FELIPE BARRETO DA SILVA Cowpea mild mottle virus EM SOJA: INTERAÇÃO VÍRUS X VETOR, INCIDÊNCIA E VETOR ASSOCIADO, VARIABILIDADE DO VÍRUS E IMPACTOS NA QUALIDADE FISIOLÓGICA DE SEMENTES Botucatu 2022 FELIPE BARRETO DA SILVA Cowpea mild mottle virus EM SOJA: INTERAÇÃO VÍRUS X VETOR, INCIDÊNCIA E VETOR ASSOCIADO, VARIABILIDADE DO VÍRUS E IMPACTOS NA QUALIDADE FISIOLÓGICA DE SEMENTES Tese apresentada à Faculdade de Ciências Agronômicas da Unesp Câmpus de Botucatu, para obtenção do título de Doutor em Agronomia (Proteção de Plantas). Orientador: Profa. Dra. Renate Krause Sakate Coorientador: Dra. Cristiane Müller Botucatu 2022 S586c Silva, Felipe Barreto da Cowpea mild mottle virus em soja: interação vírus x vetor, incidência e vetor associado, variabilidade do vírus e impactos na qualidade fisiológica de sementes / Felipe Barreto da Silva. -- Botucatu, 2022 78 p. : tabs., fotos, mapas Tese (doutorado) - Universidade Estadual Paulista (Unesp), Faculdade de Ciências Agronômicas, Botucatu Orientadora: Renate Krause Sakate Coorientadora: Cristiane Müller 1. Virologia vegetal. 2. Bemisia tabaci. 3. Carlavirus. 4. Glycine max. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca da Faculdade de Ciências Agronômicas, Botucatu. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. A meus amados pais, Adhemar e Lidia. AGRADECIMENTOS Agradeço a Deus pelo dom da vida, e por nunca me abandonar nos momentos mais desafiadores e difíceis, tanto no âmbito acadêmico quanto no pessoal. Agradeço com muito amor e gratidão a minha orientadora Profa. Dra. Renate Krause Sakate, que há cinco anos me acolheu em seu laboratório e nunca me deixou desistir desse sonho. À ela, meus mais sinceros obrigado, pela orientação, ensinamentos, conselhos, paciência, pedais e que além de ser exemplo de professor é uma grande amiga. À Corteva Agriscience pelo apoio técnico e à dra. Cristiane Muller pela coorientação. Agradeço o Programa de Pós-Graduação em Agronomia (Proteção de Plantas) e ao Departamento de Proteção Vegetal da FCA, e todos os professores em especial ao Prof. Dr. Carlos Gilberto Raetano, Prof. Dr. Marcelo Agenor Pavan (in memorian) e a Profa. Dra. Regiane Cristina de Oliveira por todos os ensinamentos, apoio e amizade, e ao Prof. Dr. Edson Luiz Lopes Baldin (in memorian) pelos conselhos. Aos funcionários do Departamento de Proteção Vegetal da FCA, o querido Nivaldo Diez e a Dra. Vanessa de Carvalho e ao Dr. Marcelo Soman por sempre me socorrerem nas mais diversas questões. À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), pelo apoio financeiro, concedido por meio de bolsa de doutorado (Processo n° 2019/24642-8). Também gostaria de agradecer os membros do Laboratório de Virologia Vegetal e Interação Vírus-Vetor-Planta (LAVIV) da Unesp e do prédio da Fito, obrigado aos que me apoiaram e pelos anos de convivência. Um agradecimento especial aos Doutores Luis Watanabe, Bruno De Marchi e Marcos Ribeiro Junior pelo companheirismo e aos estagiários que me acompanharam nessa caminhada, Rodrigo Raposo, Sarah Forlani e Geovanne Luchini. E gostaria de agradecer com muito amor os meus amados pais Adhemar e Lidia, por sempre acreditarem e confiarem em mim e me apoiarem em tudo incondicionalmente. À minha família e meus amigos que estiveram juntos e distantes durante todos esses anos, e agradeço imensamente a minha companheira e amiga Natalia Bevilaqua por sempre estar ao meu lado. Eu jamais conseguiria esse feito sem vocês. “Conheça todas as teorias, domine todas as técnicas, mas ao tocar uma alma humana, seja apenas outra alma humana”. JUNG, C. G. Contribution to analytical psychology. London: Kegan Paul, 1928. p. 361. RESUMO Doenças causadas por vírus podem impactar negativamente a cultura da soja (Glycine max L. Merril). Entre elas podemos citar a necrose da haste causada pelo cowpea mild mottle virus (CPMMV), cujo vetor é a mosca-branca Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) atualmente caracterizada como um complexo de espécie crípticas, das quais as mais invasivas são Middle East Asia Minor 1 (MEAM1, também conhecida por biótipo B) e Mediterranean (MED, conhecida por biótipo Q). Com o objetivo de verificar a distribuição destas espécies crípticas em áreas produtoras de soja no Estado de São Paulo, bem como a incidência do CPMMV nestas regiões, foram realizadas coletas de plantas de soja e mosca-branca por três anos consecutivos e avaliada a presença do vírus e caracterizada a espécie críptica de B. tabaci associada a plantas de soja. Isolados representativos de CPMMV tiveram o seu genoma completo sequenciado por sequenciamento de nova geração (HTS). Além disto foram estudados aspectos da transmissão do CPMMV por B. tabaci MED e MEAM1 e o efeito do CPMMV nos aspectos agronômicos e qualidade fisiológica das sementes de soja. Os resultados indicaram que MEAM1 ainda é a espécie predominante em áreas de soja, entretanto algumas áreas, como em Buritãma, tiveram predominância de MED na primeira safra de soja avaliada (2019/2020) com redução nas safras posteriores (2020/2021 e 2021/2022). Em Santa Cruz do Rio Pardo e Óleo houve um aumento de incidência de MED ao longo das safras de soja avaliadas. Os ensaios de transmissão com um único espécimen de mosca-branca revelaram que MED é um melhor vetor de CPMMV comparado com MEAM1, sendo capaz de transmitir o vírus com apenas dois minutos de período de acesso à inoculação. A análise filogenética das sequencias do genoma completo de isolados de CPMMV coletados nos Estados da Bahia e de São Paulo, mostraram que eles pertencem ao grupo BR2, que compreendem os isolados de CPMMV mais representativos do Brasil. O CPMMV afetou drasticamente a qualidade fisiológica das sementes de soja produzidas a partir de plantas infectadas, reduzindo a germinação das sementes. Os resultados deste trabalho reforçam os impactos negativos do CPMMV em soja e a importância do monitoramento de espécies crípticas de B. tabaci como forma de subsidiar as táticas de manejo desta praga e vetor de virus. Palavras-chave: carlavirus; levantamento; transmissão; Bemisia tabaci. ABSTACT Diseases caused by viruses are important constraints to soybean (Glycine max L. Merril) production. Among them, stem necrosis caused by cowpea mild mottle virus (CPMMV), transmitted by the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Two B. tabaci cryptic species were already detected in soybean-growing areas in São Paulo state, the Middle East Asia Minor 1 (MEAM1, formerly known as biotype B) and the Mediterranean (MED, biotype Q). Here we evaluate the distribution of CPMMV and B. tabaci cryptic species in the main soybean growing areas from São Paulo state for three years. CPMMV isolates were also sequenced by High Throughput Sequencing (HTS) and properties of CPMMV transmission by MED and MEAM1, and the effect of CPMMV on the physiologic seed quality in soybean were evaluated. Results indicated that MEAM is still the predominant whitefly species in soybean, but MED was also found in different locations where soybean is produced in the state. Some areas, like Buritãma, evolved to a predominance of MED over the three years but others where MED was previously detected, showed a reduction of the insect during the same period. In Santa Cruz do Rio Pardo and Óleo, MED incidence increased during the evaluated years. Transmission assays with one single insect revealed that MED is a better vector compared to MEAM1, transmitting the virus within two minutes of the inoculation access period. Phylogenetic analysis of the complete genome sequence of CPMMV isolates collected in Sao Paulo and Bahia State showed that they belong to BR2 clade that includes Brazilian CPMMV isolates collected from soybean. Studies of the effects of CPMMV on the agronomic aspects of soybean and its effects on the seed physiology quality showed that there are negative effects of the virus in the plants and in the seeds. Our results suggest that the continuous survey of B. tabaci cryptic species and CPMMV should be frequent to better whitefly and CPMMV management in soybean. Keywords: carlavirus; survey; transmission; Bemisia tabaci. SUMÁRIO INTRODUÇÃO GERAL........................................................................... 17 CAPÍTULO 1 - COWPEA MILD MOTTLE VIRUS AND Bemisia tabaci ON SOYBEAN IN SÃO PAULO STATE (BRAZIL) AND TRANSMISSION PROPERTIES OF MEDITERRANEAN AND MIDDLE EAST ASIA-MINOR CRYPTIC SPECIES................................................ 22 1.1 INTRODUCTION..................................................................................... 23 1.2 MATERIAL E METHODS........................................................................ 26 1.3 RESULTS................................................................................................ 29 1.4 DISCUSSION.......................................................................................... 40 REFERENCES........................................................................................ 44 CAPÍTULO 2 – EFFECTS OF COWPEA MILD MOTTLE VIRUS ON SOYBEAN SEED QUALITY AND YIELD COMPONENTS...................... 