citrus leprosis: a complex and multietiologic...

Pedro Luis Ramos-González

Instituto Biológico de São [email protected]

Citrus leprosis: a

complex and

multietiologic disease

1907

Citrus leprosis:

Florida, USA

historical review and distribution

Rusty nail head

1907 201720111920-30 20041960 1999

Central America

Florida, USA

Mexico

Hawaii, USA

Paraguay, Argentina, Brazil Venezuela, Colombia

Citrus leprosis:historical review and distribution

2000

Viral particles

Mites (genus Brevipalpus)Frezzi 1940 & Vergani 1945

Kitajima et al. 1972

Viral genomes

Locali et al. 2006

Citrus leprosis: serious viral disease

São Paulo, 2017. Pictures by Daniel Junior de Andrade

1990:

control of vector mite costs

Brazilian growers around US$ 80

million

(Bastianel et al., 2010)

2010:

10-20% cost of consumables

5-10% total expenditures

(Fundecitrus, citrus grower, pers. comm.)

Mite preference fruits>stems>leaves

Local necrotic or chlorotic lesions

Citrus leprosis: symptoms

Citrus leprosis: two disease types

C. sinensis. Enveloped-short bacilliform virions (50-60 nm X 110-120 nm).

(Kitajima et al., 1974).

Cytoplasmic type

C. sinensis. Rodlike virions (40-50 nm x 100-110 nm) in vesicles, in the

nucleus and cytoplasm. Membrane-associated virions.

(Kitajima et al., 1972).

Nuclear type

C. volkameriana. Short, bacilliform virions of ≈30 x 50 nm.

Cytoplasm of symptomatic leaf tissues

(Melzer et al., 2012).

Family: Rhabdoviridae

Order: Mononegavirales

Genus: Dichorhavirus

Citrus leprosis-Nuclear Citrus leprosis-Cytoplasmic

• Single strand

• Negative sense RNA

• Bipartite genome

• Transmitted by Brevipalpus

• mites

(Dietzgen et al., 2014)

(Afonso et al., 2016)

Genus: Cilevirus Genus: Higrevirus

• Single strand

• Positive sense RNA

(Locali et al., 2012) (Melzer et al., 2013)

• Bipartite genome

• Transmitted by Brevipalpusmites

• Tripartite genome

• Transmission not

confirmed

• Floating genera

Citrus leprosis: viral diversity

Citrus leprosis Nuclear type

Country Virus Ref.

Mexico OFV-citrus(orchid fleck virus)

Roy et al., 2013

Cruz-Jaramillo et al., 2014

Colombia OFV-citrus Roy et al., 2014

Florida,

USA*

Partial sequence80% of nt identity

> 92% aa identity

OFV-citrus

Hartung et al., 2015

Panama ? Dominguez et al., 2001

Brazil CiLV-N and CiCSV(citrus leprosis virus N, citrus chlorotic spot virus

Kitajima et al., 1972., Ramos-

González et al., 2017; Chabi et al., submitted.


*Knorr, 1948

61% of the genomeSmall RNA fraction

N P MP M G

L

RNA16.7 Kb

RNA26.0 Kb

3´ 5´

3´ 5´

51 < nt < 63

44 < aa < 64

CiCSV

CiLV-N

Citrus leprosis Nuclear type in Brazil

N P MP M G

L

RNA16.5 Kb

RNA25.9 Kb

3´ 5´

3´ 5´

Four isolates, one speciesRamos-González et al., 2017; Chabi et al., submitted.

*

• State of São Paulo

• Four isolates from four counties (20-170 km)

• Altitude: > 800 m

• Average annual temperature: 18-20 °C, including frost

• Small necrotic and chlorotic spots

• Sweet oranges

• State of Piauí

• Two isolates from an urban zone / Teresina

• Altitude: 72 m

• Average annual temperature: 27.1 °C

• Large chlorotic spots

• Sweet oranges



Brevipalpus n. yothersi

Brevipalpus phoenicis ss

• Only association

• Confirmed viral

transmission to

healthy plants

(Beard et al. 2015)dorsal ventral microplates spermateca

Ramos-González et al., 2017; Chabi et al., submitted

Citrus leprosis Cytoplasmic type

1967 2006

Diversity and

variability studies

in BrazilBrazil

Panama


Small RNA fraction

CiLV-C 99%

2015

Locali et al., 2006Pascon et al., 2006

Country Virus Ref.

