1. health and fitness
2. disease
3. aids and hiv

Orchids
Detection, Characterization, and
Management of Pineapple
Mealybug Wilt-Associated Viruses
John Hu
University of Hawaii
Pineapple
in Hawaii
Hawaii’s number one
agricultural
commodity
Hawaii Agricultural Statistics Service (2002)
Symptoms of MWP
Healthy
•
•
•
MWP
•
•
Reddening of the leaves
Downward curling of the
leaf margins
Loss of turgidity, leaves
reflex downwards
Leaf tip dieback
Plants either recover or
endure further leaf tip
dieback resulting in death
Association of Mealybugs
with the Disease
Dysmicoccus brevipes (pink)
Dysmicoccus neobrevipes (gray)
•
In 1931 Illingworth directly
associated mealybugs with
wilting pineapple plants
•
Psuedococcus brevipes:
Dysmicoccus brevipes
(pink)
Dysmicoccus neobrevipes
(gray)
Association of Ants
with the Disease
•
Caretakers of mealybugs
•
Protection against predators
Search for the Latent Virus
•
•
•
In 1989, U.B. Gunasinghe
and T.L. German isolated a
closterovirus from MWPaffected pineapple
Named the Pineapple
mealybug wilt-associated
virus (PMWaV)
Based on mealybug
transmissibility, placed in
Ampelovirus genus
Control Strategies
• Amdro ®, applied as a broadcast bait
(ants)
• Diazinon
● Pre-plant dip (mealybugs)
● Overhead application (mealybugs)
Potential Problems
• Amdro®
– Inactivated by moisture
– Not effective against some ant
species such as Technomyrmex
albipes
• Diazinon
– Use in pre-planting dips has been
eliminated
Research Areas
•
•
•
•
Detection
Epidemiology
Etiology
Management
Detection Assays
1. dsRNA analyses
2. EM & ISEM
3. ELISA
4. Tissue blot Immunoassay*
5. RT-PCR*
Multiple Closteroviruses
in Pineapple?
•
•
200nm
ISEM revealed that not all
virus particles were being
decorated by monoclonal
antibodies
At least two serotypes exist
Tissue blot immunoassay:
- distinct signal
- robust
- minimal sample
preparation
- can process 100’s of
samples per day
PMWaV-Specific RT-PCR Assays
RT-PCR Products
Southern Hybridization
PMWaV-1
PMWaV-2
Epidemiology
1. Virus diversity*
2. Mealybug transmission*
3. Interactions between PMWaV and
other stress factors
4. Host range
Multiple Closteroviruses
in Pineapple?
kb
23.1
9.4
6.6
4.4
2.3
2.0
M
1
2
•
•
A doublet of dsRNA was
often resolved by agarose
gel electrophoresis
May represent the
replicative forms of two
viruses with different
genome sizes
Lane 1 - dsRNAs extracted from 100 g of TBIA-positive pineapple tissue
2 - dsRNAs extracted from 5 g of citrus bark infected with Citrus tristeza virus
Multiple Closteroviruses
in Pineapple?
hsp70h
Clone
nt
Homology
•
pC15
100%
pC16
100%
pC18
47%
pC12
•
•
Initial cloning and
sequencing revealed two
distinct hsp70h genotypes
in viral dsRNA
PMWaV-1
PMWaV-2
PMWaV-2 Monoclonal Antibody Selection
PMWaV
2
1 and 2
1
None
dsRNA Analysis of PMWaV-1and PMWaV-2-Infected Plants
kb
23.1
M
1
2
3
Lane
1 dsRNAs isolated from
PMWaV-1-infected plants
9.4
6.6
4.4
2 dsRNAs isolated from
PMWaV-2-infected plants
3 dsRNAs isolated from
PMWaV-free plants
Genome Organization of
PMWaV-1 and PMWaV-2
PMWaV-2
HSP70
CP
p20 p5
p5/6
p46/61
CPd p21 3’
RdRp
5’ P-PRO MTR
HEL
?
0
?
