Potential of Secondary Metabolites of Diaporthe Species Associated with Terrestrial and Marine Origins

Diaporthe species produce versatile secondary metabolites (SMs), including terpenoids, fatty acids, polyketides, steroids, and alkaloids. These structurally diverse SMs exhibit a wide range of biological activities, including cytotoxic, antifungal, antibacterial, antiviral, antioxidant, anti-inflammatory, and phytotoxic activities, which could be exploited in the medical, agricultural, and other modern industries. This review comprehensively covers the production and biological potencies of isolated natural products from the genus Diaporthe associated with terrestrial and marine origins. A total of 275 SMs have been summarized from terrestrial (153; 55%) and marine (110; 41%) origins during the last twelve years, and 12 (4%) compounds are common to both environments. All secondary metabolites are categorized predominantly on the basis of their bioactivities (cytotoxic, antibacterial, antifungal, and miscellaneous activity). Overall, 134 bioactive compounds were isolated from terrestrial (92; 55%) and marine (42; 34%) origins, but about half the compounds did not report any kind of activity. The antiSMASH results suggested that Diaporthe strains are capable of encoding a wide range of SMs and have tremendous biosynthetic potential for new SMs. This study will be useful for future research on drug discovery from terrestrial and marine natural products.


Introduction
Diaporthe is an important fungal genus of plant pathogens [1] belonging to the family Diaporthaceae, order Diaporthales, and class Sordariomycetes [2]. It is isolated mainly from plant hosts, which are distributed worldwide; many of them have been reported as plant pathogens, nonpathogenic endophytes, or saprobes, and human and other mammalian pathogens [3,4]. Diaporthe sp. is a widespread fungal genus that colonizes a wide range of hosts. It consists of nearly 800 described species, with around 950 species being attributed to its asexual state (Phomopsis) [5]. It is often isolated from above-ground plants, especially tropical and temperate woody plants [6]. Among numerous endophytic fungi, the genus Diaporthe is known for its potent biosynthetic ability to produce bioactive metabolites [7,8]. Secondary metabolites (SMs) isolated from Diaporthe sp. have shown a wide range of biological activities and chemical structures [9,10]. Chemical studies on some Diaporthe spp. have revealed a variety of bioactive natural products [11], such as cytotoxic diapolic acids [12], antifungal compounds [5,13], antibacterial agents [14,15], anti-candidal ketone derivatives [16], and anti-tubercular metabolites [17]. In the last twelve years, a total of 106 bioactive SMs have been reported from the genus Diaporthe [18].
Endophytic communities that develop inside the host plants are influenced by various parameters, such as environmental conditions (terrestrial and marine), host type, etc. [19].
Fungal endophytes are asymptomatic inhabitants of plant tissues that have the capability to colonize all parts of plants and determine their functional aspects, including increasing plant growth, acting as a biocontrol agent, naturally protecting the host from pests, and enduring tolerance against numerous biotic/abiotic stresses [20,21]. In return, they benefit from host plants in several ways, including providing nutrients, protection from desiccation, spatial structure, and passing on reproductive fungal propagules to the next generation of hosts in the case of vertical transmission [22]. Due to the vast diversity of endophytic fungal communities, the characterization of the SMs of each endophytic fungal community is difficult; therefore, the current review aims to describe the SMs species from the genus Diaporthe from two main origins (terrestrial and marine) and, furthermore, to classify them on the basis of their biological potency.  (1), a known (1R,2R,4R)-trihydroxy-p-menthane (2), three linear furanopolyketides (3)(4)(5), and four lovastatin analogues, oblongolides D (6), H (7), P (8), and V (9), from Diaporthe sp. SXZ-19 on C. acuminate. These compounds showed weak cytotoxic activities against HCT 116 cells at a concentration of 10 µM [23]. Two bioactive metabolites, emodin (10) and arbutin (11), were isolated from an endophytic fungus D. lithocarpus. Compound 10 exhibited remarkable cytotoxic activity against P-388 murine leukemia cells (IC 50 = 0.41 µg/mL), and 11 showed moderate cytotoxicity against murine leukemia P-388 cells and had an IC 50 value at 2.91 µg/mL [15]. Two cytoskyrin-type bisanthraquinones, cytoskyrin C (12), and (+)-epicytoskyrin (13), were isolated from Diaporthe sp., an endophytic fungus derived from Anoectochilus roxburghii. Both compounds showed dose-dependent cytotoxicities against SMMC-7721 cells [24]. A new compound, vochysiamide B (14), and the known 2,5dihydroxybenzyl alcohol (15) were derived from D. vochysiae LGMF1583 on the medicinal plant Vochysia divergens and showed cytotoxic activities against A549 human non-small cell lung and PC3 human prostate cell lines [8]. Mycoepoxydiene (16) and eremofortin F (17) were obtained from the endophytic fungus Diaporthe sp. SNB-GSS10 on Sabicea cinerea and showed cytotoxic activity against KB and MRC5 cells [6].
FOR PEER REVIEW 7 of 27 molide C (88) from a Diaporthe sp. on Aucuba japonica var. borealis inhibited the proliferation of human colon adenocarcinoma cells at a concentration of 50 µg/mL [45]. Compound 18-des-hydroxy cytochalasin H (22) from the endophytic fungus D. phaseolorum-92C inhibited leishmanicidal activity and moderate antioxidant activity against the breast cancer cells MDA-MB-231 and MCF-7 [26]. Studies of the strain Diaporthe sp. JC-J7 from the stems of Dendrobium nobile led to the isolation of a new compound, diaporthsin E (89). It showed low antihyperlipidemic activity on triglycerides (TG) in steatotic L-02 cells with an inhibition rate of 26% at a concentration of 5 µg/mL [46]. Two dibenzopyrones, 2-hydroxyalternariol (90) and alternariol (91), were isolated from the endophytic fungus Diaporthe sp. CB10100. Both compounds significantly reduced the production of NO to as low as 10 µM in LPS-induced RAW264.7 cells [47]. A new metabolite, phomentrioloxin (92), was isolated from the liquid culture of Phomopsis sp. (asexual state of Diaphorte), which showed phytotoxic activity, and caused growth and chlorophyll content reduction in fronds of Lemna minor and inhibition of tomato rootlet elongation [48]. Structures of compounds 67-92 are shown in Figure 4.

