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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Pumpkin powdery mildew disease severity influences the fungal diversity of the phyllosphere</title><author><name sortKey="Zhang, Zhuo" sort="Zhang, Zhuo" uniqKey="Zhang Z" first="Zhuo" last="Zhang">Zhuo Zhang</name><affiliation><nlm:aff id="aff-1"><institution>Hunan Academy of Agricultural Sciences, Hunan Plant Protection Institute</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Luo, Luyun" sort="Luo, Luyun" uniqKey="Luo L" first="Luyun" last="Luo">Luyun Luo</name><affiliation><nlm:aff id="aff-1"><institution>Hunan Academy of Agricultural Sciences, Hunan Plant Protection Institute</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff-2"><institution>College of Bioscience & Biotechnology, Hunan Agricultural University</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Tan, Xinqiu" sort="Tan, Xinqiu" uniqKey="Tan X" first="Xinqiu" last="Tan">Xinqiu Tan</name><affiliation><nlm:aff id="aff-1"><institution>Hunan Academy of Agricultural Sciences, Hunan Plant Protection Institute</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Kong, Xiao" sort="Kong, Xiao" uniqKey="Kong X" first="Xiao" last="Kong">Xiao Kong</name><affiliation><nlm:aff id="aff-3"><institution>Chinese Academy of Sciences Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences</institution>,<city>Beijing</city>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Yang, Jianguo" sort="Yang, Jianguo" uniqKey="Yang J" first="Jianguo" last="Yang">Jianguo Yang</name><affiliation><nlm:aff id="aff-4"><institution>Vegetable Research Institute, Hunan Academy of Agricultural Science</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Wang, Duanhua" sort="Wang, Duanhua" uniqKey="Wang D" first="Duanhua" last="Wang">Duanhua Wang</name><affiliation><nlm:aff id="aff-4"><institution>Vegetable Research Institute, Hunan Academy of Agricultural Science</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Zhang, Deyong" sort="Zhang, Deyong" uniqKey="Zhang D" first="Deyong" last="Zhang">Deyong Zhang</name><affiliation><nlm:aff id="aff-1"><institution>Hunan Academy of Agricultural Sciences, Hunan Plant Protection Institute</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Jin, Decai" sort="Jin, Decai" uniqKey="Jin D" first="Decai" last="Jin">Decai Jin</name><affiliation><nlm:aff id="aff-3"><institution>Chinese Academy of Sciences Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences</institution>,<city>Beijing</city>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Yong" sort="Liu, Yong" uniqKey="Liu Y" first="Yong" last="Liu">Yong Liu</name><affiliation><nlm:aff id="aff-1"><institution>Hunan Academy of Agricultural Sciences, Hunan Plant Protection Institute</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">29629242</idno><idno type="pmc">5885987</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885987</idno><idno type="RBID">PMC:5885987</idno><idno type="doi">10.7717/peerj.4559</idno><date when="2018">2018</date><idno type="wicri:Area/Pmc/Corpus">000B25</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000B25</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Pumpkin powdery mildew disease severity influences the fungal diversity of the phyllosphere</title><author><name sortKey="Zhang, Zhuo" sort="Zhang, Zhuo" uniqKey="Zhang Z" first="Zhuo" last="Zhang">Zhuo Zhang</name><affiliation><nlm:aff id="aff-1"><institution>Hunan Academy of Agricultural Sciences, Hunan Plant Protection Institute</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Luo, Luyun" sort="Luo, Luyun" uniqKey="Luo L" first="Luyun" last="Luo">Luyun Luo</name><affiliation><nlm:aff id="aff-1"><institution>Hunan Academy of Agricultural Sciences, Hunan Plant Protection Institute</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff-2"><institution>College of Bioscience & Biotechnology, Hunan Agricultural University</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Tan, Xinqiu" sort="Tan, Xinqiu" uniqKey="Tan X" first="Xinqiu" last="Tan">Xinqiu Tan</name><affiliation><nlm:aff id="aff-1"><institution>Hunan Academy of Agricultural Sciences, Hunan Plant Protection Institute</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Kong, Xiao" sort="Kong, Xiao" uniqKey="Kong X" first="Xiao" last="Kong">Xiao Kong</name><affiliation><nlm:aff id="aff-3"><institution>Chinese Academy of Sciences Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences</institution>,<city>Beijing</city>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Yang, Jianguo" sort="Yang, Jianguo" uniqKey="Yang J" first="Jianguo" last="Yang">Jianguo Yang</name><affiliation><nlm:aff id="aff-4"><institution>Vegetable Research Institute, Hunan Academy of Agricultural Science</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Wang, Duanhua" sort="Wang, Duanhua" uniqKey="Wang D" first="Duanhua" last="Wang">Duanhua Wang</name><affiliation><nlm:aff id="aff-4"><institution>Vegetable Research Institute, Hunan Academy of Agricultural Science</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Zhang, Deyong" sort="Zhang, Deyong" uniqKey="Zhang D" first="Deyong" last="Zhang">Deyong Zhang</name><affiliation><nlm:aff id="aff-1"><institution>Hunan Academy of Agricultural Sciences, Hunan Plant Protection Institute</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Jin, Decai" sort="Jin, Decai" uniqKey="Jin D" first="Decai" last="Jin">Decai Jin</name><affiliation><nlm:aff id="aff-3"><institution>Chinese Academy of Sciences Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences</institution>,<city>Beijing</city>,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Yong" sort="Liu, Yong" uniqKey="Liu Y" first="Yong" last="Liu">Yong Liu</name><affiliation><nlm:aff id="aff-1"><institution>Hunan Academy of Agricultural Sciences, Hunan Plant Protection Institute</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></nlm:aff></affiliation></author></analytic><series><title level="j">PeerJ</title><idno type="eISSN">2167-8359</idno><imprint><date when="2018">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Phyllosphere microbiota play a crucial role in plant-environment interactions and their microbial community and function are influenced by biotic and abiotic factors. However, there is little research on how pathogens