Supplemental data
Table S1. Plate counting (aliquot 0.1 mL) of H. seropedicae SmR1 culture (OD600nm
0.8) after serial dilution.
Serial dilution
Plate count
CFU/mL
10-4
>500
-
475
503
68
50
6
5
4.75 x 108
5.03 x 108
6.77 x 108
5.03 x 108
6.00 x 108
5.00 x 108
10-5
10-6
10-7
(n = 6)
Table S2. Ct and Tm values generated by qPCR specificity assay with two annealing temperatures (61 or 63°C) using primers HERBAS1 and
DNA extracted from Herbaspirillum seropedicae SmR1 or other bacteria as template DNA
Annealing temperature
a
61 °C
63 °C
Template DNAa
Mean Ct
Tm1
Tm2
Tm3
Mean Ct
Tm1
Tm2
Tm3
H. seropedicae SmR1
13.75
83.46
-
-
13.46
83.56
-
-
H. hiltneri
33.35
68.28
76.69
82.73
32.91
69.02
76.24
85.75
H. huttiense
30.48
78.34
-
-
30.82
78.26
70.94
87.31
H. lusitanum
30.19
74.32
84.19
-
30.73
70.12
84.20
-
H. rubrisubalbicans
24.92
71.21
83.46
88.40
26.52
71.40
83.47
88.31
H. frisingense
30.63
71.12
79.07
86.75
31.57
69.84
79.08
84.84
A. brasilense
33.21
69.65
77.15
83.64
33.57
69.02
77.16
83.65
Bacillus cereus
-
-
-
-
-
-
-
-
Bacillus subtilis
-
-
-
-
-
-
-
-
E.coli
38.37
64.53
77.24
86.48
38.16
62.16
81.36
88.13
Rhizobium sp.
34.65
67.00
77.24
85.20
35.78
66.00
77.89
86.85
Microbacterium sp.
36.33
66.09
77.06
86.39
36.00
65.91
88.13
-
Pseudomonas sp.
30.67
68.74
86.29
-
31.70
68.38
86.30
-
Burkholderia tropica
32.70
68.65
81.26
34.38
Samples were analyzed at a final concentration of 6 ng DNA per reaction for all samples (n=4).
67.37
81.18
85.39
(-) = Ct or Tm not detected
Table S3. Parameters of qPCR standard curves for Herbaspirillum seropedicae SmR1
detection with two annealing temperatures (61 or 63°C) conditions using primers
HERBAS1 and bacterial DNA serial dilution.
Annealing temperature
61 °C
63 °C
DNA Sample
Efficiency (%)
Slope
R2
Efficiency (%)
Slope
R2
A
79.52
-3.94
0.998
78.26
-3.98
0.998
B
80.98
-3.88
0.999
82.60
-3.82
0.998
C
81.34
-3.87
0.996
77.34
-4.02
0.999
Mean
80.62
-3.89
0.997
79.40
-3.94
0.998
SD
0.96
0.04
0.001
2.81
0.10
0.001
CV (%)
1.20
0.91
0.10
3.54
2.63
0.10
Figure S1. Melting curve of qPCR using primer pair HERBAS1 of Herbaspirillum
seropedicae strain SmR1 (red line) and 12 bacteria: Herbaspirillum hiltneri,
Herbaspirillum huttiense, Herbaspirillum lusitanum, Herbaspirillum rubrisubalbicans,
Herbaspirillum frisingense, Bacillus cereus, Bacillus subtilis, Escherichia coli,
Azospirillum brasilense, Rhizobium sp, Microbacterium sp and Pseudomonas sp.,
Figure S2. Melting curve of qPCR using primer pair HERBAS2 of Herbaspirillum
seropedicae strain SmR1 (red line) and 6 bacteria: Herbaspirillum frisingense,
Herbaspirillum
hiltneri,
Herbaspirillum
huttiense,
Herbaspirillum rubrisubalbicans, Azospirillum brasilense,.
Herbaspirillum
lusitanum,
Figure S3. Electrophoresis gel of qPCR amplicon using primers HERBAS1 (76 bp;
lanes 2 to 7) and HERBAS2 (63 bp; lanes 8 to 13). Samples: 1 – 50 bp DNA ladder; 2 –
H. hiltneri, 3 – H. huttiense, 4 – H. lusitanum, 5 – H. rubrisubalbicans, 6 – H.
frisingense, 7 – H. seropedicae SmR1; 8 – H. hiltneri, 9 – H. huttiense, 10 – H.
lusitanum, 11 – H. rubrisubalbicans, 12 – H. frisingense, 13 – H. seropedicae SmR1;
14, 15 – Water.
Figure S4. Melting curve of qPCR using primer pair HERBAS1 and DNA from samples
inoculated with H. seropedicae strain SmR1 cultivated in vitro. Inoculated samples
were collected 1, 4, 7 and 10 days after inoculation (DAI).
Figure S5. Melting curve of qPCR using primer pair HERBAS1 and DNA samples
inoculated with H. seropedicae strain SmR1 cultivated in pots. Inoculated samples were
collected 4, 7 and 10 days after inoculation (DAI).
A
B
Figure S6. A) Amplification plot and B) melting curve of real-time PCR using primer
pair HERBAS1 and DNA from samples inoculated with H. seropedicae strain SmR1
and cultivated in natural soil.