Cómo las nuevas tecnologías están mejorando el
manejo integrado de virus transmitidos por insectos
Robert L. Gilbertson
Department of Plant Pathology
University of California-Davis
XV Internacional Congress of Integrated Pest Management:
An indispensable component for sustainable agriculture
Agosto 16-18, 2017
Enfermedades de plantas están
causadas por factores bióticos y
abióticos
•Agentes causantes de enfermedades
son bióticos
(e.g., hongos, viruses, bacterias,
fitoplasmas y nemátodos)
o abióticos (contaminación del aire,
daños por herbicidas y excesos y
deficiencias de agua y nutrición)
• Elementos genéticos parasíticos (AR N o
ADN)
cubiertos por una proteina protectora
• El patógeno mas importante de seres
humanos.
(Ebola, HIV, influenza, Zika); el segundo
mas importante patógeno de plantas
Viruses pueden aumentarse
sistmáticamente por la planta, encargarse
de la maquinaria celular y causar un rango
amplio de síntomas de enfermedades
• Latin word virus=poison
Viruses
Particulas de virus que atacan a
plantas (virions)
Síntomas de virus en plantas
Dispersión de viruses entre plantas
• Plantas no son móviles
• Viruses de plantas han
evolucionado maneras de mover entre
plantas
• La mayoría utilizan artropodos
(insectos) como vectores, tales como
áfidos, escarabajos, saltahojas, moscas
blancas y trips
• Otros vectores incluyen hongos, ácaros,
nemátodos y seres humanos
• Viruses pueden dispersarse via polen,
semillas o vegetativamente
Síntomas de infección por viruses
• Muchas tipos diferentes de síntomas
• El síntoma más común es crecimiento
reducido o distorcionado
• Síntomas más conspicuos son hojas
mosaicos o manchas
• Otros síntomas incluyen
hojas arrugadas o enrolladas,
hojas alargadas y delgadas
(shoestrings) y amarillamiento
• Síntomas en frutas incluyen
malformación, coloración por manchas,
manchas anilladas concéntricas
Una gran gama de viruses infeccionan a la plantas
• Hay una diversidad de formas y
tamaños de viruses
• La mayoría de tipos de viruses de
plantas tienen genomas (+) ssARN
• Pocos tipos tienen genomas
ss- o dsADN
• Genomas ADN son mucho más
comunes entre viruses de animals y
de bacterias
Otros factores que semejan
síntomas de viruses
• Factores bióticos
-Candidatus Liberibacter
• Factores abióticos
-Daño por herbicida
-Deficiencia nutricional
-Contaminación del aire
• Daño por insectos/ácaros
Virus emergentes
• Virus no son entidades estáticas
• Enfermedades virales emergentes: enfermedades
nuevas o previamente conocidas que se
convirtieron en económicamente importantes,
debido a un cambio en algún aspecto del virus,
vector, huésped y/o ambiente
• Mutación, reacondicionamiento, recombinación
conducen a la evolución y emergencia de
nuevos viruses
•Actividades humanas resultan en un movimiento
a larga distancia de virus existentes y sus vectores
Tomato yellow leaf curl virus
Squash vein yellowing virus
• Alerta de que el problema existe:
visitas regularmente al campo
•Adecuada identificación
• Recursos humanos
-Consultores profesionales de cultivo
(privado)
-Personal de extensión y Universidad
(público/gobierno/académico)
• Servicios de diagnóstico
-Clínicas de enfermedades de plantas
-Compañías privadas/organizaciones
• Materiales de referencia
-Guías de campo, compendio de enfermedades
-Recursos en línea
Diagnóstico de enfermedades de virus de plantas
El Modelo de California
Growers and/or professional crop consultants (PCAs)
University of California
Cooperative Extension
County-based Farm Advisors
University of California
Academic Research
Grower organizations:
California Melon
Research Board
Private companies
e.g., seed and chemical
companies and diagnostic
clinics
On-line resources Printed materials
Síntomas y presencia de insectos vectores es una clave
diagnóstica• Complejo de áfido-virus transmitido
-Potyviruses (many species)
-Cucumber mosaic virus (CMV)
-Alfalfa mosaic virus (AMV)
• Tospoviruses transmitidos por trips
-Tomato spotted wilt virus (TSWV)
-Tomato chlorotic spot virus (TCSV)
-Iris yellow spot virus (IYSV)
• Mtobamovirus transmitidos mecánicamente
-Pepper mild mottle virus
