Londrina 2021 MARIANA SANCHES SANTOS Azospirillum brasilense ESTIRPES CNPSo 2083 (=Ab-V5) E CNPSo 2084 (=Ab-V6): PROMOÇÃO DO CRESCIMENTO DE PLANTAS E AVALIAÇÃO DA COMPATIBILIDADE COM AGROTÓXICOS UTILIZADOS NO TRATAMENTO DE SEMENTES DE MILHO (Zea mays L.) Londrina 2021 Azospirillum brasilense ESTIRPES CNPSo 2083 (=Ab-V5) E CNPSo 2084 (=Ab-V6): PROMOÇÃO DO CRESCIMENTO DE PLANTAS E AVALIAÇÃO DA COMPATIBILIDADE COM AGROTÓXICOS UTILIZADOS NO TRATAMENTO DE SEMENTES DE MILHO (Zea mays L.) Tese apresentada ao Programa de Pós- graduação em Biotecnologia da Universidade Estadual de Londrina - UEL, como requisito à obtenção do título de Doutora. Orientadora: Drª. Mariangela Hungria MARIANA SANCHES SANTOS BANCA EXAMINADORA Londrina, 15 de março de 2021 Orientadora: Dra. Mariangela Hungria Empresa Brasileira de Pesquisa Agropecuéria – Embrapa Soja Dr. Marco Antonio Nogueira Empresa Brasileira de Pesquisa Agropecuéria – Embrapa Soja Dra. Paula Cerezini Biotrop Dra. Amanda Letícia Pit Nunes Biotrop ____________________________________ Dr. Artur Berbel Lirio Rondina Faculdades Integradas de Ourinhos MARIANA SANCHES SANTOS Azospirillum brasilense ESTIRPES CNPSo 2083 (=Ab-V5) E CNPSo 2084 (=Ab-V6): PROMOÇÃO DO CRESCIMENTO DE PLANTAS E AVALIAÇÃO DA COMPATIBILIDADE COM AGROTÓXICOS UTILIZADOS NO TRATAMENTO DE SEMENTES DE MILHO (Zea mays L.) Tese apresentada ao Programa de Pós- graduação em Biotecnologia da Universidade Estadual de Londrina - UEL, como requisito parcial para a obtenção do título de Doutora. AGRADECIMENTOS A Deus, pelo dom da vida, pela proteção, providência e por me fortalecer diante de todas as dificuldades. À Universidade Estadual de Londrina, em especial ao Programa de Pós-Graduação em Biotecnologia, pela oportunidade de crescimento profissional, científico e humano. À Embrapa Soja, pela oportunidade de realização desse trabalho. À Fundação Araucária, pela concessão da bolsa de estudos. À querida orientadora Dra Mariangela Hungria, sou grata por ter confiado em mim, por ter me apoiado na decisão de fazer o doutorado morando a 100 km de distância. Agradeço à atenção e ao carinho concedido, aos “puxões de orelha” que me fizeram crescer. Para mim foi uma honra aprender com seu exemplo como profissional e pessoa. Será lembrada com muito amor e gratidão por toda minha vida. Aos membros da banca, pela disponibilidade em avaliar e colaborar para com a melhoria do nosso trabalho. Aos colegas de laboratório que sempre estiveram prontos para ajudar de alguma maneira e por tornarem o trabalho e a rotina mais divertidos. Me sinto privilegiada em participar dessa equipe. À minha família, que são a base de tudo, por terem sido fonte de constante incentivo, por todo amor e união. Por tudo que me ensinaram e por nunca medirem esforços para que eu chegasse até aqui. Ao meu marido, Anderson, pelo carinho, amor e paciência. Por sempre me incentivar e tranquilizar com palavras sábias. Por fim, chego à conclusão de que não há fim, apenas novos começos e possibilidades para seguir caminhando. “Não fui eu quem ordenou a você que seja forte e corajoso? Não tenha medo e não se sinta acovardado, porque Javé seu Deus vai estar com você por onde você andar”. Josué 1:9. SANTOS, Mariana Sanches. Azospirillum brasilense estirpes CNPSo 2083 (=Ab- V5) e CNPSo 2084 (=Ab-V6): Promoção do crescimento de plantas e avaliação da compatibilidade com agrotóxicos utilizados no tratamento de sementes de milho (Zea mays L.). 2021. 138 f. Tese (Doutorado em Biotecnologia) – Universidade Estadual de Londrina, Londrina, 2021. RESUMO A humanindade discute a preservação ambiental, que é um desafio frente ao desenvolvimento há pelo menos um século. Nas últimas décadas o setor agrícola tem buscado criar novas práticas que aliam sustentabilidade ambiental com altos índices de produtividade e de qualidade dos alimentos. Os inoculantes são um exemplo de bioinsumo de aplicação agrícola que reúnem essas características, garantindo produtividade elevada com redução no uso de fertilizantes químicos, altamente poluentes. O Brasil tem se destacado no uso de inoculantes em diversas culturas, como a soja, o feijão e o milho. Por décadas, os inoculantes usados no país eram à base de rizóbios para leguminosas mas, há pouco mais de uma década, entraram e ganharam mercado inoculantes com as estirpes Ab-V5 (=CNPSo 2083) e Ab-V6 (=CNPSo 2084) da bactéria promotora do crescimento de plantas (BPCP) Azospirillum brasilense. Contudo, ainda há poucos estudos sobre esses inoculantes. Para garantir um bom desempenho do inoculante é necessário que as células estejam viáveis e em uma concentração elevada. Diante disso, pesquisadores e produtores têm questionado se o tratamento de sementes com agrotóxicos, prática comum e, na maioria dos casos, indispensável para a proteção contra pragas e doenças, poderia interferir nos resultados da inoculação. Os objetivos deste trabalho foram descrever aspectos importantes referentes à tecnologia da inoculação e avaliar os impactos que o tratamento de sementes com agrotóxicos pode causar às células de A. brasilense e, consequentemente, na sua contribuição à agricultura. O primeiro trabalho, uma revisão, descreve o cenário mundial de uso de inoculantes nas mais diversas culturas, os microrganismos utilizados e técnicas de inoculação. Na segunda revisão foram discutidos os principais estudos realizados no Brasil, a situação comercial atual e as expectativas sobre o uso das estirpes Ab-V5 e Ab-V6 como inoculantes nas diversas culturas. O terceiro trabalho constou no desenvolvimento de uma nova metodologia para verificar a recuperação de células viáveis de A. brasilense em sementes de milho inoculadas, impressindível para a avaliação dos efeitos do tratamento de sementes com agrotóxicos. No quarto trabalho, a nova metodologia foi aplicada e foram descritos os efeitos que o uso de agrotóxicos pode desencadear no desenvolvimento inicial e na morfologia das raízes de milho inoculado ou não com A. brasilense. No quinto trabalho foram revisados e discutidos trabalhos relatando níveis de compabitilidade ou incompatibilidade entre inoculantes e agrotóxicos (fungicidas, inseticidas e herbicidas). Os resultados obtidos evidenciaram a relevância crescente que os inoculantes microbianos vêm adquirindo na agricultura nacional e internacional, e o impacto positivo que, em uma década de uso, as estirpes Ab-V5 e Ab-V6 promoveram tanto em leguminosas, como em gramíneas. Contudo, os resultados indicam também que agrotóxicos utilizados no tratamento de sementes representam uma ameaça aos benefícios que podem ser obtidos pela inoculação com Azospirillum, afetando negativamente desde a sobrevivência das células, ao desenvolvimento radicular e rendimento das culturas. Em cada um dos cinco estudos são sugeridas pesquisas futuras e estratégias para incrementar a contribuição dos inoculantes na agricultura brasileira. Palavras-chave: Inoculantes. BPCP. Fixação biológica de nitrogênio. Agrotóxicos. SANTOS, Mariana Sanches. Azospirillum brasilense strains Ab-V5 and Ab-V6: Plant growth-promotion and compatibility with pesticides used in the seed treatment of maize (Zea mays L.). 2021. 138 pp. Thesis (Doctorate degree in Biotechnology) – Universidade Estadual de Londrina, Londrina, 2021. ABSTRACT The environment preservation has been discussed for at least a century. In the last decades, the agricultural sector has sought to create new practices that combine environmental sustainability with high yields and food quality. The inoculants are examples of bio-inputs for agricultural application that combine these characteristics, guaranteeing high yield with less use of chemical fertilizers, which are highly polluting. Brazil has excellency in the use of inoculants in several crops, such as soybeans, common beans and maize. For decades, the inoculants used in the country were based exclusively on rhizobia for legumes, but a decade ago, the strains Ab-V5 (= CNPSo 2083) and Ab-V6 (= CNPSo 2084) of the plant-growth-promoting bacterium (PGPB) Azospirillum brasilense were released and started to occupy an important space in the commercial market. However, there are still few studies on these inoculants. In order to guarantee a good performance of the inoculant, the cells must be viable at high concentration. Therefore, researchers and farmes have argued if seed treatment with pesticides, a common and indispensable practice against pests and diseases can interfere in the results of inoculation. The objectives of this study were to describe important aspects related to the inoculation technology and to evaluate the impacts that seed treatment with pesticides can cause on A. brasilense cells and, consequently, on its contributions in agriculture. In the first review work, the world scenario of the use of inoculants in a variety of crops, of inoculation techniques and microorganisms used was described. In the second review, the main studies carried out in Brazil, the current commercial situation and expectations about the use of strains Ab-V5 and Ab-V6 as inoculants in different crops were discussed. The third study describes the development of a new methodology to verify the recovery of viable cells of A. brasilense in inoculated seeds of maize, essential for evaluating the effects of seed treatment with pesticides. In the fourth study, the new developed methodology was applied to verify the effects of pesticides on the initial development and on the morphology of maize roots inoculated or not with A. brasilense. In the fifth work, papers reporting levels of compatibility or incompatibility between inoculants and pesticides (fungicides, insecticides and herbicides) were reviewed and discussed. The results obtained show the growing relevance that microbial inoculants have played in the Brazilian agriculture, and the impact that, in a decade of use, the strains Ab-V5 and Ab-V6 promoted as inoculants of both legumes and non-legumes. However, the results also indicate that pesticides used in seed treatment represent a threat to the benefits that can be obtained by inoculation with Azospirillum, affecting not only cell survival but also root development and crop yield. In each study, strategies are suggested to increase the contribution of inoculants to the agriculture in Brazil. Key-words: Inoculant. PGPB. Biological nitrogen fixation. Pesticides SUMÁRIO INTRODUÇÃO.............................................................................................................8 REFERÊNCIAS..........................................................................................................11 CAPÍTULO 1 Inoculantes microbianos: revisando o passado, discutindo o presente e prevendo um futuro brilhante para o uso de bactérias benéficas na agricultura...............................................................13 CAPÍTULO 2 Impacto excepcional das estirpes de Azospirillum brasilense Ab-V5 e Ab-V6 na agricultura brasileira: lições de que os agricultores estão receptivos para adotar novos inoculantes microbianos...................................................................................37 CAPÍTULO 3 Método para recuperação e contagem de células viáveis de Azospirillum brasilense inoculadas em sementes de milho..............................................................................................71 CAPÍTULO 4 Compatibilidade de