Metabolites from Clonostachys Fungi and Their Biological Activities

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Journal of Fungi

Review Metabolites from Clonostachys Fungi and Their Biological Activities

Peipei Han, Xuping Zhang, Dan Xu, Bowen Zhang, Daowan Lai and Ligang Zhou *

Department of Plant Pathology, College of Plant Protection, China Agricultural University, Beijing 100193, China; [email protected] (P.H.); [email protected] (X.Z.); [email protected] (D.X.); [email protected] (B.Z.); [email protected] (D.L.) * Correspondence: [email protected]; Tel.: +86-10-6273-1199

Received: 9 September 2020; Accepted: 12 October 2020; Published: 16 October 2020

Abstract: Clonostachys (teleomorph: Bionectria) fungi are well known to produce a variety of secondary metabolites with various biological activities to show their pharmaceutical and agrochemical applications. Up to now, at least 229 secondary metabolites, mainly including 84 nitrogen-containing metabolites, 85 polyketides, 40 terpenoids, and 20 other metabolites, have been reported. Many of these compounds exhibit biological activities, such as cytotoxic, antimicrobial, antileishmanial, antimalarial activities. This mini-review aims to summarize the diversity of the secondary metabolites as well as their occurrences in Clonostachys fungi and biological activities.

Keywords: secondary metabolites; Clonostachys fungi; Bionectria; Gliocladium; Nectria; structural diversity; biological activities

1. Introduction The fungal genus Clonostachys (formerly named Gliocladium), teleomorph Bionetria (formerly named Nectria or Nectriopsis), belongs to the family Bionectriaceae of Sordariomycetes in Ascomycota [1]. The Clonostachys fungi are widely distributed all over the world. They are saprotrophs, destructive mycoparasites, lichenicoles, or inhabitants of recently dead trees and decaying leaves. At present, there are about 44 species in the genus Clonostachys [1], and among them, about 18 species have been studied for their secondary metabolites, including B. byssicola, B. ochroleuca, B. pityrodes, C. candelabrum, C. compactiuscula, C. rogersoniana, C. rosea, G. roseum, N. coccinea, N. coryli, N. erubescens, N. fuckeliana, N. galligena, N. haematococca, N. inventa, N. lucida, N. pseudotrichia, and N. viridescens. Clonostachys fungi are abundant in many classes of secondary metabolites, mainly including nitrogen-containing compounds, polyketides, and terpenoids. Many metabolites exhibit biological activities, such as antimicrobial, insecticidal, nematocidal, antiparasitic, phytotoxic and cytotoxic activities. Until now, secondary metabolites of Clonostachys fungi and their biological activities have not been reviewed. This mini-review describes the classiﬁcation, occurrences, and biological activities of the secondary metabolites from Clonostachys fungi.

2. Nitrogen-Containing Metabolites and Their Biological Activities The nitrogen-containing metabolites from Clonostachys fungi mainly include linear oligopeptides, cyclopeptides, and piperazines. The nitrogen-containing metabolites, their isolated Clonostachys fungi and biological activities are shown in Table1.

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Table 1. Nitrogen-containing metabolites in Clonostachys fungi and their biological activities.

Metabolite Class Metabolite Name Fungal Species Biological Activity Ref. Linear Inhibition on platelet Clonostachin (1) Clonostachys sp. F5898 [2] oligopeptides aggregation Bionectria sp. MSX 47401 - [3] Clonostachin B (2) Bionectria sp. MSX 47401 - [3] Pullularin F (3) Bionectria ochroleuca -[4] Inhibitory activity on Cyclopeptides Argadin (4) Clonostachys sp. FO-7314 [5] chitinase Inhibitory activity on Argifin (5) Gliocladium sp. FTD-0668 [6,7] chitinase Arthrichitin (6) Nectria sp. - [8] Antifungal activity on [9] the yeasts Anti-dinoflagellate Clonostachysin A (7) Clonostachys rogersoniana [10] activity Anti-dinoflagellate Clonostachysin B (8) Clonostachys rogersoniana [10] activity Cyclo-(Gly- -Leu- -allo-Ile- - D D L Clonostachys rosea Cytotoxic activity [11] Val-L-Val-D-Trp-β-Ala) (9) Cyclo-(Gly- -Leu- -Val- -Val- - D L L L Clonostachys rosea -[11] Val-D-Trp-β-Ala) (10) Cyclo-(Gly- -Leu- -allo-Ile- - D D D Clonostachys rosea -[11] allo-Ile-L-Val-D-Trp-β-Ala) (11) Cyclosporin A (12) Nectria sp. F-4908 - [12] Immunosuppressive and antifungal [13] activities Cyclosporin C (13) Nectria sp. F-4908 - [12] Immunosuppressive and antifungal [13] activities IB-01212 (14) Clonostachys sp. ESNA-A009 Cytotoxic activity [14] Antileishmanial [15] activity Pullularin A (15) Bionectria ochroleuca Cytotoxic activity [4] Pullularin C (16) Bionectria ochroleuca Cytotoxic activity [4] Pullularin E (17) Bionectria ochroleuca -[4] Piperazines Bionectin A (18) Bionectria byssicola F120 Antibacterial activity [16] Bionectin B (19) Bionectria byssicola F120 Antibacterial activity [16] Bionectin C (20) Bionectria byssicola F120 - [16] Bionectin D (21) Bionectria sp. Y1085 Antibacterial activity [17] Bionectin E (22) Bionectria sp. Y1085 Antibacterial activity [17] 3,6-Bis(methylthio)-cyclo Nectria inventa Trypanocidal activity [18] (alanyltryptophyl) (23) Chaetocin (24) Nectria inventa Trypanocidal activity [18] Chetoseminudin B (25) Nectria inventa Trypanocidal activity [18] Clonostachys compactiuscula Clonocoprogen A (26) Antimalarial activity [19] FKR-0021 Clonostachys compactiuscula Clonocoprogen B (27) Antimalarial activity [19] FKR-0021 Clonostachys compactiuscula Clonocoprogen C (28) Antimalarial activity [19] FKR-0021 J. Fungi 2020, 6, 229 3 of 31

Table 1. Cont.

Metabolite Class Metabolite Name Fungal Species Biological Activity Ref. Clonostachys compactiuscula N14-Plmitoylcoprogen (29) Antimalarial activity [19] FKR-0021

Cyclo (L-Pro-L-Leu) (30) Bionectria sp. Y1085 - [17] Dioxopiperazine (31) Bionectria sp. Y1085 - [17] Gliocladicillin A (32) Bionectria sp. Y1085 - [17] Gliocladicillin C (33) Bionectria sp. Y1085 Antibacterial activity [17] Clonostachys rogersoniana Cytotoxic activity [20] Gliocladin A (34) Glicladium roseum OUPS-N132 - [21] Gliocladin B (35) Glicladium roseum OUPS-N132 - [21] Gliocladin C (36) Glicladium roseum OUPS-N132 Cytotoxic activity [21] Gliocladium roseum Antinematodal activity [22] YMF1.00133 Gliocladine A (37) Gliocladium roseum 1A Antinematodal activity [23] Gliocladium roseum Gliocladine B (38) Antinematodal activity [23] YMF1.00133 Bionectria sp. Y1085 - [17] Gliocladine C (39) Gliocladium roseum 1A Antinematodal activity [23] Gliocladine D (40) Gliocladium roseum 1A Antinematodal activity [23] Gliocladine E (41) Gliocladium roseum 1A Antinematodal activity [23] Gliocladium roseum Glioclatine (42) Antinematodal activity [24] YMF1.00133 Glioperazine (43) Gliocladium sp. OUPS-N132 - [21] Bionectria byssicola F120 - [25] Clonostachys rosea -[11] Glioperazine B (44) Bionectria byssicola F120 Antimicrobial activity [25] Glioperazine C (45) Bionectria byssicola F120 - [25] Haematocin (46) Nectria haematococca Antifuangl activity [26] Lasiodipline D (47) Bionectria sp. Y1085 - [17] Sch52900 (48) Gliocladium roseum 1A Antinematodal activity [23] Sch52901 (49) Gliocladium roseum 1A Antinematodal activity [23] Verticillin A (50) Gliocladium roseum 1A Antinematodal activity [23] Bionectria sp. Y1085 - [17]

110-Deoxyverticillin A (51) Gliocladium roseum 1A Antinematodal activity [23]

11,110-Dideoxyverticillin A (52) Bionectria sp. Y1085 - [17] Verticillin B (53) Nectria inventa Trypanocidal activity [18] Verticillin D (54) Bionectria byssicola F120 - [16] Bionectria ochroleuca Cytotoxic activity [4] Clonostachys rosea Cytotoxic activity [11] Verticillin G (55) Bionectria byssicola F120 Antibacterial activity [25] Bionectriaceous strains MSX Verticillin H (56) Cytotoxic activity [27] 64546 and MSX 59553 Other Clonostachys compactiuscula nitrogen-containing N-Benzyl-3-phenyllactamide (57) -[19] FKR-0021 metabolites N-Benzyl-3-phenylpropanamide Clonostachys compactiuscula -[19] (58) FKR-0021 Co-cultivation of Bionectria sp. Bionectriamine A (59) with Bacillus subtilis or -[28] Streptomyceslividans J. Fungi 2020, 6, 229 4 of 31

Table 1. Cont.

Metabolite Class Metabolite Name Fungal Species Biological Activity Ref. Co-cultivation of Bionectria sp. Bionectriamine B (60) with Bacillus subtilis or -[28] Streptomyceslividans Cinnacidin (61) Nectria sp. DA060097 Phytotoxic activity [29] Clonostalactam (62) Clonostachys rosea -[11] Weak leishmanicidal Cytochalasin D (63) Nectria pseudotrichia [30] activity Fusarin C (64) Nectria coccinea A56-9 Antifungal activity [31] (5Z)-Fusarin C (65) Nectria coccinea A56-9 Antifungal activity [31] (7Z)-Fusarin C (66) Nectria coccinea A56-9 Antifungal activity [31] verM disruption mutant of Gliocladiosin A (67) Antibacterial activity [32] Clonostachys rogersoniana verM disruption mutant of Gliocladiosin B (68) Antibacterial activity [32] Clonostachys rogersoniana verG disruption mutant of Rogersonin A (69) -[33] Clonostachys rogersoniana verG disruption mutant of Rogersonin B (70) -[33] Clonostachys rogersoniana Ilicicolin H (71) Nectria sp. B-13 - [34] (S)-Phenopyrrozin (72) Bionectria sp. - [28] (S)-p-Hydroxyphenopyrrozin (73) Bionectria sp. - [28] 1,2-Dihydrophenopyrrozin (74) Bionectria sp. - [28] 1,2-Dehydrovirgineone (75) Bionectria sp. MSX 47401 Antibacterial activity [3] Antibacterial and Virgineone (76) Bionectria sp. MSX 47401 [3] antifungal activities Virgineone aglycone (77) Bionectria sp. MSX 47401 Antibacterial activity [3] Indole-3-acetic acid (78) Bionectria sp. Y1085 - [17]

L-Tryptophan (79) Bionectria sp. Y1085 - [17] Immunostimulatory FR-900483 (80) Nctria lucida F-4490 [35] activity Bostrycoidin (81) Nectria haematococca -[36] 5-Deoxybostrycoidin (82) Nectria haematococca -[37] Penicolinate A (83) Bionectria sp. Cytotoxic activity [28] Scorpinone (84) Nectria pseudotrichia -[38]