• Geminivirus transmitidos por moscablanca
-begomoviruses (muchas especies)
Aphid-transmitted
Whitefly-
transmitted
Thrips-transmitted
No vector-human activity
Diagnosis and identification of plant virus diseases
• Symptoms and presence of insect vectors
provide the first indication of a virus disease problem
• Transmission of the disease to healthy plants
(by mechanical inoculation, grafting or insects)
• Modern biotechnology-based methods are the most definitive
-Serological (antibody-based)
Enzyme-linked immunosorbent assay (ELISA)
Lateral flow devices
-Nucleic acid-based
DNA probes
Polymerase chain reaction (PCR) and DNA sequencing
-Rolling circle amplification (RCA)
-Next generation sequencing (NGS)
ELISA
• Prueba de laboratorio que requiere
un anticuerpo hacia el virus que se está
evluando y algún equipo
• Relativamente cara y require
personal entrenado
• Muestras infectadas por virus son
amarillas
• Mejor método para muchos virus de ARN
Lateral flow devices
(immunostrips) offer a rapid
in field diagnostic test for
some plant viruses
• Based on antibodies
• Solid matrix ELISA test
• Requires some idea of
the virus that might be involved
• Rapid and precise
• Easy to use
• No equipment needed
• Examples: Cucumber mosaic
virus (CMV), Tobacco/tomato
mosaic virus, Tomato spotted wilt
virus (TSWV)
DNA probes: Squash blot hybridization
to detect begomoviruses (‘old school’)
Prior to hybridization with a
begomovirus DNA probeAfter hybridization with a
begomovirus DNA probe
PCR/RT-PCR and quantitative PCR
• Test that amplifies viral genetic material (DNA or
RNA)
• Requires ‘primers’, virus-specific-
oligonucleotides, to initiate the reaction
• Primers are now available for many viruses
• Positive result is the amplification
of specific-sized virus DNA fragment
• Relatively expensive and requires trained
personnel, equipment and reagents
• Best method for DNA viruses
(geminiviruses) and many RNA viruses
• Quantitative PCR (qPCR) allows for
quantification
PCR detection of
Squash leaf curl virus
Real-time or qPCR allows for
quantification
New methods for sample preparation
•Allows for preparation of samples from
diverse geographical locations and conducting
PCR and RT-PCR tests at a distant lab
• Examples:
-Absorption strips (AgDia)
-FTA cards (Whatman)
Samples applied to
absorption strips
in Mali, West Africa
Samples applied to
FTA cards in the Dominican Republic
Rolling circle amplification (RCA) has revolutionized
detection of circular DNA viruses
• Utilizes the DNA polymerase of
bacteriophage Φ29
(infects Bacillus subtilis)
• Provides very efficient amplification of
circular ss- and ds-DNA
• First used for DNA viruses in 2004 with
human papillomaviruses and
geminiviruses
• In vitro systems use random primers, dNTPs,
Φ29 DNA polymerase and template
• Now widely used for characterization, detection
generation of infectious clones of geminiviruses
Next generation sequencing (NGS)
• Newer technology based on
high throughput DNA sequencing
• Provides a comprehensive picture
of the complete ‘virome’
•Allows for identification of diseases of
unknown etiology
• Highly technical and requires sophisticated
equipment, bioinformatics expertise and is
fairly expensive
• VirFind: an online tool for virus detection and
discovery from NGS sequences
NGS sequencing technologies
Bioinformatics
Next generation sequencing (NGS)-Viruses
• NGS commonly used for virus detection and
characterization
-Detection of new species and
strains of viruses and mixed infections
-Discovery of novel viruses
-Sequencing of complete viral genomes
-Epidemiological studies
-Viral evolution
• Cost going down, more bioinformatics resources
• What NGS does not do
-Biological studies and Koch’s postulates
-Which viruses are pathogenic and which are
cryptic
Geminiviruses
•A family of plant viruses (Geminiviridae)
characterized by having:
-Small twinned icosahedral virions (18 X 30 nm)
-Genome composed of one (monopartite)
or two (bipartite) ~2.6 kb circular
single-stranded (ss) DNA molecules
-Transmitted by whiteflies,
