Daiani Cristina Savi

Diaporthe endophytica and D. terebinthifolii from medicinal plants for biological control of Phyllosticta citricarpa

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.

Antibacterial Activity of Endophytic Actinomycetes Isolated from the Medicinal Plant Vochysia divergens (Pantanal, Brazil)

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.

MAT gene idiomorphs suggest a heterothallic sexual cycle in the citrus pathogen Phyllosticta citricarpa

Sexual reproduction in fungi is controlled by mating type genes, which are located at the MAT locus. In this study, we investigated the structure of this locus in the phytopathogenic fungus Phyllosticta citricarpa, the causal agent of citrus black spot disease. Despite intensive study, its sexual state has never been observed in single-spore culture. Through analysis of the genome sequences of two individual P. citricarpa isolates, the sequence of the DNA lyase gene was identified and, as previously reported in the literature, the mating type genes were located in the 3′ flanking region of this gene. The results suggested that P. citricarpa is heterothallic, owing to the exclusive presence of the MAT1–1 or MAT1–2 gene in individual strains. In order to characterize the MAT locus, we designed primers to amplify this region. P. citricarpa was found to have complete and apparently functional copies of MAT genes, containing α-1 and HMG domains, present in different isolates. In addition to MAT1–2-1 and MAT1–1-1 genes, the MAT1–1-4 gene was located in the 5′ flanking region of the MAT1–1-1 gene and the MAT1–2-5 gene was located in 5′ flanking region of the MAT1–2-1 gene. A multiplex PCR protocol was also developed to differentiate P. citricarpa idiomorphs, which can be used in distribution and incidence studies of mating type strains, in order to determine the occurrence of sexual reproduction and to facility crossing studies. Furthermore, in Brazil, the two idiomorphs occur in a 1:1 ratio, which is expected in sexually reproducing populations.

Biological activity of Diaporthe terebinthifolii extracts against Phyllosticta citricarpa

Abstract Citrus black spot disease, caused by the phytopathogen Phyllosticta citricarpa, depreciates the market value of citrus fruits and prevents their exportation to disease‐free regions. It may also reduce the productivity of citrus fruit orchards. To identify an alternative to conventional disease control measures, isolates of Diaporthe terebinthifolii, active against P. citricarpa, were selected from an endophytic fungal population of Schinus terebinthifolia leaves. Different culture media were screened to identify the culture medium that afforded the most efficient production of biologically active extracts. A particular fraction (fraction VI) of the extract completely protected orange leaves by inhibiting the germination of P. citricarpa conidia with a minimum inhibitory concentration of 0.003 &mgr;g.mL−1. The active constituents in D. terebinthifolii extract fractions were identified by gas chromatography coupled to mass spectrometry as verbanol, phenylethyl alcohol, verbenyl acetate and methyl hexadecanoate. The results obtained strongly suggest the existence of a synergistic effect among the metabolites produced. Thus, these fungal metabolites could be used to control the CBS disease. As the asexual spores of P. citricarpa play an important role in fruit lesion development and disease dispersion, fungal extracts that inhibit the spore germination can be used as an effective alternative for directional disease control.

Identification of Colletotrichum species associated with postbloom fruit drop in Brazil through GAPDH sequencing analysis and multiplex PCR

The genus Colletotrichum comprises a group of important fungal pathogens that can infect a wide variety of host plants worldwide. Postbloom fruit drop (PFD) of citrus plants is responsible for extensive crop losses annually, and is particularly detrimental to Brazilian citrus production. The disease was first associated with Colletotrichum gloeosporioides and subsequently linked to Colletotrichum acutatum. However, a new species, C. abscissum, was described in 2015 as the causative agent of PFD in Brazil. The species description used a small number of strains and the distribution of the pathogen remains unclear. The proportion of PFD caused by this species is also unclear, because both C. abscissum and C. gloeosporioides are associated with the disease as well. By analyzing sequences of the GAPDH intron region, we identified 227 isolates of Colletotrichum associated with PFD in orchards of São Paulo state, 172 isolates were identified as C. abscissum and 55 as C. gloeosporioides. Morphological characters and multilocus sequencing confirmed species C. abscissum was the only species in the C. acutatum complex associated with PFD disease in Brazil. Although described as sensitive to benzimidazole-based fungicides, 20% of C. gloeosporioides isolates were found in regions with high use of this class of fungicide. Evaluated strains exhibited resistance to this fungicide in vitro. Because previously described primers differentiate between C. acutatum and C. gloeosporioides complexes, but not the particular species associated with PFD, we proposed and validated primers for a single multiplex PCR that specifically distinguished the C. abscissum and C. gloeosporioides sensu stricto.

Identification of genes associated with asexual reproduction in Phyllosticta citricarpa mutants obtained through Agrobacterium tumefaciens transformation.

Phyllosticta citricarpa is the epidemiological agent of Citrus Black Spot (CBS) disease, which is responsible for large economic losses worldwide. CBS is characterized by the presence of spores (pycnidiospores) in dark lesions of fruit, which are also responsible for short distance dispersal of the disease. The identification of genes involved in asexual reproduction of P. citricarpa can be an alternative for directional disease control. We analyzed a library of mutants obtained through Agrobacterium tumefaciens transformation system, looking for alterations in growth and reproductive structure formation. Two mutant strains were found to have lost the ability to form pycnidia. The flanking T-DNA insertion regions were identified on P. citricarpa genome by using blast analysis and further gene prediction. The predicted genes containing the T-DNA insertions were identified as Spindle Poison Sensitivity Scp3, Ion Transport protein, and Cullin Binding proteins. The Ion Transport and Cullin Binding proteins are known to be correlated with sexual and asexual reproduction in fungi; however, the exact mechanism by which these proteins act on spore formation in P. citricarpa needs to be better characterized. The Scp3 proteins are suggested here for the first time as being associated with asexual reproduction in fungus. This protein is associated with microtubule formation, and as microtubules play an essential role as spindle machinery for chromosome segregation and cytokinesis, insertions in this gene can lead to abnormal formations, such as that observed here in P. citricarpa. We suggest these genes as new targets for fungicide development and CBS disease control, by iRNA.