Azospirillum brasilense com agrotóxicos usados no tratamento de sementes de milho.............................83 CAPÍTULO 5 O Desafio de combinar altos rendimentos com bioprodutos ambientalmente amigáveis: uma revisão sobre a compatibilidade de pesticidas com inoculantes microbianos...................................................................................93 CONCLUSÃO..........................................................................................................138 8 INTRODUÇÃO Os inoculantes, bioprodutos agrícolas capazes de substituir total ou parcialmente os fertilizates químicos, têm ganhado cada vez mais notoriedade no agronegócio. Contendo microrganismos vivos, esses produtos favorecem o desenvolvimento de plantas por meio de diversos mecanismos, permitindo elevada produtividade a baixo custo. Desde quando chegou ao mercado pela primeira vez, em 1896, o número de doses comercializadas aumentou consideravelmente e o cenário atual indica continuidade de crescimento, devido à divulgação dos excelentes resultados e do desenvolvimento de novas formulações para diversas culturas. O gênero Azospirillum abrange um grupo de bactérias Gram- negativas, que fazem parte da subclasse α das proteobactérias. Diazotróficas associativas, também podem viver livremente no solo na forma de cistos (MOREIRA et al., 2010). Bactérias do gênero Azospirillum estão distribuídas em diversas regiões do mundo, em condições tropicais, subtropicais e temperadas (HUNGRIA, 2011; SIVASAKTHIVELAN; SARANRAJ, 2013), em associação com plantas monocotiledôneas e eudicotiledôneas (STEENHOUDT; VANDERLEYDEN, 2000). O Brasil foi pioneiro em estudos do gênero Azospirillum, que compreende atualmente 26 espécies (DSMZ, 2020). Estudos têm mostrado que Azospirillum brasilense pode favorecer o desenvolvimento das plantas às quais se associa de diversas maneiras, entre elas pela produção de fitormônios que atuam diretamente no crescimento radicular, na fixação de nitrogênio atmosférico, na solubilização de fosfatos, no controle biológico de insetos e fitopatógenos e no aumento da resistência do vegetal aos estresses salino, hídrico e oxidativo (DÖBEREINER, 1979; DUCA et al., 2014; CEREZINI et al., 2016; FUKAMI et al., 2017, 2018a,b; HUNGRIA et al., 2018; RONDINA et al., 2020). Esses múltiplos mecanismos, tanto em A. brasilense, como em outras espécies, levaram à denominação dessas bactérias como promotoras do crescimento de plantas (BPCP). Em 2009, após um extenso trabalho para seleção de estirpes realizado pela Embrapa Soja, foi lançado no mercado o primeiro inoculante brasileiro para milho (Zea mays L.) e trigo (Triticum aestivum L.), contendo as estirpes Ab-V5 e Ab-V6 de Azospirillum brasilense. Um ano após foram comrcializadas 300 mil doses e, nos anos seguintes, esse número cresceu consideravelmente atingindo, em 2019, 9 o valor expressivo de 10,5 milhões de doses. Esse salto nas vendas pode ser atribuído aos excelentes resultados obtidos pela inoculação, ao aumento do rendimento das culturas a baixo custo e à recomendação de uso em outras culturas, como nas leguminosas e nas gramíneas forrageiras.. De forma geral, a obtenção de altos rendimentos pelas culturas pode ser limitada tanto pela disponibilidade de nutrientes quanto pela ocorrência de pragas e doenças. Algumas práticas são fundamentais para amenizar prejuízos e, consequentemente, contribuir para a fitossanidade, como por exemplo, o uso de cultivares resistentes, sementes livres de patógenos e o tratamento químico (MERTZ; HENNING; ZIMMER, 2009). A manipulação genética para o desenvolvimento de cultivares resistentes é a maneira mais eficaz no controle de fitopatógenos, entretanto, ainda não foram desenvolvidas cultivares resistentes para a maioria das doenças. Atualmente, o uso de agrotóxicos como tratamento químico, seja nas sementes, no sulco, ou diretamente na planta representa a alternativa mais viável para o controle dessas doenças e redução de perdas (WORDELL FILHO et al., 2016). No Brasil, estima-se que 98% das sementes de soja (Glycine max (L.) Merr.) e de milho híbrido sejam tratadas com fungicidas e inseticidas (SPADOTTO et al., 2004; COTA et al., 2013; NUNES, 2016). O manejo de doenças com o auxílio de agrotóxicos, aliado à inoculação de sementes com BPCPs podem ser considerados tecnologias muito importantes para garantir elevada produtividade do milho. Entretanto, recentemente estudos têm apontado incompatibilidade entre as bactérias presentes nos inoculantes com a prática de tratamento de sementes com agrotóxicos. A presença do agrotóxico pode prejudicar a viabilidade e o metabolismo da bactéria, causando prejuízos na contribuição e/ou produção de fitormônios e reduzindo os benefícios da inoculação sobre o desenvolvimendo das plantas. Especificamente para o milho, ainda são poucas as informações sobre os efeitos da combinação do tratamento químico de sementes dessa cultura com as estirpes Ab-V5 e Ab-V6 presentes nos inoculantes, bem como sua influência sobre o desenvolvimento do vegetal. Um fator que limita estudos com essa abordagem é a ausência de um método que permita a recuperação de células de A. brasilense inoculadas em sementes de milho tratadas com agrotóxicos, a fim de se verificar a viabilidade após diferentes períodos de exposição. Um método semelhante é bastante 10 consolidado para recuperação de Bradyrhizobium spp. inoculado em sementes de soja (Glycine max (L.) Merr), o que possibilita que mais infomações sobre a combatibilidade entre esses produtos sejam obtidas. Devido à sua grande adaptabilidade e diversificada aplicabilidade, o milho ocupa, atualmente, o terceiro lugar entre os cereais mundialmente mais cultivados (AWIKA, 2011; SANTOS et al., 2019). No Brasil, seu cultivo é realizado em todos os 27 estados, com destaque para a região sul e estima-se que a safra 2020/21, deverá apresentar uma área total de 19,09 milhões de ha, com produção de 105,4 milhões de toneladas, e produtividade média de 5.525 kg ha-1 (CONAB, 2021). Apesar da grande demanda nacional pelo milho (aproximadamente 72 milhões de toneladas em 2020/2021), o Brasil também é um importante exportador dessa commodity e ocupa o segundo lugar no ranking dos principais países exportadores (FIESP, 2019). Segundo dados da Conab (2021), na safra 2020/2021, espera-se que o volume de embarques seja de aproximadamente 35 milhões de toneladas. Os objetivos desse trabalho foram: (i) caracterizar a situação mundial quanto ao denvolvimento e aplicação de inoculantes; (ii) caracterizar a eficácia das estirpes Ab-V5 e Ab-V6 de A. brasilense em estudos conduzidos no Brasil; (iii) desenvolver uma metodologia para recuperar células viáveis de A. brasilense após a inoculação de sementes de milho; (iv): avaliar o impacto de agrotóxicos utilizados no tratamento de sementes de milho para a sobrevivência de estirpes de A. brasilense, na promoção do crescimento de milho; (v): evidenciar os principais resultados de estudos que investigaram os efeitos de fungicidas, inseticidas e herbicidas sobre inoculantes microbianos. 11 REFERÊNCIAS AWIKA, J. M. Major cereal grains production and use around the world. In: AWIKA, J. M.; PIIRONEN, V.; BEAN, S. Advances in cereal science: implications to food processing and health promotion, 1 ed. 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Antonie van Leeuwenhoek, v. 106, p. 85–125, 2014. https://doi.org/10.1007/s10482-013-0095-y DSMZ- Leibniz Institut DSMZ-Deutsche Sammiung von Mikroorganismen und Zellkulturen GmbH. Prokaryotic nomenclature up-to-date. Disponível em: https://www.dsmz.de/bacterial-diversity/prokaryotic-nomenclature-up-to- date/prokaryotic-nomenclature. Acesso em: 19 jun. 2020. FIESP - Safra Mundial de Milho 2019/20 - 8º Levantamento do USDA. Informativo de dezembro de 2019. Disponível em: https://www.fiesp.com.br/indices-pesquisas-e- publicacoes/safra-mundial-de-milho-2/. Acesso em: 05 fev. 2020. FUKAMI, J.; CEREZIN, P.; HUNGRIA, M. Azospirillum: benefits that go far beyond biological nitrogen fixation. AMB Express, v. 8, p. 1-12, 2018a. https://doi.org/10.1186/s13568-018-0608-1 FUKAMI, J.; OLLERO, F. J.; DE LA OSA, C. DE L. A.; VALDERRAMA-FERNÁNDEZ, R.; NOGUEIRA, M. A.; MEGÍAS, M.; HUNGRIA, M. Antioxidant activity and induction of mechanisms of resistance to stresses related to the inoculation with Azospirillum brasilense. Archives of Microbiology, v. 200, p. 1191–1203, 2018b. https://doi.org/10.1007/s00203-018-1535-x FUKAMI, J.; OLLERO, F. J.; MEGÍAS, M.; HUNGRIA, M. Phytohormones and https://doi.org/10.1021/bk-2011-1089.ch001 https://doi.org/10.1016/j.fcr.2016.06.017 https://doi.org/10.1007/s10482-013-0095-y https://www.dsmz.de/bacterial-diversity/prokaryotic-nomenclature-up-to-date/prokaryotic-nomenclature https://www.dsmz.de/bacterial-diversity/prokaryotic-nomenclature-up-to-date/prokaryotic-nomenclature https://www.fiesp.com.br/indices-pesquisas-e-publicacoes/safra-mundial-de-milho-2/ https://www.fiesp.com.br/indices-pesquisas-e-publicacoes/safra-mundial-de-milho-2/ https://doi.org/10.1186/s13568-018-0608-1 12 induction of plant-stress tolerance and defense genes by seed and foliar inoculation with Azospirillum brasilense cells and metabolites promote maize growth. AMB Express, v. 7, n. 153, p. 1-8, 2017. https://doi.org/10.1186/s13568-017-0453-7 HUNGRIA, M. Inoculação com Azospirillum brasilense: inovação em rendimento a baixo custo. Londrina: Embrapa Soja, 2011. (Embrapa Soja. Documentos, n. 325). HUNGRIA, M.; RIBEIRO, R. A.; NOGUEIRA, M. A. Draft genome sequences of Azospirillum brasilense strains Ab-V5 and Ab-V6, commercially used in inoculants for grasses and legumes in Brazil. Genome Announcements, v. 6, p. 1-2, 2018. https://doi.org/10.1128/genomeA.00393-18. MERTZ, L. M.; HENNING, F; A.; ZIMMER, P. D. Bioprotetores e fungicidas químicos no tratamento de sementes de soja. Ciência Rural, v. 39, n. 1 p. 13-18, 2009. MOREIRA, F. M. DE S.; SILVA, K.; NÓBREGA, R. S.; CARVALHO, F. Bactérias diazotróficas associativas: diversidade, ecologia e potencial de aplicações. Comunicata Scientiae, n. 1, v. 2, p. 74-99, 2010. NUNES, J. C. S. Tratamento de sementes de soja como um processo industrial no Brasil. 2016. Disponível em: . Acesso em: 21 jul. 2020. RONDINA, A. B. L.; SANZOVO, A. W. DOS S.; GUIMARÃES, G. S.; WENDLING, J. R.; NOGUEIRA, M. A.; HUNGRIA, M. Changes is root morphological traits in soybean co-inoculated with Bradyrhizobium spp. and Azospirillum brasilense or treated with A. brasilense exudates. Biology and Fertily of Soils, v. 56, p. 537-549, 2020. https://doi.org/10.1007/s00374-020-01453-0 SANTOS, M. S.; NOGUEIRA, M. A.; HUNGRIA, M. Microbial inoculants: reviewing the past, discussing the present and previewing an outstanding future for the use of beneficial bacteria in agriculture. AMB Express, v. 9, n. 205, p. 1-22, 2019. SIVASAKTHIVELAN, P.; SARANRAJ, P. Azospirillum and its Formulations: A Review. International Journal of Microbiological Research, v. 4, n. 3, p. 275-287, 2013 SPADOTTO, C. A.; GOMES, M. A. F.; LUCHINI, L. C.; ANDRÉA, M. M. Monitoramento do risco ambiental de agrotóxicos: princípios e recomendações. Jaguariúna: Embrapa Meio Ambiente, 2004. (Embrapa Meio Ambiente. Documentos, n. 42). STEENHOUDT, O.; VANDERLEYDEN, J. Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiology Reviews, v. 24, p. 487-506, 2000. WORDELL FILHO, J. A.; RIBEIRO, L. P.; CHIARADIA, L. A.; MADALÓZ, J. C.; NESI, C. N. Pragas e doenças do milho: Diagnose, danos e estratégias de manejo. Florianópolis: Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina (Epagri), 2016. (Epagri. Boletim Técnico, n. 170). https://doi.org/10.1186/s13568-017-0453-7 https://doi.org/10.1128/genomeA.00393-18 https://doi.org/10.1007/s00374-020-01453-0 13 CAPÍTULO 1 INOCULANTES MICROBIANOS: REVISANDO O PASSADO, DISCUTINDO O PRESENTE E PREVENDO UM FUTURO BRILHANTE PARA O USO DE BACTÉRIAS BENÉFICAS NA AGRICULTURA 14 INOCULANTES MICROBIANOS: REVISANDO O PASSADO, DISCUTINDO O PRESENTE E PREVENDO UM FUTURO BRILHANTE PARA O USO DE BACTÉRIAS BENÉFICAS NA AGRICULTURA RESUMO Mais de cem anos se passaram desde o desenvolvimento do primeiro inoculante microbiano para plantas. Atualmente, o uso de inoculantes microbianos na agricultura é globalmente adotado para diferentes culturas e inclui diferentes microrganismos. Nas últimas décadas, foram alcançados progressos impressionantes na produção, comercialização e uso de inoculantes. Atualmente, os agricultores são mais receptivos ao uso desse bioinsumo, principalmente devido à alta qualidade, visto que muitos deles contêm estirpes elite de múltiplos propósitos, melhorando os rendimentos das culturas a baixo custo em comparação com os fertilizantes químicos. No contexto de uma agricultura mais sustentável, os inoculantes microbianos também ajudam a mitigar os impactos ambientais causados pelos agrotóxicos. Os desafios consistem em realizar uma produção de inoculantes microbianos para uma ampla variedade de culturas e na expansão da área inoculada em todo o mundo, além da busca por soluções microbianas inovadoras em áreas sujeitas a episódios crescentes de estresse ambiental. Nesta revisão, exploramos o mercado mundial de inoculantes, mostrando quais bactérias são proeminentes como inoculantes em diferentes países e discutimos as principais estratégias de pesquisa que podem contribuir para melhorar o uso de inoculantes microbianos na agricultura. Palavras-chave: Fixação biológica de nitrogênio, bactérias promotoras de crescimento de plantas, Azospirillum, inoculação, adubos químicos. 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 CAPÍTULO 2 IMPACTO EXCEPCIONAL DAS ESTIRPES DE Azospirillum brasilense Ab-V5 e Ab-V6 NA AGRICULTURA BRASILEIRA: LIÇÕES DE QUE OS AGRICULTORES ESTÃO RECEPTIVOS PARA ADOTAR NOVOS INOCULANTES MICROBIANOS 38 IMPACTO EXCEPCIONAL DAS ESTIRPES DE Azospirillum brasilense Ab- V5 e Ab-V6 NA AGRICULTURA BRASILEIRA: LIÇÕES DE QUE OS AGRICULTORES ESTÃO RECEPTIVOS PARA ADOTAR NOVOS INOCULANTES MICROBIANOS RESUMO Durante décadas, pesquisadores de todo o mundo buscam estratégias visando uma maior sustentabilidade na agricultura. Os inoculantes microbianos ou biofertilizantes são configurados como produtos biotecnológicos com a principal função de substituir, total ou parcialmente, os fertilizantes químicos, com ênfase nos fertilizantes nitrogenados, reduzindo os custos de produção e diminuindo a contaminação do solo, da água e da atmosfera. Embora os estudos de inoculação e o uso de inoculantes pelos agricultores ocorram há mais de um século, na década passada ganharam mais notoriedade. O Brasil tem uma longa tradição no uso de inoculantes contendo rizóbios, especialmente para a cultura da soja, mas foi apenas em 2009 que o primeiro inoculante comercial com as estirpes Ab-V5 e Ab-V6 de Azospirillum brasilense, promotoras do crescimento de plantas identificadas pela pesquisa, chegou ao mercado. Uma década depois, foram comercializadas 10,5 milhões de doses para gramíneas, incluindo milho, trigo, arroz e pastagens de braquiárias e, também, para a coinoculação de leguminosas. Resultados impactantes de incrementos no crescimento radicular, na produção de biomassa e de grãos, na absorção de nutrientes e água, e aumento da tolerância a estresses abióticos devido à inoculação com Ab-V5 e Ab-V6 foram apresentados por vários grupos de pesquisa no Brasil. Nesta revisão, reunimos os resultados obtidos até o momento com essas duas estirpes em várias culturas leguminosas e não leguminosas, confirmando sua versatilidade e indicando que, com resultados convincentes, confiáveis e consistentes, os agricultores estão ansiosos por adotar tecnologias sustentáveis baseadas em microorganismos. Palavras-chave: Inoculação, Bactérias promotoras do crescimento vegetal, Milho, Soja, Trigo, Triticum aestivum, Urochloa, Zea mays. 39 GRAPHIC ABSTRACT 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 CAPÍTULO 3 MÉTODO PARA RECUPERAÇÃO E CONTAGEM DE CÉLULAS VIÁVEIS DE Azospirillum brasilense INOCULADAS EM SEMENTES DE MILHO 72 MÉTODO PARA RECUPERAÇÃO E CONTAGEM DE CÉLULAS VIÁVEIS DE Azospirillum brasilense INOCULADAS EM SEMENTES DE MILHO RESUMO A inoculação de sementes com bactérias fixadoras de nitrogênio e promotoras de crescimento de plantas é uma prática agrícola bem estabelecida, cada vez mais adotada em todo o mundo, diminuindo os custos e os impactos ambientais da produção de alimentos. A maioria dos inoculantes comercializados globalmente é para a cultura da soja, e um método para recuperação de células de Bradyrhizobium de sementes de soja inoculadas para contagem subsequente foi adotado por vários laboratórios da América do Sul, especialmente para investigar a sobrevivência bacteriana em sementes tratadas com pesticidas. Entretanto, o uso de inoculantes contendo Azospirillum brasilense em culturas de cereais aumentou exponencialmente, exigindo investigação sobre a recuperação e contagem de células de sementes inoculadas. Neste trabalho, verificamos que o método utilizado para recuperação e contagem de células viáveis de Bradyrhizobium em sementes de soja inoculadas com Bradyrhizobium não era aplicável a sementes de milho inoculadas com A. brasilense. Em seguida, modificamos várias etapas do método, com o objetivo de recuperar células viáveis de Azospirillum. A principal limitação foi identificada na natureza do tegumento das sementes de milho, seco e pobre em nutrientes, resultando na agregação celular de A. brasilense. A pré-hidratação das sementes por 2 h em água destilada estéril, seguida de agitação por 30 minutos em água destilada estéril com Tween 80, permitiu a contagem adequada de células de A. brasilense recuperadas das sementes de milho. O método foi aplicado com sucesso para contar células de Azospirillum recuperadas de sementes de milho pré-inoculadas e para estimar o impacto do tratamento de sementes com pesticidas na sobrevivência celular. Palavras-chave: Inoculante, recuperação celular, Zea mays, Azospirillum, pesticidas. 73 74 75 76 77 78 79 80 81 82 83 CAPÍTULO 4 COMPATIBILIDADE DE Azospirillum brasilense COM AGROTÓXICOS UTILIZADOS NO TRATAMENTO DE SEMENTES DE MILHO 84 COMPATIBILIDADE DE Azospirillum brasilense COM AGROTÓXICOS UTILIZADOS NO TRATAMENTO DE SEMENTES DE MILHO RESUMO O tratamento de sementes com agrotóxicos é comumente usado como procedimento inicial de proteção de plantas contra pragas e doenças. No entanto, o uso de tais produtos químicos pode prejudicar a sobrevivência e o desempenho de microrganismos benéficos introduzidos via inoculação, como a bactéria Azospirillum brasilense, promotora de crescimento de plantas. Neste estudo, foi avaliada a compatibilidade entre o agrotóxico mais comumente utilizado no Brasil para o tratamento de sementes de milho, composto por dois fungicidas e um inseticida, com as estirpes comerciais Ab-V5 e Ab-V6 de A. brasilense, e os consequentes impactos nas desenvolvimento de plantas. A toxicidade do agrotóxico para A. brasilense foi confirmada, com aumento da mortalidade celular após apenas 24 h de exposição in vitro. A germinação das sementes e o crescimento das plântulas não foram afetados por A. brasilense, nem pelo agrotóxico. Entretanto, em casa de vegetação, o agrotóxico afetou negativamente o volume radicular, a massa radicular seca e a incidência de pelos radiculares, mas a toxicidade foi atenuada pela inoculação com A. brasilense para os parâmetros de volume e incidência de pelos radiculares. Em sementes de milho inoculadas com A. brasilense, o agrotóxico afetou negativamente o número de ramificações das raízes, a incidência de pelos radiculares e o comprimento de pelos radiculares. Consequentemente, novas formulações de inoculantes com protetores celulares e o desenvolvimento de agrotóxicos compatíveis devem ser pesquisados para garantir os benefícios da inoculação com bactérias promotoras de crescimento de plantas. Palavras-chave: Inoculação, compatibilidade, desenvolvimento de raizes, agrotóxicos, Azospirillum. 85 86 87 88 89 90 91 92 93 CAPÍTULO 5 O DESAFIO DE COMBINAR ALTOS RENDIMENTOS COM BIOPRODUTOS AMBIENTALMENTE AMIGÁVEIS: UMA REVISÃO SOBRE A COMPATIBILIDADE DE PESTICIDAS COM INOCULANTES MICROBIANOS 94 O DESAFIO DE COMBINAR ALTOS RENDIMENTOS COM BIOPRODUTOS AMBIENTALMENTE AMIGÁVEIS: UMA REVISÃO SOBRE A COMPATIBILIDADE DE PESTICIDAS COM INOCULANTES MICROBIANOS RESUMO Inoculantes ou biofertilizantes com o objetivo de substituir parcial ou totalmente os fertilizantes químicos estão se tornando cada vez mais importantes na agricultura, pois há uma percepção global da necessidade de aumentar a sustentabilidade. Nesta revisão, discutimos alguns resultados importantes da inoculação de uma variedade de culturas com rizóbios e outras bactérias promotoras de crescimento de plantas (BPCP). Melhorias importantes na qualidade dos inoculantes e na identificação de novas estirpes e formulações foram alcançadas. No entanto, a agricultura continuará a demandar agrotóxicos e sua baixa compatibilidade com inoculantes, principalmente quando aplicados via sementes, representa uma grande limitação para o sucesso da inoculação. As diferenças na compatibilidade entre agrotóxicos e inoculantes dependem de seu princípio ativo, formulação, época de aplicação e tempo de contato com os microrganismos; entretanto, em geral, eles têm um alto impacto na sobrevivência e no metabolismo das células microbianas, afetando sua contribuição para o crescimento das plantas. São necessárias novas estratégias para superar o problema da incompatibilidade entre agrotóxicos e inoculantes, pois as que foram propostas até agora ainda são muito modestas quanto ao cenário atual de uso de agrotóxicos nas culturas. Palavras-chave: rizóbios, bactérias promotoras de crescimento de plantas, fungicida, inseticida, herbicida, fixação biológica de nitrogênio, inoculação 95 Review – Agronomy The challenge of combining high yields with environmentally-friendly bioproducts: A review on the compatibility of pesticides with microbial inoculants Mariana Sanches Santos1,2, Thiago Fernandes Rodrigues, Marco Antonio Nogueira1, Mariangela Hungria1,2* 1Embrapa Soja, C.P. 231, 86001-970, Londrina, Paraná, Brazil. 