Metabolites from Alternaria Fungi and Their Bioactivities

Alternaria is a cosmopolitan fungal genus widely distributing in soil and organic matter. It includes saprophytic, endophytic and pathogenic species. At least 268 metabolites from Alternaria fungi have been reported in the past few decades. They mainly include nitrogen-containing metabolites, steroids, terpenoids, pyranones, quinones, and phenolics. This review aims to briefly summarize the structurally different metabolites produced by Alternaria fungi, as well as their occurrences, biological activities and functions. Some considerations related to synthesis, biosynthesis, production and applications of the metabolites from Alternaria fungi are also discussed.


Introduction
Alternaria fungi, belonging to the Dematiaceae of the Hyphomycetes in the Fungi Imperfecti, have a widespread distribution in Nature. They act as plant pathogens, weak facultative parasites, saprophytes and endophytes [1]. Some metabolites from Alternaria fungi are toxic to plants and animals, and are designated as phytotoxins and mycotoxins, respectively [2][3][4]. Alternaria metabolites exhibit a variety of biological activities such as phytotoxic, cytotoxic, and antimicrobial properties, which have drawn the attention of many chemists, pharmacologists, and plant pathologists in research programs as well as in application studies [5,6]. For examples, porritoxin (21, Table 1) from endophytic Alternaria species has been studied as the candidate of cancer chemoproventive agent [7].
In the early 1990s, about 70 metabolites from Alternaria fungi were reviewed [13]. Several reviews on Alternaria phytotoxins have been published over the last few decades [6,14,15]. In recent years, more and more metabolites with bioactivities from Alternaria fungi have been isolated and structurally characterized. This review mainly presents classification, occurrences, biological activities and functions of the metabolites from Alternaria fungi. We also discussed and prospected the synthesis, biosynthesis, production and applications of the metabolites from Alternaria fungi.

Nitrogen-Containing Metabolites
The nitrogen-containing compounds such as amides, amines, and cyclopeptides have been isolated from Alternaria fungi. Some of them belong to the host-selective phytotoxins in host-parasite interactions [39].
Two cyclopeptides destruxins A (49) and B (50) were isolated from A. linicola [31]. Destruxin B (50) was also found in A. brassicae as the major phytotoxin [45]. Other cyclopeptides along with their distributions in Alternaria fungi are shown in Table 1. Other nitrogen-containing metabolites isolated from Alternaria fungi are shown in Figure 3. Two nucleosides namely uridine (56) and adenosine (57) were isolated from A. alternata [49].

Steroids
Some steroids (62-68) have been isolated from Alternaria fungi ( Figure 4 and Table 1). These findings are consistent with the considerations that ergosterol (62) and their derivatives are common to all fungi and occur widely among the fungi [143].

Terpenoids
Most of terpenoids from Alternaria fungi have been found as the mixed terpenoids which have a multiple biogenesis . Other Alternaria terpenoids include diterpenoids 106-114, sesquiterpenoids 115,116 and a triterpenoid 117, which are shown in Figure 5.
Nineteen tricycloalternarenes (TCAs) were isolated from the culture filtrate of the phytopathogenic fungus A. alternata from Brassica sinensis (Cruciferae). Tricycloalternarenes are closely related to ACTG toxins 87,88. Structural differences mainly occur in the isoprenoid side chain and the substitution pattern of the C-ring of the tricycloalternarenes [57][58][59][60].

Quinones
Two groups of quinones, anthraquinone and perylenequine derivatives have been isolated in Alternaria fungi so far.

Phenolics
The phenolic metabolites 219-256 from Alternaria fungi are shown in Figures 12 and 13. Most of them have a polyketide origin. One phenylpropanoid component was identified as methyl eugenol (223) by GC-MS from the volatile oil obtained by hydrodistillation from the Alternaria species isolated as the endophyte of rose (Rosa damascaena) [111]. Methyl eugenol (223) has been used as a flavouring agent in jellies, baked goods, non-alcoholic beverages, chewing gum, candy, pudding, relish, and ice cream [144].
One Alternaria species MG1 as the endophytic fungus from Vitis vinifera L. cv. Merlot could produce resveratrol (3,5,4'-trihydroxystilbene, 252) [130]. Resveratrol has been known for preventing and slowing the occurrence of some human diseases, including cancer, cardiovascular disease, and ischemic injuries. It has also been shown that resveratrol (252) can enhance stress resistance and extend the lifespan of various organisms ranging from yeasts to vertebrates [145]. Resveratrol has been found in a variety of plant species such as Vitis vinifera, Polygonum cuspidatum, and Glycine max [141]. Endophytic Alternaria species for producing plant-derived resveratrol should be an important and novel resource with its potential application in pharmaceutical industry [146].

Curvularin
Phthalides are considered as a special group of phenolic compounds. Four phthalates 253-256 were isolated from Alternaria fungi that are shown in Figure 13, and their occurrences are shown in Table 1.

