FELIPE BARRETO DA SILVA

EFEITOS DO CARLAVÍRUS Cowpea mild mottle virus EM CULTIVARES

DE SOJA

Botucatu

2019
 FELIPE BARRETO DA SILVA

EFEITOS DO CARLAVÍRUS Cowpea mild mottle virus EM CULTIVARES

DE SOJA

Dissertação apresentada à Faculdade de

Ciências Agronômicas da Unesp Câmpus
de Botucatu, para obtenção do título de
Mestre em Agronomia (Proteção de
Plantas).

Orientadora: Dra. Renate Krause Sakate

Coorientadora: Dra. Cristiane Muller
Coorientadora: Dra. Márcia Maria Pereira
Sartori

Botucatu

2019
 Silva, Felipe Barreto da
S586e Efeitos do carlavírus Cowpea mild mottle virus em cultivares
de soja / Felipe Barreto da Silva. -- Botucatu, 2019
69 p. : tabs., fotos, mapas

Dissertação (mestrado) - Universidade Estadual Paulista

(Unesp), Faculdade de Ciências Agronômicas, Botucatu
Orientadora: Renate Krause Sakate
Coorientadora: Cristiane Muller

1. Doenças de soja. 2. Necrose da haste. 3. Queima do

Broto. 4. CPMMV. 5. TSV. 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.

Aos meus amados pais,

Lidia e Adhemar,

Dedico
 AGRADECIMENTOS

Agradeço a Deus por sempre colocar em meu caminho oportunidades que me

fazem crescer profissionalmente e espiritualmente.

Aos meus amados pais por todo amor, apoio e por nunca pouparem esforços
para minha educação. A minha irmã Selma e meu irmão André pelo apoio e
compreensão.

A Prof. Dra. Renate Krause Sakate, pela orientação, ensinamentos, amizade,

paciência e por sempre confiar a mim responsabilidades que foram essenciais na
minha formação acadêmica, e sendo fundamental importância para a conclusão dessa
etapa.

A Corteva Agrosciences, pelo apoio técnico, estrutural e financeiro para a

realização dessa dissertação, e a Dra. Cristiane Muller pela coorientação, amizade e
por sempre apoiar e acreditar no meu trabalho.

A Prof. Dra. Marcia Maria Pereira Sartori pela coorientação e pelas ideias que
enriqueceram esse trabalho.

A Faculdade de Ciências Agronômicas (FCA – UNESP Botucatu) e ao

Programa de Pós-Graduação em Agronomia – Proteção de Plantas pelo apoio,
oportunidade e formação.

Aos professores do Departamento de Proteção Vegetal por todo os

ensinamentos durante a disciplina. A Profa. Dra. Regiane de Freitas Bueno por
sempre me incentivar.

Ao CNPQ – Conselho Nacional de Desenvolvimento Científico e Tecnológico,

pela bolsa de estudos concedida.

Aos amigos e servidores do Departamento de Proteção Vegetal, Prof. Dr.

Tadeu Antonio, Nivaldo Diez, Vanessa Carvalho por toda a ajuda durante o curso,
especialmente ao Marcelo Soman pelo apoio técnico e pela edição das pranchas de
imagens.

Aos amigos do Laboratório de Virologia Vegetal, em especial ao Luis Fernando

Maranho Watanabe, Vinícius Henrique Bello por encararem milhares de quilômetros
e horas de viagens para a condução dos experimentos de campo e Bruno Rossitto De
Marchi que além das viagens foi muito solicito para a construção das árvores
filogenéticas contidas nesse trabalho; Marcos Roberto Ribeiro Junior por desenhar
ótimos primers; Eduardo Vicentin, Eduardo da Silva Gorayeb e Giovana Crusciol pela
colaboração, paciência e pelos momentos de descontração.

A minha amiga e companheira Natalia Bevilaqua por sempre estar ao meu lado
nos momentos de alegria e de dificuldades.

Aos meus amigos Raimundo Bevilaqua, Gizebel Cunha, Flavio Silva, Alisson
Mello, Marcos Godoy, Elliton Souza e Bruno Paião que apesar da distância sempre
me apoiaram e vibraram com minhas conquistas.

Enfim, a todos que de alguma forma participaram dessa jornada.

“Passarei por esse caminho apenas uma só vez; por isso, se existe qualquer bem ou
gesto de bondade que eu possa fazer em benefício do ser humano, que eu faça já.
Que eu não o adie ou negligencie, pois por aqui jamais passarei.”

Autor Desconhecido
 RESUMO

Doenças causadas por vírus são importantes fatores contrários a produção de soja.
Entre elas, a doença da necrose da haste causada pelo Cowpea mild mottle virus
(CPMMV) e transmitida pela mosca-branca Bemisia tabaci já foi observada em todas
as principais regiões produtoras de soja do Brasil e a queima do broto (Tobacco streak
virus – TSV) que têm ocorrência mais restrita nas regiões dos Estados do Paraná e
de São Paulo. Os impactos causados por ambas doenças à sojicultura brasileira ainda
são desconhecidos. Nesse contexto, o objetivo do presente trabalho foi avaliar os
efeitos do CPMMV nas cultivares de soja mais utilizadas nas principais áreas
produtoras da região sudeste e centro-oeste do Brasil e avaliar, por sequenciamento
de nova geração (NGS) a ocorrência de vírus em uma área comercial de soja na
região de Mogi Mirim. Para avaliar os efeitos do CPMMV em cultivares de soja, os
experimentos foram conduzidos durante a safra 2017/2018 em quatro regiões:
Botucatu e Mogi Mirim no estado de São Paulo, Pedra Preta no estado do Mato
Grosso e Planaltina no Distrito Federal. O delineamento experimental foi feito em
blocos casualizados, com dois tratamentos: plantas infectadas artificialmente com
CPMMV e plantas sadias, e cinco repetições. As parcelas compreenderam 6 linhas (5
m), 0,45 m entre linhas e uma média de 14 plantas por metro. Seis cultivares
comerciais foram utilizadas e distribuídas de acordo com a frequência que elas são
utilizadas nessas regiões. Para a infecção artificial, o vírus foi transmitido via tampão
fosfatado 0.01 M, pH 7 em 200 plantas por parcela. A presença do vírus nos dois
tratamentos foi avaliada através de técnicas de análise molecular usando primers
específicos para o CPMMV. Foram avaliados: altura da planta na época da colheita,
número de vagens por plantas, peso de mil grãos (g) e produtividade. Embora algumas
variedades testadas apresentaram-se assintomáticas à infecção do vírus, o CPMMV
reduziu significantemente os parâmetros agronômicos avaliados, causando perdas na
produtividade de 174 a 638 kg ha-1 sacos por hectare, dependendo da cultivar.
Evidência de transmissão do vírus por sementes também foi observada. Para a
detecção e identificação de vírus presentes em uma área comercial de soja na região
de Mogi Mirim, o estudo com sequenciamento de última geração identificou infecção
mista de ilarvirus/carlavirus e carlavirus/tospovirus. Os resultados desse trabalho
sugerem que o CPMMV pode ter um importante papel na redução da produtividade
da soja no Brasil e infecções assintomáticas podem estar mascarando o atual impacto
desse patógeno na cultura. O uso de sequenciamento de última geração como
ferramenta nos estudos de doenças que afetam a cultura da soja se mostrou eficaz
para identificar vírus insuspeitos.

Palavras-chave: Doenças de soja. Necrose da haste. Queima do broto. CPMMV.

TSV. Transmissão.
 ABSTRACT

Diseases caused by virus are important constraints to soybean production. Among

then, stem necrosis caused by Cowpea mild mottle virus (CPMMV) a whitefly-Bemisia
tabaci transmitted were observed in all the main soybean producer areas in Brazil and
the bud blight (Tobacco streak virus – TSV) occurs strictly in regions Parana and Sao
Paulo States. The impacts caused by both diseases on soybean production are still
unknown. Therefore, the goal of this study was to evaluate the effects of CPMMV on
the major soybean cultivars used in the main growing areas of the Southern and the
Midwestern regions of Brazil, and evaluate through next generation sequencing (NGS)
the presence of virus in a commercial field of soybean in Mogi Mirim County. To
evaluate the effects of CPMMV in soybean cultivars, the experiments were conducted
during the 2017/2018 season in four regions: Botucatu and Mogi Mirim Counties, in
the state of Sao Paulo, Pedra Petra County in the state of Mato Grosso and in
Planaltina County, in the Federal District. The experimental design was completely
randomised, with two treatments (plants infected by sap transmission and healthy
plants) with five replications. The plot was comprised of six rows (5m), 0.45 m between
rows and an average of 14 plants per meter. Six commercial cultivars were used and
distributed according to the frequency that they are planted in those regions. For the
field inoculation, the virus was readily sap-transmitted in soybean plants 30 d after
sowing using infected leaves ground in phosphate buffer 0.01 M, pH 7, using
approximately two hundred soybean plants per plot. The presence of the virus was
detected by RT-PCR using primers specific for CPMMV. Plant height (cm), number of
pods per plant, 1.000-grain weight and grain yield were evaluated during harvest time.
Although some cultivars tested presented asymptomatic infection for the virus,
CPMMV reduced significantly the agronomic traits evaluated, causing losses in the
yield ranging from 3.9 to 10.7 bags per hectare, depending on the cultivar. Evidence
of seed transmission of CPMMV was also observed. For the detection and
identification of viruses presence in a commercial field in Mogi Mirim County, the study
with next generation sequencing identified mixed infection of ilarvirus/carlavirus and
calarvirus/tospovirus. The results of this study suggest that CPMMV can have an
important role in yield reduction of soybean in Brazil, furthermore, asymptomatic
infections might be hiding the actual impact of this pathogen in this crop. The use of
next generation sequencing as a tool in the studies of viral diseases that affects the
soybean has showed to be a powerful to detect unsuspected viruses.

