Bioactive Secondary Metabolites of the Genus Diaporthe and Anamorph Phomopsis from Terrestrial and Marine Habitats and Endophytes: 2010–2019

The genus Diaporthe and its anamorph Phomopsis are distributed worldwide in many ecosystems. They are regarded as potential sources for producing diverse bioactive metabolites. Most species are attributed to plant pathogens, non-pathogenic endophytes, or saprobes in terrestrial host plants. They colonize in the early parasitic tissue of plants, provide a variety of nutrients in the cycle of parasitism and saprophytism, and participate in the basic metabolic process of plants. In the past ten years, many studies have been focused on the discovery of new species and biological secondary metabolites from this genus. In this review, we summarize a total of 335 bioactive secondary metabolites isolated from 26 known species and various unidentified species of Diaporthe and Phomopsis during 2010–2019. Overall, there are 106 bioactive compounds derived from Diaporthe and 246 from Phomopsis, while 17 compounds are found in both of them. They are classified into polyketides, terpenoids, steroids, macrolides, ten-membered lactones, alkaloids, flavonoids, and fatty acids. Polyketides constitute the main chemical population, accounting for 64%. Meanwhile, their bioactivities mainly involve cytotoxic, antifungal, antibacterial, antiviral, antioxidant, anti-inflammatory, anti-algae, phytotoxic, and enzyme inhibitory activities. Diaporthe and Phomopsis exhibit their potent talents in the discovery of small molecules for drug candidates.


Introduction
Diaporthe is an important fungal genus of plant pathogens [1] belonging to the family Diaporthaceae, order Diaporthales, class Sordariomycetes [2]. It is mainly isolated from various hosts distributed in tropical and temperate zones and can cause diseases to a wide range of plant hosts, as well as humans and other mammals [3,4]. The ascomycetes of Diaporthe Nitschke 1870 and Phomopsis (Sacc.) Bubák 1905 are regarded to form a genus [5,6]. In Index Fungorum (2020), more than 1120 records of Diaporthe and 986 of Phomopsis are listed (http://www.indexfungorum.org/, accessed December 2020). There is a common understanding that, in these ascomycetes, the teleomorph states are named as Diaporthe and the anamorph states called as Phomopsis [7][8][9][10]. For a long time, a dispute has remained concerning whether the generic name should be defined as Diaporthe or Phomopsis. Due to the importance of this genus as plant pathogens, the classification of Diaporthe has been discussed by many researchers. Since Diaporthe was cited earlier and represents most of the species described in nature, more mycologists suggest that the use of Diaporthe as a generic name have more priority and is more suitable for the current study of this fungal group [11][12][13]. In recent years, the previous classification methods based on morphological characteristics are no longer applicable to the genus Diaporthe and advanced molecular techniques will replace them to solve the classification problem of Diaporthe [13,14]. In this review, we use the older name Diaporthe as the generic name.
Based on the existing literature investigations, more secondary metabolites have been separated from Phomopsis than Diaporthe. To date, a large number of compounds have been isolated from endophytic fungi of terrestrial plants in Diaporthe and Phomopsis, some of which originate from the marine environment (mainly mangroves and sediments). Most of compounds are classified as polyketides, which is the main structural type of secondary metabolites in this genus. The reported compounds showed various bioactivities, such as cytotoxic [15], antifungal [16], antibacterial [17], antiviral [18], antioxidant [19], antiinflammatory [20], phytotoxic [21], and enzyme inhibition [22]. Up to now, there are 26 known species and various unidentified species of Diaporthe and Phomopsis have been studied for their metabolites. Our current review comprehensively summarize a total of 335 bioactive natural products from Diaporthe and Phomopsis between 2010 and 2019, covering their detailed chemical structures with classifications in structural types, as well as their bioactivities and habitats.

Bioactive Secondary Metabolites from Phomopsis
The Phomopsis fungi are important resource of bioactive compounds in the field of drug discovery, and have remarkable medical application value. According to the literature reports in recent ten years, a total of 246 bioactive compounds are summarized from Phomopsis herein. These substances have rich and diverse biological activities, such as cytotoxic, antifungal, antibacterial, antiviral, antioxidant, anti-inflammatory, phytotoxic, antimalarial, antialgae, antimigratory, pro-apoptotic, accelerating, and inhibiting the growth of subintestinal vessel plexus (SIV) branches, protecting effects on pancreatic β-cells, motility inhibitory and zoosporicidal potential, and enzyme inhibitory activities (Table 1). Among them, some interesting and promising bioactive compounds might be used in pharmaceutical and agricultural fields. The derived habitats of the Phomopsis strains can also be found in Table 1, which shows that there are 174 (accounting for 71%) and 66 (accounting for 27%) compounds obtained from terrestrial and marine environments, respectively, while six compounds (accounting for 2%) were not mentioned their habitats. Antimicrobial, Antioxidant, Inhibiting acetylcholinesterase and α-glucosidase [33]         A. annua (T) Cytotoxic [87] 244 Luteolin A. annua (T) Cytotoxic [87] 245 Naringenin P. castaneaemollissimae GQH87 A. annua (T) Cytotoxic [87] 246 A. annua (T) Cytotoxic [87] a T: terrestrial environment; M: marine environment; b The habitat was not mentioned.

