{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,21]],"date-time":"2025-10-21T15:15:35Z","timestamp":1761059735228,"version":"build-2065373602"},"reference-count":54,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2014,5,20]],"date-time":"2014-05-20T00:00:00Z","timestamp":1400544000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Bioinsecticides with lower concentrations of endospores\/crystals and without loss of efficiency are economically advantageous for pest biocontrol. In addition to Cry proteins, other Bacillus thuringiensis (Bt) toxins in culture supernatants (SN) have biocontrol potential (e.g., Vip3A, Cry1I, Sip1), whereas others are unwanted (\u03b2-exotoxins), as they display widespread toxicity across taxa. A strain simultaneously providing distinct toxin activities in crystals and SN would be desirable for bioinsecticides development; however, strains secreting \u03b2-exotoxins should be discarded, independently of other useful entomotoxins. Entomotoxicity of crystals and SN from a Brazilian Bt tolworthi strain (Btt01) was tested against Spodoptera frugiperda to assess the potential for biocontrol-product development based on more than one type of toxin\/activity.  Tests showed that 107 endospores mL\u22121 caused &gt;80% of larvae mortality, suggesting Btt01 may be used in similar concentrations as those of other Bt-based biopesticides. When it was applied to cornfields, a significant 60% reduction of larvae infestation was observed. However, bioassays with Btt01 SN revealed a thermostable toxic activity. Physicochemical characterization strongly suggests the presence of unwanted \u03b2-exotoxins, with  isolate-specific temporal variation in its secretion. Knowledge of the temporal pattern of secretion\/activity in culture for all forms of toxins produced by a single strain is required to both detect useful activities and avoid the potential lack of identification of undesirable toxins. These findings are discussed in the contexts of commercial Bt product development, advantages of multiple-activity strains, and care and handling recommended for large-scale fermentation systems.<\/jats:p>","DOI":"10.3390\/toxins6051598","type":"journal-article","created":{"date-parts":[[2014,5,20]],"date-time":"2014-05-20T11:41:17Z","timestamp":1400586077000},"page":"1598-1614","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Requirement of Simultaneous Assessment of Crystal- and Supernatant-Related Entomotoxic Activities of Bacillus thuringiensis Strains for Biocontrol-Product Development"],"prefix":"10.3390","volume":"6","author":[{"given":"Ronaldo","family":"Arg\u00f4lo-Filho","sequence":"first","affiliation":[{"name":"Department of Biological Sciences (DCB), State University  of Santa Cruz (UESC), Rod. BR 415, Km-16, Ilh\u00e9us-BA 45662-900, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robson","family":"Costa","sequence":"additional","affiliation":[{"name":"Farroupilha Laboratory, Av. Cica No. 555, Patos de Minas-MG 38706-420, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniele","family":"Pinheiro","sequence":"additional","affiliation":[{"name":"Embrapa Maize and Sorghum, Rod. MG 424, Km 65, Sete Lagoas-MG 35701-970, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"F\u00e1bio","family":"Corr\u00eaa","sequence":"additional","affiliation":[{"name":"Department Exact and Technological Sciences (DCET), State University of Santa Cruz (UESC), Rod. BR 415, Km-16, Ilh\u00e9us-BA 45662-900, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fernando","family":"Valicente","sequence":"additional","affiliation":[{"name":"Embrapa Maize and Sorghum, Rod. MG 424, Km 65, Sete Lagoas-MG 35701-970, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alan","family":"Pomella","sequence":"additional","affiliation":[{"name":"Farroupilha Laboratory, Av. Cica No. 555, Patos de Minas-MG 38706-420, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leandro","family":"Loguercio","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences (DCB), State University  of Santa Cruz (UESC), Rod. BR 415, Km-16, Ilh\u00e9us-BA 45662-900, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2014,5,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1128\/mr.50.1.1-24.1986","article-title":"Bacillus thuringiensis and related insect pathogens","volume":"50","author":"Aronson","year":"1986","journal-title":"Microbiol. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"807","DOI":"10.1128\/MMBR.62.3.807-813.1998","article-title":"Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins","volume":"62","author":"Crickmore","year":"1998","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1016\/j.tim.2010.02.006","article-title":"Bacillus thuringiensis: An impotent pathogen?","volume":"18","author":"Raymond","year":"2010","journal-title":"Trends Microbiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1128\/MMBR.62.3.775-806.1998","article-title":"Bacillus thuringiensis and its pesticidal crystal proteins","volume":"62","author":"Schnepf","year":"1998","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"8426","DOI":"10.1073\/pnas.0409324102","article-title":"Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect adaptation to pyramided plants","volume":"102","author":"Zhao","year":"2005","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1111\/j.1432-1033.1989.tb15096.x","article-title":"Improved purification and biochemical properties of phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis","volume":"185","author":"Kupke","year":"1989","journal-title":"Eur. J. Biochem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"620","DOI":"10.1016\/S0006-291X(03)01228-2","article-title":"Constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1","volume":"307","author":"Arora","year":"2003","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"713","DOI":"10.1007\/s00253-006-0332-7","article-title":"Discovery and characterization of Sip1A: A novel secreted protein from Bacillus thuringiensis with activity against coleopteran larvae","volume":"72","author":"Donovan","year":"2006","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"5389","DOI":"10.1073\/pnas.93.11.5389","article-title":"Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects","volume":"93","author":"Estruch","year":"1996","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1118","DOI":"10.1135\/cccc19691118","article-title":"The structure of exotoxin of Bacillus thuringiensis var. gelechaiae","volume":"34","author":"Farkas","year":"1969","journal-title":"Collect. Czech. Chem. Commun."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3172","DOI":"10.1128\/jb.172.6.3172-3179.1990","article-title":"Identification of \u03b2-exotoxin production, plasmids encoding \u03b2-exotoxin, and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography","volume":"172","author":"Levinson","year":"1990","journal-title":"J. Bacteriol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"619","DOI":"10.1042\/bj1270619","article-title":"Differential inhibition of mammalian ribonucleic acid polymerases by an exotoxin from Bacillus thuringiensis: The direct observation of nucleoplasmic ribonucleic acid polymerase activity in intact nuclei","volume":"127","author":"Beebee","year":"1972","journal-title":"Biochem. J."},{"key":"ref_13","unstructured":"Demarchi, M. (2011). An\u00e1lise da Conjuntura Agropecu\u00e1ria, Safra 2011\/12: Milho, Secretary of Agriculture and Food Supply\u2014Department of Rural Economy."},{"key":"ref_14","unstructured":"Cruz, I. (1995). A Lagarta-do-Cartucho na Cultura do Milho, Embrapa-CNPMS. 21 Technical Circular."},{"key":"ref_15","first-page":"46","article-title":"Milho Bt: Alternativa biotecnol\u00f3gica para controle biol\u00f3gico de insetos-praga","volume":"24","author":"Loguercio","year":"2002","journal-title":"Biotecnolog. Ci\u00eanc. Desenvolv."},{"key":"ref_16","first-page":"21","article-title":"Susceptibility of fall armyworm collected from different plant hosts to selected insecticides and transgenic Bt cotton","volume":"1","author":"Adamczyk","year":"1997","journal-title":"J. Cotton Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1653\/024.094.0221","article-title":"Laboratory toxicity and field efficacy of selected insecticides against fall armyworm (Lepidoptera: Noctuidae)","volume":"94","author":"Hardke","year":"2011","journal-title":"Fla. Entomol."},{"key":"ref_18","first-page":"56","article-title":"Pesticide application and its adverse impact on health: Evidences from kerala","volume":"1","author":"Tholkappian","year":"2011","journal-title":"Int. J. Sci. Technol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"396","DOI":"10.1046\/j.1439-0418.2003.00775.x","article-title":"Antifeedant and toxic activity of Adhatoda vasica leaf extract against Spodoptera littoralis (Lep., Noctuidae)","volume":"127","author":"Sadek","year":"2003","journal-title":"J. Appl. Entomol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1590\/S1519-566X2005000100010","article-title":"Effect of Baculovirus spodoptera isolates in Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) larvae and their characterization by RAPD","volume":"34","author":"Barreto","year":"2005","journal-title":"Neotrop. Entomol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"759","DOI":"10.1653\/0015-4040(2007)90[759:POFALN]2.0.CO;2","article-title":"Parasitoids of fall armyworm (Lepidoptera: Noctuidae) from a traditional maize crop in the mexican state of yucatan","volume":"90","year":"2007","journal-title":"Fla. Entomol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1590\/S1516-89132008000200018","article-title":"Effect of the Aqueous extracts of the seeds of Talisia esculenta and Sapindus saponaria on fall armyworm","volume":"51","author":"Santos","year":"2008","journal-title":"Braz. Arch. Biol. Technol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"466","DOI":"10.1046\/j.1365-2672.2002.01545.x","article-title":"Diversity of Bacillus thuringiensis strains from Colombia with insecticidal activity against Spodoptera frugiperda (Lepidoptera: Noctuidae)","volume":"92","author":"Arango","year":"2002","journal-title":"J. Appl. Microbiol."