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<label for="email-citation-format" class="action-panel-label">
Format:
</label>
<select id="email-citation-format" name="citation-format" class="action-panel-selector email-citation-format">
<option selected="selected" value="summary">Summary</option>
<option value="summary-text">Summary (text)</option>
<option value="abstract">Abstract</option>
<option value="abstract-text">Abstract (text)</option>
</select>
</div>
<div class="include-supplemental-container">
<input type="checkbox" aria-label="Include MeSH and other data" name="include-supplemental" id="email-include-supplemental" class="email-include-supplemental">
<label for="email-include-supplemental" class="email-include-supplemental-label">MeSH and other data</label>
</div>
<div class="form-field recaptcha ">
<div class="g-recaptcha" id="id-recaptcha" data-sitekey="6LfsWHMdAAAAAClKbtOpjQ2pMjgsGxvv7NdZW9uI"></div>
</div>
<div id="captcha-error-message" class="usa-input-error-message captcha-validation-message" role="alert"></div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="send">
Send email
</button>
<button class="action-panel-cancel"
aria-label="Close 'Email citations' panel"
ref="linksrc=close_email_panel"
aria-controls="email-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="cancel">
Cancel
</button>
</div>
<input type="hidden" name="email-search-details" value="" />
<input type="hidden" name="email-search-details-hash" value="0e42663a6c3bd85498fcb88798998fed7bfdc45d457db35281e41afe13cc0524" />
</form>
</div>
</div>
<div id="collections-action-panel"
class="collections-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-collections-open-panel-enabled="false"
data-collections-open-panel-url-hash="#open-collections-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to Collections
</h3>
<form id="collections-action-panel-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-myncbi-max-collection-name-length="100"
data-add-to-collection-max-amount="1000"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/">
<input type="hidden" name="csrfmiddlewaretoken" value="HNyOxwxNkkiZw7lAdPg1GXVUWoH8oUN7YAiTam09UV5tqyrqQ8T3a5ryuspkhvlr">
<div class="choice-group" role="radiogroup">
<ul class="radio-group-items">
<li>
<input type="radio"
id="collections-action-panel-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_new">
<label for="collections-action-panel-new">Create a new collection</label>
</li>
<li>
<input type="radio"
id="collections-action-panel-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_existing">
<label for="collections-action-panel-existing">Add to an existing collection</label>
</li>
</ul>
</div>
<div class="controls-wrapper">
<div class="action-panel-control-wrap new-collections-controls">
<label for="collections-action-panel-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label>
<input
type="text"
name="add-to-new-collection"
id="collections-action-panel-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="save_share"
data-ga-action="create_collection"
data-ga-label="non_favorties_collection">
<div class="collections-new-name-too-long usa-input-error-message selection-validation-message">
Name must be less than 100 characters
</div>
</div>
<div class="action-panel-control-wrap existing-collections-controls">
<label for="collections-action-panel-add-to-existing" class="action-panel-label">
Choose a collection:
</label>
<select id="collections-action-panel-add-to-existing"
class="action-panel-selector collections-action-add-to-existing"
data-ga-category="save_share"
data-ga-action="select_collection"
data-ga-label="($('#collections-action-add-to-existing').val() === 'Favorites') ? 'Favorites' : 'non_favorites_collection'">
</select>
<div class="collections-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your collection due to an error<br>
<a href="#">Please try again</a>
</div>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="add">
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to Collections' panel"
ref="linksrc=close_collections_panel"
aria-controls="collections-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="bibliography-action-panel"
class="bibliography-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-bibliography-open-panel-enabled="false"
data-bibliography-open-panel-url-hash="#open-bibliography-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to My Bibliography
</h3>
<form id="bibliography-action-panel-form"
class="bibliography-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-add-to-bibliography-max-amount="100"
data-add-to-bibliography-batch-size="10"
data-bibliography-delegates-url="/list-bibliography-delegates/"
data-add-to-bibliography-url="/add-to-bibliography/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-mybib-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/mybibliography/">
<input type="hidden" name="csrfmiddlewaretoken" value="HNyOxwxNkkiZw7lAdPg1GXVUWoH8oUN7YAiTam09UV5tqyrqQ8T3a5ryuspkhvlr">
<div class="action-panel-control-wrap bibliographies-controls">
<div class="choice-group">
<ul class="bibliographies-action-add radio-group-items">
<li>
<input name="bibliography" id="my-bibliography" class="my-bibliography" type="radio" checked/>