64 2.1 INTRODUCTION..................................................................................... 64 2.2 MATERIAL AND METHODS................................................................... 66 2.3 RESULTS AND DISCUSSION................................................................ 68 2.4 CONCLUSIONS...................................................................................... 71 LITERATURE CITED.............................................................................. 72 CONSIDERAÇÕES FINAIS.................................................................... 74 REFERÊNCIAS ...................................................................................... 75 17 INTRODUÇÃO GERAL A soja [Glycine max (L.) Merr.] está entre os cinco produtos agrícolas mais importantes do mundo, sendo principal fonte de óleo vegetal e proteína para alimentação de animais na pecuária e para o consumo humano (SAVARY et al., 2019). Além da sua utilização como fonte de alimento, a soja é altamente empregada como material base na produção de biodiesel, produtos industriais e farmacêuticos (HILL et al., 2006). Atualmente, o Brasil é líder mundial no ranking de países produtores do grão de soja, com uma produção de 126,000 milhões de toneladas na safra de 2021/2022. Nessa mesma safra, o país exportou 89,000 milhões de toneladas que corresponde a 35,64% da exportação mundial de soja, se destacando também como o atual maior exportador mundial dessa comodity (USDA, 2022). Dos 127,824 milhões de hectares de área plantada com a cultura da soja no mundo, o Brasil destina 38,502 milhões de hectares para a sua produção. O estado do Mato Grosso ocupa o primeiro lugar do país na produção de soja, concentrando cerca de 28% da produção nacional, seguido dos estados do Paraná (19%), Rio Grande do Sul (14%), Goiás (10%) e Mato Grosso do Sul (7%). Estados como a Bahia, Maranhão, Minas Gerais, Pará, Piauí, Santa Catarina, São Paulo e Tocantins, vêm crescendo anualmente suas áreas de produção. No estado de São Paulo, por exemplo, estima-se que na safra 2021/2022, houve um aumento de 3,4% na área de plantio, chegando a 1,2 milhões de hectares (IEA, 2022). Embora a sojicultura esteja em ascensão, nacionalmente e internacionalmente (USDA, 2022), assim como a produção de diversas culturas agrícolas de importância econômica, a produção de soja vem sendo negativamente impactada por pragas e doenças (BANDARA et al., 2020). As doenças causadas na cultura da soja são um dos principais desafios enfrentados pelos produtores, pois além de diminui a produção e a qualidade dos grãos, elas contribuem com o aumento nos custos de produção causando perdas mundiais de bilhões de dólares (BRADLEY et al., 2021). Entre as doenças que afetam a cultura da soja, as doenças causadas por vírus são muito desafiadoras. Diferentemente de outros patógenos como bactérias e fungos que podem ser manejados com produtos químicos, o controle do vírus é complexo, necessitando de múltiplas estratégias que se baseiam na prevenção da infecção. 18 Outro fator que dificulta o manejo destas doenças é a necessidade de uma rápida identificação e diagnose do agente viral, que necessita de técnicas específicas e sensíveis que geralmente não são de fácil acesso. Sintomas causados por vírus em plantas podem variar de leves mosqueados, mosaicos, cloroses até mesmo podendo ocasionar a morte da planta. Em soja, há doenças de etiologia viral que podem ser assintomáticas ou com seus sintomas frequentemente confundidos com aqueles causados por injúrias de herbicidas (BARRETO DA SILVA et al., 2020; MUELLER et al., 2016). Embora a cultura da soja seja suscetível à diversos vírus, no Brasil, poucas são conhecidas por causar perdas econômicas significantes. No mundo, cerca de 46 diferentes vírus foram descritos infectando naturalmente a cultura da soja (HILL; WHITHAM, 2014). No Brasil, há relatos de pelo menos 15 espécies de vírus (ALMEIDA, 2008; DE MARCHI et al., 2018; KITAJIMA, 2020), pertencente a diferentes gêneros, tais como Alfamovirus, Begomovirus, Carlavirus, Comovirus, Ilarvirus, Potyvirus e Orthotospovirus (KITAJIMA, 2020). O carlavirus cowpea mild mottle virus (CPMMV), que foi objeto de estudo desta tese, possui como vetor a mosca-branca Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), recentemente reconhecida pelo Ministério da Agricultura e do Meio Ambiente (MAPA) como uma das principais pragas para a cultura da soja no Brasil (Instrução Normativa n°16, de 4 de junho de 2019), e também está entre as 100 espécies mais invasivas do mundo na listadas pelo IUCN/SSC “Invasive Species Specialist Group” (http://www.issg.org) e pelo “Global Invasive Database” (http://www.issg.org/database/welcome/). O CPMMV foi relatado pela primeira vez no Brasil em 1983 infectando feijoeiro (COSTA; GASPAR; VEGA, 1983), e foi considerado um vírus de baixa importância até seu relato em soja, que foi feito somente duas décadas depois, quando se tornou um sério problema na sojicultura nacional ocasionando os sintomas de necrose da haste em soja (ALMEIDA et al., 2003). No início dos anos 2000, o CPMMV se tornou um fator limitante na produção de soja no estado de Goiás (ALMEIDA et al., 2003), e nos anos seguintes o vírus foi identificado nos estados da Bahia, Mato Grosso, Maranhão e Paraná (ALMEIDA, 2008; ALMEIDA et al., 2003, 2005). O único estado produtor de soja no qual o CPMMV ainda não foi relatado até o momento é o Rio Grande do Sul (ZANARDO; CARVALHO, 2017). 19 O CPMMV pertence ao gênero Carlavirus, um dos 5 gêneros da subfamília Quinvirinae, família Betaflexiviridae (ordem Tymovirales) (WALKER et al., 2022). Assim como as espécies membro deste gênero, o CPMMV possui um RNA senso positivo de fita simples, com um genoma de 8.200 nucleotídeos e composto por seis open reading frames (ORFs) (MENZEL; WINTER; VETTEN, 2010; ZANARDO et al., 2014). A ORF 1 codifica a replicase com quatro motivos conservados: metiltransferase, C23 Peptidase, RNA helicase e RNA dependente da RNA polimerase (RdRp) (ZANARDO et al., 2014). As ORFs 2, 3 e 4 codificam três proteínas (TGB 1-3p), que são essenciais para o movimento viral. A ORF 5 codifica a capa proteica (CP) e por fim, a ORF 6 codifica uma proteína rica em cisteína (CRP) com atividade de ligação ao ácido nucleico (NABP) (MENZEL; WINTER; VETTEN, 2010). Os carlavirus, em sua grande maioria, são transmitidos por afídeos (KING et al., 2011), sendo o CPMMV e o melon yellowing-associated virus (MYaV) uma exceção à regra pois são transmitidos por Bemisia tabaci (MARUBAYASHI; YUKI; WUTKE, 2010; MUNIYAPPA; REDDY, 1983; NAGATA et al., 2005; NAIDU et al., 1998), um inseto altamente polífago e muito bem-sucedido na transmissão de vírus de plantas (GILBERTSON et al., 2015), e amplamente distribuído em praticamente todas as regiões do Brasil (MORAES et al., 2018). Atualmente a B. tabaci é considerada um complexo de espécies crípticas composto de pelo menos 44 espécies morfologicamente indistinguíveis, porém distintas (DE BARRO et al., 2011; KANAKALA; GHANIM, 2019). Essas espécies divergem em diversos aspectos como: habilidade em transmitir vírus, plantas hospedeiras, endossimbiontes, suscetibilidade a inseticidas, tolerância a estresses ambientais, taxa de desenvolvimento e incompatibilidade reprodutiva entre grupos genéticos (HOROWITZ; ISHAAYA, 2014; PAN et al., 2012; PERRING, 2001; WATANABE et al., 2019; XU; DE BARRO; LIU, 2010). As espécies crípticas são identificadas com base em variações no gene mitocondrial da cytochrome oxidase I (mtCOI) (BOYKIN; DE BARRO, 2014; DE BARRO et al., 2011; KANAKALA; GHANIM, 2019). As espécies crípticas Middle East Asia Minor 1 (MEAM1) e Mediterranean (MED) são as espécies invasivas presentes em várias regiões do mundo sob diversas condições climáticas. No Brasil, estão presentes quatro espécies crípticas: duas espécies conhecidas como indígenas ou nativas das Américas denominadas de New Word 1 e New World 2 (NW1 e NW2), (BARBOSA et al., 2014; MARUBAYASHI et al., 2014) e as espécies MEAM1 e MED (MORAES et al., 2018). 