Biological Activities and Functions
Alternaria metabolites with diverse chemical properties have been clarified (Figures 1-14, Table 1). Some of them act as phytotoxins to plants or as mycototoxins to humans and animals. They have been examined to have a variety of biological activities and functions, which mainly include the effects on plants, cytotoxic and antimicrobial activities.

Effects on Plants
Plant pathogenic Alternaria species can affect cereals, vegetables and fruit crops in the field and during storage. Alternaria fungi contamination is responsible for some of the world's most devastating plant diseases, causing serious reduction of crop yields and considerable economic losses. The metabolites from plant pathogenic fungi are usually toxic to plants and are called phytotoxins. They were further divided into host-specific and host non-specific toxins. The host-specific toxins (HSTs) are toxic only to host plants of the fungus that produces the toxin [6,13]. Another definition seems to be more acceptable that the host-specific toxins are toxic to plants that host the pathogen, but have lower phytotoxicity on non-host plants [147,148]. Most HSTs are considered to be pathogenicity factors, which the fungi producing them require to invade tissue and induce disease [149] All isolates of the pathogen that produce an HST are pathogenic to the specific host. All isolates that fail to produce HSTs lose pathogenicity to the host plants. Plants that are susceptible to the pathogen are sensitive to the toxin. Such correlations between HST production and pathogenicity in the pathogens, and between toxin sensitivity and disease susceptibility in plants provide persuasive evidence that HSTs can be responsible for host-specific infection and disease development. Johnson and coworkers revealed that the genes involved in HST synthesis such as the cyclopeptide synthetase gene, whose product catalyzed AM toxin production in A. alternata apple pathotype, might reside on a conditionally dispensable (CD) chromosome. The loss of the CD chromosome led to loss of both toxin production and pathogenicity without affecting fungal growth [150]. On the other hand, the exact roles of non-specific toxins in pathogenesis are largely unknown, but some are thought to contribute to the features of virulence, such as the symptom development and in planta pathogen propagation [6]. The virulence and host-specificity of these pathogens are based on production of the distinctive HSTs [13]. For Alternaria pathogens, there are now at least nine diseases caused by Alternaria species in which HSTs are responsible for fungal pathogenicity ( Table 2). Most of Alternaria HSTs are nitrogencontaining metabolites. Table 2. Host-specific phytotoxins from Alternaria fungi.