Keywords: Soybean disease. Stem necrosis. Bud blight. CPMMV. TSV. Transmission
 SUMÁRIO

INTRODUÇÃO GERAL .................................................................................... 17

REVISÃO DE LITERATURA ............................................................................ 18
CAPÍTULO 1 - Effects of the carlavirus Cowpea mild mottle virus on
soybean cultivars in Brazil ............................................................................. 24

ABSTRACT ....................................................................................................... 24
1.1 INTRODUCTION .............................................................................................. 25
1.2 MATERIAL AND METHODS ............................................................................ 27

1.3 RESULTS ......................................................................................................... 31

1.4 DISCUSSION ................................................................................................... 37

CONCLUSIONS................................................................................................ 40

ACKNOWLEDGMENTS ................................................................................... 41

REFERENCES ................................................................................................. 41

CAPÍTULO 2 - Natural infection of soybean plants by tospovirus,

carlavirus and ilarvirus in Brazil .................................................................... 44

ABSTRACT ....................................................................................................... 44

AKNOWLEGMENTS......................................................................................... 54

REFERENCES ................................................................................................. 55

CAPÍTULO 3 - Uma ameaça oculta à soja: o carlavírus transmitido por

mosca-branca, da necrose à ausência de sintomas .................................... 57

CONSIDERAÇÕES FINAIS.............................................................................. 64
REFERÊNCIAS ................................................................................................ 65
 17

INTRODUÇÃO GERAL

A soja [Glycine max (L.) Merrill] é uma cultura de grande importância

econômica no Brasil, com um mercado interno de R$ 2.7 bilhões e de US$ 31 bilhões
de exportação. O país é o segundo maior produtor mundial do grão, produzindo
114,095 milhões de toneladas em uma área cultivada de 33,914 milhões de hectares
na safra 2016/2017 (HIRAKURI & LAZZAROTTO, 2014; CONAB, 2018). Na última
década, houve um acréscimo em torno de 63,9% da área cultivada do grão no Brasil,
com aumento da produtividade de 2.823 kg ha-1 para 3.363 kg ha-1 (CONAB, 2018).
Diversas doenças causadas por fungos, bactérias, nematoides e vírus podem
comprometer a produção de soja. Já foram identificadas cerca de 40 doenças na
cultura da soja no Brasil (EMBRAPA SOJA, 2013). No mundo, ao menos 34 viroses
foram descritas infectando naturalmente a soja, e no Brasil, há relatos de pelo menos
15 delas (ALMEIDA, 2008; DE MARCHI et al., 2018). Os vírus descritos em soja no
Brasil pertencem aos gêneros: Alfamovirus, Begomovirus, Carlavirus, Comovirus,
Ilarvirus e mais recentemente também um orthotospovirus foi descrito nesta cultura
(DE MARCHI et al., 2018; FONTES et al., 2019).
Há poucos estudos no Brasil acerca dos principais prejuízos ocasionados por
estes vírus, e os trabalhos publicados são, em geral, do final dos anos 1990 e início
dos anos 2000. O melhoramento de soja é bastante dinâmico e novos cultivares são
lançados anualmente visando aumento do potencial produtivo e resistência a
doenças. Deste modo, este trabalho teve como finalidade estudar o impacto da
espécie Cowpea mild mottle virus – CPMMV (Carlavirus, Betaflexiviridae) na produção
dos cultivares atualmente mais plantados de soja nas regiões Sudeste e Centro-Oeste
do Brasil (Capítulo 1), bem como identificar os vírus presentes em uma área comercial
de soja na região de Mogi Mirim, com a detecção por sequenciamento de última
geração. Foi constatada a infecção mista de ilarvirus/carlavirus e carlavirus/tospovirus
(Capítulo 2). No capítulo 3 estão descritos os principais sintomas atualmente
associados ao CPMMV em soja, uma vez que o CPMMV, cuja doença é conhecida
como necrose da haste, não ocasiona mais este sintoma típico nos cultivares
atualmente plantados no Brasil e sua presença no campo pode estar sendo
subestimada.
18

REVISÃO DE LITERATURA

A cultura da Soja e suas doenças virais

Considerada uma das culturas mais antigas, a soja cultivada (Glycine max [L.]
Merril) originária do leste da Ásia, chegou ao ocidente no final do século XV e início
do século XVI. Com o aumento de sua importância e do comércio, essa leguminosa
foi levada para o sul da China, Coréia, Japão e sudeste da Ásia. No Brasil, o primeiro
relato da introdução da soja foi feito no Estado da Bahia (DUTRA, 1882), mas foi nos
estados do sul que essa cultura se expandiu de forma comercial (MIYASAKA;
MEDINA, 1981).
A expansão da cultura da soja no Brasil ocorreu por vários fatores, tais como
obtenção de genótipos adaptados a regiões de baixa latitude, e resistência a doença
(SPECHT et al., 2014). Assim, observou-se um incremento de produção partir do ano
de 1970, passando de 1,5 milhão de toneladas para 119,281 milhões de toneladas em
2018, em uma área cultivada de 35 milhões de hectares (CONAB, 2018).
A soja é afetada por diversas doenças, das quais ao menos 14 doenças
associadas a vírus foram descritos no Brasil infectando-a naturalmente (ALMEIDA,
2008). Segundo este mesmo autor, as doenças de maior impacto para a cultura da
soja são: o mosaico comum da soja (espécie Soybean mosaic virus (SMV), gênero
Potyvirus), a necrose da haste (espécie Cowpea mild mottle virus (CPMMV), gênero
Carlavirus) e a queima do broto (espécie Tobacco streak virus (TSV), gênero Ilarvirus).
Recentemente foi detectada também a espécie Groundnut ringspot orthotospovirus
(GRSV) pertencente ao gênero Orthotospovirus infectando naturalmente soja e pouco
ainda se sabe sobre seu impacto na cultura (DE MARCHI et al., 2018; FONTES et al.,
2019).

O SMV, tem distribuição global, além de ser facilmente transmitido por

sementes provenientes de plantas infectadas e por várias espécies de afídeos
(ALMEIDA & MIRANDA, 1979; LIMA NETO & COSTA, 1979). Os principais danos
ocasionados pelo SMV são a redução no rendimento e manchas no tegumento da
semente, causando seu sintoma típico, conhecido por mancha café. Em 1994, 64%
das cultivares de soja utilizadas por produtores brasileiros eram suscetíveis ao SMV
(ALMEIDA et al., 1994). Apesar dos relatos da variabilidade de estirpes do SMV e de
quebra de resistência (SILVA et al., 2003), não há informações de perdas associadas
 19

a este vírus atualmente no Brasil, uma vez que as cultivares comerciais apresentam
resistência a ele.

A seguir serão abordados os gêneros Carlavirus e Ilarvirus foco de estudo da

presente dissertação.

Gênero Carlavirus, espécie Cowpea mild mottle virus

O gênero Carlavirus compreende 53 espécies sendo o mais numeroso em
termos de espécies na família Betaflexiviridae (ICTV, 2019). O genoma dos carlavírus
é composto por uma milécula RNA de fita simples, senso positivo, possui 6500 a 8600
nt, e poliadenilado (cauda poli A) na região terminal 3’, encapsidado por partículas
filamentosas flexuosas com dimensões de aproximadamente 610-700 nm de
comprimento e 12.15 nm de diâmetro (KING et al., 2011).
A organização típica do gênero Carlavirus é de 6 ORF’s (“Open Reading
Frames” ou fases abertas de leitura) (Figura 1) (King et al., 2011). A ORF1 codifica
uma proteína correspondente a replicase, RNA dependente de RMNA polimerase
(RdRp); as ORFs 2, 3 e 4 formam as proteínas do TGB (triplo gene block) que
corresponde a uma região conservada de genes, envolvidas no movimento do vírus
célula-a-célula e a longa distância; a ORF 5 codifica a proteína responsável pela
proteína capsidial, e a ORF 6 codifica uma proteína com função ainda não
determinada (MARTELLI et al., 2007; MENZEL et al., 2010).
20

Figura 1. Organização genômica dos carlavírus.

Fonte: Adaptado de King et al, 2012.