2Department of Biochemistry and Biotechnology, Universidade Estadual de Londrina, C.P. 60001, 86051-990, Londrina, Paraná, Brazil. * Corresponding author Number of text pages 38 Number of Tables 02 Number of Figures 02 Emails: M.S. Santos, mari_sanches_s@hotmail.com; T.F. Rodrigues, thiagoferrodrigues@hotmail.com; M.A. Nogueira, marco.nogueira@embrapa.br; M. Hungria, mariangela.hungria@embrapa.br *Corresponding author: Mariangela Hungria Embrapa Soja Cx. Postal 231 86001-970, Londrina, Paraná, Brazil Fax: (+55) 4333716206 Telephone: (+55)4333716206 E-mail: mariangela.hungria@embrapa.br; biotecnologia.solo@hotmail.com mailto:thiagoferrodrigues@hotmail.com mailto:mariangela.hungria@embrapa.br 96 The challenge of combining high yields with environmentally-friendly bioproducts: A review on the compatibility of pesticides with microbial inoculants ABSTRACT: Inoculants or biofertilizers aiming to partially or fully replace chemical fertilizers are becoming increasingly important in agriculture, as there is a global perception on the need to increase sustainability. In this review, we discuss some important results of inoculation of a variety of crops with rhizobia and other plant growth-promoting bacteria (PGPB). Important improvements in the quality of the inoculants and on the release of new strains and formulations have been achieved. However, agriculture will continue to demand chemical pesticides, and their low compatibility with inoculants, especially when applied to seeds, represents a major limitation to the success of inoculation. The differences in the compatibility between pesticides and inoculants depend on their active principle, formulation, time of application, and period of contact with living microorganisms; however, in general they have a high impact on cell survival and metabolism, affecting microbial contribution to plant growth. New strategies to solve the incompatibility between pesticides and inoculants are needed. The ones that have been proposed to date are still very modest in terms of demand. Keywords: rhizobia, plant-growth-promoting bacteria, fungicide, insecticide, herbicide, biological nitrogen fixation, inoculation 97 Introduction Technologies and agricultural inputs currently applied for food production are essential for large-scale production and are mandatory to feed a population of more than 7 billion people [1]. Years of research and experiments continually performed facing challenges and technological evolution result in inputs in several fields of science. The 1950s was known as the “Green Revolution” period, marked by the intense modernization of agriculture [2, 3]. Products including new machines and synthetic fertilizers, with an emphasis on nitrogen (N) fertilizers, pesticides, seeds of better quality, improvements in the water supply systems, breeding and genetic engineering are examples of technologies developed at that time and that have gained prominence in agriculture [4, 5]. The main positive result of the Green Revolution was the global increase in food production, thus contributing to the reduction of hunger in the world. However, in the following years, the side-effect of this revolution unfolded [6]. The accumulation of pesticides and chemical fertilizers contributes to the pollution of groundwater and cultivated land, soil degradation, and reduction of biodiversity in different ecosystems [5, 7]. Increased deforestation, soil degradation, and emission of polluting gases into the atmosphere have been increasingly observed [8-10]. Despite the undeniable benefits of the Green Revolution, many of the technologies and inputs generated during that period are broadly criticized today. Currently, efforts have been made towards the development of new technologies and inputs focused on more sustainable systems. Contemporary scientists have pointed out that we are living in a “New Green Revolution” whose main characteristic, and which differs from that experienced in the 1950s, is the development of environmentally-friendly technologies and products [3, 11-13]. Examples of this new concept include the development of products and techniques such as crop rotation, plant genetic engineering for resistance to pests, diseases, and abiotic stresses such as drought, 98 the use of bio-inputs as activators of soil biota, biopesticides and microbial inoculants, also known as biofertilizers in some countries, with the purpose of partially or fully replacing the use of chemical fertilizers, favoring the growth of plants [14-17]. Although the current movement towards agricultural sustainability has force worldwide, the use of agrochemicals is and will continue to be the reality of most farmers [18]. As such, the common scenario towards improving agricultural sustainability with feasible yields to guarantee food security includes the increasing use of bioproducts, such as microbial inoculants, together with pesticides, which are still indispensable for controlling pests and diseases. Therefore, the compatibility between inoculants and pesticides must be understood. In general, pesticides contain molecules that are potentially toxic to living cells. Depending on the specificity, pesticides can cause toxicity to cells of microorganisms, animals, and plants, often resulting in death after contact with the product. In agriculture, they are commonly applied to the seeds, soil, and leaves of plants to prevent or control pests and diseases [19]. Usually, pesticides and inoculants are added together on the seeds. Thus, it is necessary to verify whether microbial cells in the inoculants are affected by pesticides, impairing the benefits of inoculation. We should also mention that anticipated inoculation or pre-inoculation, i.e., treating seeds with pesticides and inoculants several days before sowing, has become increasingly adopted by farmers [20]. However, the microorganisms are subjected to long-term exposure to pesticides, increasing the pernicious effect on the bacteria and resulting, for example, in decreased nodulation in legumes [21-23], lower N accumulation in grains [24, 25], and negatively impacting root development of grasses [26]. These losses may be due to microbial cell death caused by pesticides, as demonstrated in some studies [25-28], in which the longer the contact between bacteria and pesticides, the greater is mortality. Besides, changes in cell metabolism, such as formation of smaller colonies and decreased nitrogenase activity, have 99 been reported [25]. The world will need more food, and to meet this increased demand, there is no doubt that pesticides should be employed. However, there is also an increasing demand for environmentally-friendly inputs, including inoculants, to replace chemical fertilizers. One major challenge is to make the chemicals and biologicals compatible. In this review, we gathered information on the use of pesticides and inoculants, starting with the history, current situation, and results of studies that investigated the effects of fungicides, insecticides, and herbicides on microbial inoculants. The use of pesticides Studying planting and cultivation practices in ancient times, historians have reported that civilizations were already searching for effective approaches to protect and preserve their food. For millennia, methods such as burning sulfur, using arsenic, growing toxic species together with the crop of interest, and using salts and ashes against weeds were used to protect crops [29, 30]. In one of the oldest documents, from approximately 1550 BC, called Ebers Papyrus, interesting information about techniques used to eliminate insects from food planting areas has been described [30]. The report describes a mix of mercury and arsenic that was used for pest control [30], and a century later, arsenic was used along with honey, especially against ants. In 1867, during a potato beetle [Leptinotarsa decemlineata (Say)] outbreak in Colorado, USA, arsenic was used for pest control [29]. During the 1850s, a vineyard owner from Bordeaux, France, applied a mixture of copper and lime to grapes. Initially, the objective was to keep thieves away from his vineyards, but the wine producer realized that the application resulted in lower incidence of diseases. Notably, this mixture is still used today as a fungicide. Over the years, insecticides derived from plants have been discovered, such as pyrethrins and nicotine, the latter used specially to control aphids [19]. 100 In 1939, Paul Müller discovered dichlorodiphenyltrichloroethane (DDT), which was the first modern pesticide. This product played an important role during World War II when it was broadly used to control diseases transmitted by insects, such as malaria and typhus, ensuring soldiers´ health. The discovery of DDT resulted in considerable benefits to agriculture and human health, resulting in Müller being awarded the Nobel Prize in Medicine in 1948. Numerous other cheap and effective synthetic organic pesticides have been developed, contributing to a breakthrough in the market and starting a new era in pest and disease control [19, 29-31]. Fungicides such as captan and glyodin, the insecticide malathion, and the herbicide triazine were introduced in the following decades [29]. The use of pesticides increased until 1962 when the development of new products began to slow down because of the first studies and reports on the environmental and health risks associated with the indiscriminate use of pesticides. The book “Silent Spring” (1962), authored by the American scientist Rachel Carson, played an important role in the history of pesticides. In the book, the author discusses the harmful effects caused by the field spraying of several pesticides containing chlorinated hydrocarbons, among them the most important at the time, DDT. The effectiveness of these products is closely related to their stability and persistence in the environment, but they are also able to accumulate in the adipose tissue of some animals, a process known as bioaccumulation, which in some cases results in biomagnification, factors that make these compounds highly dangerous [19, 29, 30, 32]. Another important finding was the confirmation of the housefly (Musca domestica, L). resistance to DDT in Sweden only after a few years of application, another negative point for the use of this product [19]. As a result, in 1972, the US Environmental Protection Agency (EPA) banned the use of DDT in the country, and several pesticides were classified as restricted use, for example, endosulfan, dieldrin, and lindane. Organophosphorus and carbamates, which have lower risk, had been suggested as alternatives [19, 29]. DDT was banned in several other countries and, in 101 2001; during the Stockholm Convention, 179 nations signed a treaty that agreed to ban 12 persistent organic pollutants (POPs). It is interesting to note that since the 1960s, when both pesticides production and use became strictly regulated, alternative methods for pest and disease control began to be studied, with an emphasis on biological control (BC) [19, 29, 30] and integrated pest management (IPM) [33]. The principle of IPM is based on understanding population dynamics and using actions compatible with the environment to minimize the incidence of pests. In 1998, Kogan defined IPM as “the intelligent choice and use of control tactics that will produce favorable consequences from an economic, ecological, and sociological point of view.” In conclusion, the principle is to use several compatible techniques to keep the pest population at levels below capacity of causing economic, social, and environmental damages. IPM is currently widely used in crops around the world and is responsible for excellent results like reduced use of chemicals and increase in yields, in addition to other traditional methods such as mechanical and physical control and use of resistant plants, but they are not able to fully replace the use of chemical pesticides, which are still used on a large scale [34]. In 1990, the average worldwide use of pesticides by cultivation area was OF 1.5 kg ha- 1. Almost 30 years later, this value grew considerably, reaching an average of 2.63 kg ha-1 in 2018. The continents that applied more pesticides in 2018 were Asia and America, reaching 3.67 kg ha-1 and 3.52 kg ha-1, respectively. Europe applied 1.66 kg ha-1 of pesticides, while in Africa the average application was 0.3 kg ha-1 [18]. Among Asian countries, China, Japan, and Korea had the highest averages of pesticides per hectare in 2018, reaching 13.07 kg ha-1, 11.84 kg ha-1, and 11.73 kg ha-1 respectively. High rates were also reported in 2018 for South American countries, including Ecuador (25.8 kg ha-1), Uruguay (8.16 kg ha-1), Brazil (5.94 kg ha-1), Chile (5.86 kg ha-1), and Argentina (4.29 kg ha-1), while the United States of America (USA) applied an average of 2.54 kg ha-1 of pesticides in the same year [18]. 