A mosaic disease infecting yambean; Molecular detection and transmission

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WELCOME TO

BOGOR AGRICULTURAL UNIVERSITY

WELCOME TO

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*Department of Plant Protection,

Bogor Agricultural University

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1.

COSMETICS

2.

SALADS

3.

SNACK

4.

PICKLES

5.

Leaves - soil covers at plantations

6.

Fertilizer - form rhizobium to

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SYMPTOMS

Leaf green vein-banding

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Bean malformation

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14.16

93.33

81.43

26.61

100.00

2126

60

8138

5051

4610

301

56

6627

1344

4610

4

16

12

8

20

Babakan

Raya

Cibeureum

1*

Cibeureum

2

Cifor

Situgede

Incidence

(%)

Total

Plants

(N)

Infected

Plant

(n)

Plant

Age

(WAP)

Location

INCIDENCE in YAMBEAN FIELDS at BOGOR (WEST JAVA)

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INCIDENCE in YAMBEAN FIELDS at PREMBUN

(CENTRAL JAVA)

100

20

35

100

60

2.500

7.200

2.250

6.000

3.840

2.500

1.440

788

6.000

2.304

16

8

12

10

14

Kedung

Bulus

1

Kedung

Bulus

2

Kedung

Bulus

3

Mulyo

Sri

Sembir

Kadipaten

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A. craccivora,

Koch.

A. glycines,

Mats.

A. gossypii,

Glov.

2

mm

1,5

mm

b. Insect Vector (Aphids)

1 mm

BIOLOGICAL CHARACTERS

1. TRANSMISSION MODES

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Aphid free virus

Overnight in Taro

New

nymph

H

o

t

p

e

p

e

r

S

o

y

b

e

a

n

C

o

w

p

e

a

A. gossypii

A. glycines

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Virus Transmission

Aphid

Starving 1 hr

Acquisition feeding

Inoculation feeding

Inocula

Healthy yam bean

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10/10

13-17

A. gossypii

7/10

14-28

A. glycines

10/10

12-17

A. craccivora

0/10

0

Control

*

Incidence

(n/N)

Incubation period

(dpi)

Treatment

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c

Healthy

Inocula

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Healthy

Inocula

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Healthy

Inocula

Trans

mission by

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c. Seed detection

0,556

East Java

0,622

West Java 3

0,690

West Java 2*

0,487

West Java 1

0,506

Infected leaves

0,324

Healthy

0,227

Bufer

Absorbance

Value ELISA**

Source

Result

+

* Seed taken from infected plants

* * DAS-ELI SA using BCMV antisera (DSMZ) Positively if AVE 1.5 x healthy

100 seeds/ location

Seedling

Young leaf sampling

Dot blot

Positive samples

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2. SEROLOGYCAL TEST

+

2,359

0,141 0,141 2,356 2,356 0,138 0,138 General

General PotyvirusPotyvirus

‐ ‐ 0,392 0,392 0,389 0,389 0,613 0,613 0,169 0,169 TuMV TuMV

+

0,834

0,268 0,268 4.400 4.400 0,263 0,263

PStV

‐ ‐ 0,243 0,243 0,371 0,371 0,978 0,978 0,152 0,152 PRSV

PRSV--WW

+

1,086

0,276 0,276 2,429 2,429 0,176 0,176

WMV

‐

2

‐ ‐ 0,127 0,127 0,136 0,136 0,372 0,372 0,124 0,124 PVY PVY

+

0,328

0,193 0,193 1,543 1,543 0,150 0,150

ZYMV

‐ ‐ 0,191 0,191 0,193 0,193 1,738 1,738 0,184 0,184 ChiVMV ChiVMV Potyvirus Potyvirus ‐ ‐ 0,355 0,355 0,266 0,266 0,447 0,447 0,135 0,135 SqMV SqMV Comovirus Comovirus ‐ ‐ 0,199 0,199 0,188 0,188 0,347 0,347 0,196 0,196 CMV CMV Cucumovirus Cucumovirus ‐ ‐ 0,195 0,195 0,185 0,185 0,517 0,517 0,184 0,184 TMV TMV Tobamovirus Tobamovirus Results Results Sample Sample Negative Negative Control Control Positive Positive control control Buffer Buffer AVE* AVE* ANTISERA ANTISERA VIRUS VIRUS GROUP GROUP

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4. HOST RANGE TEST

-Ageratum

Ageratum conyzoidesconyzoides

+

Lt Lt

-Gomphrena

Gomphrena globosaglobosa

-C.