Para demarcação de espécies, um dos critérios utilizados é a análise dos genes

da polimerase ou da proteína capsidial, em que isolados da mesma espécie
apresentam identidade de nucleotídeos superior a 70% (ou 80% de identidade de
aminoácidos). Vírus pertencentes a diferentes gêneros possuem identidade de
nucleotídeos inferior a 45% (KING et al., 2011). Além desses critérios de classificação
para uma espécie de carlavirus, a transmissão pelo vetor é importante, pois há
espécies de carlavirus transmitidas por afídeos e outras por mosca-branca (King et
al., 2011).
No Brasil, o CPMMV foi primeiramente identificado causando mosaico em
feijoeiro cv. Jalo (COSTA; GASPAR; VEGA; 1983) e em soja na safra 2000/2001, com
plantas exibindo sintomas de nanismo, severa necrose da haste ou até mesmo morte
de plantas em grandes áreas do centro oeste do Brasil (Almeida et al., 2003). Nos
anos seguintes (2002 e 2003), sintomas de necrose da haste foram relatados nos
estados do Maranhão e Paraná e em 2008 a doença foi reportada em diversas regiões
produtoras de soja dos estados de Minas Gerais, Goiás, Mato Grosso, Paraná, Bahia,
Tocantins e Maranhão (ALMEIDA, 2008). O CPMMV ocasiona em soja sintomas de
clorose, mosaico, nanismo, necrose das nervuras e da haste ou até mesmo a morte
dependendo do genótipo, de onde provém o nome da doença “necrose da haste”
(IWAKI et al., 1982; ALMEIDA et al., 2003, 2005).
Assim como os representantes do gênero, o CPMMV é um vírus de RNA senso
positivo de fita simples, possui um genoma de 8200 nucleotídeos com uma estrutura
cap [m7GpppG] ligada ao terminal 5´ e uma cauda poliadenilada no terminal 3´ (KING
et al., 2011; ZANARDO et al., 2014a). A gama de hospedeiros inclui espécies
 21

relatadas nas famílias Fabaceae (Arachis hypogaea, Arachis repens, Canavalia

ensiformis, Desmodium tortuosum, Glycine max, Phaseolus vulgaris, Vigna
subterranean, Vigna unguiculata), Laminacea (Salvia hispanica) e Solanacea
(Solanum lycopersicum) (ALMEIDA et al., 2003; MENZEL et al., 2010; ZANARDO et
al., 2014; CELLI et al., 2016).
Geralmente, os carlavírus são transmitidos por afídeos (KING et al., 2011),
porém o CPMMV e o Melon yellowing-associated virus (MYaV) são exceções do
gênero, pois são transmitidos por Bemisia tabaci (Gennadius) (Hemiptera:
Aleyrodidae) (COSTA; GASPAR; VEGA, 1983; NAVAS-CASTILLO; FIALLO-OLIVE;
SANCHEZ-CAMPOS, 2011). O CPMMV é transmitido por B. tabaci de forma não-
persistente (MUNIYAPPA; REDDY, 1983; MARUBAYASHI et al., 2010) e também
pode ser transmitido por extrato vegetal tamponado. Há relatos de transmissão do
CPMMV por sementes em algumas leguminosas como G. max, P. vulgaris e V.
unguiculata (THOUVENEL; MONSARRAT, FAUQUET et al., 1882; BRUNT; KENTEN,
1973; HORN et al., 1991; BRITO et al., 2012). A transmissão por semente parece ser
dependente do isolado viral, como ocorreu com um isolado de CPMMV de Gana, o
qual houve transmissão por sementes em soja, feijão de corda e em feijão comum
(BRUNT; KENTEN, 1973), e recentemente foi relatado na Venezuela a transmissão
do CPMMV por sementes de V. unguiculata subsp. sesqui pedalis (BRITO et al.,
2012). No Brasil, ainda não há relatos de transmissão do CPMMV por sementes de
soja (ALMEIDA et al., 2005).
Apesar dos diversos relatos desse vírus, não há dados recentes sobre a
incidência do CPMMV nas principais regiões produtoras e os danos ocasionados à
cultura.

Gênero Ilarvirus, espécie Tobacco streak virus

O gênero Ilarvirus compreende 22 espécies sendo o de maior número dentro
da família Bromoviridae, que possui 6 gêneros e um total de 36 espécies (ICTV, 2019).
Os ilarvirus possuem um genoma tripartido de RNAs de fita simples, senso positivo
(ssRNAs) sem a presença de cauda poli A, separados e encapsidados por partículas
esféricas de aproximadamente 36 nm de diâmetro (PALLAS et al., 2013; KING et al.,
2012). O genoma típico dos ilarvírus possui um comprimento total de
aproximadamente 8622 nt. O RNA 1 (~3400 nt) codifica uma proteína da replicase
viral. O RNA 2 (~2900 nt) codifica a RNA dependente de RNA polimerase. O RNA 3
22

(~2200 nt) codifica a proteína de movimento (PM) (PALLAS et al., 2013). A proteína
capsidial (CP) é expressada via um RNA 4 subgenômico, colinear com o final 3’ do
RNA 3 (Figura 2) (BOL, 1999).

Figura 2. Organização genômica dos ilarvírus.

Fonte: Adaptado de Foster e Taylor, 1998.

Os critérios utilizados para a demarcação das espécies no gênero incluem:
sorologia, gama de hospedeiros e similaridade das sequências de nucleotídeos,
porém as porcentagens de identidade não foram definidas para a diferenciação das
espécies (KING et al., 2011).
O Tobacco streak virus é o membro tipo do gênero Ilarvirus e foi identificado
pela primeira vez no Brasil em 1940 em plantas de fumo (Nicotiana tabaccum L.)
(COSTA, 1945). Em soja, o primeiro relato foi feito em 1955 (COSTA; MIASAKA;
D'ANDREA PINTO, 1955), e desde então, sua ocorrência em campos de produção de
soja é muito variável estando mais restrita a regiões dos Estados do Paraná e de São
Paulo (ALMEIDA et al., 1995), devido a presença natural de hospedeiros do vírus
como a cravonara (Ambrosia polystachya). O TSV tem como hospedeiros o algodoeiro
(Gossypium hirsutum L.), tomateiro (Solanum lycopersicum L.), amendoim (Arachis
hypogaea L.), girassol (Helianthus annuus L.) (COSTA; CARVALHO, 1961).
Os sintomas de curvatura e necrose do broto da soja são característicos do
TSV, e dão origem ao nome da doença “queima do broto da soja” (Almeida et al.,
1995). Os sintomas aparecem logo nos estádios iniciais, os trifólios ficam amarelados
e as folhas mais velhas apresentam sintomas de mosqueado e mosaico. Em estádios
mais avançados as plantas apresentam nanismo e atraso da maturação
 23

(RABEDEAUX et al., 2005; ALMEIDA, 2008). O superbrotamento de vagens e trifólios

também é comum em plantas infectadas por TSV (IRIZARRY et al., 2016).
A transmissão do TSV envolve o movimento físico do pólen infectado de uma
planta para outra tendo os tripes como vetor. No Brasil, a transmissão do TSV foi
associada a presença de insetos do gênero Frankliniella (Thysanopetera: Thripidae)
(COSTA & LIMA NETO, 1976). Em outros países, a transmissão por espécies de F.
occidentalis, F. schultzei, Trips tabaci, T. parvispinus e Microcephalothrips
abdominalis já foi descrita (KAISER et al., 1982; GREBER et al., 1991; KLOSE et al.,
1996). O TSV também é transmitido por sementes de soja (GHANEKAR; SCHWENK,
1973).
24

CAPÍTULO 1 - Effects of the carlavirus Cowpea mild mottle virus on soybean

cultivars in Brazil

F. B. Silvaa, C. Mullerb, V. H. Belloa, L. F. M. Watanabea, B. R. De Marchia, L. M.

Fuscoa, M. R. Ribeiro-Juniora, G. B. Minozzib, L. M. Vivanc, M. A. Tamaid, J. R. Fariase,
M. M. P. Sartoria, R. Krause-Sakatea*

aFaculdade de Ciências Agronômicas, Universidade Estadual Paulista “Júlio de

Mesquita Filho” (UNESP), Botucatu, SP 18610-034, Brazil; bCorteva™ Agrisciences;
cFundação de Apoio a Pesquisa à Pesquisa Agropecuária de Mato Grosso/Fundação
MT; dUniversidade do Estado da Bahia/UNEB, Departamento de Ciências Humanas,
Campus IX, Barreiras, Bahia; and eInstituto Phytus, Departamento de Entomologia,
Santa Maria, Rio Grande do Sul.

*E-mail: [Link]@[Link]

Article type: Research Article

Soybean stem necrosis is caused by Cowpea mild mottle virus (CPMMV), transmitted

by the whitefly Bemisia tabaci. CPMMV has already been recorded in all major

soybean-producing areas of Brazil. The impacts caused by CPMMV to Brazilian

soybean production are unknown. Thus, the goal of this study was to evaluate the

effects of CPMMV on the major soybean cultivars grown in the Southern and

Midwestern regions of Brazil. Although asymptomatic in some of the tested cultivars,

CPMMV infection significantly reduced the plant height, the number of pods per plant

and the 1,000-grain weight. In addition, estimated yield losses ranged from 174 to 642

kg ha-1, depending on the cultivar. Evidence of seed transmission of CPMMV was

observed in the BMX POTENCIA RR cultivar. These results suggest that CPMMV

could have an important role in the reduction of soybean productivity in Brazil.

Symptomless infections might be hiding the actual impact of this pathogen in

 25

commercial fields and infected seed could be the primary inoculum source of the virus

in the field.

Keywords: CPMMV; stem necrosis; soybean disease; transmission

INTRODUCTION

Soybean [Glycine max (L.) Merril] is an important crop worldwide as a source of oilseed

and protein. Brazil is the second largest producer of soybean in the world, producing

119.23 million tons in a cultivated area of 35.1 million hectares in the last season

(2017/2018). In Brazil, soybean is the most important economic crop, generating

approximately 675 million US$ to the internal market and 31 billion US$ to exportation

(Hirakuri & Lazzarotto, 2014; Conab, 2018). In the last decade in Brazil, the cultivated

area and production increased 64.9% and 98.7%, respectively, between the

2007/2008 and 2017/2018 crop seasons, while the productivity increased from 2,800

to 3,400 kg ha-1 over the same time (Conab 2018).

