**3. Discussion**

Mangrove-associated fungi are rich in diversity and can produce impressive quantities of metabolites with promising biological activities that may be useful to humans as novel physiological agents [13–17]. The phylogenetic diversity of culturable fungi derived mangrove species *Rhizophora stylosa* and *R. mucronata* collected from the South China Sea

has been reported [32]. The endophytic fungi derived from *A. ilicifolius* areamong the most favored to be studied. Up to December 2020, only 22 strains associated with *A. ilicifolius* belonging to 9 genera have been reported [13]. Investigation on phylogenetic diversity of *A. ilicifolius* associated fungi is relatively rare. In this study, 84 of the 102 isolates were successfully classified at the genus level based on ITS sequences with relatives in the NCBI database (Table S1). The identified fungi belonged to 22 genera, of which four genera *Verticillium*, *Neocosmospora*, *Valsa*, and *Pyrenochaeta* were first isolated from mangroves. (Table S1). Two strains HS-G-02 (97%) and HS-G-06 (95%) with low similarity indicated that they should be new species, which further enriched the diversity of mangrove fungi. The new strains may produce a variety of commercially interesting and potentially useful products. The above results indicated that a high diversity of fungi can be recovered from *A. ilicifolius* in the South China Sea.

Further analysis of the isolated fungi showed that *Eurotiales* was the dominant group with identified fungi accounted for 45.1%, followed by *Hypocreales*. The fungal community comprising *Penicillium* accounted for 20.6%, followed by *Fusarium*, and *Aspergillus*. It was reported that *Penicillium* (283, 20%), *Aspergillus* (246, 18%), and *Pestalotiopsis* (88, 6%) are the dominant producers of new natural products (1384) isolated from mangrove-associated fungi, comprising more than 45% of the total molecules [13]. The fungi obtained from *A. ilicifolius* could provide abundant microbial resources for the discovery of new compounds.

Natural product researchers face the challenge of maximizing the discovery of new or potent compounds from a microbial resource library. Combining activity-guided strategy with fingerprint analysis as a discovery tool will be implemented as a systematic strategy for quick discovery of active compounds. The crude extracts of 56.4% fungi displayed strong cytotoxicity. Interestingly, the remaining 10 fungi belonging to the two orders *Eurotiales* and *Hypocreales*, displayed a broad-spectrum strong cytotoxic activity. Furthermore, integrating cytotoxic activity-guided strategy and fingerprint analysis, a strong cytotoxic active compound brefeldin A was isolated from the target active strain HS-N-27. Brefeldin A is a well-known natural Golgi-disruptor and Arf-GEFs inhibitor, and shows strong anticancer activity in a variety of cancers [8–11]. BFA is considered as a promising leading molecule for developing anticancer drugs. As the metabolites of the fungi *Penicillium* sp. (HS-N-27) are relatively simple and BFA is easily separated and purified, this provides the source of compounds for the study of the medicinal properties of BFA. A series of BFA derivatives with antileukemia activity had been reported in terms of the semi-synthesis, cytotoxic evaluation, and structure-activity relationships [9]. This method, combining activity-guided strategy with fingerprint analysis, could improve the efficiency of discovering active compounds.

#### **4. Materials and Methods**

#### *4.1. Sampling Site and Plant Material*

The medicinal mangrove *A. ilicifolius*, which was authenticated by Prof. Fengqin Zhou (Shandong University of Traditional Chinese Medicine) was collected from the South China Sea. The samples were stored at the Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.

#### *4.2. Isolation of Cultivable Fungi*

To obtain the fungi associated with medicinal mangrove *A. ilicifolius* within different parts of the plant, the surface sterilization of each part from *A. ilicifolius* was carried out following an isolation as Qin et al. described with some modifications [33]. The root, stem and leaf of *A. ilicifolius* samples were washed with sterile artificial seawater for three times to remove the microorganisms and sediment attached to the surface. Appropriate samples were taken, using scissors or scalpel to cut all parts, including root, stem and leaf, with attention to the integrity of sampling. Then, the sample was soaked in 75% alcohol for 30 s, and the water on the sample was sucked up with sterile filter paper. The sample was cut into 1 cm<sup>3</sup> pieces for fungal isolation.