102 Each country has its own laws and regulations regarding the production, commercialization, and use of pesticides, designed mainly to protect human and environmental health. Among the actions of regulatory agencies are, for example, limitation of species to which a certain pesticide can be used, the requirement to use protective equipment, and the total prohibition of any product that is proven to be dangerous, which cannot be reliably mitigated [35]. Estimates point that since 1970, 508 types of active ingredients have been used in the USA, 134 of which were banned, with the majority of the cancelations taking place voluntarily by the manufacturers; only 37 prohibitions came from judiciary decisions. The United States are behind when banning pesticides, probably due to deficiencies in the legislation. From the list of pesticides still in use in the United States, 72 of them have been banned in the European Union, 17 in Brazil, and 11 in China [35]. Several studies have been carried out in the past few decades to understand the damage to human health and environment caused by of certain chemical pesticides [36-38]. These studies are very important because they generate information to guarantee food security. However, crop management still requires the application of pesticides to achieve high yields to meet the world´s increasing demand for food. Given this scenario, the indications are the use of pesticides, but with a trend toward more environmentally-friendly formulations as the replacement by biological control. Inoculants or biofertilizers Following the trend of agricultural production and concern about environmental sustainability, an innovative biotechnological product based on living microorganisms capable of making nitrogen available to plants was patented in 1896 and launched in 1898 by the first inoculant producing company, Nitragin; thus, replacing the application of potentially polluting N fertilizers. The first inoculant contained nitrogen-fixing rhizobia for soybean [Glycine max 103 (L.) Merr.] crop [17, 39-41]. Rhizobia are diazotrophic bacteria with an enzymatic apparatus to realize the biological nitrogen fixation (BNF) process, in which atmospheric nitrogen (N2) is converted into ammonia (NH3) and further to organic compounds that are easily assimilated by plants (Fig. 1). Therefore, when diazotrophic microorganisms are associated with specific plants, they supply nitrogen to their host, contributing to their development, and in return receive photosynthates from the host plant for their metabolism [42, 43]. Since the first inoculant was launched, a variety of inoculants has been produced, including rhizobia and other plant growth-promoting bacteria (PGPB) [17]. It has been necessary to research on several fronts, including the selection of microorganisms for each plant species [44-49], development of the culture media [50, 51], search for inoculant vehicles [52- 55], development of large-scale production, product distribution logistics, methods of inoculation [56-58], among others. Such studies have been responsible for expanding, diversifying, and improving the quality of inoculants. There are several reports on the contribution of inoculants increasing yields of crops at a low cost and mitigating environmental impact [15, 17]. As the production and commercialization of inoculants have increased, some countries have created laws to standardize, supervise, and guarantee the safety and quality of these bioproducts. In 1954, a microbiology professor at the University of Sydney, Australia, listed basic recommendations for quality control and use of legume inoculants, establishing the first quality control laboratory in the country. The Australian Inoculant Research Group (AIRG) is responsible for quality control. Since 2010, Australian inoculants that meet quality standards have exhibited a trademark called “Green Tick Logo,” which certifies that the product contains the correct strain, the number of living cells equal to or above the standard, and the minimum number of contaminating organisms [59]. Australian legislation served as a basis for legislation in other countries, such as in Uruguay and Brazil. In Brazil, legislation was established in 2004 104 and updated in 2011 [60-62]. Australia has moved to voluntary participation in the quality control of commercial inoculants, while in other countries such as France and Canada, as well as in many South American countries, participation is mandatory. The third group, including the US, comprises only internal control by the industry [17, 63]. Brazilian standards include a list of bacterial strains authorized for inoculation in different plant species, establishing a minimum cell concentration, and the limits for contaminants. Inoculants containing rhizobial species must present 1 × 109 viable cells per gram or mL until the expiration date, which must be at least 6 months, but other species may register with lower concentrations [61, 62]. The maintenance of cell concentration contributes for achieving the desired performance and ensuring product quality, but it is a challenge for the industry, as several factors can affect cell survival such as temperature, pH, drought, light, and availability of nutrients [50, 51, 53, 63, 64]. Soybean inoculation is certainly the most successful example worldwide, with an emphasis in South America. For example, in Brazil, it is well known that inoculation with elite Bradyrhizobium spp. strains fully supplies the plant´s demand for nitrogen, avoiding the use of N-fertilizers even with high-yielding genotypes [15, 42]. In the 2019/20 crop season, approximately 36 million hectares were cropped with soybean in Brazil, and even though most of the area had been inoculated in previous seasons, about 80% of the farmers adopted annual inoculation [65, 66]. Traditionally, soybean in Brazil has been inoculated only with Bradyrhizobium spp. strains [15]; however, in the past five years, co-inoculation of Bradyrhizobium spp. with the PGPB Azospirillum brasilense has been increasingly used, so that in a short period it has been adopted by 25% of the farmers [65, 66]. The main trait of the A. brasilense strains used for co-inoculation of soybean in Brazil has been recognized as the synthesis of phytohormones [67, 68]. Besides phytohormones synthesis, beneficial properties associated with PGPB include 105 BNF, phosphate and potassium solubilization, production of siderophores, detoxification of heavy metals, induction of plant systemic tolerance to abiotic and biotic stresses, production of hydrolytic enzymes, and production of exopolysaccharides [69-75] (Fig. 1). Such properties have been reported in several microorganisms, and the most commonly cited carrying one or more of these properties are Azospirillum spp. [72, 76], Pseudomonas spp. [77-79], and Bacillus spp. [80, 81], among others [71, 82]. With such a range of important properties and taking advantage of different microbial processes, the inoculation with mixes of bacteria has gained increased attention [67, 68, 82-84]. Some proposed mixes combine several species [75, 82], but one knows that it is difficult to grow and maintain proper concentrations of bacteria with different metabolic needs, In addition, a thorough preliminary study must be carried out so that the chosen species are compatible with each other. Before being used as a co-inoculant for legumes, PGPB such as A. brasilense has long been used in the inoculation of non-legumes, especially grasses such as maize (Zea mays L.), wheat (Triticum aestivum L.), and rice (Oryza sativa). Inoculants containing A. brasilense have been commercialized for more than 20 years; since 1996 in Argentina [76], 2002 in Mexico [85], and 2009 in Brazil [17, 44, 66]. A. brasilense can fix nitrogen, but in much lower quantities than rhizobia, making necessary supplemental N-fertilizer. Possibility of reduction of approximately 25% of the N-fertilized has been observed when A. brasilense is used as an inoculant in maize and this is environmentally and economically important [44, 57, 66, 86]. One of the greatest benefits of A. brasilense is its ability to produce phytohormones, such as auxins, that stimulate root development [72, 87] and gibberellins [87]. In addition, A. brasilense can induce plant defense mechanisms under abiotic and biotic stresses [69, 70, 72, 73]. Due to excellent results of inoculation with A. brasilense in several crops [88], it is expected that this practice will continue to grow in the coming years. In Brazil, in a short period of 10 years, since the first commercial inoculant containing A. brasilense was launched, the number of 106 commercialized doses reached 10.5 million in 2020 [66]. Rhizobial inoculants have been traditionally used in pasture-growing legumes, mainly alfalfa (Medica sativa L.) [89]. However, as most pastures worldwide have grasses, the use of other PGPB in important pastures such as brachiarias (Urochloa spp.) have increasingly attracted attention, with results showing improvement in soil fertility, biomass yield, and nutrient content in the forages (Fig. 1) [12, 90-92]. The inoculation pastures of grasses with PGPB has environmental and economic importance because most of the pastures worldwide are at some stage of degradation [93, 94]. By offering better quality fodder to cattle, the pastures can hold a larger number of animals, making saving of other areas from transformation into pastures. There are different ways to deliver the inoculants to the crops. Seed inoculation is the most common and easy method, as it can be carried out using solid or liquid inoculants. In this process, the inoculant is applied to the surface of the seeds, with or without stickers, which are then shaken so that the product spreads evenly. Other methods of applying liquid inoculants are possible, such as spraying on the soil surface or applying the product in the sowing furrow. Another alternative is leaf spraying, which can be done during certain periods of plant growth. Regardless of the strategy of inoculation, it is extremely important to apply the correct dose of the product, according to the manufacturer's recommendations, as lower or higher cell concentrations may decrease inoculation efficiency [56, 57, 67, 95]. Evidences of Incompatibility between Pesticides and Inoculants As the