4
8
12
PMWaV-1
16 kb
% Sequence Homology
Between PMWaV-1 & -2
Gene
Helicase
Polymerase
p5/p6
HSP70h
p46/p61
Coat Protein
Nucleotide
Amino Acid
Identity Similarity Identity
47
66
70
62
36
41
59
49
61
56
51
49
33
23
25
37
20
21
More Than Two?
•
•
•
Degenerate primers targeting conserved motifs in
the Hsp70h were designed.
Screening of field selections as well as pineapple
accessions at the USDA-ARS pineapple
germplasm repository
Two clones distinct from PMWaV-1 and -2 were
identified and tentatively named PMWaV-3 and -4.
Sequence Homology in the
Hsp70h region of PMWaVs
PMWaV
1
2
3
4
1
2
3
4
38
75
88
47
37
37
67
48
76
75
39
66
-
% amino acid identity
% nucleotide
identity
PMWaV-Specific RT-PCR Assays
M
bp
1353
1078
872
603
310
1
2
PMWaV
3
4
-
H2O
0
4
2
6
8
10
12
14
16
5’
p4 3’
p7
p21
p59
GLRaV-3
PMWaV-2
18kb
p22
?
PMWaV-1
p20
p46
?
PMWaV-3
protease domain
(polyprotein processing)
methyltransferase domain
(replication)
helicase domain
(replication)
RNA polymerase
(replication)
p20 p20
p61
?
hydrophobic protein
(movement)
heat shock 70 homolog
(structure, movement)
see above
(structure, movement)
major coat protein
(structure, movement)
p61
p24
p23
p6
?
?
minor coat protein
(structure, movement)
see above
(unknown function)
see above
(unknown function)
see above
(unknown function)
Genome organization of PMWaV-1 and -3 in comparison to that of the GLRaV-3 and PMWaV-2.
Boxes represent sequence domains or open reading frames (ORFs), and orthologs are colorcoordinated.
+1
+2
+3
52
91
Beet yellow stunt
virus (BYSV)
Beet yellows virus (BYV)
Genus Closterovirus
(aphid transmissible)
Citrus tristeza virus (CTV)
53
76
97
Sweet potato chlorotic
stunt virus (SPCSV)
Cucurbit yellow stunt
disorder virus (CYSDV)
Genus Crinivirus
(whitefly transmissible)
Lettuce infectious
yellows virus (LIYV)
97
95
Grapevine leafroll-associated
virus 3 (GLRaV-3)
Pineapple mealybug wilt-associated
virus 2 (PMWaV-2)
Genus Ampelovirus
(mealybug transmissible)
Little cherry virus 2 (LChV-2)
The three current genera in the family Closteroviridae are supported by vector and phylogenetic data.
Dendrogram was generated using TreePuzzle 5.2 with coat protein sequence data in a maximum likelihood
model. Numbers represent branch support in percentage following 10,000 puzzling steps.
Genus
S
A
L
F
PMWaV-2 ugc gcg uua uuuc
GLRaV-3 gcu ggu ugc uuuc
A
G
C
F
Q
Q
C
V
cag
cag
ugc
guuu
PMWaV-1,-3
ccg cag cgg guuu
BYV P
Q
R
V
E
gag
gag
E
N
aac
agc
S
Major Coat Protein (kDa)
Closterovirus
22-25
Crinivirus
28-31
Ampelovirus
PMWaV-1, -3
35-38
28-29
The +1 ribosomal frameshift sequences of PMWaV-1 and -3 more closely resemble that of Beet
yellows virus of the genus Closterovirus than other ampeloviruses.
The major coat protein of PMWaV-1 and -3 is more similar in size to the criniviruses than the
ampeloviruses.
Closterovirus
BYSV
GLRaV-2
BYV
CTV
86
LChV-1
100
Crinivirus
100
OLYaV
LIYV
100
100
100
100
SPCSV
65
MVBaV
CYSDV
100
100
PMWaV-2
100
68
100
PBNSPaV (p)
100
100
GLRaV-3
GLRaV-1
LChV-2
100
100
100
PMWaV-3
GLRaV-6 (p)
GLRaV-9
GLRaV-5 (p)
GLRaV-4 (p)
PMWaV-1
Ampelovirus
Phylogenetic assessment of the family Closteroviridae using full-length or partial (p) Hsp70h sequences as generated
by Bayesian analysis using the BLOSUM fixed rate amino acid model. Numbers on branches are posterior
probabilities and indicate branch support. LChV-1, MVBaV and OLYaV are unassigned members of the family. Viral
abbreviations as in Fig. 1 or: MVBaV, Mint vein banding-associated virus; OLYaV, Olive leaf yellowing-associated
virus; PBNSPaV, Plum bark necrotic stem pitting-associated virus.
