diagnosis and transmission of huanglongbing and …€¦ · chiang rai c. reticulata hybrid orah...

DIAGNOSIS AND TRANSMISSION OF HUANGLONGBING AND CITRUS

TRISTEZA DISEASES

Department of Pest Management Faculty of Natural Resources

Prince of Songkla University, Hat Yai, Thailand

Ratana Sdoodee

HUANGLONGBING (HLB) AND CITRUS TRISTEZA DISEASES

Why are we concerned about HLB & Tristeza•They are the destructive diseases of citrus

• Affect most of citrus cultivars

• Cause rapid decline of citrus trees

HUANGLONGBING TRISTEZA

Introduction• HLB and its vector (Diaphorina citri )was first reported in

Thailand in 1973 • No official record for first incidence of citrus tristeza and brown citrus

aphid, Toxoptera citricida (early 1900)• Widely spread (North, Central, East and South)• Host (Mandarin,Orange,Pomelo,Calamondin, Acid lime etc)• Mean of spread

- citrus propagation materials- insect vector

Diagnosis of HLB and Citrus Tristeza Disease

• Field diagnosis based on symptom expression and present of insect vector

• Graft transmission to indicator host• Vector transmission • Electron microscopy• Serological assay (ELISA, Immunoblotting, Immunostrip )• Molecular detection and identification ( PCR, RFLP, SSCP etc.)

Citrus Tristeza Closterovirus

Candidatus Liberibacter asiaticus

Table 1. Detection of citrus tristeza virus (CTV) in citrus samples collected from the North of Thailand

X

Location Citrus spp. Cultivar ELISANo. of infected samples/

Total testOD405 ( SD)

North

Chiang Mai C. reticulata hybrid Orah mandarin 4/4 0.413 0.124

C. reticulata Shogun mandarin 11/12 0.329 0.169

C. sinensis Navel 4/7 0.195 0.150

Chiang Rai C. reticulata hybrid Orah mandarin 4/4 0.248 0.099


Total 25/29 (86.2%)

Table 2. Detection of citrus tristeza virus (CTV) in citrus samples collected from the Central and the East of Thailand

X

Location Citrus spp. Cultivar ELISA

No. of infected samples/Total test

OD405 ( SD)

Central

Lop Buri C. reticulata Siam mandarin 2/2 0.932 0.325

Pathum Thani C. reticulata Siam mandarin 6/6 0.260 0.059

Samut Songkhram C. reticulata Siam mandarin 3/3 0.270 0.064

C. maxima Pomelo 0/2 0.015 4.546

Subtotal 11/13

East

Trat C. reticulata Shogun mandarin

3/6 0.347 0.146

C. reticulata Siam mandarin 0/6 0.011 2.920

C. reticulata hybrid Orah mandarin 5/5 0.450 0.168

Rayong C. reticulata Shogun mandarin

6/6 0.356 0.122

C. reticulata hybrid Orah mandarin 5/5 0.417 0.532

Total Subtotal 22/28

Total Total 33/41 (80.5 %)

Table 3. Detection of citrus tristeza virus (CTV) in citrus samples collected fromthe South of Thailand

Location Citrus spp. Cultivar ELISA

No. of infected samples/Total test

OD405 ( SD)

Songkhla C. madurensis Calamondin 2/2 0.158 0.0311

C. reticulata Som Juk tangerine 13/14 0.248 0.130


C. aurantifolia Mexican lime 5/5 0.486 0.071

Trang C. reticulata Som Juk tangerine 1/2 0.125 0


Krabi C. reticulata Shogun mandarin 9/9 0.286 0.078

Nakhon Si Thammarat C. reticulata Shogun mandarin 6/7 0.173 0.026

C. reticulata Keawan mandarin 4/4 0.356 0.059

Phatthalung C. reticulata Shogun mandarin 2/2 0.252 0.019

Yala C. reticulata Shogun mandarin 8/8 0.331 0.0

C. reticulata Som Juk tangerine 1/1 1.213 0.001

Total 63/73

X

CTV SYMPTOMS OF CITUS INFECTED FIELD TREES

CITRUS TRISTEZA SYMPTOM OF FIELD TREES

Yellow and stunt of pomelo cv. Tong Dee


Yellow and stunt of mandarin hybrid cv. Orah, North


Yellow and stunt of pomelo cv. Tong Dee, Central

Yellow and stunt of mandarin cv. Shogun, South


Distinctive stem pitting on trunk and limb of mandarin hybrid cv. Orah, North



Distinctive stem pitting of mandarin cv. Shogun, South

A

B

C

CITRUS TRISTEZA SYMPTOM OF FIELD TREESStunt, die back and stem pitting of Pomelo cv Tong Dee, South

