Chemistry and Biodiversity of Rhizophora -Derived Endophytic Fungi Chemistry and Biodiversity of Rhizophora -Derived Endophytic Fungi

Rhizophora are salt-tolerant mangrove flora located in tropical and subtropical inter tidal coastal regions. This review summarizes frequently occurring fungal endophytes in Rhizophora . In total, 41 families and 64 genera belonging to 23 taxonomic orders of Ascomycota have been reported. Among those discussed here, Pestalotiopsis , Penicillium , and Mucor are the most abundant fungal genera, and they are widely studied. In previ ous studies, 195 metabolites were encountered in Rhizophora -derived endophytic fungi, and their structures are reported within a biogenetic context. Bioassays showed antitu - mor, antimicrobial, as well as anti-H1N1 activities to be the most notable bioactivities of the secondary metabolites discussed. Some secondary metabolites with unusual structures were identified in Rhizophora endophytic fungi. Novel hybrid sesquiterpene-cyclopaldic acid metabolites with unusual carbon skele -tons, pestalotiopens A and B ( 83, 84 ), were obtained from the endophytic fungus Pestalotiopsis sp. JCM2A4 isolated from the leaves of the Chinese mangrove, R. mucronata . Bioassays revealed that antitumor, antimicrobial, and anti-H1N1 activities are the most notable bioactivities of the secondary metabolites from Rhizophora endophytic fungi. Some compounds had significant bioactivities, as exemplified by pestalpolyol 1 ( 119 ), a novel polyketone derivative isolated from P. clavispora . Compound 119 has a strong inhibitory effect on mouse lymphoma cell line L5178Y with an IC 50 value of 4.10 μM. The indole diterpene alkaloids, rhizovrin A, B, and F ( 46, 47, 50 ), isolated from endophytic fungi Mucor irregularis QEN-189, have strong inhibitory effects on lung cancer cells A549, with IC 50 values of 11.5, 6.3, and 9.2 μM, respectively, as well as inhibitory effects on leukemia myeloid cells HL-60, with IC 50 values of 9.6, 5.0, and 7.0 μM, respectively. These findings suggest that Rhizophora endophytic fungi offering numerous use ful products with medicinal and pathogenic significance have yet to be established.


Introduction
Endophytic fungi, a polyphyletic group of highly diverse, primarily ascomycetous fungi that spend all or at least for a part of their life cycle inter-or intracellularly colonizing healthy tissues of plants without causing visible disease symptoms [1]. They are found in almost all vascular plants and grass plants [2]. It is worth noting that of the nearly 300,000 plant species that exist on Earth, any given plant is colonized by several to few hundreds of endophytic fungal species. Only a few of these plants have ever been completely studied relative to their endophytic biology [3]. Until recently, extensive work has been conducted on traditionally investigated terrestrial endophytic fungi with biological significance, and these studies mostly concentrated on the tropical and rainforest regions of the world. However, systematic Fungi colonized in mangrove forests, which comprise the second largest ecological group of the marine fungi, have specially adapted their own morphological structures and physiological mechanisms to promote the survival of host plants in harsh environmental conditions through long-term endophyte-host interactions [52]. Most mangrove endophytic fungi are facultative halophiles and euryhaline in nature. Since they do not require added salt for growth, they are able to grow at high salt concentrations and show a balanced symbiotic continuum of mutualism with host mangroves [5]. For instance, the halotolerant Rhizophora stylosa endophytic Pestalotiopsis sp. is isolated and capable of producing lignin-degrading enzymes. This species secretes over 400 salt-adapted lignocellulolytic enzymes, which enhance the salt adaptation of mangrove hosts [18].

Plants species Distribution
Ref.
Identification of biologically interesting metabolites from these endophytic fungi is an important initial step in understanding the role of endophytes to host mangrove plants. According to the previous studies, the identification and phylogenetic diversity of mangrove endophytic fungi was largely associated with mangroves located in China

The secondary metabolites of endophytic fungi of Rhizophora
There is a wide range of endophytic fungi in mangroves, and their growing environment is unique. Thus, in the formation of special fungal communities, they will certainly metabolize compounds with rich structures, unlike that of terrestrial fungi. Many of these metabolites provide a rich model structure for the screening of new drugs, which have become increasingly valuable in drug-lead research [5]. A total of 195 metabolites were discovered from Rhizophora-derived endophytic fungi reported so far are included. The secondary metabolites of endophytic fungi of mangrove are classified as alkaloids, terpenes, coumarins, chromones, quinones, anthraquinones, peptides, phenolic acids, lactones, and other compounds.

Plants species Isolated endophytic fungi Sampling location
Ref.