leafhoppers, treehoppers and aphids*
-Not seed-transmitted*
-Some are sap-transmissible
• Latin word geminus=twin
• Largest group of plant viruses (367!) species)
• Cause economically important diseases in a
wide range of crop plants
Twinned icosahedral virions
Circular ssDNA genome
Geminiviruses cause
numerous diseases of
economic importance
•Abutilon mosaic
•African cassava mosaic
• Maize streak
• Bean golden mosaic
• Beet curly top
• Tomato yellow leaf curl
Geminiviruses are a diverse group of viruses: four genera were
initially recognized in the family Geminiviridae based on genome
structure, insect vector and host
Genome
Vector
Host
Mastrevirus Curtovirus Begomovirus
Dicots
DicotsMonocots
A
Leafhoppers
Topocuvirus
Dicots
Treehopper
Leafhoppers
Whitefly
(B)
Rolling circle amplification (RCA)
revolutionizes detection
of circular DNA viruses
• Utilizes the DNA polymerase of
bacteriophage Φ29 (infects Bacillus subtilis)
• Provides very efficient amplification of
circular ss- and ds-DNA
• Commercial In vitro systems use random
primers, dNTPs, Φ29 DNA polymerase and
template
• Now widely used for diagnosis, detection
generation of infectious clones, etc.
Geminivirdae family
Genus Begomovirus Curtovirus Mastrevirus Topocuvirus Becurtovirus Eragrovirus Turncurtovirus Capulavirus Grablovirus Unassigned
Genome
Vector
Bemisia tabaci
Circulifer tenellus
Leafhoppers Treehopper Circulifer haematoceps
Circulifer haematoceps
Aphis craccivora
Spissistilus festinus
Host Dicot Dicot Monocot/Dicot
Dicot Dicot Eragrostis curvula
Dicot (Family: Brassicaceae)
Dicot Vitis vinifera
Distribution NW and OW NW and OW OW NW NW and OW OW OW OW NW
Number of species
322 3 32 1 2 1 1 4 1
Begomoviruses
• Whitefly (Bemisia tabaci)-
transmitted geminiviruses
• Recently (last 20-25 years) emerged
as one of the largest and most
economically important groups of
plant viruses (now 322 species)
• Cause severe diseases in
many dicotyledonous crop plants
(e.g., beans, cassava, cotton
cucurbits, okra, pepper,
tomato) in tropical and sub-
tropical regions of the world
Begomoviruses are
transmitted by Bemisia
tabaci whiteflies
Begomoviruses cause severe
disease symptoms
Differences Between Old vs. New World Begomoviruses
• Old World types
-Usually monopartite
-Not sap-transmissible
-Often cause leaf curl or
yellow leaf curl symptoms
-May be associated with satellite DNAs
• New World types
-Bipartite
-Some are sap-transmissible
-Symptoms are more variable:
mottle/mosaic, chlorosis,
purpling, crumple/curl/crinkle
-No satellite DNAs
• Effective resistance genes vary depending
on the virus and geographic location
IR
C2
C3
V1C4
C1
AC3
AC2
AC1
AV1
(CP)DNA- A
CRAC4
BC1
BV1DNA- B
CR
TYLCV is an exotic invasive virus
spread by an exotic invasive whitefly
• Tomato yellow leaf curl virus (TYLCV) was
first described in the Middle East around 1940
• Causes tomato yellow leaf curl disease
• Vectored by invasive Bemisia tabaci biotype
B (Middle East-Asia Minor 1 [MEAM1])
• Introduced into the New World (NW) in the
early 1990s in the Dominican Republic
• Has spread extensively, e.g., Caribbean Basin,
USA (SE states; Arizona, Texas and California;
and Hawaii), Mexico, Guatemala, and South
America (Venezuela)
IR
C2
C3
V1C4
C1
TYLCV has a
monopartite genome
Emergence of indigenous New World
monopartite begomoviruses
• In the mid-1990s a new leaf curl disease of
tomato appeared in the Ica Valley of Peru
•Associated with high populations of the invasive
whitefly, Bemisia tabaci biotype B (MEAM1)
• Begomovirus DNA detected by NASH and PCR
•A new bipartite begomovirus or the further
spread of the invasive TYLCV? Ica Valley of Peru
Emergence of indigenous New World
monopartite begomoviruses
• Restriction digestion of RCA products
indicated a single genomic DNA and no
DNA-B detected
• Most similar to DNA-A components
of NW bipartite begomoviruses
• Named Tomato leaf deformation virus (ToLDeV)
• PA98-1 and PA98-2 variants (1998)
and PA10 strain (2010) identified
• Variation in the IR/C4 ‘hot-spot’
~2.6 kb
Msp I digest
0.7 kb
RCA/RFLP
How to prove ToLDeV is a monopartite begomovirus?