The methods of tissue sectioning and tissue homogenization were used to isolate fungi. Tissue sectioning method: The tissues of 1 cm<sup>3</sup> pieces were inoculated into PDA medium (200.0 g of potato extract, 20.0 g of glucose in 1 L of seawater with four salinities of 3%, 5%, 7% and 10% respectively) in a sterile environment. In order to improve the utilization of the plate and to separate more microorganisms, the medium plate was generally divided into three areas, and 2–3 pieces of tissue were placed in each area of the PDA medium with four salt gradients of 3%, 5%, 7% and 10%. Tissue homogenization method: The tissue was ground in 2 mL of sterile artificial seawater with a mortar in a sterile environment, and then the resulting homogenate was diluted with sterile artificial seawater at three dilutions (1:10, 1:100, and 1:1000). 100 μL of each dilution was plated in quadruplicate onto corresponding medium for fungal cultivation. The inoculated plates were cultured at 25 ◦C for 2 days. The fungi were replated onto new PDA plates several times until the morphology of the fungi could be distinguished. The obtained fungal strains were deposited at the Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.

#### *4.3. Genomic DNA Extraction, PCR Amplifcation, Sequencing and Phylogenetic Analysis*

The genomic DNA extraction was conducted using the Fungal DNA kits (E.Z.N.A., Omega, Norcross, GA, USA) according to the manufacturer's protocol. The internal transcribed spacer (ITS1-5.8S-ITS2) regions of the fungi were amplified with the universal ITS primers, ITS1F (5--CTTGGTCATTTAGAGGAAGTAA-3-) and ITS4 (5--TCCTCCGCTTATTG ATATGC-3-) using the polymerase chain reaction (PCR) [34]. The PCR was performed through the following cycle: initial denaturation at 94 ◦C for 5 min, 30 cycles of 94 ◦C denaturation for 40 s min, 52 ◦C annealing for 40 s, 72 ◦C extension for 1 min; with a final extension at 72 ◦C for 10 min. Finally, the amplified products were submitted for sequencing (Invitrogen, Shanghai, China) and a BLASTN search was used to search for sequences of the closest match in the GenBank by Basic Local Alignment Search Tool (BLAST) programs database.

The sequences of fungal ITS regions obtained from *A. ilicifolius* were compared with the related sequences in the National Center for Biotechnology Information (NCBI). Each of these sequences was then aligned to sequences available in the NCBI database to determine the identity of the sequence, which further determined the species and genera of fungi. All fungal ITS sequences were aligned using the BioEdit software, applying the default parameters. The phylogenetic tree was generated using neighbor-joining (NJ) algorithms in the MEGA 7 software (version 7.0, Mega Limited) combined with bootstrap analysis using 1000 replicates incorporating fungal sequences showing the highest homology to sequences amplified.

## *4.4. General Experimental Procedures*

The Agilent DD2 NMR spectrometer (JEOL, Tokyo, Japan) at 500 MHz and 125 MHz frequency was used for 1H and 13C NMR spectra respectively. The vacuum column chromatography silica gel (200–300 mesh, Qing Dao Hai Yang Chemical Group Co, Qingdao, China), silica gel plates for thin layer chromatography (G60, F-254, and Yan Tai Zi Fu Chemical Group Co, Yan Tai, China), and reverse phase octadecylsilyl silica gel column were used for the separation of compounds. UPLCMS spectra were measured on Waters UPLC ®system (Waters Ltd., Milford, MA, USA) using a C18 column (ACQUITY UPLC ®BEH C18, 2.1 × 50 mm, 1.7 μm; 0.5 mL/min) and ACQUITY QDA ESIMS scan from 150 to 1000 Da was used for the analysis of fungal extracts and ESI-MS spectra of the compounds. Semipreparative HPLC was performed on a Hitachi L-2000 system (Hi-tachi Ltd., Tokyo, Japan) using a C18 column (Kromasil 250 × 10 mm, 5 μm, 2.0 mL/min).

#### *4.5. Fungal Fermentation and Chemical Extraction*

The 55 fungal isolates were fermented in a 500 mL conical flask containing 250 mL PDA liquid medium. The fungi were shaken at 28 ◦C, 120 rpm for 7 days. Each exper-iment was conducted in three parallels. The fermentation broth was extracted three times with an equal volume of EtOAc and the whole EtOAc solutions were evaporated under reduced pressure to give the dried extracts.
