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!