Table 1.
Primers and probes used in this study specific to B. firmus I-1582 and B. amyloliquefaciens QST713.
Fig 1.
qPCR standard curves of B. firmus I-1582 and B. amyloliquefaciens QST713.
Standard curves were plotted from Cq values obtained by qPCR of spiked spore suspensions of B. firmus I-1582 and B. amyloliquefaciens QST713. Spiked spore suspensions were prepared from adding 5 g of corn roots and associated soil into 2 ml of spore suspensions with known CFU.
Fig 2.
Quantification of root associated bacteria in corn seedlings grown under sterile conditions in growth chambers.
Corn seeds were treated with 107 (BF-107), 105 (BF-105), or 103 (BF-103) spores of B. firmus I-1582; and 106 (BA-106), 104 (BA-104), or 102 (BA-102) spores of B. amyloliquefaciens QST713 per seed. Root sampling was carried out at 14 and 21 days after planting (n = 2). Number of cells at t = 0 days was estimated based on the initial seed treatment. ND = non-detectable.
Fig 3.
Quantification of root associated bacteria in corn seedlings grown in natural soil in the greenhouse.
Corn seeds were treated on each case with: (A) 107 spores of B. firmus I-1582 alone (T1), mixed with 107 spores of B. amyloliquefaciens QST713 (T2) or non-treated (T3); (B) 107 spores of B. firmus I-1582-Rif alone (T1), mixed with 107 spores of B. amyloliquefaciens QST713-Sm (T2), or non-treated (T3); (C) 107 spores of wild-type B. amyloliquefaciens QST713 alone (T1), mixed with 107 spores of B. firmus I-1582 (T2), or non-treated (T3). (D) 107 spores of B. amyloliquefaciens QST713-Sm alone (T1), mixed with 107 spores of B. firmus I-1582-Rif (T2), or non-treated (T3). In (A) and (B) B. firmus I-1582 specific primers and probe were used for qPCR bacterial population determination; while in (C) and (D) B. amyloliquefaciens QST713 specific primers and probe were used for qPCR (n = 3 seedlings/time point and error bars show SE). Repeated measures analysis of variance was performed at each data point comparing T1 and T2, Tukey’s HSD post hoc test was used to compare the means of statistically significant factors. Symbol * indicates time points that do not overlap 95% confidence intervals between T1 and T2 treatments.
Fig 4.
Comparison of bacteria colony morphology on TSA plates.
(A) B. firmus I-1582 colonies from pure culture (B) B. amyloliquefaciens QST713 colonies from pure culture (C) Rhizosphere bacteria from untreated corn seedlings (D) Rhizosphere bacteria from B. firmus I-1582 treated corn seedlings (E) Rhizosphere bacteria from B. amyloliquefaciens QST713 treated corn seedlings.
Fig 5.
Comparison of bacteria root colonization of greenhouse grown corn seedlings by qPCR and dilution-plating.
(A), B. firmus I-1582-Rif and (B), B. amyloliquefaciens QST713-Sm were quantified by qPCR and dilution-plating on TSARif and TSASm plates, respectively. Corn seeds were treated with 107 spores of each strain inoculated alone (T1), 107 spores of each strain mixed together (T2) or non-treated (T3) as described in the methods. Values represent means and error bars show standard error (n = 3 seedlings/time point). Repeated measures analysis of variance was performed separately for T1 and T2 for each data point comparing qPCR and plating measurements. Symbol * indicates time points that do not overlap 95% confidence intervals between qPCR and plating for both T1 and T2 treatments. Symbol + indicates time points that do not overlap 95% confidence intervals between qPCR quantification and dilution-plating only for T2.
Fig 6.
Root colonization of field grown corn seedlings by B. firmus I-1582 and B. amyloliquefaciens QST713.
Corn seeds were treated with (A) 106 spores of B. firmus I-1582 (T1 and T2) and (B) 106 spores of B. amyloliquefaciens QST713 (T1 and T2). T3 = uninoculated. qPCR quantification was performed with B. firmus I-1582 or B. amyloliquefaciens QST713 specific primers and probe using a standard curve. Error bars show Standard Error (n = 5 seedlings). Same letters on top of bars represent non-significant differences (P = 0.387) for each variety according to two-way ANOVA.
Fig 7.
Root colonization of field grown soybean seedlings B. firmus I-1582 and B. amyloliquefaciens QST713.
Soybean seeds were treated with (A) 106 spores of B. firmus I-1582 (T1 and T2) and (B) 106 spores of B. amyloliquefaciens QST713 (T1 and T2). T3 = uninoculated. qPCR quantification was performed with B. firmus I-1582 or B. amyloliquefaciens QST713 specific primers and probes using a standard curve. Error bars show Standard Error. (n = 5 seedlings). Same letters on top of bars represent non-significant differences (P = 0.665) for each variety according to two-way ANOVA.