figure s1 morphological features of 58 endophytic strains...
TRANSCRIPT
Figure S1 Morphological features of 58 endophytic strains (Red box: SH-1.2-R-15).
72
68
74
69
54
65 66
62 53
47
38
44 43 42 41
51
40 39
49 50
27
30
28
36 34
29 21
31
26
20
25
15 16
14
18 17
6 4 5 3 2 1
46 52
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59 60 58
7 9 10 11 13
19
29
70
71 73
67
Table S1 16S rRNA analysis of 58 endophytic strains.
NO. Strains Host plant Parts Closest sequences by BlastN Percent
Identity
Genbank accession
1 SH-1.1-R-1 Dendrobium
officinale -
one year
old-1
Root Bacillus sp. (in: Bacteria) strain 12D6 97.14% MN784185
2 SH-1.1-R-2 Root Stenotrophomonas rhizophila strain PgBE58 99.72% MN784186
3 SH-1.1-R-3 Root Bacillus sp. BS98 99.90% MN784187
4 SH-1.1-R-4 Root Streptomyces sp. strain SHP 1-2 99.80% MN784188
5 SH-1.1-R-5 Root Stenotrophomonas sp. strain H9 100.00% MN784189
6 SH-1.1-R-6 Root Streptomyces sp.SR3-82 gene 99.85% MN784190
7 SH-1.1-R-7 Root Bacillus thuringiensis strain ZLynn1000-39 97.59% MN784191
8 SH-1.1-R-8 Root Same as 5 - -
9 SH-1.1-R-9 Root Aerococcus viridans strain NBRC 12219 99.78% MN784192
10 SH-1.1-L-1 Leaf Bacillus cereus strain BC2 99.90% MN784193
11 SH-1.1-L-2 Leaf Bacillus thuringiensis strain GA-A07 99.90% MN784194
12 SH-1.1-L-3 Leaf Same as 58 - -
13 SH-1.1-S-1 Stem Stenotrophomonas sp. Strain FB14 99.93% MN784195
14 SH-1.2-R-1 Dendrobium
officinale -
one year
old-2
Root Streptomyces sp. NK04104 96.58% MN784196
15 SH-1.2-R-2 Root Streptomyces sp. CdTB01 97.13% MN784197
16 SH-1.2-R-3 Root Pseudomonas sp. BMS12 99.33% MN784198
17 SH-1.2-R-4 Root Streptomyces sp. g2b 99.79% MN784199
18 SH-1.2-R-5 Root Streptomyces sp. HF-2 96.82% MN784200
19 SH-1.2-R-6 Root Pseudomonas sp. Strain IAE244 99.92% MN784201
20 SH-1.2-R-7 Root Streptomyces sp. LCB 0297 97.94% MN784202
21 SH-1.2-R-8 Root Streptomyces sp. strain QJSt8 97.47% MN784203
22 SH-1.2-R-9 Root Same as 7 - -
23 SH-1.2-R-10 Root Same as 8 - -
24 SH-1.2-R-11 Root Same as 21 - -
25 SH-1.2-R-12 Root Bacillus cereus strain ULT15 99.60% MN784204
26 SH-1.2-R-13 Root Streptomyces sp. TM-74 97.51% MN784205
27 SH-1.2-R-14 Root Streptomyces fulvissimus strain DSM 40593 97.64% MN784206
28 SH-1.2-R-15 Root Streptomyces chartreusis strain ISP 5085 99.21% MN784207
29 SH-1.2-R-16 Root Streptomyces variabilis strain NRRL B-3984 97.52% MN784208
30 SH-1.2-R-17 Root Streptomyces sp. strain TM-A158 99.86% MN784209
31 SH-1.2-R-18 Root Bacillus thuringiensis strain GA-A07 chromosome
99.93% MN784210
32 SH-1.2-R-19 Root Same as 58 - -
33 SH-1.2-L-1 Leaf Micromonospora aurantiaca strain IMB16-201 99.92% MN784211
34 SH-3.1-R-1 Dendrobium
officinale -
three years
old-1
Root Bacillus sp. (in: Bacteria) strain 12D6 98.67% MN784212
35 SH-3.1-R-2 Root Same as 36 - -
36 SH-3.1-R-3 Root Streptomyces sp. GSENDO-0578 96.80% MN784213
37 SH-3.1-R-4 Root Same as 50 - -