PMWaV-3 amino acid identity (similarity)
with other PMWaVs
Open reading frame
Amino acid identity (similarity)
Virusa
RdRp
Hydro
HSP70
HSP70
P46
complete
Coat
Protein
PMWaV-1
63.9
(70.6)
72.5
(82.4)
79.2
(84.7)
72.0
(78.2)
63.2
(71.5)
63.7
(70.2)
PMWaV-2
30.4
(38.1)
12.8
(31.9)
44.0
(51.0)
34.9
(43.3)
21.1
(29.8)
25.8
(37.7)
PMWaV-4
70.3
(70.5)
Amino acid identity (similarity) of PMWaV-3
with other Ampeloviruses
Open reading frame -- Amino acid identity (similarity)
Virus
RdRp
Hydro
HSP70
P46
Coat
GLRaV-1
Australia
34.5 (45.4) 15.7
(23.5)
35.0
(43.0)
GLRaV-3 NY1
37.6 (47.1) 25.6
(39.5)
36.6
(45.8)
20.7
(30.2)
26.0 (32.0)
GLRaV-5
58.1
(67.0)
21.4
(29.9)
59.3 (70.1)
GLRaV-9 CA
59.3
(67.5)
23.9
(33.6)
27.5 (33.3)
LChV-2USA6b
32.2 (45.0) 16.3
(34.7)
34.2
(43.8)
19.5 (26.4)
Amino acid identity and (similarity) of PMWaV-3 with
other Closteroviridae members
Open reading frame -- Amino acid identity (similarity)
RdRp
Hydro
HSP70
P46
Coat
Tentative Ampeloviruses
GLRaV-4 CA
55.4 (65.1) 22.2 (31.1)
59.5 (67.7)
48.1 (58.8)
57.1 (67.2)
GLRaV-6 CA
23.8 (33.3)
58.2 (67.4)
49.5 (59.2)
60.2 (68.8)
22.2 (50.0)
17.9 (23.4) CPd
22.6 (33.9) CP
PBNSPaV
46.6 (55.2)
Closterovirus
GLRaV-2 Italy
34.8 (45.8) 28.5 (49.0)
33.7 (40.6)
Unassigned
GLRaV-7 VAA42
LChV-1
35.3 (43.9)
30.0 (42.9) 23.3 (33.3)
OLYaV
OLYaV Sicilian
26.0 (37.0)
30.9 (39.7)
32.6 (46.6) 22.7 (38.6)
27.3 (37.5)
27.3 (29.5)
USDA National Clonal
Germplasm Repository
Of 35 Tested
By TBIA and RT-PCR
φX
φX
PMWaV-3 Only
12 (34%)
PMWaV-1 and –3
2 (6%)
PMWaV-2 and -3
4 (11%)
PMWaV-1, -2, -3
2 (6%)
Total
20 (57%)
Pineapple mealybug wilt associated virus
1 only
2 only
3 only
Selection 1
28 ± 4
1±1
0
0
0
0
Selection 2
28 ± 4
19 ± 3
0
0
0
0
Selection 3
45 ± 7
2±1
0
0
0
0
Selection 4
82 ± 5
1±1
0
0
0
0
Selection 5
99 ± 1
0
0
0
0
0
Selection 6
43 ± 7
<1 ± 1
0
0
0
0
Hybrid 4
12 ± 5
9±7
0
<1 ± 1
0
0
Hybrid 5
16 ± 10
5±4
0
5±3
2±1
0
Hybrid 6
2±2
1±1
0
2±1
0
0
Hybrid 7
<1 ± 1
<1 ± 1
0
0
0
0
Hybrid 8
0
0
0
0
0
0
Hybrid 9
31 ± 7
5±2
9±1
3±3
5±1
5±1
Clone
1 and 3
2 and 3
1, 2, and 3
PMWaV incidence, Hybrid 1, Oahu island
PMWaV incidence (Mean ± S.E. )
Source Loc
Costa
Rica
Mean
+1
+2
+3
1&2
2& 3
1&3 1,2,3
1
42±8
17±11
18
2±4
8±6
3±4
2±2
2
31±10
16±5
3
4±3
5±6
3±4
0±2
36 ±10
16±10
10
3 ±4
6±6
3 ±4
1 ±2
What is the role of the pineapple
mealybugs in PMWaV dissemination
Dysmicoccus brevipes
D. neobrevipes
Transmission of PMWaV
No. of PMWaV infected plants/ total no. exposed
Experimental
Initial Days after initial mealybug introduction