Phytotoxin name
Alternaria species

Sunflower (Helianthus annuus)
Necrotic spots on sunflower leaves [40] Maculosin ( Alternaria balck spot of strawberry [134,135] Among the HSTs, AAL toxins from tomato stem canker pathogen (A. alternata f.sp. lycopercici) have received a special attention. They were toxic to all tissues of sensitive tomato cultivars at low concentrations and induced apoptosis in sensitive tomato plants [151], and were found to inhibit de novo sphingolipid (ceramide) biosynthesis in vitro. Therefore, AAL toxins are called sphinganineanalog mycotoxins (SAMs). It has been reported that the tomato Alternaria stem canker locus mediated resistance to SAMs-induced apoptosis [152].
Destruxins are another group of HSTs produced both in vitro and in planta by A. brassicae, the causal agent of Alternaria blackspot disease of rapeseed and canola [148]. These cyclodepsipeptides exhibited a wide variety of biological activities such as antitumor, antiviral, insecticidal, cytotoxic, immunosuppressant, and antiproliferative effects except their phytotoxicity [153].
Interactions between Alternaria species and cruciferous plants were studied in detail by the Pedras group [51]. Nectrophic phytopathogens such as A. alternata and A. brassicae are known to synthesize phytotoxins that damage plant tissues and facilitate colonization, while in response to pathogen attack crucifers biosynthesize phytoanticipins and phytoalexins. Phytoalexins are secondary metabolites produced de novo by plants in response to diverse forms of stress including microbial infection, UV irradiation, and heavy metal salts, whereas phytoanticipins are constitutive defenses whose concentrations can increase upon stress [154]. To the detriment of cruciferous plants, the phytopathogens can overcome phytoanticipins and phytoalexins by producing detoxifying enzymes.  Host non-specific Alternaria phytotoxins can affect many plants regardless of whether they are a host or non-host of the pathogen [6,13]. Host non-specific nitrogen-containing phytotoxins include tenuazonic acid (15), porritoxin (21) and tentoxin (53). Tentoxin (53), a cyclic tetrapeptide from A. alternata, inhibited chloroplast development, which phenotypically manifests itself as chlorotic tissue [157]. Tentoxin (53) was suggested to exert its effect on chlorophyll accumulation through overenergization of thylakoids [158]. Tenuazonic acid (TeA, 15) was investigated in Chlamydomonas reinhardtii thylakoids which revealed that TeA inhibited photophosphorylation with the action site at Q B level [159].
Host non-specific quinone phytotoxins included bostrycin (182), 4-dexoybostrycin (183), and altersolanols A (185), B (186) and C (187) [93][94][95]. Altersolanol A (185), a tetrahydroanthraquinone phytotoxin from the culture broth of A. solani, inhibited the growth of cultured cells of Nicotiana rustica. It acted as a potent stimulator of NANH oxidation in the mitochondria isolated from N. rustica cells. Altersolanols acted as electron acceptors in an enzyme preparation of diaphorase. The capacity of altersolanols A, B, C, D, E and F to act as electron acceptors was in the order of A > E > C > B > F > D [160].
Host non-specific phenolic phytotoxins include zinniol (226) and its analogues 227-237. Zinniol (226) from the liquid cultures of A. tagetica induced leaf tissue necrosis in a number of unrelated plant species (Avena sativa, Cucumis sativus, Daucu carota, Hordeum vulgare, Triticum aestivum) from different families which demonstrated that zinniol acted as a non host-specific phytotoxin [161]. However, Qui et al. evaluated the effects of zinniol at the cellular level and showed that pure zinniol was not obviously phytotoxic at concentrations known to induce necrosis in leaves of Tagetes erecta, which indicated that the classification of zinniol as a host non-specific phytotoxin should be further investigated [162].
Other host non-specific phytotoxins include α,β-dehydrocurvularin (250) and brefeldin A (259) from A. zinniae. They showed phytotoxic activity on Xanthium occidentale, a widespread noxious weed of Australian summer crops and pastures. The fungus A. zinniae and its toxins may be used as the mycoherbicides in integrated weed management programs [129].
Some fungal phytotoxins were toxic to weed species to show their herbicidal potentials in agriculture and forestry [10,[163][164][165]. Some examples are shown in Table 3. Weed pathogens should be a very promising source of bioactive natural products for weed control. Tentoxin (53) was transformed to isotentoxin (54) by UV irradiation. Isotentoxin (54) had stronger wilting effects than tentoxin against the weed Galium aparine [11].
Of Alternaria dibenzopyranones, alternariol (157) was the most active metabolite to have cytotoxic activity on L5178Y mouse lymphoma cells [84], as well as to have inhibitory activity on protein kinase and xanthine oxidase [55]. Further investigation showed that alternariol (157) has been identified as a topoisomerase I and II poison which might contribute to the impairment of DNA integrity in human colon carcinoma cells [167]. It induced cell death by activation of the mitochondrial pathway of apoptosis in human colon carcinoma cells [168]. Alternariol and its 9-methyl ether induced cytochrome P450 1A1 and apoptosis in murine heptatoma cells dependent on the aryl hydrocarbon receptor [169]. Other alternariol derivatives such as alternariol 5-O-sulfate (158), alternariol 9-methyl ether (159), 3'-hydroxyalternariol (161), altenuene (162), 4'-epialtenuene (164) and dehydroaltenusin (156) were also screened to be cytotoxic [84]. Dehydroaltenusin (156), isolated from A. tenuis, was found to be a specific inhibitor of eukaryotic DNA polymerase α to show its strong cytotoxic activity on tumor cells [83,170].

Other Bioactivities
Altenusin (241) isolated from the endophytic fungus Alternaria sp. (UFMGCB55) in Trixis vauthieri (Compositae) was screened to show inhibitory activity on trypanothione reductase (TR), which is an enzyme involved in the protection of the parasitic Trypanosoma and Leishmania species against oxidative stress, and has been considered to be a validated drug target. Altenusin (241) had an IC 50 value of 4.3 μM in the TR assay [122].

Conclusions and Future Perspectives
We just clarified one part of metabolites from the known Alternaria fungi. The rest of metabolties in Alternaria species need to be investigated in detail. In fact, many other Alternaria species remain unexplored for their metabolites. In most cases, both the biological activities and modes of action of the metabolites from Alternaria fungi have been studied very primarily. The structure-activity relationship has been established only for a few classes of Alternaria metabolites. This review mainly focused on the metabolites with low molecular weight from Alternaria fungi. Bioactive proteins, saccharides and glycoproteins are also important metabolites. Typical examples included a lipase from A. brassicicola [176], an endopolygalacturonase from the rough lemon pathotype of A. alternata [177], a protein elicitor (Hrip1) from A. tenuissima [178], and a polyketide synthase from A. alternata [179]. Some bioactive saccharides and glycoproteins have also been isolated such as β-1,3-, 1,6-oligoglucan elicitor from A. alternata [180] and a glycoprotein elicitor from A. tenuissima [181].
In recent years, more and more Alternaria fungi have been isolated as plant endophytic fungi from which large amounts of bioactive compounds have been structurally characterized. Another approach is to discovery novel bioactive compounds from the Alternaria fungi isolated from marine organisms. These Alternaria fungi could be the rich sources of biologically active compounds that are indispensable for medicinal and agricultural applications [191].
After comprehensive understanding of biosynthetic pathways of some Alternaria metabolites in the next few years, we can effectively not only increase yields of the bioactive metabolites, but also prohibit biosynthesis of some toxic metabolites (i.e., phytotoxins and mycotoxins) by treatment with some special fungicides.