Contribution of Hydrogen Cyanide to the Antagonistic Activity of Pseudomonas Strains Against Phytophthora infestans

Abstract

Plants face many biotic and abiotic challenges in nature; one of them is attack by disease-causing microbes. Phytophthora infestans, the causal agent of late blight is one of the most prominent pathogens of the potato responsible for multi-billion-dollar losses every year. We have previously reported that potato-associated Pseudomonas strains inhibited P. infestans at various developmental stages. A comparative genomics approach identified several factors putatively involved in this anti-oomycete activity, among which was the production of hydrogen cyanide (HCN). Here, we report the relative contribution of HCN emission to the overall anti-Phytophthora activity of two cyanogenic Pseudomonas strains, P. putida R32 and P. chlororaphis R47. To quantify this contribution, we generated HCN-negative mutants (Δhcn) and compared their activities to those of their respective wild types in different experiments assessing P. infestans mycelial growth, zoospore germination, and infection of potato leaf disks. Using in vitro experiments allowing only volatile-mediated interactions, we observed that HCN accounted for most of the mycelial growth inhibition (57% in R47 and 80% in R32). However, when allowing both volatile and diffusible compound-mediated interactions, HCN only accounted for 1% (R47) and 18% (R32) of mycelial growth inhibition. Likewise, both mutants inhibited zoospore germination in a similar way as their respective wild types. More importantly, leaf disk experiments showed that both wild-type and Δhcn strains of R47 and R32 were able to limit P. infestans infection to a similar extent. Our results suggest that while HCN is a major contributor to the in vitro volatile-mediated restriction of P. infestans mycelial growth, it does not play a major role in the inhibition of other disease-related features such as zoospore germination or infection of plant tissues.

Control of stripe rust of wheat using indigenous endophytic bacteria at seedling and adult plant stage

“… 14 , 22 . Anand et al 23 reported reducing P. infestance mycelial growth by BCA producing HCN. The exact mechanism of how HCN reduces the growth is not yet confirmed.…”

Biological hydrogen cyanide emission globally impacts the physiology of both HCN-emitting and HCN-perceivingPseudomonas

“…At the transition phase between exponential and stationary growth, the experiment was stopped, the OD600 was measured using Cytation5 automated plate reader and NaOH was collected. In a 96-well plate aliquots of 100 μl of collected NaOH sample were mixed with 100 μl of 1 M HCl (Fisher Scientific U.K. Ltd) and immediately covered with HCN detection filter paper 13 . After 30 min incubation at room temperature, the filter paper was scanned using Epson Perfection V370 Photo scanner and analysed using ImageJ image analysis software.…”

“…Moreover, we observed that the mutants had gained additional antimicrobial capacities, e.g. inducing higher inhibition of the pathogen's zoospore germination 13 . This prompted us to quantify the mutants' secretion of siderophores, as these iron-chelators are well-known mediators of antifungal activity 14 .…”

“…Pseudomonas chlororaphis (R47) and Pseudomonas putida (R32) and their derived Δhcn mutants were grown/maintained at 30 ˚C on Lysogeny broth (LB) agar medium (20 g/L of LB mix (Lennox, Fisher Bioreagents) with 15 g/L of agar (Agar-agar, Kobe I, Carl Roth)) 28 . Generation of Δhcn strains has been previously described 13 . King's B (KB) medium was prepared by mixing 20 g/L of proteose peptone #3 (Bacto TM Proteose Peptone No.…”

New Polyfunctional Biorationals Use to Achieve Competitive Yield of Organic Potatoes in the North-West Russian Ecosystem

“…In addition, polyfunctional biologics application improves the crop quality contributing to carbohydrates, proteins, vitamins, macro- and microelements accumulation in agricultural products [ 53 , 54 , 55 ]. In particular, this applies to the quantitative and qualitative indicators of the potato harvest [ 56 , 57 , 58 , 59 ].…”