Soybean can be affected by several pests regardless of growth stage. In Brazil, the

occurrence of at least 15 viral diseases have been reported (Almeida 2008; De Marchi

et al. 2018). The Cowpea mild mottle virus (CPMMV; family Betaflexiviridae, genus

Carlavirus), which is the agent of soybean “stem necrosis disease”, was reported

infecting soybean in the 2000/2001 season in the state of Goias. CPMMV is considered

an emergent virus and has been already recorded in soybean fields across Brazil in

the states of Bahia, Mato Grosso, Maranhao, and Parana (Almeida et al. 2003; Almeida

2008; Almeida et al. 2005). In 2008, the virus was observed in the Minas Gerais and

Tocantins states, which are important soybean growing areas (Almeida 2008).

Although steam necrosis is the common name of this disease, in the last years, mild

mottle and mosaic were the most common symptoms observed for this virus infection
26

(Zanardo et al. 2014; Zanardo e Carvalho 2017). Thus, the symptoms of this disease

do not match with its common name, what brought us the interest to study this

pathosystem.

In general, carlaviruses are transmitted by aphids (King et al., 2011). However,

CPMMV is one of the two exceptions of the genus that are transmitted in a non-

persistent manner by whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae)

(Almeida 2008; Marubayashi et al. 2010), pest that has been an important factor in the

reduction in soybean production (Lourenção et al.,1999; Tamai et al., 2006) and

causing losses to several crops such as vegetables, fibres and ornamentals (De Barro

et al. 2011; Lapidot et al. 2014) by virtue of its ability to transmit numerous viruses

(Navas-Castillo et al. 2011). Bemisia tabaci is widely spread in Brazil, and the Middle

East Asia Minor 1 (MEAM1, known as B biotype) is the prevalent species on major

crops across the country, and it is mainly associated with CPMMV infection in soybean

(Moraes et al. 2018). The species Mediterranean (MED, also known as biotype Q),

which was recently related in the country and it is distributed in the southeast and south

region, is associated with insecticide resistance which has been caused its rapid

invasion and dominance in new niches and countries (Moraes et al. 2018; Horowitz e

Ishaaya 2014; Yao et al. 2017). Currently, B. tabaci is between the ten most important

pest in Brazil (Brasil 2019).

CPMMV is also easily transmitted by sap (Brunt e Kenten 1973), one characteristic

that in artificial conditions is very helpful to study the virus. The seed transmission of

CPMMV has also been reported in different plant species such as soybean, cowpea

(Vigna unguiculata) and common bean (Phaseolus vulgaris) in Africa (Brunt e Kenten

1973), in addition to yardlong bean (Vigna unguiculata subsp. sesquipedalis) seed in

Venezuela (Brito et al. 2012), and by some soybean cultivars in India (Yadav et al.
 27

2013). To date, there have been no reports of CPMMV isolates transmitted by soybean

seed in Brazil (Almeida et al. 2005).

The effects caused by CPMMV to Brazilian soybean production have never been

estimated (Zanardo e Carvalho 2017), and the ability of Brazilian CPMMV to be

transmitted by seed is still unknown. Thus, the goal of this study was to evaluate the

damage of CPMMV on the major soybean varieties used in the main growing areas of

the Southern and Midwestern regions of Brazil. Additionally, the seed transmission

ability of one CPMMV isolate from the São Paulo State was assessed.

MATERIAL AND METHODS

Obtaining CPMMV isolate and partial genome characterisation

The CPMMV isolate was collected from soybeans in Casa Branca County, São Paulo

State, Brazil (2016/2017). Total RNA was extracted from the leaf tissue of symptomatic

soybean plants using the RNA Purification Kit (NORGEN) followed by reverse

transcription-polymerase chain reaction (RT-PCR) One Step using AMV reverse

transcriptase (Promega, Brazil). The specific primers were CPMMV 1280-F (5’-GGC

GTT CCA AAA GCT GCC GAT-3’) and CPMMV 1696-R (5’-GGA GCC ACC TTT CCA

ATC AA-3’) (De Marchi et al. 2017). All amplifications consisted of an initial step of

42°C for 30 min, a second step of 94°C for 2 min, 30 cycles of 94°C for 54 sec,

annealing at 54°C for 50 sec and elongation at 72°C for 50 sec, followed by a final

extension step at 72°C for 10 min. In order to obtain the partial genome

characterisation of the CPMMV isolate of Casa Branca – SP (called CPMMV-Casa

Branca), the ORF 2 to the 3’ terminus was amplified with a RT-PCR using the following

four primer pairs: 5,722 F (5’-ATG GGG GAA CGA GTA AC TGC-3’) and 6,528 R (5’-

TCA CTG TGC CTG GTC AA ACA-3’); 6,240 F (5’-ACA GGT TCG GTA AGA GC
28

ACG-3’) and 7,173 R (5’-GCT TTT GGA ACG CCA GT TTT-3’); 7,152 F (5’-CAA AAA

CTG GCG TTC CAA A AGC-3’) and 8,003 R (5’-AGG GAA ACG TGA ATG GC GAT-

3’); 7,921 F (3’-AGG GCC TGA AAT ATC TCG CA-3’) and 8,322 R (3’-GGA CTC TTT

ATA CTC TTT CCT T GGT-3’). The amplifications consisted of an initial step of 42°C

for 30 min, second step of 95°C for 2 min, 35 cycles of 94°C for 1 min, annealing at

57°C for 1 min, and elongation at 72°C for 1 min, followed by a final extension step at

72°C for 10 min. The amplified PCR products were purified (QIAquick Gel Extraction

Kit, Qiagen) and directly sequenced in both forward and reverse directions. The

sequence obtained was analysed using the software Geneious v9.1.3. (Kearse et al.

2012) and compared to different CPMMV isolates described by Zanardo et al., (2014).

After virus identification and characterisation, the isolate was maintained in common

bean (Phaseolus vulgaris L.) cv. Jalo by whitefly transmission. Virus transmission was

performed by transferring whitefly specimens (MEAM1) in cages containing infected

soybean leaves for a viral acquisition access period (AAP) of 24 h. Following virus

acquisition, whiteflies were transferred to cages containing healthy bean plants at the

VC (cotyledon leaves) growth stage, for a 24-h inoculation access period (IAP) under

controlled conditions at 30°C. After inoculation, insecticides (Oberon and Cartap) were

sprayed on plants to eliminate all the whitefly adults, nymphs and eggs. Thirty days

after the IAP, plants were analysed for the virus presence. After virus confirmation, the

plants were used as source of inoculum for the field inoculation.

Experimental areas

Field experiments were conducted during the 2017/2018 growing season with six

cultivars distributed in four different growing areas: cv. BMX POTENCIA RR in

Botucatu, State of São Paulo (coordinates: 22°48’25.4”S, 48°25’46.4”W, elevation: 739

m, sowing date 01/11/2017), cv. M 6410 IPRO and TMG 7062 IPRO in Mogi Mirim,
 29

State of São Paulo (coordinates: 22°26’42.8”S 47°04’10.8”W, elevation: 687 m, sowing

date 15/12/2017) , cv. M 7739 IPRO and M 8372 IPRO in Pedra Preta, State of Mato

Grosso (coordinates: 16°50’30.3”S 54º02’39.8”W, elevation: 744 m, sowing date

23/11/2017), and cv. M 9144 RR in Planaltina, Federal District (coordinates:

15°39’51.7”S 47°20’02.0”W, elevation: 887 m, sowing date 27/11/2017).

The four sites represented some of the major soybean producing areas in Brazil. They

have contrasting environmental conditions, and the cultivars were selected according

to the frequency that they were planted in each region. Parameters such as the

occurrence of weeds, diseases and insect pressure, especially B. tabaci, were

monitored throughout the season.

Experimental design and field inoculation

The experimental design was a randomised block, with two treatments (plants infected

by sap transmission and healthy plants) with five replications. The plot was comprised

of six rows (5m), 0.45 m between rows and an average of 14 plants per meter.

For the field inoculation, the virus was readily sap-transmitted in soybean plants 30 d

after sowing using infected leaves ground in phosphate buffer 0.01 M, pH 7, using

approximately two hundred soybean plants per plot. The presence of the virus was

detected by RT-PCR using primers specific for CPMMV.

Field sampling and evaluation of agronomic traits

A total of 100 soybean samples collected in each plot were evaluated for virus infection

30 days after inoculation. When the plants were at physiological maturity (R8), the plant

height was evaluated by the distance from the soil to the apex of the plant (cm) and

the number of pods per plants was obtained by counting the total number of pods per

plant. At the harvest, all plants of the plot were hand harvested and run through a
30

thresher. For each plot, the 1,000-grain weight (g) was determined, which was

obtained by weighing 1,000 grains from the plants in the plot, and adjusting to 13%

moisture in addition to grain productivity, which was obtained by weighing the grains

produced, and adjusting to 13% moisture, then converting into kg ha-1.

Soybean seed transmission

To study the seed-borne capacity of CPMMV, a random sample of seed was collected

from the CPMMV-infected BMX POTENCIA RR plot, harvested in the Botucatu field.