2.1. Linear Oligopeptides The oligopeptides from fungi include linear and cyclic peptides. Two linear tetradecapeptides, named clonostachin (1) and clonostachin B (2) were isolated from Clonostachys fungi (Figure1). Clonostachin (1) was ﬁrst isolated from Clonostachys sp. F5898, and both clonostachin (1) and clonostachin B (2) were then isolated from Bionectria sp. MSX 47401, and each oligopeptide contained an N-terminal acetyl group and a C-terminal mannitol unit [3]. Clonostachin (1) inhibited ADP-induced aggregation of human platelets by 80% at 150 µM[2]. Pullularin F (3) was isolated from the endophytic fungus Bionectria ochroleuca from the mangrove plant Sonneratia caseolaris [4]. J. Fungi 2020, 6, x FOR PEER REVIEW 5 of 30

Clonostachin (1) was first isolated from Clonostachys sp. F5898, and both clonostachin (1) and clonostachinJ. Fungi 2020 , B6, x( FOR2) werePEER REVIEW then isolated from Bionectria sp. MSX 47401, and each oligopeptide5 of 30 containedClonostachin an N-terminal (1) was acetyl first isolated group fromand aClonostachys C-terminal sp.mannitol F5898, unit and both[3]. Clonostachin clonostachin (1)) inhibited and ADPclonostachin-induced aggregation B (2) were ofthen human isolated pla t fromelets Bionectria by 80% atsp. 150 MSX μM 47401,[2]. Pullularin and each F oligopeptide (3) was isolated fromcontained the endophytic an N-terminal fungus acetyl Bionectria group ochroleucaand a C-terminal from themannitol mangrove unit [3]. plant Clonostachin Sonneratia (1 caseolaris) inhibited [ 4]. ADP-induced aggregation of human platelets by 80% at 150 μM [2]. Pullularin F (3) was isolated J. Fungi 2020, 6, 229 5 of 31 from the endophyticOH fungus Bionectria ochroleuca from the mangrove plant Sonneratia caseolaris [4]. H O N N OH O H O OH N H O O NN N O OH H H ON O OH O O N N H O H N O O N N OH O H H O N OH O N N O O H N H OH OH N R HN O O N O OH H N R N OH O H N OH OH HN O N O H N O O OH H N O H O O H O NO OH N O O O OH O N H OH O O N H N O N O O O OH O H O N O N N HO H N HO OH H HO OH 1. Clonostachin, R=CH3 HO 1. Clonostachin, R=CH3 3. Pullularin F 2. Clonostachin B, R=CH2CH3 3. Pullularin F 2. Clonostachin B, R=CH2CH3

FigureFigureFigure 1.1. 1.LinearLinear Linear oligopeptidesoligopeptides oligopeptides isolatedisolated from fromfrom Clonostachys ClonostachysClonostachys fungi. fungi.fungi.

2.2.2.2. CyclopeptidesCyclope2.2. Cyclopeptidesptides CyclopeptidesCyclopeptidesCyclopeptides are are are cyclic cyclic cyclic compounds compounds compounds formed formed mainly mainly mainly by by by the the the amide amide amide bonds bonds bonds between between between either either either proteinogenicproteinogenicproteinogenic oror non-proteinogenic nonor non-proteinogenic-proteinogenic aminoamino amino acidsacids [[1313,3,39,399].].]. The TheThe stru structuresstructuresctures of oftheof the thecyclopeptides cyclopeptidescyclopeptides isolated isolatedisolated from Clonostachys fungi are shown in Figure 2. fromfrom ClonostachysClonostachysfungi fungi areare shownshown inin FigureFigure2 .2.

CH3 O HOOC CH CH3 HN O HOOC O CH NH NH O O OC HN NH O NH O CHNH N N H CHO C NH C HN H2C H H N C NH O CH N N H O CH NH CH N N H C C O H H N HNC H H HOOC CH2 N C O C O O CH N N N CH C CH H H HOOCHCNH 3 N O CN O CH N C N CH CH CH C 3 HO OHNH2C CH N COH C N O CH N N C H COH C HO H OHHO2C COH N CH C H O 4N. Argadin H O H5.O ArgifinO O 4. Argadin 5. Argifin R O O O O H H N OR N OH N N HN O O O N OHNO O H H N O O N OOH N NO N O N HNO O O O O N HN HN O N OH N O O O N O N O N O O O O H6N. Arthrichitin 7. ClonostachysinN A R=H OH N O 8. Clonostachysin B R=CHN3 O 6. Arthrichitin 7. Clonostachysin A R=H 8. Clonostachysin B R=CH3 Figure 2. Cont. J. Fungi 2020, 6, 229 6 of 31 J. Fungi 2020, 6, x FOR PEER REVIEW 6 of 30

O O O O HN N HN N H HN H HN O O HN HN O HN O O HN O HN O HN O H H N N N N H H O O

9. Cyclo-(Gly-D-Leu-D-allo-Ile-L-Val-L-Val-D-Trp--Ala) 10. Cyclo-(Gly-D-Leu-L-Val-L-Val-L-Val-D-Trp--Ala) O O N O HN N H N N H HN N O O O HN O O HN O HN O O O H O N O O N H N O N N H O N

11. Cyclo-(Gly-D-Leu-D-allo-Ile-D-allo-Ile-L-Val-D-Trp--Ala) 14. IB-01212

O O OH N N N O N O HN O R O HN O O N O HN N N O H N N O N HN HN O R O O O O O O O O O N O O N O O O O 12. Cyclosporin A, R=H N N 13. Cyclosporin C, R=OH N N H H OH 15. Pullularin A, R=CH3 16. Pullularin C, R=H 17. Pullularin E Figure 2. Cyclopeptides isolated from Clonostachys fungi. Figure 2. Cyclopeptides isolated from Clonostachys fungi. Argadin (4), a cyclic pentapeptide, was isolated from Clonostachys sp. FO-7314. It showed Argadin (4), a cyclic pentapeptide, was isolated from Clonostachys sp. FO-7314. It showed inhibitory activity against blowfly (Lucilia cuprina) chitinase with IC50 values of 150 nM at 37 ◦C and inhibitory activity against blowfly (Lucilia cuprina) chitinase with IC50 values of 150 nM at 37 C and 3.4 nM at 20 C, respectively [5]. Another cyclic pentapeptide, namely argifin (5), from Gliocladium sp. 3.4 nM at 20◦ C, respectively [5]. Another cyclic pentapeptide, namely argifin (5), from Gliocladium also exhibited inhibitory activity against blowfly chitinase [6,7]. sp. also exhibited inhibitory activity against blowfly chitinase [6,7]. Arthrichitin (6) was a cyclic tetradepsipeptide isolated from Nectria sp. [8]. This lipodepsipeptide Arthrichitin (6) was a cyclic tetradepsipeptide isolated from Nectria sp. [8]. This was also isolated from other fungi to show inhibitory activity on the yeasts Schizosaccharomyces pombe lipodepsipeptide was also isolated from other fungi to show inhibitory activity on the yeasts and Rhodotorula glutinis [9]. Schizosaccharomyces pombe and Rhodotorula glutinis [9]. Clonostachysins A (7) and B (8) were two cyclic nonapeptides isolated from Clonostachys Clonostachysins A (7) and B (8) were two cyclic nonapeptides isolated from Clonostachys rogersoniana. They exhibited a selectively inhibitory effect on a dinoflagellate Prorocentrum micans at 30 rogersoniana. They exhibited a selectively inhibitory effect on a dinoflagellate Prorocentrum micans at µM but had no effect on other microalgae and bacteria, even at 100 µM[10]. 30 μM but had no effect on other microalgae and bacteria, even at 100 μM [10]. Three cyclic heptapeptides, named cyclo-(Gly-D-Leu-D-allo-Ile-L-Val-L-Val-D-Trp-β-Ala) (9), Three cyclic heptapeptides, named cyclo-(Gly-D-Leu-D-allo-Ile-L-Val-L-Val-D-Trp-β-Ala) (9), cyclo-(Gly-D-Leu-L-Val-L-Val-L-Val-D-Trp-β-Ala) (10), and cyclo-(Gly-D-Leu-D-allo-Ile-D-allo-Ile-L- cyclo-(Gly-D-Leu-L-Val-L-Val-L-Val-D-Trp-β-Ala) (10), and Val-D-Trp-β-Ala) (11), were isolated from the soil-derived fungus Clonostachys rosea. Among them, cyclo-(Gly-D-Leu-D-allo-Ile-D-allo-Ile-L-Val-D-Trp-β-Ala) (11), were isolated from the soil-derived cyclo-(Gly-D-Leu-D-allo-Ile-L-Val-L-Val-D-Trp-β-Ala) (9) exhibited significant cytotoxic activity against fungus Clonostachys rosea. Among them, cyclo-(Gly-D-Leu-D-allo-Ile-L-Val-L-Val-D-Trp-β-Ala) (9) the L5178Y mouse lymphoma cell line with an IC50 value of 4.1 µM[11]. exhibited significant cytotoxic activity against the L5178Y mouse lymphoma cell line with an IC50 value of 4.1 μM [11]. Two cyclic undecapeptides cyclosporins A (12) and C (13) were isolated from Nectria sp. F-4908 [12]. They showed immunosuppressive and antifungal activities [13]. J. Fungi 2020, 6, 229 7 of 31

Two cyclic undecapeptides cyclosporins A (12) and C (13) were isolated from Nectria sp. F-4908 [12]. J. Fungi 2020, 6, x FOR PEER REVIEW 7 of 30 They showed immunosuppressive and antifungal activities [13]. IBIB-01212-01212 ( 14), a cyclic hexadepsipeptide from the marine fungus Clonostachys sp. ESNA ESNA-A009,-A009, exhibited antitumor activity activity on on the the cell lines of LN LN-caP-caP (prostate cancer), SK SK-BR3-BR3 (breast (breast cancer), cancer), HT29 (colon (colon cancer), cancer), and and HeLa HeLa (cervix (cervix cancer) [cancer)14]. In addition, [14]. In IB-01212 addition, (14 ) IB showed-01212 antileishmanial (14) showed antileishmanialactivity [15]. activity [15]. Three cyclic hexadepsipeptides pullularins pullularins A A ( (1515),), C C ( (1616)) and and E E ( (17)) were isolated from from the endophytic fungus fungusBionectria Bionectria ochroleuca ochroleuca. Both. Both pullularins pullularins A (15 A) and (15) C and (16) showed C (16) showedmoderate moderate cytotoxic cytotoxicactivity against activity mouse against lymphoma mouse lymphoma cells [4]. cells Furthermore, [4]. Furthermore, pullularin pullularin A (15) fromA (15 another) from another fungus fungusPullularia Pullulariasp. BCC sp. 8613 BCC exhibited 8613 exhibited antimalarial, antimalarial, antiviral antivira and antitubercularl and antitubercular activities activities [40]. [40]. 2.3. Piperazines 2.3. Piperazines The piperazines (also called 2,5-diketopiperazines) are formed by the condensation of two amino The piperazines (also called 2,5-diketopiperazines) are formed by the condensation of two acids [41]. Piperazines are the common nitrogen-containing metabolites as monomers or dimers in amino acids [41]. Piperazines are the common nitrogen-containing metabolites as monomers or Clonostachys fungi, and most of them contain disulﬁde bonds. The structures of piperazines isolated dimers in Clonostachys fungi, and most of them contain disulfide bonds. The structures of from Clonostachys fungi are shown in Figure3. piperazines isolated from Clonostachys fungi are shown in Figure 3.