20 Até o início dos anos 1990, só havia sido relatado no Brasil a presença das espécies nativas do grupo NW. As populações destes insetos causaram surtos esporádicos de doenças como o mosaico dourado do feijoeiro nos anos 60. Além disso, infecção por vírus eram observadas em plantas daninhas sendo, o inseto considerado uma praga secundária (COSTA, 1965; INOUE-NAGATA; LIMA; GILBERTSON, 2016). Posteriormente, verificou-se que o grupo NW no Brasil era constituído de duas espécies crípticas denominadas de NW1 e NW2 (MARUBAYASHI et al., 2013). Por conseguinte, a partir da década de 1990, com a introdução de MEAM1 no Brasil através do comércio de plantas ornamentais no estado de São Paulo, o cenário nacional de B. tabaci mudou drasticamente (LOURENÇÃO; NAGAI, 1994). Assim como em outras regiões do mundo, MEAM1 se dispersou rapidamente se tornando uma praga chave em diversas culturas, além de ser considerada um excelente vetor de vírus de plantas. Com a introdução de MEAM1, ao menos 18 novas espécies de begomovirus em tomateiro foram relatadas (RIBEIRO et al., 1998), assim como emergência de crinivirus em tomateiro, pimentão e batata (FREITAS et al., 2012; MACEDO et al., 2019); CPMMV em feijoeiro e soja (ALMEIDA, 2008; ALMEIDA et al., 1995), e surtos de BGMV em feijoeiro se tornaram frequentes (INOUE-NAGATA et al., 2016a). Atualmente, MEAM1 é a espécie de B. tabaci predominante no Brasil (MORAES et al., 2018). Duas décadas depois da introdução de MEAM1, no ano de 2013, MED foi relatada pela primeira vez em território nacional na região sul (BARBOSA et al., 2015). Provavelmente estes insetos migraram de países vizinhos como Argentina e Uruguai, que já haviam relatado a presença de MED em seus territórios (GRILLE et al., 2011). Um levantamento de mosca-branca realizado após a introdução de MED no Brasil, em 2015 e 2016, relatou uma segunda invasão de MED nos estados de São Paulo e Paraná, associada a plantas ornamentais (MORAES et al., 2017). Um levantamento feito em 2018 nos estados do Paraná e São Paulo, relatou surtos de MED em cultivos de pimentão, tomate, pepino e beringela cultivadas tanto em casa de vegetação, quanto em campo aberto (BELLO et al., 2020). E o estudo mais recente realizado por Bello et al., (2021), relatou pela primeira vez MED em soja em campo, também nos estados do Paraná e São Paulo. De forma geral, B. tabaci pode causar danos diretos e indiretos às plantas. Em soja, o dano direto mais comum é através da ingestão de fotoassimilados do floema. A ingestão de seiva por B. tabaci resulta na excreção de honeydew, uma substância 21 açucarada, sobre folhas e vagens que pode servir de substrato para o crescimento de fungos do gênero Capnodium que cobre a superfície foliar prejudicando a fotossíntese e, consequentemente, o desenvolvimento da planta e a qualidade dos grãos (KANAKALA; GHANIM, 2015; SUEKANE et al., 2013). Contudo, o principal dano causado por B. tabaci é através da transmissão de vírus pertencentes aos gêneros Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus e Polerovírus (GHOSH et al., 2019; NAVAS-CASTILLO; FIALLO-OLIVÉ; SÁNCHEZ-CAMPOS, 2011; POLSTON; DE BARRO; BOYKIN, 2014). No Brasil, já foram relatados vírus pertencentes aos gêneros Begomovirus, Carlavirus, Crinivirus e Polerovirus (BARBOSA et al., 2008; COSTA; GASPAR; VEGA, 1983; COSTA et al., 2020; INOUE- NAGATA et al., 2016b; INOUE-NAGATA; LIMA; GILBERTSON, 2016). Como observado com a invasão de MEAM1 no início dos anos 1990 no Brasil, a introdução de uma praga exótica em um território traz grandes preocupações para o setor agrícola, principalmente quando se trata de um vetor de vírus (INOUE- NAGATA; LIMA; GILBERTSON, 2016). A recente constatação de MED em campo aberto em soja no país levanta questões sobre o futuro do manejo de mosca-branca no Brasil, dada a menor suscetibilidade deste inseto aos inseticidas comumente utilizados na agricultura visando o manejo deste inseto. Sendo assim, esta tese foi dividida em dois capítulos: Capítulo 1, “COWPEA MILD MOTTLE VIRUS AND Bemisia tabaci ON SOYBEAN IN SÃO PAULO STATE (BRAZIL) AND TRANSMISSION PROPERTIES OF MEDITERRANEAN AND MIDDLE-EAST ASIA MINOR 1 CRYPTIC SPECIES”, redigido em inglês nas normas da revista PLANT PATHOLOGY, fator de impacto 2.772 e Capítulo 2, “EFFECTS OF COWPEA MILD MOTTLE VIRUS ON SOYBEAN SEED QUALITY AND YIELD COMPONENTS”, redigido em inglês nas normas da revista PLANT HEALTH PROGRESS, fator de impacto 0.939. 22 1Capítulo redigido de acordo com as normas da revista Plant Pathology (1365-3059). Capítulo 1 Cowpea mild mottle virus AND Bemisia tabaci ON SOYBEAN IN SÃO PAULO STATE (BRAZIL) AND TRANSMISSION PROPERTIES OF MEDITERRANEAN AND MIDDLE EAST-ASIA MINOR 1 CRYPTIC SPECIES1 Felipe Barreto da Silva1*, Rodrigo de Sarandy Raposo1, Sarah de Campos Forlani1, Juliana Uzan1, Julio Massaharu Marubayashi1, Marcos Roberto Ribeiro-Junior2,3, Angélica Maria Nogueira1, João Paulo da Silva4, Cristiane Müller5, Renate Krause- Sakate1. 1Department of Plant Protection, São Paulo State University, School of Agriculture, Botucatu, SP, Brazil. 2Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, USA. 3Institute of Biosecurity and Microbial Forensics, Oklahoma State University, Stillwater, OK, USA. 4Department of Agricultural Engineering, Campinas State University, Campinas, SP, Brazil. 5Corteva™ Agrisciences, Mogi Mirim, São Paulo, Brazil. *Corresponding author (e-mail: felipe.barreto@unesp.br) https://orcid.org/0000-0003- 3225-4323 Abstract Soybean (Glycine max L. Merril), an important commodity for Brazilian agriculture can be infected by the carlavirus cowpea mild mottle virus (CPMMV) transmitted by Bemisia tabaci. B. tabaci is a cryptic species complex and Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED) species are the most invasive and they have 23 already been found infesting soybean in São Paulo state, Brazil. To understand the distribution of CPMMV and whitefly-associated species, a survey was done on major soybean growing areas in São Paulo state. Our results indicated that CPMMV is widely distributed in most of the surveyed areas. The MEAM1 was still the predominant whitefly species in soybean, but MED was also found in different locations where soybean is produced in São Paulo state. Some areas evolved to a predominance of MED over the three years but others where MED was previously detected, showed a reduction of the insect during the same period. Transmission assays with one single insect revealed that MED is a better vector compared to MEAM1, transmitting the virus within two minutes of inoculation access period. Additionally, phylogenetic analysis of the complete genome sequences of CPMMV isolates collected in Sao Paulo and Bahia State showed that they belong to BR2 clade that include Brazilian CPMMV isolates collected from soybean, bean, and a CPMMV from the United States. This clade is clearly separated from isolates of CPMMV collected from Vigna mungo (India) and cowpea (Ghana). Keywords: MED, MEAM1, plant virus, survey, carlavirus 1. INTRODUCTION Soybean, one of the world’s most important sources of animal feed and vegetable oil, stands out as the most economic crop in Brazil, the leading worldwide producer. In the 2021 summer season, Brazilian production reached 125.00 million metric tons, corresponding to 35.64% of global soybean production, followed by the United States (USDA, 2022). In São Paulo state, soybean is becoming an important agricultural crop. In 2021, the total cultivated area in São Paulo state reached 1.15 million hectares (IEA, 2021). The crop hosts a wide variety of pathogens and pest that causes significant yield losses, including a growing number of viruses and their insect vectors. Although soybean is susceptible to many viruses, only a small number of them cause economic problems to soybean production. The whitefly-transmitted cowpea mild mottle virus (CPMMV; family Betaflexividae, genus Carlavirus) was detected in Brazil first infecting 24 common beans in 1983 (Costa et al., 1983) and lately in soybean in the 2000/2001 season (Almeida et al., 2003) when the disease became an economic problem (Almeida, 2008). CPMMV causes the disease called “stem necrosis virus”, and the virus has a single-stranded positive-sense RNA flexuous filamentous particles (approx. 650 nm in length). The genome of 8,200 nucleotides has a cap structure (m7GpppG) linked to the 5´ terminus and a polyadenylated tail at the 3´ end (Menzel et al., 2010; King et al., 2011; Zanardo et al., 2014a) and six open reading frames (ORFs), typical of the genus Carlavirus (Menzel et al., 2010). Although CPMMV was very well documented in the main soybean-producing areas of the south, southeast, mid-west, and northeast regions of Brazil (Zanardo & Carvalho, 2017), little is known about its incidence in field conditions, since symptoms of steam necrosis are not common on the soybean cultivars recently used (Barreto et al., 2020). Cowpea mild mottle virus is transmitted by the polyphagous whitefly B. tabaci (Gennadius) (Hemiptera: Aleyrodidae) (Muniyappa & Reddy, 1983; Jeyanandarajah & Brunt, 1993; Marubayashi et al., 2010). B. tabaci is a major threat to several cultivated plants and ranks as one of the world’s 100 most invasive pests (Lowe et al., 2000) and it is listed by the Ministry of Agriculture, Livestock, and Supply (MAPA) as one of the most important invader pest in Brazil (https://www.gov.br/agricultura/pt- br/assuntos/sustentabilidade/tecnologiaagro pecuaria/recursos-geneticos-1/especies- introduzidas). Molecular phylogenic analysis based on the mitochondrial cytochrome oxidase I (mtCOI) gene indicate that B. tabaci is a species complex composed of at least 44 morphologically indistinguishable species (De Barro et al., 2011; Boykin & De Barro, 2014; Kanakala & Ghanim, 2019). The species in the complex also differ in characteristics such as host plant range, the capacity to cause plant disorders, attraction by natural enemies, endosymbionts, expression of resistance, and plant virus transmission capabilities (Bedford et al., 1994; Brown et al., 1995; Sánchez- Campos et al., 1999; Perring, 2001; Horowitz et al., 2005). B. tabaci Middle East Asia-Minor 1 (MEAM1) and the B. tabaci Mediterranean (MED) species are globally distributed and considered the most invasive cryptic species of the complex as well as the most challenging to control (Hu et al., 2011; Gauthier et al., 2014). In Brazil, MEAM1, which was introduced in the early 1990s (Lourenção & Nagai, 1994), is predominant (Moraes et al., 2018) and MED, was first reported in the southmost region of Brazil in 2014 on sweet pepper crops (Barbosa et https://www.gov.br/agricultura/pt-br/assuntos/sustentabilidade/tecnologiaagro%20pecuaria/recursos-geneticos-1/especies-introduzidas https://www.gov.br/agricultura/pt-br/assuntos/sustentabilidade/tecnologiaagro%20pecuaria/recursos-geneticos-1/especies-introduzidas https://www.gov.br/agricultura/pt-br/assuntos/sustentabilidade/tecnologiaagro%20pecuaria/recursos-geneticos-1/especies-introduzidas 25 al., 2015), and is spreading across the country mainly associated with ornamental plants and vegetable crops (Moraes et al., 2017, 2018; Bello et al., 2020; Krause- Sakate et al., 2020). Recently, MED was found colonizing soybean plants in open field conditions (Bello et al., 2021), which raised concerns for soybean production considering the less susceptibility of MED to some conventional insecticides used (Pan et al., 2015; Wang et al., 2017) and previous studies have shown that B. tabaci MED was considered a better vector of CPMMV, transmitting the virus with higher efficiency compared to MEAM1 (Bello et al., 2021). CPMMV has also been reported to be transmitted by seed in different plant species such as cowpea (Vigna unguiculata) and common bean (Phaseolus vulgaris) in Africa (Brunt & Kenten, 1973), yardlong bean (Vigna unguiculata) in Venezuela (Brito et al., 2012), soybean in India (Yadav et al., 2013) and Brazil (Barreto da Silva et al., 2020). The seed transmission for CPMMV seems to be dependent on the viral isolate and the host cultivar, which would make it more difficult to control the disease and can contribute to the primary inoculum source of the virus in the field. Herein, we present an updated overview of the whitefly species as well as the distribution of CPMMV in commercial soybean production fields of São Paulo state. Our study indicated that MEAM1 was the predominant whitefly species in soybean plants, but MED was also found in different locations where soybean is produced in São Paulo state. Some areas showed an increase (Óleo and Santa Cruz do Rio Pardo) and others (Araçatuba, Buritãma, and Canitar), a decrease in MED population over the three years. In addition, we obtained five complete genome sequences of CPMMV collected from soybean in São Paulo and the Bahia states. The phylogenetic analysis reveals that the isolates clustered together with CPMMV isolates from soybean and beans previously characterized in Brazil and in the USA belonging to the BR2 group. Transmission assays using one single insect showed that MED is more efficient to transmit CPMMV than MEAM1, needing less inoculation time on plants to start to transmit CPMMV and also transmitting the virus with higher efficiency to plants. 26 2. MATERIAL AND METHODS 2.1. Virus and whitefly sampling During the growing summer seasons 2019/2020, 2020/2021 and 2021/2022, field surveys were carried out in major soybean growing areas around São Paulo state. 100 soybean leaves were randomly collected from commercial soybean fields (with or without symptoms). Single leaf samples were individually placed and labeled in plastic bags and transported to a -80 ºC storage ultra-freezer until further analysis. Adults’ specimens of whiteflies were collected from the underside of leaves selected at random across the soybean fields with a hand-held aspirator, and the specimens were immediately transferred to a tube containing absolute ethanol and stored at -20°C until further molecular identification of the whitefly species. The sites, times of collection, and geographical coordinates of each sample are summarized in Table 1. 2.2. Cowpea mild mottle virus identification Total RNA was extracted from each sample following the method described by Bertheau et al. (1998), followed by a one-step reverse transcription-polymerase chain reaction (RT-PCR) using AMV reverse transcriptase (Promega, São Paulo, Brazil) with the specific primer pair CPMMV1280-F (5´-GGCGTTCCAAAAGCTGCCGAT-3´) and CPMMV1696-R (5´-GGAGCCACCTTTCCAATCAA-5´), amplifying a region of the coat protein (De Marchi et al., 2017). All amplifications consisted of an initial step of 42 ºC for 30 min, a second step of 94 ºC for 2 min, 30 cycles of 94 ºC for 54 s, annealing at 54 ºC for 50 s, and elongation at 72 ºC for 50 s, followed by a final extension step at 72 ºC for 10 min. The amplified DNA was visualized by electrophoresis in 2% agarose gel stained with ethidium bromide. 2.3. DNA extraction and MEAM1 and MED species identification Total nucleic acids were extracted from each individual whitefly, following a modified Chelex protocol (Walsh et al., 1991). Ten insects for each population were individually macerated and homogenized in 50 µl 5% Chelex solution in a 1.5 ml tube. The tube was vortexed for a few seconds and then incubated at 56 °C for 15 min and 27 99 °C for 8 min. After centrifugation at 13000 rpm for 5 min, the supernatant was then collected and used as a template for the PCR amplification. All individual DNA samples were subjected to PCR analysis to differentiate MEAM1 and MED using the Bem23 primer pair: Bem23F (5´-CGGAGCTTGCGCCTTAGTC-3´) and Bem23R (5´-CGGCTTTATCATAGCTCTCGT-3´) (De Barro et al., 2003). This primer pair amplifies a microsatellite locus of about 200 bp and 400 bp for MEAM1 and MED, respectively (Skaljac et al., 2010). The amplified DNA was visualized by electrophoresis in 2% agarose gel stained with ethidium bromide. 2.4. Complete genomes characterization and phylogenetic analysis. CPMMV isolate from Buritãma, Rubiácea, and Mogi Mirim, São Paulo state, and two isolates from Luís Eduardo Magalhães, Bahia state, were collected and selected to obtain their complete genome sequence. Total RNA was extracted from the leaf tissue of symptomatic soybean plants using the PureLink RNA/DNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. A one- step RT-PCR using AMV reverse transcriptase (Promega, São Paulo, Brazil) was performed using the specifical primers CPMMV 1280-F/1696-R as described in topic 2.2. In order to obtain the complete genome of the CPMMV from the samples, each RNA was used for the construction of a cDNA library using the Complete ScriptSeq Kit (Epicenter; Illumina, San Diego, CA, USA) and transcriptome sequencing with Illumina HiSeq2500 platform (Roossinck et al., 2015), at the Center of Functional Genomics (ESALQ/USP, Piracicaba, Brazil). Adapter sequence removal and quality trimming were performed with CLC Genomics Workbench software version 9.0.3. The Sequences obtained were analyzed using the software Geneious v11.1.5 (Kearse et al., 2012) and compared dataset composed of nine CPMMV complete genome isolates from Ghana (Menzel et al., 2010), Florida (Rosario et al., 2014), and different CPMMV isolates from Brazil describe by Zanardo et al., (2014b) and Barreto da Silva et al. (2020). Two independent runs were conducted simultaneously using 1 million generations and excluding 25% from the resulting tree as burning. The phylogenetic tree was visualized, edited, and rooted using FigTree v1.4.4. (tree.bio.ed.ac.uk/software/figtree/). Pairwise comparison between the sequences was 28 performed with the program SDT v.1.2. (Muhire et al., 2014) using the MUSCLE alignment option (Edgar, 2004). 2.5. Transmission of CPMMV by one single B. tabaci MEAM1 and MED insect To perform the transmissions assays, a population of MEAM1 was collected from cabbage (Brassica oleracea) in Campinas, São Paulo, Brazil, and maintained in cabbage. The MED population was initially collected in bell pepper (Capsicum annuum) in Óleo, São Paulo, Brazil, and maintained in cotton plants. For species identification, DNA from whiteflies of each colony was extracted using the Chelex protocol (Walsh et al., 1991) and used as a template for PCR analysis as described in topic 2.3. The samples were purified (QIAquick Gel Extraction Kit Qiagen), sequenced, and confirmed as MEAM1 (GenBank access: MF624473) and MED (GenBank access: KX673609). The populations were maintained in insect-proof rearing cages in greenhouses, and colony purity was monitored every 6 months via molecular analyses. Whiteflies from each species used in these experiments were newly emerged (48h after emergence). Healthy and CPMMV-infected soybean cv. TMG 7062 IPRO were used. Soybean plants at the first three-true-leaf were sap inoculated with CPMMV, the infection of inoculated plants was confirmed by molecular analyses and then they were used as a source of inoculum. For the acquisition assay, about 200 newly emerged whiteflies were released from the rearing to cages containing CPMMV-infected soybean plants. After acquisition access periods (AAPs) of 5, 10, 15, 20, 30 min, and 1, 3, 6, and 24 h, adults were collected randomly from the CPMMV-infected soybean leaves, and then one adult was transferred into a clip-cage placed in a health soybean plant. An inoculation access period (IAP) of 24 h was assessed for each plant. After the IAP was assessed, all the whiteflies were removed, and the plants were kept in insect-proof cages. Thirty replicated plants were used for each AAP. Thirty days after the IAP each plant was tested for CPMMV infection via molecular analyses. For the transmission assay, newly emerged whitefly adults were placed on CPMMV-infected plants in cages. After 24h AAP, adults were collected and placed in clip-cages attached to healthy soybean plants with the first-true-leaf. An IAP of 2, 5, 10, 20, 30 min, and 1, 2, 3, 6, 12, and 24 h was assessed. For each IAP, thirty replicated plants were used. After each IAP was assessed, all the whiteflies were removed, and the plants were 29 kept in insect-proof cages. Thirty replicated plants were used for each AAP. Thirty days after the IAP each plant was tested for CPMMV infection via molecular analyses. 