Figure 1. Phylogenetic tree constructed based on the sequences of ITS1-5.8S rRNA gene-ITS2 (a) and the D1/D2 domain of the 28S rRNA gene (b) using the maximum likelihood method and the Tamura-Nei model [8]. The percentage of trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths indicating the number of substitutions per site. This analysis involved 22 nucleotide sequences. The green dots/pyramids indicate the isolates from this study. The others are previously identified sequences of antimicrobial substance-producing fungi. There were a total of 1318 positions in the final dataset. Evolutionary analyses were conducted by applying MEGA X v10.2.4.

Figure 2. Structures of nodulisporone sodium (1) and daidzein (2) detected by LC-MS/MS in the EtOAc extract of the fungus A. stygium WL1B5. Chemical structures were drawn using the ChemDraw Professional 18.1 software (PerkinElmer Informatics, Waltham, Massachusetts, United States).

Table 1. Mean values and standard deviation of the limnological parameters measured at the various sampling sites.

Table 1. Mean values and standard deviation of the limnological parameters measured at the various sampling sites.

Limnological ParametersSampling Sites at Mindú StreamUnitHead WaterCentral PointEstuaryTemperature°C27 ± 0.127 ± 0.127 ± 0.1pH-6.3 ± 0.17. 3 ± 0.26.1 ± 0.1Dissolved oxygen (DO)(Sat) %3.2 ± 0.10.1 ± 0.10.1 ± 0.1Chemical oxygen demand O2 (Conc.)mgO2/L57 ± 0.1107 ± 0.2151 ± 0.3TurbidityNTU3.4 ± 0.23.9 ± 0.210 ± 0.3Eletric conductivityµScm−10.4 ± 0.147.6 ± 0.4106.8 ± 0.1Nitratemg/L0.1 ± 0.10.2 ± 0.12 ± 0.1Nitritemg/L0.1 ± 0.10.1 ± 0.10.2 ± 0.1Amoniamg/L0.1 ± 0.10.2 ± 0.01.2 ± 0.1Total nitrogenmg/L1.4 ± 0.12.4 ± 0.68.7 ± 0.1Total phosphorusmg/L0.1 ± 0.10.2 ± 0.20.4 ± 0.1Orthophosphatemg/L0.1 ± 0.10.1 ± 0.10.3 ± 0.1

Table 2. Number of CFU (colony forming units)/mL, the total number of fungal isolates and the total number of isolates of aerobic fungi from three different sampling sites on the Mindú stream, Manaus, Amazonas. CFU = colony forming units.

Table 2. Number of CFU (colony forming units)/mL, the total number of fungal isolates and the total number of isolates of aerobic fungi from three different sampling sites on the Mindú stream, Manaus, Amazonas. CFU = colony forming units.

Sample SiteCFU/mLTotal IsolatesGeneraNumber of Isolates by GenusHead water (03 01′07.31″ S 59 55′84″ W)1.4 × 1014Colletotrichum sp.
Oidiodendrum sp.
Mycelia sterilia1
1
12Central point (03 04′28.4″ S 59 58′57.7″ W)2.4 × 1024Annulohypoxylon sp.
Chloridium sp.
Clonostachys sp.
Oidiodendrum sp.
Paecilomyces sp.
Rodothorula sp.
Trichoderma sp.
Mycelia sterillia1
1
1
1
1
1
1
17Estuary (03 06′58.4″ S 60 02′01.5″ W)2.9 × 1029Aspergillus sp.
Clonostachys sp.
Fusarium sp.
Penicillium sp.
Rodothorula sp.
Trichoderma sp.
Mycelia sterillia1
1
5
1
1
4
17

Table 3. Antibacterial activity of different solvent extracts based on hexane, EtOAc and crude extraction of fungal isolates from the Mindú stream assessed with E. coli ATCC 25922 and an ESBL-positive E. coli NCTC 13353.

Table 3. Antibacterial activity of different solvent extracts based on hexane, EtOAc and crude extraction of fungal isolates from the Mindú stream assessed with E. coli ATCC 25922 and an ESBL-positive E. coli NCTC 13353.

Fungal SpeciesZones of Inhibition (mm)Bacterial StrainsE. coli ATCC 25922E. coli NCTC 13353 HexEtOAcCEHexEtOAcCEAnnulohypoxylon stygium WL1B5-35.9 ± 0.1--14 ± 1.45.95 ± 0.1Colletotrichum fructicola WL3B97.9 ± 0.17.25 ± 0.46 mm---Clonostachys rosea WL5B18-19.8 ± 0.313.9 ± 0.1-4 ± 1.4-Clonostachys rosea WL8B281 ± 1.413.15 ± 1.226.95 ± 0.1-8 ± 1.418.9 ± 0.1Trichoderma harzianum WL9B49-4.4 ± 0.8----Positive control (amoxillin 50 µg/mL)20 mm0 mmNegative control (10% DMSO)0 mm0 mm

Table 4. Minimum inhibitory concentrations (MIC, µg/mL) of extracts of selected fungi with antimicrobial activity isolated from the Mindú stream.

Table 4. Minimum inhibitory concentrations (MIC, µg/mL) of extracts of selected fungi with antimicrobial activity isolated from the Mindú stream.

ExtractMinimum Inhibitory Concentration of EtOAc Extracts (µg/mL)Fungal Species and Strain IdentityBacterial StrainsE. coli ATCC 25922E. coli NCTC 13353EtOAcAnnulohypoxylon stygium WL1B5400 µg/mL400 µg/mLClonostachys rosea WL5B18400 µg/mL400 µg/mLClonostachys rosea WL8B28400 µg/mL400 µg/mLPositive controlamoxillin (12.5 µg/mL)Negative control10% DMSO

Table 5. TLC analysis and contact bioautography of mobile phases tested against E. coli ATCC 25922 with Rf values.

Table 5. TLC analysis and contact bioautography of mobile phases tested against E. coli ATCC 25922 with Rf values.

EtOAc ExtractMobile PhaseRf of Bands with Antimicrobial ActivityA. stygium WL1B5Hex (100%)0.15MeOH (100%)0.86C. rosea WL5B18EtOAc/MeOH (1:1)0.71C. rosea WL8B28EtOAc/MeOH (1:1)0.78