<label for="my-bibliography">My Bibliography</label>
</li>
</ul>
</div>
</div>
<div class="bibliographies-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your delegates due to an error<br>
<a href="#">Please try again</a>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore>
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to bibliography' panel"
ref="linksrc=close_bibliography_panel"
aria-controls="bibliography-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="mybib"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="saved-search-action-panel" class="saved-search-action-panel action-panel " aria-hidden="true"
data-saved-search-open-panel-enabled="false"
data-saved-search-open-panel-url-hash="#open-saved-search-panel">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your saved search
</h2>
<form id="saved-search-action-panel-form"
class="saved-search-action-panel-form action-panel-content action-form"
data-create-saved-search-url="/create-saved-search/"
data-try-search-terms-url="/try-search-term/"
data-saved-search-root-url="https://www.ncbi.nlm.nih.gov/myncbi/searches/">
<input type="hidden" name="csrfmiddlewaretoken" value="HNyOxwxNkkiZw7lAdPg1GXVUWoH8oUN7YAiTam09UV5tqyrqQ8T3a5ryuspkhvlr">
<div class="action-panel-control-wrap">
<label for="saved-search-name" class="action-panel-label saved-search-name-label required-field-asterisk">
Name of saved search:
</label>
<input maxlength="200"
type="text"
name="saved-search-name"
id="saved-search-name"
class="saved-search-name"
value=""
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-term" class="action-panel-label required-field-asterisk">
Search terms:
</label>
<textarea name="saved-search-term" id="saved-search-term" class="saved-search-term" required></textarea>
</div>
<div class="test-search-term-wrap">
<a href="#" class="try-search-term">Test search terms</a>
</div>
<div class="choice-group action-panel-extra-margin-top">
<span class="action-panel-label" id="fieldset-label">
Would you like email updates of new search results?
</span>
<fieldset id="saved-search-alert" aria-describedby="fieldset-label">
<legend class="usa-sr-only">Saved Search Alert Radio Buttons</legend>
<ul class="radio-group-items">
<li>
<input type="radio" id="saved-search-alert-yes" class="saved-search-alert-yes" name="saved-search-alert" value="yes" checked>
<label for="saved-search-alert-yes" class="action-panel-label">Yes</label>
</li>
<li>
<input aria-label="No radio input" type="radio" id="saved-search-alert-no" class="saved-search-alert-no" name="saved-search-alert" value="no">
<label for="saved-search-alert-no" class="action-panel-label">No</label>
</li>
</ul>
</fieldset>
</div>
<div class="alert-schedule-wrap">
<div class="action-panel-control-wrap">
<label class="action-panel-label">
Email:
</label>
<span aria-label="Email address" id="saved-search-email" class="action-panel-label"><span class="action-panel-label-bold"></span> (<a class="myncbi-account-settings" href="https://www.ncbi.nlm.nih.gov/account/settings/">change</a>)</span>
</div>
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="saved-search-frequency" class="action-panel-label">
Frequency:
</label>
<select id="saved-search-frequency" class="no-border-panel-selector saved-search-frequency">
<option value="monthly">Monthly</option>
<option value="weekly">Weekly</option>
<option value="daily">Daily</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-monthly-additional">
<label for="saved-search-monthly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-monthly-on-day" class="no-border-panel-selector">
<option value="Sunday">The first Sunday</option>
<option value="Monday">The first Monday</option>
<option value="Tuesday">The first Tuesday</option>
<option value="Wednesday">The first Wednesday</option>
<option value="Thursday">The first Thursday</option>
<option value="Friday">The first Friday</option>
<option value="Saturday">The first Saturday</option>
<option value="day">The first day</option>
<option value="weekday">The first weekday</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-weekly-additional">
<label for="saved-search-weekly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-weekly-on-day" class="no-border-panel-selector saved-search-weekly-on-day">
<option value="Sunday">Sunday</option>
<option value="Monday">Monday</option>
<option value="Tuesday">Tuesday</option>
<option value="Wednesday">Wednesday</option>
<option value="Thursday">Thursday</option>
<option value="Friday">Friday</option>
<option value="Saturday">Saturday</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-report" class="action-panel-label">
Report format:
</label>
<select id="saved-search-report" class="no-border-panel-selector saved-search-report">
<option value="DocSum">Summary</option>
<option value="DocSumText">Summary (text)</option>
<option value="Abstract">Abstract</option>
<option value="AbstractText">Abstract (text)</option>
<option value="MEDLINE">PubMed</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-amount" class="action-panel-label">
Send at most:
</label>
<select id="saved-search-amount" class="no-border-panel-selector saved-search-amount">
<option value="1">1 item</option>
