The citrus industry is severely affected by citrus black spot (CBS), a disease caused by the pathogen Phyllosticta citricarpa. This disease causes loss of production, decrease in the market price of the fruit, and reduction in its export to the European Union. Currently, CBS disease is being treated in orchards with various pesticides and fungicides every year. One alternative to CBS disease control without harming the environment is the use of microorganisms for biological control. Diaporthe endophytica and D. terebinthifolii, isolated from the medicinal plants Maytenus ilicifolia and Schinus terebinthifolius have an inhibitory effect against P. citricarpa in vitro and in detached fruits. Moreover, D. endophytica and D. terebinthifolii were transformed by Agrobacterium tumefaciens for in vivo studies. The transformants retained the ability to control of phytopathogenic fungus P. citricarpa after transformation process. Furthermore, D. endophytica and D. terebinthifolii were able to infect and colonize citrus plants, which is confirmed by reisolation of transformants from inoculated and uninoculated leaves. Light microscopic analysis showed fungus mycelium colonizing intercellular region and oil glands of citrus, suggesting that these two new species are capable of colonizing citrus plants, in addition to controlling the pathogen P. citricarpa.

Endophytic actinomycetes from medicinal plants produce a wide diversity of secondary metabolites (SM). However, to date, the knowledge about endophytes from Brazil remains scarce. Thus, we analyzed the antimicrobial potential of 10 actinomycetes isolated from the medicinal plant Vochysia divergens located in the Pantanal sul-mato-grossense, an unexplored wetland in Brazil. Strains were classified as belonging to the Aeromicrobium, Actinomadura, Microbacterium, Microbispora, Micrococcus, Sphaerisporangium, Streptomyces, and Williamsia genera, through morphological and 16S rRNA phylogenetic analyzes. A susceptibility analysis demonstrated that the strains were largely resistant to the antibiotics oxacillin and nalidixic acid. Additionally, different culture media (SG and R5A), and temperatures (28 and 36°C) were evaluated to select the best culture conditions to produce the active SM. All conditions were analyzed for active metabolites, and the best antibacterial activity was observed from metabolites produced with SG medium at 36°C. The LGMB491 (close related to Aeromicrobium ponti) extract showed the highest activity against methicillin-resistant Staphylococcus aureus (MRSA), with a MIC of 0.04 mg/mL, and it was selected for SM identification. Strain LGMB491 produced 1-acetyl-β-carboline (1), indole-3-carbaldehyde (2), 3-(hydroxyacetyl)-indole (4), brevianamide F (5), and cyclo-(L-Pro-L-Phe) (6) as major compounds with antibacterial activity. In this study, we add to the knowledge about the endophytic community from the medicinal plant V. divergens and report the isolation of rare actinomycetes that produce highly active metabolites.

Medicinal plants have been recognized as hosts of high diverse endophytic microorganisms, including fungi that produce secondary metabolites with biological activity. Two biomes in Brazil, Pantanal (wetland), and Cerrado (savannah), are known as biodiversity hotspots, and despite their importance as a reservoir for several species, knowledge about the fungal biodiversity in these biomes is very limited. Fungal endophytic communities associated with leaves and petioles of the medicinal plants Vochysia divergens (from Pantanal) and Stryphnodendron adstringens (from Cerrado) were analyzed and studied for their antimicrobial activity against human and plant pathogens. A total of 1,146 isolates of endophytic fungi were obtained from plants collected in January and June of 2016 and grouped into 124 morphotypes. One isolate of each morphotype was identified by sequencing of internal transcribed spacer (ITS) region of the rDNA gene, which revealed the presence of 24 genera, including 3 possible new genera, and 48 taxa. Differences in the endophytic community according to the biomes were observed concerning the analyzed morphotypes. However, when we analyzed the diversity of genera and richness, they were similar for both plants, with Diaporthe, Phyllosticta, and Neofusicoccum as dominant genera. In addition, the community composition of V. divergens differs according to the analyzed plant tissues (petiole and leaf). These data suggested that both, the plant species and plant tissues play a role in the composition of endophytic community. As regards the biotechnological potential, 5 isolates showed activity against the phytopathogens Phyllosticta citricarpa, Colletotrichum abscissum, and Fusarium verticilioides, and 8 isolates showed high activity against clinical pathogens and were selected for the production of crude extract in different culture media. Extract from cultivation of Diaporthe sp. LGMF1548 and LGMF1583 and Neofusicoccum brasiliense LGMF1535 showed activity against methicillin-resistant Staphylococcus aureus, Klebssiella pneumonia, and Candida albicans. In addition, extracts of Diaporthe cf. heveae LGMF1631 inhibited 90% of the mycelial growth of the P. citricarpa and 70% of C. abscissum and may represent an alternative to be used in the biological control of these phytopathogens. Future research will focus on the chemical characterization and structural elucidation of these bioactive compounds.

Endophytic actinomycetes encompass bacterial groups that are well known for the production of a diverse range of secondary metabolites. Vochysia divergens is a medicinal plant, common in the “Pantanal” region (Brazil) and was focus of many investigations, but never regarding its community of endophytic symbionts. During a screening program, an endophytic strain isolated from the V. divergens, was investigated for its potential to show biological activity. The strain was characterized as Microbispora sp. LGMB259 by spore morphology and molecular analyze using nucleotide sequence of the 16S rRNA gene. Strain LGMB259 was cultivated in R5A medium producing metabolites with significant antibacterial activity. The strain produced 4 chemically related β-carbolines, and 3 Indoles. Compound 1-Vinyl-β-carboline-3-carboxylic acid displayed potent activity against the Gram-positive bacterial strains Micrococcus luteus NRRL B-2618 and Kocuria rosea B-1106, and was highly active against two human cancer cell lines, namely the prostate cancer cell line PC3 and the non-small-cell lung carcinoma cell line A549, with IC50 values of 9.45 and 24.67 µM, respectively. 1-Vinyl-β-carboline-3-carboxylic acid also showed moderate activity against the yeast Saccharomyces cerevisiae ATCC204508, as well as the phytopathogenic fungiPhyllosticta citricarpa LGMB06 and Colletotrichum gloeosporioides FDC83.