*3.4. Fermentation, Extraction, and Purification*

*B. dothidea* was cultured in 80 conical flasks (1 L) containing 500 mL of potato dextrose broth and incubated at 27 ◦C for 14 days on an orbital shaker at 100 rpm. The mycelium was separated by filtration, and the broth was extracted with an equal amount of EtOAc (×3). The EtOAc extract was evaporated to give a black residue (3.15 g).

A portion of the EtOAc extract (3 g) was chromatographed over silica gel and eluted with a gradient of hexanes, CH2Cl2 and MeOH to yield 15 fractions. Fractions which showed antimalarial activity were combined (550 mg), chromatographed over Sephadex LH-20 (105 × 3 cm), and eluted with MeOH to give 12 fractions. A white precipitate observed in subfraction 11 was separated and washed with Et2O to yield a 3:1 mixture (10 mg) of **1** and **2** as white amorphous powders. Their identity was confirmed by comparing 1H and 13C NMR, and HRESIMS data with the literature data [22–26].

Subfractions 6–10, which showed antimalarial activity, were combined (60 mg) and further separated using a C18 reversed-phase preparative HPLC column (Luna 10μ C18 250 × 10 mm, 10 micron) and was eluted with MeOH-H2O (1:4) at a flow rate of 3.0 mL/min to give two major peaks. Peak one, which showed no antimalarial activity, was further purified by Sephadex LH-20 (60 × 2 cm) gel filtration with MeOH (100%) to give a new compound (**3**, 1.5 mg).

Peak 2, which showed moderate antimalarial activity, was purified by using C18 reversed-phase analytical HPLC chromatography (Luna 10μ C18 250 × 4.6 mm, 10 micron) and was eluted with MeOH-H2O (1:4) at a flow rate of 1.5 mL/min to give **4** (1.0 mg) and two additional components.

Compound **<sup>3</sup>**: HRESIMS [M + H]<sup>+</sup> *m/z* 388.2184 (calcd for [C22H29NO5 + H]+ 388.2124), 1H- and 13C-NMR data: see Table 2.

Compound **<sup>4</sup>**: HRESIMS [M + H]<sup>+</sup> *m/z* 448.2268 (calcd for [C24H33NO7 + H]+ 448.2335), 1H- and 13C-NMR data: see Table 2.

#### *3.5. In Vitro Antiplasmodial Assay*

The antiplasmodial assay was performed against D6 (chloroquine sensitive) and W2 (chloroquine resistant) strains of *P. falciparum* using the in vitro assay as reported earlier [41]. Artemisinin and chloroquine were included as the drug controls, and IC50 values were computed from the dose-response curves.

#### *3.6. In Vitro Phytotoxicity Assay*

Herbicidal or phytotoxic activity of the extract and compounds was performed according to a published procedure [42] using bentgrass (*Agrostis stolonifera*) and lettuce (*Lactuca sativa* cv. L., Iceberg), in 24-well plates. Phytotoxicity was ranked visually. The ranking of phytotoxic activity was based on a scale of 0 to 5 with 0 showing no effect and 5 showing no growth.

#### *3.7. In Vitro Cytotoxicity Assay*

In vitro cytotoxicity was determined against a panel of mammalian cells that included kidney fibroblast (Vero), kidney epithelial (LLC-PK11), malignant melanoma (SK-MEL), oral epidermal carcinoma (KB), breast ductal carcinoma (BT-549), and ovarian carcinoma (SK-OV-3) cells [12]. Cells were seeded to the wells of a 96-well plate at a density of 25,000 cells/well and incubated for 24 h. Samples at different concentrations were added and plates were again incubated for 48 h. The number of viable cells was determined by using neutral red dye and IC50 values were obtained from dose response curves. Doxorubicin was used as a positive control.

#### **4. Conclusions**

Bioactivity-guided fractionation of the EtOAc extract of the broth of *B. dothidea* isolated from a seed of a diseased *T. taxifolia* plant afforded a mixture of two known isomeric 2,4 pyridione epoxides, FRT-A (**1**) and flavipucine (**2**) (or their enantiomers, sapinopyridione and (−)-flavipucine) and two new *α*-alkyl-*γ*-lactam alkaloids, dothilactaene A (**3**), and dothilactaene B (**4**). The mixture of 2,4-pyridione epoxides, displayed strong phytotoxicity against both a dicot and a monocot and moderate cytotoxicity against a panel of cell lines but no antiplasmodial activity. Dothilactaene A showed no activity. Dothilactaene B was isolated from the active fraction, which showed moderate in vitro antiplasmodial activity with high selectivity index. In spite of this activity, its instability and various other biological activities shown by related compounds would preclude it from being a viable antimalarial lead.

**Supplementary Materials:** The following are available online. Figure S1: Constructed tree using Neighbor-Joining method using MEGA X software to match UM124 to already published sequences of family *Botryosphaeriaceae* taxa to help identifying close relatives. Figure S2: Constructed tree using Neighbor-Joining method using MEGA X software to match UM124 to already published sequences to help identifying close relatives. Figures S3–S24: NMR spectra and HRMS data of compounds **1**–**4**, Figures S25–S38: NMR spectra and HRMS data of two additional components (compounds **7** and **8**) isolated from the active fraction. Table S1: Best nineteen hits 100% sequence identity, Table S2: ITS sequences of taxa of the *Botryosphaeriaceae* used for alignment. Table S3: 1H and 13C NMR data for mixture of compounds **1** & **2**.

**Author Contributions:** N.P.D.N., B.L.T., and S.O.D. conceived and designed experiments and reviewed the manuscript. M.K. carried out fungal culture, extraction, isolation and identification of compounds and wrote the original draft. L.H.R. isolated fungi from the infected *T. taxifolia* seeds and reviewed the manuscript. S.K. supervised in vitro antiplasmodial and cytotoxic assays, analyzed the data, and reviewed the manuscript. D.F. assisted with determination of structures of the compounds critically and reviewed the manuscript. E.M.C.J. identified and provided the infected plant material and reviewed the manuscript. N.T. identified the fungi using ITS DNA analysis and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was support in part, by the United State of Department of Agriculture, ARS, Specific Cooperative Agreement No. 58-6060-6-015. L.H.R. received funding from the National Council for Scientific and Technology Development (CNPq), Brazil.

**Data Availability Statement:** The data presented in this study are available in this article or supplementary material.

**Acknowledgments:** We thank Bharathi Avula and Frank Wiggers, NCNPR, University of Mississippi, for recording MS and NMR spectra, respectively, and Marsha Wright, John Trott, and Robert Johnson for biological testing.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Sample Availability:** Samples of the compounds **1**–**4** are not available from the authors.