Healthy

Stem pitting


Stem pitting of West Indian (Mexican) lime, South

A

B

C

Decline and over flowering of Pomelo cv. Kao Num Peuk, Central


Biological index

Vein clearing of West Indian lime (index host) caused by CTV

A. SM4-2 isloate

B. YS 2-3

A B

A

B

Severe corky vein on index host ,West Indian lime, caused by TS 12 (A), CmO2 (B)

BA

West Indian lime infected with CTVA.TS12-3 isolateB. CmO2-5 isloate

CitrusSymptom 1/ (No. week after inoculation)

4 5 6 7 8 9 10 11 12 13 14 15 16 17

West Indian lime TS1West Indian lime SM1West Indian lime CmO2Siam mandarin TS1Siam mandarin CmO2Orah mandarin TS1Orah mandarin SM1Orah mandarin CmO2Shogun mandarin TS1Shogun mandarin CmO2

VCVC

VC,YVC,Y

VCVC,YVC,Y

VC

VCVC,YVC,Y

VC

VC

VCVC,YVC,Y

VC

VC

VCVC,YVC,Y

VC

VC

VCVC,YVC,Y

VC

VC

VC

VCVC,YVC,Y

VCVC

VC

VC

VCVC,YVC,Y

VCVC

VC

VC

VCVC,YVC,Y

VCVC

VC

VC

VCVC,Y,CV

VC,Y

VCVC

VC

VC

VCVC,Y,CV

VC,Y

VCVC

VC

VC

VC, CVVC,Y,CVVC,Y,CV

VCVC

CV,YVC

VC

1/ VC = vein clearing, SP = stem pitting, Y = yellowing, CV = corky vein

Table 4. Symptoms induction by CTV Stem pitting isolate (TS1, SM1, CmO2) in different

Citrus spp.

Growth (height) of CTV-SP infected citrus seedlings

Height (cm)

height increase, week 9‐12



TS12 TS1 SM4 SN7 SN17 CMO1 TtO3 SsKw1 NkW1

SSCP (Single Strand Conformation Polymorphism) pattern of CTV

Restriction digestion of coat protein gene (P25) of 12 CTV isolates analyzed by Hinf I/RFLP.Lane M 1 kb markerLanes 1, 2 showing Hinf I/RFLP pattern of gr.1+ 2 + 6 Lane 3, showing pattern of gr 5 + 7Lane 4, showing pattern of gr 3Lane 5 and 7 showing pattern of gr 4+6Lane 6 showing pattern of gr 6Lanes 8 and 9 showing pattern of gr 3+6

Table 5. Identity of P27 nucleotide sequence of CTV, Thai isolates

CTV Identity (%)

Isolate SN7 TS12 KS1 PS2 YS1 RYS5 TtO3

SN7 100

TS12 90 100

KS1 89 94 100

PS2 89 98 94 100

YS1 89, 83 97, 86 95, 81 98, 83 100

RYS5 88 97 94 97 98, 84 100

TtO3 89, 83 94, 84 92, 86 95, 85 97, 86 97, 86 100

Table 6. Comparesion of P27 nucleotide (nt) identity and amino acid (aa) identity and similarity (%) of CTV Thai isolates CmO1, TS12 with 7 CTV isolates from different geographical location

CTV Isolate1/

CmO1 TS12

Identitiesa a similarities

Identitiesa a similarities

nt2/ a a3/ nt2/ a a3/

Capao BonitoT36T30T385KPS27SY568VT

98%92%92%92%88%87%87%

99%98%96%96%96%95%94%

100%99%97%97%97%97%96%

97%92%92%92%88%87%87%

99%97%96%96%95%96%94%

99%98%97%97%97%97%96%

1/ Capao Bonito Sweet orange stem pitting isolate from BrazilT36 Severe decline using sour orange rootstock from FloridaT30 Mild isolate from FloridaT385 Mild isolate from SpainKPS27 Citrus hystrix isolate from ThailandSY568 Sweet orange stem pitting isolate from CaliforniaVT Severe isolate from Israel