These soybean seeds were planted in Styrofoam seedling trays containing Tropstrato

HA Hortaliças (Vida Verde Tecnologia em Substratos, Mogi Mirim, SP). The seedling

trays were kept in an insect-proof cage. Germination was greater than 90%, and the

seedlings did not show any typical disease symptoms. For virus detection, leaf

samples were tested using RT-PCR. To compose a sample, leaves of ten plants were

collected and combined. There were 80 samples tested, totalling 800 plants. Once the

presence of CPMMV was detected in a sample, the ten plants were tested individually

to identify the infected plant. The positive plants were kept in an insect-proof cage for

60 days in order to observe the symptoms.

Data Analysis

Because of the interdependency and interrelationship of agronomic traits, principal

component analysis (PCA) was performed to investigate the data collected in the

current study. PCA was performed using Minitab 17 Statistical Software (2010). Data

were also submitted to analysis of variance (ANOVA) using Statview software

(Concepts and StatView, 1987) to determine whether significant differences (p < 0.05)

occurred between treatments. Then means were compared using Tukey’s test (α =

5%).
 31

RESULTS

Virus characterisation

The partial genome of the CPMMV-Casa Banca isolate (ORF 2 to the 3’ terminus)

(Accession number MK900736) obtained is this study showed 99% of nucleotide

identity with the CPMMV:BR:MT:02:1 and Florida_Bean_2011 isolates retrieved from

GenBank (Accession numbers: KC884246.1 and KC774020.1, respectively).

According to the classification used by Zanardo et al. (2014), the CPMMV-Casa

Branca isolate belongs to the BR2 group, which encompasses the most common

CPMMV strains from soybean in Brazil. This isolate was used in all field experiments.

Virus incidence and symptoms on inoculated plots

The symptoms observed were variable among the cultivars. BMX POTÊNCIA RR, M

7739 IPRO and M 8372 IPRO cultivars were symptomless to CPMMV infection. In

contrast, the M 6410 IPRO, TMG 7062 IPRO and M 9144 RR cultivars showed the

most severe symptoms. The most common symptoms were chlorosis, mottling and

mild symptoms (Figure 1). Due to the variation in symptoms, the estimate of virus

infection was carried out by sampling the plots followed by molecular analysis. All the

six sap-inoculated soybean cultivars had an infection from the CPMMV-Casa Branca

isolate greater than 70% in field conditions (Table 1). The virus transmission efficiency

was 70% in cv. M 8372 IPRO, 85% in cv. BMX POTÊNCIA RR, 87% in cv. M 9144

RR, 89% in cv. M 7739 IPRO, 90% in cv. TMG 7062 IPRO and 92% cv. M 6410 IPRO.
32

Figure 1. Symptoms observed in the six cultivars tested in the field experiment: (a)
BMX POTÊNCIA RR, (d) M 7739 IPRO and (e) M 8372 IPRO showed to be
symptomless; (c) M 7062 IPRO, (d) M 8372 IPRO and (f) M 9144 RR showed chlorosis,
mild and mottling symptoms

Field plot experiments and agronomic performance of the cultivars

The PCA of soybean cultivars comparing CPMMV-infected and healthy plants showed

the proportion of the variance retained by the first principal component (PC1) was

67.5% and for the second principal component (PC2) corresponded to 22.3% of the

original remaining variance (Figure 2).

The exploratory analysis allowed the evaluation of the virus influence on the groups of

cultivars and cultivated areas (Figures 2 and 3). Among the cultivars, the cv. M 9144

RR healthy plants had the greatest plant height and were among the varieties that have

the highest number of pods per plants, which set this cultivar and treatment apart from

the rest. Regarding the 1,000-grain weight, the cultivar that showed the best
 33

performance was TMG 7062 IPRO followed by M 7739 IPRO, M 6410 IPRO and BMX

POTÊNCIA IPRO. The greatest productivity was reached by the cultivar BMX

POTÊNCIA IPRO, followed by M 7739 IPRO and M 8372 IPRO, being the cv. BMX

POTÊNCIA the most affected by the presence of the virus.

Figure 2. Projection of vectors of traits: plant height in cm, number of pods per plants,
1000-grain weight in g and productivity in bags ha-1.
34

Figure 3. Biplot graph with dispersion of six soybean cultivars according to the principal
components (PC1 and PC2). ● cv. BMX POTÊNCIA RR healthy plants, ○ cv. BMX
POTÊNCIA RR CPMMV-Infected plants, ■ cv. M 6410 IPRO healthy plants, □ cv. M
6410 IPRO CPMMV-Infected plants, ♦ TMG 7062 IPRO healthy plants, ◊ TMG 7062
IPRO CPMMV-Infected plants, ▲ cv. M 7739 IPRO healthy plants, ∆ cv. M 7739 IPRO
CPMMV-infected plants, ► cv. M 8372 IPRO healthy plants, > cv. M 8372 IPRO
CPMMV-infected plants, ◄ cv. M 9144 RR healthy plants, < cv. M 9144 RR CPMMV-
infected plants.

As expected, each cultivar had distinct performance once they have particular

characteristics and they were cultivated in areas with contrasting environmental

conditions (Figure 3). The exploration of the data also demonstrated that the cultivars

M 6410 IPRO and TMG 7062 IPRO cultivated in Mogi Mirim - SP had a close

performance to all evaluated traits as well as the cultivars M 7739 IPRO and M 8372

IPRO which were cultivated in Pedra Preta – MT. The cv. M 9144 RR tested in

Planaltina – DF showed the most distant performance data comparing to the other

cultivars (Figure 4).

 35

Figure 4. Biplot graph with dispersion of four areas according to the principal
components (PC1 and PC2). ● Botucatu, São Paulo, ■ Mogi Mirim, São Paulo, ♦ Pedra
Preta, Mato ▲ Planaltina, Federal District.

Analysis of variance showed that cultivar tested in this work presented different

response to the virus infection, showing a significant (p<0.05) or non-significant

reduction in the traits evaluated (Table 1).

There was a significant effect (p<0.05) of CPMMV-infection in all agronomic traits

evaluated in the cv. BMX POTÊNCIA RR. CPMMV-infected plants have reduction in

plant height (p<0.01, F=467.76), number of pods per plant (p<0.01, F=36.53) and

1,000-grain weight (p<0.01, F=11.64) that reflected directly in the productivity (p=0.01,

F=11.20), which had a loss of 638 kg ha-1, this cultivar being the one that had the

greater yield loss, approximately 16%.

 36

Table 1. Percentage of infected plants and mean of agronomic traits of fields assays according to their cultivar.
Plant Height (cm) Pods per plant 1,000-grain weight Yield (kg ha-1)
Infected (g)
Location Cultivar
Plants (%) Healthy CPMMV- Healthy CPMMV- Healthy CPMMV- Healthy CPMMV-
infected infected infected infected
Botucatu BMX 85
78.80a 60.60b 82.10a 40.30b 149.60a 132.60b 4,029a 3,391b
POTENCIA RR
Mogi Mirim M 6410 IPRO 92 68.71a 59.71b 70.15a 41.26b 139.37a 129.13b 2,564a 2,330b
TMG 7062
90 67.13a 66.86a 34.22a 30.92b 186.05a 169.01b 2,393a 2,145b
IPRO
Pedra Preta M 7739 IPRO 89 74.76a 70.84b 45.68a 42.89a 145.54a 144.97a 3,323a 2,953a
M 8372 IPRO 70 86.84a 84.48a 50.82a 44.40b 123.66a 119.00a 3,230a 3,056a
Planaltina M 9144 RR 87 123.13a 121.07a 61.60a 42.45b 148.71a 105.45b 2,278a 1,962b
Mean followed by the same letter within rows indicate no significant (p < 0.05) difference between healthy and CPMMV-infected plants according to an ANOVA.
 37

Regarding the cultivar M 6410 IPRO, there was also a significant reduction (p < 0.05)

in all traits evaluated, and only the plant height (p=0.74, F=0.1) did not differ to the cv.

TMG 7062 IPRO. The cultivars tested in Rondonopolis differed significantly only in one

parameter each, plant height (p<0.01, F=17.92) and number of pods per plant (p<0.01,

F=8.97), for cv. M 7739 IPRO and M 8372 IPRO, respectively. Although no significant

effect of treatment in the yield occurred in both cultivars, there was a reduction of 370

kg ha-1 for cv. M 7739 IPRO (p=0.07, F=4.08), and 174 kg ha-1 for cv. M 8372 IPRO

(p=0.33, F=1.07).

All traits in the cv. M 9144 RR, except for plant height, were affected significantly

(p<0.05) due to CPMMV-infection. Although the height was significantly the same for

the treatments (p=0.51, F=0.47), the reduction caused in number of pods per plant

(p=0.25, F=7.57) and 1000-grain weight (p<0.01, F=182328.14) in the diseased plants

directly affected the in productivity (p=0.01, F=9.85) with a reduction of 316 kg ha-1, or

approximately 14%.

Seed transmission

From 800 seedlings obtained from seeds harvested in the cv. BMX POTÊNCIA RR

CPMMV-infected field plot, three plants were found to be infected by CPMMV. The RT-

PCR confirmed the presence of the systemic infection of BMX POTÊNCIA RR plants

coming from seeds collected from CPMMV-infected soybean plants. Not a single plant

developed typical disease symptoms after emergence until their senescence. The

observed percentage of plants infected with the virus was 0.375%.