affect the microbial community of phyllosphere fungi. In this study, we collected 16 pumpkin (<italic>Cucurbita moschata</italic>) leaf samples which exhibited powdery mildew disease, with a severity ranging from L1 (least severe) to L4 (most severe). The fungal community structure and diversity was examined by Illumina MiSeq sequencing of the internal transcribed spacer (ITS) region of ribosomal RNA genes. The results showed that the fungal communities were dominated by members of the Basidiomycota and Ascomycota. The <italic>Podosphaera</italic> was the most dominant genus on these infected leaves, which was the key pathogen responsible for the pumpkin powdery mildew. The abundance of Ascomycota and <italic>Podosphaera</italic> increased as disease severity increased from L1 to L4, and was significantly higher at disease severity L4 (<italic>P</italic> < 0.05). The richness and diversity of the fungal community increased from L1 to L2, and then declined from L2 to L4, likely due to the biotic pressure (i.e., symbiotic and competitive stresses among microbial species) at disease severity L4. Our results could give new perspectives on the changes of the leaf microbiome at different pumpkin powdery mildew disease severity.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Abarenkov, K" uniqKey="Abarenkov K">K Abarenkov</name></author><author><name sortKey="Nilsson, Rh" uniqKey="Nilsson R">RH Nilsson</name></author><author><name sortKey="Larsson, Kh" uniqKey="Larsson K">KH Larsson</name></author><author><name sortKey="Alexander, Ij" uniqKey="Alexander I">IJ Alexander</name></author><author><name sortKey="Eberhardt, U" uniqKey="Eberhardt U">U Eberhardt</name></author><author><name sortKey="Erland, S" uniqKey="Erland S">S Erland</name></author><author><name sortKey="H Iland, K" uniqKey="H Iland K">K Høiland</name></author><author><name sortKey="Kj Ller, R" uniqKey="Kj Ller R">R Kjøller</name></author><author><name sortKey="Larsson, E" uniqKey="Larsson E">E 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PeerJ</journal-id><journal-id journal-id-type="iso-abbrev">PeerJ</journal-id><journal-id journal-id-type="publisher-id">peerj</journal-id><journal-id journal-id-type="pmc">peerj</journal-id><journal-title-group><journal-title>PeerJ</journal-title></journal-title-group><issn pub-type="epub">2167-8359</issn><publisher><publisher-name>PeerJ Inc.</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">29629242</article-id><article-id pub-id-type="pmc">5885987</article-id><article-id pub-id-type="publisher-id">4559</article-id><article-id pub-id-type="doi">10.7717/peerj.4559</article-id><article-categories><subj-group subj-group-type="heading"><subject>Agricultural Science</subject></subj-group><subj-group subj-group-type="heading"><subject>Mycology</subject></subj-group></article-categories><title-group><article-title>Pumpkin powdery mildew disease severity influences the fungal diversity of the phyllosphere</article-title></title-group><contrib-group><contrib id="author-1" contrib-type="author" equal-contrib="yes"><name><surname>Zhang</surname><given-names>Zhuo</given-names></name><xref ref-type="aff" rid="aff-1">1</xref></contrib><contrib id="author-2" contrib-type="author" equal-contrib="yes"><name><surname>Luo</surname><given-names>Luyun</given-names></name><xref ref-type="aff" rid="aff-1">1</xref><xref ref-type="aff" rid="aff-2">2</xref></contrib><contrib id="author-3" contrib-type="author"><name><surname>Tan</surname><given-names>Xinqiu</given-names></name><xref ref-type="aff" rid="aff-1">1</xref></contrib><contrib id="author-4" contrib-type="author"><name><surname>Kong</surname><given-names>Xiao</given-names></name><xref ref-type="aff" rid="aff-3">3</xref></contrib><contrib id="author-5" contrib-type="author"><name><surname>Yang</surname><given-names>Jianguo</given-names></name><xref ref-type="aff" rid="aff-4">4</xref></contrib><contrib id="author-6" contrib-type="author"><name><surname>Wang</surname><given-names>Duanhua</given-names></name><xref ref-type="aff" rid="aff-4">4</xref></contrib><contrib id="author-7" contrib-type="author"><name><surname>Zhang</surname><given-names>Deyong</given-names></name><xref ref-type="aff" rid="aff-1">1</xref></contrib><contrib id="author-8" contrib-type="author" corresp="yes"><name><surname>Jin</surname><given-names>Decai</given-names></name><email>dcjin@rcees.ac.cn</email><xref ref-type="aff" rid="aff-3">3</xref></contrib><contrib id="author-9" contrib-type="author" corresp="yes"><name><surname>Liu</surname><given-names>Yong</given-names></name><email>haoasliu@163.com</email><xref ref-type="aff" rid="aff-1">1</xref></contrib><aff id="aff-1"><label>1</label><institution>Hunan Academy of Agricultural Sciences, Hunan Plant Protection Institute</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></aff><aff id="aff-2"><label>2</label><institution>College of Bioscience & Biotechnology, Hunan Agricultural University</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></aff><aff id="aff-3"><label>3</label><institution>Chinese Academy of Sciences Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences</institution>,<city>Beijing</city>,<country>China</country></aff><aff id="aff-4"><label>4</label><institution>Vegetable Research Institute, Hunan Academy of Agricultural Science</institution>,<city>Changsha</city>,<state>Hunan</state>,<country>China</country></aff></contrib-group><contrib-group><contrib contrib-type="editor"><name><surname>Amend</surname><given-names>Anthony</given-names></name></contrib></contrib-group><pub-date pub-type="epub" date-type="pub" iso-8601-date="2018-04-02"><day>2</day><month>4</month><year iso-8601-date="2018">2018</year></pub-date><pub-date pub-type="collection"><year>2018</year></pub-date><volume>6</volume><elocation-id>e4559</elocation-id><history><date date-type="received" iso-8601-date="2017-09-19"><day>19</day><month>9</month><year iso-8601-date="2017">2017</year></date><date date-type="accepted" iso-8601-date="2018-03-09"><day>9</day><month>3</month><year iso-8601-date="2018">2018</year></date></history><permissions><copyright-statement>©2018 Zhang et al.</copyright-statement><copyright-year>2018</copyright-year><copyright-holder>Zhang et al.</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.