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Morphological and Molecular Characterization of Curvularia

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Morphological and Molecular Characterization of Curvularia DOI: http://dx.doi.org/10.1590/1678-4499.2017258 P. R. R. Santos et al. PLANT PROTECTION - Note Morphological and molecular characterization of Curvularia lunata pathogenic to Andropogon grass Patrícia Resplandes Rocha dos Santos, Evelynne Urzêdo Leão, Raimundo Wagner de Souza Aguiar, Maruzanete Pereira de Melo, Gil Rodrigues dos Santos* Universidade Federal do Tocantins - Agronomia - Departamento de Produção Vegetal - Gurupi (TO), Brazil. ABSTRACT: The fungal genus Curvularia is associated with a The disease transmission was evaluated from seed sowing, in which number of diseases in plants, commonly producing foliar spots in after 40 days typical symptoms of Curvularia sp. were observed. forage grasses. The objective of this study was to characterize the Pathogenicity was evaluated by inoculating conidial suspension morphological and molecular diversity of the isolates of Curvularia sp. into the leaves of healthy plants, and after ten days, inspecting for associated with Andropogon seeds, and to assess both their capacity pathogenic symptoms. Based on morphological and molecular to transmit disease and the pathogenicity of this fungus to crop. Ten features, the pathogen associated with Andropogon seeds was isolates of Curvularia sp. were sourced from Andropogon seeds from identified asCurvularia lunata, which, as such, is transmitted through agricultural producing regions in the Brazilian states Tocantins and the Andropogon plants via its seeds and is pathogenic to this species Pará. Morphological characterization was achieved by observing of forage grass. fungus colonies and conidia and molecular characterization by Key words: Andropogon L., Curvularia lunata, diagnosis, phylogeny, DNA extraction and amplification with sequence-specific primers. pathogen. *Corresponding author: [email protected] Received: Aug. 4, 2017 – Accepted: Sept. 18, 2017 326 Bragantia, Campinas, v. 77, n. 2, p.326-332, 2018 Leaf spot of Curvularia in Andropogon The genus Curvularia consists of more than 40 species of Tocantins and Santa Maria das Barreiras (SMB) in the distinguished by differences that are very often evident in State of Pará. All cities are important agricultural producers. the conidia morphology, number of septa and the colony Morphological identification was carried out drawing morphology (Zhang et al. 2004; Chung and Tsukiboshi 2005). on micro and macromorphological observations, based The species of this fungal genus consists of saprophytes, on characteristics described by Ellis (1971) and Sivanesan endophytes and pathogens (Sanchez-Marquez et al. 2008). (1987) for the Curvularia genus. The isolates were grown Most of these species are pathogenic and give rise to losses in Petri dishes in a potato-dextrose-agar medium (BDA). in agricultural production. In vegetables, there are several Subsequently, the characteristics of the colonies and the examples of diseases caused by this genus responsible in the conidia were observed. The analyzed colonies morphological main part for necrotic spots in the leaves of several plant characteristics were as follows: mycelial aspect (cottony or families (Dasgupta et al. 2005), especially Poaceae (Toledo thin), border shape (regular or irregular), colony coloration et al. 1990; Yago et al. 2011; Silva et al. 2014; Santos et al. (black, moss green or grey), colony reverse side (with or 2014; Kusai et al. 2016). without dark pigmentation) and absence or presence of In the Poaceae family, the Andropogoneae group has 85 conidia in BDA after ten days of incubation in a chamber at a genera and 960 species, of which the most representative genus temperature of 25 °C ± 2 °C and a 12-hour photoperiod. The is Andropogon, which belongs to the Panicoideae subfamily. conidia were collected with a platinum loop and transferred to This genus is characterized by its wide distribution in the slides, where 50 conidia were randomly counted per sample, tropical and subtropical regions of the Planet (Clayton and using an optical microscope (400X) coupled to a camera, Renvoize 1986). In Brazil, Andropogoneae comprise 10% of which was designed and measured by the TSView7 software all species that make up the family and are found in all regions program. From the data obtained by conidia measuring, a (Hartley 1958). This species is for the most part perennial, Scott-Knott test (p < 0.05) was performed to generate an often spawns multiple shoots, and is especially noted for its isolates clustering according to the conidia characteristics, feathery inflorescences (Zanin and Longhi-Wagner 2006). using the SISVAR software program (Ferreira 2014). The state of Tocantins has an area of approximately 7.5 DNA extraction was carried