DISCUSSION

The data obtained in this study revealed that CPMMV causes reduction of agronomic

traits such as productivity, plant height, 1000-grain weight and pods per plant in the
38

main soybean cultivars used in Brazil, suggesting that this virus may be responsible

for major economic losses for soybean crop in our country. Additionally, the CPMMV-

seed transmission data for a Brazilian isolate highlights the seed importance as a

primary inoculum source in the field, especially in areas with low whitefly population,

such as in Parana south region, Santa Catarina and Rio Grande do Sul States.

CPMMV was first recorded in Brazil in common bean in the 1980s (Costa et al.,1983),

and reported as a threat to soybean production in 2002 (Almeida et al. 2003). The

losses reported at that time were higher than 85%, since the cultivars used were

developing the stem necrosis symptom that affected the whole plant (Almeida et al.

2003). The new cultivars has reduced the impact of this disease in soybean (Arias et

al. 2015). However, although some soybean cultivars are symptomless when infected

by CPMMV, they can have great yield losses, as our results suggest.

In addition, B. tabaci MEAM1 is the predominant species in the country, and MED is

distributed in the southeast and south region (Moraes et al. 2018). More recently, MED

has reported in the middle-western region (data not published). The Brazilian middle-

western region is the largest soybean and common bean producer, and both crops are

cultivated near to each other. It is already known that MEAM1 can colonize soybean

and common bean as well as transmit viruses (Marubayashi et al. 2010; Inoue-Nagata

et al. 2016), but recent studies revealed that MED has a great reproduction in both

crops, especially common bean (Watanabe et al. 2019) and is a good vector of many

Brazilian viruses (Bello et al. 2019). Then, the combination of these conditions may

help the CPMMV to spread, to cause damage in plants and to be present in the field

during the whole year. Currently, the virus management is based on the chemical

control of B. tabaci (Yao et al. 2017). However, another important parameter that

should be included in the management is the Brazilian agriculture system, which is

 39

complex and has contributed to increase the whitefly population as well as the virus

dissemination (Inoue-Nagata et al. 2016). In addition, as soybean and common bean

are grown in nearby areas, common bean can also serve as inoculum source to

soybean crop, contributing to spread the virus and to reduce the soybean yield. In our

study, the highest reduction in productivity was observed for cv. BMX POTENCIA RR

(638 kg ha-1) followed by cv. M 7739 IPRO (370 kg ha-1) and M 9144 RR (316 kg ha-1)

(Table 1). It is also important to highlight that even a reduction in productivity of 174 kg

ha-1 observed for cv. M 8372 IPRO may cause a huge economic impact, since the bag

(60 kg) price is, on average, 20.00 US$ (CEPEA, 2019). As Brazil is the largest

soybean oilseed exporter in the world (USDA. 2019), the amount of bags reduction

impacts directly not only for the farmers, but also for the Brazilian economy.

Seed-borne transmission has already been reported for CPMMV isolates worldwide,

but here we provide the first evidence of seed-transmission of a CPMMV Brazilian

isolate in soybean. Seed transmission was observed in cowpea, soybean and common

bean seeds in Ghana, even though at a low incidence (Brunt e Kenten 1973). In

Thailand, the virus was also observed to be transmitted by soybean seeds at a

frequency lower than 1% (Iwaki et al., 1982). In Venezuela seed transmission was

observed for yardlong bean seeds (Brito et al. 2012). In India, the seed-borne nature

of the virus was detected in several soybean cultivars with higher rates of transmission,

ranging from 0.62% to 14.2% (Yadav et al. 2013). The seed transmission ability for

CPMMV seems to be dependent on the viral isolate and the host cultivar and is an

important component of the epidemiology of the virus in the field. A few infected seeds

can provide enough CPMMV inoculum to be disseminated by the efficient B. tabaci

vector. Then, the virus can be easily disseminated in the cultivated area, making its

management even harder in tropical countries. In the world scale, the CPMMV isolates
40

from Florida, Puerto Rico and Brazil have shown to be phylogenetically close,

indicating a common origin (Zanardo & Carvalho, 2017). Our data reinforce that the

CPMMV capacity to be transmitted by seeds might have disseminated the virus in

different countries through the international trade.

In summary, soybean productivity was decreased by CPMMV infection and losses of

more than 637.8 kg ha-1 were observed for the symptomless cv. BMX POTENCIA RR,

one of the most popular cultivars used in Brazil. Also, the Brazilian agriculture system

helps the CPMMV and the whitefly to be in the field during the whole year as well as

to be disseminated, increasing the concern about this disease. Seed transmission of

CPMMV was also observed for this cultivar, even in low frequency. CPMMV is a

serious concern for soybean production in Brazil and it cannot be underestimated as it

has been in the last years. Thus, because of these conditions, it is important to improve

the management, including whiteflies populations, soybean cultivars and seed analysis

in order to avoid the CPMMV spread. Then, the amount of losses might be reduced.

In addition, there is a need for implementing CPMMV in soybean breeding programs

in order to identify varieties with higher tolerances to this virus.

CONCLUSIONS

Soybean productivity was decreased by CPMMV infection and losses of more than

637.8 kg ha-1 were observed for the symptomless cv. BMX POTENCIA RR, one of the

most popular cultivars used in Brazil. Seed transmission of CPMMV was also observed

for this cultivar, even in low frequency.

 41

ACKNOWLEDGMENTS

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal

de Nível Superior – Brasil (CAPES) – Finance Code 001 and by grants from Corteva™

Agrisciences and FAPESP (process number 2017/21588-7). F.B.S. is recipient of

CNPq scholarships. R.K.S. is a CNPq research fellow.

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 43

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44

CAPÍTULO 2 - Natural infection of soybean plants by tospovirus, carlavirus and

ilarvirus in Brazil

Felipe Barreto da Silva1, Cristiane Muller2, Marcos Roberto Ribeiro Junior1, Vinicius

Henrique Bello1, Eduardo P. de Sousa1, Bruno Rossitto De Marchi3, Marcelo Agenor

Pavan1, Renate Krause Sakate1

1 Faculdade de Ciências Agronômicas, Universidade Estadual Paulista “Júlio de

Mesquita Filho” (UNESP), Botucatu, SP 18610-034, Brazil;

2Corteva™ Agrisciences, Mogi Mirim Research Station

3University of Florida Gulf Coast Research & Education Center

Corresponding author: Dra. Renate Krause-Sakate, e-mail: [Link]@[Link]

Article type: Short Communication

Abstract
Soybean is a major food crop in Brazil and the primary source of protein and

vegetable oil worldwide. Although during the lasts decades soybean production has

increased through development of new varieties, this crop hosts a wide range of

pathogens that causes significant yield losses. Among pathogens that affects soybean,

the viruses have been drawing attention due to their diversity of symptoms and the
 45

lack of information about their actual impacts in soybean in Brazil. In this work, we have

detected and identified three viruses in a commercial soybean field in the County of

Mogi Mirim in the State of Sao Paulo by high throughput sequencing analysis.

Sequences of a Carlavirus, Tospovirus and Ilarvirus in a pooled sample of 10 plants

suggests a mix infection of Tobacco streak virus (TSV) and Cowpea mild mottle virus

(CPMMV), and CPMMV and Groundnut ringspot virus (GRSV) and reinforce the

importance of whiteflies and thrips as vector of viruses in soybean. Based on the

complete genome sequence analysis of the TSV isolate obtained in this study we

conclude that our isolate is phylogenetic close to a TSV isolate from Australia.

Keywords: TSV, CPMMV, GRSV, soybean disease, bud blight

Soybean [Glycine max (L.) Merril] is a major food crop in Brazil and its

production has been improved significantly in the past decades (Conab, 2018).

Although the Brazilian soybean production is in evidence, growers of this leguminous

face the raise of several diseases, and among the pathogens affecting this crop, viral

diseases has an important role as a reducing factor (Almeida 2008).

Until the beginning of the 2000s, about fourteen diseases in soybean associated

with virus were reported in Brazil (Almeida 2008). During the last two decades other

virus affecting soybean has been identified in Brazil as the tospovirus Groundnut

ringspot virus (GRSV) (DeMarchi et al., 2018; Fontes et al., 2019), as well as other

known diseases has re-emerged like the carlavirus Cowpea mild mottle virus

(CPMMV) (Barreto da Silva et al., Unpublished Data).

46

In the 2018/2019 season, a commercial soybean field in Mogi Mirim County,

State of São Paulo, began to draw attention since plants showed severe stunting,

reduced leaf size and delayed maturity. High population of thrips was also associated

to the plants. To investigate the occurrence of viruses in this field, a pooled sample of

ten leaves, collected from individual plants, was characterized by high throughput

sequence analysis. Total RNA extraction was performed for the pooled sample with

the PureLink Viral RNA/DNA kit (Invitrogen), followed by library preparation and

transcriptome sequencing using the Illumina HiSeq2500 platform. De novo assembly

of the 30,636,554 reads was carried out using the CLC Genomics Workbench software

v7.0.3. The 13,345 contigs obtained were submitted to a BLASTn search using the

Geneious software v9.1.5.

RNA seq analysis revealed the presence of the carlavirus CPMMV, the

tospovirus GRSV and the ilarvirus Tobacco streak virus (TSV) in the pooled sample.

RT-PCR using tospovirus universal primers BR60/BR65 (Eiras et al. 2001), the

carlavirus CPMMV specific primers CPMMV 1280-F/CPMMV 1696-R (De Marchi et al.

