*Agriculture* **2020**, *10*, 286

**Table 1.** *Cont.*

70


 in last column are linked to endophyte genera, activities, and metabolites.

#### *Agriculture* **2020** , *10*, 286

#### **4. Fungal Endophytes Associated with Asteraceae—Biochemistry**

#### *4.1. Plant Growth Promoting Secondary and Anti-Stress Metabolites*

Asteraceae are leading examples of the synergistic effect of fungal endophytes in improving biotic and abiotic stress resistance and promoting plant growth because numerous species of this family possess extraordinary tolerance and competition skills. For example, Khan et al. [115] determined the growth-promoting ability of endophytic *Penicillium citrinum* in helping its plant host *Ixeris repens* in rapid colonization of the sand dunes. *P. citrinum* stimulated competition skills of the host plant through the production of secondary metabolites promoting plant growth, like gibberellins, and protective compounds, like mycotoxins, citrinin, and cellulose digesting enzymes [115]. *P. citrinum* and *Aspergillus terreus* were found to stimulate *H. annuus* growth and improve disease resistance due to the higher content of plant-defense hormones, salicylic, and jasmonic acids. The mentioned endophytes regulated oxidative stress of the host plant through activation of glutathione and polyphenol oxidases, alteration of catalase and peroxidase, as well as secretion of organic acids [88]. The individual or co-inoculation of endophytes increased amino acid content in sunflower (*H. annuus*) diseased leaves, delaying cell death, and consequently disturbing pathogen progression in plant tissues [88]. Ren et al. [75] showed that endophyte *Gilmaniella* sp. induced jasmonic acid production, which was recognized to be a signal compound promoting the accumulation of volatile oils in the Chinese medicinal plant *Atractylodes lancea*. The jasmonic acid acted as a downstream signal of nitric oxide and hydrogen peroxide-mediated production of volatile oil in the host. Various strains of *Penicillium* and *Aspergillus* species associated with Asteraceae were reported for gibberellins production [116]. *Penicillium* strains, especially MH7, produced nine gibberellins which significantly increased the growth and development of the host plant crown daisy (*Chrysanthemum coronarium*, synonym of *Glebionis coronaria*) [117]. The reactive oxygen species (ROS) production together with increased siderophore excretion by endophytes contributed towards improved growth and resistance against sunflower pathogens. Endophyte-origin ROS in plant roots are tackled by internal physiological plant apparatus resulting in an acute resistance against present and future stresses [89]. Huang et al. [58] compared the antioxidant capacity of plants used in Chinese traditional medicine, including mugworts: *Artemisia capillaris*, *A. indica*, and *A. lactiflora* (Asteraceae) and their endophytes. A fungal endophyte strain isolated from the flower of *A. capillaris* showed the strongest total antioxidant capacity. The antioxidant compounds detected in the highest amounts in both endophytic fungus and its host *A. indica* were chlorogenic and di-O-caffeoylquinic acids, and the volatile compound artemisinin. Both chlorogenic acid and artemisinin acted as antioxidant, antimutagenic, immunomodulatory, and antiviral. The production of the same bioactive natural compounds, as well as some of those found in *A. indica* and its fungal endophytes, was suggested. In general, phenolic compounds, including phenolic acids, flavonoids, tannin constituents, hydroxyanthraquinones, and phenolic terpenoids as well as volatile or aliphatic constituents were major substances in the fungal endophyte cultures and host plant extracts responsible for high antioxidant activity of all investigated Chinese medicinal plants [58]. In terms of abiotic and biotic stress, fungal endophytes conferred resistance against drought, salinity, heat stress, and enhanced resistance against pathogens and insects. The different mechanisms can stay behind the competitive success of invasive Asteraceae species like crofton weed (*A. adenophora*). The most abundant endophytic fungus isolated from this species was *Colletotrichum* sp. which has pathogenic effects on other plants. Spreading *Colletotrichum* spores could be a competitive advantage for *A. adenophora* as it was hypothesized by Fang et al. [62]. The recognition of endophyte roles in host plant expansion and competition mechanisms enables the application or modification of cultivation techniques dedicated to particular medicinal Asteraceae species, especially those with promising therapeutic and economical potential.

