Canadian Journal of Microbiology Estimating genetic structure and diversity of cyanobacterial communities in Atlantic forest phyllosphere Journal: Canadian Journal of Microbiology Manuscript ID cjm-2016-0229.R1 Manuscript Type: Article Date Submitted by the Author: 07-Jun-2016 Complete List of Authors: Rigonato, Janaina; CENA-USP, DVPROD Gonçalves, Natalia; University of Sao Paulo Andreote, Ana; University of São Paulo, Center for Nuclear Energy in AgricultureDraft Lambais, Marcio; ESALQ Fiore, Marli; CENA-USP, Keyword: Tropical forest, Clone library, DGGE, qPCR https://mc06.manuscriptcentral.com/cjm-pubs Page 1 of 23 Canadian Journal of Microbiology 1 Estimating genetic structure and diversity of cyanobacterial communities in Atlantic forest phyllosphere [Running title: Cyanobacteria in Atlantic forest phyllosphere] Janaina Rigonato 1,*, Natalia Gonçalves 1* , Ana Paula Dini Andreote 1, Marcio Rodrigues Lambais 2, Marli Fátima Fiore 1# 1University of São Paulo, Center for Nuclear Energy in Agriculture, CENA/USP, Piracicaba-SP, Brazil 2 University of São Paulo, Department of Soil Science, ESALQ/USP, Piracicaba-SP, Brazil Draft * Janaina Rigonato and Natalia Gonçalves share the first authorship #Corresponding author : Marli Fátima Fiore, Center for Nuclear Energy in Agriculture, University of São Paulo. Av. Centenário 303, Postal Code 96, CEP 13400-970, Piracicaba, SP, Brazil, Tel.: +55 19 3429-4657, Fax: +55 19 3429-4610, e-mail: [email protected] https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 2 of 23 2 Abstract Cyanobacterial communities on the phyllosphere of four plant species inhabiting the endangered Brazilian Atlantic Forest biome was evaluated using cultivation-independent molecular approaches. Total genomic DNA was extracted from cells detached from leaves surface of Euterpe edulis, Guapira opposita, Garcinia gardneriana and Merostachys neesii sampled in two Brazilian Atlantic Forest locations along an elevational gradient, i.e., lowland and montane forest. The DNA fingerprinting method PCR-DGGE revealed that the cyanobacterial phyllosphere community structures were mainly influenced by the plant species, with a low effect of the geographical location of the plant. The 16S rRNA gene sequences obtained by clone libraries showed a predominance of nitrogen-fixing cyanobacteria of the order Nostocales, even though the majority of retrieved OTUs (~60% of the sequences) showed similarity only to uncultured cyanobacteria phylotypes.Draft The leaf surfaces of Guapira opposita had the highest richness and diversity of cyanobacteria, whereas the M. neesii (bamboo) had the largest number of copies of cyanobacterial 16S rRNA gene per cm 2 of leaf. This study investigated cyanobacteria diversity and its distribution pattern in Atlantic forest phyllosphere. The results indicated that plant species is the main driver of cyanobacteria community assemblage in the phyllosphere, and that these communities are comprised by a high diversity of cyanobacterial taxa to be discovered. Key words: Tropical forest, Clone library, DGGE, qPCR. https://mc06.manuscriptcentral.com/cjm-pubs Page 3 of 23 Canadian Journal of Microbiology 3 Introduction The Atlantic Forest bears high levels of biological diversity and endemism (Ribeiro et al., 2009, Colombo and Joly, 2010, Scarano and Ceotto, 2015). Nonetheless, its original area (~150 million ha) was reduced to approximately 11 to 16 % due to the agricultural expansion and urbanization (Ribeiro et al. 2011). The largest remaining area of continuous forest is located mainly along the coastal mountains of the São Paulo State (Ribeiro et al . 2011). Associated with forest, there is a rich diversity of epiphytic and endophytic microorganisms with essential functions for maintenance and survival of such ecosystems. The leaf surface (phyllosphere) is a dynamic and stressful environment, subjected to variations of temperature, humidity and incidence of UV radiation (Lindow and Brandl, 2003). Despite these conditions, microenvironments provided by the leaves can harbor a diverse and underexplored microbial communityDraft (Lambais et al. 2006, Baldoto and Olivares 2008, Fürnkranz et al. 2008, Peñuelas and Terradas, 2014). Bacterial structure communities from phyllosphere can be determined by plant species and/or its geographical position (Yadav et al. 2004, Lambais et al. 2006, Finkel et al . 