**6. Secondary Metabolites and Enzymes of Endophytic** *Lasiodiplodia theobromae*

As introduced above, the biological properties of culture extracts of endophytic *L. theobromae* might be linked to the capacity of the fungus to produce bioactive compounds (Figure 2). In fact, *L. theobromae* is a proficient producer of compounds belonging to different classes of secondary metabolites, such as diketopiperazines, indoles, jasmonates, melleins, lactones and phenols [146].

Biotic and abiotic stimuli influence the capacity of *L. theobromae* to grow and produce secondary metabolites, with implications for its physiology, lifestyle and pathogenic aptitude [146–148]. Studies on fungal genomes have shown that the capability of fungi to produce secondary metabolites has been underestimated, because many secondary metabolite biosynthetic gene clusters are silent under standard cultivation conditions [149,150]. In fact, different metabolomic profiles have been reported for *L. theobromae* strains according to variation in growth conditions, with reference to temperature [147,148], nutrient availability [151,152], presence of signal molecules [153] and incubation period [122].

Metabolomic investigations of *L. theobromae* have pointed out that some compounds are produced by endophytic strains only. This is the case of preussomerins and cloropreussomerins, compounds with an unusual structure isolated from the culture extract of a strain from leaves of the mangrove *Acanthus ilicifolius* and characterized for their cytotoxicity against five human cancer cell lines [112]. Moreover, endophytic strains from *Aquilaria sinensis* have been reported to produce 2-(2-phenylethyl)chromones, which are among the most abundant constituents of agarwood [154]. The coumarins meranzine and monocerin could be responsible for the antimicrobial activity of the

culture extract of an endophytic strain from *Dracaena draco*, displaying characteristic inhibition zones against Gram-positive and Gram-negative bacteria [36].

**Figure 2.** Representative secondary metabolites produced by endophytic *Lasiodiplodia theobromae*.

Lasiodiplodins were frequently, although not exclusively, reported as products of endophytic strains of *L. theobromae* [47,69,113]. These macrolides are relevant for a variety of biological properties including cytotoxic, antimicrobial and anti-inflammatory activities [69,155]. Within this class, lasiodiplactone A was obtained from a mangrove endophytic strain showing anti-inflammatory activity [113]. Furthermore, desmethyl-lasiodiplodin was isolated, together with cladospirone B and (-)-mellein, from the crude extract of a strain from leaves of *Vitex pinnata*. Interestingly, cladospirone B and desmethyl-lasiodiplodin showed good activity against *Trypanosoma brucei* [122].

An endophytic strain from the medicinal plant *Bidens pilosa* yielded four depsidones, botryorhodines A-D, and the auxin 3-indolecarboxylic acid, which are not exclusively produced by endophytic strains. Botryorhodines A and B show moderate cytotoxic activity against cervical cancer cells (i.e., HeLa) and antifungal activity against pathogenic fungi, such as *Aspergillus terreus* and *Fusarium oxysporum* [126].

The fact that two of the leading natural products, namely camptothecin and taxol, in cancer chemotherapy were originally extracted from plants is quite interesting from an applicative perspective [144]. The first compound has been detected as a secondary metabolite of strains isolated from the leaves and stem of *Nothapodytes nimmoniana* in the Western Ghats, India [103]. One of these strains (L-6) was investigated in depth with reference to the common phenomenon of attenuation of bioactive

metabolite production in axenic cultures. It was found that its re-inoculation in the host promoted higher production of camptothecin, indicating that the fungus receives eliciting signals from the host tissues, or some factors which prevent silencing of the genes responsible for biosynthesis [156].

Taxol, the first billion-dollar natural antitumor product [157], has been reported as a secondary metabolite of several endophytic strains of *L. theobromae*, from *Taxus baccata*, *Morinda citrifolia*, *Salacia oblonga* and *Piper nigrum* [32,35,57,110]. Investigational activity carried out on the product extracted from these strains pointed out its ability to counteract the carcinogenic effects of dimethylbenzanthracene [158]. Moreover, valuable studies have disclosed the capacity by non-*Taxus* endophytic strains to produce the compound through a similar biosynthetic pathway as the one reported from the plant. In fact, the gene encoding 10-deacetylbaccatin-III-*O*-acetyltransferase, as well as the open reading frame of WRKY1 transcription factor, were cloned and sequenced and found to share high similarity with deposited sequences from *Taxus chinensis*, *T. cuspidata* and *T. celebica* [35].

Of great interest in endophytic *L. theobromae* is the production of phytohormones, such as indole derivatives and jasmonic acid analogues [146]. It is known that 3-indoleacetic acid and 3-indolecarboxylic acid are the most studied auxins regulating plant growth and development. These compounds have been frequently reported as fungal metabolites [144] and have also been documented as being produced by *L. theobromae* strains. The biological role of 3-indolecarboxylic acid has not been fully investigated, but some studies address its biosynthesis [159–161] and toxicity [147]. Several *L. theobromae* strains with different lifestyles are in vitro producers of jasmonic acid and analogues. Jasmonic acid is one of the most important signal molecules involved in several plant processes including seed germination, senescence and blooming. Hence, investigations of the bioactive properties of jasmonic acid and related compounds are essentially focused on their role in the interaction between host and pathogen.

The great ability of adaptation to different environments, the capacity to colonize a high number of hosts and the expression of high amounts of extracellular enzymes make *L. theobromae* a producer of relevant enzymes (Table 2) to be considered for biotechnological applications [162]. The most recognized extracellular enzymes used to penetrate the plant host include cellulases, proteases and lipases. Endophytic strains colonizing *C. nucifera*, *Pongamia pinnata* and *A. indica* exhibited great lipase activity [43]. Moreover, endophytic strains from *Terminalia catappa* and *T. mantaly* were found to produce amylases and cellulases [86]. Finally, *L. theobromae* isolated from *Psychotria flavida* turned out to be able to degrade irradiated polypropylene thanks to the production of laccases [62].
