Abstract
This study was carried out to investigate the use of different substrates for the production of Escovopsis conidia and verify the virulence of four different isolates cultured on four types of substrates using a novel bioassay. Escovopsis isolates were molecularly identified, based on Internal Transcribed Spacer (ITS) nucleotide sequences. To evaluate conidial production, suspensions (1 × 106 conidia mL−1) of each Escovopsis isolate were inoculated onto four substrates (parboiled rice, white rice, rolled oats, and corn grits). After 14 days, conidial yields were assessed. The virulence of each isolate cultured on the four substrates was tested against Leucoagaricus fungus garden fragments, by directly applying 500 µL of each conidial suspension (1 × 107 conidia mL−1), and the development of the parasite was monitored daily until it completely colonized the fungus garden. It was observed that rolled oats were the best substrate for conidial production, with a yield of 1.7 × 107 to 2.0 × 108 conidia mL−1. Furthermore, isolate AT-01 produced the highest number of conidia when compared with the other isolates. Regardless of the substrate used to produce AT-01 conidia, this isolate completely colonized the fungus garden 6 days post inoculation (dpi), followed by AT-02, AC-01, and AC-2. High levels of both conidial production and virulence against the leaf-cutting ant fungus garden were observed here.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
All data will be made available on request.
References
Augustin JO, Simões TG, Dijksterhuis J, Elliot SL, Evans HC (2017) Putting the waste out: a proposed mechanism for transmission of the mycoparasite Escovopsis between leafcutter ant colonies. R Soc Open Sci 4:161013. https://doi.org/10.1098/rsos.161013
Aylward FO, Khadempour L, Tremmel DM, McDonald BR, Nicora CD, Wu S, Moore RJ, Orton DJ, Monroe ME, Piehowski PD, Purvine SO, Smith RD, Lipton MS, Burnum-Johnson KE, Currie CR (2015) Enrichment and broad representation of plant biomass-degrading enzymes in the specialized hyphal swellings of Leucoagaricus gongylophorus, the fungal symbiont of leaf-cutting ants. PLoS ONE 10:e0134752. https://doi.org/10.1371/journal.pone.0134752
Barra-Bucarei L, Vergara P, Cortes A (2016) Conditions to optimize mass production of Metarhizium anisopliae (Metschn.) Sorokin 1883 in different substrates. Chil J Agric Res 76:448–454. https://doi.org/10.4067/S0718-58392016000400008
Batey SFD, Greco C, Hutchings MI, Wilkinson B (2020) Chemical warfare between fungus-growing ants and their pathogens. Curr Opin Chem Biol 59:172–181. https://doi.org/10.1016/j.cbpa.2020.08.001
Bigelis R, He H, Yang HY, Chang LP, Greenstein M (2006) Production of fungal antibiotics using polymeric solid supports in solid-state and liquid fermentation. J Ind Microbiol Biotechnol 33:815–826. https://doi.org/10.1007/s10295-006-0126-z
Birnbaum SS, Gerardo NM (2016) Patterns of specificity of the pathogen Escovopsis across the fungus-growing ant symbiosis. Am Nat 188:52–65. https://doi.org/10.1086/686911
Cafaro MJ, Poulsen M, Little AE, Price SL, Gerardo NM, Wong B, Stuart AE, Larget B, Abbot P, Currie CR (2011) Specificity in the symbiotic association between fungus-growing ants and protective Pseudonocardia bacteria. Proc Biol Sci 278:1814–1822. https://doi.org/10.1098/rspb.2010.2118
Cando-Narvaez A, Loera O, Méndez-Hernández JE (2022) Rice recycling: a simple strategy to improve conidia production in solid-state cultures. Lett Appl Microbiol 74:385–394. https://doi.org/10.1111/lam.13614
Carolino AT, Teodoro TBP, Gomes SA, Silva CP, Samuels RI (2021) Production of conidia using different culture media modifies the virulence of the entomopathogenic fungus Metarhizium against Aedes aegypti larvae. J Vector Borne Dis 4:346–351. https://doi.org/10.4103/0972-9062.318315
