Vanessa Kava

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.

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.

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

Differential colonization by bioprospected rhizobial bacteria associated with common bean in different cropping systems

In this study, we evaluated the diversity of rhizobia isolated from root nodules on common bean (Phaseolus vulgaris) derived from Andean and Mesoamerican centers and grown under field and greenhouse conditions. Genetic characterization of isolates was performed by sequencing analyses of the 16S rRNA gene and 2 housekeeping genes, recA and glnII, and by the amplification of nifH. Symbiotic efficiency was evaluated by examining nodulation, plant biomass production, and plant nitrogen (N) accumulation. The influence of the environment was observed in nodulation capacity, where Rhizobium miluonense was dominant under greenhouse conditions and the Rhizobium acidisoli group prevailed under field conditions. However, strain LGMB41 fit into a separate group from the type strain of R. acidisoli in terms of multilocus phylogeny, implying that it could belong to a new species. Rhizobium miluonense LGMB73 showed the best symbiotic efficiency performance, i.e., with the highest shoot-N content (77.7 mg/plant), superior to the commercial standard strain (56.9 mg/plant). Biodiversity- and bioprospecting-associated studies are important to better understand ecosystems and to develop more effective strategies to improve plant growth using a N-fixation process.

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.