benefits of inoculation are closely related to the establishment of a plant- microorganism interaction, the survival and maintenance of microbial properties are crucial and mandatory. Therefore, the evaluation of microbial survival at the time of inoculation, and of the effectiveness on the inoculated crop are critical. The most common situation in commercial 107 crops is the combination of several products for different purposes, such as soybean seed treatment with microbial inoculants for nitrogen fixation, fertilizers and pesticides for prevention or treatment of pests and diseases. In many cases, depending on the mode of application, one product ends up being exposed to the other, or interacting one another either on the seeds, propagated material, in the soil, or leaf surface. It is important to know whether the contact of pesticides with microorganisms in the inoculant can affect cell survival and metabolism. Concerns about the compatibility of agrochemicals with microbial inoculants have been raised for decades, and several studies have shown that the impact of chemicals on the inoculant depends on the active ingredient, presence of other chemical substances that make up the formulation of pesticides, mechanism of action (systemic or contact), and method of application. The effects of incompatibility also depend on the bacterial species present in the inoculant, which may have different responses. However, few species have been evaluated for this purpose. The most recurring ones belong to the genres Rhizobium spp., Bradyrhizobium spp. and Azospirillum sp. Compatibility with Fungicides Fungicides are chemical products formulated to prevent the infection of plant tissues by phytopathogenic fungi, and in some cases, capable of extending the control of diseases caused by bacteria and viruses. The control exerted by fungicides can be mediated by killing the pathogen, temporarily inhibiting its germination and growth, or by affecting the production of spores [96]. Over the years, several fungicides have made it to the market; some have stood out and remained at the top in the list of the most used fungicides until today, more effective products have replaced others, and some have been banned. Fungicides of contact generally do not have a specific mode of action, are highly toxic, and when applied to seeds, soil, and plant leaves limit the pathogen survival. The most common are thiram (dithiocarbamate), captan 108 (quinone and heterocyclic), exon (aromatic), and guazatine. Upon entering microbial cells, the molecules promote a series of chemical reactions in nucleic acids and their precursors, and metabolic routes, affecting cell survival [96]. The majority of studies on compatibility have been performed with fungicides and microbial inoculants carrying rhizobia. Fungicides may affect several steps of the symbiosis, from the survival of the rhizobia on the seed to nodule formation and N2 fixation efficiency; in general, studies have been performed with soybean (e.g., 53, 97-99). The use of pesticides intensified in the past two decades, and so did concerns about their compatibility with inoculants [17]. Brikol et al. [21] evaluated the effects of applying different concentrations of the fungicide Thiram (10 to 750 µg mL-1) on soybean seeds inoculated with B. japonicum, which were grown under greenhouse conditions for 75 days. They observed that concentrations above 100 µg mL-1 reduced nodule number and dry weight, as well as the activity of the bacterial enzyme nitrogenase, responsible for the nitrogen fixation process. Similarly, there are reports [24, 100] of decrease in soybean nodulation and N accumulation in plants when seeds were inoculated with B. elkanii (SEMIA 5019) and B. japonicum (SEMIA 5079) and treated with different fungicides, benomyl, captan, carbendazin, carboxin, difenoconazole, thiabendazole, thiram, and tolylfluanid. Changes in nodule number were also observed in a field trial by Zilli et al. [101] when soybean seeds were treated with either carbendazin + thiram or carboxin + thiram. Interestingly, results of some studies indicate differences between strains in their tolerance to fungicides. For example, in soybean seeds treated with carbendazim + thiram, the lowest tolerance was observed in B. elkanii amongst four soybean Bradyrhizobium strains used in commercial inoculants in Brazil [B. elkanii (SEMIA 5019 and SEMIA 587), B. japonicum (SEMIA 5079), and B. diazoefficiens (SEMIA 5080)] [101]. In another study, Gomes et al. 109 [102] observed no effects on nodulation when seeds were inoculated with B. japonicum SEMIA 5079 + B. diazoefficiens SEMIA 5080 and treated with carbendazin + thiram. More recently, when the compatibility of B. japonicum SEMIA 5079 and B. elkanii SEMIA 587 was verified with Standak®Top, composed of a mixture of two fungicides and one insecticide (piraclostrobin, thiophanatemethyl, and fipronil), the effects were also more drastic for B. elkanii [25]. Altogether, indications are that B. elkanii is less tolerant to fungicides than B. japonicum or B. diazoefficiens. It is reasonable to postulate that the main effects of the fungicides used in seed treatment would be the decrease of rhizobial survival or inhibition of the root infection process, consequently affecting nodulation and BNF, and as a result grain yield, as observed by Zilli et al. [101]. In that study, the grain yield reduced by 20%, in addition to a decrease in the N content in grains when seeds were treated with B. elkanii SEMIA 587 + carbendazin + thiram (Fig. 2). However, some reports have indicated that the effects of fungicides may appear later. For example, in a study by Gomes et al. [102], although fungicides (carbendazin + thiram) did not affect nodulation, plants had lower number of pods per plant, grains per plant, and yield. Intriguingly, in two field experiments performed with soybean in Brazil, seed treatment with Standak®Top affected the total N accumulated in the grains of plants relying on both BNF and N fertilizer, indicating the negative impact of the pesticide on N metabolism [25]. In another study, a decrease in both protein and oleic acid contents was observed in soybean inoculated and treated with mefenoxan + fludioxonil [103]. As mentioned previously, negative effects may be related to the toxicity of fungicides on microbial cells, followed by impacts on microbial metabolism, reducing the effectiveness of the inoculant. Ahmed et al. [104] evaluated the growth of Bradyrhizobium sp. and Rhizobium sp. in Petri dishes containing solid culture medium and different concentrations of fungicides (captan, thiram, luxan, milcurb, and frernasan-D), soaked in discs placed on the medium. The 110 bacteria were tolerant to concentrations below 100 µg L-1, but at 1000 µg L-1 inhibition of growth and a decrease in the colony diameter of the surviving cells were observed (Fig. 2). Rathjen et al. [105] also reported that Rhizobium leguminosarum bv. viciae (WSM1455) was sensitive to the fungicides Thiram 600 and P-Pickel T (PPT), and their active ingredients (thiram and thiabendazole) at concentrations above 200 µg disc-1, with growth halos greater than 10 mm around the disks containing the fungicide. To verify the survival of B. elkanii (SEMIA 5019) and B. japonicum (SEMIA 5079) on seeds treated with fungicides, Campo et al. [24] recovered and counted viable cells from seeds inoculated and treated with benomyl, captan, carbendazin, carboxin, difenoconazole, thiabendazole, thiram, or tolylfluanid. After only 2 h of contact with carbendazin + thiram, viable bacterial cells were reduced by up to 64%, and reached 83% after 24 h. Mortality was verified with all other fungicides, reaching 95% with the mixture of thiabendazole and tolylfluanid after 24 h of contact. In addition to rhizobia, fungicides can also impair the contribution of other PGPB. The toxic effect of the combination of carbendazin + thiram was also verified for A. brasilense strains Ab-V5 and Ab-V6 by Santos et al. [27]. In that study, a decrease in the recovery of viable cells from seeds only after 2 h of contact was observed, and the viable cell count drastically decreased after 24 h, in comparison with the seeds not treated with fungicides (Fig. 2). Compatibility with Insecticides Many herbivorous insects feed on plants during their larval and adult stages, and/or some are important vectors of plant diseases. In both cases, insects may cause serious damages to crops. Insecticides, usually synthetic chemicals, acting as ovicidal, larvicidal, and adulticidal agents are used to prevent growth or kill insects [106]. Neonicotinoids, organophosphates, diamides, pyrethroids, and carbamates act on nerves and muscles; phosphides, cyanides, and 111 carboxamides on respiration, and cyclic ketoenols and ecdysone are agonists that interfere with insect growth and development [107]. Rathjen et al. [105] evaluated the in vitro toxicity of an imidacloprid-based insecticide on R. leguminosarum bv. viciae (WSM1455), and Mesorhizobium ciceri (CC1192). The strains were applied on solid culture medium in Petri dishes and sterile filter discs containing different concentrations (0, 100, 200, and 300 µg discs-1) of the insecticide. The inhibition of R. leguminosarum growth was observed at all concentrations. In alfalfa (Medicago sativa L.), Fox et al. [22] reported that seed treatment with the insecticides methyl parathion, DDT and pentachlorophenol affected the symbiosis with Ensifer (syn. Sinorhizobium) meliloti. The insecticides not only reduced nodule number and dry weight but also nitrogenase activity in nodules and plant biomass production. Ahemad [23] also reported several negative effects with the application of pyriproxifene, at the recommended dose of 1,300 μg kg-1 soil, in chickpeas (Cicer arietinum L.), peas (Pisum sativum L.), mung beans (Vigna radiata L. Wiclzek), and lentils (Lens esculentus, = Lens culinaris Medik) grown in pots that remained in an open field. Although the plants had not been inoculated, inferring that nodulation was related to indigenous rhizobia, pyriproxifer resulted in a 44% decrease in nodule number in peas, 14% in mung beans, and 5% in chickpeas and lentils, resulting in decreases in nodule dry weight compared with the controls not treated with insecticide. There was also a 17% decrease in the concentration of nitrogen in the roots of chickpeas, 15% in peas, 14% in mung beans, and 18% in lentils, and a 5% decrease in the protein contents in grains of chickpeas, 4% in mung beans, 3% in lentils, and 1% in peas, compared with the controls not treated with insecticide. Insecticides also affect PGPB other than rhizobia. For example, Fernandes et al. [108] studied the effects of five insecticides (imidacloprid, fipronil, fenamethoxam, endosulfan, and carbofuran) indicated for sugarcane (Saccharum spp.) on the diazotrophic bacterium Herbaspirillum seropedicae. In vitro evaluations of cell growth after 33 h indicated that the 112 insecticides that most interfered with bacterial growth were endosulfan and carbofuran. Madhaiyan et al. [109] evaluated the effect of different insecticides (monocrotophos, malathion, chlorpyriphos, diclorvos, lindane and endosulfan) on Gluconacetobacter diazotrophicus, another PGPB found in association with sugarcane. Except for malathion, all other