Castrillo ML, Bich GA, Zapata PD, Villalba L (2016) Biocontrol of Leucoagaricus gongylophorus of leaf-cutting ants with the mycoparasitic agent Trichoderma koningiopsis. Mycosphere 7:810–819. https://doi.org/10.5943/mycosphere/7/6/12
Currie CR, Bot ANM, Boomsma JJ (2003) Experimental evidence of a tripartite mutualism: bacteria protect ant fungus gardens from specialized parasites. Oikos 101:91–102. https://doi.org/10.1034/j.1600-0706.2003.12036.x
Decker EA, Rose DJ, Stewart D (2014) Processing of oats and the impact of processing operations on nutrition and health benefits. Br J Nutr 112:S58–S64. https://doi.org/10.1017/S000711451400227X
Della Lucia TMC, Gandra LC, Guedes RN (2014) Managing leaf-cutting ants: peculiarities, trends and challenges. Pest Manag Sci 70:14–23. https://doi.org/10.1002/ps.3660
Dhodary B, Schilg M, Wirth R, Spiteller D (2018) Secondary metabolites from Escovopsis weberi and their role in attacking the garden fungus of leaf-cutting ants. Chemistry 24:4445–4452. https://doi.org/10.1002/chem.201706071
Dong M, Gong Y, Guo J, Ma J, Li S, Li T (2020) Optimization of production conditions of rice α-galactosidase II displayed on yeast cell surface. Protein Expr Purif 171:105611. https://doi.org/10.1016/j.pep.2020.105611
Folgarait P, Gorosito N, Poulsen M, Currie CR (2011) Preliminary in vitro insights into the use of natural fungal pathogens of leaf-cutting ants as biocontrol agents. Curr Microbiol 63:250–258. https://doi.org/10.1007/s00284-011-9944-y
Fowler HG, Della Lucia TMC, Moreira DDO (1993) Posição taxonomica das formigas cortadeiras. In: Della Lucia TMC (ed) as formigas cortadeiras, 1st edn. Folha de Viçosa, Viçosa, pp 4–25
Francoeur CB, May DS, Thairu MW, Hoang DQ, Panthofer O, Bugni TS, Pupo MT, Clardy J, Pinto-Tomás AA, Currie CR (2021) Burkholderia from fungus gardens of fungus-growing ants produces antifungals that inhibit the specialized parasite Escovopsis. Appl Environ Microbiol 87:e0017821. https://doi.org/10.1128/AEM.00178-21
Gao L, Liu X (2010) Sporulation of several biocontrol fungi as affected by carbon and nitrogen sources in a two-stage cultivation system. J Microbiol 6:767–770. https://doi.org/10.1007/s12275-010-0049-2
Garza-López PM, Konigsberg M, Gómez-Quiroz LE, Loera O (2012) Physiological and antioxidant response by Beauveria bassiana Bals (Vuill.) to different oxygen concentrations. World J Microbiol Biotechnol 1:353–359. https://doi.org/10.1007/s11274-011-0827-y
Jenkins NE, Heviefo G, Langewald J, Cherry AJ, Lomer CJ (1998) Development of mass production technology for aerial conidia for use as mycopesticides. Bioc N Inform 19:21N-32N
Jiménez-Gómez I, Barcoto MO, Montoya QV, Goes AC, Monteiro LSVE, Bueno OC, Rodrigues A (2021) Host susceptibility modulates Escovopsis pathogenic potential in the fungiculture of higher attine ants. Front Microbiol 12:673444. https://doi.org/10.3389/fmicb.2021.673444
Karthikeyan A, Shanthi V, Nagasathya A (2008) Effect of different media and pH on the growth of Beauveria bassiana and its parasitism on leaf-eating caterpillars. Res J Agric Sci 4:117–119
Khadempour L, Kyle JE, Webb-Robertson BM, Nicora CD, Smith FB, Smith RD, Lipton MS, Currie CR, Baker ES, Burnum-Johnson KE (2021) From plants to ants: fungal modification of leaf lipids for nutrition and communication in the leaf-cutter ant fungal garden ecosystem. mSystems 6:e01307–e01320. https://doi.org/10.1128/mSystems.01307-20
Lane BS, Trinci AP, Gillespie AT (1991) Endogenous reserves and survival of blastospores of Beauveria bassiana harvested from carbon-and nitrogen-limited batch cultures. Mycol Res 95:821–828. https://doi.org/10.1016/S0953-7562(09)80045-2
Leão MP, Tiago PV, Andreote FD, de Araújo WL, de Oliveira NT (2015) Differential expression of the pr1A gene in Metarhizium anisopliae and Metarhizium acridum across different culture conditions and during pathogenesis. Genet Mol Biol 38:86–92. https://doi.org/10.1590/S1415-475738138120140236
