Corresponding author: Yasmina Marin Felix (
Academic editor: Thorsten Lumbsch
During the course of a study on the biodiversity of endophytes from Cameroon, a fungal strain was isolated. A multigene phylogenetic inference using five DNA loci revealed that this strain represents an undescribed species of
Matio Kemkuignou B, Schweizer L, Lambert C, Anoumedem EGM, Kouam SF, Stadler M, Marin-Felix Y (2022) New polyketides from the liquid culture of
The genus
We have recently isolated and studied a new endophytic species of
The fungus was isolated from fresh twigs of an apparently healthy plant belonging to
Herbarium type material and the ex-type strain of the new species are maintained at the collection of the Westerdijk Fungal Biodiversity Institute (
For cultural characterization, the isolate was grown for 15 days on malt extract agar (
DNA of the fungus was extracted and purified directly from colony growing in yeast malt agar (
In order to restrict the phylogenetic inference to the relevant species to compare with, a first phylogenetic analysis was carried out based on the combination of the five loci sequences (ITS,
Electrospray ionization mass (
High-resolution electrospray ionization mass spectrometry (
Optical rotations were recorded in methanol (Uvasol, Merck, Darmstadt, Germany) by using an Anton Paar MCP-150 polarimeter (Seelze, Germany) at 20 °C. UV/Vis spectra were recorded using methanol (Uvasol, Merck, Darmstadt, Germany) with a Shimadzu UV/Vis 2450 spectrophotometer (Kyoto, Japan).
Isolated and reference strains of
Species | Isolates1 | GenBank accession numbers2 | References | ||||
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ITS |
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MFLUCC 17-0956T |
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CFCC 52556T |
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MFLU 19-2880T |
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BRIP 54792T |
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ICMP 20657T |
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Present study | |
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JZB 320143T |
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CFCC 52563T |
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NCYU 19-0357T |
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CMRP 4331T |
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SCHM 3614T |
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MFLUCC 19-0101T |
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MFLUCC 17-1422T |
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MFLUCC 17-1023T |
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CFCC 52569T |
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LC3083T |
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DAOM 42078T |
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ICMP 20662T |
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VTCC 930005T |
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MFLU 18-2588T |
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MAFF 246408T |
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SCHM 3622T |
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BRIP 55657aT |
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ZHKUCC20-0014T |
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BRIP 54025T |
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CGMCC 3.20100T |
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SAUCC 194.85T |
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JZB 320123T |
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BRIP 54033T |
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BRIP 54031T |
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FAU 599T |
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SAUCC 194.111T |
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BRIP 57892aT |
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SCHM 3603 |
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BRIP 54884eT |
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URM 7972T |
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MFLUCC 14-1136T |
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ICMP 20659T |
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COAD 2074T |
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MFLUCC 17-2658T |
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COAD 2913T |
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CAA 762T |
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BRIP 54669bT |
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CFCC 51986T |
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MFLU 15-1279T |
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BRIP 55665aT |
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MFLUCC 10-0573a |
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CGMCC 3.19521T |
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KUMCC 17-0153T |
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MFLUCC 12-0777T |
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MFLUCC 13-0471T |
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MFLUCC 12-0033T |
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BRIP 62248a |
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FAU 656 |
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BRIP 54736j (type of |
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CGMCC 3.17569T |
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STE-U 2675 |
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LGMF 1583T |
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CGMCC 3.18289T |
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1BRIP: Queensland Plant Pathology Herbarium, Brisbane, Australia;
The fungus was cultivated in three different liquid media (
The small-scale cultivation of
Preliminary results obtained from small-scale screening suggested that the fungus grew and produced best in ZM ½ medium (Suppl. material
The mycelial and the supernatant extracts from shake flask batch fermentation dissolved in methanol were centrifuged by means of a centrifuge (Hettich Rotofix 32 A, Tuttlingen, Germany) for 10 min at 4000 rpm. Afterwards, the mycelia and supernatant extracts were fractionated separately using preparative reverse phase HPLC (Büchi, Pure C-850, 2020, Switzerland). VP Nucleodur 100-5 C18ec column (150 × 40 mm, 7 µm: Machery-Nagel, Düren, Germany) was used as stationary phase. Deionized water (Milli-Q, Millipore, Schwalbach, Germany) supplemented with 0.1% formic acid (FA) (solvent A) and acetonitrile (
Note: The given retention times were obtained from HPLC-
The antifungal and antibacterial activities (Minimum Inhibition Concentration,
The
The lengths of the fragments of the first phylogenetic inference using the five previously mentioned loci used in the combined dataset for the tree including all
Name refers to the host genus that this fungus was isolated from,
Not sporulated.