2017) and the designed primers TSV ORF2a 899 F (CAC AGG GTC CCA ACT CAG

C) and ORF2a 1200 R (ACT GGC TTC AAG GTG GTC TTT) was performed for the

10 plants separately. The presence of CPMMV was confirmed for all the plants tested.

The GRSV was present in one plant and the TSV was identified in seven plants. The

presence of CPMMV and GRSV was also confirmed for soybean plants collected in

nearby fields to Mogi Mirim.

Plants infected with TSV and CPMMV showed reduced plant height, excessive

axillary branching, reduced leaf size and mottling symptoms while the plants infected

solely with CPMMV presented mosaic symptoms but reduction of grown was not
 47

observed. Plant infected with GRSV + CPMMV presented necrotic areas on leaves,

which was not observed for plants infected solely by CPMMV. (Figure 1).
48
 49

Figure 1. Symptoms observed for: A and B, Soybean plant mix infected with Cowpea

mild mottle virus and Tobacco streak virus exhibiting symptoms of (A) stunt, (B) leaf

size reduction and excessive axillary branching; (C and D) soybean plant infected with

Cowpea mild mottle virus exhibiting symptoms mosaic; and ( E and F) soybean plant

infected Cowpea mild mottle virus and Groundnut ringspot virus exhibiting necrotic

spots.

The tospovirus was recently reported infecting soybean in the state of Bahia and

Federal District, Brazil (De Marchi et al. 2018; Fontes et al. 2019). The CPMMV was

detected on soybean in the 2000-2001 growing season (Almeida et al. 2003) and it

seems to be an emergent virus in this crop with a variability of isolates detected in

Brazil (Zanardo et al. 2014a, b). A recent study also reinforced that CPMMV can be a

silent virus in soybean, since cultivars have not shown evident symptoms of the

disease, but production can be significantly affected (Barreto da Silva et al., in press).

TSV was first detected in Brazil in 1940 infecting tobacco (Nicotiana tabacum

L.) (Costa, 1945) and soybean in 1955 (Costa et al., 1955). Since then, TSV incidence

varies in functions of the year and the region, and its occurrence is more restrict to the

states of Sao Paulo and Parana. TSV infects also cotton (Gossypium hirsutum L.),

tomato (Solanum lycopersicum L.), peanut (Arachis hypogaea L.), sunflower

(Helianthus annuus L.) and some weeds, such as Ambrosia poystachia that is a known

host of the virus and serves as a source of inoculum in the field (Costa e Carvalho

1961). TSV belongs to the Ilarvirus genus and is transmitted mechanically by the thrips

feeding on pollen grains containing the virus. Viruses are present in/on the pollen and

may be transmitted when wind-blown pollen and populations of vectors thrips are

coincident on a susceptible host (Sdoodee e Teakle 1987; Klose et al. 1996; King et

al. 2011). Five thrips species can be involved in the transmission of TSV: Frankliniella
50

occidentalis, F. schultzei, Trips tabaci, T. parvispinus and Microcephalothrips

abdominalis (KAISER et al., 1982; GREBER et al., 1991; KLOSE et al., 1996). In Brazil

it was demonstrated the association of TSV with the presence of Frankliniella schulzei

(Costa & Lima Neto, 1976; Moscardi & Almeida, 1980). Seed transmission was also

reported in soybean (Ghanekar e Schwenk 1973) and in others host species (Johnson

et al., 1984). The disease associated to TSV in soybean plants is characterized as bud

blight of soybean, also referred as Brazilian bud blight that causes symptoms of

chlorosis, necrosis if leaves, stem, and buds, read node, stunting, leaf size reduction,

mosaic, mottle, rugosity and delay of maturation.

Here we obtained the complete genome of the Brazilian soybean isolate of TSV

that is composed of a positive-sense single-stranded RNA tripartite genomes (RNA 1

to 3) with a total length of 8622 nucleotides (nt) (King et al. 2011). TSV is the type

species of the genus Ilarvirus, family Bromoviridae. RNA 1 (~3400 nt) encode for the

viral replication protein and the RNA 2 (~2900 nt) is coding for RNA-dependent RNA

polymerase (RdRp). RNA 3 (~2200 nt) code for the movement protein (MP) (King et

al. 2011; Pallas et al. 2013). The viral coat protein (CP) is expressed via a subgenomic

RNA 4, collinear with the 3’ end of the RNA 3 (Bol, 1999).

Contigs obtained for the RNA 1, RNA2 and RNA3 of the TSV soybean Mogi

Mirim isolate aligned with the GenBank database using BLAST 2.7 showed the highest

nucleotide identity (98%) with the Australian TSV isolate from sunflower (Acession

numbers: JX463334, JX463335 and JX463336, respectively). The bayesian

phylogenetic analysis performed with Mr. Bayers 3.2.2 for the three RNAs confirmed

the closest relationship with this isolate indicating a possible common origin. It is

interesting to note that according to the CP analysis, TSV Soybean Mogi Mirim share
 51

very high identity with the Brazilian isolate TSV-BR collected from soybean, Accession

number AY354406, previously described by (Almeida et al. 2005) (Figure 2).

52
53
54

Figure 2. Phylogenetic trees constructed based on complete sequences of RNA 1 (A),

RNA 2 (B), RNA 3 (C) and CP (D) for the Tobacco_streak_virus_Soybean Mogi Mirim

isolate and other isolates of Tobacco streak virus. The putative novel ilarvirus peanut

virus C was used as on out-group.

In the USA there was a sudden revival of TSV in 2016 that might have been

influenced by hot and dry weather that is preferred by thrips (Irizarry et al. 2016). In

Brazil, the 2018/2019 season was hot and dry, that might have contributed to high

populations of thrips and helped to spread GRSV and TSV in the field, as well as for

the whitefly Bemisia tabaci which might contribute to spread CPMMV.

Diagnosis can be difficult in cases of mix infection as we observed in this study.

Therefore, the application of next generation high throughput sequencing technology

is a powerful and sensitive tool to detect unsuspected or even unknown viruses

(Kreuze et al. 2009; Dutta et al. 2015; Adams et al. 2018; De Marchi et al. 2018).

Study of virus diseases that may affect soybean is important to keep up the

soybean yield and further studies are necessary to evaluate the impacts of the TSV in

soybeans in Brazil, once that there is not much is knowledge about its impact on

soybean productivity. This is the first complete genome determined of a Brazilian TSV

soybean isolate.

AKNOWLEGMENTS
This work was supported by grants received from Coordenação de Aperfeiçoamento

de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001 and FAPESP

(process number 2017/21588-7). F.B.S. is recipient of CNPq scholarship. R.K.S. is a

CNPq research fellow.

 55

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Tobacco Streak Ilarvirus by Different Thrips Species Using Virus-infected Pollen.
J Phytopathol 144:281–284
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discovery of novel viruses by deep sequencing of small RNAs : A generic
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 57

CAPÍTULO 3 - Uma ameaça oculta à soja: o carlavírus transmitido por mosca-

branca, da necrose à ausência de sintomas

Felipe Barreto da Silva, Cristiane Muller, Luís Fernando Maranho Watanabe,

Vinicius Henrique Bello, Eduardo Vicentin, Bruno Rossitto De Marchi, Lucia Madalena
Vivan, Marco Antonio Tamai, Juliano Ricardo Faria, Marcelo Agenor Pavan, Renate
Krause Sakate

Tipo de artigo: Notícia Técnica

Revista Cultivar Grandes Culturas

A cultura da soja é de grande importância a nível mundial para produção de

óleo e como fonte de proteína. O Brasil é o segundo maior produtor de soja no mundo,
ficando atrás apenas dos Estados Unidos. No Brasil, a área plantada de soja alcançou
35,1 milhões de hectares, com produção de 119,23 milhões de toneladas na safra
2017/2018.

Com esse aumento do cultivo, várias pragas e doenças se destacaram como

agentes preocupantes na produção de soja, incluindo a mosca-branca Bemisia tabaci
Gennadius (Hemiptera: Aleyrodidae). Bemisia tabaci é uma das espécies mais
invasivas e disseminadas a nível global, causando grandes prejuízos para diversas
espécies vegetais (Foto 1). Por sua polifagia, ou seja, por colonizar várias espécies
de plantas, pode estar presente durante o ano todo nos campos brasileiros.
58

Figura 1. Bemisia tabaci MEAM1.

Composta por um complexo de até 43 espécies crípticas morfologicamente

indistinguíveis, B. tabaci pode ser diferenciada apenas pelo uso da tecnologia
molecular, baseando-se em uma parte do genoma mitocondrial, denominado de
“mitochondrial cytochrome oxidase I” (mtCOI). Dentre as espécies crípticas, as que
ganham maior destaque são as denominadas Middle East-Asia Minor 1 (MEAM1 ou
biótipo B) e Mediterranean (MED ou biótipo Q).

No Brasil, a espécie MEAM1 (biótipo B) foi relatada pela primeira vez no início
da década de 90, no estado de São Paulo, associada a plantas ornamentais. A
espécie MED, por sua vez, foi relatada em 2014 no estado do Rio Grande do Sul em
pimentão e, posteriormente, nos estados de São Paulo e Paraná associada a plantas
ornamentais. A espécie MEAM1 é a predominante no país inteiro, enquanto que a
espécie MED restrita a algumas áreas e culturas nos estados do Rio Grande do Sul,
São Paulo, Paraná, Santa Catarina, Minas Gerais e Espírito Santo.
 59

Figura 2. Mapa de distribuição de Bemisia tabaci no Brasil.