O R1 O O O O N H H OH N S S N N HO N O N HN OH NH O R O 2 H R2 O N 30. Cyclo(L-Pro- L-Leu) R2 R1 OH O HO N N O S O H N N O S S N NH H H N HN 26. Clonocoprogen A, R1=OH, R2= CO(CH2)13CH3 H O OH 27. Clonocoprogen B, R =H, R = CO(CH ) CH 1 2 2 13 3 O S 28. Clonocoprogen C, R =OH, R = CO(CH ) CH 24. Chaetocin, R1=CH2OH, R2=H 1 2 2 14 3 14 53. Verticillin B, R1=CH3, R2=OH 29. N -Palmitoylcoprogen, R1=H, R2= CO(CH2)14CH3 31. Dioxopiperazine

Figure 3. Cont. J. Fungi 2020, 6, 229 8 of 31 J. Fungi 2020, 6, x FOR PEER REVIEW 8 of 30

O O O R1 S H O OH HN N O N NH N HN S H O OH H S N SN S O H O O 44. Glioperazine B, R =OCH , R =SCH O 1 3 2 3 46. Haematocin 47. Lasiodipline D 45. Glioperazine C, R1=H, R2=H

HO O O O O O H H N H H S S N N H H N H H NS S N N S S N N NS S N N O O O O HO OH HO OH OH O O O O N N S N NS S N S N N N S H H H H N N S S N S N H H H H O O OH O O 52. 11,11'-Dideoxyverticillin A 54. Verticillin D 55. Verticillin G 56. Verticillin H

Figure 3. Piperazines isolated from Clonostachys fungi.fungi.

Bionectins A (18), B (19) and C (20), and verticillin D (54) were isolated from the liquid fermentation Bionectins A (18), B (19) and C (20), and verticillin D (54) were isolated from the liquid cultures of Bionectria byssicola F120. Both bionectins A (18) and B (19) exhibited antibacterial fermentation cultures of Bionectria byssicola F120. Both bionectins A (18) and B (19) exhibited activity against Staphylococcus aureus including methicillin-resistant Staphylococcus aureus (MRSA) antibacterial activity against Staphylococcus aureus including methicillin-resistant Staphylococcus and quinolone-resistant Staphylocossu aureus (QRSA), with MIC values of 10-30 µg/mL [16]. Bionectins aureus (MRSA) and quinolone-resistant Staphylocossu aureus (QRSA), with MIC values of 10-30 D(21) and E (22), cyclo (L-Pro-L-Leu) (30), dioxopiperazine (31), and gliocladicillins A (32) and C μg/mL [16]. Bionectins D (21) and E (22), cyclo (L-Pro-L-Leu) (30), dioxopiperazine (31), and (33) were isolated from Bionectria sp. Y1085. Among them, bionectins D (21) and E (22), as well gliocladicillins A (32) and C (33) were isolated from Bionectria sp. Y1085. Among them, bionectins D as gliocladicillin C (33) showed antibacterial activity on Escherichia coli, Staphylococcus aureus and (21) and E (22), as well as gliocladicillin C (33) showed antibacterial activity on Escherichia coli, Salmnonella typhimurium [17]. Staphylococcus aureus and Salmnonella typhimurium [17]. Four diketopiperazines: 3,6-bis(methylthio)-cyclo(alanyltryptophyl) (23), chaetocin (24), Four diketopiperazines: 3,6-bis(methylthio)-cyclo(alanyltryptophyl) (23), chaetocin (24), chetoseminudin B (25) and verticillin B (53) from deep water marine-derived fungus Nectria inventa chetoseminudin B (25) and verticillin B (53) from deep water marine-derived fungus Nectria inventa showed trypanocidal activity on Trypanosoma brucei [18]. showed trypanocidal activity on Trypanosoma brucei [18]. Four siderophore analogs, clonocoprogens A (26), B (27) and C (28) and N14-plmitoylcoprogen (29), were isolated from Clonostachys compactiuscula FKR-0021. They exhibited antimalarial activity J. Fungi 2020, 6, 229 9 of 31

Four siderophore analogs, clonocoprogens A (26), B (27) and C (28) and N14-plmitoylcoprogen (29), were isolated from Clonostachys compactiuscula FKR-0021. They exhibited antimalarial activity against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum strains, with J. Fungi 2020, 6, x FOR PEER REVIEW 9 of 30 IC50 values ranging from 1.7 µM to 9.9 µM[19]. Gliocladins A (34), B (35) and C (36) and glioperazine (43) were isolated from Gliocladium sp. against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum strains, with originally separated from the sea hare (Aplysia kurodai). Gliocladin C (36), which was a structurally IC50 values ranging from 1.7 μM to 9.9 μM [19]. unique trioxopiperazine,Gliocladins A (34 showed), B (35) signiﬁcantand C (36) cytotoxicityand glioperazine against (43) the were murine isolated P388 from lymphocytic Gliocladium leukemia sp. cellsoriginally with IC50 separatedvalue of from 2.4 µ theg/mL sea [21hare]. Gliocladin(Aplysia kurodai C ().36 Gliocladin) from Gliocladium C (36), which roseum was aYMF1.00133 structurally was furtherunique screened trioxopiperazine, to show antinematodal showed signific activityant against cytotoxicity nematodes against Panagrellus the murine redivivus P388 lymphocytic, Caenothabditis elegansleukemiaand Bursaphelenchus cells with IC50 xylophilus value of [22 2.4]. μg/mL [21]. Gliocladin C (36) from Gliocladium roseum NineYMF1.00133 epipolysulfanyldioxopiperazines was further screened to show antinematodal isolated from activityGliocladium against nematodes roseum Panagrellus1A displayed antinematodalredivivus, Caenoth activityabditis against elegans andCaenorhabditis Bursaphelenchus elegans xylophilusand [22].Panagrellus redivivus. The dimers, includingNine gliocladine epipolysulfanyldioxopiperazines A (37), gliocladine B (38 isolated), sch52900 from (Gliocladium48), sch52901 roseum (49 ),1A verticillin displayed A (50), antinematodal activity against Caenorhabditis elegans and Panagrellus redivivus. The dimers, including and 11 -deoxyverticillin A (51) are more active than the monomers with the indole moiety, namely, gliocladine0 A (37), gliocladine B (38), sch52900 (48), sch52901 (49), verticillin A (50), and gliocladines C (39), D (40) and E (41). Among them, 11 -Deoxyverticillin A (51) was the most potent 11′-deoxyverticillin A (51) are more active than the monomers0 with the indole moiety, namely, antinematodalgliocladines compound C (39), D (40 [23) and]. E (41). Among them, 11′-Deoxyverticillin A (51) was the most potent Threeantinematodal dioxopiperazines: compound [23 glioperazine]. (43), glioperazine B (44) and glioperazine C (45) were isolated fromThreeBionectria dioxopiperazines: byssicola glioperazinF120. Amonge (43 them,), glioperazine glioperazine B (44) B and (44 ) glioperazine showed weak C (45 antibacterial) were activityisolated against fromStaphylococcus Bionectria byssicola aureus F120.[25 ].Among them, glioperazine B (44) showed weak antibacterial Haematocinactivity against (46 Staphylococcus) was isolated aureus from [25]. the culture broth of Nectria haematococca, the blight disease pathogenHaematocin of ornamental (46) was plants. isolated Haematocin from the culture (46) broth inhibited of Nectria the germ-tubehaematococca elongation, the blight disease and spore pathogen of ornamental plants. Haematocin (46) inhibited the germ-tube elongation and spore germination of rice blast pathogen Pyricularia oryzae at the IC50 values of 30 and 160 µg/mL, germination of rice blast pathogen Pyricularia oryzae at the IC50 values of 30 and 160 μg/mL, respectively [26]. respectively [26]. Verticillins were the dimeric epipolythiodioxopiperazines widely distributed in Bionectriaceous Verticillins were the dimeric epipolythiodioxopiperazines widely distributed in fungi.Bionectriaceous Most of verticillins fungi. exhibited Most of cytotoxic verticillins activities exhibited [11 cytotoxic,27]. Among activities them, [11,27]. verticillin Among A ( 50 them,) showed obviouslyverticillin cytotoxic A (50) acitivity showed by obviously causing cytotoxic apoptosis acitivity and reducing by causing tumor apoptosis burden and in reducing high-grade tumor serious ovarianburden cancer in high by- inducinggrade serious DNA ovarian damage cancer [42 by]. inducing Verticillins DNA D damage (54) and [42]. G Verticillins (55) were D isolated (54) and from BionectriaG (55 byssicola) were , isolated and verticillin from Bionectria G (55) was byssicola screened, and to verticillin have antibacterial G (55) was activity screened on Staphylococcus to have aureusantibacterialwith MIC ac valuestivity on of Staphylococcus 3–10 µg/mL [aureus25]. Verticillin with MIC Dvalues (54) fromof 3–10 the μg/mL endophytic [25]. Verticillin fungus DBionectria (54) ochroleucafrom showed the endophytic pronounced fungus cytotoxic Bionectria activity ochroleuca against showed mouse pronounced lymphoma cytotoxic cells [4 activity]. against mouse lymphoma cells [4]. 2.4. Other Nitrogen-Containing Metabolites 2.4. Other Nitrogen-Containing Metabolites The structures of the other nitrogen-containing metabolites, including amides and amines isolated The structures of the other nitrogen-containing metabolites, including amides and amines from Clonostachys fungi are shown in Figure4. isolated from Clonostachys fungi are shown in Figure 4.

O O OH OH O N H H N OH N R N H H OH OH O HO O HO O 57. N-Benzyl-3-phenyllactamide, R=OH 58. N-Benzyl-3-phenylpropanamide, R=H 59. Bionectriamine A 60. Bionectriamine B

OH O

H O O H H O N HN HO NH O O O O HO OH O OH O 61. Cinnacidin 62. Clonostalactam 63. Cytochalasin D

Figure 4. Cont. J. Fungi 2020, 6, 229 10 of 31

J. Fungi 2020, 6, x FOR PEER REVIEW 10 of 30

O O O O O O O O O O O O HO HO HO O O N N N O HO H HO H HO H 64. Fusarin C 65. (5Z)-Fusarin C 66. (7Z)-Fusarin C H N O O _ _ + + O N O N O O O O HN HN HO NNH H O OH O OH O O R O O O O O O O OH O O 67. Gliocladiosin A, R=H 69. Rogersonin A 70. Rogersonin B 68. Gliocladiosin B, R=CH3

FigureFigure 4. Other 4. Other nitrogen-containing nitrogen-containing metabolitesmetabolites isolated isolated from from ClonostachysClonostachys fungi.fungi.

Both N-benzyl-3-phenyllactamide (57) and N-benzyl-3-phenylpropanamide (58) were isolated from Clonostachys compactiuscula FKR-0021 [19]. J. Fungi 2020, 6, 229 11 of 31

Fusarin C (64), (5Z)-fusarin C (65) and (7Z)-fusarin C (66) were isolated from Nectria coccinea A56-9. They showed antifungal activity against Pyricularia oryzae by inhibiting dihydroxynaphthalene-melanin biosynthesis [31]. Gliocladiosins A (67) and B (68), the dipeptides conjugated with macrolides, were isolated from an O-methyltransferase gene, verM disruption mutant of the Cordycep-colonizing fungus Clonostachys rogersoniana. These two compounds showed moderate antibacterial activity on Klebsiella pneumonia and Bacillus subtlilis [32]. Similarly, rogersonins A (69) and B (70) were two indole-polyketide hydrids isolated from verG disruption mutant of Clonostachys rogersoniana [33]. Blocking the biosynthesis of secondary metabolites through the disuption of the biosynthesis-related genes provide a method to activate cryptic or silent secondary metabolites in fungi. Three tetramic acid derivatives namely 1,2-dehydrovirgineone (75), virgineone (76) and virgineone aglycone (77) were isolated from Bionectria sp. MSX 47401. They showed obviously antibacterial activity against Staphylococcus aureus and several MRSA isolates. In addition, virgineone (76) showed moderate antifungal activity against Candida albicans, Cryptococcus neoformans, and Aspergillus niger with an MIC value of 14.4 µg/mL [3]. FR-900483 (80), which was called nectrisine or 3-(R)-4-(R)-dihydroxy-5-(R)-hydroxymethyl-1- pyrroline, was an immunoactive substance produced by Nectria lucida F-4490. FR-900483 (80) could restore the capacity of immunosuppressed mice to produce antibody against sheep red blood cells [35]. Penicolinate A (83) was induced from the endophytic fungus Bionectria sp. through bacterial co-culture. Penicolinate A (83) exhibited potent cytotoxic activity against the human ovarian cancer cell line A2780 with an IC50 value of 4.1 µM[28].