</license-p></license></permissions><self-uri xlink:href="https://peerj.com/articles/4559"></self-uri><abstract><p>Phyllosphere microbiota play a crucial role in plant-environment interactions and their microbial community and function are influenced by biotic and abiotic factors. However, there is little research on how pathogens affect the microbial community of phyllosphere fungi. In this study, we collected 16 pumpkin (<italic>Cucurbita moschata</italic>) leaf samples which exhibited powdery mildew disease, with a severity ranging from L1 (least severe) to L4 (most severe). The fungal community structure and diversity was examined by Illumina MiSeq sequencing of the internal transcribed spacer (ITS) region of ribosomal RNA genes. The results showed that the fungal communities were dominated by members of the Basidiomycota and Ascomycota. The <italic>Podosphaera</italic> was the most dominant genus on these infected leaves, which was the key pathogen responsible for the pumpkin powdery mildew. The abundance of Ascomycota and <italic>Podosphaera</italic> increased as disease severity increased from L1 to L4, and was significantly higher at disease severity L4 (<italic>P</italic> < 0.05). The richness and diversity of the fungal community increased from L1 to L2, and then declined from L2 to L4, likely due to the biotic pressure (i.e., symbiotic and competitive stresses among microbial species) at disease severity L4. Our results could give new perspectives on the changes of the leaf microbiome at different pumpkin powdery mildew disease severity.</p></abstract><kwd-group kwd-group-type="author"><kwd>Disease severity</kwd><kwd>Powdery mildew</kwd><kwd>Illumina MiSeq</kwd><kwd>Phyllosphere microbiota</kwd><kwd>Fungal community</kwd></kwd-group><funding-group><award-group id="fund-1"><funding-source>National Science and Technology Pillar Program</funding-source><award-id>2014BAD05B04-4</award-id></award-group><award-group id="fund-2"><funding-source>National Natural Science Foundation of China</funding-source><award-id>31501696</award-id><award-id>31471831</award-id></award-group><award-group id="fund-3"><funding-source>Agriculture Research System of China</funding-source><award-id>CARS-25-B-05</award-id></award-group><award-group id="fund-4"><funding-source>Innovation Platform and Talent Plan</funding-source><award-id>2016RS2019</award-id></award-group><funding-statement>This work was supported by the National Science and Technology Pillar Program during the 12th Five-year Plan Period (2014BAD05B04-4), the National Natural Science Foundation of China (31501696 and 31471831), the Agriculture Research System of China (CARS-25-B-05) and the Innovation Platform and Talent Plan (2016RS2019). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</funding-statement></funding-group></article-meta></front><body><sec sec-type="intro"><title>Introduction</title><p>Powdery mildew is a common fungal disease of cucurbits and the major cause of losses in cucurbit production worldwide (<xref rid="ref-6" ref-type="bibr">Bellon-Gómez et al., 2011</xref>). <italic>Golovinomyces cichoracearum</italic> (syn. <italic>Erysiphe cichoracearum</italic>) and <italic>Podosphaera xanthii</italic> (syn. <italic>Sphaerotheca fuliginea</italic>) are the two main pathogenic fungi that cause powdery mildew in the cucurbits (<xref rid="ref-27" ref-type="bibr">Lebeda, Mggrath & Sedlakova, 2010</xref>). Impacts of powdery mildew on crop production include reduced photosynthesis, impaired growth, premature senescence, and yield loss (<xref rid="ref-27" ref-type="bibr">Lebeda, Mggrath & Sedlakova, 2010</xref>). The powdery mildew pathogen lives with the obligate biotrophic lifestyle (<xref rid="ref-20" ref-type="bibr">Hacquard, 2014</xref>). Powdery mildew symptoms first appear as pale, chlorotic spots on leaves that soon turn powdery-white in appearance (fungal spores) and start on the crown and lower leaves, mainly on the under-leaf shaded surface (<xref rid="ref-27" ref-type="bibr">Lebeda, Mggrath & Sedlakova, 2010</xref>). Young plants may turn yellow, stunted, and may die, and then severely infected leaves become brown and brittle, resulting in foliage loss (<xref rid="ref-27" ref-type="bibr">Lebeda, Mggrath & Sedlakova, 2010</xref>).</p><p>The phyllosphere or leaf surface is an important microbial habitat for members of the major bacterial and fungal groups, and Archaea (<xref rid="ref-30" ref-type="bibr">Lindow & Leveau, 2002</xref>; <xref rid="ref-29" ref-type="bibr">Lindow & Brandl, 2003</xref>). These microorganisms play a crucial role in helping their host against pathogens (<xref rid="ref-26" ref-type="bibr">Lacava et al., 2006</xref>; <xref rid="ref-36" ref-type="bibr">Mejía et al., 2008</xref>; <xref rid="ref-39" ref-type="bibr">Rajendran et al., 2011</xref>). In past years, many studies focused on screening plant growth-promoting microorganisms which can help us manage plant diseases (<xref rid="ref-11" ref-type="bibr">Compant et al., 2005</xref>; <xref rid="ref-17" ref-type="bibr">Everett et al., 2005</xref>; <xref rid="ref-22" ref-type="bibr">Hirano & Upper, 2000</xref>; <xref rid="ref-51" ref-type="bibr">Whipps et al., 2008</xref>). However, not all the microbes in the natural environment are considered culturable (<xref rid="ref-25" ref-type="bibr">Kimura, 2006</xref>). In the past few years, the development of next-generation rRNA sequencing techniques has enabled us to obtain in-depth descriptions of the composition of the microbial communities associated with leaves of <italic>Arabidopsis thaliana</italic> (<xref rid="ref-41" ref-type="bibr">Reisberg et al., 2013</xref>), potatoes (<xref rid="ref-5" ref-type="bibr">Becker et al., 2008</xref>), rice (<xref rid="ref-37" ref-type="bibr">Mwajita et al., 2012</xref>), spinach (<xref rid="ref-32" ref-type="bibr">Lopez-Velasco et al., 2011</xref>; <xref rid="ref-31" ref-type="bibr">Lopez-Velasco et al., 2013</xref>), grape tree (<xref rid="ref-28" ref-type="bibr">Leveau & Tech, 2011</xref>), and various tree species including salt cedar (<xref rid="ref-40" ref-type="bibr">Redford et al., 2010</xref>; <xref rid="ref-18" ref-type="bibr">Finkel et al., 2011</xref>).