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      Metagenomic Analysis of Fungal Diversity on Strawberry Plants and the Effect of Management Practices on the Fungal Community Structure of Aerial Organs

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          Abstract

          An amplicon metagenomic approach based on the ITS2 region of fungal rDNA was used to identify the composition of fungal communities associated with different strawberry organs (leaves, flowers, immature and mature fruits), grown on a farm using management practices that entailed the routine use of various chemical pesticides. ITS2 sequences clustered into 316 OTUs and Ascomycota was the dominant phyla (95.6%) followed by Basidiomycota (3.9%). Strawberry plants supported a high diversity of microbial organisms, but two genera, Botrytis and Cladosporium, were the most abundant, representing 70–99% of the relative abundance (RA) of all detected sequences. According to alpha and beta diversity analyses, strawberry organs displayed significantly different fungal communities with leaves having the most diverse fungal community, followed by flowers, and fruit. The interruption of chemical treatments for one month resulted in a significant modification in the structure of the fungal community of leaves and flowers while immature and mature fruit were not significantly affected. Several plant pathogens of other plant species, that would not be intuitively expected to be present on strawberry plants such as Erysiphe, were detected, while some common strawberry pathogens, such as Rhizoctonia, were less evident or absent.

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          Rapid denoising of pyrosequencing amplicon data: exploiting the rank-abundance distribution

          We developed a fast method for denoising pyrosequencing for community 16S rRNA analysis. We observe a 2–4 fold reduction in the number of observed OTUs (operational taxonomic units) comparing denoised with non-denoised data. ~50,000 sequences can be denoised on a laptop within an hour, two orders of magnitude faster than published techniques. We demonstrate the effects of denoising on alpha and beta diversity of large 16S rRNA datasets.
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            The Colletotrichum acutatum species complex

            Colletotrichum acutatum is known as an important anthracnose pathogen of a wide range of host plants worldwide. Numerous studies have reported subgroups within the C. acutatum species complex. Multilocus molecular phylogenetic analysis (ITS, ACT, TUB2, CHS-1, GAPDH, HIS3) of 331 strains previously identified as C. acutatum and other related taxa, including strains from numerous hosts with wide geographic distributions, confirmed the molecular groups previously recognised and identified a series of novel taxa. Thirty-one species are accepted, of which 21 have not previously been recognised. Colletotrichum orchidophilum clusters basal to the C. acutatum species complex. There is a high phenotypic diversity within this complex, and some of the species appear to have preferences to specific hosts or geographical regions. Others appear to be plurivorous and are present in multiple regions. In this study, only C. salicis and C. rhombiforme formed sexual morphs in culture, although sexual morphs have been described from other taxa (especially as laboratory crosses), and there is evidence of hybridisation between different species. One species with similar morphology to C. acutatum but not belonging to this species complex was also described here as new, namely C. pseudoacutatum. Taxonomic novelties: New combinations - Colletotrichum limetticola (R.E. Clausen) Damm, P.F. Cannon & Crous, C. lupini (Bondar) Damm, P.F. Cannon & Crous, C. salicis (Fuckel) Damm, P.F. Cannon & Crous. New species - C. acerbum Damm, P.F. Cannon & Crous, C. australe Damm, P.F. Cannon & Crous, C. brisbanense Damm, P.F. Cannon & Crous, C. cosmi Damm, P.F. Cannon & Crous, C. costaricense Damm, P.F. Cannon & Crous, C. cuscutae Damm, P.F. Cannon & Crous, C. guajavae Damm, P.F. Cannon & Crous, C. indonesiense Damm, P.F. Cannon & Crous, C. johnstonii Damm, P.F. Cannon & Crous, C. kinghornii Damm, P.F. Cannon & Crous, C. laticiphilum Damm, P.F. Cannon & Crous, C. melonis Damm, P.F. Cannon & Crous, C. orchidophilum Damm, P.F. Cannon & Crous, C. paxtonii Damm, P.F. Cannon & Crous, C. pseudoacutatum Damm, P.F. Cannon & Crous C. pyricola Damm, P.F. Cannon & Crous, C. rhombiforme Damm, P.F. Cannon & Crous, C. scovillei Damm, P.F. Cannon & Crous, C. sloanei Damm, P.F. Cannon & Crous, C. tamarilloi Damm, P.F. Cannon & Crous, C. walleri Damm, P.F. Cannon & Crous. Typifications: Epitypifications - C. acutatum J.H. Simmonds, C. limetticola (R.E. Clausen) Damm, P.F. Cannon & Crous, C. nymphaeae (Pass.) Aa, C. phormii (Henn.) D.F. Farr & Rossman, C. salicis (Fuckel) Damm, P.F. Cannon & Crous. Lectotypifications - C. nymphaeae (Pass.) Aa, C. orchidearum Allesch.
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              Bacterial Communities Associated with the Surfaces of Fresh Fruits and Vegetables