3. Polyketides and Their Biological Activities A variety of polyketides occur widely in the Clonostachys fungi. According to the structure characteristics, these metabolites were classifed into aromatic, alipahtic and mixed biogenic polyketides [43]. The aromatic polyketides mainly include pyranones, quinones, sorbicillinoids, and others. The polyketides, their isolated Clonostachys fungi and biological activities are shown in Table2.

Table 2. Polyketides in Clonostachys fungi and their biological activities.

Metabolite Metabolite Name Fungal Species Biological Activity Ref. Class Pyranones AGI-7 (85) Bionectria sp. MSX 47401 - [3] Alternariol (86) Clonostachys rosea YRS-06 - [44] Alternariol 5-O-methyl ether (87) Clonostachys rosea YRS-06 - [44] Cephalochromin (88) Nectria viridescens -[45] Cytotoxic activity [46] Clonostachys compactiuscula Coniochaetone G (89) -[19] FKR-0021 Inhibitory activity Citreoisocoumarinol (90) Nectria sp. HN001 [47] on α-glucosidase 12-Epicitreoisocoumarinol (91) Nectria sp. HN001 - [47] Inhibitory activity Citreoisocoumarin (92) Nectria sp. HN001 [47] on α-glucosidase Co-cultivation of Bionectria sp. 6,8-Dihydroxyisocoumarin with Bacillus subtilis or -[28] -3-carboxylic acid (93) Streptomyceslividans Inhibitory activity Macrocarpon C (94) Nectria sp. HN001 [47] on α-glucosidase 3-(3-Chloro-2-hydroxypropyl)-8- hydroxy-6-methoxyisochromen-1-one Clonostachys sp. AP4.1 - [48] (95) J. Fungi 2020, 6, 229 12 of 31

Table 2. Cont.

Metabolite Metabolite Name Fungal Species Biological Activity Ref. Class Dichlorodiaportin (96) Clonostachys sp. AP4.1 - [48] Mellein (97) Nectria fuckeliana -[49] 3,4-Dimethyl-6,8- Nectria pseudotrichia 120-1NP - [50] dihydroxyisocoumarin (98) Nectriapyrone A (99) Nectria pseudotrichia 120-1NP - [50] Nectriapyrone B (100) Nectria pseudotrichia 120-1NP - [50] (S)-4-Methoxy-6-pentanoyl-5,6- Nectria sp. - [51] dihydro-2H-pyran-2-one (101) Necpyrone A (102) Nectria sp. - [51] Necpyrone B (103) Nectria sp. - [51] Necpyrone C (104) Nectria sp. - [51] Necpyrone D (105) Nectria sp. - [51] Necpyrone E (106) Nectria sp. - [51] LL-P880α (107) Nectria sp. - [51] LL-P880β (108) Nectria sp. - [51] (1S, 2R)-1-Hydroxy-1-((S)-4- methoxy-6-oxo-3,6-dihydro-2H- Nectria sp. - [51] pyran-2-yl)-pentan-2-yl acetate (109) LL-P880γ (110) Nectria sp. - [51] PC-2 (111) Nectria sp. - [51] Quinones Anhydrofusarubin lactone (112) Nectria haematococca -[52] Aurantiogliocladin (113) Clonostachys candelabrum -[53] 2,5-Dimethyoxy-3,6-dimethyl- Antibacterial Nectria coryli [54] 1,4-benzoquinone (114) activity Nectria fuckeliana -[49] Fusarubin (115) Nectria haematococca -[36] 4-Deoxyfusarubin (116) Nectria haematococca -[55] 4-Deoxyanhydrofusarubin (117) Nectria haematococca -[55] 5-Deoxyfusarubin (118) Nectria haematococca -[55] 5-Deoxyanhydrofusarubin (119) Nectri haematococca -[55] Fusarubinoic acid (120) Nectria haematococca -[56] 13-Hydroxynorjavanicin (121) Nectria haematococca -[56] Antibacterial and Herbarin (122) Nectria pseudotrichia 120-1NP phytotoxic [50] activities Nectria pseudotrichia Cytotoxic activity [38] O-Methylherbarin (123) Nectria pseudotrichia 120-1NP Cytotoxic activity [50] Dehydroherbarin (124) Nectria pseudotrichia Cytotoxi activity [38] 2-Acetoxyl-5,7-dimethoxy-3- Nectria pseudotrichia Cytotoxic activity [38] methyl-1,4-naphthoquinone (125) Pseudonectrin A (126) Nectria pseudotrichia Cytotoxic activity [38] Pseudonectrin B (127) Nectria pseudotrichia Cytotoxic activity [38] Pseudonectrin C (128) Nectria pseudotrichia Cytotoxic activity [38] Pseudonectrin D (129) Nectria pseudotrichia -[38] Nectriafurone (130) Nectria haematococca -[52] Nectriaquinone A (131) Nectria pseudotrichia 120-1NP Cytotoxic activity [50] Antibacterial and Nectriaquinone B (132) Nectria pseudotrichia 120-1NP [50] cytotoxic activities P-Toluquinone (133) Nectria erubescens -[57] J. Fungi 2020, 6, 229 13 of 31

Table 2. Cont.

Metabolite Metabolite Name Fungal Species Biological Activity Ref. Class Sorbicillinoids Sorbicillin (134) Clonostachys rosea YRS-06 - [44] Antibacterial Dihydrotrichodimer ether A (135) Clonostachys rosea YRS-06 [44] activity Antibacterial Dihydrotrichodimer ether B (136) Clonostachys rosea YRS-06 [44] activity Tetrahydrotrichodimer ether (137) Clonostachys rosea YRS-06 - [44] Antibacterial Dihydrotrichodimerol (138) Clonostachys rosea YRS-06 [44] activity Antibacterial Tetrahydrotrichodimerol (139) Clonostachys rosea YRS-06 [44] activity Trichodimerol (140) Clonostachys rosea YRS-06 - [44] Other polyketides 3,5-Dihydroxyfuran-2(5H)-one Gliocladium roseum 1A - [23] (141) Clonostachys rosea -[11] Sapinofuranone B (142) Gliocladium roseum 1A - [23] Clonostachys rosea -[11] (-)-Vertinolide (143) Clonostachys rosea B5-2 - [58] (-)-Dihydrovertinolide (144) Clonostachys rosea B5-2 Phytotoxic activity [58] Clonostachys cylindrospora FH-A Anthelmintic Clonostachydiol (145) [59] 6607 activity Bionectriol A (146) Bionectria sp. - [60] Bionectriol B (147) Bionectria ochroleuca -[61] Bionectriol C (148) Bionectria ochroleuca Antifungal activity [61] Bionectriol D (149) Bionectria ochroleuca -[61] Rogerson A (150) Clonostachys rogersoniana -[62] Rogerson B (151) Clonostachys rogersoniana -[62] Nectriacid A (152) Nectria sp. HN001 - [47] Inhibitory activity Nectriacid B (153) Nectria sp. HN001 [47] on α-glucosidase Inhibitory activity Nectriacid C (154) Nectria sp. HN001 [47] on α-glucosidase Clonostachys compactiuscula Curvularin (155) -[19] FKR-0021 Cytotoxic and α,β-Dehydrocurvularin (156) Nectria glligena phytotoxic [63] activities Nectriatone B (157) Nectria sp. B-13 - [64] Nectriatone C (158) Nectria sp. B-13 - [64] TMC-151A (159) Clonostachys rosea -[65] Moderate Gliocladium catenulatum cytotoxicity to [66] tumor cells Bionectria ochroleuca -[67] TMC-151B (160) Clonostachys rosea -[67] Moderate Gliocladium catenulatum cytotoxicity to [66] tumor cells TMC-151C (161) Clonostachys rosea -[67] Moderate Gliocladium catenulatum cytotoxicity to [66] tumor cells J. Fungi 2020, 6, 229 14 of 31

Table 2. Cont.

Metabolite Metabolite Name Fungal Species Biological Activity Ref. Class TMC-151D (162) Clonostachys rosea -[67] Moderate Gliocladium catenulatum cytotoxicity to [66] tumor cells TMC-151E (163) Clonostachys rosea -[67] Moderate Gliocladium catenulatum cytotoxicity to [66] tumor cells Bionectria ochroleuca Antifungal activity [61] TMC-151F (164) Clonostachys rosea -[67] Moderate Gliocladium catenulatum cytotoxicity to [66] tumor cells Bionectria ochroleuca Antifungal activity [61] TMC-154 (165) Gliocladium roseum -[67] TMC-171A (166) Gliocladium roseum -[67] TMC-171B (167) Gliocladium roseum -[67] TMC-171C (168) Gliocladium roseum -[67] Antibacterial Usnic acid (169) Bionectria ochroleuca Bo-1 [68] activity

3.1. Pyranones Pyranones (also named pyrones) from fungi include α-, β- and γ-pyranones [69]. Most pyranones produced by Clonostachys fungi belong to α-pyranones. Their structures are shown in Figure5. Cephalochromin (88), a bisnaphtho-γ-pyrone, was isolated from Nectria viridescens [45]. This compound was screened to exhibit cytotoxic activity by inducing G0/G1 cell cycle arrest and apoptosis in A549 human non-small-cell lung cancer cells by inﬂicting mitochondrial disruption [46]. Citreoisocoumarinol (90), citreoisocoumarin (92) and macrocarpon C (94) showed moderate inhibitory activity on α-glucosidase with IC50 values ranging from 300 to 600 µM[47]. Two isocoumarin derivatives, 3-(3-chloro-2-hydroxypropyl)-8-hydroxy- 6-methoxyisochromen-1-one (95) and 3-[(R)-3,3-dichloro-2-hydroxypropyl]- 8-hydroxy-6- methoxy-1H-isochromen-1-one (dichlorodiaportin, 96), were identiﬁed from Clonostachys sp. AP4.1 [48]. J. Fungi 2020, 6, x FOR PEER REVIEW 14 of 30

Usnic acid (169) Bionectria ochroleuca Bo-1 Antibacterial activity [68]

3.1. Pyranones Pyranones (also named pyrones) from fungi include α-, β- and γ-pyranones [69]. Most pyranones produced by Clonostachys fungi belong to α-pyranones. Their structures are shown in Figure 5. Cephalochromin (88), a bisnaphtho-γ-pyrone, was isolated from Nectria viridescens [45]. This compound was screened to exhibit cytotoxic activity by inducing G0/G1 cell cycle arrest and apoptosis in A549 human non-small-cell lung cancer cells by inflicting mitochondrial disruption [46]. Citreoisocoumarinol (90), citreoisocoumarin (92) and macrocarpon C (94) showed moderate inhibitory activity on α-glucosidase with IC50 values ranging from 300 to 600 μM [47]. Two isocoumarin derivatives, 3-(3-chloro-2-hydroxypropyl)-8-hydroxy- 6-methoxyisochromen-1-one (95) and 3-[(R)-3,3-dichloro-2-hydroxypropyl]- 8-hydroxy-6- methoxyJ. Fungi 2020-1,H6,- 229isochromen-1-one (dichlorodiaportin, 96), were identified from Clonostachys sp. AP4.115 of 31 [48].

HO HO HO O

O OH O O

OH O OH O OH O 92. Citreoisocoumarin 93. 6,8-Dihydroxyisocoumarin- 94. Macrocarpon C 3-carboxylic acid

J. Fungi 2020, 6, x FOR PEER REVIEW 15 of 30

Figure 5. Pyranones isolated from Clonostachys fungi. Figure 5. Pyranones isolated from Clonostachys fungi.

