Citrus Virus Diseases

Bill Dawson

All graft-transmissible diseases initially were considered to be caused by viruses

Examples of changes Viroids citrus exocortis cachexia xyloporosis

Phytoplasmas, Spiroplasmas stubborn new phytoplasma in Brazil that causes greening symptoms Bacteria greening citrus varigated chlorosis

http://www.apsnet.org/online/common/names/citrus.asp
VIRAL DISEASES Citrus mosaic Satsuma dwarf-related virus Bud union crease Virus for some combinations, otherwise genetic or unknown Citrus leaf rugose genus Ilarvirus, Citrus leaf rugose virus (CLRV) Citrus yellow mosaic genus Badnavirus Crinkly leaf Crinkly leaf ilarvirus (strain of citrus variegation virus) Infectious variegation genus Ilarvirus, Citrus variegation virus (CVV) Navel infectious mottling Satsuma dwarf-related virus Psorosis Spirovirus (ophiovirus) proposed new group Satsuma dwarf Satsuma dwarf virus (SDV) Tatter leaf = citrange stunt genus Capillovirus, Citrus tatter leaf virus (probably a closely related strain of apple stem grooving capillovirus rather than a distinct virus Tristeza = decline and stem pitting, seedling yellows genus Closterovirus, Citrus tristeza virus (CTV)

VIROIDS AND GRAFT-TRANSMISSIBLE PATHOGENS (GTP) Algerian navel orange virus Impietratura GTP GTP Blight = young tree decline, rough lemon decline Indian citrus ringspot GTP GTP Blind pocket Leaf curl GTP GTP Cachexia Leathery leaf Citrus cachexia viroid (Hostuviroid) GTP Chlorotic dwarf Leprosis White-fly transmitted GTP GTP associated with Brevipalpus spp. mites Citrus dwarfing Measls Varios viroids GTP Citrus yellow mottle Milam stem-pitting GTP GTP Citrus yellow ringspot Multiple sprouting disease GTP GTP Concave gum Nagami kumquat disease GTP GTP Cristacortis Ringspot diseases GTP Various GTPs Exocortis Vein enation = woody gall Citrus exocortis viroid (CEVd) (Pospiviroid) GTP (possible luteovirus) Fatal yellows Xyloporosis = cachexia GTP Citrus cachexia viroid (Hostuviroid) Gummy bark Yellow vein GTP, possible viroid GTP Gum pocket and gummy pittings Yellow vein clearing of lemon GTP, possible viroid GTP

Edited by
J.O. Whiteside, S.M. Garnsey, and L.W. Timmer

APS Press
The American Phytopathological Society 1988

Endemic Viruses of Concern

Tatterleaf / Citrange Stunt Infectious Variegation Citrus Leaf Rugose Psorosis complex Citrus leaf blotch Citrus tristeza decline

Tatter Leaf and Citrange Stunt virus complex

latent infections in Meyer lemons apparently wide spread in China Host Range and Symptoms when infected cultivars are propagated on trifoliate or hybrids (Swingle citrumelo, Carrizo, Troyer, and Kuharske citrange), trees are stunted and chlorotic, virus-induced incompability can infect a wide range of citrus cultivars causes mild symptoms if not on trifoliate rootstock can infect a wild range of herbaceous hosts

Tatter Leaf and Citrange Stunt virus complex continued Causal Agent one positive-sense RNA two subgenomic RNAs
ORF 1 CP

A(n) A(n) A(n)

ORF 2

Transmission and Epidemiology graft transmitted by infected budwood mechanically transmitted to a range of hosts thought to be aphid transmitted?