C. amaranticoloramaranticolor

-Cucumis

Cucumismelomelo

-Cucumis

Cucumissativussativus

-Arachis

Arachis hypogeahypogea

Cucurbitaceae Cucurbitaceae

+

MS MS 10

10--1212

Phaseolus

Phaseolusvulgarisvulgaris

+

CS

21

21--2323

Vigna

Vignaunguiculataunguiculata

-Nicotiana

Nicotiana tabacumtabacum

Leguminoseae Leguminoseae

+

CS CS 13

13--1515

Lycopersicon

Lycopersicon esculentumesculentum

-Solanaceae Solanaceae Capsicum

Capsicum annuumannuum

ELISA ELISA test test Symptom Symptom Incubation periods Incubation periods (day) (day) Plant Species Plant Species Chenopodiaceae Compositae

-* CS= Chlorosis systemic; MS = mosaic systemic; Lt = latent infection

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-CP

NIb

Nia-Pro

VPg 6

K 2

CI

6 K 1

P3

HC-PRO

P1

Sprimer

M4T

Potyvirus Genome Structure

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~1.7 kb

564

λHindIII

DNA isolated from gel

DNA Purification

Bulk Sequencing

Yb

Seq. analysis

(

DNASIS software for Mac. System

)

2027 2322

Infected leaves

Total RNA Extraction

(Qiagen kit)

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35 45

PROTEIN ANALYSIS BY

SDS-PAGE

YbMV Marker

I nfected leaves

Viral Purification

Virion 30

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CP nucleotide alignment (75.6%)

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CP Amino Acid Alignment (75.6%)

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3’-UTR nucleotide Alignment (76.9%)

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ABU60100 NC003397 AY864314 AJ312437 AJ312438 AY968604 NC003492 NC004013 AJ132156 AJ132157 AJ132158 NC001616 AY294044 NC002509 AB218280 NC007216 NC004752 NC003224 75.1 75.8 56.6 75.4 74.7 76.7 52.0 48.0 78.6 78.2 78.7 43.7 59.8 44.6 61.8 63.4 52.4 51.6 73.7 74.6 49.1 51.7 52.1 75.4 60.1 65.8 77.4 77.2 77.2 59.4 72.3 56.4 70.8 72.8 57.1 66.1 75.1 76.0 69.2 76.0 75.7 75.7 49.8 64.3 76.8 76.4 76.4 54.6 74.3 47.1 68.8 72.1 46.3 68.8 AzMV BCMV-gp1 K R Y NL1 BYMV CabMV PStV I13 I14 I15 PVY SMV TuMV WMV WVMV YMV ZYMV Nucleotide Amino acid Accession no.

3’ UTR

Coat Proteins

Virus

Seq. identities of CPs and 3’UTR between YbMV and other potyviruses

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Species demarcation in Potyvirus

Shukla & Ward (1989) & Frenkel (1989) similarity between strain of a potyvirus in CP nucleotide sequences showed over 90% and 3’UTR 83-99% homology

Adam et al (2005) reviewed from many published papers; demarcation of CP Nucleotide identity optimal was 76-77% and CP amino acid identity over 79.6% I n same species

“YbMV” CP nucleotide, amino acid, 3’UTR (77.4%,

76.8%

and

78.6%)

closely with BCMV strain peanut stripe

New strain of BCMV (BCMV strain yam bean)

and/or new potyvirus proposed as

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1. Competition Research fund B of Department of Plant Protection, BAU from Directorate of Higher Education (Partially)

2. Fundamental research grant from Directorate of Higher Education

3. Prof. Dr. Tetsuro Okuno and Dr. K. Mise, Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University for research facilities, suggestions and technical supports

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