<option value="5" selected>5 items</option>
<option value="10">10 items</option>
<option value="20">20 items</option>
<option value="50">50 items</option>
<option value="100">100 items</option>
<option value="200">200 items</option>
</select>
</div>
<div>
<input type="checkbox" id="saved-search-send-if-no-result" class="saved-search-send-if-no-result" name="saved-search-send-if-no-result">
<label for="saved-search-send-if-no-result" class="action-panel-label smaller-checkbox">
Send even when there aren't any new results
</label>
</div>
<div class="action-panel-control-wrap option-text-in-email-wrap">
<label for="saved-search-email-text" class="action-panel-label">
Optional text in email:
</label>
<textarea name="saved-search-email-text"
id="saved-search-email-text"
class="saved-search-email-text"></textarea>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Saving..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Save
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your saved search' panel"
ref="linksrc=close_saved_search_panel"
aria-controls="saved-search-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="citation-manager-action-panel" class="citation-manager-action-panel action-panel" aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Create a file for external citation management software
</h2>
<form id="citation-manager-action-panel-form"
class="action-panel-content action-form"
action="/results-export-ids/"
data-by-search-action="/results-export-search-data/"
data-by-ids-action="/results-export-ids/"
method="post"
data-by-search-method="post"
data-by-ids-method="post">
<input type="hidden" name="csrfmiddlewaretoken" value="HNyOxwxNkkiZw7lAdPg1GXVUWoH8oUN7YAiTam09UV5tqyrqQ8T3a5ryuspkhvlr">
<input name="results-format" type="hidden" value="pubmed"/>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="save">
Create file
</button>
<button class="action-panel-cancel"
aria-label="Close 'Send citations to citation manager' panel"
ref="linksrc=close_citation_manager_panel"
aria-controls="citation-manager-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="rss-action-panel" class="rss-action-panel action-panel " aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your RSS Feed
</h2>
<form id="rss-action-panel-form"
class="rss-action-panel-form action-panel-content action-form"
data-create-rss-feed-url="/create-rss-feed-url/"
data-search-form-term-value="">
<input type="hidden" name="csrfmiddlewaretoken" value="HNyOxwxNkkiZw7lAdPg1GXVUWoH8oUN7YAiTam09UV5tqyrqQ8T3a5ryuspkhvlr">
<div class="action-panel-control-wrap">
<label for="rss-name" class="action-panel-label required-field-asterisk">
Name of RSS Feed:
</label>
<input maxlength="200"
placeholder="Name"
type="text"
name="rss-name"
id="rss-name"
class="rss-name"
value=''
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="rss-limit-wrap">
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="rss-limit" class="action-panel-label">
Number of items displayed:
</label>
<select id="rss-limit" class="no-border-panel-selector rss-limit">
<option value="5">5</option>
<option value="10">10</option>
<option value="15" selected="selected">15</option>
<option value="20">20</option>
<option value="50">50</option>
<option value="100">100</option>
</select>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Creating..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Create RSS
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your RSS' panel"
ref="linksrc=close_rss_panel"
aria-controls="rss-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
<div class="action-panel-control-wrap rss-link-copy-wrap">
<label for="rss-link" class="usa-sr-only">RSS Link</label>
<input placeholder="Your RSS Feed Link" type="text" name="rss-link" id="rss-link" class="rss-link" title="RSS Link">
<button
type="button"
disabled
class="rss-link-copy-button disabled"
data-ga-category="save_share"
data-ga-action="rss"
data-ga-label="copy">
Copy
</button>
</div>
</form>
</div>
</div>
</div>
</div>
<div class="article-page" id="article-page" data-article-pmid="27223617">
<aside class="page-sidebar">
<div class="inner-wrap">
<div class="full-text-links">
<div class="full-view">
<h3 class="title">
Full text links
</h3>
<div class="full-text-links-list">
<a class="link-item
dialog-focus"
href="https://dx.plos.org/10.1371/journal.pone.0156247"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=False&amp;PrId=4656&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=27223617&amp;db=pubmed&amp;nlmid=101285081"
title="See full text options at Public Library of Science"
data-ga-category="full_text"
data-ga-action="Public Library of Science"
data-ga-label="27223617"
><img src="https://cdn.ncbi.nlm.nih.gov/corehtml/query/egifs/https:--journals.plos.org-resource-img-external-pone_120x30.png" alt="Public Library of Science full text link"><span class="text">
Public Library of Science
</span></a><a class="link-item
pmc
"
href="https://pmc.ncbi.nlm.nih.gov/articles/pmid/27223617/"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=True&amp;PrId=3494&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=27223617&amp;db=pubmed&amp;nlmid=101285081"
title="Free full text at PubMed Central"
data-ga-category="full_text"