</p><p>Historically, scholars began to study the rhizosphere as a microbial habitat as early as 100 years ago (<xref rid="ref-21" ref-type="bibr">Hartmann, Rothballer & Schmid, 2008</xref>) and the importance of microbial communities is well recognized in plant health and growth. Although the root–rhizosphere microbiome has been well-studied now, much remains to be understood about the plant-microbe interactions occurring in the phyllosphere. Now, the development of new high-throughput sequencing technologies enables researchers to better understand the microbiome fields, especially the phyllosphere microbiome. Not only can it help us understand the communities better, but it can also help us study the interactions between host plants and the environment deeply.</p><p>As sessile organisms, plants are affected by environmental stresses during their growth period (<xref rid="ref-54" ref-type="bibr">Zhang et al., 2014</xref>). The phyllosphere microorganisms are influenced by both biotic and abiotic factors, some of which are fairly stable and constant, such as habitat conditions (<xref rid="ref-52" ref-type="bibr">Yang et al., 2016</xref>; <xref rid="ref-19" ref-type="bibr">Fonsecagarcía et al., 2016</xref>), the host genotype (<xref rid="ref-44" ref-type="bibr">Sapkota et al., 2015</xref>; <xref rid="ref-8" ref-type="bibr">Bodenhausen et al., 2014</xref>; <xref rid="ref-23" ref-type="bibr">Hunter, Pink & Bending, 2015</xref>), elevation gradient (<xref rid="ref-13" ref-type="bibr">Cordier et al., 2012</xref>; <xref rid="ref-53" ref-type="bibr">Zhang et al., 2015</xref>), and seasonal variation (<xref rid="ref-12" ref-type="bibr">Copeland et al., 2015</xref>; <xref rid="ref-24" ref-type="bibr">Jackson & Denney, 2011</xref>; <xref rid="ref-14" ref-type="bibr">Davey et al., 2012</xref>). Microbial interactions in the phyllosphere play an important role in the agroecosystem, not only affecting the health and growth of plants in natural communities, but also the productivity of agricultural crops (<xref rid="ref-51" ref-type="bibr">Whipps et al., 2008</xref>). The phyllosphere is constituted of a high proportion of plant-beneficial microorganisms such as antagonists, diazotrophs, and plant growth-promoting bacteria (PGPB) that colonized plant-associated habitats, but also plant pathogens and potential human pathogens (<xref rid="ref-7" ref-type="bibr">Berg, Eberl & Hartmann, 2005</xref>). Plants can protect themselves against pathogenic fungal infection by natural means which include biological and non-biological inducers (<xref rid="ref-48" ref-type="bibr">Shi et al., 2007</xref>). However, less is known about the colonization and persistence of nonpathogenic microbes on this extensive habitat, as well as about their interactions with pathogenic microorganisms, and impact of single strains in the microbial community. The rhizosphere community of specific biocontrol agents have shown minor and only transient effects according to the risk assessment and colonization studies (<xref rid="ref-45" ref-type="bibr">Scherwinski, Wolf & Berg, 2007</xref>; <xref rid="ref-2" ref-type="bibr">Adesina et al., 2009</xref>; <xref rid="ref-10" ref-type="bibr">Chowdhury et al., 2013</xref>; <xref rid="ref-47" ref-type="bibr">Schmidt et al., 2012</xref>), while impacts of pathogens on the phyllosphere microbiome are largely underexplored. To the best of our knowledge, although there are fewer studies about the relationship between the phyllosphere microbiome and pathogen using Illumina sequencing technology, the existing results still showed that microbes present on the plant surface play an important role in the resistance to the pathogen (<xref rid="ref-42" ref-type="bibr">Ritpitakphong et al., 2016</xref>; <xref rid="ref-49" ref-type="bibr">Vogel et al., 2016</xref>; <xref rid="ref-9" ref-type="bibr">Busby, Peay & Newcombe, 2016</xref>).</p><p>In this study, we intended to (1) further explore the interaction between the pathogen <italic>Podosphaera</italic> and other dominant microorganisms and (2) gain a better understanding of the theoretical basis for disease control in agroecological systems by evaluating whether the diversity and community structure of pumpkin (<italic>Cucurbita moschata</italic> Duchesne ex Poir) phyllosphere microbiota is influenced by the abundance of the pumpkin powdery mildew pathogen <italic>Podosphaera</italic>. We analyzed the fungal communities of 16 pumpkin leaf samples showing symptoms of powdery mildew disease with different disease severity levels ranging from L1 (least severe) to L4 (most severe) by sequencing the ITS regions of fungal rRNA genes using Illumina MiSeq. The richness and diversity of the fungal community were compared across disease severity levels, and statistical analyses based on OTUs or taxonomic classification were also performed. These results are indicative for new perspectives on the changes of leaf microbiome at different pumpkin powdery mildew disease severity.</p></sec><sec sec-type="materials|methods"><title>Materials and Methods</title><sec><title>Site and sampling</title><p>Leaf samples were randomly collected from pumpkin (<italic>C. moschata</italic>:<italic>nen zao 1</italic>) plants showing symptoms of powdery mildew disease. The samples were collected in June 2015 in the base of Vegetable Research Institute, Changsha, Hunan Province, China. The field was divided into four adjacent experiment areas, planted with the same type of pumpkin NZ number 1. The leaf samples were divided into four groups (L1–L4) based on the proportion of lesion area every leaf; L1 (no lesions), 6%<sup>2</sup>. Leaf samples were collected in separate bags at refrigerated temperature, and were transferred to the laboratory for processing. Each of the 10 leaves in each bag were cut into tiny pieces and mixed. To harvest microbes on the leaf surface, 10 g of leaf were submerged in 100 mL of PBS with 0.01% Tween-80 in a 250 mL sterile conical flask. The flask was shaken at 250 rpm for 30 min at 28 °C, and then subjected to ultrasound for 10 min. The microbes were then harvested using air pump filtration using a 0.22 µm filter. The microfiltration membrane was stored at −20 °C until used.