38 SH-3.1-R-5 Root Streptomyces sp. 13-22 96.92% MN784214
39 SH-3.1-R-6 Root Streptomyces sp. MI02-7b 99.58% MN784215
40 SH-3.1-R-7 Root Streptomyces anulatus strain 174456 97.06% MN784216
41 SH-3.1-R-8 Root Same as 38 - -
42 SH-3.1-R-9 Root Streptomyces sp. DSM 40835 clone K12 99.70% MN784217
43 SH-3.1-R-10 Root Bacillus thuringiensis strain JW-1 99.81% MN784218
44 SH-3.1-R-11 Root Bacillus thuringiensis strain GA-A07 100.00% MN784219
45 SH-3.1-R-12 Root Same as 66 - -
46 SH-3.1-R-13 Root Bacillus sp. JAS24-2 chromosome 100.00% MN784220
47 SH-3.1-R-14 Root Streptomyces sp. strain MUSC11 98.46% MN784221
48 SH-3.1-R-15 Root Same as 66 - -
49 SH-3.1-R-16 Root Streptomyces sp. strain BOR09 93.69% MN784222
50 SH-3.1-R-17 Root Viridibacillus sp. strain TM-B117 99.69% MN784223
51 SH-3.1-R-19 Root Streptomyces sp. SR3-82 96.17% MN784224
52 SH-3.1-R-20 Root Bacillus thuringiensis strain GCU1 99.79% MN784225
53 SH-3.1-R-21 Root Streptomyces prunicolor NBRC 13075 99.80% MN784226
54 SH-3.1-R-22 Root Streptomyces sp. strain GDMCC 60254 96.51% MN784227
55 SH-3.1-L-1 Leaf Same as 59 - -
56 SH-3.1-L-2 Leaf Same as 65 - -
57 SH-3.1-L-3 Leaf Same as 60 - -
58 SH-3.1-S-1 Stem Streptomyces sp. strain GDMCC 60254 96.92% MN784228
59 SH-3.1-S-2 Stem Bacillus cereus strain NRRL B-23957 98.73% MN784229
60 SH-3.1-S-3 Stem Streptomyces sp. SM17 99.31% MN784230
61 SH-3.1-S-4 Stem Same as 65 - -
62 SH-3.2-R-1 Dendrobium
officinale -
three years
old-2
Root Bacillus sp. HT-Z74-B2 98.55% MN784231
63 SH-3.2-R-2 Root Same as 50 - -
64 SH-3.2-R-3 Root Same as 60 - -
65 SH-3.2-R-4 Root Bacillus cereus strain GE16 98.78% MN784232
66 SH-3.2-R-5 Root Streptomyces sp. GKU 867 100.00% MN784233
67 SH-3.2-R-6 Root Bacillus thuringiensis strain QZL38 chromosome
100.00% MN784234
68 SH-3.2-R-7 Root Streptomyces rochei 7434AN4 DNA 99.79% MN784235
69 SH-3.2-R-8 Root Streptomyces sp. Endophyte N2 chromosome 100.00% MN784236
70 SH-3.2-R-9 Root Bacillus cereus strain Sneb2000 100.00% MN784237
71 SH-3.2-R-10 Root Brevundimonas sp. strain 7002-176 100.00% MN784238
72 SH-3.2-R-11 Root Corynebacterium sp. strain ABYHD3-2 99.85% MN784239
73 SH-3.2-R-12 Root Streptomyces sp. TJ-27 97.98% MN784240
74 SH-3.2-R-13 Root Bacillus sp. AR4-2 chromosome 100.00% MN784241
75 SH-3.2-L-1 Leaf Bacillus thuringiensis strain GA-A07 100.00% MN784242
Figure S2 Scatterplot of 75 cultured endophytic strains screened in triplicate at 25 or 50 µg/mL in Hep3B2.1-
7 cell viability assay. Data are shown normalized to percent max response of the high control (Medium only
group). Each data point represents the mean and standard deviation of three replicates in 96 wells. The hit
cutoff calculated as the average percent inhibition plus three times the standard deviation of the low control
wells, is shown as the black line at 11.7%.
Table S2. Strains showed inhibition against Hep3B2.1-7 cell more than the hit cutoff (>11.7%).