3.2. Quinones The quinones isolated from Clonostachys fungi were mainly naphthoquinones except for three p-benzoquinones. Their structures are shown in Figure 6.

O OH O O O OH O O O O O O O O O OH OH O O O OH O 114. 2,5-Dimethyoxy-3,6- 112. Anhydrofusarubin lactone 113. Aurantiogliocladin dimethylbenzoquinone 115. Fusarubin

J. Fungi 2020, 6, x FOR PEER REVIEW 15 of 30

J. Fungi 2020, 6, 229 Figure 5. Pyranones isolated from Clonostachys fungi. 16 of 31

3.2. Quinones 3.2. Quinones The quinones isolated from Clonostachys fungi were mainly naphthoquinones except for three The quinones isolated from Clonostachys fungi were mainly naphthoquinones except for three p-benzoquinones. Their structures are shown in Figure 6. p-benzoquinones. Their structures are shown in Figure6.

O OH O O O OH O O O O O O O O O OH OH O O O OH O 114. 2,5-Dimethyoxy-3,6- 112. Anhydrofusarubin lactone 113. Aurantiogliocladin dimethylbenzoquinone 115. Fusarubin

J. Fungi 2020, 6, x FOR PEER REVIEW 16 of 30

FigureFigure 6.6. Quinones isolated from ClonostachysClonostachys fungi.fungi.

2,5-Dimethoxy-3,6-dimethyl-1,4-benzoquinone2,5-Dimethoxy-3,6-dimethyl-1,4-benzoquinone ( 114) from Nectria corylicoryli inhibitedinhibited thethe growthgrowth ofof StaphylococcusStaphylococcus aureusaureus atat aa concentrationconcentration ofof 11µ μg/mLg/mL [54[54]].. HerbarinHerbarin ( (122122)) and and nectriaquinone nectriaquinone B B ( (132132)) isolatedisolated from from the the brown brown rice rice culture culture of of NectriaNectria pseudotrichiapseudotrichia 120-1NP120-1NP exhibitedexhibited antibacterialantibacterial activitiesactivities againstagainst StaphylococcusStaphylococcus aureus andand Pseudomonas aeruginosaaeruginosa [[5050]].. HerbarinHerbarin ((122122),), O O-methylherbarin-methylherbarin ( 123(123),), nectriaquinone nectriaquinone A A (131 (131),) and, and nectriaquinone nectriaquinone B (B132 (132) displayed) displayed cytotoxic cytotoxic activity activity against against human human promyelocytic promyelocytic leukemia leukemia HL60 cells HL60 with cells IC50 withvalues IC of50 values of 11.9, 1.33, 1.93, and 11.6 μM, respectively. The structure-function relationship elucidated that the higher cytotoxicity of herbarin (122) and nectriaquinone B (132), compared to that of the related compounds O-methylherbarin (123) and nectriaquinone A (131) was attributed to their increased cell membrane permeability due to the presence of the hydroxyl group [38,50]. In addition, herbarin (122) showed a significant inhibition on lettuce seedling growth [50]. Seven naphthoquinones, named pseudonectrins A (126), B (127), C (128), D (129), herbarin (122), dehydroherbarin (124) and 2-acetoxyl-5,7-dimethoxy-3-methyl-1,4-naphthoquinone (125) were isolated from Nectria pseudotrichia. They all showed cytotoxic activity except for pseudonectrin D (129). In addition, pseudonectrins A (126), B (127) and C (128) had a skeleton of pyranonaphthoquinone [38].

3.3. Sorbicillinoids Sorbicillinoids are important hexaketide metabolites produced by fungi [70]. Six dimeric and one monomeric sorbicillinoids were extracted from culture broth of Clonostachys rosea YRS-06 [44]. Their structures are shown in Figure 7. Dihydrotrichodimer ether A (135), dihydrotrichodimer ether B (136) and tetrahydrotrichodimer ether (137) are rare bisorbicillinoids with a γ-pyrone moiety. Dihydrotrichodimer ether A (135), dihydrotrichodimer ether B (136), dihydrotrichodimerol (138) and tetrahydrotrichodimerol (139) showed antibacterial activity against Bacillus subtilis, Clostridium perfringens, and Escherichia coli [44].

Figure 7. Sorbicillinoids isolated from Clonostachys fungi. J. Fungi 2020, 6, x FOR PEER REVIEW 16 of 30

Figure 6. Quinones isolated from Clonostachys fungi.

2,5-Dimethoxy-3,6-dimethyl-1,4-benzoquinone (114) from Nectria coryli inhibited the growth of Staphylococcus aureus at a concentration of 1 μg/mL [54]. Herbarin (122) and nectriaquinone B (132) isolated from the brown rice culture of Nectria pseudotrichia 120-1NP exhibited antibacterial activities against Staphylococcus aureus and Pseudomonas aeruginosa [50]. Herbarin (122), O-methylherbarin (123), nectriaquinone A (131), and nectriaquinone J. Fungi 2020, 6, 229 17 of 31 B (132) displayed cytotoxic activity against human promyelocytic leukemia HL60 cells with IC50 values of 11.9, 1.33, 1.93, and 11.6 μM, respectively. The structure-function relationship elucidated 11.9,that 1.33,the higher 1.93, and cytotoxicity 11.6 µM, respectively. of herbarin The(122 structure-function) and nectriaquinone relationship B (132),elucidated compared that to that the higherof the cytotoxicityrelated compounds of herbarin O- (methylherbarin122) and nectriaquinone (123) and B ( nectriaquinone132), compared to A that (131 of) was the related attributed compounds to their Oincreased-methylherbarin cell membrane (123) and permeability nectriaquinone due to A the (131 presence) was attributed of the hydroxyl to their group increased [38,50]. cell In membrane addition, permeabilityherbarin (122 due) showed to the a presence significant of theinhibition hydroxyl on grouplettuce [ 38seedling,50]. In growth addition, [50]. herbarin (122) showed a signiﬁcantSeven inhibition naphthoquinones on lettuce, named seedling pseudonectrins growth [50]. A (126), B (127), C (128), D (129), herbarin (122),Seven dehydroherbarin naphthoquinones, (124 named) and pseudonectrins2-acetoxyl-5,7-dimethoxy A (126), B (-1273-methyl), C (128-1,4),-naphthoquinone D (129), herbarin (122 (125),) dehydroherbarinwere isolated from (124 Nectria) and 2-acetoxyl-5,7-dimethoxy-3-methyl-1,4-naphthoquinonepseudotrichia. They all showed cytotoxic activity except ( 125for )pseudonectrin were isolated fromD (129Nectria). In pseudotrichia addition, . pseudonectrins They all showed A cytotoxic (126), B activity (127) except and C for ( pseudonectrin128) had a skeleton D (129). In of addition,pyranonaphthoquinone pseudonectrins [38]. A (126 ), B (127) and C (128) had a skeleton of pyranonaphthoquinone [38].

3.3.3.3. SorbicillinoidsSorbicillinoids SorbicillinoidsSorbicillinoids areare importantimportant hexaketidehexaketide metabolitesmetabolites producedproduced byby fungifungi [[7070].]. SixSix dimericdimeric andand oneone monomericmonomeric sorbicillinoidssorbicillinoids werewere extractedextracted from cultureculture brothbroth ofof ClonostachysClonostachys rosea YRS-06YRS-06 [4444].]. TheirTheir structures are are shown shown in in Figure Figure 7.7 .Dihydrotrichodimer Dihydrotrichodimer ether ether A (135 A (),135 dihydrotrichodimer), dihydrotrichodimer ether etherB (136 B)( 136 and) and tetrahydrotrichodimer tetrahydrotrichodimer ether ether (137 (137) are) are rare rare bisorbicillinoids bisorbicillinoids with with a aγγ--pyronepyrone moiety. DihydrotrichodimerDihydrotrichodimer ether ether A A ( (135135),), dihydrotrichodimer dihydrotrichodimer ether ether B B ( (136136),), dihydrotrichodimeroldihydrotrichodimerol ( (138138)) andand tetrahydrotrichodimeroltetrahydrotrichodimerol ((139139)) showedshowed antibacterialantibacterial activityactivity againstagainst BacillusBacillus subtilissubtilis,, ClostridiumClostridium perfringensperfringens,, andand EscherichiaEscherichia colicoli[ [4444].].

FigureFigure 7.7. SorbicillinoidsSorbicillinoids isolatedisolated fromfrom ClonostachysClonostachysfungi. fungi. 3.4. Other Polyketides The structures of the other polyketides isolated from Clonostachys fungi are shown in Figure8. These metabolites mainly belong to aliphatic polyketides. Some of them contain a glycosyl group and exist as glycosides. Four α-furanones were obtained. Both 3,5-dihydroxyfuran-2(5H)-one (141) and sapinofuranone B (142) were isolated from Gliocladium roseum 1A [23]. Both (-)-vertinolide (143) and (-)-dihydrovertinolide (144) were isolated from Clonostachys rosea B5-2. (-)-Dihydrovertinolide (144) displayed phytotoxic activity against lettuce seedlings at a concentration of 50 µg/mL [58]. Clonostachydiol (145) was a 14-membered macrodiolide isolated from the fungus Clonostachys cylindrospora (strain FH-A 6607). It exhibited anthelimintic activity against abomasum nematode Haemonchus cortorus in artiﬁcially infected lambs [59]. Four stereocenters in clonostachydiol were revised later [71]. Polyketide glycosides bionectriols A (146), B (147) and C (148) were isolated from Bionectria chroleuca [61]. TMC-151E (163), TMC-151F (164) and bionectriol C (148) moderately inhibited Candida albicans bioﬁlm formation with IC50 values of 36.3, 41.0 and 24.1 µM, respectively [61]. Nectriacids B (153) and C (154) showed stronger α-glucosidase inhibitory activity than positive control (acarbose, IC50, 815.3 µM) with IC50 values of 23.5 and 42.3 µM, respectively. J. Fungi 2020, 6, 229 18 of 31

α,β-Dehydrocurvularin (156) from Nectria glligena was proved to be cytotoxic to human lung ﬁbroblasts with IC50 value less than 12 µg/mL. In addition, α,β-dehydrocurvularin (156) signiﬁcantly reduced radicle length and epicotyl growth in Lactuca sativa at 100 and 200 µg/disk [63]. Both nectriatones B (157) and C (158) were cyclohexanone derivatives from Nectria sp. B-13 [64]. J. FungiA 20 series20, 6, x of FOR polyketides PEER REVIEW TMC-151 (159–164), TMC-154 (165) and TMC-171 (166–168) were found17 of 30 exclusively in Gliocladium and Clonostachys species [67]. They contained D-mannopyranoside and D3.-mannitol4. Other Polyketides or D-arabitol and showed moderate cytotoxicity on several tumor cells [66]. UsnicThe structures acid (169 of) is the a unique other polyketides polyketide fromisolatedBionectria from C ochroleucalonostachysBo-1 fungi which are shown was isolated in Figure as an8. endophyticThese metabolites fungus mainly from rice. belong It showed to aliphatic antibacterial polyketides. activity Some against of themXanthomonas contain a oryzae glycosylwith group MIC µ valueand exist of 200 as glycosides.g/mL [68].