              Fresh fruits and vegetables can harbor large and diverse populations of bacteria. However, most of the work on produce-associated bacteria has focused on a relatively small number of pathogenic bacteria and, as a result, we know far less about the overall diversity and composition of those bacterial communities found on produce and how the structure of these communities varies across produce types. Moreover, we lack a comprehensive view of the potential effects of differing farming practices on the bacterial communities to which consumers are exposed. We addressed these knowledge gaps by assessing bacterial community structure on conventional and organic analogs of eleven store-bought produce types using a culture-independent approach, 16 S rRNA gene pyrosequencing. Our results demonstrated that the fruits and vegetables harbored diverse bacterial communities, and the communities on each produce type were significantly distinct from one another. However, certain produce types (i.e., sprouts, spinach, lettuce, tomatoes, peppers, and strawberries) tended to share more similar communities as they all had high relative abundances of taxa belonging to the family Enterobacteriaceae when compared to the other produce types (i.e., apples, peaches, grapes, and mushrooms) which were dominated by taxa belonging to the Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria phyla. Although potentially driven by factors other than farming practice, we also observed significant differences in community composition between conventional and organic analogs within produce types. These differences were often attributable to distinctions in the relative abundances of Enterobacteriaceae taxa, which were generally less abundant in organically-grown produce. Taken together, our results suggest that humans are exposed to substantially different bacteria depending on the types of fresh produce they consume with differences between conventionally and organically farmed varieties contributing to this variation.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                4 August 2016
                2016
                : 11
                : 8
                : e0160470
                Affiliations
                [1 ]Dipartimento di Agraria, Università Mediterranea di Reggio Calabria, Località Feo di Vito, Reggio Calabria, 89122, Italy
                [2 ]USDA-ARS-AFRS, 2217 Wiltshire Road, Kearneysville, West Virginia, United States of America
                [3 ]Dipartimento di Agricoltura, Alimentazione e Ambiente, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy
                The National Orchid Conservation Center of China; The Orchid Conservation & Research Center of Shenzhen, CHINA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: AA LS.

                • Data curation: AA LS.

                • Formal analysis: AA LS.

                • Funding acquisition: LS.

                • Investigation: AA LS SOC.

                • Methodology: AA LS MGLDN.

                • Project administration: LS.

                • Software: AA.

                • Supervision: LS.

                • Validation: AA LS MW.

                • Writing - original draft: AA MW MGLDN SOC LS.

                • Writing - review & editing: AA MW MGLDN SOC LS.

                Author information
                http://orcid.org/0000-0002-9737-2593
                Article
                PONE-D-15-48750
                10.1371/journal.pone.0160470
                4973904
                27490110
                3ad95d00-4b5b-4d10-9880-f5803e392e99

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 7 November 2015
                : 20 July 2016
                Page count
                Figures: 6, Tables: 3, Pages: 17
                Funding
                Funded by: MIUR
                Award ID: Modelli sostenibili e nuove tecnologie per la valorizzazione delle filiere vegetali mediterranee" - PON Ricerca e competitività 2007-2013 (PON03PE_00090_03)
                Award Recipient :
                This work was funded by The Italian Ministry of Education, University and Research (MIUR) with grant “Modelli sostenibili e nuove tecnologie per la valorizzazione delle filiere vegetali mediterranee”-PON Ricerca e competitività 2007–2013 (PON03PE_00090_03). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Agriculture
                Crop Science
                Crops
                Fruits
                Biology and Life Sciences
                Organisms
                Plants
                Fruits
                Biology and Life Sciences
                Plant Science
                Plant Anatomy
                Flowers
                Biology and Life Sciences
                Plant Science
                Plant Anatomy
                Leaves
                Biology and Life Sciences
                Plant Science
                Plant Pathology
                Plant Pathogens
                Plant Fungal Pathogens
                Research and Analysis Methods
                Database and Informatics Methods
                Biological Databases
                Sequence Databases
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Databases
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Databases
                Biology and Life Sciences
                Genetics
                Fungal Genetics
                Biology and Life Sciences
                Mycology
                Fungal Genetics
                Biology and Life Sciences
                Agriculture
                Agrochemicals
                Fungicides
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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