#### *4.2. Antibacterial Secondary Metabolites*

The best criterion for host plant selection in order to investigate the endophytes with potential antimicrobial activity is the plant traditionally used for the treatment of infections [118]. Plant-associated fungi may interact using, inter alia, antibiotic molecules, so the production of antibiotics and the parallel development of antibiotic-resistance mechanisms can spread in dynamic microbiota/plant systems by bacterial mobilization and horizontal gene transfer [119,120]. In recent years, the number of multidrug-resistant microorganisms have been a growing concern for public health worldwide. The key determinants of bacteria drug resistance are inactivation of the antibiotics, changes in bacterial targets, and restricted entry of antibiotics by less permeable drug transporters [121]. Asteraceae/fungal endophytes consortia could be a source of active compounds targeted against many drug-resistant microorganisms [122,123]. A fungus *Colletotrichum* sp. was isolated from the stems of *Artemisia annua* and characterized as a source of ergosterol derivatives (Figure 3), with inhibitory potential against both Gram-negative and -positive bacteria, such as *Pseudomonas* sp. and *Bacillus subtilis* with minimal inhibitory concentrations (MICs) ranging from 25 to 75 g mL−<sup>1</sup> [70]. *Colletotrichum* sp. can also produce plant hormones such as indole-3-acetic acid (IAA), up-regulating host growth. Both mechanisms of action, namely antibiosis and growth promotion, can enhance adaptability and pathogen resistance of a host plant. At the same time, Zou et al. [44] isolated from the stem of *Artemisia mongolica* an endophytic fungus *Colletotrichum gloeosporioides*, synthesizing colletotric acid with antibacterial activity against *B. subtilis*, *Staphylococcus aureus*, *Sarcina lutea*, and *Pseudomonas* sp. with MICs of 25, 50, and 50 μg mL<sup>−</sup>1, respectively, and inhibited a pathogenic fungus *Helminthosporium sativum* (current name *Bipolaris sorokiniana*) with a MIC of 50 μg mL−1. This was the first report of *C. gloeosporioides* as a fungal endophyte in the Asteraceae, although it was previously mentioned as an endophyte of plants belonging to the other families. The isocoumarins and naphthalene derivatives produced by *Papulaspora immersa*, a fungal endophyte isolated from the Andean tuber crop, the yacon (*S. sonchifolius*), presented antimicrobial activities and could act synergistically [99]. Interestingly, some fungal metabolites were identified as constituents of an extract derived from a healthy Asteraceae, prickly goldenfleece (*Urospermum picroides*), indicating that the production of bactericides by the fungal endophyte *Ampelomyces* sp., proceeds also in situ within the host plant [110]. Among seven phomosine derivatives isolated from *Phomopsis* sp., an endophyte of the Syrian thistle (*Notobasis syriaca*), phomosine K had strong antibacterial activity against *Legionella pneumophila* Corby, *Escherichia coli* K12 with MIC 25 and 100 μg mL<sup>−</sup>1, respectively [96]. Endophyte colonization offers protection from various stressors, such as toxins which affect plant pathogens by disrupting the cellular membrane and inducing cell death. Such ecological relationships were recorded for the mentioned Asteraceae/endophyte systems.

2+ 2+ 2

hydroxycyclohex-2-en

(+)-epiepoxydon (+)-epoxydon monoacetate (−)-phyllostine 2-hydroxymethyl-4β,5α,6β-tri

2

+

2+ (3R,4R)-4-hydroxymellein (3R)-5-hydroxymellein (4S)-4,8-dihydroxy-α-tetralone 2+ 2+ 2+ 2 


6,8-dihydroxy-3-methylisocoumarin Naphthalene-1,8-diol 2,3-epoxy-1,2,3,4-tetrahydronaphthalene

6-isoprenylindole-3-carboxylic acid

2

2+ +2

2

2

2+ 2

3β,5α-dihydroxy-6β-acetoxy-ergosta-7,22-diene (R = OCOCH3) 3β,5α-dihydroxy-6β-phenylacetyloxy-ergosta-7,22-diene (R = OCOCH2C6H5)

Colletotric acid (+AF) Phomosine K

**Figure 3.** The molecular structure of chosen specific compounds with antibacterial activity synthesized by fungal endophytes associated with Asteraceae species [44,70,96,99,110]; +AF—antifungal activity; +CA—cytotoxic activity.