Lactones
Five new compounds, including cytosporones J-N (152-156), together with known metabolites, dothiorelones A (150) and cytosporones C (151), were isolated from the Pestalotiopsis sp. from R. mucronata. Biological tests showed that compound 150 was cytotoxic to human oral epidermoid carcinoma KB cells, lymphoma cells Raji, and human osteosarcoma cells Mg-63. Compounds 151-156 had no significant antitumor activity [69]. In the further study of Pestalotiopsis sp. of R. mucronata, eight new pyrone compounds, pestalotiopyrones A-H     A and B (166, 167); and one known compound, nigrosporapyrone D (165), were found in large amounts of fermentation products in the rice culture medium [80]. Three new α-pyrone pestalotiopyrones A-C (168-170); two new seiricuprolide macrolides, pestalotioprolides A (171) and B (173); and two known compounds, seiricuprolide (174) and 2′-hydroxy-3′,4′-didehydropenicillide (172), were isolated from two endophytic fungi Pestalotiopsis sp. PSU-MA92 and Pestalotiopsis sp. PSU-MA119 of R. apiculata and R. mucronata [96]. Among these, compounds 168-170 were repetitive names of pestalotiopyrones A-C [80]. Thus far, the carbon skeleton of phenyleol lactones has been rarely found among natural products [97]. One new butenolactone, pestalolide (175), and one known phytotoxin, seiridin (176), were found in the fermentation product of endophytic fungi pestalotiopyrones sp. PSU-MA69, which was from R. apiculata. The bioactivity analysis showed that compound 175 had weak antimicrobial activity against Candida albicans and Cryptococcus neoformans, with MIC values of 653.06 μM [25]. A new phthalic acid derivative, acremonide (177), and one new depsidone, acremonone A (179), together with two known substances, (+)-brefelin A (180) and 5,7-dimethoxy-3,4-dimethyl-3-hydroxyphthalide (178), were separated from the Acremonium sp. PSU-MA70, which was isolated from R. apiculata [26]. Brefelin A (BFA) is a fungal metabolite that was originally used as an antiviral agent and is now primarily used to study protein transport. It can specifically and reversibly inhibit the Golgi membrane protein protease, prohibiting the linkage of guanine nucleotides to ADP ribosylation factor and, therefore, preventing the transport of proteins from the endoplasmic reticulum (ER) to the Golgi. BFA is also used to inhibit the secretion of cytokine and other proteins as well as enhance the immunostaining of secretory proteins. BFA can activate the neural sheath phosphoric acid cycle, inducing the apoptosis of some tumor cells [98], and it has a weak antibacterial activity against Candida albicans NCPF3153 [26]. Three known substances, macrolides pestalotiollides A and B (181, 182) and 2-epi-herbarumin II (183), were isolated from the fermentation extract of Pestalotiopsis clavispora from R. harrisonii. Bioactivity tests showed that compounds 181-183 had no antitumor effect on leukemia myeloid cells HL-60, hepatoma cell SMMC-7721, lung cancer cell line A-549, breast cancer cell MCF-7, or human colon cancer cell SW480 [43]. In order to effectively control the biosynthesis of Leucostoma persoonii from R. mangle and stimulate the production of cytosporone compounds, a known antibacterial trihydroxy lactone compound, cytosporone E (184), was induced by epigenetic modification [72]. Compound 184 showed a strong anti-infective activity against Plasmodium falciparum with an IC 50 value of 13 μM. Additionally, compound 184 showed strong inhibitory activity against human lung cancer cell A549, with an IC50 value of 437 μM, and a strong inhibitory effect on methicillin-resistant S. aureus, with an MIC value of 72 μM [97] (Figure 9).

Conclusion
In this review, we summarize the distribution of frequently occurring fungal endophytes in Rhizophora: 26 genera of mangrove endophytic fungi were isolated from R. stylosa; 27 genera were isolated from R. apiculata; 26 genera were obtained from R. mucronata; 23 genera were isolated from R. mangle; 1 genus was isolated from R. harrisonii and R. annamalayana (namely Pestalotiopsis and Fusarium); and 4 genera of endophytic fungi were isolated from R. racemosa. Until now, no studies have been conducted on R. samoensis. In total, the frequently occurring fungi entophytes in Rhizophora, including 41 families and 64 genera belonging to 23 taxonomic orders of Ascomycota have been reported. Although the biological potential of endophytic fungi from the abovementioned Rhizophora species has not been thoroughly investigated, the core group of fungi can be recognized from different geographic locations. The distribution and molecular phylogeny of the fungi are discussed as well as new findings regarding the chemistry and bioactivity of natural products found in Rhizophora endophytic fungi. The Pestalotiopsis, Penicillium, and Mucor genera of endophytic fungi were identified as the most promising fungal groups in terms of chemical diversity. In particular, the Pestalotiopsis genus constituted 42.56% of the compounds reported, as shown in Figure 11. R. apiculata (34.36%) was observed to be the most investigated host plant, followed by R. stylosa (33.85%) and R. mucronata (23.59%). The chemical identification of metabolites of R. racemosa endophytic fungi has not yet been reported (Figure 11).  Some secondary metabolites with unusual structures were identified in Rhizophora endophytic fungi. Novel hybrid sesquiterpene-cyclopaldic acid metabolites with unusual carbon skeletons, pestalotiopens A and B (83,84), were obtained from the endophytic fungus Pestalotiopsis sp. JCM2A4 isolated from the leaves of the Chinese mangrove, R. mucronata. Bioassays revealed that antitumor, antimicrobial, and anti-H1N1 activities are the most notable bioactivities of the secondary metabolites from Rhizophora endophytic fungi. Some compounds had significant bioactivities, as exemplified by pestalpolyol 1 (119), a novel polyketone derivative isolated from P. clavispora. Compound 119 has a strong inhibitory effect on mouse lymphoma cell line L5178Y with an IC 50 value of 4.10 μM. The indole diterpene alkaloids, rhizovrin A, B, and F (46, 47, 50), isolated from endophytic fungi Mucor irregularis QEN-189, have strong inhibitory effects on lung cancer cells A549, with IC 50 values of 11.5, 6.3, and 9.2 μM, respectively, as well as inhibitory effects on leukemia myeloid cells HL-60, with IC 50 values of 9.6, 5.0, and 7.0 μM, respectively. These findings suggest that Rhizophora endophytic fungi offering numerous useful products with medicinal and pathogenic significance have yet to be established.