Mexico to

Argentina

CiLV-C

(citrus leprosis virus C)Bastianel et al., 2010

Colombia CiLV-C2 strain Co Roy et al., 2013

Argentina


CiLV-C2 ORF p29 (Roy et al., 2013)

OFV-citrus ORF M (Roy et al., 2014)

CiLV-C ORF mp (Locali et al., 2003)

CiLV-C ORF p29 (Pereira et al. 2012)

cDNA-PCR

51 samples BZ, MX, CO, PA, PY, AR

CiLV-C(Locali, et al., 2006)

mpp29

Clade SJPClade CRD

Citrus leprosis virus C: variability

Ramos-González et al., 2016

B. yothersiA1 isoline

siRNA

sequencing

Complete

genome

strain SJP

Nucleotide identity

> 99%

RNA1

RNA2

RNA1

RNA2

C. sinensis

A. thalianaIsolate SJP01

Putative recombination

Strains

SJP-CRD

~ 85% nt

identity

Citrus leprosis virus C: strain detection

Strain-specific RT-PCR

Ramos-González et al., 2016Mixed infections of CiLV-C strains detected.

Relevance:

• Support for the putative RNA2

recombination predicted in silico

• Reassorment between viral RNA

components, synergism, trans-

complementation...

• New biological treats

Citrus leprosis cytoplasmic type

CiLV-C strain distributionB. yothersi variability

Sánchez-Velázquez et al., 2015

Haplotype diversity:

COI sequence

CiLV-C / vector relationship

Tassi et al., 2017

larva protonymph deutonymph female male

adult

Ca. Cardinium

AAP 4 h

IAP 2 h

Latent period 7 h

Retention period 12 d

Viruliferous

mites / colony25-60%

Brevipalpus yothersi(ex- B. phoenicis)

• Non-transovarial transmission of CiLV-C

Ph

as

eo

lus

vu

lga

ris

Citrus leprosis / vector relationship

B. yothersi

Circulative non-propagative


CiLV-C

• No viruses

inside

mite’s cells

• Detection of

Viral RNA

negative strand

in mites

Roy et al., 2015Kitajima & Alberti,

2014

≠

Citrus leprosis / vector relationship

B. yothersi

Circulative non-propagative

Citrus leprosis Cytoplasmic type Citrus leprosis Nuclear type

Circulative propagative

B. phoenicis B. californicus

CiLV-C CiLV-N OFV

• No viruses

inside

mite’s cells

• Detection of

Viral RNA

negative strand

in mites

• Viruses inside

mite’s cells

• Long latent

periods

Roy et al., 2015Kitajima & Alberti,

2014

≠

Roy et al., 2015

B. yothersiA1 isoline

After 7-10 days

A. thaliana Col-0 Arena et al., 2015

CiLV-C / plant interaction - Mite / plant interaction

ROS

productionDAB stain

6 hai 12 hai 24 hai 8 dai

Cell

deathTrypan blue stain

Arena et al., 2016

RT-qPCR

Marker genes:

SA: ICS1, EDS5, NPR1, TGA3, WRKY70, PR1, PR5

JA/ET: JAR1, ETR1, EIN2, MYC2, VSP2, PDF1,2

Gene silencing: RDR1, RDR6, DCL2, DCL4,

AGO1, AGO2, HEN1

ROS and cell death: NHL10, RBOHD

Reference genes: F-box and SAND

Mock Mites Mites + CiLV-C

CiLV-C / plant interaction - Mite / plant interaction

Arena et al., 2016

• Gene silencing

• Host manipulation by mites

• VOC emission

• Metabolite quantity and

quality changes

• Virus as an effector

• Role of each viral ORF

Working hypothesis:

CiLV-C / mite / plant interaction

p < 0.05 (*) or < 0.01 (**)

Primarily

infested leaves

Arena et al., 2016

n= 26 plants (13 infested with

non-viruliferous and 13 with

viruliferous mites).

5 mites/leaf

3 leaves/plant

Mite count: 13 dpi

Secondary

infested leaves

Citrus leprosis

CiLV-C2OFV-

citrusCiLV-N CiCSV

B. yothersi B. papayensis B. californicus B. phoenicis

Cytoplasmic type Nuclear type

CiLV-C

B. n. yothersi

Sweet oranges,

tangerines, Swinglea glutinosa, Commelina

benghalensis

HGSV2

Sweet oranges,

Hibiscus sp. Swinglea glutinosa, Dieffenbachia sp.