2011, Rigonato et al. 2012, Lambais et al. 2014). In addition, the bacterial distribution on the leaf surfaces can be associated with anatomical characteristics and availability of nutrients (micro-environment) (Leveau and Lindow 2001, Monier and Lindow 2003, Dulla et al. 2005). However, Cyanobacteria phylum may be less dependent of the plant for their nutrition since these organisms are autotrophic and some strains can also fix atmospheric nitrogen. Studies in the Atlantic Forest have revealed high levels of cyanobacterial diversity. Floristics surveys have shown huge morphological richness of cyanobacteria (Branco et al. 2009, Sant´anna et al. 2013, Gama et al. 2014) and novel species and genera have been described (Fiore te al. 2007, Sant´Anna et al. 2010, Hentschke et al. 2016) in this biome. Nevertheless, the https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 4 of 23 4 genetic diversity of the phylum Cyanobacteria inhabiting the Atlantic Forest is still poorly understood. Even though several studies have characterized the bacterial community in phyllosphere of plants of tropical forests, reports on cyanobacteria community composition in the phyllosphere are scarce (Fürnkranz et al. 2008, Rigonato et al. 2012, Alvarenga et al., 2016, Venkatachalam et al., 2016). Herein, the community structure of cyanobacteria inhabiting phyllosphere of four plant species from the Brazilian Atlantic Forest biome was accessed using cultivation- independent molecular approaches. Materials and Methods Site description and sampling: The plant leaves were collected in twoDraft locations along an elevational gradient within Atlantic Forest in Serra do Mar State Park (São Paulo State): Lowland Forest Permanent Sample Plot E (PSP E - 23º20’05” S 44º49’55”W) at the Picinguaba nucleus, municipality of Ubatuba, and Montane Forest Permanent Sample Plot N (PSP N - 23º20’36’’S 45º04’22’’W) at Santa Virginia nucleus, municipality of São Luis do Paraitinga. The elevational gradient includes a network of 14 1-ha permanent plots established in 2005-2006 to study forest diversity and dynamics, and ecosystem functioning of the Brazilian Coastal Atlantic Forest (Joly et al. 2012). The lowland forest PSP E is inserted in wavy and scarped relief with moderately steep slopes, with altitudes ranging from 64 to 89 m and an average annual rainfall over 2,200 mm, and an average annual temperature of 22 °C (Joly et al. 2012). Towards the top of the mountain, there is a gradual cooling along the slope, but there is no reduction in rainfall . The montane forest PSP N is almost daily covered by a dense fog , with altitudes ranging from 1010 to 1040 m and an average annual temperature of 17 °C (Joly et al. 2012). https://mc06.manuscriptcentral.com/cjm-pubs Page 5 of 23 Canadian Journal of Microbiology 5 The plant species were selected based on its richness according to a previous study (Joly et al. 2012) in the two different locations. Four adult individuals of Garcinia gardneriana (Planch. & Triana) Zappi , Guapira opposita (Vell.) Reitz, and Euterpe edulis Mart. in the lowland forest PSP E and Merostachys neesii Rupr., G. opposita and E. edulis in the montane forest PSP N were sampled randomly, in a total of 24 samples. Approximately 10 leaves of each individual were collected using long reach pruner under canopy (two to eight meters of height). The samples were maintained at 4°C until transported to the laboratory, where they were immediately processed to dislodge microbial cells from the leaf surface. DNA extraction: Three intact leaves of 24 samples (four individuals of each plant species and site) were placed in 500 mL Erleymeyer containing 100 mLDraft of sterilized NaCl solution (9% w/v ) and glass beads (3 mm ϕ) and were subjected to agitation (230 rpm) at 20 ºC for 1 h to promote detachment of the cells from leaf surfaces. The suspension containing detached cells was transferred to a 50 ml sterile tube and concentrated by centrifugation at 10,000 x g for 20 min. The pellet formed was used to extract total DNA using the kit MoBio Powersoil (Laboratories Inc, Carlsbad, CA, USA) according to manufacturer’s instructions. The DNA integrity was confirmed by electrophoresis in agaroses gel 1% (w/v) and this DNA was used for the subsequent analysis of PCR-DGGE, qPCR and the clone library construction. Nested PCR and DGGE: The cyanobacterial 16S rRNA gene amplicons of the 24 samples were obtained by Nested PCR using the primer sets 27F1/1494Rc (Neilan et al. 1997) and CYA359F (40 GC bases clamp)/CYA781aR and CYA781bR (equimolar concentration, Nübel et al. 1997). https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 6 of 23 6 DGGE was performed in 6% polyacrylamide gels with a denaturing gradient 25-60% of urea and formamide in a constant voltage of 100V at 60 ºC for 15 h. DGGE analysis, 300 ng of each amplified sample was used. The gel was stained with silver nitrate (Blum et al. 1987) and photographed using a digital camera. The electrophoretic profiles of DGGE were evaluated using the image analysis system Bionumerics (Applied Maths, NV, Belgium). The presence/absence