Identification characteristic bud union creasing symptoms on herbaceous hosts

Control virus-free budwood, difficult to remove decontaminate tools, knives use rootstocks other than Poncirus hybrids

Wild type virus infected (8 dpi)

Wild type virus infected (15 dpi)

Healthy

Infectious Variegation Citrus Variegation Virus (CVV)

Host Range and Symptoms can infect most citrus species lemons, sour orange, citron, and grapefruit develop chlorotic leaf symptoms and distortion mild strains exist that cause only mild mottle or chlorotic flecking trees can be stunted, some fruit may be distorted symptoms are less in warm weather can be mechanically transmitted to some cowpeas (chlorotic or necrotic lesions and beans (chlorotic mottle or veinbanding)

Infectious Variegation Citrus Variegation Virus (CVV) continued

Causal Agent ilarvirus 4 positive-sense RNAs, sperical particles Citrus crinkly leaf virus is a mild variant Transmission and Epidemiology grafting budwood mechanically inoculate seed transmitted apparently rare pollen transmission? Identification indexing by graft inoculation to lemon or citron mechanical inoculation to cowpea or bean Control infected budwood can be freed by thermotherapy or shoot tip grafting then use virus-free budwood

Leaf Rugose Citrus leaf rugose virus (CLRV)

Host Range and Symptoms infects a broad range of citrus hosts Mexican lime, Eureka lemon, and Duncan grapefruit develop leaf puckering, pinpoint chlorotic spotting, and stunting can be mechanically transmitted to a range of noncitrus hosts

Leaf Rugose Citrus leaf rugose virus (CLRV) continued

Causal agent ilarvirus 4 positive-sense RNAs, isometric particles Transmission and Epidemiology grafting and mechanical transmission no vector known, but get some local spread pollen? Identification indexing by grafting Eureka lemon or bush bean serology Control use virus-free budwood decontamination of pruning tools

Psorosis (psorosis A, mild; psorosis B, severe; concave gum; )

probably a complex of several diseases, which share common symptoms usually found present in old-line trees often bark scaling Host Range and Symptoms symptoms stronger on young leaves ranging from chlorotic flecks, to leaf mottling and chlorotic patterns scaling and flaking of bark on the trunk and limbs of sweet orange and grapefruit and occasionally mandarins -- usually not on other citrus wood becomes impregnated with gum concave gum thought to be related, mandarins and sweet oranges are affected, deep concavities in the trunk and main branches

Concave Gum

Psorosis

Psorosis (psorosis A, mild; psorosis B, severe) continued Causal Agent

Transmission and Epidemiology graft transmitted seed transmission rare apparently some unknown vector Identification field symptoms of bark scaling indexing by grafting to sweet orange or mandarin seedlings for leaf-flecking Control use virus-free budwood, use seed only from virus-free trees can use shoot-tip grafting or thermotherapy decontaminate tools

Citrus ringspot virus (CRSV) Psorosis B

Host Range and Symptoms irregular spots or large, irregular chlorotic patterns on mature leaves frequently gum impregnated and are slightly raised on the lower surface fruit may have ringspots some isolates are mechanically transmitted to Chenopodium spp

Citrus ringspot virus (CRSV) Psorosis B continued Causal Agent ophiovirus, 3 negative-sense RNAs very thin thread-like particles next slide has genome description Transmission and Epidemiology graft transmissible mechanical transmissible vector suspected but unknown Control virus-free budwood decontamination of tools

The complete nucleotide sequence of a Spanish isolate of Citrus psorosis virus: comparative analysis with other ophioviruses Brief Report S. Martn1, C. Lopez2, M. L. Garca3, G. NaumOngana3, O. Grau3, R. Flores2, P. Moreno1, and J. Guerri1

Arch Virol (2005) 150: 167176

Association of citrus psorosis B symptoms with a sequence variant of the Citrus psorosis virus RNA 2 Velazquez, K (Velazquez, K.)1; Pina, JA (Pina, J. A.)1; Navarro, L (Navarro, L.)1; Moreno, P (Moreno, P.)1; Guerri, J (Guerri, J.) PLANT PATHOLOGY 61: 448-456