Figure 8. Cont. J. Fungi 2020, 6, 229 19 of 31 J. Fungi 2020, 6, x FOR PEER REVIEW 18 of 30

R R2 2 O OH OH O OH OH R 1 O OH HO HO O R1 O O HO O O HO HO OH OH OH OH OH HO O 159. TMC-151A, R1=H, R2= HO 165. TMC-154, R1=CH3, R2= OH O OH OH O OH OH OH 166. TMC-171A, R =H, R = OH 160. TMC-151B, R1=H, R2= HO 1 2 HO OH OH OH O OH OH OH O 161. TMC-151C, R =H, R = O 167. TMC-171B, R =H, R = OH 1 2 HO 1 2 HO OH OH OH OH OH OH OH OH HO O O HO 162. TMC-151D, R1=H, R2= 168. TMC-171C, R1=H, R2= OH OH OH OH OH O O 163. TMC-151E, R1=Ac, R2=HO O OH OH HO OH OH OH 164. TMC-151F, R =Ac, R = OH O 1 2 HO O O 169. Usnic acid OH

Figure 8. Other polyketides isolated from Clonostachys fungi.fungi. 4. Terpenoids and Their Biological Activities Four α-furanones were obtained. Both 3,5-dihydroxyfuran-2(5H)-one (141) and sapinofuranone B (142The) terpenoids were isolated from Clonostachys from Gliocladiumfungi include roseum monoterpenoids, 1A [23]. Both sesquiterpenoids, (-)-vertinolide diterpenoids, (143) and triterpenpoids,(-)-dihydrovertinolide polyterpenoids, (144) were and isolated meroterpenoids. from Clonostachys The rosea terpenoids, B5-2. (-) along-Dihydrovertinolide with their isolated (144) Clonostachysdisplayed phytotoxicfungi and activity biological against activities lettuce are seedlings shown in at Table a concentration3. of 50 μg/mL [58]. Clonostachydiol (145) was a 14-membered macrodiolide isolated from the fungus Clonostachys cylindrospora (strainTable FH 3.-ATerpenoids 6607). It in exhibitedClonostachys antheliminticfungi and their activity biological against activities. abomasum nematode

HaemonchusMetabolite Class cortorus in artificially Metabolite Name infected lambs [59]. Fungal Four Species stereocenters Biological in clonostachydiol Activity Ref. were revisedMonoterpenoids later [71]. Nectriapyrone (170) Nectria sp. HLS206 - [72] Polyketide glycosidesNectriapyrone bionectriols C (171) A (146), BNectria (147sp.) and HLS206 C (148) were isolated - from Bionec [72] tria chroleuca [61]. TMC-151ENectriapyrone (163), TMC D (-172151F) (164) andNectria bionectriolsp. HLS206 C (148) moderately - inhibited Candida [72] Sesquiterpenoids 5,6-Dihydroxybisabolol (173) Bionectria sp. MSX 47401 - [3] 50 albicans biofilm formation with IC values of 36.3,Nectria 41.0 and pseudotrichia 24.1 μM, respectively [61]. Nectrianolin C (174) Cytotoxic activity [73] Nectriacids B (153) and C (154) showed stronger 120-1NPα-glucosidase inhibitory activity than positive 10-Acetyl trichoderonic acid A control (acarbose, IC50, 815.3 μM) with IC50 valuesNectria of 23.5 pseudotrichia and 42.3 μM, Leishmanicidalrespectively. activity [30] (175) α,β-DehydrocurvularinHydroheptelidic (156) acidfrom (176 Nectria) glligenaNectriapseudotrichia was proved toLeishmanicidal be cytotoxic activity to human [30 lung] Clonostachys compactiuscula fibroblasts with IC50 valueOphioceric less than acid 12 (177 μg/mL.) In addition, α,β-dehydrocurvularin-[ (156) significantly19] FKR-0021 reduced radicle length andXylaric epicotyl acid D( 178growth) in LactucaNectria sativa pseudotrichia at 100 and 200 μg/disk-[ [63]. 30] DiterpenoidsBoth nectriatones B ( Agathic157) and acid C (179 (158) ) were cyclohexanoneBionectria sp. derivatives from - Nectria sp. B-13 [28 [64].] A series of polyketidesNectriatone TMC A-151 (180 )(159-164), TMCNectria-154sp. ( B-13165) and TMC Cytotoxic-171 (166 activity-168) were [found64] Nectria pseudotrichia Zythiostromic acid C (181) -[50] exclusively in Gliocladium and Clonostachys species [67].120-1NP They contained D-mannopyranoside and D-mannitol or D-arabitol and showed moderate cytotoxicityClonostachys on rosea several tumor cells [66]. Triterpentoids Eburicol (182) Cytotoxic activity [74] Usnic acid (169) is a unique polyketide from BionectriaMMS1090 ochroleuca Bo-1 which was isolated as an endophytic fungus fromHelvolic rice. It acid showed (183) antibacterialNectria activitysp. against Xanthomonas - oryzae with [51 MIC] Antimicrobial activity [75,76] value of 200 μg/mL [68]. J. Fungi 2020, 6, 229 20 of 31

Table 3. Cont.

Metabolite Class Metabolite Name Fungal Species Biological Activity Ref. Inhibition on Polyterpenoids Glisoprenin A (184) Gliocladium sp. FO-1513 acyl-CoA:cholesterol [77] acyltransferase Inhibition on Glisoprenin B (185) Gliocladium sp. FO-1513 acyl-CoA:cholesterol [77] acyltransferase Inhibition on Gliocladium roseum Glisoprenin C (186) appressorium formation of [78] HA190-95 Magnaporthe grisea Inhibition on Gliocladium roseum Glisoprenin D (187) appressorium formation of [78] HA190-95 Magnaporthe grisea Inhibition on Gliocladium roseum Glisoprenin E (188) appressorium formation of [78] HA190-95 Magnaporthe grisea Bionectin F (189) Bionectria sp. Y1085 - [17] Ascochlorin = Ilicicolin D = LL-Z Meroterpenoids Nectria lucida -[79] 1272γ (190) Nectria sp. Antifungal activity [8] Nectria sp. B-13 - [34] Nectria sp. B-13 Cytotoxic activity [64] Dechloroilicicolin D = Cylindrol Nectria sp. Antifungal activity [8] (191) 3-Bromoascochlorin (192) Nectria coccinea -[80] Chloronectrin (193) Nectria coccinea -[80] Deacetylchloronectrin (194) Nectria sp. B-13 - [34] Dechlorodihydroascochlorin = Dechloro-12,13-dihydroascochlorin Nectria lucida -[79] = LL-Z 1272ε (195) Nectria sp. B-13 - [34] Ilicicolin C = LL-Z 1272δ (196) Nectria sp. B-13 - [34] Inhibitory activity on Nectria galligena AChE and [63] α-glucuronidase Ilicicolin E = 80,90-Dehydroascochorin = Nectria sp. B-13 Antibacterial activity [34] Cylindrochlorin (197) Nectria sp. Antifungal activity [8] Cytotoxic and antibacterial Nectria sp. B-13 [64] activities Inhibitory activity on Nectria galligena AChE and [63] α-glucuronidase Ilicicolin F = LL-Z 1272ζ (198) Nectria sp. B-13 - [34] Nectria galligena -[63] Nectria sp. Antifungal activity [8] Nectria pseudotrichia Cytotoxic activity against Nectrianolin A (199) [73] 120-1NP HL60 and HeLa cells Nectria pseudotrichia Cytotoxic activity against Nectrianolin B (200) [73] 120-1NP HL60 and HeLa cells Ascofuranone (201) Nectria sp. Antifungal activity [8] Chalmicrin (202) Nectria sp. HLS206 - [81] Colletochlorin B (203) Nectria sp. B-13 - [34] Inhibitory activity on Nectria galligena AChE and [63] α-glucuronidase Colletorin B (204) Nectria galligena -[63] Ilicicolin A (205) Nectria sp. B-13 - [34] Ilicicolin B = LL-Z 1272β (206) Nectria coccinea -[80] Nectria lucida -[79] MBJ-0009 (207) Nectria sp. f26111 Cytotoxic activity [82] MBJ-0010 (208) Nectria sp. f26111 Cytotoxic activity [82] Cytotoxicity on cancer Taxol = Paclitaxel (209) Gliocladium sp. [83] cells J. Fungi 2020, 6, x FOR PEER REVIEW 20 of 30

Nectria sp. B-13 - [34] Ilicicolin C = LL-Z 1272δ Nectria sp. B-13 - [34] (196) Inhibitory activity Nectria galligena on AChE and [63] α-glucuronidase Ilicicolin E = Antibacterial 8′,9′-Dehydroascochorin = Nectria sp. B-13 [34] activity Cylindrochlorin (197) Nectria sp. Antifungal activity [8] Cytotoxic and Nectria sp. B-13 antibacterial [64] activities Inhibitory activity Nectria galligena on AChE and [63] α-glucuronidase Ilicicolin F = LL-Z 1272ζ Nectria sp. B-13 - [34] (198) Nectria galligena - [63] Nectria sp. Antifungal activity [8] Cytotoxic activity Nectrianolin A (199) Nectria pseudotrichia 120-1NP against HL60 and [73] HeLa cells Cytotoxic activity Nectrianolin B (200) Nectria pseudotrichia 120-1NP against HL60 and [73] HeLa cells Ascofuranone (201) Nectria sp. Antifungal activity [8] Chalmicrin (202) Nectria sp. HLS206 - [81] Colletochlorin B (203) Nectria sp. B-13 - [34] Nectria galligena Inhibitory activity [63] on AChE and α-glucuronidase Colletorin B (204) Nectria galligena - [63] Ilicicolin A (205) Nectria sp. B-13 - [34] Ilicicolin B = LL-Z 1272β Nectria coccinea - [80] (206) Nectria lucida - [79] MBJ-0009 (207) Nectria sp. f26111 Cytotoxic activity [82] MBJ-0010 (208) Nectria sp. f26111 Cytotoxic activity [82] J. Fungi 2020 6 Cytotoxicity on , , 229 Taxol = Paclitaxel (209) Gliocladium sp. [2183] of 31 cancer cells

4.1. Monoterpenoids Three monoterpenoidsmonoterpenoids named nectriapyrone (170),), nectriapyrones C C (171)) and D (172) with α––pyronepyrone skeletons were isolated from thethe fungusfungus NectriaNectria sp.sp. HLS206 associated with the marine sponge Gelliodes carnosa [7272].]. Their structures are shown in Figure9 9..

J. Fungi 2020, 6, x FOR PEER REVIEW 21 of 30

4.2. Sesquiterpenoids The structures of the sesquiterpenoids isolated from Clonostachys fungi are shown in Figure 10. Nectrianolin C (174) fromFigure Nectria 9. Monoterpenoids pseudotrichia isolated 120-1NP from exhibited Clonostachys cytotoxic fungi. activity against HL60 and HeLa cells [73]. 4.2. SesquiterpenoidsThree sesquiterpene acids: 10-acetyl trichoderonic acid A (175), hydroheptelidic acid (176), and xylaricThe acid structures D (178 of) were the sesquiterpenoids isolated from the isolated endophytic from Clonostachys fungus Nectriafungi pseudotrichia are shown inof Figure the tree 10. NectrianolinCaesalpinia echinata C (174.) The from 10Nectria-Acetyl pseudotrichia trichoderonic120-1NP acid A exhibited(175) and cytotoxichydroheptelidic activity acid against (176 HL60) showed and HeLastrong cells antileishmanial [73]. activity [30].

O O R HO HO O O O OH 176. Hydroheptelidic acid, R=OH 177. Ophioceric acid 178. Xylaric acid D, R=H

Figure 10. Sesquiterpenoids isolated from Clonostachys fungi.