insecticides reduced cell concentration, and lindane lysed the cells (Fig. 2). In the same study, nitrogenase activity was fully inhibited by monocrotophos, dichlorvos, and lindane, 83.3% by chlorpyriphos, 80.9% by melation, and 33.4% by endosulfan. Concerning the synthesis of indoleacetic acid (IAA) and gibberellin A3 (GA3) by G. diazotrophicus, inhibition was observed with lindane, with decreases of 93.2% and 96.5% for IAA and GA3, respectively. The authors also described that the insecticides dichlorvos, chlorpyriphos, and lindane completely inhibited the solubilization of phosphate (P) and zinc (Zn) [109] (Fig. 2). Compatibility with Herbicides Another class of important pesticides for agriculture are herbicides used for weed control. Herbicides have different degrees of specificity based on differences in biochemical pathways in certain plant groups. The mode of action of herbicides is generally related to cell division process, which inhibits a key enzyme/protein [110]. Examples of herbicides applied worldwide include glyphosate, paraquat, and diuron. Among these, the most well-known is glyphosate; since its introduction in the 1970s, its use spread quickly, facilitated cropping, but also implied in the growing appearance of resistant weeds, resulting from a natural process of plant adaptation, and decreasing its efficacy [111-113]. An important alternative to minimize this problem, in addition to integration with other control methods, would be diversification in the use of herbicides, including others with different mechanisms of action [114]. Few studies have investigated the compatibility between herbicides and inoculants. Madhaiyan et al. [109] evaluated the effects of different herbicides (butachlor, alachlor, 113 atrazine, and 2,4-D) in liquid culture medium on the growth and metabolism of G. diazotrophicus. Cell growth was impaired only in the presence of 2,4-D, but all herbicides reduced nitrogenase activity, the production of IAA and GA3, and the solubilization of P and Zn. The highest inhibition of nitrogenase activity (73.6% and 65.3%) was observed with alachlor and atrazine, respectively, while butachlor mostly affected production of IAA (53.3%), whereas 2.4-D mostly affected the production of GA3 (78.8%). Butachlor was also responsible for the strongest reduction in the solubilization of P and Zn. Therefore, although herbicides do not affect cell survival, they significantly affect metabolism of G. diazotrophicus [109]. In an assay performed under greenhouse conditions, Angelini et al. [115] evaluated the effects of imazetapir, imazapic, S-metachlor, diclosulam, and glyphosate on diazotrophic bacteria in the soil during the cultivation of peanuts (Arachis hypogaea L.). The seeds were sown and the herbicide was sprayed on the soil surface. All herbicides caused reduction in cell concentration in both free and symbiotic diazotrophic bacteria, and this negative impact was confirmed under field conditions even one year after the application. Nitrogenase activity also reduced due to herbicides, except for glyphosate [115]. The ability of cyanobacteria to fix atmospheric nitrogen in flooded soils suitable for rice cultivation, make this group an important ally in maintaining soil fertility and contributing for cereal yield. Thus, Dash et al. [116] evaluated the responses of cyanobacteria in rice plantation soil to the exposure of different agrochemicals, including the herbicide benthiocarb that was applied in one dose at the time of puddling. The herbicide decreased cell growth, which was even worse when combined with urea (used as a fertilizer). Benthiocarb reduced nitrogenase activity between 13-27%, compared with the control without herbicide, and its combination with urea resulted in an even greater reduction in addition to a decrease in the nitrogen accumulation that reached 47% at 60 days. Concerning the symbiosis between legumes and rhizobia, in general, herbicides have 114 been considered less toxic than fungicides and insecticides [100], with glyphosate being the one with lower toxicity [117; 118]. Although the negative effects of glyphosate on B. japonicum growth were reported by King et al. [119], the doses in the experiment were far higher than those recommended for field application. With the release of genetically modified (GM) genotypes tolerant of herbicides, studies on the compatibility with the GM genotypes and herbicides have begun. In soybean, which represents the most used herbicide-tolerant species, glyphosate-resistant (Roundup Ready) pairs of nearly isogenic cultivars were evaluated in six field sites in Brazil for three crop seasons. Although the transgenic trait negatively affected some BNF variables, these effects had no significant impact on soybean grain yield, and no consistent differences between glyphosate and conventional herbicide application were observed on BNF-associated parameters [120]. Similar results were reported in 20 field trials performed with soybean with the ahas transgene, imidazolinone, and conventional herbicides [121]. Finally, it is worth mentioning the reported capacity of several rhizobia to degrade herbicides, such as glyphosates [122, 123], contributing to a decrease in their negative impact. Compatibility with mixtures (fungicides, insecticides, and herbicides) Approximately 70% of the pesticides available in the market contain mixtures of two or more types of fungicides and insecticides [124] and are often combined with herbicides at the time of application, aiming to facilitate the combined control of pests, diseases, and weeds. However, the damage to microbial inoculants increases with the number of combined chemicals. As previously mentioned, Standak® Top, one of the most used for treatments of seeds in several countries, especially for soybean, is composed of two fungicides and one insecticide; it affects the survival of B. japonicum and especially B. elkanii cells, with a drastic decrease verified after 7 days of contact [25]. It is worth mentioning that pre-inoculated soybean seeds have been in contact with Standak®Top for up to 90 days, often resulting in zero 115 recoveries of rhizobial cells from seeds [124]. Another interesting observation in the study by Rodrigues et al. [25] was the changes in colony morphology, smaller with the increase of the exposure to the pesticide. However, regular colonies were recovered after the bacteria were grown under optimal conditions, indicating an adaptive mechanism to the stressful conditions when exposed to the pesticides. Santos et al. [26] evaluated the compatibility of Standak®Top with A. brasilense strains Ab-V5 and Ab-V6. First, differences were observed between strains, with lower tolerance of Ab-V5, so that after 24 h of exposure the recovery of viable cells dropped from 4.56 x 105 to 4.37 x 102 cells seed-1. In a greenhouse experiment with the combined strains, Standak®Top decreased the number of lateral roots and root hairs and resulted in shorter root hair length. Pereira et al. [28] also reported the mortality of A. brasilense strains Ab-V5 and Ab-V6 in maize seeds treated with a mixture of fungicides and insecticides (metalaxyl-M + fludioxonil + thiamethoxam + abamectin). The cell survival rate after 12 h of inoculation of seeds treated with the pesticide was only 13.56%, whereas in untreated seeds was 65.87%. Are there alternatives to the challenges of using pesticides and microbial inoculants? Remarkable advances in genetic engineering have occurred particularly in the last decade and are advancing towards obtaining plant genotypes resistant to abiotic stresses, pests and diseases [125-127]. However, large-scale agriculture to feed the increasingly growing population still demands the heavy use of pesticides for at least two more decades. Practical agriculture without pesticides may be a dream for most of our society but is currently restricted to a few farmers, most in small properties, and there is no technology in the research pipeline to make it feasible for the majority of the cropped areas in a short time. On the other hand, the use of bioproducts aimed at the total or partial replacement of chemicals used for the control of pests, diseases, weeds, and fertilizers is growing at a rate never seen before [17]. The major 116 limitation, as we have shown in this review, is the low compatibility between pesticides and microbial inoculants applied to seeds. There is an urgent need to develop alternatives to make microbial inoculants compatible with pesticides. Ahemad and Khan [128] selected strains of R. leguminosarum that were tolerant to high concentrations of the insecticides fipronil and pyriproxifene used in peas. The tolerant strain MRP1 was characterized as the highest in the synthesis of IAA, siderophores, and EPS; in a field trial performed in soil previously treated with insecticides, the strain contributed to the significant increases in nodulation, N and P contents of roots and shoots, and grain yield. The results show the feasibility of selecting strains tolerant of pesticides. One main limitation is the increasing number of pesticides used in agriculture, which would require multiple steps of microbial selection. To develop pesticides less harmful to microbial bioinoculants, the inclusion of carriers for the active ingredients might be simpler than obtaining tolerant strains. Unfortunately, the pesticide industry has not demonstrated interest in following this strategy, probably because the chemical industry is financially more powerful than the inoculant industry, and the appeal to invest in less toxic molecules. Another strategy could be to take advantage of microbial metabolites instead of living microbes [87]; however, this is only applicable to microorganisms that produce secondary metabolites useful to the host plants, like Azospirillum sp. in grasses and pastures, and not by mechanisms that require living microorganisms, such as rhizobia, to nodulate legumes. Investment should be made on innovation in formulations, including cell protection to minimize or avoid the toxic effects of pesticides on microbial cells. The addition of protective molecules such as polymers, chemicals, or synthesized by microorganisms may help in this regard, for example, polyhydroxyburytate (PHB) [50, 129, 130] and biofilms [131]. In Brazil, with no short-term solutions for compatibility in sight, the only feasible 117 strategy is physically avoiding the contact of inoculants with pesticides. Seed treatment with pesticides, for example, for soybeans that currently may contact up to 14 chemical products, an in-furrow application of biologicals has proven to be effective in guaranteeing the benefits to the crop. Despite the need of increasing the dose of inoculant applied in-furrow, the cost of inoculant is low in comparison with the benefits. A pioneering study confirming that the in- furrow inoculation of soybean with 2.5 times the concentration used for seed inoculation alleviated the effects of seed treatment with agrochemicals was published in 2010 [56]. Despite requiring that the farmers buy new equipment, 20% of the farmers in Brazil adopted this technique by 2020. Other strategies, with an emphasis on soil-spray and leaf-spray inoculation [57, 86, 132, 133] have also been investigated and show some degree of effectiveness, but