Genetic diversity and differential in vitro responses to Ni in Cenococcum geophilum isolates from serpentine soils in Portugal

Abstract

Amplified fragment length polymorphism (AFLP) analysis was used to investigate the genetic diversity in isolates of the ectomycorrhizal fungus Cenococcum geophilum from serpentine and non-serpentine soils in Portugal. A high degree of genetic diversity was found among C. geophilum isolates; AFLP fingerprints showed that all the isolates were genetically distinct. We also assessed the in vitro Ni sensitivity in three serpentine isolates and one non-serpentine isolate. Only the nonserpentine isolate was significantly affected by the addition of Ni to the growth medium. At 30 μg g −1 Ni, radial growth rate and biomass accumulation decreased to 73.3 and 71.6% of control, respectively, a highly significant inhibitory effect. Nickel at this concentration had no significant inhibitory effect on serpentine isolates, and so the fitness of serpentine isolates, as evaluated by radial growth rate and biomass yield, is likely unaffected by Ni in the field. In all isolates, the Ni concentration in the mycelia increased with increasing Ni concentration in the growth medium, but two profiles of Ni accumulation were identified. One serpentine isolate showed a linear trend of Ni accumulation. At the highest Ni exposure, the concentration of Ni in the mycelium of this isolate was in the hyperaccumulation range for Ni as defined for higher plants. In the remaining isolates, Ni accumulation was less pronounced and seems to approach a plateau at 30 μg g −1 Ni. Because two profiles of Ni accumulation emerged among our Ni-insensitive serpentine isolates, this result suggests that different Ni detoxification pathways may be operating. The nonserpentine isolate whose growth was significantly affected by Ni was separated from the other isolates in the genetic analysis, suggesting a genetic basis for the Ni-sensitivity trait. This hypothesis is further supported by the fact that all isolates were maintained on medium without added Ni to avoid carry-over effects. However, because AFLP analysis failed to distinguish between serpentine and non-serpentine isolates, we cannot conclude that Ni insensitivity among our serpentine isolates is due to evolutionary adaptation. Screening a larger number of isolates, from different geographical origins and environments, should clarify the relationships between genetic diversity, morphology, and physiology in this important species.

Occurrence, physiological responses and toxicity of nickel in plants

“…As a result of competition, Ni at high concentrations may inhibit the absorption of these metals, decreasing their concentration and even leading to their deficiency in plants (Ahmad et al 2007;Van Assche and Glijsters 1990;Rubio et al 1994). Subsequently, this may affect important physiological processes, and ultimately result in toxic effects (Gajewska et al 2006;Gon_alves 2007). For example, Ni can decrease Mg (or Fe) uptake and its supply to aerial parts via competition, and then induce deficiencies of these elements in plants.…”

“…This can result in the retardation of germination, growth suppression, and reductions in yields (Madhava Rao and Sresty 2000;Seregin and Kozhevnikova 2006a, b). These inhibitory effects of Ni on the growth of plants can be reduced by supplying additional Mg (or Fe) ions (Genrich et al 1998;Gon_alves 2007;Ouzounidou et al 2006). Therefore, Ni toxicity in plants is partly due to interference with other essential metal ions.…”

Large and variable genome size unrelated to serpentine adaptation but supportive of cryptic sexuality in Cenococcum geophilum

“…Although multilocus genotype assessments suggest the occurrence of recombination and a possible cryptic sexual cycle (Taylor et al 1999, Lobuglio and Taylor 2002, Douhan et al 2007), a sexual stage has so far been assumed to be absent in C. geophilum (although see Fernandez-Toiran andAgueda 2007, andSpatafora et al 2012). While this absence would imply clonal reproduction and a relatively low genetic diversity, various studies have demonstrated an unexpectedly high level of genetic diversity within and between C. geophilum populations (e.g., Panaccione et al 2001;Wu et al 2005;Douhan et al 2007;Gonçalves et al 2007).…”

“…geophilum has been shown to be locally adapted to serpentine soil conditions (Gonçalves et al 2007(Gonçalves et al , 2009. Serpentine soils are widespread soils formed by the weathering of ultramafic rocks and typically have a low calcium-to-magnesium ratio, with calcium levels significantly lower compared to surrounding non-serpentine soils.…”

Growth responses and accumulation of heavy metals by fungus Agaricus bisporus

“…In one study all isolates of Paxillus involutus were less tolerant to Zn, than any of those of Amanita muscaria (Brown and Wilkins 1985) and also, it was demonstrated that while radial growth of a Suillus bovinus isolate was inhibited by 91% when exposed to 0.5 ppm cadmium, while Paxillus involutus and Rhizopogon subcaerulescens were only inhibited by approximately 26 and 28%, respectively (Kim et al 2004). Also, four isolates of C. geophilum demonstrated a range of sensitivities to nickel (Goncalves et al 2007). A similar response was observed for Cu: Paxillus involutus and Laccaria laccata grew at 2.5 ppm Cu, whereas the concentration had to be decreased 10-fold before any growth of Scleroderma citrinum…”