Secondary Metabolites Produced by Neofusicoccum Species Associated with Plants: A Review

: The genus Neofusicoccum is comprised of approximately 50 species with a worldwide dis ‐ tribution and is typically associated with plants. Neofusicoccum is well ‐ known for the diseases it causes on economically and ecologically relevant host plants. In particular, members of this genus are responsible for grapevine diseases, such as leaf spots, fruit rots, shoot dieback, bud necrosis, vascular discoloration of the wood, and perennial cankers. Many secondary metabolites, including ( − ) ‐ botryoisocoumarin A, botryosphaerones, cyclobotryoxide and isosclerone, were identified from species of Neofusicoccum and their structural variability and bioactivities might be associated with the role of these compounds in the fungal pathogenicity and virulence. In this review, we summa ‐ rize the secondary metabolites from Neofusicoccum species focusing on the role of these compounds in the interaction between the fungus and host plant.


Introduction
The genus Neofusicoccum (Dothideomycetes, Botryosphaeriales, Botryosphaeriaceae) was introduced in 2006 to reassign species that were previously included in the genus Fusicoccum and its sexual morph Botryosphaeria [1]. Initially, the genus included 13 species but it has grown considerably, due mainly to the description of a large number of cryptic species, and currently contains about 50 distinct species listed in MycoBank [2] and Index Fungorum [3].
Species in the genus Neofusicoccum are typically associated with plants, being common in a wide range of woody hosts, from forest trees to crops and ornamentals [4,5]. The majority of these species have a worldwide distribution, a plurivorous nature, and exhibit an endophytic or latent pathogen lifestyle. Some Neofusicoccum species are plant pathogens of increasing relevance, causing diseases, most commonly dieback and canker, on economically and ecologically relevant host plants, such as grapevine, olive, eucalypts, among many others [4,5]. Grapevine is a particularly relevant crop worldwide, and several species of Neofusicoccum have been reported to be pathogens of this host. In fact, N. parvum is regarded as one of the most aggressive causal agents of Botryosphaeria dieback of grapevine [6].
In the last decade, literature concerning metabolites produced by Neofusicoccum species has been significantly enriched by many reports dealing with the biosynthetic potential of this fungal genus. Members of the genus Neofusicoccum are often symbiotically associated with different plant species. Symbiotic association between a fungus and a plant is a widespread phenomenon in nature and several symbiotic lifestyles have been defined considering the benefits to or impacts on host and symbiont [7]. Recent studies have indicated that botryosphaeriaceous fungi may express different symbiotic lifestyles in response to host internal signals or environmental factors which also influence the secondary metabolites production [8][9][10][11][12]. In fact, secondary metabolites may have a crucial role in the fungal lifestyles because these compounds have a broad number of biological functions, including mediating communication, nutrient acquisition and acting as virulence factors [13,14].
Neofusicoccum species found as endophytes or pathogens of plants, produce structurally different metabolites. The variety of secondary metabolites produced by diverse species of Neofusicoccum is reported in Table 2, and many compounds showed interesting biological activities (e.g., antibacterial, cytotoxic, phytotoxic).
Diverse species, such as Neofusicoccum australe, Neofusicoccum luteum, Neofusicoccum parvum, and Neofusiccocum vitifusiforme, have been isolated from grapevine (Vitis vinifera) and shown to be pathogenic to this host [15][16][17][18][19][20][21][22][23]. In fact, a long list of secondary metabolites has been identified from these strains and the structural variability might be associated with the role of these compounds in the grapevine diseases. In particular, the capability of N. parvum to colonize woody tissue, combined with the secretion of phytotoxic compounds, is thought to underlie its pathogenicity and virulence [24]. For this reason, a section of this review is dedicated to the phytotoxic activities of secondary metabolites produced by pathogenic strains from grapevine (see Section 3).
The biosynthesis of (-)-terramutin is particularly relevant because this compound is a precursor of terreic acid, a potential anticancer drug capable of inhibiting Bruton's tyrosine kinase [35]. This polyketide is biosynthesized from the condensation of three units of malonyl CoA and a unit of acetyl CoA. 6-Methylsalicylic acid is a cyclic intermediate produced, whose subsequent decarboxylation produces 6-methylcatechol and then terremutin. Interestingly, two precursor phenolic compounds were isolated from cultures of strain producers of 1 and 2 ( Table 2).