Polyketides
Polyketides are a large and diverse family of natural products, containing various chemical structures and biological activities [104]. In this review, 171 polyketides are summarized from Phomopsis, accounting for 70% of the total compounds from Phomopsis.

Oblongolides
Oblongolides are a kind of natural active products with novel norsesquiterpene γ-lactone. At present, oblongolides are relatively less reported than other kinds of polyketides. Most of them exist in the fungi of Phomopsis, and mainly have cytotoxic activities [113]. Three new oblongolides, oblongolides Z (147) and Y (148) and 2-deoxy-4αhydroxyoblongolide X (154), were extracted from Phomopsis sp. BCC 9789 isolated from a wild banana (Musa acuminata) leaf. Compound 147 was found to have inhibitory effect on anti-herpes simplex virus type 1 (HSV-1) with IC 50

Terpenoids
Terpenoids are a kind of natural bioactive substances with isoprene as scaffold, which are widely distributed and rich in species [114,115]. Herein, a total of 38 terpenoids, including three monoterpenoids, 25 sesquiterpenoids, seven diterpenoids, and three triterpenoids, were isolated from various Phomopsis strains, accounting for 15% of all the described metabolites, second only to polyketides. It is worth noting that some terpenoids showed interesting bioactivities, such as enzyme inhibitory and anti-inflammatory activities.

Macrolides
Macrolides are a class of medicinal compounds containing macrolactone ring structures, many of which are used as antifungal and antibacterial drugs in clinic, such as erythromycins [122]. Nowadays, a large number of macrolide antibiotics are widely used in the treatment of human diseases. Eight secondary metabolites were obtained from Phomopsis and showed cytotoxic, antimicrobial, and enzyme inhibitory activities. Three cytotoxic polyketides, Sch-642305 (219), LMA-P1 (220), and benquoine (221), were found in the endophytic fungus Phomopsis sp. CMU-LMA of Alpinia malaccensis. Compounds 219 and 221 also displayed antimicrobial activities [94]. The endophytic fungus Phomopsis sp. IFB-ZS1-S4 provided a known aspergillide C (222), which had moderate inhibitory effect on neuraminidase in vitro with IC 50 value of 5.59 µM [37]. Four highly oxygenated tenellone-macrolide conjugated dimers, lithocarpins A-D (223-226), were obtained from P. lithocarpus FS508 isolated from the deep-sea sediment sample collected in the Indian Ocean. All metabolites (223-226) showed cytotoxic activities against three human tumor cells (SF-268, MCF-7, and HepG-2) with IC 50 values in the range of 17.0-52.2 µM [95]. The structures of macrolides (219-226) are shown in Figure 10.

Fatty Acids
Fatty acids are simple linear compounds that play an important role in the synthesis and catabolism of organisms [167]. Over here, six fatty acids are reported from Diaporthe. The fungus D. phaseolorum derived from Laguncularia racemose, afforded 3-hydroxypropionic acid (330), which showed antimicrobial activity against S. aureus and S. typhi [162]. A phytotoxic metabolite, 3-nitropropionic acid (331), was isolated from D. gulyae. Compound 331 was notably active in causing necroses on several weedy and crop plant species [149]. Two new fatty acids, diapolic acids A and B (332 and 333), were isolated from endophytic fungus D. terebinthifolii. They had moderate antibacterial activities against Y. enterocolitica with IC 50 values of 78.4 and 73.4 µM [156]. Studies of the strain Diaporthe sp. JC-J7 from stems of Dendrobium nobile led to the isolation of a new compound, diaporthsin E (334). It showed low antihyperlipidemic activity on triglycerides (TG) in steatotic L-02 cells with the inhibition rate of 26% at the concentration of 5 µg/mL [163]. The novel anti-candidal metabolite, 3-hydroxy-5-methoxyhex-5-ene-2,4-dione (335), was derived from Diaporthe sp. ED2 of medicinal herb Orthosiphon stamieus Benth. It showed antifungal activity against C. albicans with MIC value of 3.1 µg/mL [164]. The structures of fatty acids (330-335) are shown in Figure 19.