},{"key":"ref_24","first-page":"7029","article-title":"Genetic variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) populations from Latin America is associated with variations in susceptibility to Bacillus thuringiensis Cry toxins","volume":"77","author":"Monnerat","year":"2005","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_25","first-page":"195","article-title":"Molecular characterization of the Bacillus thuringiensis (Berliner) strains 344 and 1644, efficient against fall armyworm Spodoptera frugiperda (J.E. Smith)","volume":"7","author":"Valicente","year":"2008","journal-title":"Milho Sorgo"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1046\/j.1472-765X.2001.00920.x","article-title":"Association of PCR and feeding bioassays as a large-scale method to screen tropical Bacillus thuringiensis isolates for a cry constitution with higher insecticidal effect against Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae","volume":"32","author":"Loguercio","year":"2001","journal-title":"Lett. Appl. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1590\/S1519-566X2003000400014","article-title":"Bacillus thuringiensis survey in Brazil: Geographical distribution and insecticidal activity against Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)","volume":"32","author":"Valicente","year":"2003","journal-title":"Neotrop. Entomol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1184","DOI":"10.1002\/ps.2206","article-title":"Growth variation among Bacillus thuringiensis strains can affect screening procedures for supernatant-secreted toxins against insect pests","volume":"67","author":"Gomes","year":"2011","journal-title":"Pest Manag. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"464","DOI":"10.1139\/w99-032","article-title":"Characterization of Bacillus thuringiensis subsp. kurstaki strain S93 effective against the fall armyworm (Spodoptera frugiperda)","volume":"45","author":"Dias","year":"1999","journal-title":"Can. J. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1016\/S0021-9673(03)00422-9","article-title":"Determination of spore concentration in Bacillus thuringiensis through the analysis of dipicolinate by capillary zone electrophoresis","volume":"994","author":"He","year":"2003","journal-title":"J. Chromatogr. A"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"675","DOI":"10.1590\/S0301-80591999000400010","article-title":"Insecticidal activity of culture supernatants from Bacillus thuringiensis Berliner strains against Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) larvae","volume":"28","author":"Barreto","year":"1999","journal-title":"An. Soc. Entomol. Bras."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1046\/j.1365-2672.2002.01694.x","article-title":"Combined analysis of supernatant-based feeding bioassays and PCR as a first-tier screening strategy for Vip-derived activities in Bacillus thuringiensis strains effective against tropical fall armyworm","volume":"93","author":"Loguercio","year":"2002","journal-title":"J. Appl. Microbiol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1042\/bj1140477","article-title":"A purification and some properties of an insecticidal exotoxin from Bacillus thuringiensis Berliner","volume":"114","author":"Bond","year":"1969","journal-title":"Biochem. J."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1046\/j.1365-2672.2001.01288.x","article-title":"Update on the detection of \u03b2-exotoxin in Bacillus thuringiensis strains by HPLC analysis","volume":"90","year":"2001","journal-title":"J. Appl. Microbiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1016\/j.jip.2009.03.007","article-title":"An improved bioassay for the detection of Bacillus thuringiensis \u03b2-exotoxin","volume":"101","author":"Innes","year":"2009","journal-title":"J. Invertebr. Pathol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"4782","DOI":"10.1128\/AEM.64.12.4782-4788.1998","article-title":"A holistic approach for determining the entomopathogenic potential of Bacillus thuringiensis strains","volume":"64","author":"Masson","year":"1998","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"378","DOI":"10.1046\/j.1365-2672.1999.00536.x","article-title":"Specific primers for the detection of Vip3A insecticidal gene within a Bacillus thuringiensis collection","volume":"28","author":"Rice","year":"1999","journal-title":"Lett. Appl. Microbiol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1590\/S1519-566X2003000200001","article-title":"Discovery