Conditions
status
44
75
125
175
________________________________________________________
Without mealybugs
PMWaV “-”
0/40
0/40
0/40
0/40
0/40
PMWaV “+”
20/20
20/20
20/20
20/20
20/20
Virus-free mealybugs
PMWaV “-”
0/40
0/40
0/40
0/40
0/40
Viruliferous mealybugs
PMWaV “-”
0/40
7/40
21/40
31/40
40/40
PMWaV “+”
20/20
20/20
20/20
20/20
20/20
Effect of Mealybug Densities
# of PMWaV infected plants/ total # exposed
Days after
Number of “crawlers”
introduction 1
5
10
20
40
_____________________________________________
20
0/45
0/15
1/15
2/15
5/15
30
0/45
1/15
6/14
7/15
8/15
50
1/45
3/15
10/14 14/15 13/15
75
2/45
3/15
10/14 15/15 14/15
Effect of Mealybug Age
# of PMWaV infected plants/ total # exposed
Days
Prelarvaposition period Larvaposition Postafter
1st
2nd
3rd
young old
larvapos.
feeding
gravid gravid nonfeed.
___________________________________________________
30
1/20
7/20
13/20 2/20
1/20
0/15
55
5/20
11/20 16/20 7/20
1/20
0/15
80
6/20
15/20 20/20 8/20
1/20
0/15
Virus Transmission
•
•
•
•
PMWaV 1 and 2 can be transmitted by
mealybugs.
1 mealybug can cause transmission; 20
mealybugs/plant = 100% transmission.
1 month after transmission, virus infection
can be detected by tissue blotting.
Instars are better vectors than adults.
Etiology
1. Symptom induction
2. Mealybug transmission of
PMWaVs*
Symptom Induction
Mealybugs
PMWaV
+
-
+
no MWP
no MWP
no MWP
YES !
Mealybug-free
PMWaVPMWaV
free
infected
Mealybug-inoculated
PMWaVPMWaVfree
infected
MWP Susceptibility
Pineapple
Selection 1
Selection 2
Selection 3
Selection 4
Selection 5
X/X
0/10
0/10
0/10
0/10
0/10
V/X
0/10
0/10
0/10
0/10
0/10
V/M
17/20
20/20
18/20
18/20
10/10
Transmission of PMWaVs
and Symptom Induction
Mealybugs
D. brevipes
D. neobrevipes
D. brevipes
D. neobrevipes
D. brevipes
D. neobrevipes
Acquisition
Source
PMWaV-2
PMWaV-2
PMWaV-1
PMWaV-1
PMWaV-free
PMWaV-free
No. infected/ MWP
No. exposed
54/72
20/20
28/30
20/20
7/10
0/10
10/10
0/10
0/10
0/10
0/10
0/10
Vector Transmission and MWP
Acquisition source
Virus
combination
Infection incidence
Symptom incidence
S2
H5
S2
HY5
D. brevipes
Accession 100
1 and 3
4/5
5/5
0/5
0/5
Accession 111
2 and 3
4/5
5/5
4/5
5/5
Accession 126
Hybrid 9
Selection 1
2 and 3
3
-
3/5
4/5
0/5
4/5
5/5
0/5
3/5
0/5
0/5
4/5
0/5
0/5
D. neobrevipes
Accession 100
1 and 3
5/5
5/5
0/5
0/5
Accession 111
2 and 3
5/5
5/5
5/5
5/5
Accession 126
2 and 3
5/5
5/5
5/5
5/5
Hybrid 9
3
5/5
5/5
0/5
0/5
Selection 1
-
0/5
0/5
0/5
0/5
PMWaV-3 can be acquired
and transmitted by pink and
grey pineapple mealybugs.
