Antifungal Secondary Metabolites Produced by the Fungal Endophytes: Chemical Diversity and Potential Use in the Development of Biopesticides

Plant diseases caused by phytopathogenic fungi can lead to huge losses in the agricultural fields and therefore remain a continuous threat to the global food security. Chemical-based fungicides contributed significantly in securing crop production. However, indiscriminate application of fungicides has led to increased chemical resistance and potential risks to human health and environment. Thus, there is an urgent need for searching for new bioactive natural products and developing them into new biopesticides. Fungal endophytes, microorganisms that reside in the fresh tissues of living plants, are regarded as untapped sources of novel natural products for exploitation in agriculture and/or medicine. Chemical examination of endophytic fungi has yielded enormous antifungal natural products with potential use in the development of biopesticides. This review summarizes a total of 132 antifungal metabolites isolated from fungal endophytes in the past two decades. The emphasis is on the unique chemical diversity of these metabolic products, together with their relevant antifungal properties. Moreover, some “star molecules,” such as griseofulvin and trichothecene, as well as their synthetic derivatives that possess high potential as candidates of new natural fungicides, are also presented herein.


INTRODUCTION
Plant diseases caused by phytopathogenic fungi are continuing to be a huge threat in the agricultural fields. It is estimated that the global loss caused by plant diseases is more than 20% of the crop yield in the major food and cash crops worldwide (Kim et al., 2004). Fungal pathogens were responsible for the considerable postharvest losses of grain crops, fruits, and vegetables, which, in addition to causing decay, can produce mycotoxins that are harmful to humans and animals (Bai et al., 2013;Shi et al., 2017). Therefore, effective and sustained control of these fungal pathogens has become an urgent task. Chemical control has been widely adopted in crop production. During the past years, a great variety of chemical fungicides (agrochemicals), such as thiophanate-methyl, carbendazim, and imazalil, are designed and applied to control these diseases. It is indisputable that these chemicals have resulted in substantial increases in productivity and contributed significantly in agricultural industry. However, these most-applied chemical fungicides have been limited by many serious problems. Indiscriminate use of these fungicides has led to the appearance of pathogens with multiple fungicide resistances, which further complicated the management of the diseases (Talibi et al., 2014). Furthermore, repeated and exclusive use of fungicides is increasingly restricted owing to their undesirable effects on non-target organisms (carcinogenicity, high and acute residual toxicity) and potential risks to environmental pollution (long degradation period) (Bai et al., 2013;Talibi et al., 2014). Thus, these synthetic chemical fungicides are subject to registration and permission for use, and are even no longer authorized in various countries.
Based on above questions, the challenge is to develop new and eco-friendly alternatives for the safe control of these pathogens, which pose low risk to human health and environment. Nowadays, many promising biological approaches were considered to be potential alternatives to synthetic fungicides, including (i) use of biocontrol microorganisms, (ii) application of naturally sourced metabolites, and (iii) induction of natural resistance (Talibi et al., 2014). Among them, naturally sourced secondary metabolites from microbes have attracted great attention. Microorganisms are well known for their ability to synthesize bioactive secondary metabolites, which have provided abundant chemical entities for pharmaceuticals and agrochemicals. Many natural antifungal fungicides, such as kasugamycin, polyoxins, validamycin, and blasticidin-S, have been obtained from microbial resources (Copping and Duke, 2007;Wang et al., 2016).
Fungal endophytes, microorganisms that asymptomatically reside in the internal tissues of plants, have been widely distributed in almost every plant and are rich in diversity (Jia et al., 2016). Endophytic fungi have a close and complex interaction with their hosts, which involve mutualism, antagonism and rarely parasitism (Gouda et al., 2016). These species are known to promote host growth and gain essential nutrition. They also provide tolerance to plants against various types of abiotic and/or biotic stresses. Most importantly, they possess the ability to produce plenty of structurally diverse and biologically active secondary metabolites to protect their hosts from pathogenic microorganisms and pests. In this sense, endophytic fungi are a treasure source of searching for novel secondary metabolites with immense potential agricultural applications. It has been reported that a large number of metabolites with different chemical skeletons have been deciphered from endophytic fungi, such as alkaloids, terpenoids, steroids, peptides, benzopyranones, quinones, and isocoumarins (Gouda et al., 2016). Chemically speaking, the discovery of these metabolites provided impressive chemical basis in the development of agrochemicals. Moreover, most of them exhibited promising bioactivities, such as antifungal, antibacterial, herbicidal, nematocidal, insecticidal, and other agricultural activities.
Therefore, the topical subject of antifungal secondary metabolites produced by fungal endophytes has been searched and analyzed. The literature search was conducted using the combined keywords "antifungal, " "secondary metabolites, " and "endophytic fungus" in the databases such as Web of Science, Google Scholar, and SciFinder Scholar, with a previously reported search method (Zhang et al., 2020). As a result, a total of 132 metabolites with anti-phytopathogenic activities from fungal endophytes in the past two decades (covering from 2000 to 2020) were included in this review. The present compounds possess diverse chemical structures, which were classified into alkaloids (including cytochalasins, indoles, diketopiperazines, and other nitrogen-containing compounds), terpenoids, polyketides (quinones, macrolides, benzopyrones and unsaturated lactones), and other miscellaneous compounds within a biogenetic context. Moreover, we also describe the sources, the producing strains, target plant pathogenic fungi, and their potential use as lead compounds in the development of biopesticides.