2

2+

## *4.3. Antifungal Secondary Metabolites*

Colonization of the host plant by endophytes and pathogens depends on their adaptations to the host environment but also the innate host defense mechanism and variation in virulence. A few reports on endophytic fungi, protecting against other fungal infection, found in association with Asteraceae species, especially *Viguiera* spp. (syn. *Aldama* spp.) were published, and several new compounds were described but their biological action needs future research [17,22,68,75,78,101]. *Ampelomyces* spp. were widely studied as the first fungi used as biocontrol agents of powdery mildews [122]. Chagas et al. [100] investigated the interactions between the fungal endophytes that cohabit *S. sonchifolius*. They found that *Alternaria tenuissima* synthesized some polyketides, including antifungal stemphyperylenol in the presence of endophytic *Nigrospora sphaerica* (Figure 4)*. A. tenuissima* is characterized by a slower growth rate than *N. sphaerica*, so specific antifungal compounds might control the growth rate of *N. sphaerica* during host plant colonization, without any damage to the host plant tissues. The competition of fungal endophytes colonizing the same host plant stimulates the production of metabolites that could decrease the growth of particular fungi species without damaging the host plant and maintaining the symbiosis [100]. A closer metabolome relationship was found for *S. sonchifolius* and endophytic fungus *Coniochaeta ligniaria*. Both symbionts produced the same antifungal fatty acids: caproic, caprylic, and palmitic acids at high concentrations which might raise the resistance of *S. sonchifolius* to fungal pathogenic attacks and *C. ligniaria* to fungi competing within the host tissues [101]. *B. trimera* is a native medicinal plant of the Brazilian savannah. Vieira et al. [53] isolated from the leaves of this species 23 fungal taxa, inter alia, *Epicoccum* sp., *Pestalotiopsis* sp., *Cochliobolus lunatus*, and *Nigrospora* sp., which showed antifungal activity against *Paracoccidioides brasiliensis.* Additionally, the fungi isolated from different host plants displayed distinct antimicrobial activities, so the endophytic richness and the antimicrobial activity were closely correlated. The endophyte fungus *Preussia* sp. revealed strong antifungal activity, related to the synthesis of anthraquinones, auranticins, culpin, cycloartane triterpenes diphenyl ether, spirobisnaphthalenes, and thiopyranchromenones [53,124]. However, metabolome analysis of *Preussia* sp. isolated from Asteraceae herb carqueja (*B. trimera*) confirmed antioxidant but not antifungal activity of isolated compounds, namely preussidone, 1 ,5-dimethoxy-3,5 -dimethyl-2,3 -oxybiphenyl-1,2 -diol, 5-methoxy-3,5 -dimethyl-2,3 -oxybiphenyl-1,1 ,2 -triol, and cyperin [124]. Waqas et al. [88,89] determined the inhibitory effect of two fungal endophytes, *P. citrinum* and *A. terreus*, against *Sclerotium rolfsii*, a soilborne plant pathogen which causes root rot, stem rot, collar rot, wilt, and foot rot diseases in *H. annuus*. The antifungal activity of *Penicillium* and *Aspergillus* strains was linked with synthesis of gibberelins, organic acids, and siderophores. Two new fatty acid amides, bipolamides A and B, were isolated from endophytic fungus *Bipolaris* sp., but only bipoliamide B revealed bioactivity against *Cladosporium cladosporioides*, *C. cucumerinum*, *Saccharomyces cerevisiae*, *Aspergillus niger*, and *Rhisopus oryzae* [85]. Fungal endophytes possess multiple balanced antagonisms, namely with the other microbial inhabitants of the host plant and with the host plant itself, to support the growth conditions enabling reproduction. Most genes involved in secondary metabolite synthesis in fungi are activated while being co-cultured in plant and/or with other microbes, but they are generally silent in cultures, confirming that multiple antagonisms are involved in endophytism [22]. Three strains of endophytic fungus *Diaporthe citri* isolated from Brazilian medicinal vine, guaco (*Mikania glomerata*) presented 60% inhibition index of mycelia growth against *Fusarium solani* and 66% against *Didymella bryoniae* [94]. The mechanisms of inhibition were not tested in the cited reference, but the authors stated that endophytic microorganisms with the highest inhibition indices were considered candidates for tests involving the production of secondary metabolites with potential antimicrobial activity.

**Figure 4.** The molecular structure of chosen specific compounds with antifungal activity synthesized by fungal endophytes associated with Asteraceae species [85,100].