Dysm icoccus brevipes
D. neobrevipes
Plants infected with PMWaV-3
and exposed to mealybugs did
not develop MWP.
Back row: ‘Smooth Cayenne’ infected with PMWaV-3 only
Front row: Hybrid 9 infected with PMWaV-3 only
All plants were exposed to Dysmicoccus brevipes
Left: Plants
infected with
PMWaV-3 only
that were
exposed to
Dymiscoccus
brevipes
Right: Plants
infected with
PMWaV-3 and
PMWaV-2
that were
exposed to
Dymiscoccus
brevipes
Working Hypothesis
of the Etiology of MWP
Pineapple plants have developed tolerance to
infection by PMWaVs and do not develop wilt
symptoms when infected by PMWaVs. When
mealybugs feed on these plants, the insects
inject an agent that suppresses this tolerance.
As a result, MWP symptoms develop. This
hypothesis
also
explains
the
recovery
phenomenon: if the mealybug factor is
removed, plants regain tolerance to PMWaV
infection and MWP symptoms disappear.
BADNAVIRUSES
•Family Caulimoviridae
Genus Badnavirus
• Circular dsDNA
(7.35 kb – 8.3 kb)
• Possible synergistic effects
with other viruses
Host plants :
MWP DISEASE COMPLEX
Vector
PMWaV-2 +
Mealybug
feeding
PMWaV-2
Synergistic?
Badnavirus
MWP
PCR with degenerate oligonucleotide Badna1a & Badna 4
using total DNA from pineapple plants representing different
hybrids.
Expected target size = 600 bp
Products were cloned and sequenced.
Many products are similar to retro-like elements
such as dea1, gypsy. gag, etc.
Several were similar to badnavirus sequences.
Neighbor joining using PAUP.
Based on 200 amino acids
Optimized alignment using ClustalX.
Badnavirus Detection
Polymerase chain reaction assays (PCR)
• Nucleic acid extraction (DNeasy® kit)
Badnavirus
Primer sets
Amplicon
size
A B C M
500
A
642/573
505 bp
B
654/655
553 bp
C
656/657
563 bp
M
652/653
573 bp
Agarose gel analysis
Purify, purify, purify……………………………….
100 nm
100 nm
100 nm
Badnavirus incidence
Source
No. of
plants
sampled
Badnavirus incidence
(Mean percentage)
+A
+B
+C
+M
Hybrid 1
(Costa Rica)
30
0
100
100
47
Hybrid 1
(Philippines)
30
0
100
100
23
12
100
100
100
100
12
10
100
100
100
12
50
100
100
100
Hybrid 2
Hybrid 2
Hybrid 3
Objective 1. Develop universal and specific polymerase chain reaction
assays to detect, differentiate, and determine the distribution of
badnaviruses in pineapple and other potential host plants
Identification of badna-like viruses
Detection of integrated viral sequences
Development of reliable specific and universal detection assays
Objective 2. Evaluate the roles of PMWaVs, PBVs, and mealybugs in the
etiology of MWP
Vector transmissibility
MWP etiological studies
Functional assays used to identify suppressors of RNA
Transient expression assays
A. Assay for suppressors of local silencing
B. Assay for suppressors of systemic silencing
silencing
Identification of p20 as suppressor of RNA silencing by the
Agrobacterium coinfiltration assay. Leaves of the 16c GFP plants
were infiltrated with an A. tumefaciens EHA105 carrying GFP
together with an A. tumefaciens EHA105 carrying the empty binary
plasmid GFP:-- (left), GFP:TBSVp19(middle) and GFP: PMWaV-II
(right)；
The green fluorescence images of the coinfiltrated leaves were
taken 13 days postinfiltration under a long-wave UV lamp.
Strategies for Reducing the
Incidence of PMWaVs and MWP
1. Use virus-free planting material
2. Use physical-based methodologies (ie. “edge
quarantines”, roguing, planting bed spacing,
etc.)