data-ga-action="PMC"
data-ga-label="27223617"
><span class="text">
Free PMC article
</span></a>
</div>
</div>
<div class="short-view">
<a href="#" class="full-text-links-button full-text-links-dialog-trigger">
Full text links
</a>
</div>
</div>
<div class="actions-buttons sidebar"><h3 class="title">Actions</h3><div class="inner-wrap"><button class="citation-button citation-dialog-trigger"
aria-label="Open dialog with citation text in different styles"
data-ga-category="save_share"
data-ga-action="cite"
data-ga-label="open"
data-all-citations-url="/27223617/citations/"
data-citation-style="nlm"
data-pubmed-format-link="/27223617/export/"><span class="button-label">Cite</span></button><link type="text/css" href="ncbi-overlay-block/src/overlay-block.css"><div class="collections-button-container" data-article-id="27223617" data-article-db="pubmed"><button class="collections-button collections-dialog-trigger"
aria-label="Save article in MyNCBI collections."
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_button"
data-collections-open-dialog-enabled="false"
data-collections-open-dialog-url="https://account.ncbi.nlm.nih.gov/?back_url=https%3A%2F%2Fpubmed.ncbi.nlm.nih.gov%2F27223617%2F%23open-collections-dialog"
data-in-collections="false"><span class="button-label">Collections</span></button><div class="overlay" role="dialog"><div id="collections-action-dialog"
class="dialog collections-dialog"
aria-hidden="true"><div class="title">Add to Collections</div><div class="collections-action-panel action-panel"><form id="collections-action-dialog-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-myncbi-max-collection-name-length="100"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/"><input type="hidden" name="csrfmiddlewaretoken" value="HNyOxwxNkkiZw7lAdPg1GXVUWoH8oUN7YAiTam09UV5tqyrqQ8T3a5ryuspkhvlr"><div class="choice-group" role="radiogroup"><ul class="radio-group-items"><li><input type="radio"
id="collections-action-dialog-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_new"><label for="collections-action-dialog-new">Create a new collection</label></li><li><input type="radio"
id="collections-action-dialog-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_existing"><label for="collections-action-dialog-existing">Add to an existing collection</label></li></ul></div><div class="controls-wrapper"><div class="action-panel-control-wrap new-collections-controls"><label for="collections-action-dialog-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label><input
type="text"
name="add-to-new-collection"
id="collections-action-dialog-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="collections_button"
data-ga-action="create_collection"
data-ga-label="non_favorties_collection"><div class="collections-new-name-too-long usa-input-error-message selection-validation-message">
Name must be less than 100 characters
</div></div><div class="action-panel-control-wrap existing-collections-controls"><label for="collections-action-dialog-add-to-existing" class="action-panel-label">
Choose a collection:
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</a><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China." href="#short-view-affiliation-2" ref="linksrc=author_aff">
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data-ga-label="Xiaoyu Wang">Xiaoyu Wang</a><sup class="affiliation-links"><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China." href="#short-view-affiliation-2" ref="linksrc=author_aff">
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data-ga-label="Rongsheng Zhang">Rongsheng Zhang</a><sup class="affiliation-links"><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China." href="#short-view-affiliation-2" ref="linksrc=author_aff">
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data-ga-label="Zhiyi Chen">Zhiyi Chen</a><sup class="affiliation-links"><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="College of Plant Protection, Nanjing Agriculture University, Nanjing 210095, China." href="#short-view-affiliation-1" ref="linksrc=author_aff">
1
</a><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China." href="#short-view-affiliation-2" ref="linksrc=author_aff">
2
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<div class="affiliations">
<h3 class="title">
Affiliations
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<li data-affiliation-id="short-view-affiliation-1"
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><sup class="key">1</sup> College of Plant Protection, Nanjing Agriculture University, Nanjing 210095, China.</li>
<li data-affiliation-id="short-view-affiliation-2"
id="short-view-affiliation-2"
><sup class="key">2</sup> Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.</li>
<li data-affiliation-id="short-view-affiliation-3"
id="short-view-affiliation-3"
><sup class="key">3</sup> School of Life Science and Chemical Engineering, Huaiyin Institute of Technology, Huaiyin 223003, China.</li>
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Abstract
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<p>
<strong class="sub-title">
Aims:
</strong>
This study examined the contribution of GltB on biofilm formation and biocontrol efficiency of B. subtilis Bs916.