</p></sec><sec><title>DNA extraction and purification</title><p>The MP FastDNA<sup>®</sup> SPIN Kit for soil (MP Biochemicals, Solon, OH, USA) was used to extract DNA from the leaf surface samples according to the manufacturer’s protocol. DNA was extracted from the microbes harvested from the leaf surface. PCR amplicon libraries were prepared for each sample (DNA concentration at 30 ng/µL) using the eukaryotic primers ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) with the forward primer modified to contain a unique 6 nt barcode at the 5′ end. Fungal ITS1 regions were amplified in a total volume of 50 µL that contained 1 µL (5 µM) of each forward and reverse primer, 1.5 µL of dNTP mix (30 mM each), 0.5 µL of 5 U <italic>Taq</italic> DNA polymerase (TaKaRa), 5 mL of 10 × PCR buffer (with Mg<sup>2+</sup>) and 1 µL of DNA. Reaction conditions consisted of an initial denaturation step at 94 °C for 1 min, followed by 35 cycles of denaturation at 94 °C for 20 s, primer annealing at 57 °C for 25 s, and extension at 68 °C for 45 s, and then a final extension at 68 °C for 10 min. PCR products with a bright band of between 250 and 450 bp were collected by agarose gel electrophoresis and purified with an E.Z.N.A.<sup>®</sup> Gel Extraction Kit. The purified PCR amplicons were pooled in equimolar amounts using Qubit (CA, USA) and paired-end sequenced (2 × 250 bp) on an Illumina MiSeq platform by ANNOROAD Gene Technology Co., Ltd. (Beijing, China) according to standard protocols.</p></sec><sec><title>Processing of sequence data</title><p>After the MiSeq sequencing, the raw sequence data reads in fastq format were collected. Separate files were generated based on the forward and reverse directions and the barcodes. Paired end reads were merged using the FLASH program (<xref rid="ref-33" ref-type="bibr">Mago & Salzberg, 2011</xref>). Sequences containing ambiguous ‘N’ were removed. Chimera sequences were detected and removed using UCHIME (<xref rid="ref-15" ref-type="bibr">Edgar et al., 2011</xref>). All sequences with 97% similarity were clustered using the USEARCH software to yield operational taxonomic units (OTUs). Low abundance OTUs (≤2 counts) were eliminated from the OTU table. Representative sequences for each OTU were assigned to taxonomic groups using UNITE database (Version 07.04.2014) (<xref rid="ref-1" ref-type="bibr">Abarenkov et al., 2010</xref>). In this study, all the sequences obtained were deposited in the SRA database short-read archive <ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/sra?term=SRR5075731">SRR5075731</ext-link>–<ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/sra?term=SRR5075746">SRR5075746</ext-link>.</p></sec><sec><title>Statistical analysis</title><p>The Mothur software was used to calculate rarefaction and diversity indices of all the leaf samples based on resampling of OTUs generated by USEARCH (<xref rid="ref-46" ref-type="bibr">Schloss et al., 2009</xref>). Detrended correspondence analysis (DCA) and Venn diagram analysis were performed in subsequent analyses using the vegan package in R (v.3.2.5) (<xref rid="ref-38" ref-type="bibr">Oksanen et al., 2007</xref>). Community differences among the treatments were tested by using Nonparametric multi-response permutation procedures (MRPP, 999 permutations) based on Bray-Curtis distance methods in the R software package using the vegan package (v.3.2.5) (<xref rid="ref-3" ref-type="bibr">Anderson, 2001</xref>; <xref rid="ref-38" ref-type="bibr">Oksanen et al., 2007</xref>). The statistical significance of differences between groups (including Shannon index, inverse Simpson index and relative abundance of the taxonomic subgroups) was assessed by performing a one-way ANOVA followed by Tukey’s multiple comparison post hoc test when comparing several groups. The data are presented as the mean ± SE. Besides, a <italic>P</italic> value of <0.05 was considered to be statistically significant. The software IBM SPSS for Windows, version 22.0 was used to perform statistical analyses.</p><p>To determine whether the overall microbial communities present in the phyllosphere of pumpkin leaves with different disease levels were significantly different, nonparametric multi-response permutation procedures (MRPP) and <bold>Adonis</bold> were used based on Bray–Curtis distance methods in R package vegan (v.3.2.5) (<xref rid="ref-3" ref-type="bibr">Anderson, 2001</xref>; <xref rid="ref-38" ref-type="bibr">Oksanen et al., 2007</xref>).</p></sec></sec><sec sec-type="results"><title>Results</title><sec><title>Fungi communities of the pumpkin phyllosphere</title><p>In total, 797,077 quality sequences were obtained for the four disease severity groups. The mean number of sequences per sample was 49,817, with a range of 39,028–62,150 sequences per sample. In total, 399 operational taxonomic units (OTUs) were detected using the UPARSE-OTU algorithm at the 97% identity cut-off (<xref ref-type="supplementary-material" rid="supp-3">Tables S1</xref> and <xref ref-type="supplementary-material" rid="supp-4">S2</xref>). Rarefaction analysis and the Chao1 estimator indicated that the diversity in these leaf samples was within the same range (<xref ref-type="supplementary-material" rid="supp-1">Fig. S1</xref>).</p><p>The four-way Venn diagrams in <xref ref-type="supplementary-material" rid="supp-2">Fig. S2</xref> show the distribution of the OTUs in the four disease severity groups. One hundred and fifty-five shared OTUs (38.8% of the total eukaryal OTUs) were found among four different groups. There were 10 (2.5%), 21 (5.2%), 14 (3.5%), and 5 OTUs (1.2%) of total eukaryal OTUs were only found in disease severity group L1, L2, L3 and L4, respectively (<xref ref-type="supplementary-material" rid="supp-2">Fig. S2</xref>).</p><p>Four fungal phyla, 15 classes and 36 orders were detected in the phyllosphere of the pumpkin samples (<xref ref-type="table" rid="table-1">Table 1</xref>). The relative abundance of the main fungal phyllospheric populations at the taxonomic levels of Phyla and Class is shown in <xref ref-type="fig" rid="fig-1">Figs. 1A</xref> and <xref ref-type="fig" rid="fig-1">1B</xref>, respectively. The abundance of Fungi_unidentified decreased while Ascomycota increased as disease severity increased with leaf. The heatmap of genus level indicated the most dominant genus was <italic>Podosphaera</italic> (<xref ref-type="fig" rid="fig-2">Fig. 2</xref>), which showed different abundance among four disease severity groups.</p><table-wrap id="table-1" orientation="portrait" position="float"><object-id pub-id-type="doi">10.7717/peerj.4559/table-1</object-id><label>Table 1</label><caption><title>Number of detected phylotypes classified at different taxonomic levels.