No. Endophytic strains %Inhibition No. Endophytic strains %Inhibition
2 SH-1.1-R-2 22.14±4.46 54 SH-3.1-R-22 35.74±2.31
4 SH-1.1-R-4 44.24±1.39 56 SH-3.1-L-2 18.64±1.16
16 SH-1.2-R-3 14.67±1.99 58 SH-3.1-S-1 14.87±3.17
18 SH-1.2-R-5 97.24±0.38 59 SH-3.1-S-2 47.80±0.99
19 SH-1.2-R-6 89.27±9.58 61 SH-3.1-S-4 12.00±1.38
24 SH-1.2-R-11 23.97±4.63 62 SH-3.2-R-1 33.77±2.22
27 SH-1.2-R-14 98.44±0.36 63 SH-3.2-R-2 41.40±1.71
28 SH-1.2-R-15 20.30±3.76 64 SH-3.2-R-3 31.27±4.01
31 SH-1.2-R-18 34.47±2.74 65 SH-3.2-R-4 12.30±3.31
33 SH-1.2-L-1 14.47±1.28 66 SH-3.2-R-5 47.07±1.86
42 SH-3.1-R-9 23.14±1.77 67 SH-3.2-R-6 62.20±6.76
43 SH-3.1-R-10 26.37±2.21 68 SH-3.2-R-7 98.67±0.62
44 SH-3.1-R-11 12.17±4.47 69 SH-3.2-R-8 89.50±2.46
47 SH-3.1-R-14 45.34±1.71 73 SH-3.2-R-12 49.97±1.41
Figure S3 Scatterplot of 75 cultured endophytic strains screened in triplicate at 25 or 50 µg/mL in S. aureus
inhibitory assay. Data are shown normalized to percent max response of the high control (Bacterial treated
with 100 µg/mL Ampicillin). Each data point represents the mean and standard deviation of three replicates
in 96 wells. The hit cutoff calculated as the average percent inhibition plus three times the standard deviation
of the low control wells, is shown as the black line at 36.2%.
Table S3 Strains showed inhibition against S. aureus more than the hit cutoff (>36.2%).
No. Endophytic strains %Inhibition No. Endophytic strains %Inhibition
12 SH-1.1-L-3 41.86±3.50 37 SH-3.1-R-4 51.50±1.96
13 SH-1.1-S-1 50.13±5.59 44 SH-3.1-R-11 41.81±8.64
14 SH-1.2-R-1 36.70±6.74 48 SH-3.1-R-15 40.45±21.15
15 SH-1.2-R-2 40.17±5.94 50 SH-3.1-R-17 48.24±15.86
18 SH-1.2-R-5 95.12±3.34 52 SH-3.1-R-20 47.21±7.18
23 SH-1.2-R-10 54.04±7.21 56 SH-3.1-L-2 46.37±3.41
24 SH-1.2-R-11 72.75±6.14 57 SH-3.1-L-3 38.59±3.27
26 SH-1.2-R-13 51.38±3.27 68 SH-3.2-R-7 100.00±0.24
27 SH-1.2-R-14 99.66±0.58 69 SH-3.2-R-8 99.32±0.53
28 SH-1.2-R-15 100.12±0.09 70 SH-3.2-R-9 51.01±6.67
29 SH-1.2-R-16 40.21±5.97
Figure S4 Scatterplot of 75 samples screened in triplicate at 25 or 50 µg/mL in E. coli inhibitory assay. Data
are shown normalized to percent max response of the high control (Bacterial treated with 100 µg/mL
Ampicillin). Each data point represents the mean and standard deviation of three replicates in 96 wells. The
hit cutoff calculated as the average percent inhibition plus three times the standard deviation of the low
control wells, is shown as the black line at 26.4%.
Table S4 Strains showed inhibition against E. coli more than the hit cutoff (>26.4%).
No. Endophytic strains %Inhibition
59 SH-3.1-S-2 31.49±6.86
Scheme S1 Work-up scheme for the Streptomyces sp. SH-1.2-R-15.
Figure S5. (+) and (‒)-ESI-MS of compound 1.