</p>
<p>
<strong class="sub-title">
Methods and results:
</strong>
The gltB gene was identified through a biofilm phenotype screen and a bioinformatics analysis of serious biofilm formation defects, and then a gltB single knockout mutant was constructed using homologous recombination. This mutant demonstrated severe deficits in biofilm formation and colonisation along with significantly altered production ofγ-polyglutamate (γ-PGA) and three lipopeptide antibiotics (LPs) as measured by a transcriptional analysis of both the wild type B. subtilis Bs916 and the gltB mutant. Consequently, the mutant strain retained almost no antifungal activity against Rhizoctonia solani and exhibited decreased biocontrol efficiency against rice sheath blight. Very few gltB mutant cells colonised the rice stem, and they exhibited no significant nutrient chemotaxis compared to the wild type B. subtilis Bs916. The mechanism underlying these deficits in the gltB mutant appears to be decreased significantly in production of γ-PGA and a reduction in the production of both bacillomycin L and fengycin. Biofilm restoration of gltB mutant by additionγ-PGA in the EM medium demonstrated that biofilm formation was able to restore significantly at 20 g/L.
</p>
<p>
<strong class="sub-title">
Conclusions:
</strong>
GltB regulates biofilm formation by altering the production ofγ-PGA, the LPs bacillomycin L and fengcin and influences bacterial colonisation on the rice stem, which consequently leads to poor biocontrol efficiency against rice sheath blight.
</p>
<p>
<strong class="sub-title">
Significance and impact of study:
</strong>
This is the first report of a key regulatory protein (GltB) that is involved in biofilm regulation and its regulation mechanism and biocontrol efficiency by B. subtilis.
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Conflict of interest statement
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<p xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:p1="http://pubmed.gov/pub-one"><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p>
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<div class="figures" id="figures">
<h2 class="title">
Figures
</h2>
<div class="figures-list" id="slides-container"
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data-image-width="765"
data-image-height="554"
data-image-alt="Fig 1"
data-pmc-id="PMC4880196"
data-figure-id="pone.0156247.g001">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-0"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ffc/4880196/de2a320328c4/pone.0156247.g001.gif"
alt="Fig 1" />
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<strong class="figure-label">
<p> Fig 1. Changes in biofilm formation due… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Fig 1. Changes in biofilm formation due to the <i> B </i> . <i> subtilis </i> Bs916 and Δ… </p>
</strong>
</div>
<figcaption id="figure-caption-0" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 1. Changes in biofilm formation due to the <i>B</i>. <i>subtilis</i> Bs916 and Δ<i>gltB</i> mutants in MSgg culture medium with 20 μg/mL Congo Red and 10 μg/mL Coomassie brilliant blue.
</strong>
<div class="figure-caption-contents">(1) <i>B</i>. <i>subtilis</i> Bs916 biofilm was dense and solid with clear lines. In contrast, the Δ<i>gltB</i> mutant formed an uneven biofilm with an irregular shape. (2) The net weight of the Δ<i>gltB</i> mutant biofilm was more than three times less than the net weight of the WT <i>B</i>. <i>subtilis</i> Bs916 biofilm.</div>
</figcaption>
</figure>
<figure class="figure-item "
itemscope itemtype="http://schema.org/ImageObject"
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data-label-slug="fig-2">
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href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ffc/4880196/a9f55568cc43/pone.0156247.g002.jpg"
itemprop="contentUrl"
aria-describedby="figure-caption-1"
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data-image-width="660"
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data-image-alt="Fig 2"
data-pmc-id="PMC4880196"
data-figure-id="pone.0156247.g002">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-1"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ffc/4880196/5fc6a6523f4e/pone.0156247.g002.gif"
alt="Fig 2" />
</a>
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<strong class="figure-label">
<p> Fig 2. Differences in antibacterial activity against… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Fig 2. Differences in antibacterial activity against <i> R </i> . <i> solani </i> between WT <i> B </i> . <i> subtilis… </i> </p>
</strong>
</div>
<figcaption id="figure-caption-1" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 2. Differences in antibacterial activity against <i>R</i>. <i>solani</i> between WT <i>B</i>. <i>subtilis</i> Bs916 and the Δ<i>gltB</i> mutant.