</title></caption><alternatives><graphic xlink:href="peerj-06-4559-g005"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col></colgroup><thead><tr><th rowspan="1" colspan="1">Disease severity groups</th><th rowspan="1" colspan="1">Phylum</th><th rowspan="1" colspan="1">Class</th><th rowspan="1" colspan="1">Order</th><th rowspan="1" colspan="1">Family</th><th rowspan="1" colspan="1">Genus</th></tr></thead><tbody><tr><td rowspan="1" colspan="1">No. of detected phylotypes</td><td rowspan="1" colspan="1">4</td><td rowspan="1" colspan="1">15</td><td rowspan="1" colspan="1">36</td><td rowspan="1" colspan="1">70</td><td rowspan="1" colspan="1">101</td></tr><tr><td rowspan="1" colspan="1">L1</td><td rowspan="1" colspan="1">3</td><td rowspan="1" colspan="1">14</td><td rowspan="1" colspan="1">35</td><td rowspan="1" colspan="1">63</td><td rowspan="1" colspan="1">86</td></tr><tr><td rowspan="1" colspan="1">L2</td><td rowspan="1" colspan="1">3</td><td rowspan="1" colspan="1">15</td><td rowspan="1" colspan="1">35</td><td rowspan="1" colspan="1">66</td><td rowspan="1" colspan="1">92</td></tr><tr><td rowspan="1" colspan="1">L3</td><td rowspan="1" colspan="1">3</td><td rowspan="1" colspan="1">14</td><td rowspan="1" colspan="1">31</td><td rowspan="1" colspan="1">68</td><td rowspan="1" colspan="1">87</td></tr><tr><td rowspan="1" colspan="1">L4</td><td rowspan="1" colspan="1">4</td><td rowspan="1" colspan="1">13</td><td rowspan="1" colspan="1">30</td><td rowspan="1" colspan="1">53</td><td rowspan="1" colspan="1">67</td></tr></tbody></table></alternatives></table-wrap><fig id="fig-1" orientation="portrait" position="float"><object-id pub-id-type="doi">10.7717/peerj.4559/fig-1</object-id><label>Figure 1</label><caption><title>Relative abundance of fungal classification at the phylum and class level.</title></caption><graphic xlink:href="peerj-06-4559-g001"></graphic></fig><fig id="fig-2" orientation="portrait" position="float"><object-id pub-id-type="doi">10.7717/peerj.4559/fig-2</object-id><label>Figure 2</label><caption><title>Heat map of the top 30 genera detected in all the samples.</title><p>R01–R04, four replicate samples of the L1 level, R11–R14, four replicate samples of the L2 level, R21–R24, four replicate samples of the L3 level, R31–R34, four replicate samples of the L2 level. Different colors represent different relative abundances, red represents the high relative abundance, and green represents the low relative abundance. L1, L2, L3, and L4 are expressed in purple, green, pink, and blue, respectively.</p></caption><graphic xlink:href="peerj-06-4559-g002"></graphic></fig><p>Results of the MRPP analysis of fungal community composition showed an overall significant difference among four treatment levels based on the OTU table (<italic>p</italic> < 0.05) (<xref ref-type="supplementary-material" rid="supp-5">Table S3</xref>). Adonis analysis also indicates that there was a significant difference between groups (<italic>p</italic> < 0.05) (<xref ref-type="supplementary-material" rid="supp-5">Table S3</xref>). The detrended correspondence analysis (DCA) plot in <xref ref-type="fig" rid="fig-3">Fig. 3</xref> shows that the communities detected in leaves with different disease levels were clearly separated.</p><fig id="fig-3" orientation="portrait" position="float"><object-id pub-id-type="doi">10.7717/peerj.4559/fig-3</object-id><label>Figure 3</label><caption><title>Detrended correspondence analysis (DCA).</title><p>L1–L4 indicate the severity level of powdery mildew disease in each pumpkin leaf. <italic>N</italic> = 4.</p></caption><graphic xlink:href="peerj-06-4559-g003"></graphic></fig></sec><sec><title>Correlation between fungal communities and disease severity</title><p>We compared the fungal alpha diversity of the pumpkin leaves using the Shannon and Inverse Simpson diversity indices and OTU numbers (richness). The Shannon index ranged from 0.90 ± 0.09 to 1.87 ± 0.19, the Inverse Simpson index ranged from 1.61 ± 0.10 to 3.12 ± 0.53, and the richness ranged from 110.25 ± 6.85 to 217.00 ± 20.84 for the four disease severity groups. The results indicated that the fungal alpha diversity of the pumpkin leaves decreased significantly with increased disease severity from L2 to L4 (<italic>p</italic> < 0.05) (<xref ref-type="table" rid="table-2">Table 2</xref>). However, alpha diversity in L2 leaves was higher than in L1 leaves.</p><p>The fungal communities were dominated by members of the Ascomycota and the most dominant genus was <italic>Podosphaera</italic> (<xref ref-type="fig" rid="fig-4">Fig. 4</xref>). The abundance of Ascomycota and <italic>Podosphaera</italic> increased with increased disease severity. When the disease severity was greatest (L4), there was less fungal diversity but a greater number of OTUs showed a high level of abundance. 38.8% (155) were present in the phyllosphere of all the groups. The OTU_2, OTU_3, OTU_5, and OTU_9 were identified as Fungi_sp—SH234328.06FU (<ext-link ext-link-type="uri" xlink:href="https://blast.ncbi.nlm.nih.gov/Blast.cgi">https://blast.ncbi.nlm.nih.gov/Blast.cgi</ext-link>, it is matched the sequence NCBI accession <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF800560.1">KF800560.1</ext-link>, as an uncultured eukaryote clone CMH469 18S ribosomal RNA gene, partial sequence, and the sequence similarity reached 98%) at the species level, and accounted for 92.42%, 75.41%, 75.85%, and 14.76% of the sequence reads detected in leaves at disease severity levels L1, L2, L3 and L4, respectively (<italic>p</italic> < 0.05) (<xref ref-type="fig" rid="fig-4">Fig. 4</xref>). OTU_1 was identified as <italic>Podosphaera_fusca</italic>—SH194415.06FU, and accounted for 1.05%, 1.11%, 10.64%, and 77.9% of the sequence reads detected in leaves at disease severity levels L1, L2, L3 and L4, respectively (<italic>p</italic> < 0.05).</p><table-wrap id="table-2" orientation="portrait" position="float"><object-id pub-id-type="doi">10.7717/peerj.4559/table-2</object-id><label>Table 2</label><caption><title>Diversity indices of the communities on leaf surface showed different disease severity.</title><p>The data are presented as the mean ± SE, a <italic>P</italic> value of <0.05 was considered to be statistically significant. The same letter indicates that there were no differences between groups, and different letters (a, b, c) indicate statistically significant differences.