CONFUSED ISSUES NEED TO BE ADDRESSED
Prior to the beginning of this review, there are three issues that need to be addressed. (i) The level of antifungal potency should be standardized. As we can see from literatures, the literatures are replete with uncalibrated potency descriptors, including 'mild, moderate, strong, pronounced, significant, remarkable, and potent.' Apparently these descriptions confused the readers that which level means strong and which should be defined as weak. The unified standardization has not yet formed since the definition of the level of bioactivity just represents the individual judgment of the authors. However, in this review, the antifungal potency was distinguished as 'potent, ' 'moderate, ' and 'weak' based on the comparison with positive controls. We discretionarily define 'potent' as the antifungal activity higher than that of the positive controls, 'moderate' as the bioactivity equal to that of the positive controls, and 'weak' as that of lower than positive controls (or inactive) throughout this review. (ii) Quantitative comparisons of the results of the bioactivity tests between different studies are problematic. As the fungal pathogens used in bioassays may have different provenance and viability/sensitivity with different assay protocols, the comparisons should be qualified by consideration of positive/negative controls, and even then should be best limited within individual studies, rather than between different studies. (iii) The selection of different positive controls may lead to radically different results. Commercial fungicides were generally chosen as positive controls. However, since they belong to different chemical classes, they may present distinct potency and mechanisms of action.