are not as effective as in-furrow and seed inoculations. Final Remarks The critical analysis of this review points to some certainties: (i) considering the technologies available today and those that should be available in the next few years, large- scale agriculture to meet the increasing food demand will require high inputs of pesticides; (ii) the demand for higher sustainability in agriculture, with bioproducts aiming at partially or fully replacing pesticides and fertilizers is increasing; (iii) inoculants or biofertilizers have been increasingly adopted by farmers, but their compatibility with pesticides, especially when used for seed treatment, is very low; (iv) strategies to solve the incompatibility between pesticides and inoculants are needed, as those proposed until now are still very modest with regards to their feasibility. Incompatibility between pesticides and inoculants affects cell survival and metabolism. The level of incompatibility with the pesticides depends on the active principle, formulation, doses, time of contact with the cells, and may vary with the bacterial species or strain. Despite 118 the increasing market for biological products aiming at more sustainable agriculture [134], very few intellectual and economic investments have been made to search for compatibility of biological products with chemicals. Therefore, there is an urgent need to emphasize studies and development of innovative strategies to mitigate the incompatibility between pesticides and microbial inoculants. ACKNOWLEDGEMENTS M.S.S. acknowledges a PhD fellowship from Araucaria Foundation of support to the Scientific and Technological Development of the State of Paraná. M.A. Nogueira and M. Hungria are research fellows of CNPq. We also thank the financial support given by the National Institute of Science and Technology, INCT-Plant-Growth Promoting Microorganisms for Agricultural Sustainability and Environmental Responsibility (CNPq 465133/2014-4, Fundação Araucária-STI 043/2019, CAPES). Abbreviations All abbreviations have been cited in their complete forms when mentioned for the first time in the manuscript. Ethics Approval and consent of participation The study has not involved any human or animal participation or data Consent for publication All authors gave the consent for publication Availability of Data and Materials 119 All data and materials cited in the manuscript are freely available for the scientific community. Competing Interests Authors declare no competing interests regarding the data or the manuscript Ethical interests Authors declare no ethical problems. Funding information National Institute of Science and Technology, INCT-Plant-Growth Promoting Microorganisms for Agricultural Sustainability and Environmental Responsibility (CNPq 465133/2014-4, Fundação Araucária-STI 043/2019, CAPES) and Embrapa (20.19.02.009.00.01) REFERENCES 1. UNITED NATIONS, Department of Economic and Social Affairs, Population Division. World Population Prospects 2019 Highlights. Available online: https://population.un.org/wpp/Publications/Files/WPP2019_Highlights.pdf (accessed on 04 Jan 2021). 2. Ameen, A.; Raza, S. Green Revolution: A Review. IJASR 2017, 3, 129-137, doi:10.7439/ijasr. 3. Armanda, D.T.; Guinée, J.B.; Tukker, A. The second green revolution: Innovative urban agriculture’s contribution to food security and sustainability – A review. Glob Food Sec 2019, 22, 13-14, doi:10.1016/j.gfs.2019.08.002 4. Arora, N.K. Agricultural sustainability and food security. Environ Sustain 2018, 1, 217–219, doi:10.1007/s42398-018-00032-2 5. 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Available online: http://dunhamtrimmer.com/wpcontent/uploads/2019/05/TOCDT_Global_Biocontrol_Overvie w_Links.pdf (accessed on 02 Fev 2021). https://doi.org/10.5539/jas.v12n1p194 http://dunhamtrimmer.com/wpcontent/uploads/2019/05/TOCDT_Global_Biocontrol_Overview_Links.pdf http://dunhamtrimmer.com/wpcontent/uploads/2019/05/TOCDT_Global_Biocontrol_Overview_Links.pdf 131 Figures Legends Figure 1 - Main benefits of inoculation with rhizobia and plant-growth-promoting bacteria (PGPB). Biological Nitrogen Fixation (BNF), phosphate (P) and potassium (K) solubilization, phytohormone production (auxins and gibberellins), and induced plant systemic tolerance to abiotic and biotic stresses are represented. As benefits, there are increases in biomass production, yield, and improvement in soil fertility. Figure 2 - Representation of the effects reported on the incompatibility between pesticides and inoculants, from the moment of contact with microbial cells to damages to plant development. 132 Table 1 – Effect of pesticides on cell viability and/or morphology and on the metabolism of microorganisms used as inoculants Pesticide Concentration Microorganism Effect Reference Monocrotophosi, Malathioni, Chlorpyriphoi, Dichlorvosi, Lindanoi e Endosulphani Recommended dose of each product Gluconacetobacter diazotrophicus With the exception of Malathion, all insecticides reduced cell viability. Nitrogenase activity was totally inhibited by Monocrotophos, Dichlorvos and Lindano. Production of IAA and GA3, and solubilization of P and Zn were impaired [109] Butachlorh, Alachlorh Atrazineh and 2,4-Dh Recommended dose of each product Cell growth was hindered by 2,4-D. All herbicides reduced the activity of nitrogenase, the production of IAA and GA3, and the solubilization of P and Zn Captanf, Thiramf, Luxanf and Fernasan-Df 1000 µg L-1 Bradyrhizobium sp. and Rhizobium sp. Decreased colony diameter and inhibited growth in areas close to the fungicide application site [104] Benomylf, Captanf, Carbendazinf, Carboxinf, Difenoconazolef, Thiabendazolef, Thiramf, Tolylfluanidf Recommended dose for soybean B. elkanii and B. japonicum All fungicides caused mortality of microorganisms [24] Imidaclopridi, Fipronili, Thiamethoxami, Endosulphani e Carbofurani 250 g ha-1, 400 g ha-1, 480 g ha-1, 2.800 g ha-1, 1.650 g ha-1, respectively Herbaspirillum seropedicae Endosulphan increased the lag phase. Carbofuran increased generation time and reduced lag phase [108] Pyraclostrobinf, thiophanato-methylf e fipronili 2 mL kg-1 maize seed A. brasilense (Ab- V5 and Ab-V6) Drastic reduction in cell concentration 24 h after inoculation in treated seeds [26] Carbendazinf+Thiramf 40-60 mL 20 Drastic reduction in cell concentration 24 h after [27] 133 kg-1 maize seed inoculation in treated seeds metalaxil-m + fludioxonil + tiametoxame + abamectin Recommended dose for maize Drastic reduction in cell concentration 12 h after inoculation in treated seeds [28] Thiramf, Thiram+Thiabendazolef and PPTf > 200 µg L-1 Rhizobium leguminosarum bv. viciae Formation of growth inhibition halos greater than 10 mm around the fungicide [105] Imidaclopridi 0, 100, 200 e 300 µg L-1 Formation of growth inhibition halos greater than 10 mm around the insecticide for all concentrations evaluated Pyraclostrobinf, thiophanato-methylf e fipronili Recommended dose for soybean B. elkanii and B. japonicum Drastic decrease in cell concentration after 7 days of exposure. Colony formation with smaller diameter [25] ffungicide; iinseticide, hherbicide 134 Table 2 – Effect of pesticides on the development of plants whose seeds had been inoculated. Culture Fungicide Microorganism local Effect Reference Soybean (Glycine max) Thiramf B. japonicum Greenhouse Lower nodule number, nodule dry weight and nitrogenase activity [21] Benomylf, Captanf, Carbendazinf, Carboxinf, Difenoconazolef, Thiabendazolef, Thiram, Tolylfluanidf B. elkanii (SEMIA 5019) + B. japonicum (SEMIA 5079 ) Greenhouse and field Reduction in the number of nodules and in the total N in grains [24] Carbendazinf+Thiramf; Carboxinf+Thiramf B. elkanii (STRAIN 5019 and STRAIN 587), B. japonicum (STRAIN 5079 and STRAIN 5080) Field Reduction of nodulation efficiency. Reduction of N content and grain yield to SEMIA 587 with Carbendazin + Thiram [101] Carbendazinf+Thiramf B. japonicum (STRAIN 5079 and STRAIN 5080) Field Reduction in the number of pods per plant and grains per plant [102] Mefenoxamf+Fludioxonilf B. japonicum Field Reduced grain yield and protein and oleic acid content [103] Pyraclostrobinf, thiophanato-methylf and fipronili B. japonicum (STRAIN 5079) and B. diazoefficiens (STRAIN 5080) Field Less N accumulation in grains [25] Alfafa (Medicago sativa) Methyl parathioni, DDTi e pentachlorophenoli Sinorhizobium meliloti, Greenhouse Reduction of nitrogenase activity, number of nodules and plant dry weight [22] Peanut (Arachis hypogaea). Imazetapirh, Imazapich, S- metachloroh, Diazotrophic bacteria present in Greenhouse Reduction of cell concentration of free and symbiotic diazotrophic [115] 135 ffungicide; iinseticide, hherbicide Dichlosulamh and Glyphosateh the soil bacteria Field Reduced cell concentration of free and symbiotic diazotrophic bacteria and reduced nitrogenase activity except for glyphosate Chickpeas (Cicer arietinum L.), pea (Pisum sativum L.), Mung beans (Vigna radiata L. Wiclzek) and lentil (Lens esculentus, = Lens culinaris Medik). Pyriproxyfeni Bacteria commonly present in the soil used Pots in the field Reduction in the number of nodules, in the dry weight of nodules, in the concentration of N in roots and in protein concentration in the grains [23] Rice (Oryza sativa) Benthicarbh Cyanobacteria naturally found in the rice paddy soil Field Decreased cell growth, nitrogenase activity and Naccumulation [116] Maize (Zea mays) Pyraclostrobinf, thyophanato-methylf and fipronili A. brasilense (Ab- V5 and Ab-V6) Greenhouse Fewer branch roots, root hair and shorter root hair length [26] 136 Fig. 1. 137 Fig. 2 138 CONCLUSÃO Com base nos resultados obtidos nos trabalhos apresentados, pode-se concluir que a inoculação de culturas é uma prática capaz de gerar aumento significativo de produtividade. O Brasil é destaque nas pesquisas relacionadas a inoculantes, portanto espera-se que cada vez mais agricultores comecem a aderir e que novas formulações, ainda mais eficientes, cheguem ao mercado. Apesar dos benefícios resultantes do uso dos inoculantes, quando associados a agrotóxicos pode-se observar que a viabilidade celular de Azospirilum brasilense inoculado em sementes de milho cai drasticamente em 24 horas quando as sementes também recebem tratamento com agrotóxicos. Com o desenvolvimento da metodologia para recuperação de células inoculadas em sementes de milho, mais trabalhos poderão ser realizados a fim de se compreender mais detalhadamente esse processo, para diferentes ingredientes ativos usados no tratamento de sementes dessa cultura. Além disso, os benefícios da inoculação do milho com as estirpes Ab- V5 e Ab-V6 podem ser prejudicados, principalmente quanto ao número de ramificações e pelos radiculares, apesar de não parecer interferir no desenvolvimento inicial da planta. Estudos futuros são necessários para observar se há interferência na produtividade do milho. Levando em consideração que o uso de pesticidas ainda é a forma mais comum para se combater pragas e doenças nas lavouras, torna-se evidente a necessidade de que novas formulações de inoculantes sejam desenvolvidas levando em consideração a vulnerabilidade das células bacterianas em contato com esses produtos químicos, visando a proteção dessas células, a fim de que todo potencial biotecnológico dos inoculantes seja explorado.