and development of biological agents to control crop pests","volume":"32","author":"Slininger","year":"2003","journal-title":"Neotrop. Entomol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1111\/j.1523-1739.2003.00172.x","article-title":"The strength of biological control in the battle against invasive pests: A reply","volume":"18","author":"Hoddle","year":"2004","journal-title":"Conserv. Biol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1111\/j.1523-1739.2004.00070.x","article-title":"The doubled-edged sword of biological control in conservation and restoration","volume":"18","author":"Louda","year":"2004","journal-title":"Conserv. Biol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"242","DOI":"10.1128\/mr.53.2.242-255.1989","article-title":"Insecticidal crystal proteins of Bacillus thuringiensis","volume":"53","author":"Whiteley","year":"1989","journal-title":"Microbiol. Rev."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"4965","DOI":"10.1128\/AEM.64.12.4965-4972.1998","article-title":"Characterization of cry genes in mexican Bacillus thuringiensis strain collection","volume":"64","author":"Bravo","year":"1998","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1128\/AEM.65.2.457-464.1999","article-title":"Toxicity, binding, and permeability analyses of four Bacillus thuringiensis Cry1 \u03b4-endotoxins using brush border membrane vesicles of Spodoptera exigua and Spodoptera frugiperda","volume":"65","author":"Luo","year":"1999","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1590\/S0301-80592000000100018","article-title":"PCR identification of Cry I genes in Bacillus thuringiensis strains that are efficient against fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)","volume":"29","author":"Valicente","year":"2000","journal-title":"An. Soc. Entomol. Bras."},{"key":"ref_45","unstructured":"Cornell Guide for Integrated Field Crop Management 2012. Available online:http:\/\/www.ipmguidelines.org\/FieldCrops\/."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1016\/0965-1748(95)00102-6","article-title":"Digestion of delta-endotoxin by gut proteases may explain reduced sensitivity of advanced instar larvae of Spodoptera littoralis to Cry1C","volume":"26","author":"Keller","year":"1996","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"2141","DOI":"10.1128\/jb.178.7.2141-2144.1996","article-title":"Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis: The cryV-encoded protein is expressed early in stationary phase","volume":"178","author":"Kostichka","year":"1996","journal-title":"J. Bacteriol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1146\/annurev.en.39.010194.000403","article-title":"Evolution of resistance to Bacillus thuringiensis","volume":"39","author":"Tabashnik","year":"1994","journal-title":"Annu. Rev. Entomol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"4182","DOI":"10.1128\/AEM.68.9.4182-4186.2002","article-title":"Correspondence of high levels of beta-exotoxin I and the Presence of cry1B in Bacillus thuringiensis","volume":"68","author":"Espinasse","year":"2002","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_50","unstructured":"WHO\u2014World Health Organization (1999). Microbial Pest Control Agent: Bacillus thuringiensis, World Health Organization."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1007\/BF01748152","article-title":"Microbial control of flies in latrines in Dares Salaam with a Bacillus thuringiensis (serotype 1) preparation, Muscabac","volume":"1","author":"Carlberg","year":"1985","journal-title":"World J. Appl. Microbiol. Biotechnol."},{"key":"ref_52","first-page":"112","article-title":"Solid-state fermentation of Bacillus thuringiensis tolworthi to control fall armyworm in maize","volume":"4","author":"Capalbo","year":"2001","journal-title":"Electron. J. Biotechnol."},{"key":"ref_53","unstructured":"Nakano, O., Silveira-Neto, S., and Zucchi, R.A. (1981). Entomologia Econ\u00f4mica, Luiz de Queiroz College of Agriculture (ESALQ-USP)."},{"key":"ref_54","first-page":"62","article-title":"Bacillus thuringiensis is an environmental pathogen and host-specificity has developed as an adaptation to human-generated ecological niches","volume":"5","author":"Loguercio","year":"2014","journal-title":"Insects"}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/6\/5\/1598\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T21:11:36Z","timestamp":1760217096000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/6\/5\/1598"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,5,20]]},"references-count":54,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2014,5]]}},"alternative-id":["toxins6051598"],"URL":"https:\/\/doi.org\/10.3390\/toxins6051598","relation":{},"ISSN":["2072-6651"],"issn-type":[{"type":"electronic","value":"2072-6651"}],"subject":[],"published":{"date-parts":[[2014,5,20]]}}}