{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T10:27:47Z","timestamp":1787740067399,"version":"build-2784847793"},"reference-count":17,"publisher":"Wiley","issue":"8","license":[{"start":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T00:00:00Z","timestamp":1786665600000},"content-version":"vor","delay-in-days":13,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T00:00:00Z","timestamp":1785542400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,8]]},"abstract":"Abstract<\/jats:title>\n \n Callose is a \u03b2\u20101,3\u2010glucan polysaccharide deposited at the plant cell wall interface. It is involved in numerous plant physiological processes and responses to both biotic and abiotic stresses. Callose deposition under biotic stress conditions is typically associated with pattern\u2010triggered immunity, which is activated upon recognition of pathogen\u2010associated molecular patterns or damage\u2010associated molecular patterns at the cell wall interface. These depositions reinforce compromised and damaged cell walls caused by pathogen invasion. The standard method for visualizing callose deposition in various plant tissues involves aniline blue staining. Aniline blue fluorochrome preferentially binds to \u03b2\u20101,3\u2010glucans, which enables this staining technique to specifically locate callose deposition. Although multiple protocols for callose detection using aniline blue are available in various model plants, such as\n Arabidopsis<\/jats:italic>\n , there is no optimized method for lignified lateral root tissues after fungal infection. Lignification of roots can hinder the clear visualization of callose depositions; therefore, it is essential to remove them for improved callose detection. Here, we have optimized a robust and reliable method for detecting callose deposition in soybean lateral roots during\n Macrophomina phaseolina<\/jats:italic>\n infections.\n M. phaseolina<\/jats:italic>\n is a filamentous, soil\u2010borne, necrotrophic fungus that causes charcoal rot disease in soybean and other crop plants. Here, we also provide a detailed methodology for soybean root infection with\n M. phaseolina<\/jats:italic>\n using the root\u2010dip method of inoculation, followed by aniline blue staining. Furthermore, we provide a detailed workflow for employing open\u2010source Fiji software together with the Trainable Weka Segmentation (TWS) plugin to detect and count callose structures in fungal\u2010infected root tissues. This protocol may be applicable for detecting callose deposition in other crop plants during fungal infections. \u00a9 2026 Wiley Periodicals LLC.\n <\/jats:p>\n \n Basic Protocol 1<\/jats:bold>\n : Infection assay with\n M. phaseolina<\/jats:italic>\n using root dip method of inoculation\n <\/jats:p>\n \n Basic Protocol 2<\/jats:bold>\n : Staining of\n M. phaseolina\u2010<\/jats:italic>\n infected soybean roots with aniline blue and imaging of stained soybean roots using fluorescence microscopy\n <\/jats:p>\n \n Support Protocol<\/jats:bold>\n : Callose quantification using ImageJ software combined with the TWS plugin\n <\/jats:p>","DOI":"10.1002\/cpz1.70435","type":"journal-article","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T13:36:26Z","timestamp":1786714586000},"source":"Crossref","is-referenced-by-count":0,"title":["Detecting Callose Deposition in Soybean Lateral Roots During Fungal Infections"],"prefix":"10.1002","volume":"6","author":[{"given":"Chetan Veeraganti Naveen","family":"Prakash","sequence":"first","affiliation":[{"name":"Department of Biological Sciences Birla Institute of Technology and Science Pilani (BITS Pilani) India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0600-4534","authenticated-orcid":false,"given":"Muthusaravanan","family":"Sivaramakrishnan","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences Birla Institute of Technology and Science Pilani (BITS Pilani) India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5696-6777","authenticated-orcid":false,"given":"Balakumaran","family":"Chandrasekar","sequence":"additional","affiliation":[{"name":"Department of Bioscience and Bioengineering Indian Institute of Technology Jodhpur (IIT Jodhpur) Jodhpur India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,8,14]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.4236\/abb.2013.48106"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1004496"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.4161\/psb.4.6.8359"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00425\u2010008\u20100812\u20103"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1104\/pp.112.211011"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms21113853"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1104\/pp.19.00560"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.3791\/61702"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1105\/tpc.111.087742"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molp.2019.10.013"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1086716"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1111\/pce.70302"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2025.146005"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1002\/cpz1.869"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2216397120"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1105\/tpc.113.110676"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12864\u2010024\u201010523\u20108"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70435","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpz1.70435","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70435","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T10:16:14Z","timestamp":1787739374000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70435"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8]]},"references-count":17,"journal-issue":{"issue":"8","published-print":{"date-parts":[[2026,8]]}},"alternative-id":["10.1002\/cpz1.70435"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70435","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8]]},"article-number":"e70435"}}