Lopez-Perez M, Rodriguez-Gomez D, Loera O (2015) Production of conidia of Beauveria bassiana in solid-state culture: current status and future perspectives. Crit Rev Biotechnol 35:334–341. https://doi.org/10.3109/07388551.2013.857293
Marfetán JA, Romero AI, Folgarait PJ (2015) Pathogenic interaction between Escovopsis weberi and Leucoagaricus sp.: mechanisms involved and virulence levels. Fungal Ecol 17:52–61
Masangkay RF, Paulitz TC, Hallett SG, Watson AK (2000) Solid substrate production of Alternaria alternata f. sp. sphenocleae conidia. Biocontrol Sci Technol 10:399–409. https://doi.org/10.1080/09583150050114990
Mascarin GM, Jackson MA, Kobori NN, Behle RW, Dunlap CA, Delalibera Júnior Í (2015) Glucose concentration alters dissolved oxygen levels in liquid cultures of Beauveria bassiana and affects formation and bioefficacy of blastospores. Appl Microbiol Biotechnol 99:6653–6665. https://doi.org/10.1007/s00253-015-6620-3
Mascarin GM, Lopes RB, Delalibera Í Jr, Fernandes ÉKK, Luz C, Faria M (2019) Current status and perspectives of fungal entomopathogens used for microbial control of arthropod pests in Brazil. J Invertebr Pathol 165:46–53. https://doi.org/10.1016/j.jip.2018.01.001
Mattoso TC, Moreira DD, Samuels RI (2012) Symbiotic bacteria on the cuticle of the leaf-cutting ant Acromyrmex subterraneus subterraneus protect workers from attack by entomopathogenic fungi. Biol Lett 8:461–464. https://doi.org/10.1098/rsbl.2011.0963
Meirelles LA, Solomon SE, Bacci M Jr, Wright AM, Mueller UG, Rodrigues A (2015) Shared Escovopsis parasites between leaf-cutting and non-leaf-cutting ants in the higher attine fungus-growing ant symbiosis. R Soc Open Sci 2:150257. https://doi.org/10.1098/rsos.150257
Mendonça DMF, Caixeta MCS, Martins GL, Moreira CC, Kloss TG, Elliot SL (2021) Low virulence of the fungi Escovopsis and Escovopsioides to a leaf-cutting ant-fungus symbiosis. Front Microbiol 12:673445. https://doi.org/10.3389/fmicb.2021.673445
Miller MA, Holder MT, Vos R, Midford PE, Liebowit T, Chan L, Hoover P, Warnow T (2010) The CIPRES Portals CIPRES http://www.phylo.org/sub-sections/portal Accessed 14 Jun 2023
Mina Mejia SY, Rodríguez J, Montoya-Lerma J (2018) Euphorbia cotinifolia (Euphorbiaceae): a promising alternative for leaf cutting ant Atta cephalotes (Hymenoptera: Formicidae) control. Biocontrol Sci Technol 28:486–495. https://doi.org/10.1080/09583157.2018.1460315
Montoya QV, Martiarena MJS, Danilo AP, Akazu S, Rodrigues A (2019) More pieces to a huge puzzle: two new Escovopsis species from fungus gardens of attine ants. MycoKeys 46:97–118. https://doi.org/10.3897/mycokeys.46.30951
Nascimento VC, Rodrigues-Santos KC, Carvalho-Alencar KL, Castro MB, Kruger RH, Lopes FAC (2022) Trichoderma: biological control efficiency and perspectives for the Brazilian Midwest states and Tocantins. Braz J Biol 82:e260161. https://doi.org/10.1590/1519-6984.260161
Nylander JAA (2004) MrModeltest2. v.2.3. Evolutionary Biology Centre, Uppsala University https://github.com/nylander/MrModeltest2 Accessed 14 Jun 2023
Oli P, Ward R, Adhikari B, Torley P (2014) Parboiled rice: understanding from a materials science approach. J Food Eng 124:173–183
Oliveira F, Buzato JB, de Melo MR (2020) Effect of agro-industrial residues mixtures on the production of endoglucanase by Aspergillus niger in solid state fermentation. Acta Sci Technol 42:1–9. https://doi.org/10.4025/actascitechnol.v42i1.41358
Osman M, Stigloher C, Mueller MJ, Waller F (2020) An improved growth medium for enhanced inoculum production of the plant growth-promoting fungus Serendipita indica. Plant Methods 16:1–7. https://doi.org/10.1186/s13007-020-00584-7
Ottati-de-Lima EL, Batista Filho A, Almeida JEM, Gassen MH, Wenzel IM, de Almeida AMB, Zapellini LO (2010) Produção semissólida de Metarhizium anisopliae e Beauveria bassiana em diferentes substratos e efeito da radiação ultravioleta e da temperatura sobre propágulos desses entomopatógenos. Arq Inst Biol 77:651–659. https://doi.org/10.1590/1808-1657v77p6512010
