*Department of Plant Protection,
Bogor Agricultural University
1.
COSMETICS
2.
SALADS
3.
SNACK
4.
PICKLES
5.
Leaves - soil covers at plantations
6.
Fertilizer - form rhizobium to
SYMPTOMS
Leaf green vein-banding
Bean malformation
Objective
Objective
•
•
Karakterisasi
Karakterisasi
bioekologi
bioekologi
dan
dan
biomolekuler
biomolekuler
penyebab
penyebab
mosaik
mosaik
pada
pada
bengkuang
bengkuang
•
•
Respon
Respon
beberapa
beberapa
kultivar
kultivar
bengkuang
bengkuang
terhadap
terhadap
infeksi
infeksi
virus
virus
14.16
14.16
93.33
93.33
81.43
81.43
26.61
26.61
100.00
100.00
2126
2126
60
60
8138
8138
5051
5051
4610
4610
301
301
56
56
6627
6627
1344
1344
4610
4610
4
4
16
16
12
12
8
8
20
20
Babakan
Babakan
Raya
Raya
Cibeureum
Cibeureum
1*
1*
Cibeureum
Cibeureum
2
2
Cifor
Cifor
Situgede
Situgede
Incidence
Incidence
(%)
(%)
Total
Total
Plants
Plants
(N)
(N)
Infected
Infected
Plant
Plant
(n)
(n)
Plant
Plant
Age
Age
(WAP)
(WAP)
Location
Location
INCIDENCE in YAMBEAN FIELDS at BOGOR (WEST JAVA)
INCIDENCE in YAMBEAN FIELDS at PREMBUN
(CENTRAL JAVA)
100
100
20
20
35
35
100
100
60
60
2.500
2.500
7.200
7.200
2.250
2.250
6.000
6.000
3.840
3.840
2.500
2.500
1.440
1.440
788
788
6.000
6.000
2.304
2.304
16
16
8
8
12
12
10
10
14
14
Kedung
Kedung
Bulus
Bulus
1
1
Kedung
Kedung
Bulus
Bulus
2
2
Kedung
Kedung
Bulus
Bulus
3
3
Mulyo
Mulyo
Sri
Sri
Sembir
Sembir
Kadipaten
Kadipaten
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
Aphid free virus
Overnight in Taro
New
nymph
H
o
t
p
e
p
p
e
r
S
o
y
b
e
a
n
C
o
w
p
e
a
A. gossypii
A. glycines
Virus Transmission
Virus Transmission
Aphid
Starving 1 hr
Acquisition feeding
Inoculation feeding
Inocula
Healthy yam bean
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
c
Healthy
Inocula
Healthy
Inocula
Healthy
Inocula
Trans
mission by
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
2. SEROLOGYCAL TEST
+
+
2,359
2,359
0,141 0,141 2,356 2,356 0,138 0,138 GeneralGeneral PotyvirusPotyvirus
‐ ‐ 0,392 0,392 0,389 0,389 0,613 0,613 0,169 0,169 TuMV TuMV
+
+
0,834
0,834
0,268 0,268 4.400 4.400 0,263 0,263PStV
PStV
‐ ‐ 0,243 0,243 0,371 0,371 0,978 0,978 0,152 0,152 PRSV PRSV-W-W+
+
1,086
1,086
0,276 0,276 2,429 2,429 0,176 0,176WMV
WMV
‐
‐
2
2
‐ ‐ 0,127 0,127 0,136 0,136 0,372 0,372 0,124 0,124 PVY PVY
+
+
0,328
0,328
0,193 0,193 1,543 1,543 0,150 0,150ZYMV
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 GROUPElectron micrograph of “YbMV” particles from infected
Leaf (leaf dip method), negatively stained with PTA
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 1010--1212
Phaseolus
Phaseolusvulgarisvulgaris
+
+
CS
CS
21
21--2323
Vigna
Vignaunguiculataunguiculata
-Nicotiana
Nicotiana tabacumtabacum
Leguminoseae Leguminoseae
+
+
CS CS 1313--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
-CP
NIb
Nia-Pro
VPg 6
K 2
CI
6 K 1
P3
HC-PRO
P1
Sprimer
M4T
Potyvirus Genome Structure
~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)
35
25 30
Marker YbMV
PROTEIN ANALYSIS
SDS-PAGE
kda
Infected leaves
Viral Purification
CP nucleotide alignment (75.6%)
CP Amino Acid Alignment (75.6%)
3’-UTR nucleotide Alignment (76.9%)
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
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
Response 5 Yam-bean cultivars Against Viral Infection
+ + + + + + + + + + + + + + + + + + + + + + 0.9666b 0.9666b 1.7990a 1.7990a 1.9983a 1.9983a 2.2070a 2.2070a 2.1517a 2.1517a 1.7778c 1.7778c 2.6923b 2.6923b 2.7222b 2.7222b 3.6364a 3.6364a 2.3077bc 2.3077bc 18/ 25 18/ 25 15/ 25 15/ 25 18/ 25 18/ 25 12/ 25 12/ 25 13/ 25 13/ 25 JawaJawa TengahTengah Hideung Hideung Porselen Porselen Paris Paris Kapas Kapas 1. 1. 2. 2. 3. 3. 4. 4. 5. 5.
Response
Response
NAE
NAE
Severity
Severity
Incidence
Incidence
Cultivar
Cultivar
No.
No.
CONCLUSI ON
1. The mosaic disease on yam bean caused by a Potyvirus, with proposed
name Yam bean Mosaic Virus (YbMV) based on nucleotide sequences
and biological characters
2. YbMV transmitted mechanically, through seed, and at least via 3
species of aphids
3. Long bean, tomato, P.vulgaris
and G. globosa
able to infected by virus
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