4.3. DiterpenoidThree sesquiterpenes acids: 10-acetyl trichoderonic acid A (175), hydroheptelidic acid (176), and xylaric acid D (178) were isolated from the endophytic fungus Nectria pseudotrichia of the tree Caesalpinia echinataThree. The diterpenoids 10-Acetyl trichoderonic (179–181) have acid been A ( isolated175) and from hydroheptelidic Clonostachys acidfungi (176 so) far showed (Figure strong 11). antileishmanialNectriatone A (180 activity) from [30 Nectria]. sp. B-13 exhibited cytotoxic activity against the human cancer cell lines, including SW1990, HCT-116, MCF-7 and K562 [64]. 4.3. Diterpenoids OH

Three diterpenoids (179–181O ) have been isolatedO from Clonostachys fungi so far (Figure 11). O Nectriatone A (180) from Nectria sp. B-13 exhibitedH H cytotoxic activity against theH humanOH cancer cell lines, includingO SW1990, HCT-116, MCF-7 and K562 [64]. HO OH H OH O OH O OH 179. Agathic acid 180. Nectriatone A 181. Zythiostromic acid C Figure 11. Diterpenoids isolated from Clonostachys fungi.

4.4. Triterpenoids Only two triterpenoids (182, 183) were described with their structures shown in Figure 12. Eburicol (182) exhibited cytotoxic activities on the four human cancer cell lines, which included MCF-7, MDA-MB-231, NSCLC-N6-L16 and A549 cells with IC50 values lower than 40 μM [74]. Helvolic acid (183) was a nortriterpenoid isolated from many other fungi, such as Pichia guilliermondii [75], and Aspergillus fumiatus [76]. This compound exhibited obvious antimicrobial activity [75,76]. J. Fungi 2020, 6, x FOR PEER REVIEW 21 of 30

4.2. Sesquiterpenoids The structures of the sesquiterpenoids isolated from Clonostachys fungi are shown in Figure 10. Nectrianolin C (174) from Nectria pseudotrichia 120-1NP exhibited cytotoxic activity against HL60 and HeLa cells [73]. Three sesquiterpene acids: 10-acetyl trichoderonic acid A (175), hydroheptelidic acid (176), and xylaric acid D (178) were isolated from the endophytic fungus Nectria pseudotrichia of the tree Caesalpinia echinata. The 10-Acetyl trichoderonic acid A (175) and hydroheptelidic acid (176) showed strong antileishmanial activity [30].

O O R HO HO O O O OH 176. Hydroheptelidic acid, R=OH 177. Ophioceric acid 178. Xylaric acid D, R=H

Figure 10. Sesquiterpenoids isolated from Clonostachys fungi.

4.3. Diterpenoids Three diterpenoids (179–181) have been isolated from Clonostachys fungi so far (Figure 11). J. Fungi 2020, 6, 229 22 of 31 Nectriatone A (180) from Nectria sp. B-13 exhibited cytotoxic activity against the human cancer cell lines, including SW1990, HCT-116, MCF-7 and K562 [64]. OH

O O O H H H OH O HO OH H OH O OH O OH 179. Agathic acid 180. Nectriatone A 181. Zythiostromic acid C FigureFigure 11. 11. DiterpenoidsDiterpenoids isolated isolated from from ClonostachysClonostachys fungi.fungi.

4.4.4.4. Triterpenoid Triterpenoidss OnlyOnly two two triterpenoids triterpenoids (182 (182, ,183183) )were were described described with with their their structures structures shown shown in in Figure Figure 12. 12 . EburicolEburicol (182) exhibited exhibited cytotoxic cytotoxic activities activities on on the the four four human human cancer cancer cell lines, cell lines which, which included included MCF-7, MDA-MB-231, NSCLC-N6-L16 and A549 cells with IC values lower than 40 µM[74]. Helvolic acid MCF-7, MDA-MB-231, NSCLC-N6-L16 and A549 cells50 with IC50 values lower than 40 μM [74]. Hel(183volic) was acid a nortriterpenoid (183) was a isolated nortriterpenoid from many isolated other fungi, from suchmany as otherPichia fungi guilliermondii, such as[75 Pichia], and Aspergillus fumiatus [76]. This compound exhibited obvious antimicrobial activity [75,76]. guilliermondiiJ. Fungi 2020, 6, x[ 7FOR5], PEER and REVIEWAspergillus fumiatus [76]. This compound exhibited obvious antimicrobi22 ofal 30 activity [75,76].

Figure 12. Triterpenoids isolated from Clonostachys fungi.fungi.

4.5. Polyterpenoids 4.5 Polyterpenoids The polyterpenes in Clonostachys fungi were tetraterpenes or pentaterpenes whose structures are The polyterpenes in Clonostachys fungi were tetraterpenes or pentaterpenes whose structures shown in Figure 13. Five polyprenol polyterpenoids, glioprenins A–E (184–188) were isolated from are shown in Figure 13. Five polyprenol polyterpenoids, glioprenins A-E (184-188) were isolated Gliocladium 184 185 Gliocladium from Gliocladiumspecies species [77,78 ].[7 Glisoprenins7,78]. Glisoprenins A ( ) A and (184 B) ( and) fromB (185) from Gliocladiumsp. FO-1513 sp. FOshowed-1513 186 187 inhibitoryshowed inhibitory activity on activity acyl-CoA:cholesterol on acyl-CoA:cholesterol acyltransferase acyltransferase [77], and glioprenins [77], and glioprenins C ( ), D ( C (18) and6), D E 188 Gliocladium roeum ((187)) fromand E the (188 submerged) from the cultures submerged of cultures of GliocladiumHA190-95 roeum showed HA190 inhibition-95 showed on appressoriuminhibition on Magnaporthe grisea formationappressorium of formation of Magnaporthe[78]. grisea [78]. 189 Bionectin F ((189),), anotheranother polyprenolpolyprenol polyterpenoid,polyterpenoid, waswas isolatedisolated fromfrom thethe endophyticendophytic fungusfungus Bionectria Bionectria sp.sp. Y1085 [[17].17].

OH OH OH OH OH OH OH OH

O HO HO OH OH 186. Glisoprenin C 187. Glisoprenins D

Figure 13. Polyterpenoids isolated from Clonostachys fungi.

4.6. Meroterpenoids Meroterpenoids are metabolites that are partially derived from terpenoid biosynthetic pathways. The structures of meroterpenoids isolated from Clonostachys fungi are shown in Figure 14. J. Fungi 2020, 6, x FOR PEER REVIEW 22 of 30

Figure 12. Triterpenoids isolated from Clonostachys fungi.

4.5 Polyterpenoids The polyterpenes in Clonostachys fungi were tetraterpenes or pentaterpenes whose structures are shown in Figure 13. Five polyprenol polyterpenoids, glioprenins A-E (184-188) were isolated from Gliocladium species [77,78]. Glisoprenins A (184) and B (185) from Gliocladium sp. FO-1513 showed inhibitory activity on acyl-CoA:cholesterol acyltransferase [77], and glioprenins C (186), D (187) and E (188) from the submerged cultures of Gliocladium roeum HA190-95 showed inhibition on appressorium formation of Magnaporthe grisea [78]. J. FungiBionectin2020, 6, 229 F (189), another polyprenol polyterpenoid, was isolated from the endophytic fungus23 of 31 Bionectria sp. Y1085 [17].

OH OH OH OH OH OH OH OH

O HO HO OH OH 186. Glisoprenin C 187. Glisoprenins D

Figure 13. Polyterpenoids isolated from Clonostachys fungi. Figure 13. Polyterpenoids isolated from Clonostachys fungi. 4.6. Meroterpenoids 4.6. Meroterpenoids Meroterpenoids are metabolites that are partially derived from terpenoid biosynthetic pathways. The structuresMeroterpenoids of meroterpenoids are metabolites isolated that from areClonostachys partially fungiderived are shown from terpenoid in Figure 14 biosynthetic. pathwaysAscochlorin. The structures (also named of meroterpenoids illicolin D or LL-Z isolated 1272 γfrom, 190 ),Clonostachys dechlorodihydroascochlorin fungi are shown in (195 Figure) and 14.ilicicolin B (or called LL-Z 1272β, 206) were isolated from Nectria sp. [79]. Ilicicolins D (190), E (197) and F (198), dechloroilcicolin D (191) and ascofuranone (201) showed antifungal activity against plant pathogens Neurospora crassa, Botrytis cinerea, Fusariumculmorum, Pyricularia oryzae, and Penicillium digitarum [8]. Ilicicolins C (196) and E (197), and colletochlorin B (203) from the phytopathogenic fungus Nectria galligena displayed inhibitory activity toward acetylcholinesterase (AChE) and α-glucuronidase with IC50 values of 30-36 µg/mL in the AChE assay and 32-43 µg/mL in the α-glucuronidase test [63]. Ilicicolin E (197) obtained from soil-derived fungus Nectria sp. B-13 showed antibacterial activities against Escherichia coli, Bacillus subtilis and Staphylococcus aureus with MIC values of 4.0, 2.0 and 4.0 µg/mL, respectively [64]. Ilicicolin C (196) and ilicicolin F (198) obtained from phytopathogenic fungus Nectria galligena were active against Pseudomonas syringae with IC50 values of 28.5 and 28.5 µg/mL, respectively [63]. Both nectrianolins A (199) and B (200) were sesquiterpene-epoxyclohexenone conjugates isolated from Nectria pseudotrichia 120-1NP [73]. Chalmicrin (202), a mannitol ether of methylated monocyclofarnesol, was isolated from Nectria sp. HLS206 that was associated with the marine sponge Glliodes carnosa [81]. This compound was previously isolated from the fungus Chalara microspora [84]. Both MBJ-0009 (207) and MBJ-0010 (208), which were related to the eremophilane class and isolated from the saprobic fungus Nectria sp. f26111, showed moderate cytotoxic activity against human ovarian adenocarcinoma SKOV-3 with the IC50 values of 24.7 and 11.2 mM, respectively [82]. Taxol (generic name paclitaxel, 209), the well-known anticancer agent, was isolated from the endophytic fungus Gliocladium sp. from Taxus baccata [83]. The backbone of taxol (209) is a diterpenoid, and the side chain is phenylalanine-derived. Both diterpenoid and phenylpropanoid pathways are J. Fungi 2020, 6, x FOR PEER REVIEW 23 of 30

Ascochlorin (also named illicolin D or LL-Z 1272γ, 190), dechlorodihydroascochlorin (195) and ilicicolin B (or called LL-Z 1272β, 206) were isolated from Nectria sp. [79]. Ilicicolins D (190), E (197) and F (198), dechloroilcicolin D (191) and ascofuranone (201) showed antifungal activity against plant pathogens Neurospora crassa, Botrytis cinerea, Fusariumculmorum, Pyricularia oryzae, and Penicillium digitarum [8]. Ilicicolins C (196) and E (197), and colletochlorin B (203) from the phytopathogenic fungus Nectria galligena displayed inhibitory activity toward acetylcholinesterase (AChE) and α-glucuronidase with IC50 values of 30-36 μg/mL in the AChE assay and 32-43 μg/mL in the α-glucuronidase test [63]. Ilicicolin E (197) obtained from soil-derived fungus Nectria sp. B-13 showed antibacterial activities against Escherichia coli, Bacillus subtilis and Staphylococcus aureus with MIC values of 4.0, 2.0 and 4.0 μg/mL, respectively [64]. Ilicicolin C (196) and ilicicolin F (198) obtained from phytopathogenic fungus Nectria galligena were active against Pseudomonas syringae with IC50 values of 28.5 and 28.5 μg/mL, respectively [63]. Both nectrianolins A (199) and B (200) were sesquiterpene-epoxyclohexenone conjugates isolated from Nectria pseudotrichia 120-1NP [73]. Chalmicrin (202), a mannitol ether of methylated monocyclofarnesol, was isolated from Nectria sp. HLS206 that was associated with the marine sponge Glliodes carnosa [81]. This compound was previously isolated from the fungus Chalara microspora [84]. Both MBJ-0009 (207) and MBJ-0010 (208), which were related to the eremophilane class and isolated from the saprobic fungus Nectria sp. f26111, showed moderate cytotoxic activity against human ovarian adenocarcinoma SKOV-3 with the IC50 values of 24.7 and 11.2 mM, respectively [82].