Figure S6. HRESI-MS spectrum of compound 1.
m / z1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
*M S D 1 S P C , tim e= 5 .0 0 0 o f D :\u s er1 \d a ta \Z H A O H U IM IN \Z H A O H U IM IN 2 0 1 9 -0 4 -2 3 1 5 -1 0 -0 2 \D J 6 0 -7 L H 7 P 3 .D E S -A P I, P os , S c an , F r
M a x : 1 0 3 4 0 0
431.2
292.2
410.2
409.2
m / z1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0
0
2 0
4 0
6 0
8 0
1 0 0
*M S D 2 S P C , tim e= 5 .0 1 0 o f D :\u s er1 \d a ta \Z H A O H U IM IN \Z H A O H U IM IN 2 0 1 9 -0 4 -2 3 1 5 -1 0 -0 2 \D J 6 0 -7 L H 7 P 3 .D E S -A P I, N eg , S c an , F r
M a x : 1 4 4 1 2 8
407.2
249.0
227.2
113.2
200 250 300 350 400 450 500 550 600 650 700
m/z
0
10
20
30
40
50
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70
80
90
100
0
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40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
409.2444
431.2260274.2740
318.3000230.2478197.0921 447.1993362.3262 475.2771 552.3053
391.2328
504.2688 638.4754574.2767
407.2294
507.1544284.0769 473.2618255.1341 341.1707 439.2177394.2227 550.2842
421.2029
719.4003663.3711215.0092176.0700 599.3121
NL: 3.03E9
60-7LH7P3#549 RT: 3.66 AV: 1 T: FTMS + p ESI Full ms [100.0000-1500.0000]
NL: 1.18E9
60-7LH7P3#548 RT: 3.65 AV: 1 T: FTMS - p ESI Full ms [100.0000-1500.0000]
Figure S7. 1H NMR (500 MHz, DMSO-d6) spectrum of compound 1.
Figure S8. 1H NMR (500 MHz, CDCl3) spectrum of compound 1.
Figure S9. 13C NMR (125 MHz, CDCl3) spectrum of compound 1.
Figure S10. 1H-1H COSY (500 MHz, CDCl3) spectrum of compound 1.
Figure S11. HSQC (500 MHz, CDCl3) spectrum of compound 1.
Figure S12. HMBC (500 MHz, CDCl3) spectrum of compound 1.
Figure S13. ROESY (500 MHz, CDCl3) spectrum of compound 1.
Figure S14. (+) and (‒)-ESI-MS of compound 2.
m / z1 0 0 2 0 0 3 0 0 4 0 0 5 0 0
0
2 0
4 0
6 0
8 0
1 0 0
*M S D 1 S P C , tim e= 5 .1 7 1 o f D :\u s er1 \d a ta \Z H A O H U IM IN \Z H A O H U IM IN 2 0 1 9 -0 4 -2 3 1 5 -1 0 -0 2 \D J 6 0 -7 L H 7 P 4 .D E S -A P I, P os , S c an , F r
M a x : 3 0 5 2 8 0
425.2
445.2
306.2 4
24.2
423.2
m / z1 0 0 2 0 0 3 0 0 4 0 0 5 0 0
0
1 0
2 0
3 0
4 0
*M S D 2 S P C , tim e= 5 .1 6 2 o f D :\u s er1 \d a ta \Z H A O H U IM IN \Z H A O H U IM IN 2 0 1 9 -0 4 -2 3 1 5 -1 0 -0 2 \D J 6 0 -7 L H 7 P 4 .D E S -A P I, N eg , S c an , F r
M a x : 1 3 6 8 3 2
249.0
227.2
422.4
113.2
Figure S15. HRESI-MS spectrum of compound 2.
Figure S16. 1H NMR (500 MHz, DMSO-d6) spectrum of compound 2.
200 250 300 350 400 450 500 550 600 650 700
m/z
0
10
20
30
40
50
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90
100
0
10
20
30
40
50
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Re
lative
Ab
un
da
nce
423.2597
445.2412306.1807278.1851
211.1075 461.2160331.1863240.0871 646.3412545.3363 567.3140405.2542356.2169 481.1740 668.3198
421.2451
521.1702407.2104
484.2415339.1921 375.1832286.0928255.1342 457.2207226.9763 557.2133439.2525
644.3322616.3126
NL: 4.19E9
60-7LH7P4#565 RT: 3.77 AV: 1 T: FTMS + p ESI Full ms [100.0000-1500.0000]
NL: 1.68E9
60-7LH7P4#568 RT: 3.79 AV: 1 T: FTMS - p ESI Full ms [100.0000-1500.0000]
Figure S17. 1H NMR (500 MHz, CDCl3) spectrum of compound 2.
