Figure 1. Direct and indirect effect of pseudomonad strains on the growth of A. thaliana Col-0 roots (A&B). Severe reduction of primary root length (A&B) followed by the death of the seedlings was observed 5 days post-inoculation in the case of PAO1, PA14 and CHAO cultured at a distance of 1.5 cm from the primary root tip. The data also shows less reduction in primary root length in the case of PAO6344, CHAO77, when compared to PA01, PA14 and CHAO, Different letters indicate a statistically significant difference between treatments (Fisher’s LSD, P,0.05). Data is the mean6SD of 12 replicates and the experiment was repeated on two independent occasions. doi:10.1371/journal.pone.0002073.g001 software. Each experiment was repeated twice with three replicates each. Statistical analysis All data were averaged from two separate experiments and further analyzed for variance using Microsoft Excel, followed by a Student’s t test. The data means were considered significantly different at the probability of P,0.05 according to Fisher’s least significant difference (LSD) test. Results Pseudomonads inhibit primary root growth of Arabidopsis thaliana Col-0 seedlings The direct effect of pseudomonads on primary root growth was studied by inoculating 3–4 day old A. thaliana (Col-0) seedlings with different strains of bacteria at a distance of 1.5 cm from the primary root tip. The results presented in Figure 1A show that five days after inoculation, P. aeruginosa (strains PAO1&PA14) and P. fluorescens (wild type strain CHAO) caused significantly shorter primary root lengths in A. thaliana compared to control treatments (Fig. 1A&B) (P,0.05; n = 12). A less drastic (but not significant) reduction in the primary root length was observed with the Pseudomonad Cyanogenesis cyanide synthase (DhcnB) mutant (PAO6344) of P. aeruginosa (Figure 1A&B) and CHAO77 (DhcnABC) of P. fluorescens. Apart from the wild type strains and cyanide mutants, we also examined a quorum sensing (QS) mutant PAO210 (DrhlI) of P. aeruginosa to evaluate if another known virulence pathway, such as QS played a role in the observed root phenotype. The data showed significant inhibition of primary root growth by the mutant PAO210 compared to the cyanide mutant indicating that the inhibitory effect is more of a result of the cyanide synthase mutation. These results also suggest that an external factor produced by pseudomonad strains is involved in the inhibition of A. thaliana Col-0 seedlings. However, the relatively less inhibition of root length by the cyanide mutant strains of P. aeruginosa (PAO6344) and P. fluorescens (CHAO77) indicated a possible role for cyanide in the inhibition of root growth. Indirect effect of Pseudomonad cyanogenesis causes the suppression of primary root growth of A. thaliana Col-0 seedlings 2 Most pseudomonad strains produce cyanide (CN ), which is subsequently converted to the diffusible HCN in aqueous media [24,38]. To account for the effect of possible HCN emission from pseudomonad strains on A. thaliana Col-0 root growth, we conducted an indirect assay using compartment plates (see materials and methods for details). The data presented in Figure 1B shows that the P. aeruginosa strains PAO1, PA14, PAO210 and P. fluorescens strain CHAO caused a significantly higher suppression of primary root growth of A. thaliana Col-0 plants (P,0.05; n = 12) when compared to the untreated control plants even when the plants are spatially separated. Significantly less inhibition of primary root length was also observed with cyanide mutants PAO6344 and CHAO77 (P,0.05; n = 12). These results together with the previous direct assay results (Figures 1A) show that the pseudomonad exometabolites, specifically cyanide, inhibited the primary root growth of A. thaliana Col-0 plants. Pseudomonads produce cyanide (CN2) in cultures Based on our earlier direct and indirect compartment plate bioassay results, we speculated that the primary root growth inhibition of A. thaliana Col-0 by different pseudomonad strains was due to cyanide production by these bacterial strains. Therefore, to check whether these strains produce cyanide in cultures, we estimated the total cyanide produced in the cultures of different strains of P. aeruginosa and P. fluorescens. The cyanide in the culture filtrate as CN2 ion was estimated using a specific cyanide ion electrode at different time intervals from 0 to 36 hours during the growth period. The results presented in Figure 2 show a significantly higher amount of cyanide production in the culture of P. fluorescens strain CHAO followed by P. aeruginosa strains PAO1 and PA14. However, significantly less cyanide production was observed in the cyanide mutants PAO6344, CHAO77 compared to the respective parental (PAO1 and CHAO) strains (Figure 2). The production of CN2 directly correlated with the root inhibition data described in Figure 1A&B. In most of the strains tested, the trend for cyanide accumulation in the cultures was highest in the early exponential phase followed by early stationary phase and accumulation declined in the late stationary phase. Cyanide inhibits primary root growth of A. thaliana Col-0 seedlings Based on our earlier results, which showed a significantly higher cyanide ion accumulation in the cultures of P. aeruginosa PAO1/ PA14 and P. fluorescens CHAO, we hypothesized that if the cyanide PLoS ONE | www.plosone.org 4 April 2008 | Volume 3 | Issue 4 | e2073