Cytochalasin Alkaloids
The cytochalasin alkaloids are a class of structurally related fungal metabolic products. To date, more than 300 cytochalasin analogs have been isolated from many genera of ascomycetes and basidiomycetes, including Aspergillus, Chaetomium, Penicillium, Phomopsis, Phoma, Spicaria, Xylaria, and so on (Wei et al., 2017). Structurally, cytochalasins are characterized by a highly substituted perhydroisoindol-1-one moiety which is fused with a 9-to 15-membered macrocyclic ring. Many of cytochalasins exhibit a wide range of biological activities, such as cytotoxic, antimicrobial, and phytotoxic properties (Wei et al., 2017;Xu et al., 2017). 14 cytochalasins (Figure 1) isolated from fungal endophytes were reported to possess moderate to potent antifungal activity. Cytochalasin D (1) was produced by various endophytic fungi Xylaria sp., which were isolated from leaves of guarana plant (Elias et al., 2018). 1 showed fungistatic activity against the phytopathogen Colletotrichum gloeosporioides, which causes the anthracnose disease, with an MIC of 2.46 mM. Commercial fungicides captan and difenoconazole were applied as positive controls (MICs 16.63 and 0.02 mM, respectively) (Elias et al., 2018). Bioassay-guided separation of Xylaria sp. XC-16, an endophyte from Toona sinensis led to the discovery of five agriculturally active cytochalasin alkaloids, including a new compound cytochalasin Z 28 (2) (Zhang Q. et al., 2014). Compound 2 showed potent fungicidal effect (MIC = 12.5 µM) against the phytopathogen Gibberella saubinetti, which was better than that of the positive control hymexazol (MIC = 25 µM) (Zhang Q. et al., 2014). Chemical investigation of the biocontrol potential endophytic fungus Aspergillus capensis CanS-34A in Brassica napus has resulted in the isolation and identification the antifungal metabolite rosellichalasin (3) . 3 inhibited the plant pathogenic fungi Botrytis cinerea, Monilinia fructicola, Sclerotinia sclerotiorum, and S. trifoliorum with the EC 50 values of 36.8, 87.1, 5.3, and 41.1 µM, respectively. Thus, S. sclerotiorum was the most sensitive target fungus . A new chaetoglobosin, penochalasin K (4) possessing a rare six-cyclic 6/5/6/5/6/13 fused ring system, was isolated from the solid culture of the mangrove endophytic fungus Penicillium chrysogenum V11 (Zhu et al., 2017b). Compound 4 displayed potent selective activities against C. gloeosporioides and Rhizoctonia solani, with MIC values of 6.13 and 12.26 µM, respectively, which were about ten-fold and two-fold better than that of the positive control carbendazim (Zhu et al., 2017b). Five metabolites, chaetoglobosins A (5), B (6), E (7), F (8), and penochalasin G (9), were obtained from endophytic Chaetomium globosum, isolating from the seeds of Panax notoginseng . Some of them exhibited remarkable inhibition against phytopathogenic fungi causing root rot disease. For example, chaetoglobosin E (7) and penochalasin G (9) indicated potent inhibition against Epicoccum nigrum with the MICs < 2 µM FIGURE 2 | Other alkaloids characterized from endophytic fungi (15-35). . A new chaetoglobosin named penochalasin J (10), as well as two known chaetoglobosins, chaetoglobosin C (11) and armochaetoglobosin I (12), were isolated from the mangrove endophytic fungus P. chrysogenum V11 . Compound 10 showed more potent antifungal activity against plant pathogen C. gloeosporioides with an MIC value of 25.08 µM, than the positive control carbendazim (MIC = 65.38 µM). Simultaneously, compounds 11 and 12 remarkably inhibited R. solani, with MIC values of 23.66 and 12.11 µM, respectively . Chaetoglobosins V (13) and G (14) were isolated from the culture of the endophytic fungus C. globosum, associated with the leaves of Ginkgo biloba tree (Xue et al., 2012). Compounds 13 and 14 exhibited potent antifungal activity against Alternaria solani, with MICs of 47.3 µM, while 14 also possessed potent activity against A. alternate, with an MIC of 47.3 µM (Xue et al., 2012). In total, the above results indicated that cytochalasin alkaloids could be used as fungicides or as leads of new fungicides to the related phytopathogenic fungi.

POTENTIAL USE IN THE DEVELOPMENT OF BIOPESTICIDES
As mentioned above, biologically active and structurally diverse fungal metabolites constitute a rich resource for drugs and pesticides discovery. These recently discovered metabolites 1-132, which possessed extensive chemical skeletons, exhibited moderate to potent anti-phytopathogenic activities. Therefore, some of them might have the potential use in the development of new biopesticides. Especially, based on these potential compounds, a series of novel derivatives with agricultural and pharmaceutical importance were designed and synthesized.