out by scraping the mycelium million hectares recognized for its stretches of pasture, and and reproductive structures formed in monocultures of is notable for the presence of the genus Andropogon (Seagro the Curvularia sp. previously identified by morphological 2015). Furthermore, this forage grass has a substantial presence analysis and cultivated in BDA for ten days in a chamber at in areas that have undergone some kind of transformation, a temperature of 25 °C ± 2 °C and a 12-hour photoperiod. along roadsides and in clearings (Zanin and Longhi-Wagner With the help of a sterile scalpel, the mycelium was transferred 2006), and takes on the role of an invasive plant thanks to to 2 mL tubes, and the biomass obtained in this process its ability to easily disseminate seeds. was macerated in liquid nitrogen with a glass rod. DNA It is commonly known that, despite the vast areas under extraction was carried out according to the CTAB-based cultivation, there is little information available relating protocol (Murray and Thompson 1980). to diseases which affect forage species of the Andropogon To amplify the DNA from the isolates, primers P1 (forward: genus. Thus, it is against this background that the chosen 5’- ATGGACGAGAACAACAGGATAACGA-3’) and P2 objective of this study was to characterize the morphological (reverse: 5’CTACCAGCATTTAAGTTTACTCCAG-3’) based and molecular diversity of Curvularia sp. isolates associated on the nucleotide sequence of the Clg2p Ras protein gene with Andropogon seeds, and to evaluate the transmissibility described by Hou et al. (2013), specific toC. lunata were capability and pathogenicity of Curvularia sp. in Andropogon used. The total volume of the amplification reactions was plants. 50 μL, comprising 25 μL of Taq DNA Polymerase 2x Master For the assay, a total of ten monosporic isolates collected Mix RED (Ampliqon III PCR Enzymes & Reagents), 5 μL of from Andropogon seeds were characterized. Each isolate each 10 μM primer oligonucleotide, 10 μL of ultrapure water corresponds to one region, i.e., Araguacema (ARA), Araguaína and 5 μL DNA of the samples. PCR reactions were run on a (ARG), Brejinho de Nazaré (BJN), Caseara (CAS), Gurupi thermal cycler (TC-5000) programmed for an initial cycle of (GUR), Lagoa da Confusão (LGC), Marianópolis (MAR), 5 min at 95 °C (initial denaturation), followed by 35 cycles of Palmas (PAL) and Sucupira (SUC), cities located in the state 30 s at 95 °C (denaturation), 40 s at 55 °C (annealing), 30 s Bragantia, Campinas, v. 77, n. 2, p.326-332, 2018 327 P. R. R. Santos et al. at 72 °C (extension) and a final cycle of 5 min at 72 °C (final tissue, the fungus was reisolated and grown in BDA medium extension). The PCR product was subjected to electrophoresis (Alfenas and Mafia 2007). in 0.8% agarose gel (TAE 1X) stained with Neotaq Brilliant The results found in the morphological characterization Green Plus DNA Stain (Neobio). DNA concentration was assay, in relation to the fungal mycelium, showed little measured at 230 and 280 nm using a spectrophotometer variation between cotonous and thin, only the LGC isolate (Biodrop – μLite). had a thin aspect. This variation of LGC isolate is common in The products used to amplify the C. lunata samples fungal isolation. The difference in mycelium color or texture were purified using a Purelink® purification kit (Invitrogen) can often occur with the fungus removed in the same Petri following the manufacturer’s recommendations and were dish, being, therefore, a characteristic that alone should not subjected to direct sequencing using the P1 forward primer. be considered in the species identification. The shape of the The DNA concentration after purification ranged from 88.06 colony borders also presented variations between regular to 192.8 ng.μL–1. The products were sent for sequencing by and irregular throughout its growth. The BJN, PAL and SMB the Sanger method to the DNA Sequencing Laboratory of the isolates presented an irregular shape. There was a prevalence Catholic University of Brasilia, Brazil. After the sequences of coloration of greyish colonies in all the isolates obtained, were obtained, they were alignment using the ClustalW dark pigmentation was observed on the reverse side of the in MEGA (version 7.0) software program, then, similar colony. After ten days of incubation, the presence of dark sequences were identified using the GenBank BLASTn tool. brown conidia was observed. According to Sun et al. (2003), After alignment, these sequences were processed using the only the morphological analysis of the Curvularia genus does MEGA software program (version 7.0) (Tamura et al. 2011) not support a safe identification, requiring analysis based on to elaborate a phylogenetic tree of the isolates, using the another marker, such as DNA region sequencing. Maximum Likelihood method and the Kimura 2 evolution
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