Colonies on
Cameroon, Kala mountain, on leaves of
Cultivation trials carried out on
Chemical structures of compounds
HR-ESI(+)MS and
HR-ESI (+) MS analysis of
13C (125 MHz) and 1H-
3 | 4 | |||
---|---|---|---|---|
No. | ||||
1 | 174.3, C | - | 175.9, C | - |
2-NH | - | 7.89, s | - | 7.57, s |
3 | 53.9, CH | 3.16, m | 53.8, CH | 3.14, q (4.9) |
4 | 50.5, CH | 2.02, t (4.1) | 50.9, CH | 2.47, t (4.4) |
5 | 34.1, CH | 2.18, m* | 34.3, CH | 2.3, m |
6 | 137.3, C | - | 137.1, C | - |
7 | 126.8, CH | 5.21* | 127.4, CH | 5.17, br s |
8 | 42.3, CH | 3.06 br d (9.9) | 40.9, CH | 3.04, br d (9.8) |
9 | 55.5, C | - | 57.2, C | - |
10 | 44.0, CH2 | 2.59, dd (13.2, 7.4) 2.74, dd (13.1, 5.3) | 43.6, CH2 | 2.65, dd (13.6, 5.2) 2.70, dd (13.6, 5.2) |
11 | 12.8, CH3 | 0.64, d (7.2) | 13.0, CH3 | 0.84, d (7.3) |
12 | 19.2, CH3 | 1.62, s | 19.3, CH3 | 1.63, s |
13 | 129.2, CH | 5.73, dd (15.7, 10.1) | 129.7, CH | 5.66, dd (15.3, 10.1) |
14 | 133.5, CH | 5.08, ddd (15.3, 10.9, 4.5) | 132.8, CH | 5.02, ddd (15.3, 11.0, 4.4) |
15 | 42.1, CH2 | 1.57, m* 1.89, br dd (12.4, 4.3) | 42.3, CH2 | 1.52, q (12.5) 1.84, br dd (12.5, 4.2) |
16 | 27.6, CH | 1.69, m | 27.7, CH | 1.69, m |
17 | 53.1, CH | 1.37, br dd (13.6, 3.2) 1.59, m* | 53.1, CH2 | 1.34, br dd (13.4, 3.3) 1.60, dd (13.6, 3.3) |
18 | 72.1, C | - | 72.2, C | - |
19 | 137.3, CH | 5.36, dd (16.6, 2.3) | 136.2, CH | 5.61, dd (16.7, 2.4) |
20 | 125.1, CH | 5.71, dd (16.9, 2.4) | 130.7, CH | 5.76, dd (16.7, 2.4) |
21 | 75.7, CH | 5.23* | 73.7, CH | 3.63, br s |
22 | 25.8, CH3 | 0.94, d (7.3) | 25.9, CH3 | 0.93, d (7.1) |
23 | 31.0, CH3 | 1.13, s | 31.5, CH3 | 1.12, s |
24 | 169.3, C | - | - | - |
25 | 20.2, CH3 | 2.18, s | - | - |
1´ | 136.8, C | - | 136.9, C | - |
2´/6´ | 129.6, CH (x2) | 7.12, d (7.0) | 129.8, CH (x2) | 7.21* |
3´/5´ | 127.9, CH (x2) | 7.29, t (7.5) | 127.7, CH (x2) | 7.29, t (7.7) |
4´ | 126.0, CH | 7.21, t (7.5) | 126.0, CH | 7.21* |
18-OH | - | 4.36, s | - | 4.17, s |
21-OH | - | - | - | 4.88, br d (5.6) |
*overlapping signals, assignments were supported by HSQC and HMBC
Selected 1H–1H COSY, NOESY and HMBC correlations of 4.