2.6. Weather data collection To evaluate and infer weather influences on the B tabaci population and CPMMV incidence, the historical data series including minimum and maximum temperatures (ºC) and precipitation (mm) from the past 30 years (1991 – 2020) were collected. The data was obtained from the POWER Project's Daily 2.2.22 version on 2022/04/15. Climatological data from sites collected in the three seasons can be found in Supplementary Figures 1 – 16. 3. RESULTS 3.1. Whitefly sampling A total of 67 collections were made over the tree soybean seasons: 2019/2020, 2020/2021, and 2021/2022. In general, a predominance of MEAM1 species was found across almost all sampling sites (Table 1, Figure 1). For the first soybean summer season surveyed (2019/2020), a total of 28 sites were sampled and MED was found in a single infestation in one site (Buritãma, 7), and co-occurring with MEAM1 in other six locations (Sites ID 5, 9, 16, 20, 26 and 27) (Figure 1a). For the second soybean summer season (2020/2021), from a total of 24 sites collected, MED was found co- occurring with MEAM1 in six sites (Sites ID 5, 7, 16 18, 25, and 26) (Figure 1b). For the last season surveyed (2021/2022), the number of sites visited was reduced to 19 areas, but B. tabaci was found in 15 of them. No whiteflies could be collected from four sites because no insects were found infesting soybean (Sites ID 13, 21, 26, and 28). MED was found co-occurring with MEAM1 in three of these sites (Sites ID 16, 19, and 24) (Figure 1c). Some areas like Araçatuba (5), where the incidence of MED was high (70%) in the first season, had a 10% incidence in the second season until the absence of MED in the last season. Buritãma (7) site had a similar dynamic where the presence of MED was 100%, 20%, and zero through the first, second, and third seasons, respectively. Otherwise, the area of Óleo (16) showed a progressive increase of MED through the three seasons going from 10% (first season) and 40% (second season) to 80% in the last season. The same was observed for Santa Cruz do Rio Pardo (26) which had a 30 progressive increase of MED in the first two seasons, 40% and 65% respectively, but in the last season, there were no presence of B. tabaci in this site. On sites like Canitar (ID 9), Palmital (ID 18), Paranapanema (19), Pindamonhangaba (20), Rubiácea (ID 24), Salto Grande (ID 25), and Taciba (27), the presence of a low number of MED insects was verified in one season. In the last season, the incidence of B. tabaci was very low in several sites, or the insect was even absent like Itaí (13), Piraju (21), Santa Cruz do Rio Pardo (26), and Taquarituba (28). This was probably related to wheatear conditions. 3 1 Table 1. Survey of Bemisia tabaci and cowpea mild mottle virus in the main soybean growing areas in São Paulo State, Brazil between the soybean summer seasons 2019/2020, 2020/2021, and 2021/2022 ID Collection site Coordinates 2019/2020 2020/2021 2021/2022 MEDa MEAM1a CPMMVb MEDa MEAM1a CPMMVb MEDa MEAM1a CPMMVb 1 Aguaí 22°04’16”S 46°57’25”W 0 100 NC 0 100 18/100 NC NC NC 2 Alvarez Florença 20°17’12”S 49°55’12”W 0 100 NC NC NC NC NC NC NC 3 Alvarez Machado 22°01’06”S 51°24’37”W 0 100 NC NC NC NC NC NC NC 4 Anhumas 22°27’03”S 51°26’01”W 0 100 NC NC NC NC NC NC NC 5 Araçatuba 21°10’33”S 50°31’33”W 70 30 NC 10 90 03/100 0 100 0/100 6 Assis 22°40’48”S 50°21’05”W 0 100 NC 0 100 NC NC NC NC 7 Buritãma 21°04’08”S 50°12’08”W 100 0 NC 20 80 95/100 0 100 11/100 8 Cândido Mota 22°44’02”S 50°21’26”W 0 100 NC 0 100 NC NC NC NC 9 Canitar 23°00’48”S 49°46’25”W 10 90 69/100 0 100 90/100 0 100 02/100 10 Casa Branca 21°55’48”S 47°04’08”W 0 100 NC 0 100 100/100 0 100 74/100 11 Guaíra 20°04’03"S 48°’04'”04"W NC NC NC 0 100 98/100 0 100 94/100 12 Ipaussu 23°05’11”S 49°33’03”W 0 100 03/03 0 100 40/100 0 100 0/100 13 Itaí 23°19’07”S 49°05’17”W 0 100 NC 0 100 1/100 NP NP 0/100 14 Jaú 22°14’42"S 48°’33'”20"W NC NC NC 0 100 10/100 0 10 04/100 15 Mogi Mirim 22°28’36”S 47°03’36”W 0 100 26/100 0 100 15/100 0 100 11/100 16 Óleo 22°56’33”S 49°26’14”W 10 90 0/100 40 60 0/100 80 20 25/100 17 Ourinhos 22°55’57”S 49°53’13”W 0 100 16/100 0 100 80/100 0 100 0/100 18 Palmital 22°45’40”S 50°11’30”W 0 100 NC 10 90 NC NC NC NC 19 Paranapanema 23°28’26”S 48°45’16”W 0 100 31/100 0 100 40/100 15 85 02/100 20 Pindamonhangaba 22°52’05”S 45°28’18”W 10 90 NC NC NC NC NC NC NC 3 2 21 Piraju 23°16’13”S 49°16’30”W 0 100 NC 0 100 0/100 NP NP NC 22 Pirassununga 22°03’45”S 47°30’01”W 0 100 NC 0 100 0/100 NC NC NC 23 Porto Ferreira 21°49’51”S 47°27’10”W 0 100 NC 0 100 NC NC NC NC 24 Rubiácea 21°25’40”S 50°49’05”W 0 100 03/05 0 100 26/100 30 70 39/100 25 Salto Grande 22°50’13”S 50°01’16”W 0 100 67/100 10 90 07/100 0 100 32/100 26 Sta. Cruz Rio Pardo 22°56’09”S 49°32’42”W 40 35 50/100 65 35 60/100 NP NP 0/100 27 Taciba 22°24’38”S 51°19’30”W 10 90 NC NC NC NC 0 100 NP 28 Taquarituba 23°31’27”S 49°16’15”W 0 100 NC 0 100 0/100 NP NP 0/100 29 Vargem Grande 21°50’07”S 46°54’40”W 0 100 NC 0 100 94/100 0 100 35/100 30 Votuporanga 20°27’30”S 50°03’52”W 0 100 NC NC NC NC NC NC NC Total of Sites Sampled 28 9 24 20 15 17 NC: Not Collected; NP: Not Present (areas where whiteflies were not found at the moment of the survey); a Data in percentage; b Number of plants infected/tested. 33 Figure 1. Sampling locations for Bemisia tabaci collected in São Paulo State on soybean summer seasons (a) 2019/2020, (b) 2020/2021, and (c) 2021/2022 with species colored in blue (Bemisia tabaci Middle East-Asia Minor 1) and red (Bemisia tabaci Mediterranean). 34 3.2. Detection of cowpea mild mottle virus in soybean in São Paulo State The carlavirus, CPMMV was detected in several collection sites in the three soybean seasons (Table 1, Figure 2). In the 2019/2020 season, nine sites were surveyed and Óleo (16) was the only site where CPMMV was not detected infecting soybean plants. For the 2020/2021 soybean season, the number of sites surveyed increased from nine to 20 sites, and CPMMV was not detected only in four sites (16, 21, 22, and 28). In three sites (5, 13, and 25) CPMMV was found in less than 10% of the plants. Otherwise, we observed more than 90% of infected plants in four sites (7, 9, 10, and 29). In Casa Branca (ID 10), 100% of the plants surveyed were infected with CPMMV in the 2020/2021 season summer season. Casa Branca and Guaíra are sites with an irrigated soybean system. In the 2021/2022 season, the number of sites surveyed for CPMMV was 17. In six of them, CPMMV was not detected (5, 12, 13, 17, 26, and 28), and in two of them (9 and 19), the incidence of CPMMV was 2%, and 4% in site 14. The other sites (7, 10,15, 16, 24, 25, and 29), ranged from 11% to 74%. Guaíra was the site with the highest incidence of CPMMV with 94% of infected plants. 35 Figure 2. Sampling locations for cowpea mild mottle virus collected in São Paulo State between the soybean summer seasons (a) 2019/2020, (b) 2020/2021, and (c) 2021/2022. The size of the circles represents the evaluated percentage of infection in each location sampled. 36 3.3. Sequence analysis and phylogeny The complete genome sequence of CPMMV MT473963-Soybean (Casa Branca_BR) (Barreto da Silva et al., 2020) served as the reference for mapping HTS data obtained in this study. The number of reads-matches and percentage of genome coverage for each isolate was: Soybean (LEM_BR1) (GenBank accession number: OP611423) 7,680,138 (100%), Soybean (LEM_BR2) (GenBank accession number: OP611425) 2,518,529 (100%), Soybean (Mogi Mirim_BR) (GenBank accession number: OP61142) 3,188,516 (100%), Soybean (Buritãma_BR) (GenBank accession number: OP611425) 2,443,113 (100%) and Soybean (Rubiácea_BR) (GenBank accession number: OP611427) 5,245,653 (100%). The CPMMV phylogenetic tree (Figure 3) of the complete nucleotide sequence analysis showed that the isolates Soybean (LEM_BR1), Soybean (Mogi Mirim_BR), Soybean (LEM_BR2), Soybean (Buritãma_BR) and Soybean (Rubiácea_BR) are closest related to the six Brazilian isolates previously described from soybean (Zanardo et al., 2014a; Barreto da Silva et al., 2020) and one from the USA (Rosario et al., 2014). These isolates belong to the BR2 group (Zanardo et al., 2014b), which encompasses the most common CPMMV strains found in soybean in Brazil. 