Antibacterial and Antifungal Metabolites
A chemical investigation into Diaporthe amygdali SgKB4, an endophytic fungal strain isolated from the West Sumatran mangrove plant Sonneratiagriffithii Kurz, led to the isolation of cytochalasin H (60). This compound showed mild antibacterial activity against some pathogenic bacteria [49]. The fungus D. phaseolorum derived from Laguncularia racemose, afforded 3-hydroxypropionic acid (166), which showed antimicrobial activity against S. aureus and S. typhi [50]. A new compound (167), named diaporthelactone, was isolated from the culture of Diaporthe sp., a marine fungus growing in the submerged decayed leaves of Kandelia candel in the mangrove, and exhibited inhibitory antifungal activity against Aspergillus niger with a MIC of 50 µg/mL [51]. Niaz

Antibacterial and Antifungal Metabolites
A chemical investigation into Diaporthe amygdali SgKB4, an endophytic fungal strain isolated from the West Sumatran mangrove plant Sonneratiagriffithii Kurz, led to the isolation of cytochalasin H (60). This compound showed mild antibacterial activity against some pathogenic bacteria [49]. The fungus D. phaseolorum derived from Laguncularia racemose, afforded 3-hydroxypropionic acid (166), which showed antimicrobial activity against S. aureus and S. typhi [50]. A new compound (167), named diaporthelactone, was isolated from the culture of Diaporthe sp., a marine fungus growing in the submerged decayed leaves of Kandelia candel in the mangrove, and exhibited inhibitory antifungal activity against Aspergillus niger with a MIC of 50 µg/mL [51]. Niaz et al. (2021) isolated a new isochromophilone G (168) along with six known azaphilones (169-174) from the endophytic fungus Diaporthe perseae on the Chinese mangrove Pongamia pinnata (L.). All compounds exhibited antibacterial potency against human pathogens [52]. Compounds 166-174 are shown in Figure 6.