The virus induces two different syndromes: psorosis A (PsA), characterized by limited bark scaling lesions in the trunk and main limbs, and a more aggressive form of the disease called psorosis B (PsB) with rampant bark lesions affecting even thin branches and chlorotic blotches in old leaves. In the greenhouse, the PsA and PsB syndromes can be induced by graft inoculating healthy citrus seedlings with non-lesion or with lesion bark inoculum from PsA-affected field trees. PsA- and PsB-inducing CPsV sub-isolates obtained by this procedure from the same tree showed segments of the RNA 2 enabled discrimination between PsA- and PsB-associated sequence variants. (i) PsA-inducing isolates contain PsB-associated sequence variants at low frequency, (ii) the PsB-associated sequence variant is predominant in blistered twigs and gummy pustules affecting old leaves, characteristic of PsB isolates, and (iii) the PsB-associated sequence variant accumulates preferentially in bark lesions of the trunk and limbs.

Citrus leaf blotch virus (CLBV)

Host Range and Symptoms first found in kumquat on Troyer citrange rootstock with bud union crease Spain, France, Japan, Australia, Florida associated with abnormal bud unions on poncirus hybrid rootstocks Causal Agent filamentous particles one positive-stranded RNA with 2 subgenomics p41 p227 p40

Poly-A Poly-A

Transmission and Epidemiology no known vector seed transmitted Control virus-free budwood alternative rootstocks if necessary

Poly-A

Citrus tristeza virus

Causes four disease syndromes: decline only on sour orange rootstocks can be quick decline can be slow seedling yellows -- usually seen in greenhouse on sour orange and grapefruit no disease mild strains stem pitting on all rootstocks

Department of Plant Industry Budwood certification Dr. Peggy Sieburth Winter Haven Budwood trees tested: Citrus tristeza virus decline isolate (MCA-13 positive) tested yearly OK to have budwood with the mild isolate of CTV Psorosis and Concave gum tested once by biological indexing now starting to test every 6 years Citrus leaf blotch virus (Dweet mottle virus) once every 6 years in budwood source trees and also in seed source trees since it is seed transmitted Citrus tatter leaf virus will start test for next year Infectious variegation virus is not tested for because it is mainly a problem for lemons which are limited in Florida Also test for viroids I, II, III, IV, V, and CEV every 6 years

EXOTIC VIRUSES

Exotic Viruses of Concern

Citrus chlorotic dwarf (CCD) Citrus yellow mosaic Leprosis Satsuma Dwarf Tristeza stem pitting Vein enation Sudden death

Vein Enation (Woody Gall)

important commercially only when severe galling occurs on young trees budded to rough lemon rootstocks Host Range and Symptoms enations on the veins of Mexican lime, sour orange, and rough lemon swelling or galls on the stems of rough lemon and Mexican lime

Vein Enation (Woody Gall) continued

Casual Agent spherical viruslike particles

Transmission grafting aphid transmitted Myzus persicae, Toxoptera, citricida, and Aphis gossipii not mechanically transmitted
Control clean budwood avoid use of rough lemon rootstocks

Leprosis and Zonate Chlorosis

Host Range and Symptoms observed primarily on sweet orange sour orange and mandarins are also show symptoms other citrus normally do not show symptoms symptoms are chlorotic lesions, sometimes with a necrotic center later lesions become flat or raised necrotic areas leaves and fruit abscise when lesions are abundant, can get twig dieback can mechanically transmit to Chenopodium spp

Leprosis

Leprosis and Zonate Chlorosis continued Causal Agent controversy may be two different viruses causing similar diseases rare nuclear form that is rod shaped and more prevalent cytoplasmic form of baciliform particles would be expected to be a rhabdovirus positive-sense RNA virus with 2 RNAs virus is a mite virus Brevipalpus spp. virus multiplies in mites

