized as phytoremediation plant due to production of huge biomass and high tolerance in stressed environment. Hydroponics and pot experiments were conducted to investigate mechanism of copper tolerance and to evaluate copper accumulation capacity of Moso bamboo. In hydroponics experiment there was non significant variation in MDA contents of leaves compared with control. SOD and POD initially indicated enhancing trend with application of 5 μM Cu and then decreased consistently with application of 25 and 100 μM Cu. Application of each additional increment of copper have constantly enhanced proline contents while maximum increase of proline was observed with application of 100 μM copper. In pot experiment chlorophyll and biomass initially showed increasing tendency and decreased gradually with application of each additional increment of Cu. Normal growth of Moso bamboo was observed with application of 100 mg kg−1 copper. However, additional application of 300 or 600 mg kg−1 copper had significantly inhibited growth of Moso bamboo. The concentration of Cu in Moso bamboo has attained the levels of 340, 60, 23 mg kg−1 in roots, stems and leaves respectively. The vacuoles were the main organs which accumulated copper and reduced toxicity of copper as studied by TEM-DEX technology.

A multicopper oxidase contributes to the copper tolerance of Brucella melitensis 16M.

Copper is a potent antimicrobial agent. Multiple mechanisms of copper tolerance are utilized by some pathogenic bacteria. BMEII0580, which is significantly similar to the multicopper oxidase from Escherichia coli, was predicted to be the probable blue copper protein YacK precursor in Brucella melitensis 16M, and was designated as Brucella multicopper oxidase (BmcO). A bioinformatics analysis indicated that the typical motifs of multicopper oxidases are present in BmcO. BmcO, the expression of which was up-regulated by copper, could catalyze the oxidation of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), dimethoxyphenol (DMP) and para-phenylenediamine (pPD), which are widely used as substrates for multicopper oxidase. Additionally, BmcO exhibited ferroxidase activity, which indicated that it might play an important role in the Fe(2+) uptake of B. melitensis. Importantly, the mutant strain 16MΔbmcO was more sensitive to copper than the wild-type strain B. melitensis 16M as well as its complementation strain 16MΔbmcO(bmcO). The infection assays of cells showed that similar bacterial numbers of B. melitensis 16M, 16MΔbmcO and 16MΔbmcO(bmcO) strains were recovered from the infected macrophages. This result indicated that BmcO was not essential for B. melitensis intracellular growth. In conclusion, our results confirm that BmcO is a multicopper oxidase and contributes to the copper tolerance of B. melitensis 16M.

Copper tolerance mechanisms of Mesorhizobium amorphae and its role in aiding phytostabilization by Robinia pseudoacacia in copper contaminated soil.

The legume-rhizobium symbiosis has been proposed as an important system for phytoremediation of heavy metal contaminated soils due to its beneficial activity of symbiotic nitrogen fixation. However, little is known about metal resistant mechanism of rhizobia and the role of metal resistance determinants in phytoremediation. In this study, copper resistance mechanisms were investigated for a multiple metal resistant plant growth promoting rhizobium, Mesorhizobium amorphae 186. Three categories of determinants involved in copper resistance were identified through transposon mutagenesis, including genes encoding a P-type ATPase (CopA), hypothetical proteins, and other proteins (a GTP-binding protein and a ribosomal protein). Among these determinants, copA played the dominant role in copper homeostasis of M. amorphae 186. Mutagenesis of a hypothetical gene lipA in mutant MlipA exhibited pleiotropic phenotypes including sensitivity to copper, blocked symbiotic capacity and inhibited growth. In addition, the expression of cusB encoding part of an RND-type efflux system was induced by copper. To explore the possible role of copper resistance mechanism in phytoremediation of copper contaminated soil, the symbiotic nodulation and nitrogen fixation abilities were compared using a wild-type strain, a copA-defective mutant, and a lipA-defective mutant. Results showed that a copA deletion did not affect the symbiotic capacity of rhizobia under uncontaminated condition, but the protective role of copA in symbiotic processes at high copper concentration is likely concentration-dependent. In contrast, inoculation of a lipA-defective strain led to significant decreases in the functional nodule numbers, total N content, plant biomass and leghemoglobin expression level of Robinia pseudoacacia even under conditions of uncontaminated soil. Moreover, plants inoculated with lipA-defective strain accumulated much less copper than both the wild-type strain and the copA-defective strain, suggesting an important role of a healthy symbiotic relationship between legume and rhizobia in phytostabilization.

Choice tests and neighbor effects during fungal brown rot of copper- and non-treated wood

In previous work, a ‘choice’ test was used to test wood durability in mixtures of wood types. Results demonstrated that a filamentous fungus (Gloeophyllum trabeum) made a ‘choice,’ degrading white pine significantly less when less-durable woods were readily available. For this experiment, we substituted white pine with copper-treated pine and used three fungi (G. trabeum, Serpula himantioides, and Wolfiporia cocos) representing a range of copper tolerances. Southern pine blocks treated commercially with micronized copper azole or treated in-house using copper ethanolamine were used as treated wood substrates. Trials were soil-block tests, using two-block combinations to include non-treated, non-durable pine or as single-block treatments. After 12 weeks of decay, copper tolerance by S. himantioides and W. cocos was evident while G. trabeum was not copper tolerant. Availability of non-durable pine blocks, however, had no effect on decay in copper-treated wood. This contrasts with our past results using white pine. The reason for the discrepancy may relate to fungal mechanisms of copper tolerance (e.g., constitutive oxalic acid secretion) which may differ from mechanisms to overcome extractives in white pine. More fungal research using choice trials to assess these ‘neighbor effects’ is logical, given increased use of mixed, composite, and hybrid materials.

PCR-cloning of tilapia ATP7A cDNA and its mRNA levels in tissues of tilapia following copper administrations

We are studying the toxicity of copper to tilapia and zebrafish and have found that the copper tolerance of tilapia and the sensitivity of zebrafish were due to several proteins’ regulation mechanisms that were related to the effects of reactive oxygen species, mitochondrion copper transport, and stress response. To further reveal the mechanism of copper tolerance and sensitivity in tilapia and zebrafish, a full length cDNA of ATP7A was obtained in tilapia. Using real time quantitative PCR, the differential regulations of ATP7A in tilapia and zebrafish were studied. It was found that Cu 2+ gave a higher induction of ATP7A in tilapia than zebrafish, both in vivo and in vitro. These results suggest that the copper tolerance of tilapia may be due to higher expression level of ATP7A.

Tolerance of brown-rot and dry-rot fungi to CCA and ACQ wood preservatives

Copper remains the primary biocide component used today to protect wood. Increased interest in the use of non-arsenic copper-based wood preservatives has also led to increased studies on copper-tolerant decay fungi. Oxalic acid production by brown-rot fungi is proposed as one mechanism of copper tolerance. This study evaluated oxalic acid production and copper losses from ACQ- and CCA-treated Pinus sylvestris L. and Populus × euramericana I 214 wood by brown-rot fungi: Postia placenta, Gloeophyllum trabeum, Tyromyces palustris, and 2 strains of Serpula lacrymans. There was no clear linear correlation among mass losses, oxalic acid production, or copper losses in most cases. However, T. palustris and one strain of S. lacrymans showed copper tolerance in treated wood. P. placenta caused considerable mass losses in ACQ-treated specimens only. G. trabeum produced very low oxalic acid and was inhibited by ACQ and CCA wood preservatives. We concluded that wood species and preservative formulation affected the oxalic acid production, mass losses, and copper tolerance of the tested fungi.