37 Figure 3. Phylogenetic tree based on the complete sequence of different CPMMV isolate available on GenBank using Bayesian inference (implemented in MrBayes V.3.1, with model GTR+I+G and one million generations). Cucumber vein-clearing virus (CuVCV; genus Carlavirus, family Betaflexiviridae) was used as outgroup. Based on pairwise distance comparison (Figure 4) the isolates obtained in this study, the isolate Soybean (Buritãma_BR) showed a lower identity (96,6%) with the KC884247-Soybean (Brazil_MT) inside de clade and 97,8 of identity with Soybean (Mogi Mirim_BR), Soybean (LEM_BR1) and KC884246-Soybean (Brazil_MT). The isolate Soybean (LEM_BR2) showed 99,6% of pairwise identity with the isolate MT433963-Soybean (Casa Branca_Brazil), the highest identity among the Brazilian isolates, followed by the isolate Soybean (LEM_BR1) which showed 99% of nucleotide identity with the KC884246-Soybean (Brazil_MT). Among the isolates obtained in this study, the highest identity (98,8%) was showed by isolates from LEM (Soybean (LEM_BR1) and Soybean (LEM_BR2)), and the lower identity (97%) was showed between Soybean (LEM_BR2) and Soybean (Buritãma_BR). Figure 4. Genome pairwise identity based on complete sequence of different CPMMV isolates available in GenBank using SDT v1.2. 38 3.4. Transmission of CPMMV by MEAM1 and MED using one single insect CPMMV transmission differed among MEAM1 and MED (Table 2 and Figure 5), as indicated by the number of plants infected by CPMMV exposed to one CPMMV- infected whitefly. The transmission was significantly higher for MED than for MEAM1 and with lower period of access of inoculation, MED can transmit CPMMV better compared to MEAM1. Transmission efficiency of 40% was achieved by one MED insect during 2 minutes of inoculation of a soybean plant. Table 2. Efficiency of cowpea mild mottle virus (CPMMV) transmission by one single insect of Bemisia tabaci Mediterranean (MED) and Middle East-Asia Minor 1 (MEAM1) using different inoculations access period (IAP). PAI MED MEAM1 2 min 12/30a (40b) cdA 0/30a (0b) aB 5 min 12/30 (40) cdA 0/30 (0) aB 10 min 12/30 (40) cdA 1/30 (3,33) aB 20 min 16/30 (53,3) bcA 0/30 (0) aB 30 min 18/30 (60) bcA 5/30 (16,6) aB 1 h 6/30 (20) dA 2/30 (6,6) Aba 2 h 16/30 (53,3) bcA 9/30 (30,0) aB 3 h 22/30 (73,3) abA 5/30 (16,6) aB 6 h 28/30 (93,3) aA 3/30 (10,0) aB 12 h 20/30 (66,6) abcA 8/30 (26,6) aB 24 h 20/30 (66,6) abcA 3/30 (10,0) aB aNumber of plants infected/tested. bpercentage of infection. Medians followed by different upper case within rows and lower case within columns indicate a significant difference (p < .05) for each parameter evaluated. 39 Figure 5. Efficiency of cowpea mild mottle virus (CPMMV) transmission by Bemisia tabaci Mediterranean (MED) and Middle East-Asia Minor 1 (MEAM1) species using different inoculation access periods (IAP). The acquisition of CPMMV assessed for MED showed that the higher the PAA, the transmission is more efficient (Table 3). An efficient transmission starts with only 20 min of acquisition and reaches 100% of efficiency at the maximum PAA evaluated of 24 h. Table 3. Efficiency of cowpea mild mottle virus (CPMMV) transmission by Bemisia tabaci Mediterranean (MED) and Middle East-Asia Minor 1 (MEAM1) species using different acquisition access periods (AAP). PAA MED 5 min 0/30a (0b) D 10 min 2/30 (6,6) D 15 min 4/30 (13,3) D 20 min 16/30 (53,3) BC 30 min 14/30 (46,6) C 1 h 14/30 (46,6) C 3 h 24/30 (80,0) AB 6 h 14/30 (46,6) C 24 h 30/30 (100) A 40 aNumber of plants infected/tested, bpercentage of infection. Medians followed by different upper case within columns indicate a significant difference (p < .05) for each parameter evaluated. 3.5. Weather influences on B. tabaci population and CPMMV incidence According to the frequency and regression analyses of the temperature and precipitation on MEAM1, MED and CPMMV, there is no a significant influence of the weather on the insect population and CPMMV incidence, but our data showed that there is a slightly negative correlation (-0,24641) of MED with precipitation, which means that in areas where there is the presence of MED, the greater the monthly precipitation, the occurrence of MED is lesser. In the other hand, for MEAM1, the correlation with precipitation is positive in the same intensity (0,24641). High temperature showed to have a slightly positive influence in MED population, and the population of MED was higher in years where the temperature was high in comparing the years and areas where there is the presence of MED. Temperature and precipitation did now show to have any influence on CPMMV incidence according to the data collected in this study. DISCUSSION Herein, we report the current scenario of B. tabaci in soybean fields across São Paulo state three years after MED detection in open field conditions (Bello et al., 2021). The data obtained in this study revealed that MEAM1 is still prevalent in the main soybean production areas in São Paulo state, but MED is gradually spreading to different soybean fields compared to the first survey realized in the state. In some areas (Óleo and Santa Cruz do Rio Pardo), an increase in MED population was observed during the three years of the survey, but in others, a decrease was clearly verified (Araçatuba, Buritãma, and Canitar). These different situations make it difficult to understand the behavior of MED insects in open field conditions and if the insect will completely adapt to open field conditions in Brazil since it is well characterized as an insect of greenhouse conditions (Kontsedalov et al., 2012; McKenzie et al., 2012; Parrella et al., 2012). Our data also reinforce that MED is a better vector of CPMMV compared 41 to MEAM1, and one single insect of MED is capable to transmit CPMMV with 40% of efficiency with an IAP of 2 minutes. Almost the same virus efficiency transmission (30%) was observed for MEAM1 specimens when the insect had two hours of IAP. This data indicates that MED specimens with few minutes in contact with soybean leaves can transmit CPMMV. The CPMMV isolates collected from soybean from different locations in Brazil belong all to the BR2 group, according to the classification used by (Zanardo et al., 2014b), which encompasses the most common CPMMV strains found in soybean in Brazil (Zanardo et al., 2014a; Barreto da Silva et al., 2020) and in the USA (Rosario et al., 2014). They were distantly related to CPMMV collected from Cowpea (Ghana) and Vigna mungo (India) as also observed by (Zanardo et al., 2014a; Barreto da Silva et al., 2020). Although the spreading and colonization of MED in soybean are recent in Brazil, these studies are important to understand if a possible displacement of MEAM1 by MED can occur in soybean, since it could change the pest management system of whiteflies in soybean in Brazil. It is very well known that MED species have low susceptibility to insecticides used to control whiteflies (Horowitz & Ishaaya, 2014; Krause- Sakate et al., 2020). Besides the possible increase in the cost of production, the threat of MED to the soybean system can also be related to the possible new viruses introduction to soybean and other crops, as happened in the early 1990s in Brazil when outbreaks of begomoviruses infecting Solanaceae have occurred after MEAM1 detection (Faria et al., 2000). A previous study evaluating the behavior of MED and MEAM1 on soybean in the absence of insecticides showed that MEAM1 and MED do not outcompete on this crop and can very well co-occur in a plant in mixed infestations (Watanabe et al., 2019). Otherwise, it is very well known that under conditions of high insecticide use, MED is highly competitive and causes the displacement of MEAM1 species (Gerling et al., 1980; Nauen & Denholm, 2005; Horowitz & Ishaaya, 2014; Yao et al., 2017). In our studies we observed that in most areas, MED was found in mixed infestation with MEAM1, supporting the data obtained by Watanabe et al. (2019) that both can well co-exist on soybean plants. It is also interesting to mention that MED was also found as the predominant species (in single infestation) in areas like Buritãma. If this is related to whitefly management and the variety/amount of insecticide use, we cannot affirm, and more studies are necessary to verify the influence of these parameters on the B. tabaci dynamic. 