Transmission and Epidemiology transmitted by mites -- millions of individual infections graft transmitted with difficulty virus does not move long distances, systemically Control eliminate sources of inoculum control mites, acaricides to reduce mite population

Satsuma dwarf virus (SDV)

Host Range and Symptoms wide host range in citrus widely spread in Japan stunting and narrow, boat- or spoon-shaped leaves in satsuma mandarin Chenopodium, cowpea, bean, Physalis, Gomphrena

Satsuma dwarf virus (SDV) continued

Causal Agent small isometric virus with 2 RNAs genome on next slide Transmission and Epidemiology graft transmitted by infected budwood mechanically transmitted to herbaceous hosts local spread, maybe a soilborne vector Control virus-free budwood

Nucleotide sequences and taxonomy of satsuma dwarf virus Toru Iwanami,1 Yoshiko Kondo1 and Alexander V. Karasev2 J Gen Virol. 1999 Mar;80 ( Pt 3):793-7.

Citrus Sudden Death

new disease reported in Brazil in 1999 Host Range and Symptoms decline disease limited to Rangpur lime and to limited extent on Volkamer lemon during periods of drought rapid death of trees during drought characteristic yellow stain at or below the bud union

Citrus Sudden Death -- continued

Causal Agent some controversy, a sobemovirus-like, marafivirus but CTV association genome on next slide Transmission and Epidemiology spread looks similar to aphid transmission of CTV no vector identified can be grafted? Identification decline determine whether on Rangpur lime or Volkamer lemon yellow stain at graft union Control virus-free budwood irrigation inarching

Identification and Genomic Characterization of a New Virus (Tymoviridae Family) Associated with Citrus Sudden Death Disease
Walter Maccheroni,1 Marcos C. Alegria,1 Christian C. Greggio,1 Joo Paulo Piazza,1 Rachel F. Kamla,1 Paula R. A. Zacharias,1 Moshe Bar-Joseph,1, Elliot W. Kitajima,2 Laura C. Assumpo,1 Giovana Camarotte,1 Jussara Cardozo,1 Elaine C. Casagrande,1 Fernanda Ferrari,1 Sulamita F. Franco,1 Poliana F. Giachetto,1 Alessandra Girasol,1 Hamilton Jordo Jr.,1 Vitor H. A. Silva,1 Leonardo C. A. Souza,1 Carlos I. Aguilar-Vildoso,1 Almir S. Zanca,1 Paulo Arruda,1 Joo Paulo Kitajima,1 Fernando C. Reinach,1 Jesus A. Ferro,1 and Ana C. R. da Silva1*

J Virol. 2005 Mar;79(5):3028-37.

Citrus Sudden Death

inarching

Citrus yellow mosaic virus

India, perhaps Japan Host Range and Symptoms mosaic of leaves does not reduce fruit production, but fruit have less juice and ascorbic acid wide citrus host range but apparently does not infect Mexican lime

Citrus yellow mosaic virus

Causal Agent badnavirus double-stranded circular DNA virus

Transmission and Epidemiology mechanically transmitted transmitted by aphids or mealybugs?

Citrus chlorotic dwarf virus (CCD)

found in Turkey Host Range and Symptoms causes crinkled and puckered leaves in lemon, tangelo, and sweet orange

Transmission and Epidemiology transmitted by the bayberry whitefly Parabemisia myricae grafting of infected budwood mechanically transmitted

Identification can do greenhouse experiments, but no simple identification

Control

CCD in lemon

CCD in mandarin

CCD in grapefruit

Identification of a single-stranded DNA virus associated with citrus chlorotic dwarf disease, a new member in the family Geminiviridae Giuliana Loconsolea, Pasquale Saldarellib, Harshavardhan Doddapanenic, Vito Savinoa, b, Giovanni P. Martellia, b, Maria Saponarib, Virology 432: 162-172 (2012)

Citrus Tristeza Virus (CTV) Decline

Host Range and Symptoms can infect most citrus varieties and relatives Poncirus spp are resistant to most isolates disease is limited to scions on sour orange rootstocks

