**4. Discussion**

Several mechanisms have been proposed to control *G. boninense* causing BSR disease in oil palm trees. However, none of them have successfully been treated or been shown to suppress the disease [9]. The search for antifungal alternatives is representative of a potential solution that has drawn significant interest. In this study, new compounds were identified during the isolation of anti-*Ganoderma* substances from *S. palmae* CMU-AB204T. The assessment of antimicrobial activity of four new phenyl alkenoic acids showed that AB204-A (**1**) and B (**2**) mildly inhibited the growth of fungi, *G. boninense* BCC 21330, *Mu. racemosus* IFO 4581, and *A. niger* ATCC 6275, while AB204-E (**5**) and F (**6**) displayed a positive degree of activity against Gram-positive bacteria, *B. subtilis* ATCC 6633, *K. rhizophila* ATCC 9341, *M. smegmatis* ATCC 607, and *S. aureus* ATCC 6538p. New antifungal compounds, AB204-A (**1**) and B (**2**), possessed similar structures to phenylethyl alcohol (PEA), an antifungal aromatic compound that was obtained from *Trichoderma virens* 7b, which had significant potential as a biological control agent for BSR [43]. These compounds may exhibit a mechanism in inhibiting fungi similar to PEA which inhibits protein, DNA, RNA, and aminoacyl tRNA syntheses of fungi [44,45]. However, a mode of action of novel compounds in controlling fungi should be confirmed in the future.

AB204-B (**2**) contained more C2H4 units than AB204-A (**1**), but it displayed antimicrobial activity against the same pathogenic strains with similar inhibition zone sizes. This result indicated that the presence of a longer chain of the carboxylic group in **2** had not been involved in the antimicrobial activity. Biological activities of the *E*-isomer of AB204-A (**1**), (*E*)-5-(2-methylphenyl)-4-pentenoic acid was previously reported to be an inactive compound against bacteria and fungi but the tested concentration and the strain of tested microorganisms have not been indicated [33]. However, the difference of an antimicrobial activity between (*E*)-5-(2-methylphenyl)-4-pentenoic acid and new compounds, AB204-A (**1**) and AB204-B (**2**), revealed that the existence of *Z*-olefin in **1** and **2** had been involved in their antifungal activity. The structures of a mixture of AB204-C (**3**) and AB204-D (**4**) were predicted based on HREI-MS and 1H NMR spectra. In the future, the mixture should be reseparated using other techniques, and additional data is needed to confirm their structures and antimicrobial activities. AB204-E (**5**) and F (**6**) have an 1,2-*Z*-heptene moiety connected to the aromatic ring. These metabolites have not shown antifungal activity but exhibited strong antibacterial activity when associated with this moiety. Moreover, the existence of one pair of *Z*-olefin in the chain of the carboxylic group of AB204-E (**5**), instead of the *E* and *Z*-olefins of AB204-F (**6**), increased the antibacterial activity of this compound.

Previously, Thong et al. [35] found two closely related compounds of **1**–**4**, and they were isolated from a *Streptomyces* that had been spontaneously acquired rifampicin resistance. These compounds contained *E*-olefins and have both a methylbenzene unit and a 2-amino-3-hydroxycyclopent-2-enone (C5N) moiety. The phenyl alkenoic acid-associated metabolites discovered by Thong et al. did not display antibacterial activity against *E. coli*, *M. luteus*, *S. aureus*, and *B. subtilis* in testing with a microplate assay at 100 μM or approximately 28.5 and 33.5 mg/mL, thus revealing similar results to AB204-A (**1**) and B (**2**). Notably, the presence of a carboxylic acid moiety in the novel compounds, and a C5N moiety in the known compounds, may not be involved in the antimicrobial activity. Based on draft genome sequences of the rifampicin-resistant mutant (TW-R50–13), the methylbenzene moiety may be biosynthesized by the expression of polyketide synthase (PKS) genes that are located at a different locus from the biosynthetic genes for the C5N moiety [35]. The genes encoding for PKS have been disclosed to complex biosynthetic mechanisms, which were involved in the production of many metabolites in microorganisms [46]. Genome sequences would provide the data of potential gene clusters to understand the metabolic pathways of *S. palmae* CMU-AB204T. Thus, the genome sequences of this strain should be further studied to determine the presence of both silent and cryptic secondary metabolite biosynthetic gene clusters that are able to synthesize the corresponding novel natural products.

In addition to **1** and **2**, other antifungal compounds, anguinomycin A (**7**), leptomycin A (**8**), and actinopyrone A (**9**) obtained from the same broth of *S. palmae* CMU-AB204T, also displayed anti*-Ganoderma* activity. The ability of *S. palmae* to produce a variety of antifungal compounds was proven. This strain might produce each antifungal secondary metabolite depending on the prevailing environmental conditions, such as nutritional source, incubation period, pH value, and temperature [47,48]. Hence, the optimization of culture conditions should be studied in order to obtain high yields of the antifungal metabolites. The protecting effect of *S. palmae* CMU-AB204<sup>T</sup> against BSR has also been confirmed in a glasshouse experiment [49]. The results obtained from this study strongly suggest that the antimicrobial secondary metabolites were involved in the mechanism exhibiting anti-BSR effects by this *Streptomyces* strain.

Although the new compounds obtained in this study showed moderate activity towards *G. boninense*, they inhibited clinical bacterial pathogens and other phytopathogenic fungi, suggesting a possible utility of the four new antimicrobial substances in both agricultural and medical treatments. However, cytotoxicity to mammalian cell of both new compounds and three known compounds should be tested before being applied to these compounds. The recovery of novel actinomycetes species, especially the genus *Streptomyces*, has the potential to be a rich source of both new and known natural products [50,51]. Notably, *S. palmae* CMU-AB204<sup>T</sup> was found to produce various bioactive metabolites.