• Agroinoculation, an insect vector-independent inoculation method
• Insert the cloned geminivirus genome
into a disarmed Ti-plasmid and
introduce into Agrobacterium tumefaciens
• Multimeric forms facilitate release
• Inoculate plants with syringe or infiltration
• Cells around the inoculated area
become transformed and the viral
DNA escapes from the integrated DNA
and spreads systemically
Emergence of indigenous New World
monopartite begomoviruses
• Cloned genomic ToLDeV
DNA agroinoculated into
tomato and Nicotiana
benthamiana caused leaf
curl symptoms
• Symptoms in tomato were
identical to those in the field
• Fulfilled Koch’s postulates
• Differential pathogenicity
among variants/strains
was correlated with the
IR/C4 sequence
Mock PA98-1 PA98-2 PA10-3
Beet curly top virus (BCTV) is the
type member of the genus Curtovirus
• BCTV has a very wide host range, including crops
and weeds
• Occurs in the NW and OW, especially in the
western USA and north-central Mexico
• BCTV causes curly top disease of tomato
• Curly top disease transmitted by
the beet leafhopper, Circulifer tenellus, in a
persistent circulative manner
• No curly top-resistant tomato
varieties
BCTV virions
Curly top disease of tomato
• In tomato, plants show stunted
growth and upcurled leaves with
dull green-yellow color and
purpling of the veins
• Plants infected at a young age may die
• Plants infected later are stunted with
yellow upcurled leaves with purple veins
• Fruits are small and ripen prematurely
Curly top symptoms: Tomato
Progress in developing tomato varieties with resistance to
TYLCV and other begomoviruses
• Resistance to TYLCV and other tomato-infecting
begomoviruses has been identified and introgressed from
wild tomato species including:
-Ty-1 (Solanum chilense)
-Ty-2 (S. habrochaites)
-Ty-3 (S. chilense)
-Ty-4 (S. chilense)
-Ty-5 (S. peruvianum)
-Ty-6 (S. chilense)
• Will these genes confer resistance to BCTV?
1st
Tomato
2nd
3rd
BCTV agroinoculation system developed for screening tomato
varieties and germplasm for resistance to BCTV
48 hr old liquid culture of
Agrobacterium tumefaciens
containing the viral DNA
BCTVObserve symptom development
Check for virus infection
(by PCR)
• Rapid
• Large numbers
• Highly efficient
• Uniform
• Easy to grow and
maintain
ControlLine 13 (1.2)Line 9 (3.7)
Curly top resistance identified in breeding lines with
Ty genes from the World Vegetable Center
NGS-Case Study-Background
• Sinop - Mato Grosso Brazil – 2016
• Tomato plants showing geminivirus-like symptoms: stunting,
upward leaf curling and purpling of veins and leaves
Is it the elusive Brazilian Curly Top?-Described in 1940s affecting sugarbeets and tomatoes
-Vectored by a leafhopper (Agallia albidula)-Etiology remains unknown
Initial tests:• Dellaporta total DNA extraction PCR:
• Begomovirus degenerate primers• Curtovirus degenerate primers
NEGATIVE
And now, what should we do? NGS
Symptoms
NGS Results
Virus Species Identified Length Mapped Reads
Tomato chlorosis virus RNA 1, complete genome 8539 bp 12,055
Tomato chlorosis virus RNA 2, complete genome 8225 bp 28,816
Novel geminivirus (closest to French bean severe leaf curl virus,a species of the genus Capulovirus!)