Griseofulvin
Griseofulvin (85), a secondary metabolite possessing spirocyclic benzofuran-3-one skeleton, was initially isolated from the fungus Penicillium griseofulvum in 1939 by Oxford et al. (Petersen et al., 2014). Then, this polyketide has also been found to be produced by several Ascomycetes including Penicillium sp., Aspergillus sp., and Xylaria sp. . Griseofulvin has a rich chemical diversity, and until now, more than 400 griseofulvin analogs have been isolated and synthesized (Figure 8; Petersen et al., 2014). Griseofulvin was one of the first antifungal metabolic products in filamentous fungi, offered in vitro fungistatic effect against dermatomycoses. Recently, it has gained renewed attention due to many reports of antifungal properties against plant pathogenic fungi. Zhao et al. reported that griseofulvin, produced by an endophyte Nigrospora sp., displayed clear growth inhibition of the test eight plant pathogenic fungi (B. cinerea, Colletotrichum orbiculare, F. oxysporum f.sp. cucumerinum, F. oxysporum f.sp. melonis, Pestalotia diospyri, Pythium ultimum, R. solani, and S. sclerotiorum) . Among them, it exhibited potent activity against B. cinerea and C. orbiculare with the EC 50 values of 0.6 and 1.4 µM, respectively . It should be pointed out that, its dechlorinated derivative, dechlorogriseofulvin, only showed weak activity, indicating that the chlorine played a decisive role in the antifungal activity. Tang et al. reported that griseofulvin isolated from Penicillium brasilianum displayed strong inhibitory effect on the growth of A. solani with an MIC of 3.13 µM (Tang et al., 2015). These impressive activities make this compound suitable candidate for biopesticide discovery and trigger the following synthesis studies, including semisynthesis from griseofulvin and de novo synthesis.
Bai et al. designed and synthesized 22 griseofulvin derivatives from commercially available griseofulvin (Bai et al., 2019). In vitro antifungal assay indicated that griseofulvin and its derivatives possessed remarkable activities against five phytopathogenic fungi (Cytospora sp., C. gloeosporioides, B. cinerea, A. solani, and F. solani) (Bai et al., 2019). Of significance was that, compounds numbered 6a-6f were found to have significant potential, which were superior to commercial fungicides hymexazol and thiophanate-methyl. The threedimensional quantitative structure-activity relationship analysis revealed that the modification of the 4' position, for example, the suitable bulky and electronegative acyl-substituted groups at the 4' position, can significantly improve the antifungal activity, even up to 10-fold higher than inhibitory effect of the parent compound griseofulvin (Bai et al., 2019). Kartsev et al. carried out the synthetic studies of griseofulvin derivatives (Kartsev et al., 2019). As a result, a total of 42 new griseofulvin derivatives were designed and synthesized. These newly synthesized griseofulvin derivatives exhibited potent antifungal activity against A. niger, A. ochraceus, A. fumigatus, A. versicolor, Penicillium funiculosum, P. ochrochloron, P. verucosum var. cyclopium, Trichoderma viride. All compounds showed higher activity than the commercial antifungal drugs ketoconazole (7-42 times) and bifonazole (3-16 fold) (Kartsev et al., 2019). Interestingly, the synthesized compounds were more active than the parent compound griseofulvin (up to 4 times). Therefore, in conclusion, griseofulvin especially its derivatives can be further used for the development of new agricultural fungicides.

Trichothecene
Trichothecenes, a group of sesquiterpene-based fungal metabolites with a common tricyclic 12,13-epoxytrichothec-9ene core, are found to be produced by various microorganisms such as Fusarium sp., Myrothecium sp., Spicellum sp., Stachybotrys sp., Cephalosporium sp., Trichoderma sp., and Trichothecium sp. Based on the substitution pattern, trichothecenes are classified into different families of nivalenols, neosolaniols, isotrichodermins, calonectrins, trichothecins, and trichobreols (Figure 9; Takahashi-Ando et al., 2020).  Trichothecenes are well-known as mycotoxins, causing significant negative effects on agriculture and human health. Initial study of trichothecenes was focused on the phytotoxicity and mammalian intoxications, and later emphasis was on exploring their complementary bioactivities. Some related compounds exhibited potent antitumor effect and have been used for clinical trials . Meanwhile, trichothecenes showed varied role in the field of agriculture and may act as bio-control agents, strengthen the defense system of the plants against pathogens (Kumari et al., 2016). Previous structureactivity relationships study revealed that a slight modification of trichothecenes could dramatically decrease the toxicity but still retain its bioactivity . In this case, there seems to be a special significance to search for new trichothecenes with agricultural applications.
Yamazaki et al. reported three new antifungal trichothecenes, trichobreols A-C, from the NaI-containing fermentation of the marine-derived Trichoderma cf. brevicompactum (Yamazaki et al., 2020a). Then, from the same fungus, another two new trichothecenes, trichobreols D and E, were obtained (Yamazaki et al., 2020b). Trichobreols showed good antifungal activity, especially toward yeast-like pathogenic fungi. Moreover, five semisynthetic derivatives were prepared from trichobreol A to evaluate the structure-activity relationship of antifungal trichothecenes. The results indicated that the substituents at C-3 and C-4 positions were responsible for the potency of antifungal activity (Yamazaki et al., 2020b). Li et al. reported three new macrocyclic trichothecenes possessing rare 6'-ketal moieties, roridoxins A-C, from the insect-associated fungus Myrothecium roridum . Roridoxins A and C were found to possess potent antifungal activity against Alternaria tenuissima, A. niger, Pyricularia grisea, and F. oxysporum . These findings suggested that trichothecenes are also promising leads which are applicable for the development of new agrochemicals.