Analysis of the genetic diversity of Candida isolates obtained from diabetic patients and kidney transplant recipients

Yeasts of the genus Candida have high genetic variability and are the most common opportunistic pathogenic fungi in humans. In this study, we evaluated the genetic diversity among 120 isolates of Candida spp. obtained from diabetic patients, kidney transplant recipients and patients without any immune deficiencies from Paraná state, Brazil. The analysis was performed using the ITS1-5.8S-ITS2 region and a partial sequence of 28S rDNA. In the phylogenetic analysis, we observed a consistent separation of the species C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. parapsilosis, C. metapsilosis and C. orthopsilosis, however with low intraspecific variability. In the analysis of the C. albicans species, two clades were formed. Clade A included the largest number of isolates (91.2%) and the majority of isolates from GenBank (71.4%). The phylogenetic analysis showed low intraspecific genetic diversity, and the genetic polymorphisms between C. albicans isolates were similar to genetic divergence found in other studies performed with isolates from Brazil. This low genetic diversity of isolates can be explained by the geographic proximity of the patients evaluated. It was observed that yeast colonisation was highest in renal transplant recipients and diabetic patients and that C. albicans was the species most frequently isolated.

Bioprospecting and Structure of Fungal Endophyte Communities Found in the Brazilian Biomes, Pantanal, and Cerrado

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.

Antagonistic Activity and Agrotransformation of Xylaria cubensis, Isolated from the Medicinal Plant Maytenus ilicifolia, Against Phyllosticta citricarpa

Background: Endophytic fungi isolated from medicinal plants have been recognized as promising sources of highly variable bioactive metabolites. Maytenus ilicifolia, a highly exploited medicinal plant commonly used in popular medicine, is included in the current list of endangered species. Our research group is particularly interested in bioprospecting endophytes from medicinal plants found in Brazil, to control citrus diseases. In this context, Phyllosticta citricarpa is an agronomically important fungus associated with Citrus Black Spot (CBS), a disease associated with large economic losses, mainly due the phytosanitary restrictions for CBS disease in Europe. Objective: The objective of this study was to identify and explore the activity of two endophytic isolates from the medicinal plant M. ilicifolia against P. citricarpa, and to develop a reliable and efficient transformation system for the endophytic strains. Methods: During a screening program on the biodiversity of M. ilicifolia, endophytic strains LGMF1252 and LGMF1253 were isolated and selected for bioprospecting study. The secondary metabolites from LGMF1252 and LGMF1253 were obtained by fermentation in Malt Extract Agar (MEA), and extracted with EtOAC. The antagonistic property was evaluated against mycelial growth and pycnidia formation of the phytopathogen P. citricarpa. A multi-locus sequence analysis using ITS, βtubulin, and α-actin genes was performed to identify the endophytes LGMF1252 and LGMF1253. Based on these results, the isolate LGMF1252 was transformed via Agrobacterium tumefaciens, with GFP and BAR expression cassettes, using a binary plasmid vector based on pPZP201BK. Results: The secondary metabolites from LGMF1252 and LGMF1253 showed antagonistic properties against mycelial growth and pycnidia formation of P. citricarpa. Multi-locus sequence analysis revealed that the endophytic strains belong to the Xylaria cubensis aggregate. The isolate LGMF1252 was successfully transformed via A. tumefaciens. Conclusion: The inhibition of pycnidia formation can be an alternative for CBS control, considering that there is no effective treatment for the disease and asexual spores play an important role in the development of fruits lesions. Due to the high activity observed, strain LGMF1252 was transformed via A. tumefaciens. The results of this study will facilitate future studies on plant interaction and biological control. A R T I C L E H I S T O R Y Received: January 17, 2016 Revised: November 29, 2016 Accepted: January 11, 2017 DOI: 10.2174/2211550106666170210121140

Colletotrichum gloeosporioides sensu stricto: an endophytic species or citrus pathogen in Brazil?

Postbloom fruit drop (PFD) was first associated with Colletotrichum gloeosporioides until 1990s. Thereafter, the causal agent was reclassified as a species belonging to C. acutatum complex. Recent findings suggested that among the species, within the C. acutatum complex, C. abscissum is the PFD causal agent. Moreover, previous study reported C. abscissum as the most frequent species among isolates of Colletotrichum from blooms showing PFD symptoms. Despite the recent increase in knowledge concern PFD epidemiology, strategies of pathogen survival in citrus leaves remain unclear. Therefore, the aim of this study was to identify the Colletotrichum endophytic species obtained from leaves of sweet orange and weeds from orchards located in Sao Paulo state, Brazil, and to evaluate the ability of these isolates to cause typical PFC symptoms on flowers. Species-specific PCR was used to identify 188 isolates belonging to the C. gloeosporioides complex, which were characterized as C. gloeosporioides sensu stricto based on multilocus sequence analysis. The pathogenicity test indicated that one isolate of C. gloeosporioides sensu stricto, obtained from citrus leaf, was associated with PFD symptoms on inoculated flowers. Lastly, the species associated with PFD may survive on leaf surface, but only C. gloeosporioides sensu stricto is able to colonize endophytically the sweet orange leaves in Brazil.