2/ nt = nucleotide3/ aa = amino acid

CTV SINGLE APHID TRANSMISSION

CTV isolate Test Plant No. Symptoms1/ELISA

(OD405±SD)

Rate of transmission

Infected test plant/ Total test plant %

Severe CmO2-4

TS12-3

LK1-3

MildSM4

SsKw3-4YS15-1

CmO2-4/2CmO2-4/10CmO2-4/15TS12-3/4TS12-3/21TS12-3/22LK1-3/14LK1-3/16

SM4/1SM4/7SsKw3-4/11YS15-1/1YS15-1/24

CV+, SP+++, VC++, Y+VC+++VC+, SP+++, Y+VC+CV+++, SP+++, VC+++, Y++VC+VC+VC+

VC+VC+VC+VC+, SP+VC+, SP+

1.076±0.0360.216±0.0000.046±0.0040.376±0.0062.138±0.0740.049±0.0020.026±0.0070.048±0.039

0.055±0.0050.040±0.0210.022±0.0110.024±0.0060.034±0.021

3/20

3/20

2/20

2/20

1/202/20

15

15

10

10

510

Table 7. Single aphid (Toxoptera citricida) transmission of CTV, Thai isolates to West Indian lime, a test plant

1/CV = corky vein, SP = stem pitting, VC = vein clearing, Y = yellowing

Table 8. Growth (height) deduction caused by CTV single aphid transmission

1/ Height of healthy test plant = 63.6 cm

CTV Isolate Test Plant No. Height (cm)

12 month after transmission % deduction1/

SevereCmO 2-4

TS 12-3

LK 1-3

MildSM 4

SsKw 3-4YS 15-1

CmO 2-4/2CmO 2-4/10CmO 2-4/15TS 12-3/4TS 12-3/21TS 12-3/22LK 1-3/14LK 1-3/16

SM 4/1SM 4/7SsKw 3-4/11YS15-1/1YS15-1/24

9.018.76.850.011.060.058.237.4

46.450.548.028.056.0

85.070.589.3 21.482.75.78.541.2

27.020.624.556 .011.9

X-section of West indian lime young shoot, pt= pit, x= xylem

34

CTV TEST KIT

CTV TEST KIT

A

FED

CB

HG I

Table 12. Detection of HLB by PCR1/ during 2000-2010

Citrus Location Incidence of HLB

Total tested plants

Shogun mandarin

Siam mandarin

Orah mandarin

Som Juk mandarin

Pomeloes

NorthCentralSouth

NorthCentral

North

South

Central

South

7/2414/1411/32

1/25/6

2/8

54/96

4/7

11/20Total 109/209

1/ Using primers specific 16s rDNA of Ca. L. asiaticus, HLB bacterium designed by Jagoueix et al., 1994

M 1 2 3 4 5 6 7 8 9 10

Detection of HLB bacteria (Ca. L. asiaticus) by PCRLane M 1 Kb marker Lane 5 YS4 Lane 10 HLB DNALane 1 SN13 Lane 6 YS5Lane 2 YS1 Lane 7 YS6

Lane 3 YS2 Lane 8 YS7Lane 4 YS3 Lane 9 YS8

16S rDNA sequence of Thai isolate of HLB pathogen (Ca L. asiaticus)