CONCLUSION AND FUTURE PERSPECTIVES
Fungal endophytes, which are ubiquitous in plants and symbiotic with their hosts, are well-known for producing a variety of antimicrobial metabolites and enhancing plant resistance to pathogens and pests. These bioactive metabolites play a defensive role in protecting the host plants against pathogenic attacks. Therefore, antibiotic metabolites from the endophytes have the potential to be applied as agrochemicals to control pathogens. This review summarizes the structural/biogenetic types of 132 antifungal metabolites isolated from fungal endophytes in the past two decades. These present metabolites possess diverse chemical structures. Based on their putative biogenetic origin, they were classified into alkaloids (including 1-14 for cytochalasins and 15-35 for other alkaloids), terpenoids (36-49), polyketides (50-123), and other miscellaneous compounds (124)(125)(126)(127)(128)(129)(130)(131)(132). It is worth mentioning that the structural classifications based on biogenetic categories are somewhat arbitrary, as many compounds are derived from mixed biosynthetic pathways. Taking compounds 86-89 as an example, they are new members of the chromenone-type of metabolites biogenetically derived from isoprenoids and a polyketide. As shown in Figure 10,  Frontiers in Microbiology | www.frontiersin.org among the 132 active metabolites, approximately 56% were polyketides. Molecules grouped as polyketides are significant in natural products research due to their biosynthetic complexity and high value in pharmaceutical and/or agrochemical industries. This finding revealed that polyketides are the most potential classes for the discovery of novel antifungal lead compounds. It should be pointed out that we divided cytochalasins into an individual group, since this kind of compounds constitutes 10.6% of the metabolites reported, nearly as many as terpenoids (10.6%) and other alkaloids (15.9%). Cytochalasins are a diverse group of fungal polyketide synthase-non-ribosomal peptide synthetase (PKS-NRPS) hybrid metabolites. This class of compounds are worthy of particular attention and may be applied in the field of bio-pesticides.
It is well-known that the endophytic fungi from terrestrial plants are a treasure house of bioactive secondary metabolites. Moreover, marine-derived endophytic fungi have also been considered as a non-negligible resource to search for antifungal lead compounds. In this review, compounds 4, 10-12, 55-57, and 76-78 were isolated from marine mangrove-associated endophytic fungi. The marine environment is quite different from the terrestrial environment, which indicates that marinederived endophytic fungi may possess unique metabolic pathways to produce interesting antifungal compounds with novel structures. As for the producing strains, the fungal genera Phomopsis, Aspergillus, Chaetomium, and Nigrospora are predominant genera as producers of these antifungal metabolites, with 17, 11, 10, and 10 compounds described, respectively (Figure 11). As shown in Figure 11, these metabolites are scattered across a variety of fungi belonging to 29 various genera, including some rare species. Among them, C. globosum is a creative species known for making a large number of exclusive and structurally significant bioactive chaetoglobosins. The antifungal activity of these metabolites against the phytopathogenic fungi indicated that the endophytes could protect their host plants by producing bioactive molecules, which may be toxic or even lethal to phytopathogens and highlighted the potential of endophytic fungi in producing valuable metabolites. Moreover, a chemical interaction between the endophytes and the host plants, which produces metabolites as chemical defense compounds, needs further investigation.
Overall, endophytes are considered to be a treasure house of antifungal metabolites. This review summarizes 132 metabolites with moderate to potent antifungal activities isolated from fungal endophytes. We also list some "star molecules" such as griseofulvin and its derivatives that possess high potential as candidates of new natural fungicides herein. It is believed that in the near future, research on antifungal metabolites of endophytic fungi will become more prolific and be beneficial for the development of new agrochemicals.

AUTHOR CONTRIBUTIONS
KX and X-QL performed the literature material's collection and reorganization. PZ and D-LZ wrote the manuscript. All authors reviewed the manuscript.