Molecular identification, in vitro copper resistance and antibiotics susceptibility of the causal agent of the olive knot disease in Morocco

Aims@#This study aimed to i) identify Pseudomonas savastanoi pv. savastanoi (Pss) as a causal agent of the olive knot on the basis of biochemical, pathogenicity and PCR technique ii) investigate in vitro bacterial resistance toward copper-based compounds and efficiency of some antibiotics on pathogen suppression. @*Methodology and results@#Biochemical, pathogenicity and molecular identification based on alkaline method for the DNA extraction were performed to identify possible causal agent of the olive knot. Copper resistance for Pss strains was evaluated by inoculation of bacterial suspensions into YPG medium, containing the cupric sulfate at 0, 100, 250 and 500 ppm. The efficiency of eight antibiotics on Pss strain was evaluated at different concentrations. Fifty-nine isolates caused typical knots at the site of inoculation with bacterial suspensions. All isolates have been identified as Pss using specific primers. No resistance to copper was detected with concentration of 500 ppm. In contrast, copper resistance was found during 48 h with lower concentration (100 or 250 ppm). The maximal inhibition of Pss 2102-4M was observed with the highest concentration (20 μg/mL) of the Aureomycin, Streptomycin and Novobiocin with inhibition diameters of 30, 24 and 10 mm, respectively. Whereas, Colchicine, Bacitracin, Cephalex, Ampicillin and Cycloserine have no inhibitory effect on the Pss 2102-4M strain. @*Conclusion, significance and impact of study@#The alkaline method for the DNA extraction from pure culture was reliable and rapid and can be recommended for molecular detection the causal agent of the olive knot. This is the first report determined copper resistance levels of Moroccan strains of Pss and in vitro evaluated for the susceptibility towards the antibiotics.

Copper resistance of different ectomycorrhizal fungi such as Pisolithus microcarpus, Pisolithus sp., Scleroderma sp. and Suillus sp.

Environments contaminated with heavy metals negatively impact the living organisms. Ectomy­corrhizal fungi have shown important role in these impacted sites. Thus, this study aimed to evaluate the copper-resistance of ectomycorrhizal fungi isolates Pisolithus microcarpus - UFSC-Pt116; Pisolithus sp. - UFSC-PT24, Suillus sp. - UFSM RA 2.8 and Scleroderma sp. - UFSC-Sc124 to different copper doses in solid and liquid media. The copper doses tested were: 0.00, 0.25, 0.5, 0.75, 1.0 and 1.25 mmol L-1 in the solid medium and 0.00, 0.32, 0.64 and 0.96 mmol L-1 in the liquid medium. Copper was amended as copper sulphate in order to supplement the culture medium MNM at pH 4.8, with seven replicates to each fungus-dose combination. The fungal isolates were incubated for 30 days at 28 °C. UFSC-Pt116 showed high copper-resistance such as accessed by CL50 determinations (concentration to reduce 50% of the growth) as while as UFSC-PT24 displayed copper-resistance mechanism at 0.50 mmol L-1 in solid medium. The UFSC-PT24 and UFSC-Sc124 isolates have increased copper-resistance in liquid medium. The higher production of extracellular pigment was detected in UFSC-Pt116 cultures. The UFSC-Pt116 and UFSC-PT24 isolates showed higher resistance for copper and produced higher mycelium biomass than the other isolates. In this way, the isolates UFSG-Pt116 and UFSC-PT24 can be important candidates to survive in copper-contaminated areas, and can show important role in plants symbiosis in these contaminated sites.

Copper resistance of different ectomycorrhizal fungi such as Pisolithus microcarpus, Pisolithus sp., Scleroderma sp. and Suillus sp

Environments contaminated with heavy metals negatively impact the living organisms. Ectomy­corrhizal fungi have shown important role in these impacted sites. Thus, this study aimed to evaluate the copper-resistance of ectomycorrhizal fungi isolates Pisolithus microcarpus - UFSC-Pt116, Pisolithus sp. - UFSC-PT24, Suillus sp. - UFSM RA 2.8 and Scleroderma sp. - UFSC-Sc124 to different copper doses in solid and liquid media. The copper doses tested were: 0.00, 0.25, 0.5, 0.75, 1.0 and 1.25 mmol L-1 in the solid medium and 0.00, 0.32, 0.64 and 0.96 mmol L-1 in the liquid medium. Copper was amended as copper sulphate in order to supplement the culture medium MNM at pH 4.8, with seven replicates to each fungus-dose combination. The fungal isolates were incubated for 30 days at 28 °C. UFSC-Pt116 showed high copper-resistance such as accessed by CL50 determinations (concentration to reduce 50% of the growth) as while as UFSC-PT24 displayed copper-resistance mechanism at 0.50 mmol L-1 in solid medium. The UFSC-PT24 and UFSC-Sc124 isolates have increased copper-resistance in liquid medium. The higher production of extracellular pigment was detected in UFSC-Pt116 cultures. The UFSC-Pt116 and UFSC-PT24 isolates showed higher resistance for copper and produced higher mycelium biomass than the other isolates. In this way, the isolates UFSG-Pt116 and UFSC-PT24 can be important candidates to survive in copper-contaminated areas, and can show important role in plants symbiosis in these contaminated sites.

Differential expression of pathogenicity- and virulence-related genes of Xanthomonas axonopodis pv. citri under copper stress

In this study, we used real-time quantitative PCR (RT-qPCR) to evaluate the expression of 32 genes of Xanthomonas axonopodis pv. citri related to pathogenicity and virulence that are also involved in copper detoxification. Nearly all of the genes were up-regulated, including copA and copB. Two genes homologous to members of the type II secretion system (xcsH and xcsC) and two involved in the degradation of plant cell wall components (pglA and pel) were the most expressed in response to an elevated copper concentration. The type II secretion system (xcs operon) and a few homologues of proteins putatively secreted by this system showed enhanced expression when the bacteria were exposed to a high concentration of copper sulfate. The enhanced expression of the genes of secretion II system during copper stress suggests that this pathway may have an important role in the adaptative response of X. axonopodis pv. citri to toxic compounds. These findings highlight the potential role of these genes in attenuating the toxicity of certain metals and could represent an important means of bacterial resistance against chemicals used to control diseases.