Pérez-Guzmán D, Montesinos-Matías R, Arce-Cervantes O, Gómez-Quiroz LE, Loera O, Garza-López PM (2016) Reactive oxygen species production, induced by atmospheric modification, alter conidial quality of Beauveria bassiana. J Appl Microbiol 121:453–460. https://doi.org/10.1111/jam.13156
Prasanthi PS, Naveena N, Vishnuvardhana Rao M, Bhaskarachary K (2017) Compositional variability of nutrients and phytochemicals in corn after processing. J Food Sci Technol 54:1080–1090. https://doi.org/10.1007/s13197-017-2547-2
Rahardjo YS, Tramper J, Rinzema A (2006) Modeling conversion and transport phenomena in solid-state fermentation: a review and perspectives. Biotechnol Adv 24:161–179. https://doi.org/10.1016/j.biotechadv.2005.09.002
Rannala B, Yang Z (1996) Probability distributions of molecular evolutionary trees: a new method of phylogenetic inference. J Mol Evol 43:304–311
Ronquist F, Teslenko M, Van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542. https://doi.org/10.1093/sysbio/sys029
Santa HSD, Santa OR, Brand D, Vandenberghe LPS, Soccol CR (2005) Spore production of Beauveria bassiana from agroindustrial residues. Braz Arch Biol Technol 48:51–60
Silva JN, Mascarin GM, Lopes RB, Freire DMG (2023) Production of dried Beauveria bassiana conidia in packed-column bioreactor. Biochem Eng J 198:109022. https://doi.org/10.1016/j.bej.2023.109022
Stefanelli LEP, Mota Filho TMM, Camargo RDS, Matos CAO, Forti LC (2020) Effects of entomopathogenic fungi on individuals as well as groups of workers and immatures of Atta sexdens rubropilosa leaf-cutting ants. Insects 1:10. https://doi.org/10.3390/insects12010010
Sun M, Liu X (2006) Carbon requirements of some nematophagous, entomopathogenic and mycoparasitic hyphomycetes as fungal biocontrol agents. Mycopathologia 161:295–305. https://doi.org/10.1007/s11046-006-0249-9
Teodoro TBP, Carolino AT, Queiroz RRS, Oliveira PB, Moreira DDO, Silva GA, Samuels RI (2023) Production of Escovopsis weberi (Ascomycota: Hypocreales) mycelial pellets and their effects on leaf-cutting ant fungal gardens. Pathogens 12:330. https://doi.org/10.3390/pathogens12020330
Villacide JM, Gomez DF, Perez CA, Corley JC, Ahumada R, Rodrigues Barbosa L, Furtado EL, González A, Ramirez N, Balmelli G, Souza CD, Martínez G (2023) Forest health in the Southern Cone of America: state of the art and perspectives on regional efforts. Forests 14:756. https://doi.org/10.3390/f14040756
Acknowledgements
The authors are grateful to the Brazilian governmental research agencies CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). We also wish to thank UENF (Universidade Estadual do Norte Fluminense Darcy Ribeiro) for providing the infrastructure for this research. RIS is a CNPq research fellow.
Funding
Conselho Nacional de Desenvolvimento Científico e Tecnológico, 309975/2021-2.
Author information
Authors and Affiliations
Contributions
Conceptualization, RRSQ, RIS, TBPT and ATC; methodology, RRSQ, TBPT, ATC, ROBB and MSBB; validation, RRSQ, TBPT and ATC; formal analysis, RRSQ, TBPT, ROBB, WGS and GAS; investigation, RRSQ, TBPT and ATC; data curation, RRSQ, TBPT, ROBB, MSBB and RRS; writing, original draft preparation, RRSQ and ROBB; writing—review and editing, RRSQ, ROBB and RIS; visualization, RRSQ, ROBB and RIS; project administration, RIS; supervision, RIS; funding acquisition, RIS. All authors have read and agreed to the published version of the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Informed consent
For this type of study formal consent is not required.
Additional information
Communicated by Nischitha R.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Queiroz, R.R.S., Teodoro, T.B.P., Carolino, A.T. et al. Production of Escovopsis conidia and the potential use of this parasitic fungus as a biological control agent of leaf-cutting ant fungus gardens. Arch Microbiol 206, 128 (2024). https://doi.org/10.1007/s00203-024-03862-3
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00203-024-03862-3