(Fonte: [Link]

Além da preocupação com a disseminação do inseto, é importante lembrar que

a mosca-branca não causa apenas danos diretos nas culturas pela sucção de seiva
do floema. Na verdade, o grande destaque dessa praga está na capacidade de
transmissão de diversos vírus em culturas variadas, especialmente nas solanáceas e
fabáceas. Esse vetor é capaz de transmitir vírus dos gêneros Begomovirus,
Carlavirus, Crinivirus, Ipomovirus e Torradovirus.

Dos vírus transmitidos por mosca-branca destaca-se no feijoeiro o Bean golden

mosaic virus – BGMV, Begomovirus, causador do mosaico dourado do feijoeiro,
podendo causar perdas de 100% nas lavouras de feijão. Os sintomas ocasionados
pelo BGMV no feijoeiro incluem um forte amarelecimento e redução do crescimento
60

da planta. Outro vírus que vem merecendo destaque nestes últimos anos, é o Cowpea
mild mottle vírus (CPMMV), também transmitido por mosca-branca. O CPMMV
pertence ao gênero Carlavirus, Família Betaflexiviridae. O CPMMV foi descrito pela
primeira vez no país no final da década de 70, sendo denominado causador da doença
da mancha angular do feijoeiro Jalo.

O CPMMV também infecta a soja. Em soja, essa doença foi relatada no Brasil
apenas na safra de 2000/01 na região centro-oeste, quando produtores do estado de
Goiás observaram plantas com sintomas de nanismo, queima do broto e necrose da
haste e até mesmo morte das plantas (Prancha 1 A). Nas safras seguintes a doença
foi constatada em diversas regiões produtoras do país como: Tocantins, Pernambuco,
Maranhão, Bahia, Distrito Federal, Goiás, Mato Grosso, Mato Grosso do Sul, Minas
Gerais, São Paulo e Paraná.

Através do melhoramento da soja, a doença da necrose da haste causada pelo

CPMMV teve sua importância reduzida, uma vez que os genótipos que respondiam
com a necrose sistêmica foram substituídos por outros que não exibia esse sintoma.
Nos últimos anos, porém, sintomas diversificados, característicos de vírus em
cultivares de soja começaram a surgir nas principais regiões produtoras. Após
análises moleculares, foi detectado o CPMMV em diversas regiões do Brasil,
ocasionando sintomas muito diferentes daqueles relatados na literatura no passado.

O Grupo de Pesquisa em Mosca-Branca (GPMB) da Faculdade de Ciências

Agronômicas – UNESP, campus de Botucatu em conjunto com a Fundação MT,
Instituto Phytus, Universidade Estadual da Bahia e Corteva Agrisciences, têm
trabalhado com o CPMMV em soja e sinaliza: o CPMMV pode provocar perdas
significativas para a cultura da soja. Neste estudo diversos genótipos comerciais e
bastante utilizados nos estados de SP, MT, GO e BA foram avaliados quanto a
resposta à infecção com o CPMMV (Prancha 1) e geraram informações importantes
para a identificação e entendimento dessa ameaça no campo.

Pôde-se verificar sintomas de mosqueado, clorose, redução do crescimento da

planta, redução do número de vagens, redução da massa de grãos, sintomas esses
que vão influenciar diretamente a produtividade. As respostas das cultivares não
seguiram um padrão definido, e algumas cultivares de soja não mostraram sintomas
claros da infecção viral, ou seja, foram assintomáticas. Também foi verificado que
 61

plantas podem mostrar os sintomas de clorose e mosaico nas fases vegetativas e

início da reprodutiva, e os sintomas desaparecerem na fase reprodutiva podendo
facilmente serem confundidos com fitotoxicidade de produtos aplicados. Além disso,
as condições ambientais, estirpes de vírus e infecções mistas com outros vírus
implicam em variações dos sintomas causados pelo CPMMV, dificultando assim sua
correta identificação.

É interessante ressaltar que a ausência de sintomas visíveis do CPMMV nas

folhas de soja, não garante ausência de perdas de produção. Estudos realizados com
inoculação do vírus em condições de campo, demonstraram perdas de até 11 sacas
por hectare em uma das cultivares, na qual não havia sintomas visíveis da doença,
demonstrando que essa pode ser uma doença “silenciosa”.

Essas informações remetem a reflexão de que o produtor pode enfrentar

dificuldade de identificar a presença da doença e entender possíveis perdas, uma vez
que a “necrose da haste” observada nos estudos com cultivares da época, hoje não é
o principal sintoma apresentado nas cultivares atuais.

O aumento da ocorrência do CPMMV no campo está possivelmente

relacionado com aumento populacional de mosca-branca, devido às grandes áreas
de sucessão das safras de soja e feijão, e vice-versa, em diferentes regiões do país.
Durante o ciclo da cultura, veranicos podem, também, favorecer a explosão
populacional da praga. Além disso, ambas as culturas são excelentes hospedeiras do
inseto, além de ser hospedeiras do vírus. Dessa forma, as populações de mosca-
branca e do vírus podem ser mantidas no campo durante todo o ano em soja e feijão.

A transmissão do CPMMV pela mosca-branca é do tipo não-persistente, ou

seja, curtos períodos de alimentação do inseto em uma planta infectada já possibilitam
a aquisição do vírus, bem como a inoculação em plantas sadias. Portanto, tudo pode
ocorrer muito rápido no campo.

Além da soja e do feijão, o CPMMV infecta o amendoim (Arachis hypogea) e

diversas plantas daninhas, dentre elas: guanxuma (Sida sp.), Macropitilium spp.,
fedegoso (Senna spp.), carrapicho beiço-de-boi (Desmodium glabrum), feijãozinho-
bravo (Rhynchosia mínima), maravilha (Mirabilis jalapa), mussambe (Cleome affinis),
e erva-palha (Blainville arhomboidea) que podem servir de reservatório para o vírus
62

no campo. Além destas espécies, o CPMMV infecta, experimentalmente, outros

hospedeiros, como: quinoa (Chenopodium quinoa), C. amaranthicolor, fumo
(Nicotiniana spp.), chícharo (Lathyrus sativum), ervilha (Pisum sativum) e tremoço
(Lupinusalbus). Esse cenário de abundância de hospedeiros para o vírus e também
para o inseto remete à importância do manejo adequado da lavoura levando em
consideração as demais disciplinas além de entomologia.

Além da transmissão pela mosca-branca, o CPMMV também pode ser

transmitido por sementes de soja, como já relatado em Gana e na Índia. Estudos
realizados pelo grupo demonstraram que para pelo menos em um cultivar avaliado foi
verificada transmissão via semente de soja do CPMMV o que agrava a epidemiologia,
já que a infeção primária da lavoura pode ser via semente. O percentual foi baixo mas
merece esforços para melhor entendimento, e devido a isso novos estudos incluindo
um número maior de cultivares e novos parâmetros fisiológicos envolvidos estão em
fase de execução.

Por fim, esse conjunto de componentes - inseto vetor, vírus, hospedeiros

suscetíveis à doença, ambiente climático favorável para a presença do inseto e dos
hospedeiros – compõe um patossistema que requer uma abordagem interdisciplinar
para que propostas de manejo sejam efetivas. O controle de sanidade das sementes
é um critério fundamental para evitar a infecção primária no início do ciclo aliado é
claro a cultivares mais tolerantes. As medidas adotadas no cultivo já estabelecido
partem do manejo adequado de plantas daninhas para reduzir a fonte de inóculo do
vírus, bem como a abundância de hospedeiros alternativos que promovam um
aumento exponencial do vetor. Além disso, o controle do vetor se torna fundamental
para redução da disseminação da doença.

Embora a detecção do patógeno ainda seja possível somente com emprego de

ferramentas moleculares, o grupo está desenhando um mapeamento de ocorrência
da doença em diferentes regiões e ofertar informações sobre histórico de inóculo.

O manejo desse patossistema envolve integração de conhecimento e de áreas

de atuação e esse novo desafio para a sojicultura pode promover um entendimento
mais conectado de conhecimento e profissionais, desde a academia até a extensão,
para contribuirmos de maneira efetiva com nossos produtores.
 63

Prancha 1. Sintomas observados em cultivares de soja inoculadas com Cowpea

mild mottle virus (CPMMV). A. BRS 132. B. MM 6410 IPRO. C. TMG 7062 IPRO. D.
cv. não identificada coletada em Mori Mirim. E. M 8808 IPRO. F. MM 8349 IPRO. G.
M 7739 IPRO. H. M 9144 RR I. M 9144 RR. J. M 9144 RR. K. M 8372 IPRO. L. BMX
PORTÊNCIA RR.
64

CONSIDERAÇÕES FINAIS

O CPMMV é capaz de reduzir a produtividade da soja.

Os sintomas foliares do CPMMV em soja são variáveis de acordo com a

cultivar, podendo ser de clorose, bolhas, enrugamento ou até mesmo assintomáticos.

Os danos causados pelo CPMMV devem ser observados pela avaliação dos
aspectos agronômicos da cultura e não somente através dos sintomas foliares.

O TSV encontrado infectando soja no Brasil é filogeneticamente próximo ao

TSV da Austrália.
 65

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