J. FungiTaxol2020, 6(generic, 229 name paclitaxel, 209), the well-known anticancer agent, was isolated from24 ofthe 31 endophytic fungus Gliocladium sp. from Taxus baccata [83]. The backbone of taxol (209) is a diterpenoid, and the side chain is phenylalanine-derived. Both diterpenoid and phenylpropanoid requiredpathways for are taxol required biosynthesis. for taxol Taxol biosynthesis. (209) was foundTaxol in(209 both) was plants found [85] in and both fungi plants [86]. [ It85 is] and a result fungi of the[86]. co-evolution It is a result ofof plantsthe co- andevolution fungi inof secondaryplants and metabolismfungi in secondary [75]. metabolism [75].

J. Fungi 2020, 6, x FOR PEER REVIEW 24 of 30

Figure 14. Meroterpenoids isolated from Clonostachys fungi.

5. Miscellaneous Metabolite Metabolitess and Their Biological Activities The miscellaneous metabolites mainly including phenolics and fatty acids isolated from Clonostachys fungifungi areare listedlisted inin TableTable4 4,, and and their their structures structures are are shown shown in in Figure Figure 15 1.5. Four phenolic metabolites were isolated and identifiedidentiﬁed as 44-hydroxybenzoic-hydroxybenzoic aldehyde (210),), 4-hydroxybenzoic4-hydroxybenzoic acid ( 211), 3,4-dihydroxybenzoid3,4-dihydroxybenzoid acid ( 212), and 3,5 3,5-dihydroxybenzoic-dihydroxybenzoic acid ( 213) from GliocladiumGliocladium roseum CGMCC 3.36573.3657 [[8787].]. 5-5-n--HeneicosylresorcinolHeneicosylresorcinol (217) was isolated isolated from from GliocladiumGliocladium roseumroseum YMF1.00133.YMF1.00133. After 24 h incubation, 5- 5n-n-heneicosylresorcinol-heneicosylresorcinol (217 (217) showed) showed antinematodal antinematodal activity activity against againstCaenothabditis Caenothabditis elegans atelegans 15 and at 30 15µ g and/mL, 30 against μg/mL,Panagrellus against redivivusPanagrellusat 50 redivivus and 80 atµg / 50mL, and and 80 against μg/mL,Bursaphelenchus and against xylophilusBursaphelenchusat 200 xylophilus and 180 µ gat/mL, 200 respectivelyand 180 μg/mL, [22]. respectively [22]. Five fatty acids namednamed clonostach acids A ( 219), B ( 220), and C (221) were isolated from the endophytic fungus Clonostachys rosea B5-2B5-2 [[5858].]. Three furanfuran derivatives,derivatives, named 22-furoic-furoic acid (222), 5-hydroxymethyl5-hydroxymethyl furoic acid (223) and 2-hydroxy-5-hydroxymethyl2-hydroxy-5-hydroxymethyl furanfuran ((224224),), werewere isolatedisolated fromfrom BionectriaBionectria sp.sp. Y1085Y1085 [[1717].]. Three piliformic piliformic acid acid derivatives derivatives were were isolated isolated from fromNectria Nectria pseutrichia pseutrichia. Both. Both 3-(S)-piliformic 3-(S)-piliformic acid (acid226) ( and226) 6’-acetoxy-piliformicand 6’-acetoxy-piliformic acid (acid227) ( were227) were screened screened to show to show leishmanicidal leishmanicidal activity activity [30]. [30].

Table 4. Miscellaneous metabolites in Clonostachys fungi and their biological activities.

Biological Metabolite Name Fungal Species Ref. Activity 4-Hydroxybenzoic aldehyde (210) Gliocladium roseum CGMCC 3.3657 - [87] 4-Hydroxy-benzoic acid (211) Gliocladium roseum CGMCC 3.3657 - [87] 3,4-Dihydroxy-benzoic acid (212) Gliocladium roseum CGMCC 3.3657 - [87] 3,5-Dihydroxy-benzoic acid (213) Gliocladium roseum CGMCC 3.3657 - [87] 2,5-Dimethoxy-3,6-dimethylbenzene-1,4-diol (214) Nectria coryli - [54] 3,5-Dihydroxybenzyl alcohol (215) Nectria sp. B-13 - [34] 3,5-Dihydroxytoluene (216) Nectria sp. B-13 - [34] Antinematodal 5-n-Heneicosylresorcinol (217) Gliocladium roseum YMF1.00133 [22] activity Toluquinol (218) Nectria erubescens - [57] Clonostach acid A (219) Clonostachys rosea B5-2 - [58]

Clonostach acid B (220) Clonostachys rosea B5-2 - [58]

Clonostach acid C (221) Clonostachys rosea B5-2 - [58]

2-Furoic acid (222) Bionectria sp. Y1085 - [17]

5-Hydroxymethyl furoic acid (223) Bionectria sp. Y1085 - [17]

2-Hydroxy-5-hydroxymethyl furan (224) Bionectria sp. Y1085 - [17]

3-(R)-Piliformic acid (225) Bionectria sp. - [28] Leishmanicidal 3-(S)-Piliformic acid (226) Nectria pseudotrichia [30] activity J. Fungi 2020, 6, 229 25 of 31

Table 4. Miscellaneous metabolites in Clonostachys fungi and their biological activities.

Metabolite Name Fungal Species Biological Activity Ref. 4-Hydroxybenzoic aldehyde (210) Gliocladium roseum CGMCC 3.3657 - [87] 4-Hydroxy-benzoic acid (211) Gliocladium roseum CGMCC 3.3657 - [87] 3,4-Dihydroxy-benzoic acid (212) Gliocladium roseum CGMCC 3.3657 - [87] 3,5-Dihydroxy-benzoic acid (213) Gliocladium roseum CGMCC 3.3657 - [87] 2,5-Dimethoxy-3,6-dimethylbenzene-1,4-diol Nectria coryli -[54] (214) 3,5-Dihydroxybenzyl alcohol (215) Nectria sp. B-13 - [34] 3,5-Dihydroxytoluene (216) Nectria sp. B-13 - [34] 5-n-Heneicosylresorcinol (217) Gliocladium roseum YMF1.00133 Antinematodal activity [22] Toluquinol (218) Nectria erubescens -[57] Clonostach acid A (219) Clonostachys rosea B5-2 - [58] Clonostach acid B (220) Clonostachys rosea B5-2 - [58] Clonostach acid C (221) Clonostachys rosea B5-2 - [58] 2-Furoic acid (222) Bionectria sp. Y1085 - [17] J. Fungi5-Hydroxymethyl 2020, 6, x FOR furoic PEER acid REVIEW (223) Bionectria sp. Y1085 -25 of [3017 ] 2-Hydroxy-5-hydroxymethyl furan (224) Bionectria sp. Y1085 - [17] 3-(R)-Piliformic acid (225) Bionectria sp.Leishmanicidal - [28] 6′-Acetoxy-piliformic acid (227) Nectria pseudotrichia [30] 3-(S)-Piliformic acid (226) Nectria pseudotrichia Leishmanicidalactivity activity [30] 6 -Acetoxy-piliformic acid (227) Nectria pseudotrichia Leishmanicidal activity [30] 0 5′,6′-Dehydropiliformic acid (228) Nectria pseudotrichia - [30] 50,60-Dehydropiliformic acid (228) Nectria pseudotrichia -[30] HypocrealesateHypocrealesate (229) (229) Nectria sp.Nectria HLS206 sp. HLS206 - - [81] [81]

FigureFigure 15. 1Miscellaneous5. Miscellaneous metabolitesmetabolites isolated isolated from from ClonostacClonostachyshys fungi.fungi.

6. Conclusions and Future Perspectives In this mini-review, we summarized chemical structures, occurrences and biological activities of the secondary metabolites from Clonostachys fungi. The main metabolites belong to nitrogen-containing compounds, polyketides, and terpenoids. Some piperazines (i.e., bionetins, gliocladins and gliocladins and verticillins), polyketides (i.e., nectriaquinones, pesudonectrins, bionectriols, and nectriacids) and terpenoids (i.e., glisoprenins and ilicicolins), which were only isolated from Clonostachys fungi, exhibited obvious biological activities, such as antimicrobial, cytotoxic, antinematodal, and AChE inhibitory activities (Tables1–3). Some metabolites, such as alternariol ( 86) sorbicillinoids, were also distributed in other groups of fungi. Some metabolites, such as cyclosporin A (12), cinnacidin (61), and taxol (209), have shown their medicinal and agricultural applications. In order to search for new bioactive metabolites from Clonostachy fungi, some strategies, such as gene disruption, modification of the fermentation medium, co-cultivation and synthetic modification, have been proven to be effective. Gliocladiosins A (67) and B (68), as well as rogersonins A(69) and B (70) were alkaloid–polyketide hydrids isolated from gene disruption mutants of Clonostachys rogersoniana [32,33]. Fermentation of the Clonostachys rosea on white beans instead of rice afforded one γ-lactam clonostalactam (62) and two γ-lactones 3,5-dihydroxyfuran-2(5H)-one (141) and sapinofuranone B (142) that were not detected in the former extracts [11]. The apple juice supplemented solid rice media led to significant changes in the secondary metabolism of the fungus, Clonostachys rosea B5-2, and induced the production of four new compounds, (-)-dihydrovertinolide (144), clonostach acids A (219), B (220) and C (221) together with the known compound, (-)-vertinolide (143)[58]. Co-cultivation of the Bionectria sp. with either Bacillus subtilis or Streptomyces lividans resulted in the production of bionectriamines A (59) and B (60), and 6,8-dihydroxyisocoumarin-3-carboxylic acid (93)[28]. In addition, based on the isolated compounds, more bioactive compounds can be synthesized. A typical example was the synthesis of cinnacidin (61) analogs. Two new structural analogs of cinnacidin (61), namely (2S,3S)-2-[(3RS, 3aSR,6aRS)-3-methoxy-4-oxo-3,3a,4,5,6,6a-hexahydropentalen-1-ylcarbamoyl]-3-methylvaleric acid and benzyl (2S,3S)-2-[(3RS,3aSR,6aRS)-3-methoxy-4-oxo-3,3a,4, 5,6,6a-hexahydro pentalen-1- ylcarbamoyl]-3-methylvalerate, have been synthesized. The synthetic compounds were highly phytotoxic on a range of weeds to show their potential application as an herbicide [29]. Furthermore, the phenolic sesquiterpenoids which are also called ascochlorin derivatives or ilicicolins were widely distributed in the fungi of genus Nectria (synonym: Clonostachys). The occurrence of these compounds further confirms the close chemotaxonomic relationships among the related Nectria species. Ilicicolin H(71) was considered to be of potential chemotaxonomic significance and could be used as the main chemotaxonomic marker of the Nectriaceae family [34]. Some piperazines, such as bionetins [16], gliocladins [21], gliocladines [23], glioperazines, and verticillins were also only isolated from the fungal species of Clonostachys. Their chemotaxonomic significance should be further verified. Though major fungal species of Clonostachys fungi have been studied for their metabolites [1], the remaining fungi need to be revealed in detail. Moreover, the biological activities, structure–activity relationships, mechanisms of action, as well as biosynthesis of the metabolites from Clonostachys fungi need to be further investigated. Clarification of the metabolites of Clonostachys fungi could not only be in favor of discovering more compounds with novel structures and excellent biological activities, but also better understand the chemotaxonomy of the genus Clonostachys.

Author Contributions: Conceptualization, L.Z. and P.H; Writing—original draft preparation, P.H. and L.Z.; Writing—review and editing, D.L., X.Z., D.X. and B.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Key R&D Program of China (2017YFD0201105 and 2017YFD0200501). Conﬂicts of Interest: The authors declare no conﬂict of interest. J. Fungi 2020, 6, 229 27 of 31

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