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      Antifungal potential of secondary metabolites involved in the interaction between citrus pathogens

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          Abstract

          Numerous postharvest diseases have been reported that cause substantial losses of citrus fruits worldwide. Penicillium digitatum is responsible for up to 90% of production losses, and represent a problem for worldwide economy. In order to control phytopathogens, chemical fungicides have been extensively used. Yet, the use of some artificial fungicides cause concerns about environmental risks and fungal resistance. Therefore, studies focusing on new approaches, such as the use of natural products, are getting attention. Co-culture strategy can be applied to discover new bioactive compounds and to understand microbial ecology. Mass Spectrometry Imaging (MSI) was used to screen for potential antifungal metabolites involved in the interaction between Penicillium digitatum and Penicillium citrinum. MSI revealed a chemical warfare between the fungi: two tetrapeptides, deoxycitrinadin A, citrinadin A, chrysogenamide A and tryptoquialanines are produced in the fungi confrontation zone. Antimicrobial assays confirmed the antifungal activity of the investigated metabolites. Also, tryptoquialanines inhibited sporulation of P. citrinum. The fungal metabolites reported here were never described as antimicrobials until this date, demonstrating that co-cultures involving phytopathogens that compete for the same host is a positive strategy to discover new antifungal agents. However, the use of these natural products on the environment, as a safer strategy, needs further investigation. This paper aimed to contribute to the protection of agriculture, considering health and ecological risks.

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          Most cited references37

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          Pathogenic fungus harbours endosymbiotic bacteria for toxin production.

          A number of plant pathogenic fungi belonging to the genus Rhizopus are infamous for causing rice seedling blight. This plant disease is typically initiated by an abnormal swelling of the seedling roots without any sign of infection by the pathogen. This characteristic symptom is in fact caused by the macrocyclic polyketide metabolite rhizoxin that has been isolated from cultures of Rhizopus sp.. The phytotoxin exerts its destructive effect by binding to rice beta-tubulin, which results in inhibition of mitosis and cell cycle arrest. Owing to its remarkably strong antimitotic activity in most eukaryotic cells, including various human cancer cell lines, rhizoxin has attracted considerable interest as a potential antitumour drug. Here we show that rhizoxin is not biosynthesized by the fungus itself, but by endosymbiotic, that is, intracellular living, bacteria of the genus Burkholderia. Our unexpected findings unveil a remarkably complex symbiotic-pathogenic relationship that extends the fungus-plant interaction to a third, bacterial, key-player, and opens new perspectives for pest control.
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            Importance of microbial natural products and the need to revitalize their discovery.

            Microbes are the leading producers of useful natural products. Natural products from microbes and plants make excellent drugs. Significant portions of the microbial genomes are devoted to production of these useful secondary metabolites. A single microbe can make a number of secondary metabolites, as high as 50 compounds. The most useful products include antibiotics, anticancer agents, immunosuppressants, but products for many other applications, e.g., antivirals, anthelmintics, enzyme inhibitors, nutraceuticals, polymers, surfactants, bioherbicides, and vaccines have been commercialized. Unfortunately, due to the decrease in natural product discovery efforts, drug discovery has decreased in the past 20 years. The reasons include excessive costs for clinical trials, too short a window before the products become generics, difficulty in discovery of antibiotics against resistant organisms, and short treatment times by patients for products such as antibiotics. Despite these difficulties, technology to discover new drugs has advanced, e.g., combinatorial chemistry of natural product scaffolds, discoveries in biodiversity, genome mining, and systems biology. Of great help would be government extension of the time before products become generic.
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              Co-Cultivation—A Powerful Emerging Tool for Enhancing the Chemical Diversity of Microorganisms

              Marine-derived bacteria and fungi are promising sources of novel bioactive compounds that are important for drug discovery programs. However, as encountered in terrestrial microorganisms there is a high rate of redundancy that results in the frequent re-discovery of known compounds. Apparently only a part of the biosynthetic genes that are harbored by fungi and bacteria are transcribed under routine laboratory conditions which involve cultivation of axenic microbial strains. Many biosynthetic genes remain silent and are not expressed in vitro thereby seriously limiting the chemical diversity of microbial compounds that can be obtained through fermentation. In contrast to this, co-cultivation (also called mixed fermentation) of two or more different microorganisms tries to mimic the ecological situation where microorganisms always co-exist within complex microbial communities. The competition or antagonism experienced during co-cultivation is shown to lead to a significantly enhanced production of constitutively present compounds and/or to an accumulation of cryptic compounds that are not detected in axenic cultures of the producing strain. This review highlights the power of co-cultivation for increasing the chemical diversity of bacteria and fungi drawing on published studies from the marine and from the terrestrial habitat alike.
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                Author and article information

                Contributors
                taicia@unicamp.br
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                9 December 2019
                9 December 2019
                2019
                : 9
                : 18647
                Affiliations
                [1 ]ISNI 0000 0001 0723 2494, GRID grid.411087.b, Institute of Chemistry, , University of Campinas, CP 6154, ; 13083-970 Campinas, SP Brazil
                [2 ]ISNI 0000 0004 0643 8839, GRID grid.412368.a, Center for Natural and Human Sciences, , Federal University of ABC, ; 09210-580 Santo André, SP Brazil
                [3 ]ISNI 0000 0001 0143 807X, GRID grid.418398.f, Department of Biomolecular Chemistry, , Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute, ; Jena, Germany
                [4 ]ISNI 0000 0001 1939 2794, GRID grid.9613.d, Chair of Natural Product Chemistry, , Friedrich Schiller University Jena, ; 07743 Jena, Germany
                Author information
                http://orcid.org/0000-0002-2611-3471
                http://orcid.org/0000-0002-0367-337X
                Article
                55204
                10.1038/s41598-019-55204-9
                6901458
                31819142
                819bf75e-a02e-43aa-b78b-8593e9a26d5d
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 17 September 2019
                : 23 November 2019
                Funding
                Funded by: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001
                Funded by: FundRef https://doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft (German Research Foundation);
                Award ID: SFB 1127 ChemBioSys
                Award Recipient :
                Funded by: Fundação de Amparo a Pesquisa no Estado de São Paulo [grant number 2017/24462-4 and 2019/06359-7]. TPF was recipient of a postdoctoral Fellowship from Capes/Humboldt
                Categories
                Article
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                © The Author(s) 2019

                Uncategorized
                antifungal agents,fungi
                Uncategorized
                antifungal agents, fungi

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