Roots of declining tree no feeder roots

Citrus tristeza virus decline

Causal Agent positive-stranded RNA virus ~ 20,000 nts

RdRp

p6

p61

p18

p20

PRO PRO MT

HEL p33 HSP70 p27 CP p13 p23

Gold-labeled CP antiserum

Gold-labeled CPm antiserum

CTV virions
From: Tian et al. J. Gen. Virol.80:1111 (1999)

p27 (minor capsid protein, CPm)

p25 (major capsid protein, CP)

Citrus tristeza virus

ORF PRO PRO MT 1a 1b 2 3 4 5 6 CPm 7 8 9 10 11 p18 p20 p33 HSP70h IDR HEL

5 probe

3 probe
RdRp p6
( ) () ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( )

p61

CP p13 p23

Polyprotein: Replicase

+1 Ribosomal frameshift: RdRp

p33 p6 HSP70h p61 CPm CP p18 p13 p20 p23

Proteins

Citrus tristeza virus (CTV) decline

Transmission and Epidemiology aphid transmitted by a series of aphids, brown citrus aphid best also melon, cotton aphids transmitted in a semi-persistent manner ~ 1 hr to ~ 24 hr Identification in field, identify rootstock sour orange bud union staining, scion overgrowth, thickened bark at union serology can detect CTV; can differentiate FL mild from decline biological assay in greenhouse vein clearing and leaf cupping in Mexican lime Control use alternative rootstock use virus-free or decline-free budwood

Citrus tristeza virus (CTV) stem pitting

no severe stem pitting isolates of CTV known to be in Florida every effort is being made to keep them out Host Range and Symptoms stem pitting does not kill trees reduces vigor, reduces growth, reduces yield and fruit size stem pitting is specific to virus isolate and host some isolates cause stem pitting in grapefruit some isolates cause stem pitting in sweet orange some isolates cause stem pitting in both mandarins are more tolerant Control keep stem pitting isolates out mild strain cross protection

FS674 on DG

Risk evaluation of exotic pathogens for citrus

Disease HLB Blackspot CTV-SP Impact 10 5.8 6.1 Intro 7 6.2 5.8 Estab 7.1 8.7 9.8 Spread 7 10 9.1 Estab m 7.03 8.30 8.23 Estab F 347.90 539.40 517.24 Detection 2.6 3.3 2.5 Control 10 6.9 5.5 factor 1 1828.7 1096.1 690.6 factor 2 90454.0 71236.4 43383.8 4factRel % 100.00% 59.94% 37.76% 6factRel% 100.00% 78.75% 47.96%