Fatty Acids
Fatty acids are essential storage molecules and the starting material for the synthesis of many secondary metabolites [39]. Some long chain fatty acids and their esters ( Figure  3) have been detected in culture extracts of strains of N. vitifusiforme and N. parvum [23,34]. Particularly relevant is the finding of linoleic acid (12) as a product of species of Neofusicoccum isolated from grapevine [23]. In fact, this octadecanoid acid may take part in the biosynthesis of jasmonic acid participating in the signaling pathway during pathogen attack and plant colonization [40].

Melleins
Melleins, also known as 3,4-dihydroisocoumarins, are lactonic natural compounds commonly produced by fungi, bacteria and plants representing a subgroup of the isocoumarins. A large majority of isocoumarins belong to this subgroup characterized by having one carbon substituent at C-3. (-(−)-(R)-Mellein is the founding product of this subgroup first isolated from the fungus Aspergillus melleus (1933) [41]. Several melleins with hydroxyl, methoxyl, alkyl or acetate groups in diverse positions have been subsequently isolated from natural sources [42].
Interestingly, (3R)-5-hydroxymellein (28), isolated from N. parvum, has an effect in the suppression of the oxidation of LDL and HDL through the inhibition of lipid peroxidation, the decrease in negative charges, the reduction in hyperchromicity and carbonyl contents, and the prevention of apolipoprotein A-I (ApoA-I) aggregation and apoB-100 fragmentation. Furthermore, 28 significantly reduced foam cell formation induced by oxidized LDL (oxLDL). These findings show that it could be a potential preventive agent of atherosclerosis via obvious anti-LDL and HDL oxidation and the inhibition of foam cell formation [33].

Myrtucommulones
Myrtucommulone A (32) is the founding product of the group of structurally related compounds named myrtucommulones whose structures are characterized by a phloroglucinol nucleus coupled with syncarpic acid residues ( Figure 5) [28]. Myrtucommulones and related acylphloroglucinols are frequently reported as products of plant species belonging to the family Myrtaceae (e.g., Eucalyptus globulus [43], Kunzea ericoides [44], Myrtus communis [45][46][47], Melaleuca citrina [48], Rhodomyrtus tomentosa [49][50][51]) spread in the Australasian region and these compounds are well-known for their antimicrobial, antioxidant and anti-inflammatory activities [28]. Some endophytic fungi from the genus Neofusicoccum are able to produce myrtucommulones. This finding represents a new frontier in the study of myrtucommulones because the availability of strains to be cultured in vitro may provide access to increased amounts of these products for further investigations. To date, myrtucommulones A, B and D (32)(33)(34) were identified in cultures of endophytic strains of N. australe of myrtle (Myrtus communis) [27,28]. Antiproliferative activity effects on the human prostatic cancer cell lines DU145 and PC3 were observed for a mixture of 32 and 34 [27].
Among the fungal naphthalenones producers, a strain of Botryosphaeria australis (=N. australe) originally obtained from the epidermis of the mangrove plant Sonneratia apetala, is particularly relevant for the capacity to synthesize some new tri-substituted naphthalenones named botryosphaerones A-D (35-38) [29] which showed cytotoxicity against HeLa, HepG-2, and A-549 cells at a concentration of 10 μg mL −1 , and antimicrobial activities against pathogenic bacteria or yeasts at a concentration of 50 μg mL −1 . Subsequently, naphthalenones were isolated from cultures of Neofusicoccum species involved in grapevine diseases. This is the case of a strain of N. australe involved in branch dieback of Juniperus phoenicea in Sardinia (Italy) which produced botryosphaerone D (37)