Inactive Compounds
Secondary metabolites 207-221 and 124-134 were isolated from the mangrove-associated fungus Diaporthe sp. SCSIO 41011. None of these compounds reported any kind of activity [53]. Two new polyketides, phaseolorins G and H (222 and 223), and one new phaseolorin I (224), along with two known compounds (225 and 226), were isolated from D. phaseolorum FS431. None of these compounds showed any activity [54]. Two new metabolites, diaporchromanones A and B (227 and 228), and a known compound (229) were obtained from D. phaseolorum SKS019, but showed no activity [56]. Three chloroazaphilone derivatives (230−232) were obtained from the fungus Diaporthe sp. SCSIO 41011, along with three known analogues (233−235). None of these isolated compounds were reported to have any kind of activity [57]. Two inactive compounds, diaporisoindole B (236) and diaporisoindole C (237), were isolated from the endophytic fungus Diaporthe sp. SYSUHQ3 [58]. A new arecine (238) and twenty-two known diketopiperazines (239-260) were isolated from the endophytic fungus D. arecae, but showed no activity [60]. Six new compounds, including diaporphasines A-D (261-264) and meyeroguillines C and D (265-266), and a known meyeroguilline A (267) were isolated from an endophytic fungus D. phaseolorum. None of these compounds reported any kind of activity [61]. A chemical investigation into the fungus D. longicolla FS429 led to the isolation of six metabolites, the novel longidiacid B (268), two new polyketides (269-270), a new cytochalasin analogue longichalasins A (272), and two known compounds (271 and 273). None of them showed activity [63]. Four inactive compounds, including the new diaporpenoids B and C (274 and 275), and the known diaporpyrones B and C (160 and 161), were isolated from the mangrove endophytic fungus Diaporthe sp. QYM12 [64]. The structures of compounds 207-275 are shown in Figure 8.  Secondary metabolites 207-221 and 124-134 were isolated from the mangrove-associated fungus Diaporthe sp. SCSIO 41011. None of these compounds reported any kind of activity [53]. Two new polyketides, phaseolorins G and H (222 and 223), and one new   Figure 8. Chemical structures of compounds 207-275 of marine origin. In this paper, a total of 275 secondary compounds from the genus Diaporthe are summarized. As can be seen in Figure 9, 153 secondary metabolites were isolated from terrestrial origins and 110 from marine origins, and 12 were common to both environments. These compounds are categorized on the basis of their activity and inactivity. Figures 10 and 11, and Tables 1 and 2 show that about half of all 275 compounds reported from terrestrial and marine origins were inactive, accounting for 74 (45%) and 80 (66%) metabolites, respectively. Moreover, the active compound ratios were 56% and 34%, respectively. The active secondary metabolites showed various types of bioactivities, mainly cytotoxic (34; 20%), antibacterial (18; 11%), antifungal (14; 9%), and miscellaneous activities (26; 15%) for those of terrestrial origin and antibacterial and antifungal (10; 8%) and miscellaneous activities (32; 26%) for those of marine origin.