Canker
CVC Phaeoram CCD Leprosis Swt. Scab Vein Enation SDV CYM ACD Stubborn

7.9
7 5.2 1.9 2.4 1.2 0.2 0.8 1.9 0.2 1

10
4.1 3.2 2.9 4.2 2.7 3.1 2.6 2.1 1.3 4.2

7.7
10 6.8 5.1 3.8 4.7 4.7 4.1 4.1 2.1 2.1

10
7.8 9.3 4.3 3.6 4.7 7.9 3.8 3.1 2.4 3.8

9.23
7.30 6.43 4.10 3.87 4.03 5.23 3.50 3.10 1.93 3.37

770.00
319.80 202.37 63.60 57.46 59.64 115.10 40.51 26.69 6.55 33.52

1
0.9 2 10 4 1.6 2.5 1.2 1 9 0.6

3.5
8.4 7.2 7.1 3.5 3.2 3.6 4.8 2.6 8.9 4.9

255.3
386.3 481.7 553.1 129.9 24.8 9.4 16.1 15.3 31.0 9.9

21290.5
16923.8 15153.3 8579.2 1930.5 366.4 207.2 186.7 131.9 105.0 98.5

13.96%
21.13% 26.34% 30.24% 7.10% 1.36% 0.52% 0.88% 0.84% 1.69% 0.54%

23.54%
18.71% 16.75% 9.48% 2.13% 0.41% 0.23% 0.21% 0.15% 0.12% 0.11%

Pow. Mil
LWB Mal Secco

0.6
0.7 0.4

1.3
1.3 2.1

2.3
1.8 1.1

4
1.9 1.8

2.53
1.67 1.67

11.96
4.45 4.16

0.8
1.7 1.3

2.7
1.9 1.5

3.3
3.8 1.3

15.5
10.1 3.2

0.18%
0.21% 0.07%

0.02%
0.01% 0.00%

Mild strain cross protection

Purposely inoculate trees with a mild strain of the virus to prevent infection by severe stem pitting isolate Common practice in Brazil, South Africa, Australia How does cross protection work? different strains of CTV do not cross protect against each other must find a mild strain that has close sequence to the severe strain otherwise must make one

CTV-based expression vector

ORF L1 L2 MT IDR HEL 1a 1b 2 p33 3 4 HSP70h 5 6 CPm 7 8 p18 9 10 11 p20

RdRp

p6

p61

CP

p13

p23

Green fluorescent protein from jellyfish

L1 L2 MT IDR HEL p33 HSP70h gfp p18 p20

RdRp

p6

p61

CPm

CP

p13

p23

DIFFERENTIAL SUSCEPTIBILITY OF DIFFERENT HOSTS TO T36

ML

CM

MV

SO

DG

1 mm

C mac

So Orange

No protection against GFP-expressing T36 strain-based virus:

T68-1 T3 T30 VT

T36

Protection

FS 703 protection FS672 challenge

No protection FS672 challenge

FS 703 protection FS701 challenge

No protection

FS701 challenge

What about CTV decline?

Have to have a mild isolate of the T36 strain Probably does not exist in Florida Then have to make one

Florida CTV strains

PRO PRO MT

decline

RdRp HEL

p6

p61

p18 p20

p33 HSP70 PRO PRO MT

p27 CP p13 p61 p18 p20

p23

mild

RdRp HEL

p6

p33 HSP70

p27 CP p13

p23

Genetic system for CTV

cDNA CTV Infected tree

Healthy seedling

dsRNA

Bacteria
Virus particles

in vitro transcription

Protoplast

virus RNA

Healthy

Seedling yellows reaction on sour orange plants

rCTV-T36 WT CTV-T36

T36/T30 hybrids
p33 HSP70h CPm p18 p20

3
RdRp
p33

p6
HSP70h

p61
CPm

CP
p18

p13
p20

p23

3
RdRp p33 p6 p61 CP p13 p18 p20 p23

HSP70h

CPm

3
RdRp p33 p6 HSP70h p61 CPm CP p18 p13 p20 p23

3
RdRp p33 p6 HSP70h p61 CPm CP p18 p13 p20 p23

3
RdRp p33 p6 HSP70h p61 CPm CP p18 p13 p20 p23

3
RdRp p6 p61 CP p13 p23

PRO PRO MT

decline

RdRp HEL

p6

p61

p18 p20

SY

p33 HSP70 PRO PRO MT

p27 CP p13 p61 p18 p20

p23

+ -

mild

RdRp HEL

p6

p33 HSP70 PRO PRO MT

p27 CP p13 p61 p18 p20

p23

hybrid

RdRp
HEL

p6

p33 HSP70 p27 CP p13 p23

Healthy

T30 T36

P23+NTR of
T30 in T36

Sour Orange

P65+p61 of T30

Substitution of p23 gene + NTR of T30 into genome of T36 creates cross-protecting isolate against seedling yellows

T36-infected sour orange

Healthy sour orange

In the center p23+NTR-T30/T36 hybrid-infected sour orange, later challenged with T36

field test to protect citrus on sour orange rootstocks