Characteristics of Bioactive Secondary Metabolites from the Genus Diaporthe and Anamorph Phomopsis
In this paper, a total of 335 bioactive compounds from the genus Diaporthe and Phomopsis are summarized. There are 106 secondary metabolites from Diaporthe and 246 ones from Phomopsis, in which 17 compounds were obtained from both of Diaporthe and Phomopsis. These compounds are classified into polyketides, terpenoids, steroids, macrolides, tenmembered lactones, alkaloids, flavonoids, and fatty acids. As seen in Figure 20, about two thirds of all compounds reported from Diaporthe and Phomopsis are refered to polyketides, accounting for 63% and 70%, respectively. Moreover, terpenoids (8%, 15%), alkaloids (17%, 6%), and steroids (2%, 4%) were also produced by both of Diaporthe and Phomopsis. It is worth noting that fatty acids (6%) and ten-membered lactones (4%) are only reported from Diaporthe, while flavonoids (2%) and macrolides (3%) are only found in Phomopsis. Polyketides, as the largest member of the metabolites, are widely used in the field of medicine and play an important role in the treatment of cancer diseases. The various bioactivities of the compounds isolated from Diaporthe and Phomopsis are presented in Figure 21, mainly containing cytotoxic, antibacterial, antifungal, antiviral, anti-inflammatory, antioxidant, antialgae, enzyme inhibition, and phytotoxic activities. Most of compounds have at least one kind of bioactivities. As seen in Figure 21 and Tables 1 and 2, secondary metabolites of Diaporthe and Phomopsis mainly exhibit cytotoxic, antibacterial and antifungal activities, accounting for 73% of all compounds, with 56 in Diaporthe and 200 from Phomopsis. Interestingly, in recent years, more and more compounds with anti-inflammatory, antioxidant and enzyme inhibitory activities have been studied in important human diseases.

Conclusions
This review presents the diverse chemical structures and bioactivities of 335 compounds isolated from 26 known species and various unidentified species of the genus Diaporthe and its anamorph Phomopsis between 2010-2019. Here, we can see from Tables 1 and 2, among all of the reported compounds, there are 236 (accounting for about 70%) and 92 (about 27%) compounds derived only from terrestrial and marine environments (including mangroves, sediments, deep-sea fungi and marine animals), respectively. In addition, only one compound is obtained from both of terrestrial and marine environments. In contrast, six compounds are not mentioned with their habitats in the literature. Polyketides represent the main chemical population, accounting for 64%. About 73% of all metabolites possess cytotoxic, antibacterial, and antifungal activities. The species named as Phomopsis significantly produce much more compounds than Diaporthe, and most strains have not yet been identified at the species level. In conclusion, these results illustrate that the metabolic resources of Diaporthe and Phomopsis are of great value and deserved to conduct further research. Interestingly, in the past three years, there have been more reports on the secondary metabolites of the fungi in Diaporthe and Phomopsis than before, displaying an increasing trend, which indicates that Diaporthe and Phomopsis are regarded as important sources for discovering new natural bioactive substances.
In the past many years, lots of interesting fungal bioactive metabolites had been widely developed into new drugs, like antibiotics. Although most compounds obtained from Diaporthe and Phomopsis fungi had been studied on their isolation, structures, and activities, the in-depth research on pharmacological mechanisms and development of potent active compounds in drugs are still less. According to current studies, some compounds with remarkable bioactivities may serve as potential drug candidates in the future, such as cytotoxic altersolanol A and PM181110, and antimicrobial dicerandrol A. In order to ascertain the therapeutic potential of these compounds, further studies of pharmacological and producing mechanisms are required.
The fungal species in Diaporthe and Phomopsis have been considered to be important sources that can produce diverse and novel bioactive metabolites, which has attracted many natural product chemists and pharmacologists to study in recent years. The metabolites produced by Diaporthe and Phomopsis have rich biological activities, which is enough to show the importance of its metabolic resources. Nowadays, many fungi produce interesting bioactive metabolites that have been studied for their biosynthesis pathway, while similar studies in Diaporthe and Phomopsis are performed relatively less often. In the following work, the microbial biosynthesis pathway might be considered for further developing valuable products from Diaporthe or Phomopsis, which are hoped to be used as drug molecules for disease treatment. However, it cannot be ignored that Diaporthe or Phomopsis are important plant pathogens which might cause a wide range of plant host diseases and even serious human pathogens. In the future work, we should also focus on the role of metabolites produced by these pathogens, as well as the relationships with their hosts.