</p></caption><alternatives><graphic xlink:href="peerj-06-4559-g006"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col></colgroup><thead><tr><th rowspan="1" colspan="1">Group</th><th rowspan="1" colspan="1">Richness</th><th rowspan="1" colspan="1">Shannon index</th><th rowspan="1" colspan="1">Inverse Simpson index</th><th rowspan="1" colspan="1">Chao1</th></tr></thead><tbody><tr><td rowspan="1" colspan="1">L1</td><td rowspan="1" colspan="1">182.25 ± 4.53a</td><td rowspan="1" colspan="1">1.23 ± 0.03ab</td><td rowspan="1" colspan="1">2.03 ± 0.08ab</td><td rowspan="1" colspan="1">279.47 ± 10.47a</td></tr><tr><td rowspan="1" colspan="1">L2</td><td rowspan="1" colspan="1">217.00 ± 20.84a</td><td rowspan="1" colspan="1">1.87 ± 0.19c</td><td rowspan="1" colspan="1">3.12 ± 0.53c</td><td rowspan="1" colspan="1">283.08 ± 23.85a</td></tr><tr><td rowspan="1" colspan="1">L3</td><td rowspan="1" colspan="1">192.75 ± 27.19a</td><td rowspan="1" colspan="1">1.62 ± 0.16bc</td><td rowspan="1" colspan="1">2.64 ± 0.18bc</td><td rowspan="1" colspan="1">290.35 ± 22.21a</td></tr><tr><td rowspan="1" colspan="1">L4</td><td rowspan="1" colspan="1">110.25 ± 6.85b</td><td rowspan="1" colspan="1">0.90 ± 0.09a</td><td rowspan="1" colspan="1">1.61 ± 0.10a</td><td rowspan="1" colspan="1">181.91 ± 15.71b</td></tr></tbody></table></alternatives></table-wrap><fig id="fig-4" orientation="portrait" position="float"><object-id pub-id-type="doi">10.7717/peerj.4559/fig-4</object-id><label>Figure 4</label><caption><title>Relative abundance of <italic>Podosphaera</italic> and <italic>Fungi_sp—SH234328.06FU</italic> at different severity levels of powdery mildew disease (L1–L4).</title></caption><graphic xlink:href="peerj-06-4559-g004"></graphic></fig></sec></sec><sec><title>Disscusion</title><p>A number of studies have focused on the phyllosphere microorganisms in various plants, but the fungal community composition and diversity of pumpkin leaves infected with powdery mildew has not been reported. In our study, amplicon pyrosequencing of the ITS region of rDNA was used to detect the dynamics of fungal community response to different pumpkin powdery mildew disease severity. The dissimilarity among samples might be owing to the differences in the disease severity, and the pathogenic fungi which could select the related fungi colonize pumpkin leaf surface because of the symbiotic and competitive stresses among microbial species.</p><p>Microorganisms are the largest organisms on our planet and are an important component in the biogeochemical cycling of the earth. Microorganisms also play a crucial role in keeping leaves healthy (<xref rid="ref-4" ref-type="bibr">Baker et al., 2010</xref>) and in maintaining the balance of the ecosystem. Most microorganisms participate in the ecosystem cycle as decomposers. In addition, a variety of beneficial microorganisms colonized on the plant leaves can help to afford plant nutrition and defense against pathogens. Although there are many studies on the plant rhizosphere, there still lacks considerable attention and interest in the microbiology of leaf surfaces pathogens (<xref rid="ref-50" ref-type="bibr">Vorholt, 2012</xref>). Powdery mildew is a common fungal disease that can infect a wide range of plants, including cucurbits such as cucumbers, Luffa spp., melons and watermelons, leading to huge economic losses annually (<xref rid="ref-35" ref-type="bibr">Mcgrath & Shishkoff, 1999</xref>). Among the different species of fungi in the order Erysiphales causing powdery mildew, <italic>Podosphaera xanthii</italic> (a.k.a. <italic>Sphaerotheca fuliginea</italic>) is the most commonly reported cause (<xref rid="ref-35" ref-type="bibr">Mcgrath & Shishkoff, 1999</xref>). The development of high-throughout molecular techniques has helped us understand the microbial composition and structure in different environments and know how microbial diversity changes as the disease severity changes.</p><p>Our study has provided new insights into the impact of the plant pathogen <italic>Podosphaera</italic>, a serious pathogen that also causes pumpkin powdery mildew, on the microorganisms inhabiting the pumpkin phyllosphere. Previous studies have reported that there are usually more unique OTUs in the rhizosphere of healthy soil than in diseased soil (<xref rid="ref-43" ref-type="bibr">Rosenzweig et al., 2012</xref>). In the phyllosphere, there may be the same phenomenon as the soil. In our study, the greatest number of unique OTUs was found at disease severity level L2. Fungi_sp—SH234328.06FU was negatively correlated with disease severity (<xref ref-type="fig" rid="fig-4">Fig. 4</xref>). There may be an antagonistic relationship between Fungi_sp—SH234328.06FU and <italic>Podosphaera_fusca</italic>—SH194415.06FU (<italic>Podosphaera_xanthii</italic>). We will investigate this relationship in a future study. The abundance of Ascomycota and <italic>Podosphaera</italic> was positively correlated with disease severity. As the pathogen of pumpkin powdery mildew, <italic>Podosphaera</italic> was the dominant genus in the heavy symptoms of mildew infection. DCA, MRPP and Adonis revealed significant differences in the composition and structure of the fungal assemblages observed in the four disease severity groups (<xref ref-type="fig" rid="fig-3">Fig. 3</xref>, <xref ref-type="supplementary-material" rid="supp-5">Table S3</xref>), suggesting that the composition and structure of the fungal assemblages altered as the disease severity increased.