2235 bp 117,798
Cloning
ExtractDNA
RCA Restriction Enzyme Digestion:• Eco RI• Kpn I• Spe I
Ligation to pSL1180Transformation in Escherichia coli DH5α
Clone screening
Sequencing
Based on
sequence
generated by
NGS
EcoRI KpnI SpeI
Conclusiones
CP Tomato pseudo-curly top virus 34%
Rep French bean severe leaf curl virus 60%
C3 Alfalfa leaf curl virus 50%
-¿Un Nuevo género en la familia Geminiviridae ?-¿La elusive causa de la enfermedad de la puntaacolochada (curly top) de Brasil?-¿Qué hacer después?
• Combina multiples estrategias de manejo
seleccionadas en base al conocimiento
del virus
• Meta: emanejo eficiente con
mínimas aplicaciones de pesticidas
• Tres pasos básicos:
1. Correcta ID del patógeno
2. Entender la biología del patógeno/
epidemiología de la enfermedad
3. Desarrollar y evaluar la estrategia de manejo
El Manejo Integrado de Plagas (IPM) de
virus de plantas
Entendimiento de la biología del virus es necesaria
para el efectivo manejo de la enfermedad
• Propiedades del virus (estructura del virión y
• material genético
• Rango de hospederos
• Vector (insecto u otro)
• Fuentes de inóculo
• -semilla
• -malezas/otros hospederos
-cultivos viejos
-insectos
• Medios de sobrevivencia en ausencia de los hospederos
•económicos
• Potencial para pérdidas, qué tan preocupado debe estar?
• Seleccione las estrategias de manejo apropiadas
basadas en el conocimiento de la biología del virus
-regulatorias (no introducer patógenos exóticos en/adentro
de semillas y transplantes)
-avoidance (field location, planting dates)
-disease resistance
(conventional and transgenic)
-pathogen-free propagative materials
(seeds and transplants)
-protection
(screenhouses, greenhouses, row covers)
-disease monitoring and forecasting
-vector management (insecticides)
-removal of diseased plants (roguing)
-sanitation (destroy harvested crops, weeds, volunteers)
-crop rotation
-host-free periods
Desarrolle un programa IPM
IPM for insect-transmitted viruses
•Antes de la estación de crecimiento
-Selección de las mejores variedades
(alta producción, Resistencia a la
enfermedad)
-Uso semillas de alta calidad y libre de
patógenos.
(semilla probada libre de virus)
-Producir transplantes libres de
insectos y patógenos
-Tratar pilones con sinsecticidas
sistémicos para manejar a los insectos
vectores (áfidos, trips y moscablanca)
después de transplante
IPM para insectos transmisores
de viruses• Durante la estación de crecimiento
-Plante immediatamente después del
período libre o del invierno
-No not plante cerca de campos de cultivo
viejo ya establecidos
-Row covers
-Monitoree/maneje los insectos vectores
-neonicotinoides
-reguladores del crecimiento del insecto
(IGRs)
-insecticidas de contacto u
-nuevas químicas (e.g., cyazypyr
[Verimark])
-Elimine plantas con síntomas
IPM para insectos transmisores de viruses
• Después de la estación de crecimiento
-Sanidad
remueva y destruya el cultivo
viejo/voluntarias
(arando/remoción física)
-Manejo de malezas
(alrededor de campos)
-Período libre de hospederos regional
-Unoe-tres meses
-Refuerzo voluntario o enforzado
• Virus de plantas están constantemente cambiando y respondiendo a
presión de selección (esfuerzos humanos de manejo)
•Actividades humanas (comercio global) están facilitando ldispersión
de viruses de plantas e insectos vectores a larga-distancia
• Nuevas tecnologías permiten una ID rápida y precisa e intervención
temprana
• Métodos de inoculación independiente del vector facilitan la
búsqueda de germoplasma para resistencia
• El manejo efectivo se basa en el conocimiento de la biología del virus
y el desarrollo e implementación de una estrategia IPM
Conclusiones
People-Maria Rojas
-Monica Macedo
-Ozgur Batuman
-Tatsuya Kon
-Tomas Melgarejo
-Juliana Osse de Souza
-Alice Nagata
Support
-USAID IPM Innovation Lab
-Transagricola SA
-California Tomato Research Institute
-California Dept. Food and Agriculture
-CNPq (Brazil)
¡Muchas gracias para su atención!
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