Naphthoquinones
As can be seen from Table 2, different species of Neofusicoccum show the production of 1,4-naphtoquinones. Natural naphthoquinone derivatives have been widely identified as functional metabolites from various plants, microbes, and marine organisms [55]. For their notable activities, this class of compounds is the prime target of a vigorous research activity focused on the development of new therapeutic agents [56]. Furthermore, naphtoquinones are also relevant because of their bioactivities. In fact, naphthoquinones have a very interesting spectrum of biological activities, including antibiotic, antiviral, anti-inflammatory, antipyretic, antiproliferative and cytotoxic effects, which are related to several mechanisms of action, such as redox cycles, arylation of the thiol groups of proteins, intercalation, induction of breaks in the DNA chain, generation of free radicals and other reactive oxygen species (ROS) and bioreductive alkylation via the formation of quinone methide [57].
Seven naphthoquinones have been isolated from Neofusicoccum species (Figure 7). The most significant structural differences are the presence of hydroxyl, methoxyl, alkyl or acetate groups in different positions on the 1,4-naphthalenoid ring, but these compounds share the hydroxy group at C-5 and the methoxy groups at C-2 and C-7. These groups are all present in the 5-hydroxy-2,7-dimethoxynaphthalene-1,4-dione (50) [25], which was identified as a product of an endophytic strain of B. australis (=N. australe) isolated from the mangrove plant Avicennia marina, together with botryosphaenin (45), a new naphtoquinone. This latter compound and its 6-ethyl analogue (6-ethyl-5-hydroxy-2,7-dimethoxynaphthalene-1,4-dione 47) showed antibiotic and antiproliferative activities with a minimum inhibitory concentration (MIC) ranging from 2 to 32 μg mL −1 when tested on Gram-positive bacteria and an IC50 value from 0.5 to 2 μg mL −1 when tested on different cell lines [25].
As reported above, 6-methylsalicylic acid, together with (−)-terremutin (2), takes part in the biosynthesis of the terric acid [35] and, interestingly, these compounds were both identified as products of a strain of N. parvum isolated from grapevine [21].
The volatile fraction of sesquiterpenes consists mainly of lipophilic compounds and this suggests that their principal targets are cell membranes causing toxic effects due to the loss of osmotic control [64]. Another possibility is that volatile sesquiterpenes facilitate the passage of other toxins through membranes by acting as solvents and synergizing their effects [65].

Miscellaneous
A number of products of Neofusicoccum species are placed in a miscellaneous category ( Figure 10) because they have no structural affinity with previous groups. This is the case of luteoethanones A and B (88-89), two phytotoxic 1-substituted ethanones, identified for the first time from the culture extract of N. luteum [18].
Another interesting compound is (−)-terpestacin isolated from Neofusicoccum batangarum, pathogenic to cactus pear [15]. (−)-Terpestacin has also been isolated from several fungal cultures, in particular from Rutstroemia capillus-albis, the causal agent of bleach blonde syndrome on the Bromus tectorum [70]. In fact, phytotoxic tests showed that this compound is toxic for both host and non-host (tomato) plants confirming its potential role in fungal pathogenesis [15].