3. Develop a system that can predict when
mealybug control should be instigated
4. Compare and demonstrate IPM tactics
5. Develop PMWaV-resistant transgenic pineapple
Strategy 1. Use PMWaV-free Pineapple
Material for MWP Management
1. Screen propagation material with
antibodies in tissue blot immunoassays
before or after tissue culture
propagation (hybrids)
2. Virus elimination by meristem tissue
culture
Removal of apical
meristem
Resulting plant
5122 plants were gouged
7 slips per plant
36,000 propagules
Strategy 2. Use Physical-based Methodologies
to Reduce PMWaVs and MWP in the Field
1. Selection of initial planting area
2. Spatially-based quarantines for selection of
planting material
3. Manipulation of planting bed spacing
4. Roguing of PMWaV-infected plants
Strategy 3. Develop a system that can predict
when mealybug control should be Instigated
1. Develop a quantitative mealybug detection
system
2. Monitor PMWaVs and MWP incidences
over time
Determine if correlations exist between
relative mealybug numbers detected
and
virus spread
and
mealybug wilt
Strategy 4. Compare and
demonstrate IPM tactics
Based on alternative technologies
including:
1. Virus incidence
2. Pesticide application methods
3. Pesticide application timing
The purpose is to reduce the use of the
more toxic pesticides!
Strategy 5. Develop PMWaV-resistant
Transgenic Pineapple Plants
1. Develop inverted repeat gene
constructs
2. Optimize transformation and
regeneration systems
3. Screen resistant plants
Goal
Application of RNA-mediated
virus resistance to this pathosystem
will allow for the development of
pineapple plants which are resistant to
PMWaV and MWP.
Gene Constructs
pBI121 Backbone
RB
1
2
NOS
NOS
NPT II
NPT II
NOS-T
NOS-T
UBI9
UBI9
AMV
AMV
LB
CPS
NOS-T
CPS HSP CPAS
NOS-T
pCAMBIA1300 Backbone
3
UBI9
AMV
4
UBI9
AMV
CPS
NOS-T
35S
CPS HSP CPAS
NOS-T
HYG
35S
35S
HYG
35S
Pineapple Transformation and
Regeneration Systems
Conclusions
1. There are at least three distinct PMWaVs. Specific
and sensitive assays have been developed for
detection of these viruses.
2. PMWaVs are transmitted by mealybugs.
3. PMWaV-2 and another factor associated with
mealybug feeding result in mealybug wilt of
pineapple.
4. PMWaV-2, but not PMWaV-1 and PMWaV-3, plays
an essential role in the etiology of MWP.
Conclusions
5. Badnaviruses are being characterized; PCR assays
are being developed.
6. Gene silencing suppressors are being identified
and used to study the potential involvement in
symptom development.
7. Strategies are being evaluated for control of MWP,
including PMWaV-resistant trangenic pineapple
plants.
Acknowledgments
D. Sether, E. Perez, M. Melzer, H.Ma, V. Subere,
L. Martinez, K, Cheah
A. Karasev, C. Nagai, F. Zee, B. Sipes
P. Wood, C. Hubbard, C. Oda, H. Fleisch
Acknowledgments
USDA-ARS
USDA-CSREES
Hawaii Department of Agriculture
Pineapple Growers Association of Hawaii
Banana bunchy top virus
(BBTV) is the most
important
virus disease in banana
worldwide.
Kheng Cheah, Chen Yan
Eden Perez
Impacts
1. BBTV-resistant banana plants
2. Resistance to other banana diseases
3. Improved quality of bananas
4. Vaccines for oral immunization
Citrus tristeza in Hawaii
•
•
Citrus tristeza closterovirus
(CTV), the causal agent of
citrus decline and stempitting, was first reported in
Hawaii in 1952
Brown citrus aphid
(Toxoptera citricidus), the
most efficient vector of CTV,
has been present in Hawaii
since 1907
Mike Melzer
Ph.D. student
Stem-pitting
Impacts
• Help to develop a new citrus
industry in Hawaii.
• Our research will benefit the
entire citrus industry of the
USA.