1 GACTTCGCAA CCCATTGTAA CCACCATTGT AGCACGTGTG TAGCCCAGCC

51 CATAAGGGCC ATGAGGACTT GACGTCATCC CCACCTTCCT CCGGCTTATC

101 ACCGGCAGTC CCTATAAAGT ACCCAACATC TAGGTAAAAA CCTAAACTTG

151 ATGGCAACTA GAGGCAGGGG TTGCGCTCGT TGCGGGACTT AACCCAACAT

201 CTCACGACAC GAGCTGACGA CAGCCATGCA GCACCTGTGT AAAGGTCTCC

251 GAAAAGAAAA TACCATCTCT GATATCGTCC TATACATGTC AAGGGCTGGT

301 AAGGTTCTGC GCGTTGCATC GAATTAAACC ACATGCTCCA CCGCTTGTGC

351 GGGCCCCCGT CAATTCCTTT GAGTTTTAAT CTTGCGACCG TACTCCCCAG

401 GCGGAGTGCT TAATGCGTTA GCTGCGCCAC TGAATGGTAA ACCACCCAAC

451 AGCTAGCACT CATCGTTTAC GGCGTGGACT ACCAGGGTAT CTAATCCTGT

501 TTGCTCCCCA CGCTTTCGCG CCTCAGCGTC AGTATCAGGC CAGTGAGCCG

551 CCTTCGCCAC CGGTGTTCCT CCGAATATCT ACGAATTTCA CCTCTACACT

601 CGGAATTCCA CTCACCTCTC CTAAACTCTA GACAACCAGT ATTAAAGGCA

651 GTTCCAAGGT TGAGCCCTGG GATTTCACCT CTAACTTAAT CGCCCGCCTA

701 CGCGCCCTTT ACGCCCAGTT ATTCCGAACA ACGCTCGCCC CCTTCGTATT

751 ACCGCGGCTG CTGGCACGAA GTTAGCCGGG GCTTCTTCTC CGAATACCGT

801 CATTATCTTC TCCGGCGAAA GAGCTTTACA ACCCTAAGGC CTTCTTCACT

851 CACGCGGCAT GGCTGGATCA GGGTTGCCCC CATTGTCCAA TATTCCCCAC

901 TGCTGCCTCC CGTAGGAGTC TGGGCCGTGT CTCAGTCCCA GTGTGGCTGA

951 TCGTCCTCTC AGACCAGCTA TAGATCGTAG CCTTGGTAGG CTCTTACCCT

1001 ACCAACTAGC TAATCCAACG CAGGCTCATC TCTCTCCAAT AAAATCTTTC

1051 CCCCAATAGG GCGTATACGG TATTAGCACA CGTTTCCATG CGTTATCCCG

1095 TAGAAAAAGG TAGATTCCTA CGCGTTACTC ACCCGTCTGC CGCTC

Table 13. Detection of Huanglongbing (HLB) by immunoblottinga during 1992-1994

Citrus Location (Thailand)Incidence of HLBb

Total tested plants+ -

Siam mandarin

Shogun mandarinSom Juk tangerinShogun mandarinPomeloesCalamondin

YalaPhathum ThaniChanthaburiChiang RaiSongkhlaSongkhlaSongkhlaChanthaburiChanthaburi

(South)(Central)(East)(North)(South)(South)(South)(East)(East)

89823121301209

543032

1091510

14311234231392719

Total 202 187 389

a IgG of cultured HLB antisera was previously cross absorbed with healthy citrus and purified Citrus Tristeza Virus (OD206 = 0.15). The absorbed IgG and GAR-AP were used at 1 µgml-1 and 1:7500 respectively. A total of 22 healthy citrus plants were tested but none reacted with the antisera.

b + positive reaction- negative reaction

Sectional Yellow Shoot of Pomelo cv. Tup Tim Siam

HLB SYMPTOM OF FIELD TREES


Sectional Yellow Shoot of Valencia Sweet Orange)

Zinc deficiency symptoms


Som Juk tangerin Shogun mandarin

Huanglongbing infected citrus in Songkla province, southern Thailand

Trifoliate orange Mexican lime

HLB SYMPTOM

Poor color development and lopesided fruits

healthy

healthy

HLB SYMPTOMS ON FRUIT

Yellow fruit and fruit drop

HLB SYMPTOMS ON FRUIT

Table 14. Mixed infection of CTV and HLB in citrus samples collected from the North and the South of Thailand

Location Citrus spp. CultivarMixed infection (CTV+HLB)

No. of infected samples/ Total test

NorthChiang MaiChiang MaiChiang Rai

SouthKrabiTrangSongkhla

Yala

C. reticulataC. sinensisC. reticulata

C. reticulataC. reticulataC. reticulataC. reticulataC. reticulataC. reticulata

Shogun mandarinNavel sweet orangeOrah mandarin

Shogun mandarinShogun mandarinSom Jok tangerinShogun mandarinShogun mandarinSom Jok mandarin