Compounds
The molecular formula of compound
13C and 1H-
5a | 7b | 8b | ||||
---|---|---|---|---|---|---|
No. | ||||||
1 | 14.7, CH3 | 0.88* | 14.7, CH3 | 0.87* | 14.5, CH3 | 0.87, t (6.9)* |
2 | 23.4, CH2 | 1.20 ̴ 1.31, m* | 23.4, CH2 | 1.20 ̴ 1.31, m* | 23.1, CH2 | 1.20 ̴ 1.31, m* |
3 | 32.6, CH2 | 1.20 ̴ 1.31, m* | 32.6, CH2 | 1.20 ̴ 1.31, m* | 32.3, CH2 | 1.20 ̴ 1.31, m* |
4 | 27.7, CH2 | 1.20 ̴ 1.31, m* | 27.7, CH2 | 1.20 ̴ 1.31, m* | 27.4, CH2 | 1.20 ̴ 1.31, m* |
5 | 30.3, CH2 | 1.20 ̴ 1.31, m* | 30.3, CH2 | 1.20 ̴ 1.31, m* | 30.1, CH2 | 1.20 ̴ 1.31, m* |
6 | 37.5, CH2 | 1.09, m* 1.20-1.31, m* | 37.5, CH2 | 1.09, m* 1.20 ̴ 1.31, m* | 37.3, CH2 | 1.09, m* 1.20 ̴ 1.31, m* |
7 | 33.2, CH | 1.39, m* | 33.2, CH | 1.40, m* | 32.9, CH | 1.38, m* |
7´ | 20.0, CH3 | 0.88* | 20.0, CH3 | 0.88* | 19.8, CH3 | 0.87, d (6.9)* |
8 | 36.8, CH2 | 1.20 ̴ 1.31* 1.40, m* | 36.9, CH2 | 1.20 ̴ 1.31, m* 1.40, m* | 36.6, CH2 | 1.20 ̴ 1.31, m* 1.40, m* |
9 | 27.2, CH2 | 2.19, m* | 27.2, CH2 | 2.18, m | 27.0, CH2 | 2.21, m* |
10 | 144.5, CH | 6.03, br t (7.4) | 144.5, CH | 6.03, br t (7.2) | 144.3, CH | 6.01, t (7.4) |
11 | 133.9, C | - | 140.0, C | - | 133.9, C | - |
11´ | 12.6, CH3 | 1.83, s | 12.7, CH3 | 1.83, s | 12.5, CH3 | 1.85, s |
12 | 148.4, CH | 7.54, d (15.7) | 148.3, CH | 7.56, d (15.7) | 148.2, CH | 7.55, d (15.7) |
13 | 123.7, CH | 6.40, d (15.7) | 123.8, CH | 6.40, d (15.7) | 123.6, CH | 6.45, d (15.7) |
14 | 203.8, C | - | 203.6, C | - | 204.1, C | - |
15 | 44.5, CH | 2.84, m | 44.6, CH | 2.80 ̴ 2.88, m* | 44.6, CH | 2.88, m |
15´ | 17.7, CH3 | 1.10, d (6.9) | 17.6, CH3 | 1.10, d (6.9) | 17.1, CH3 | 1.13, d (6.9) |
16 | 28.5, CH2 | 1.57, m 1.93 ̴ 2.00, m* | 28.3, CH2 | 1.54, m 1.93 ̴ 2.00, m* | 29.1, CH2 | 1.66, m 2.04, m* |
17 | 30.3, CH2 | 1.87, m 1.93 ̴ 2.00, m* | 30.2, CH2 | 1.84, m 1.93 ̴ 2.00, m* | 31.3, CH2 | 1.97, m 2.04, m* |
18 | 77.3, CH | 5.44, m | 77.2, CH | 5.48, m | 77.6, CH | 5.45, m |
19 | 44.6, CH | 3.03, quin (7.0) | 44.5, CH | 3.05, quin (7,0) | 45.6, CH | 2.95, m |
19´ | 15.8, CH3 | 1.30, d (7.0)* | 15.9, CH3 | 1.33, d (7.3)* | 14.9, CH3 | 1.35, d (7.3) |
20 | 173.9, C | - | 174.0, C | - | 173.5, C | - |
21-NH | - | 10.43, s | - | 10.55, s | - | 8.15, br s |
22 | 139.6, C | - | 139.8, C | - | 50.9, CH | 4.89, m |
22´ | 114.6, CH2 | 5.60, s 6.24, s | 114.3, CH2 | 5.59, s 6.22, s | 17.3, CH3 | 1.65, d (7.1) |
23 | 165.2, C | - | 165.3, C | 173.9, C | - | |
24-NH | - | 7.81, br s | - | 7.88, br t (6.1) | - | 7.96, br s |
25 | 43.0, CH2 | 3.81, dt (13.5, 6.9) 3.92, dt (13.3, 4.9) | 43.0, CH2 | 3.78, dt (13.5, 6.7) 3.94, m | 42.1, CH2 | 3.49, dt (13.6, 3.8) 4.04, dt (13.5, 7.9) |
26 | 42.7, CH | 2.87, m | 42.7, CH | 2.92, m | 42.8, CH | 2.85, m |
26´ | 15.5, CH3 | 1.30, d (7.0)* | 15.8, CH3 | 1.33, d (7.3)* | 14.9, CH3 | 1.22, d (7.3) |
27 | 175.0, C | - | 175.1, C | - | 175.4, C | - |
28-NH | - | 9.06, br d (7.5) | - | 9.11, br d (7.7) | - | 8.90, br d (7.7) |
29 | 53.6, CH | 5.13, dd (14.3, 7.6) | 53.4, CH | 5.18, m* | 53.6, CH | 5.06, dd (12.9, 6.2) |
30 | 172.3, C | - | 172.4, C | - | 172.5, C | - |
31 | 27.6, CH2 | 2.63, dt (13.7, 7.0) 2.69 ̴ 2.77, m* | 27.5, CH2 | 2.62, dt (13.8, 6.9) 2.71, tt (13.8, 6.9) | 27.3, CH2 | 2.51, m 2.68 ̴ 2.74, m* |
32 | 32.8, CH2 | 2.69 ̴ 2.77, m* | 32.1, CH2 | 2.80 ̴ 2.88, m* | 32.7, CH2 | 2.68 ̴ 2.74, m* |
33 | 175.7, C | - | 176.1, C | - | 176.7, C | - |
34-NH2 | - | 8.34, s | - | - | - | 8.32, br s |
*overlapping signals: assignments were supported by HSQC and HMBC, a 1H 500 MHZ, 13C 125 MHz; b 1H 700 MHZ, 13C 175 MHZ.
