Molecular and biological characterization of Cucurbit Aphid-Borne Yellows Virus (CABYV) causing the Namamarako (NMK) syndrome in Ampalaya

LOLITA M.DOLORES

University Researcher , CSC-IPB, UPLB

College, Laguna

Funded by: UPLB-BASIC RESEARCH

Duration: July 1, 2006-June 30, 2008

Molecular and Biological Characterization

of Cucurbit Aphid-Borne Yellows Virus

(CABYV) Causing the Namamarako (NMK)

Syndrome in Ampalaya

• Ampalaya – top money maker ( cash crop / commercial crop) – 7,000 ha (Golez and Caterno, 2002)

– Popular crop for its nutritive & medicinal value

– Good source of Vitamins A,B,C, iron, folic acid, phosphorus and calcium

• Production Constraint

– NMK- severely reduces yield

Significance

Causal Pathogen

CABYV virus particles

- 25-30 nm in diameter

- hexagonal

- no envelope

- icosahedral symmetry

Transmitted efficiently by

-aphids in persistent manner

1. To collect and isolate different CABYV isolates

from major ampalaya production areas

2. To characterize the different isolates using

biological and molecular methods

3. To determine the genetic variation of CABYV,

the causal pathogen of NMK disease of

ampalaya in the Philippines.

Objectives

• Virus Isolates

• Pure virus isolate

• Optimized PCR protocol

• PCR products, Clones, DNA sequences

• Terminal Report, paper for publication

Expected Output

Survey &

Collection

of virus

isolates

Purification of

isolates thru series

of inoculations from

NMK infected

ampalaya to

healthy ampalaya

seedlings & vice

versa

Biological

characterization

Symptom

Host

range

Vector

transmissibility

Transmission:

mechanical or

graft-inoculation

Through aphid

transmission

ELISA

RT-PCR

Confirmed

Confirmed

METHODOLOGY: Virus Isolation, Transmission &

Biological Characterization

METHODOLOGY: Molecular identification and

characterization

RNA extraction using Trizol

RT-PCR and PCR amplification using specific primers

(CE-9 and CE-10)

F 5’ GAATACGGTCGCGGCTAGAAATC3’

R 5’ CTATTTGGGGTTCTGGACCTGGC3’

Cloning and NA sequencing using TOPO TA cloning kit

DNA sequencing and analysis

RESULTS

Survey and Collection

Biological Characterization

Transmission and Host range

Molecular identification and characterization

Symptoms of collected infected ampalayac

c

Luzon

Pangasinan

Tarlac

Bulacan

Pampanga

Laguna

RESULTS

Ilocos sur

RESULTS

Area/ Location Symptoms Virus Infection

(%)*

1 .Bulacan:

San Ildefonso-1

Baliwag

Severe stunting, wrinkling, thickened veins,

plastic like appearance , shortened internodes

thickened veins, vein banding

90

75

2. Laguna:

Liliw -1

CES UPLB

IPB- UPLB

Liliw 2

yellowing, vein banding, thickened Veins, plastic like

appearance

yellowing, vein banding

thickening of veins, vein banding

mosaic, motlling, leaf distortion, little leaf

70

50

30

40

3. Pangasinan:

Urdaneta -1

Urdaneta 2

Asingan -1

Asingan -2

Sta. Maria

Tayug

Yellowing, thickened veins, short internodes, plastic like appearance

wrinkling, thickened veins, short internodes

yellowing, thickened veins

thickened veins, plastic like appearance

vein banding, thick veins, yellowing

wrinkled leaf, stunting, yellowing

70

80

70

80

60

60

4. Tarlac:

Bamban

Moncada

Yellowing thickened veins, vein banding


70

40

5.Pampan

Arayat Yellowing, thickened veins, short

internodes, plastic like appearance

60

Table1. Survey and collection of NMK infected ampalaya:

RESULTS

Area/ Location Symptoms Virus Infection (%)*

1 .Bulacan:

San Ildefonso-1

Baliwag

Severe stunting, wrinkling, thickened veins, plastic like appearance

, shortened internodes

thickened veins, vein banding

90

75

2. Laguna:

Liliw -1

CES UPLB

IPB- UPLB

Liliw 2

yellowing, vein banding, thickened

Veins, plastic like appearance

yellowing, vein banding

thickening of veins, vein banding

mosaic, motlling, leaf distortion, little leaf

70

50

30

40

3. Pangasinan:

Urdaneta -1

Urdaneta 2

Asingan -1

Asingan -2

Sta. Maria

Tayug

Yellowing, thickened veins, short internodes, plastic like appearance






70

80

70

80

60

60

4. Tarlac:

Bamban Yellowing thickened veins, vein banding 70


RESULTS

Area/ Location Symptoms Virus Infection

(%)*

3. Pangasinan:

Urdaneta -1

Urdaneta 2

Asingan -1

Asingan -2

Sta. Maria

Tayug

Yellowing, thickened veins, short internodes, plastic

like appearance






70

80

70

80

60

60

4. Tarlac:

Bamban

Moncada



70

40

5.Pampanga



60


RESULTS


3. Pangasinan:

Urdaneta -1

Urdaneta 2

Asingan -1

Asingan -2

Sta. Maria

Tayug

Yellowing, thickened veins, short internodes, plastic like

appearance






70

80

70

80

60

60

4. Tarlac:

Bamban

* Moncada



70

40

5.Pampannga



60


* = wilted

RESULTS


3. Pangasinan:

Urdaneta -1

Urdaneta 2

Asingan -1

Asingan -2

Sta. Maria

Tayug


appearance






70

80

70

80

60

60

4. Tarlac:

Bamban

Moncada



70

40

5.Pampanga



60


RESULTS


3. Pangasinan:

Urdaneta -1

Urdaneta 2

Asingan -1

Asingan -2

Sta. Maria

Tayug


appearance






70

80

70

80

60

60

4. Tarlac:

Bamban

Moncada



70

40

5.Pampanga



60

6. Ilocos Sur

*Cabugao wrinkling, thickened veins, short internodes 20


* = wilted

RESULTS

Fig 1. NMK infected ampalaya

from Bulacan (above) and

Pangasinan (picture below)

Fig. 2. NMK infected ampalaya

sample from UPLB compound

with green vein banding and

thickened vein symptoms.c

RESULTS

RESULTS

Fig. 3. NMK infected sample

from Tayug , Pangasinan

showing very prominent vein

banding and plastic like

appearance

Fig. 4. Ampalaya plant showing mosaic and mild to moderate curling

of leaves(left) and a close up of virus infected ampalaya leaf showing

mottling and leaf distortion (right).

Name of Isolate Place Collected (%)

Transmission

(%) ELISA

Positive

1. Bulacan 1 San Ildefonso, Bulacan 50 50

2. Bulacan 2 Baliwag 17 17

3. Pangasinan Tayug 33 50

4. Laguna Bay, Laguna 50 50

*5. Ilocos Sur Cabugao , Ilocos Sur 20 20

6. Tarlac *Moncada, Tarlac 40 40

Results of transmission and ELISA tests for some NMK virus isolates.

% transmission = total no. of plants with symptoms x 100total no of plants inoculated

% ELISA positive = total no. of samples positive to CABYV antiserum x 100

total no of samples assayed

* = wilted

View of NMK virus transmission inside an insect proof screen cage

TRANSMISSION

Bulacan isolate- Aphid transmitted Laguna isolate- Aphid transmitted

Graft-inoculated (Bulacan isolate- 3 wks) 6 wks ( right) after grafting

Reaction of Host Plant Species to Two NMK virus isolates.

• Host plant No. of Symptomatic Plants Symptoms ELISA

• Bulacan Laguna Bulacan Laguna Bulacan Laguna

• ----------------------------------------------------------------------------------------------------------------------

• Chenopodiaceae:

• C. amaranticolor 0 0 N/A N/A - -

• C. quinoa 0 0 N/A N/A - -

• Cucurbitaceae

• C. pepo 3/5 2/5 yel yel + +

• C. moschata 0 0 N/A - -

• Luffa acutangula 4/5 4/5 vb vb + +

• L. siceraria 0 0 N/A N/A - -

• Solanceae

• Solanum melongena 0 0 N/A N/A - -

• N. tabaccum 0 0 N/A N/A - -

• Caricaceae

• Carica papaya 0 0 N/A N/A - -

• ----------------------------------------------------------------------------------------------------------------------

RESULTS ( Current)

• Host Range

RESULTS

Luffa acutangula (patola ridge) –Bulacan isolateCucurbita pepo- Bulacan isolate

Cucurbita pepo- Laguna isolate Luffa acutangula (patola ridge) –Laguna isolate

1. Total RNA was extracted from 100 mg of symptomatic leaves of

infected ampalaya using Trizol (Gibco, BRL Life Technologies,

England).