Subtotal

Subtotal

1/92/42/25/15

1/41/117/81/17/81/1

18/33

Total 23/48

HLB AND CITRUS TRISTEZA MIXED INFECTION OF FIELD TREE

Stunt decline and die back of pomelo cv. Tup Tim Siam

CTV and HLB mixed infection pomelo showing yellow leaves (left) and Zn deficiency symptom (right)


Yellow, decline, dieback and stem pitting of Som Juk tangerine


Decline and thin canopy of pomelo cv. Tong Dee, Central


Host plantNo.of adult psyllid carrying HLB

Total test

Shogun mandarin (C. reticulata)Orange jasmine (Murraya paniculata)Orange jasmine (M. exotica)Curry leaf (Bergera koenigii)

5/50/51/52/5

Table 15. Detection of HLB pathogen by PCR in adult psyllid vector visiting citrus and

citrus and related trees

Murraya spp. No. of infected/Total test

M. paniculata 5/12M. exotica 1/3M. koenigii 0/ 3

Table 4. Detection of HLB pathogen by PCR in Murraya spp.

Fig 4.Jasmine orange (Murraya paniculata ) naturally infected with Ca. L. asiaticus as determined by PCR, showing typical huanglongbing symptom (interveinal chlorosis leaves), Hat Yai, Thailand

HLB Naturally Infected Murraya exotica

Fac. Of Natural Resources, Prince of Songkla University, Hat Yai, Songkla, THAILAND

Table 16. Transmission of HLB by Diaphorina citri Kuwayama from infected citrus to healthy Siam mandarin (index plant).

HLB sources Positive citrus sample code

Number of transmission per total index plants

Som Juk mandarin

Shogun mandarin

1-19-29-3

10-2

G1G2G7H7I7H8

3/32/33/32/3

2/33/33/33/31/32/3

Total 24/30

No. of insect1 No. of transmission /total test plant2

Transmission rate (%)

0 0/4 0

1 2/4 50

15 2/4 50

NT 3/4 75

Table 17. Transmission of HLB pathogen by psyllid vectorfrom naturally infected Shogun mandarinto orange jasmine

1 Number of D. citri insect vector feeding on one test plant (healthy Murraya paniculata), NT (natural transmission), healthy test plant placed under infected shogun mandarin.

2 Transmission determined by HLB symptom on test plant and followed by PCR amplificationof HLB DNA.

Symptom of HLB in Murraya paniculata transmittedby single Diaphorina citri insect vector

Symptom of HLB in M. paniculata transmitted by 15 insect vectors

Symptom of HLB in M. paniculata naturally transmitted by insect vector

Single psyllid caged on orange jasmine seedling Inoculated seedling kept in insect proved cage

HLB SINGLE PSYLLID TRANSMISSION TO ORANGE JASMINE

2/10 of either M. paniculata or M. exotica seedlings became infected by single psyllid transmission

HLB infected Murraya paniculata by single psyllid transmission

HLB infected Murraya exotica by single psyllid transmission

% Leaf flushing

% Aphid infestation

Weekly rainfall mm.

% Psyllid (Diaphorina citri) infestation

HLB and citrus tristeza insect vector monitoring of Som Juke tangerine plot,

PSU Klong Hoi Khung research station, Songkla, Thailand

CONCLUSION SCREENING BY SYMPTOMS AND INSECT VECTOR DETECTION OF SUSPECTED PATHOGEN

IDENTIFICATION OF CAUSAL PATHOGEN

STUDENT INVOLVED

PIYAWIT TOTHAUM PUTTARN TIPPENG JUTIKARN VORAPATTAMASI PREECHA SONGSERM AUSARA JUMPADUANG AMAVADEE CHAICHANA

ACKNOWLEDGEMENTS

CONFERENCE ORGANISING COMMITTEE, ACPP APPS DARWIN 2011 DEPT. OF PEST MANAGEMENT, PSU FACULTY OF NATURAL RESOUCES, PSU PRINCE OF SONGKLA UNIVERSITY, HAT YAI, THAILAND

diagnosis and transmission of huanglongbing and …€¦ · chiang rai c. reticulata hybrid orah...

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