42 Regarding CPMMV on soybean, our data indicate that the virus is widely occurring in the different areas of São Paulo state, causing mainly symptoms of mosaic, and mottle, but also being asymptomatic on soybean, as previously observed (Zanardo et al., 2014a; Barreto da Silva et al., 2020). In general, the incidence was superior during the seasons 2019/2020 and 2020/2021, when the whitefly population was visible higher compared to 2021/2022. In Guaíra, however, the incidence of CPMMV was respectively 98 and 94% during the last two seasons surveyed, indicating that this is a site under high CPMMV pressure. A high incidence was also observed in Casa Branca, where 100 and 74% of the plants surveyed were infected with CPMMV in the last two seasons surveyed. The soybean in Guaíra and Casa Branca are both produced under an irrigated system and probably this can contribute to the presence of the vector during the whole year, even when the wheatear is not so propitious for whitefly development. A recent study showed that even in asymptomatic infections, CPMMV can affect the soybean yield, reducing about 638 kg ha-1 when compared with healthy soybean plants under the same field conditions (Barreto da Silva et al., 2020). The same study also observed seed transmission of a Brazilian isolate by an important soybean cultivar (cv. MX POTÊNCIA) former used in Brazil. Even though the frequency of seed transmission is low (Brunt & Kenten, 1973; Brito et al., 2012; Barreto da Silva et al. (2020), infected seed can be an important component of CPMMV in soybean. Although the climate data have not shown significant effects on the B. tabaci species and on the CPMMV incidence, there is a slightly correlation of high temperatures and low precipitation with MED. It is worth mentioning that warming temperatures and more frequent extreme weather events associated with climate change is expected to allow pathogens and their vectors to expand into new geographic ranges, possibly causing new disease epidemics (Whitham et al., 2016). The detection of MED species of B. tabaci in soybean requires close monitoring of its expansion to other states. Moreover, its capacity to transmit CPMMV and its ability to be less susceptible to pesticides is an additional concern. ACKNOWLEDGMENTS This project was financed supported by Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (grant number: 2019/24642-8. This study was also supported 43 by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – finance code 001; F.B.S was the recipient of a FAPESP doctoral fellowship. A.M.N. was the recipient of a FAPESP Post-Doctoral fellowship (grant number: 2019/18639-4) and R.K.S. holds a CNPq Fellowship (grant number: 303411/2018-0). Data availability The nucleotide sequences reported in this manuscript have been deposited in the GenBank database under accession number OP611423, OP611424, OP611425, OP611426, and OP611427 and are available in the Electronic Supplementary Material. Corresponding author designation Felipe Barreto da Silva, email: felipe.barreto@unesp.br Declarations Ethical approval: This article does not contain any studies with human participation or animal performed by any of the authors. 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Zanardo L, Silva F, Lima A et al., 2014b. Molecular variability of cowpea mild mottle virus infecting soybean in Brazil. Archives of Virology 159, 727–737. 4 8 Supplementary Figure 1. Araçatuba climatological data. 4 9 Supplementary Figure 2. Buritama climatological data. 5 0 Supplementary Figure 3. Canitar climatological data. 5 1 Supplementary Figure 4. Casa Branca climatological data. 5 2 Supplementary Figure 5. Ipaussu climatological data. 5 3 Supplementary Figure 7. Itai climatological data. 5 4 Supplementary Figure 7. Mogi Mirim climatological data. 5 5 Supplementary Figure 8. Oleo climatological data. 5 6 Supplementary Figure 9. Ourinho climatological data. 5 7 Supplementary Figure 10. Paranapanema climatological data. 5 8 Supplementary Figure 11. Piraju climatological data. 5 9 Supplementary Figure 12. Rubiacea climatological data. 6 0 Supplementary Figure 13. Salto Grande climatological data. 6 1 Supplementary Figure 14. Santa Cruz do Rio Pardo climatological data. 6 2 Supplementary Figure 15. Taquarituba climatological data. 6 3 Supplementary Figure 16. Vargem Grande do Sul climatological data. 64 2Capítulo redigido de acordo com as normas da revista Plant Healthy Progress (1535-1025). Capítulo 2 EFFECTS OF COWPEA MILD MOTTLE VIRUS ON SOYBEAN SEED QUALITY AND YIELD COMPONENTS2 Felipe Barreto da Silva, José Eduardo Berndt De Brito, Edivaldo Aparecido Amaral da Silva and Renate Krause-Sakate, Department of Plant Protection, São Paulo State University, School of Agriculture, Botucatu, SP, Brazil Abstract Cowpea mild mottle virus (CPMMV) is spread in all major soybean production areas in Brazil and has been a threat to national soybean production since 2002. The effects of CPMMV agronomic components of soybean have been studied recently but its effect on seed quality has never been estimated. In the present study, we have evaluated the effects of CPMMV on the seed quality and agronomic traits of soybean cultivar TMG 7062 IPRO under greenhouse conditions. Our data shows that CPMMV infection on soybean significantly reduced the plant height, the number of pods per plant, and plant-grain weight in greenhouse experiments. Although there was no evidence of seed transmission, seeds obtained from CPMMV-infected plants had their germination reduced to a rate of 45 to 12%. These results suggest that CPMMV has an important role in the reduction of the agronomic components of soybean and affects seed germination quality. 2.1. Introduction Cowpea mild mottle virus (CPMMV, Carlavirus) was first detected in Brazil in 1983 infecting common bean (Phaseolus vulgaris) (Costa et al. 1983) and become a major problem in soybean (Glycine max) production in the 2000s (Almeida et al. 2003). The losses reported at that time were higher than 85% since the cultivar used developed the stem necrosis symptom that affected the whole plant taking it to death (Almeida et al. 2003). Nowadays, the impact caused by this disease is reduced in the current soybean cultivars used since there is no longer the development of systemic 65 necrosis (Arias et al. 2015, Barreto et a., 2020). Symptoms in soybean are highly variable, including mosaic, chlorosis, vein clearing, mottling, and dwarfing (Naidu et al. 1998; Almeida et al. 2003; Zanardo et al. 2014; Barreto da Silva et al. 2020). Symptomless infection was also reported in some soybean cultivars (Zanardo et al. 2014; Barreto da Silva et al. 2020) which makes it difficult to know if the virus is occurring infecting plants in the field. CPMMV infection can lead to yield losses by reducing the number of pods and, seeds per plant as well as infected plants produce lower seed weights. Although the full cost impact of CPMMV on soybean production in Brazil has never been estimated, a recent study showed that some CPMMV-infected cultivars had their yield significantly impacted by the reduction in plant height, number of pods per plant, and 1,000-grain weight (Barreto da Silva et al. 2020). CPMMV is transmitted by the whitefly Bemisia tabaci (Muniyappa and Reddy 1983; Jeyanandarajah and Brunt 1993; Naidu et al. 1998; Marubayashi et al. 2010) but the possibility of seed transmission is also a major concern for CPMMV because it can lead to early infections which is an important component of the epidemiology of the disease in the field. Seed-born transmission of CPMMV has been reported in cowpea, common bean, and soybean in Ghana (Brunt and Kenten 1973) and in yardlong bean in Venezuela (Brito et al. 2012). In Thailand, the virus was observed to be transmitted by soybean seed at a frequency lower than 1% (Iwaki 1982), but in India, the seed- born nature of CPMMV was detected in several soybean cultivars with higher rates of transmission, ranging from 0,62% to 14,2% (Yadav et al. 2013) and most recently, Barreto da Silva et al. (2020) observed seed transmission in soybean by a Brazilian isolate at a rate of 0,37% in a cultivar that did not show any symptoms of the disease. Besides seed transmission, factors like longevity, hormonal balance (Bueso et al. 2017), increase in seed protein content, and decrease in seed oil and fatty acids (Demski and Jellum 1975) as well as disposition to fungal infections (Groves et al. 2016), can be affected by a viral infection, decreasing the seed quality. The objective of this work was to evaluate the quality of seeds germination produced by CPMMV-infected and the effects of CPMMV in yield components in soybean plants under greenhouse conditions. 