2.2.2. Copper Toxicity

Copper contains antifungals that have been investigated against various fungi. In *S. cerevisiae*, cupric sulfate (CuSO4) and copper oxide nanoparticles (CuO NPs) significantly reduce growth in a dose dependent manner, with the toxicity of both potentially related to Cup2 [113,198,199]. Deletion of *CUP2* increases copper sensitivity, suggesting that a mechanism of toxicity could be reducing or inactivating its regulation, resulting in decreased Cu+/MT binding and increased cytosolic Cu<sup>+</sup> [200,201]. Giannousi et al. found that CuO NPs cause DNA damage that interferes with replication and increases lipid peroxidation, reducing membrane lipid content, resulting in porous cells [202]. In *Candida* spp., CuO NPs have also shown toxic capabilities by inducing porous cell membranes [12]. Copper(II) complexes which have been shown to exhibit fungicidal and fungistatic activity in species that have a history of azole resistance appear to have a similar mechanism by reducing ergosterol content [203–207]. In filamentous fungi, copper also has dosedependent toxicity. In the agricultural pathogen *Rhizoctonia solani*, a copper (II)–lignin hybrid had high efficacy and significantly reduced the number of plants attacked by *R. solani* [84,208]. In some instances, fungi can overcome toxicity by increasing their tolerance, which may be beneficial in the case of nanoparticle production, but can become a nuisance in pathogenic species.

#### 2.2.3. Copper Tolerance and Resistance

Since copper is implicated as an antifungal agent, its ability to evade copper toxicity must be continuously evaluated. In *S. cerevisiae*, short-term exposure to CuSO<sup>4</sup> causes significant regulation of open reading frames (ORFs) responsible for cellular detoxification and Cu<sup>+</sup> uptake [113]. Exposure results in the upregulation of *CUP1* (~20-fold,) and *CRS5* (~8-fold) and the downregulation of *FRE1, FRE7,* and *CTR1* (0.07, 0.08, and 0.10-fold, respectively) [113]. This fold change, and increased CuSO<sup>4</sup> sensitivity in *cup2*∆ mutants indicates MTs, coupled with decreased Cu2+ reduction and decreased Cu<sup>+</sup> uptake, are likely to be employed as mechanisms of copper resistance [113,200].

Less is known about copper resistance in filamentous fungi. In *Aspergillus* spp., P-type ATPase CrpA has Cu<sup>+</sup> exporting activity that aids in cellular detoxification, increasing Cu<sup>+</sup> resistance [90,193,209]. High-affinity copper importers, CtrA2 and CtrC, may be involved in resistance, but are still under investigation [37,49]. In *Fusarium graminearum*, copper exposure upregulates *FgCrpA* (ATPase exporter) and the MT *FgCrdA* as a means to prevent over accumulation, with the predominate method being Cu<sup>+</sup> export activity [14]. In *F. oxysporum*, upregulation of oxidoreductase activity may decrease susceptibility to oxidative stress that can be induced by excessive copper exposure [210]. In Basidiomycetes, some progress has been made in identifying resistance mechanisms in *F. radiculosa*, where increased production of copper oxalate increases resistance [119]. However, this is the extent of the knowledge.