Figure S18. 13C NMR (125 MHz, CDCl3) spectrum of compound 2.
Figure S19. 1H-1H COSY (500 MHz, CDCl3) spectrum of compound 2.
Figure S20. HSQC (500 MHz, CDCl3) spectrum of compound 2.
Figure S21. HMBC (500 MHz, CDCl3) spectrum of compound 2.
Figure S22. ROESY (500 MHz, CDCl3) spectrum of compound 2.
Figure S23. (+) and (‒)-ESI-MS of compound 3.
Figure S24. HRESI-MS spectrum of compound 3.
m / z2 0 0 4 0 0 6 0 0 8 0 0
0
2 0
4 0
6 0
8 0
1 0 0
*M S D 1 S P C , tim e= 4 .6 4 3 o f D :\u s er1 \d a ta \Z H A O H U IM IN \Z H A O H U IM IN 2 0 1 9 -0 3 -1 4 2 0 -1 6 -3 5 \D J -1 0 0 -4 .D E S -A P I, P os , S c an , F rag :
M a x : 3 6 0 0 8
101.2
213.2
660.0
980.0
360.6
271.2
143.2
335.2
m / z2 0 0 4 0 0 6 0 0 8 0 0
0
2 .5
5
7 .5
1 0
1 2 .5
1 5
1 7 .5
*M S D 2 S P C , tim e= 4 .6 3 3 o f D :\u s er1 \d a ta \Z H A O H U IM IN \Z H A O H U IM IN 2 0 1 9 -0 3 -1 4 2 0 -1 6 -3 5 \D J -1 0 0 -4 .D E S -A P I, N eg , S c an , F rag :
M a x : 6 6 8 5
147.0
671.0
113.2
640.0
639.2
400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200
m/z
0
10
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30
40
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100
0
10
20
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50
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100
Re
lative
Ab
un
da
nce
663.1688
989.2375384.3091
417.1764
870.4260
679.1422
641.4619431.1870 475.3294 818.2929555.5335 914.4601760.5887
969.3007
1143.2715
702.4931
639.1683
702.1633
655.1626
757.1034401.0848 789.0935491.1156 843.1236
NL: 4.39E7
CHARESINms640#535 RT: 3.66 AV: 1 T: FTMS + p ESI Full ms [100.0000-1500.0000]
NL: 7.52E8
CHARESINms640#526 RT: 3.60 AV: 1 T: FTMS - p ESI Full ms [100.0000-1500.0000]
Figure S25. 1H NMR (500 MHz, DMSO-d6) spectrum of compound 3.
Figure S26. 13C NMR (125 MHz, DMSO-d6) spectrum of compound 3.
Figure S27. 1H-1H COSY (500 MHz, DMSO-d6) spectrum of compound 3.
Figure S28. HSQC (500 MHz, DMSO-d6) spectrum of compound 3.
Figure S29. HMBC (500 MHz, DMSO-d6) spectrum of compound 3.
Figure S30. (+) and (‒)-ESI-MS of compound 4.
Figure S31. HRESI-MS spectrum of compound 4.
Figure S32. 1H NMR (500 MHz, DMSO-d6) spectrum of compound 4.
Figure S33. 13C NMR (125 MHz, DMSO-d6) spectrum of compound 4.
Figure S34. (+) and (‒)-ESI-MS of compound 5.
Figure S35. HRESI-MS spectrum of compound 5.
Figure S36. 1H NMR (500 MHz, CDOD3) spectrum of compound 5.
Figure S37. 13C NMR (125 MHz, CDOD3) spectrum of compound 5.
Figure S38. (+) and (‒)-ESI-MS of compound 6.
Figure S39. HRESI-MS spectrum of compound 6.
Figure S40. 1H NMR (500 MHz, CDOD3) spectrum of compound 6.
Figure S41. 13C NMR (125 MHz, CDOD3) spectrum of compound 6.
Figure S42. (+) and (‒)-ESI-MS of compound 7.
Figure S43. HRESI-MS spectrum of compound 7.
Figure S44. 1H NMR (500 MHz, DMSO-d6) spectrum of compound 7.
Figure S45. 13C NMR (125 MHz, DMSO-d6) spectrum of compound 7.