Phytotoxicity of Neofusicoccum Metabolites on Grapevine
A large number of species of the family Botryosphaeriaceae have been associated with canker and dieback of grapevines. Grapevine trunk diseases (GTDs) are among the prime causes worldwide of serious damages on vineyards and significant economic losses [71,72]. One of the major concerns in GTDs control is the slow progression of the host xylem colonization by pathogenic fungi or the absence of symptoms for long periods [71,73,74]. In the broad spectrum of GTDs, species of Botryosphaeriaceae are the disease causative agent of the Botryosphaeria dieback [12,58,75] and several of these species belong to genus Neofusicoccum, causing diverse symptoms in infected grapevines, such as leaf chlorosis, bud and wood necrosis, weak spring growth, and vascular cankers primarily in the shape of wedges [72,76,77]. Many symptoms, particularly the foliar ones, can be attributed to the production of toxic secondary metabolites by the fungus [11,78]. Table 3, strains of N. australe, N. luteum, N. parvum, N. vitifusiforme associated with Botryosphaeria dieback produce phytotoxic metabolites. In fact, the phytotoxic activity of some of them has been investigated on leaves, leaf discs or detached leaves of grapevines showing concentration dependent necrosis. As reported by Abou-Mansour et al. [21], (R)-(−)-3-hydroxymellein (25) is the most active mellein tested with 62% leaf disc necrosis. Among cyclohexenones, the compounds with an epoxydic ring are the most active (i.e., cyclobotryoxide (1), (−)-terremutin (2), (+)-epi-sphaeropsidone (6)), while (+)-(6R,7S)-dia-asperlin (8) is responsible for 33% of leaf disc necrosis. Among naphthalenones, the principal phytotoxic compound is botryosphaerone D (38) whose activity is photosensitive [19].
Concerning the potential involvement of phytotoxins in the symptoms expression by grapevines, a simplified model using grapevine cells (calli) from V. vinifera cv. Chardonnay was employed. In this respect (-)-terremutin and mellein are responsible for the expression of some genes involved in the defense mechanisms of grapevine (e.g. cellular detoxification, jasmonic acid pathway, synthesis of secondary metabolites of the phenylpropanoid pathway, flavonoid synthesis), after 1-6 days of exposure on calli [21,22]. Moreover, these findings confirm the presence and the phytotoxic effects of (−)-terramutin and mellein in grapevine wood from plants showing Botryosphaeria dieback symptoms [21,22]. It seems possible that there is a synergistic action between different fungal metabolites, but the different hypotheses still need to be examined.

Conclusions
In this review, the available literature on secondary metabolites produced by Neofusicoccum species has been analyzed. A total of 91 chemically defined compounds from over 20 isolates belonging to 7 species of Neofusicoccum have been reported. These compounds were classified based on their structure in nine groups (i.e., cyclohexenones, 5,6-dihydro-2-pyranones, fatty acids, melleins, myrtocommulones, naphthalenones, naphthoquinones, phenols and alcohols, sesquiterpenes). Furthermore, an additional group of miscellaneous compounds was added in order to gather metabolites that have no structural affinity with compounds present in the reported groups.
An aptitude of members of the Neofusicoccum genus to colonize different plant species, essentially as a latent pathogen, arises from an accurate examination of the existing literature. The pathogenicity and virulence of these fungi seem to be related, at least in part, to the capacity to produce secondary metabolites. Several strains from different species caused similar effects on plants, perhaps due to the production of specific metabolites. In fact, many disease symptoms (e.g., bud necrosis, vascular discoloration of the wood, and perennial cankers) might be caused by metabolites which were detected in cultures of phytopathogenic species of Neofusicoccum. For instance, mellein and its derivatives have been detected in most strains isolated from grapevines showing symptoms of grapevine trunk diseases (GTDs). This evidence is supported by the phytotoxic activity of these compounds on host and non-host plants.
Moreover, metabolites produced by Neofusicoccum species exhibit additional biological activities, including antibacterial, cytotoxic, antiproliferative and indoleamine 2,3-dioxygenase (IDO) inhibitory effects. In this respect, the genus Neofusicoccum can be regarded as a source of bioactive products to be used in diverse biotechnological fields.
The knowledge present in this review can be applied where recommendations for Neofusicoccum disease management strategies are required. In fact, enriching the existing literature with data on secondary metabolites produced by Neofusicoccum isolates from different hosts might be useful for several purposes, such as understanding the resistance mechanisms, screening of diseases, and potential applications of secondary metabolites.

Conflicts of Interest:
The authors declare no conflict of interest.