</strong>
<div class="figure-caption-contents">The Δ<i>gltB</i> mutant completely lost its antibacterial activity compared to the WT <i>B</i>. <i>subtilis</i> Bs916.</div>
</figcaption>
</figure>
<figure class="figure-item "
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href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ffc/4880196/3a58c7b36c1f/pone.0156247.g003.jpg"
itemprop="contentUrl"
aria-describedby="figure-caption-2"
role="button"
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data-pmc-id="PMC4880196"
data-figure-id="pone.0156247.g003">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-2"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ffc/4880196/201562078561/pone.0156247.g003.gif"
alt="Fig 3" />
</a>
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<div class="caption-wrap">
<strong class="figure-label">
<p> Fig 3. Colonisation of the rice plant… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Fig 3. Colonisation of the rice plant against rice sheath blight. </p>
</strong>
<div class="figure-caption-contents"><p> Over time, the number… </p></div>
</div>
<figcaption id="figure-caption-2" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 3. Colonisation of the rice plant against rice sheath blight.
</strong>
<div class="figure-caption-contents">Over time, the number of both WT <i>B</i>. <i>subtilis</i> Bs916 and Δ<i>gltB</i> mutant cells initially increased and then began to decrease. However, although WT <i>B</i>. <i>subtilis</i> Bs916 could produce normal clusters of cells, the Δ<i>gltB</i> mutant also demonstrated this same trend. By the last time point (i.e., 15 d), there were very few cells in either the WT <i>B</i>. <i>subtilis</i> Bs916 or the Δ<i>gltB</i> mutant, and the clustering of WT <i>B</i>. <i>subtilis</i> Bs916s had disappeared.</div>
</figcaption>
</figure>
<figure class="figure-item "
itemscope itemtype="http://schema.org/ImageObject"
itemprop="associatedMedia"
data-slide-index="3"
data-label-slug="fig-4">
<a class="figure-link"
href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ffc/4880196/b4f576a3bd3f/pone.0156247.g004.jpg"
itemprop="contentUrl"
aria-describedby="figure-caption-3"
role="button"
data-image-width="660"
data-image-height="437"
data-image-alt="Fig 4"
data-pmc-id="PMC4880196"
data-figure-id="pone.0156247.g004">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-3"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ffc/4880196/0287276bcf9c/pone.0156247.g004.gif"
alt="Fig 4" />
</a>
<meta itemprop="width" itemtype="http://schema.org/ImageObject" content="660">
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<div class="caption-wrap">
<strong class="figure-label">
<p> Fig 4 </p>
</strong>
<div class="figure-caption-contents"><b> Antibiotic secretion of bacillomycin L… </b></div>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Fig 4 </p>
</strong>
<div class="figure-caption-contents"><b> Antibiotic secretion of bacillomycin L (a), fengycin (c), and surfactin (b). </b> The Δ…</div>
</div>
<figcaption id="figure-caption-3" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 4
</strong>
<div class="figure-caption-contents"><b>Antibiotic secretion of bacillomycin L (a), fengycin (c), and surfactin (b).</b> The Δ<i>gltB</i> mutant no longer secreted bacillomycin L or fengycin. In contrast, the Δ<i>gltB</i> mutant produced significantly higher (approximately five times higher) levels of surfactin compared to the WT <i>B</i>. <i>subtilis</i> Bs916.</div>
</figcaption>
</figure>
<figure class="figure-item "
itemscope itemtype="http://schema.org/ImageObject"
itemprop="associatedMedia"
data-slide-index="4"
data-label-slug="fig-5">
<a class="figure-link"
href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ffc/4880196/80a7bd8412fa/pone.0156247.g005.jpg"
itemprop="contentUrl"
aria-describedby="figure-caption-4"
role="button"
data-image-width="660"
data-image-height="396"
data-image-alt="Fig 5"
data-pmc-id="PMC4880196"
data-figure-id="pone.0156247.g005">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-4"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ffc/4880196/14968346a18d/pone.0156247.g005.gif"
alt="Fig 5" />
</a>
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<div class="caption-wrap">
<strong class="figure-label">
<p> Fig 5. γ-PGA and glutamate content of… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Fig 5. γ-PGA and glutamate content of WT <i> B </i> . <i> subtilis </i> Bs916 and the Δ… </p>
</strong>
</div>
<figcaption id="figure-caption-4" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 5. γ-PGA and glutamate content of WT <i>B</i>. <i>subtilis</i> Bs916 and the Δ<i>gltB</i> mutant in the process of biofilm formation.