66 2.2. Material and Methods Experimental setup and data collection Soybean seeds (cv TMG 7062 IPRO) were sowed in 20 L pots and separated into two plots (Healthy and CPMMV-infected plots) (Figure 1). Each plot was composed of a hundred pots. Ten days after seedling emergence, only three plants were left in each pot and all plants from the CPMMV-infected plot were sap inoculated with CPMMV using as inoculum source leaves of common bean cv Jalo infected with CPMMV. Leaves were ground in phosphate buffer 0,01 M, pH 7 containing the abrasive carborundum (600 mesh). Thirty days after inoculation, total RNA was extracted from each inoculated sample following the method described by Bertheau et al. (1998), followed by one-step RT-PCR using AMV reverse transcriptase (Promega, São Paulo, Brazil) with the specific primer pair CPMMV1280-F (5´- GGCGTTCCAAAAGCTGCCGAT-3´) and CPMMV1696-R (5´- GGAGCCACCTTTCCAATCAA-5´), amplifying a region of the coat protein of the virus (De Marchi et al., 2017). All amplifications consisted of an initial step of 42 ºC for 30 min, a second step of 94 ºC for 2 min, 30 cycles of 94 ºC for 54 s, annealing at 54 ºC for 50 s, and elongation at 72 ºC for 50 s, followed by a final extension step at 72 ºC for 10 min. The amplified DNA was visualized by electrophoresis in 2% agarose gel stained with ethidium bromide. CPMMV-infected Plants Healthy Plants 67 Figure 1. Soybean seed production of the cultivar TMG 7062 IPRO for healthy and cowpea mild mottle virus-infected plants. Agronomic traits. A total of 150 soybean plants (healthy and infected with CPMMV (Figure 2) were evaluated at the physiological maturity stage (Reproductive stage 8). The plant height was obtained measuring the distance from the soil to the apex of the plant (cm) and the number of pods per plant by counting the total number of pods per plant. All plants were individually hand harvested and seeds separated for each plant and weighed. Agronomic traits for each evaluation are presented in Table 1. Figure 2. Healthy and cowpea mild mottle virus-infected soybean cultivar TMG 7062 IPRO plants. Seed transmission. To study the seed-borne capacity of CPMMV, a random sample of seeds was collected from the CPMMV-infected TMG 7062 IPRO trial in both seasons 2020/2021 and 2021/2022 (Figure 3). Soybean seeds were sowed in Styrofoam seedlings trays containing Substrato Carolina Soil (CSC, Pardinho, SP). The seedling trays were kept in an insect-proof cage. Germination was greater than 75%, and the seedling did not show any typical disease symptoms. For virus detection, leaf samples were tested using RT-PCR with primers previously described. To compose a sample, for each season, leaves of ten plants were collected and combined in 100 samples tested, totaling 1000 plants analyzed. CPMMV-infected Plants Healthy Plants 68 Figure 3. Visual evaluation of seeds obtained from health and cowpea mild mottle virus soybean infected plants. Germination. For each plot, four samples of 100 seeds were germinated on a paper towel moistened with water 2.5 times the dry paper weight at 25 ºC. Germination percentage was evaluated by counting normal seedlings in the fifth (first germination (FG)) and on the eighth day (total germination (G)), according to the rules for Seed Testing (Brasil, 2009). 2.3. Results and Discussion Differences in the number of pods per plant, and plant-grain weight varied significantly among health and CPMMV-infected plants and years evaluated (Table 1). The plant height did not show a significant difference in the first year (S1), this same tendency has already been observed for TMG 7062 IPRO in a previous study under field conditions (Barreto da Silva et al. 2020), but it showed a significant difference for the second year (S2). Although the plants were cultivated under the same greenhouse conditions, this variation in plant height over the years can be explained by some fertilizing and/or irrigation differences, but the effects of CPMMV on plant height are expressive. Recent field studies showed that the effects of CPMMV on plant height in different cultivars can be significant or non-significant depending on the cultivar tested Seeds originated from healthy plants Seeds originated from CPMMV-infected plants 69 (Barreto da Silva et al. 2020). But, even in the year that there was a non-significant difference in plant height, the number of pods per plant and grain weight were significantly different. Table 1. Agronomic traits of cultivar TMG 7062 IPRO. Mean of plant height, pods per plant ad plant-grain weight. Year Plant height (cm) Pods per plant Plant-grain weight (g) Healthy CPMMV- infected Healthy CPMMV- infected Healthy CPMMV- infected 2020/2021 (S1) 34,21a 34,54a 13,67a 9,36b 6,59a 2,18b 2021/2022 (S2) 64,58a 50,42b 14,02a 11,64b 7,65a 4,85b Mean followed by the same letter within rows indicate no significant ( 𝑝 < 0.05) difference between healthy and CPMMV-infected plants according to ANOVA. The visual aspect of healthy and CPMMV-infected soybean plants, can be observed on Figure 1 and 2, evidencing early plant defoliation and maturation in plants infected with CPMMV. Plant defoliation leads to a reduction in the photosynthetic area and consequently a reduction in the number of pods and grains. The visual quality of the seeds originated from CPMMV-infected plants was also inferior to seeds from healthy plants (Figure 3). This decrease in seed quality was also observed in germination (Figure 4). Germination is considered to be the most critical phase in the plant life cycle because of its high vulnerability to injury, disease, and water/environmental stress. Our data shows that there is a significant difference between seeds originated from healthy and CPMMV-infected plants. The germination of healthy seed reached 75 and 82%, in the first and second year evaluated, respectively. For the CPMMV-infected seeds, germination ranged from 12 to 45% in the first and second year, respectively. This reduction demonstrate that seeds originated from CPMMV-infected plants have their germination viability reduced by the virus. Some viruses have been already reported to directly affect the viability of seeds. Seeds of spurrey (Spergula arvenisis), infected by tomato black ring virus (TBRV) had slow germination compared to healthy seeds (Lister and Murant 1967), and strains of prunus necrotic ringspot virus (PNRSV) can reduce the germination of hop from 20% to 90% (Blattny and Osvald 1954). 70 Figure 4. Percentage of germination of soybean seeds cultivar TMG 7062 IPRO originated from healthy and cowpea mild mottle virus-infected plants from experiments conducted in 2020/2021(S1) and 2021/2022 (S2). From 1,000 seedlings obtained from seeds harvested in the CPMMV-infected plot for each year, neither of them was found to be infected by CPMMV, confirmed by RT-PCR. As far as we know, only one study in Brazil confirmed the seed-born ability of a CPMMV isolate (Barreto da Silva et al. 2020). The transmission of CPMMV by seed is still unclear but appears to be dependent on the viral isolate. Studies of the complete sequences from CPMMV isolate by Zanardo et al. (2014) demonstrated significant differences between isolates infecting soybean in Brazil. Seed transmission was observed in a frequency about 91% in soybean for an isolate from Ghana (Brunt and Kenten 1973). In Thailand, the virus was observed to be transmitted by soybean seeds at a frequency lower than 1% (Iwaki 1982). A study from India where eight from 27 soybean cultivars tested transmitted CPMMV by seeds in a range from 0.62% to14,2% by the same isolate and phylogenetic analyses of CP showed that the isolate used in that study were distinct from the Brazilian isolates (Yadav et al. 2013). These studies evidenced the complex relationship between CPMMV isolate and cultivar. Besides soybean, the seed-borne ability of CPMMV has also been demonstrated by cowpea, common bean (Brunt and Kenten 1973), and yardlong bean seed (Brito et al. 2012). 0 10 20 30 40 50 60 70 80 90 Healthy S1 CPMMV-infected S1 Healthy S2 CpMMV-infected S2 G er m in at io n r at e (% ) 5 days after sowing 8 days after sowing Total 71 2.4. Conclusion In summary, CPMMV affects plant height, number of pod per plant, and grain weight per plant in soybean plants under greenhouse conditions. CPMMV was not seed transmitted by soybean cv TMG 7062 IPRO. Seeds originated from CPMMV- infected plants have visual and germination quality inferior compared to seeds from healthy plants. Aknowlegments This project was financed supported by Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (grant number: 2019/24642-8. 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A., Gowda, S., Satyanarayana, T., Boyko, V., Reddy, A. S., Dawson, W. O., et al. 1998. Evidence that whitefly-transmitted cowpea mild mottle virus belongs to the genus Carlavirus. Arch Virol. 143:769–780. Yadav, M. K., Biswas, K. K., Lal, S. K., Baranwal, V. K., and Jain, R. K. 2013. A Distinct Strain of Cowpea mild mottle virus Infecting Soybean in India. Journal of Phytopathology. 161:739–744. Zanardo, L. G., Silva, F. N., Bicalho, A. A. C., Urquiza, G. P. C., Lima, A. T. M., Almeida, A. M. R., et al. 2014. Molecular and biological characterization of Cowpea mild mottle virus isolates infecting soybean in Brazil and evidence of recombination. Plant Pathol. 63:456–465 Available at: http://doi.wiley.com/10.1111/ppa.12092 [Accessed March 26, 2018]. 74 CONSIDERAÇÕES FINAIS B. tabaci MEAM1 ainda é a espécie críptica prevalente nos campos de produção de soja do Estado de São Paulo. 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