Selected 1H–1H COSY and HMBC correlations of
Compound
Compounds
Phomopchalasin N (
Fusaristatin G (
Fusaristatin H (
The extracts obtained from the fungal culture in ZM ½ exhibited activities against
Minimum Inhibitory Concentrations (
Test organisms | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 9 | 10 | 12 | 13 | References |
---|---|---|---|---|---|---|---|---|---|---|---|---|
|
- | - | - | - | - | - | - | - | - | - | - | 0.26c |
|
- | - | 16.7 | 66.7 | 16.7 | 16.7 | 1.7 | 1.7 | 66.7 | 8.3° | ||
|
- | - | - | - | - | - | 66.7 | - | - | - | 16.6n | |
|
- | - | - | - | - | - | - | - | - | - | - | 0.83° |
|
- | - | - | - | - | - | - | - | - | - | 1.7° | |
|
66.7 | - | 66.7 | 66.7 | 66.7 | 66.7 | 66.7 | 16.7 | 66.7 | 66.7 | 66.7 | 8.3n |
|
- | - | - | - | - | - | - | 66.7 | - | - | - | 1.7k |
|
- | - | - | - | - | - | - | - | - | - | - | 8.3n |
|
- | - | - | - | - | - | - | - | - | - | - | 0.21g |
|
66.7 | - | - | - | - | - | - | - | - | - | - | 4.2n |
|
16.7 | 66.7 | 66.7 | 66.7 | - | - | - | - | 66.7 | - | - | 8.3n |
|
- | - | 66.7 | 66.7 | 66.7 | 66.7 | 4.2 | 4.2 | 66.7 | - | 0.83° |
(-): No inhibition, cCiprobay 2.54 mg/mL, gGentamycin 1 mg/mL, kKanamycin 1 mg/mL, nNystatin 1 mg/mL, °Oxytetracyclin 1 mg/mL. Starting concentration for antimicrobial assay were 66.7 μg/mL.
The cytotoxicity of all the isolated compounds except
Cytotoxic activity of compounds
IC50 (µM) | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Cell lines | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 12 | 13 | Epothilone B |
KB3.1 | 0.064 | 0.33 | 1.7 | 5.8 | 10.6 | - | - | 30.3 | 0.36 | 0.91 | - | - | 6.5×10-5 |
L929 | 0.19 | 1.5 | 1.3 | 10.8 | >30.4 | - | - | - | 1.06 | 5.6 | - | - | 6.5×10-4 |
A431 | 0.085 | 0.33 | 14.3 | 11.0 | 12.0 | n.t | n.t | n.t | 0.04 | 0.17 | n.t | n.t | 1.2×10-4 |
MCF-7 | 0.14 | 3.1 | 7.3 | 19.3 | 7.44 | n.t | n.t | n.t | 0.02 | 0.36 | n.t | n.t | 8.2×10-5 |
A549 | 0.16 | 0.73 | 3.1 | 10.3 | 19.7 | n.t | n.t | n.t | 0.43 | 1.0 | n.t | n.t | 6.1×10-5 |
SKOV-3 | 0.073 | 0.33 | 13.6 | 45.9 | 13.9 | n.t | n.t | n.t | 0.15 | 0.65 | n.t | n.t | 2.9×10-4 |
PC-3 | 0.14 | 0.29 | 4.2 | 9.4 | 7.3 | n.t | n.t | n.t | 1.1 | 9.7 | n.t | n.t | 9.5×10-4 |
n.t: not tested, (-): no activity. Starting concentration for cytotoxicity assay was 37 µg/mL
The genus
We are grateful to W. Collisi for conducting the cytotoxicity assays, C. Kakoschke for recording
Figures S1–S100, Tables S1–S5
Docx file.
The following are available online: 1D, 2D