Analysis of Secondary Metabolite Biosynthetic Potential
Despite the numerous compounds isolated from Diaporthe species, recent advances in genome sequencing and bioinformatics analysis indicate that the number of biosynthetic gene clusters (BGCs) of SMs exceeds the number of SMs identified so far [65]. To

Analysis of Secondary Metabolite Biosynthetic Potential
Despite the numerous compounds isolated from Diaporthe species, recent advances in genome sequencing and bioinformatics analysis indicate that the number of biosynthetic gene clusters (BGCs) of SMs exceeds the number of SMs identified so far [65]. To Inactive [47] 155 Inactive [29] 164 Phomopthane B D. unshiuensis YSP3 Inactive [30] 165 Phomopyrone B − Inactive [30]      250

Analysis of Secondary Metabolite Biosynthetic Potential
Despite the numerous compounds isolated from Diaporthe species, recent advances in genome sequencing and bioinformatics analysis indicate that the number of biosynthetic gene clusters (BGCs) of SMs exceeds the number of SMs identified so far [65]. To fully understand SMs' biosynthetic potential, we used the "antibiotics and secondary metabolite analysis shell-antiSMASH" tool to predict BGCs from the genomes of Diaporthe species available in the NCBI database (National Center for Biotechnology Information, http://www. ncbi.nlm.nih.gov/, accessed on 1 February 2023). A total of 19 species were analyzed, and the antiSMASH 7 beta was applied using the "relaxed" detection strictness. As is shown in Figure 12, most species encoded~90 BGCs to 110 BGCs except for Diaporthe aspalathi (46 BGCs) and Diaporthe helianthi (65 BGCs).
i 2023, 9, x FOR PEER REVIEW 23 of 2 anticancer agent in tumors [72]. FR901512 is an HMG-CoA reductase inhibitor that ha the potential to lower cholesterol and fat [73].  The BGCs were characterized as polyketide (PKSs), non-ribosomal peptides (NRPSs), terpenes, hybrid PKS-NRPSs, ribosomally synthesized and post-translationally modified peptides (RiPPs), and indole-related compounds. PKSs and NRPSs are the most abundant BGCs of all species (Figure 12). Some BGCs show high similarity with known BGCs, and their SMs are common to different species ( Figure 13). A number of Diaporthe species were predicted to synthesize alternariol, mellein, and nectriapyrone C, which were noted for their phytotoxic and antimicrobial activities [66][67][68]. These metabolites may allow organisms to inhibit competitors that occupy the same niches and facilitate invasion when organisms are acting as phytopathogens. The BGCs of enniatin, ochratoxin A, and culmorin are present in several Diaporthe genomes [69][70][71]. These compounds are described as "emerging mytotoxins" and are mainly produced by the Fusarium species, which are wheat pathogens. This indicates that not only the Fusarium, but also the Diaporthe strains can produce contaminants in food and feed. Certain compounds with medicinal potential were also observed. Clavaric acid is an inhibitor of FPTase and may be effective as an anticancer agent in tumors [72]. FR901512 is an HMG-CoA reductase inhibitor that has the potential to lower cholesterol and fat [73].

Conclusions
This review highlights the potential of the secondary metabolites of the genus Diaporthe. A total of 275 secondary metabolites associated with terrestrial and marine environments have been isolated from this genus during the last twelve years. We can see in Figure 9 that of the 275 compounds reported, 153 (accounting for about 55% of the total) and 110 (about 41% of the total) were derived from terrestrial and marine origins, respectively, and 12 (about 4%) were isolated in both environments. After the comprehensive literature review, we found that active metabolites (56% and 34%, respectively) are less common than inactive metabolites (45% and 66%, respectively) in terrestrial and marine environments. Moreover, a total of 92 bioactive compounds (approximately 56%) were found in terrestrial samples, while 42 (about 34%) were found in marine samples. Current studies suggest that compounds with strong bioactivities could be used as potential drug candidates in the future, but more in-depth studies are needed to explore the mechanisms involved. This study also confirms the potential of terrestrial habitats for drug discovery and will help researchers find novel natural, potent fungal products. Genomic analyses suggested that Diaporthe species have great potential to produce more SMs. Therefore, future efforts should be focused on activating these silent BGCs via various methods, such as changing fermentation conditions, transcriptional regulation, using chemical elicitors, and heterologous gene expression.