2. Briefly, tissue was ground to a fine powder in liquid nitrogen and

homogenized in 1.0 ml of Trizol reagent and 20 µl mercaptoethanol.

3. After vortexing for 15-20 sec, the homogenate was incubated at

56 C for 5 min then centrifuged at 4,000 rpm for 10 min.

4. The aqueous phase was collected and incubated at room

temperature for 2-3 min then centrifuged at 4,000 rpm at 4 C for 15

min.

5. The aqueous phase was collected and added 750 µl isopropanol

and salt mixture (0.8 M sodium citrate, 1.2 M sodium chloride).

6. Total RNA was precipitated by centrifugation at 14,000 rpm at 4

C for 10 min.

7. The RNA pellet was washed with 1.0 ml of 75% ethanol and

dissolved in 50 µl of diethyl pyrocarbonate (DEPC) treated water

(Ambion, Austin, Texas).

RNA extraction and RT PCR Optimization

RNA EXTRACTION ( Trizol)

Total RNA extracted from 2 NMK virus

isolates (Bulacan and Laguna).

PCR amplification was conducted with 0.8ul cDNA template using 2

different program conditions for PCR as follows;

Program 1: initial denaturation of template DNA at 95 C for 2 min,

denaturation at 94C for 30sec, then primer annealing at 55 C for 30

sec and extension at 60C for 1min, in 30 cycles before a final

extension at 60C for 10 min.

Program 2: consisted of initial denaturation at 95 C -2 min;

denaturation at 94 C 1 min ; annealing at 57C for 30 sec; and a 60 C

extension for 1min for 30 cycles before a final extension at 60C for

15 min.

The results of PCR showed the expected 600 bp for both NMK

samples 1 and 2 in a 1.5% agarose gel with good DNA fragments

obtained using an annealing temperature of 57C under the PCR

program profile number 2.

RT PCR amplification

RT- PCR amplification of 2 NMK isolates with PCR Program profile -1

(left) and Program profile-2 (right).

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

~600bp

~600bp

~600bp

~600bp

1 – 1kb plus ladder 5 – NMK-Laguna – undilute

2 – NMK-Bulacan – undiluted 6—NMK– laguna ( 1:10)

3 – NMK- Bulacan – 1:10 7—NMK Laguna ( 1:50)

4 – NMK-Bulacan – 1:50 8 -- water

~600bp

RTPCR amplification of Laguna isolates showing 600 bp DNA fragment

with CE9/CE10 primers .

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

1 – 1kb plus ladder 5 – NMK-Laguna 2 – undiluted

2 – NMK-Laguna 1 – undiluted 6—NMK Laguna 2- 1:10

3 – NMK- Laguna 1 – 1:10 7—NMK Laguna 2 – 1:50

4 – NMK-Laguna 1 – 1:50 8 --water

600bp

Conclusion & Recommendations• NMK disease syndrome consisting of yellowing, crinkling,

thickened veins, dark greening , plastic-like, and

shortened internodes caused by CABYV luteovirus

• NMK was transmitted by aphids ( persistently) (Aphis

gosyppii L.) and grafting

• NMK infection was higher in Bulacan than in Laguna

• NMK can be transmitted to other cucurbits ( can serve as

alternate hosts of the virus)

• Isolates maybe differentiated based on symptom severity

• NMK isolates used in this study can be amplified with

primers derived from CABYV luteovirus DNA sequence

• Studies on biological and molecular aspects can be very

important parameters for epidemiological studies and in

virus disease management

PUBLICATIONS:1. Refereed journalDOLORES, L.M., M LJ SISON and MG N. YEBRON, Jr. 2007. Host Range and

Virus vector relationship of luteoviruscausing the namamarako syndrome (

NMK) of ampalaya. Journal Of Tropical Plant Pathology, Volume 42 : 1&2, Jan-

Dec. 2006, pp51-62

2. Terminal ReportDOLORES, L.M. 2008. Molecular and Biological Characterization of Cucurbit

Aphidborne Yellows Virus (CABYV) causing the Namamarako Syndrome in

Ampalaya. Research Terminal Report funded by UPLB Basic Research,

UPLB, College Laguna.

Thank You!

Documents

Molecular and biological characterization of Cucurbit Aphid-Borne Yellows Virus (CABYV) causing the Namamarako (NMK) syndrome in Ampalaya