Sweet oranges Sweet oranges

Sweet oranges,

grapefruit, lemon, lime,

mandarin, sour orange,

sweet or navel lime, and

Persian lime

C.volkameriana, Hibiscus sp.

Camila Chabi Gabriela Arena Juliana Freitas-

Astúa

Elliot W. KitajimaAline Tassi

CollaboratorsRenato Bassanezi - Fundecitrus

Daniel Junior de Andrade - UNESP / Jaboticabal

Ricardo Harakava - IB

Valdenice Morerira - CCSM

Marines Bastianel - CCSM

Marcos Machado -CCSM

Mariani Rodrigues

Mateo Postcam

Citrus leprosis management

• Reduction of the inoculum source: pruning

• Use of mite- and virus-free seedlings

• Avoiding the presence of alternative virus

hosts in fields

• Control of viruliferous mite populations by

using acaricides

Effect of gene silencing during CiLV-C infection in Arabidopsis

n= 15 plants per mutant genotype

Three independent experiments

5 mites/leaf

3 leaves/plant

0

20

40

60

80

100

120

5 6 7 8 12

Pe

rce

nta

ge

of

sy

mp

tom

ati

cle

av

es

dcl2/4 rdr2/6 wt

dpi

dcl2/4 rdr2/6 wt

Genotype

Ro

se

tte

lea

ve

s(1

0 d

pi)

*

*

0

0,5

1

1,5

2

2,5

wild type dcl2/4 rdr2/6

Re

lati

ve

vira

l ti

ters

Inoculated leaves

Systemic leaves

Non symptoms

observed neither

viral RNA detected

by RT-qPCR

Ca

uli

ne

lea

ve

s(1

5 d

pi)


Total RNA

NGS/bioinformatics

RACE/Secondary re-

sequencing

N*

*

N*

*

I- CiLV-C (MP gene)

II- CiLV-C2 (p29 gene)

III-OFV-citrus (N gene)

IV- OFV (N gene),

VI- degenerate primer pair

for the detection of L gene

Nucleorhabdovirus CytorhabdovirusDichorhavirus

In summary: viruses associated to Citrus leprosis

Protein L

Cytoplasmic type Nuclear type

Cilevirus

Tobamovirus

Negevirus

Blunervirus

Tobravirus

Virgaviridae

Higrevirus

Closteroviridae

RdRP2 (motif)

VTB Sequência dos iniciadores

CiLV-CMP-F: GCGTATTGGCGTTGGATTTCTGAC

MP-R: TGTATACCAAGCCGCCTGTGAACT

CiLV-C CRDp29-F: CAGAAGGCCGAGGTTGTAAAG

p29-R: GTAGTGATCACTGAACTCGAATACC

CiLV-C SJPp29-F: GTAARCAAAAGGTCGAGGTTGTCC

p29-R: TCTGTTGTCTAGCAGCRAGTAATG

CiLV-C2p29-F: ATGAGTAACATTGTGTCGTTTTCGTTGT

p29-R: TCACTCTTCCTGTTCATCAACCTGTT

PfGSV

MP-F: CGATATTTGATCAATCCGTT

MP-R: CACCTTAAAATTCGAGGGTT

RpdR-F : CTGTTGTGCCAAATCATCAA

RpdR R : TTCATCGCAAGTTCATATACCT

SvRSVRpdR-F: TGTCGAACTTTGGTATGAGTCG

RpdR R:CCGGTTCGTCAAATAACTCC

OFV-citrusN-F: ATGGCTAACCCAAGTGAGATCGATTA

N-R: AGTTGCCTTGAGATCATCACATTGGT

OFVN-F: TGTCATAGCCGACATAAACACC

N-R: TGTAGAGCTTGCGAGATACAG

ClCSVL-F: ATATCACCGTTTAAGCAAGC

L-R: TCCTTGTTACAACTCCTTGC

Diagnostic tools: PCR primers

Cileviruses: diversity

Cilevirus

Tobamovirus

Negevirus

Floating genera

Blunervirus

Tobravirus

Virgaviridae

Higrevirus

ClosteroviridaeRdRp2 (protein motif)

citrus leprosis: a complex and multietiologic...

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