</strong>
<div class="figure-caption-contents">For glutamate detection, in addition to standard L-glutamate and double dilution to <i>B</i>. <i>subtilis</i> Bs916, all treatments were diluted tenfold for detection. For γ-PGA detection, all treatments were also diluted tenfold for detection.</div>
</figcaption>
</figure>
<figure class="figure-item "
itemscope itemtype="http://schema.org/ImageObject"
itemprop="associatedMedia"
data-slide-index="5"
data-label-slug="fig-6">
<a class="figure-link"
href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ffc/4880196/de1708700b1f/pone.0156247.g006.jpg"
itemprop="contentUrl"
aria-describedby="figure-caption-5"
role="button"
data-image-width="773"
data-image-height="991"
data-image-alt="Fig 6"
data-pmc-id="PMC4880196"
data-figure-id="pone.0156247.g006">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-5"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ffc/4880196/a8aaaa4c1368/pone.0156247.g006.gif"
alt="Fig 6" />
</a>
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<strong class="figure-label">
<p> Fig 6. Biofilm restoration in the Δ… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Fig 6. Biofilm restoration in the Δ <i> gltB </i> mutant. </p>
</strong>
<div class="figure-caption-contents"><p> A final concentration of 10 g/L, 20… </p></div>
</div>
<figcaption id="figure-caption-5" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 6. Biofilm restoration in the Δ<i>gltB</i> mutant.
</strong>
<div class="figure-caption-contents">A final concentration of 10 g/L, 20 g/L, and 30 g/Lγ-PGA solution were added into the Δ<i>gltB</i> mutant in 4mL EM medium. The final concentration of 10 g/L and 20 g/Lγ-PGA were able to recover biofilm formation in the Δ<i>gltB</i> mutant, however, high final concentration of 30 g/Lγ-PGA inhibited biofilm formation in WT <i>B</i>. <i>subtilis</i> Bs916 and the Δ<i>gltB</i> mutant.</div>
</figcaption>
</figure>
<figure class="figure-item tail"
itemscope itemtype="http://schema.org/ImageObject"
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data-label-slug="fig-7">
<a class="figure-link"
href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ffc/4880196/3bff413cb63f/pone.0156247.g007.jpg"
itemprop="contentUrl"
aria-describedby="figure-caption-6"
role="button"
data-image-width="580"
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data-image-alt="Fig 7"
data-pmc-id="PMC4880196"
data-figure-id="pone.0156247.g007">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-6"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ffc/4880196/da2424a3de3c/pone.0156247.g007.gif"
alt="Fig 7" />
</a>
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<strong class="figure-label">
<p> Fig 7. Biofilm restoration by <i> B </i> .… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Fig 7. Biofilm restoration by <i> B </i> . <i> subtilis </i> Bs916sγ-PGA in the Δ <i> gltB </i> mutant. </p>
</strong>
<div class="figure-caption-contents"><p> The Δ <i> gltB… </i> </p></div>
</div>
<figcaption id="figure-caption-6" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 7. Biofilm restoration by <i>B</i>. <i>subtilis</i> Bs916sγ-PGA in the Δ<i>gltB</i> mutant.
</strong>
<div class="figure-caption-contents">The Δ<i>gltB</i> mutant grew next to <i>B</i>. <i>subtilis</i> Bs916 on solid EM plate with 1.5% agar to observe their biofilm formation. Biofilm formation of Δ<i>gltB</i> mutant was no significant restoration before 36 h. But in 72h, its biofilm formation had been restored to most parts.</div>
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