</p><p>The leaf fungal alpha diversity decreased significantly with increasing disease severity from L2 to L4 (<xref ref-type="table" rid="table-2">Table 2</xref>). This result agrees with findings reported by <xref rid="ref-34" ref-type="bibr">Manching, Balintkurti & Stapleton (2014)</xref>, who analyzed the relationship between southern leaf blight disease severity and maize leaf epiphytic bacterial species richness. It was found that lower species richness (alpha diversity) was correlated with an increase of southern leaf blight disease severity when disease pressure was higher. The decline in overall fungal diversity was enhanced after pathogen stimulation, which also agrees with the results reported by <xref rid="ref-16" ref-type="bibr">Erlacher et al. (2014)</xref>. Interestingly, leaf fungal alpha diversity increased with increasing disease severity from L1 to L2, which suggests that the pathogen may have caused an increase in the fungal community richness at first and then a decrease when disease pressure was higher. It is well known that powdery mildew fungi are obligate biotrophs and will therefore compete for host nutrient reserves and suppress host defense responses. The growth and reproduction of other fungus could be inhibited when disease pressure was higher in the phyllosphere. This study further increases our understanding of the effect of powdery mildew disease on the microbial communities that inhabit the phyllosphere of pumpkin leaves. But there were some limitations in our study: first of all, a relative quantification of phyllosphere microbial populations by high-throughout sequencing unable to accurately determine the content of the species; second, a lack of greenhouse experiment control. In the subsequent experiments, we plan to study phyllosphere microbial communities’ response to different disease severity of pumpkin powdery mildew in the field and greenhouse by artificial inoculation and qPCR quantitative detection.</p></sec><sec sec-type="conclusions"><title>Conclusions</title><p>In our current study, we demonstrated that the plant pathogen <italic>Podosphaera_fusca</italic> can affect the phyllosphere fungal communities of pumpkin. The pathogen caused an increase in the fungal community richness at first and then a decrease when disease pressure was higher. The decline in overall fungal diversity was enhanced after pathogen stimulation. The abundance of an unidentified genus as Fungi_sp—SH234328.06FU was inversely proportional to pathogen community of <italic>Podosphaera</italic>. It could give new perspectives on the changes in the leaf microbiome at different pumpkin powdery mildew disease severities.</p></sec><sec sec-type="supplementary-material" id="supplemental-information"><title> Supplemental Information</title><supplementary-material content-type="local-data" id="supp-1"><object-id pub-id-type="doi">10.7717/peerj.4559/supp-1</object-id><label>Figure S1</label><caption><title>Rarefaction curves for the operational taxonomic units (OTUs)</title><p>R01–R04: four replicate samples of the L1 level; R11–R14: four replicate samples of the L2 level; R21–R24: four replicate samples of the L3 level; R31–R34: four replicate samples of the L2 level. L1, L2, L3, and L4 are expressed in red, green, blue and purple, respectively.</p></caption><media xlink:href="peerj-06-4559-s001.jpeg"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="supp-2"><object-id pub-id-type="doi">10.7717/peerj.4559/supp-2</object-id><label>Figure S2</label><caption><title>Venn diagram showing unique and shared OTUs detected in the phyllosphere of the four disease severity groups (L1, L2, L3 and L4)</title></caption><media xlink:href="peerj-06-4559-s002.eps"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="supp-3"><object-id pub-id-type="doi">10.7717/peerj.4559/supp-3</object-id><label>Table S1</label><caption><title>The unique and shared OTUs and their taxonomic annotation in the four different samples</title></caption><media xlink:href="peerj-06-4559-s003.docx"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="supp-4"><object-id pub-id-type="doi">10.7717/peerj.4559/supp-4</object-id><label>Table S2</label><caption><title>Taxonomic information of each OTU ID</title></caption><media xlink:href="peerj-06-4559-s004.xlsx"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="supp-5"><object-id pub-id-type="doi">10.7717/peerj.4559/supp-5</object-id><label>Table S3</label><caption><title>Statistical analysis of the microbial community composition and structure detected at different disease severity levels</title></caption><media xlink:href="peerj-06-4559-s005.docx"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><sec sec-type="additional-information"><title>Additional Information and Declarations</title><fn-group content-type="competing-interests"><title>Competing Interests</title><fn id="conflict-1" fn-type="COI-statement"><p>The authors declare there are no competing interests.</p></fn></fn-group><fn-group content-type="author-contributions"><title>Author Contributions</title><fn id="contribution-1" fn-type="con"><p><xref ref-type="contrib" rid="author-1">Zhuo Zhang</xref>, <xref ref-type="contrib" rid="author-2">Luyun Luo</xref> and <xref ref-type="contrib" rid="author-4">Xiao Kong</xref> performed the experiments, analyzed the data, prepared figures and/or tables.</p></fn><fn id="contribution-3" fn-type="con"><p><xref ref-type="contrib" rid="author-3">Xinqiu Tan</xref> performed the experiments, analyzed the data, authored or reviewed drafts of the paper.</p></fn><fn id="contribution-5" fn-type="con"><p><xref ref-type="contrib" rid="author-5">Jianguo Yang</xref> performed the experiments.</p></fn><fn id="contribution-6" fn-type="con"><p><xref ref-type="contrib" rid="author-6">Duanhua Wang</xref> performed the experiments, authored or reviewed drafts of the paper.</p></fn><fn id="contribution-7" fn-type="con"><p><xref ref-type="contrib" rid="author-7">Deyong Zhang</xref> analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper.</p></fn><fn id="contribution-8" fn-type="con"><p><xref ref-type="contrib" rid="author-8">Decai Jin</xref> conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the paper, approved the final draft.</p></fn><fn id="contribution-9" fn-type="con"><p><xref ref-type="contrib" rid="author-9">Yong Liu</xref> conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft.</p></fn></fn-group><fn-group content-type="other"><title>Data Availability</title><fn id="addinfo-1"><p>The following information was supplied regarding data availability:</p><p>The sequences obtained were deposited in the SRA database short-read archive <ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/sra?term=SRR5075731">SRR5075731</ext-link>–<ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/sra?term=SRR5075746">SRR5075746</ext-link>.</p></fn></fn-group></sec><ref-list content-type="authoryear"><title>References</title><ref id="ref-1"><label>Abarenkov et al. 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