# A Smart and Sustainable Future for Viticulture Is Rooted in Soil: How to Face Cu Toxicity
## 2 Current Challenges
### Copper Effects on Soil Agrobiodiversity  
Soil contains a vast underground world inhabited by many fungal and bacterial species. It is estimated that 1 g of soil contains a number of bacteria ranging between 100,000 to 1,000,000, and, within this fraction, there may exist different micro-niches in which several ecotypes may live [64; 65]. Although only a small fraction of the fungal and bacterial diversity is known, certain fungal and bacterial groups play a crucial role in several agroecosystem services, including plant-growth promotion and cycling of nutrients (e.g., nitrogen, N, and phosphorus, P) [66; 67; 68]. However, the accumulation and the excessive availability of heavy metals such as Cu can impact the soil microbial biomass and activity, thus affecting some microbial-driven soil processes, including N fixation and mineralization [69]. Moreover, Cu has been demonstrated to impact microbial communities' composition and functionality, with higher effects on bacterial than fungal communities [70; 71; 72; 73; 74]. Fungi and bacteria have evolved different strategies against high Cu concentrations in soils (Figure 3). For instance, some filamentous fungal and yeasts species act against the overload soil Cu, directly at the cytosol level [75]. Herein, the sequestration of the free Cu ions occurs due to metallothioneeins, a family of cysteine-rich proteins (e.g., Cup1 and Crs5) that chelate Cu [76; 77; 78]. Furthermore, when the metal in the cytosol is in excess, fungal cells act through organellar compartmentation of the metal, mainly directing it to the vacuole. Concerning this latter strategy, no specific Cu transporter catalyzing the transmembrane transport into the vacuole has yet been identified [79]. In addition, cytoplasmic chaperones' activity is essential for mitigating Cu stress in the cytosol. Indeed, while the chaperone Atx1 delivers Cu to the trans-Golgi network, Ccs1 delivers Cu to the Cu-Zn Superoxide dismutase Sod1 [80; 81]. Figure 3: Exposure of soil microorganisms to Cu in vineyards. (**a**) Selection of Cu-resistant and antibiotic-resistant genes due to the Cu application (**b**) Schematic representation of the bacterial and fungal strategies for coping with excessive Cu inputs;  
In contrast, in bacteria, the detoxification strategy relies mainly on a Cu export system based on the functionality of two different mechanisms for Gram-positive and Gram-negative bacteria [82]. In Gram-positive bacteria, proteins belonging to the Cop family are at the basis of cytosolic Cu disposition. The system includes a Cu chaperone (CopZ) that delivers Cu first to CopY (copper responsive repressor), activating the transcription of _copA_ and _copB_ (genes encoding for ATPase pumps), which extrude Cu\({}^{+}\) and Cu\({}^{2+}\) from the cell, respectively. In Gram-negative bacteria, the presence of periplasmic space determines the evolution of specific mechanisms for extruding Cu in the extracellular space. Specifically, the Cop-protein system is located on the inner membrane and in the inner cell, and its activation is mediated by the MerR-type Cu-responsive transcriptional activator CueR [83]. Additionally, while Gram-positive bacteria mainly use the Cop system, Gram-negative bacteria exploit a trans-envelope extrusion system (CusABC ATPase pumps system) [82]. Additionally, the periplasmic multicopper oxidases (PcoA, CueO), oxidizing Cu\({}^{+}\) to Cu\({}^{2+}\) and activating the production of siderophores to sequester the cytoplasmic Cu, play a crucial role in the Cu detoxification process [84; 85]. Moreover, the Cu accumulation in agricultural soils other than bacterial Cu resistance can also promote the occurrence of antibiotic resistance, with these traits being linked together [86; 87; 88; 89; 90] (Figure 3). Therefore, at the molecular level, the Cu-resistant and antibiotic-resistant genes can be reasonably co-selected. Thus, they can commonly occur on the same mobile genetic element (i.e., plasmid, integron or transposon) [91; 92].