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
• Reddening of the leaves
• Downward curling of the
leaf margins
• Loss of turgidity, leaves
MWP
reflex downwards
• Leaf tip dieback
• Plants either recover or
endure further leaf tip
dieback resulting in death
 Association of Mealybugs
with the Disease
• In 1931 Illingworth directly
associated mealybugs with
wilting pineapple plants

• Psuedococcus brevipes:
Dysmicoccus brevipes (pink) Dysmicoccus brevipes
(pink)
Dysmicoccus neobrevipes
(gray)

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 MWP-
affected 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?

• ISEM revealed that not all

virus particles were being
decorated by monoclonal
antibodies
• At least two serotypes exist

200nm
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 M 1 2
• A doublet of dsRNA was
23.1
often resolved by agarose
9.4
6.6 gel electrophoresis
4.4
• May represent the
2.3 replicative forms of two
2.0 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 nt
Clone Homology • Initial cloning and
sequencing revealed two
pC15 distinct hsp70h genotypes
100%
in viral dsRNA
pC16
100% • PMWaV-1
pC18 • PMWaV-2
47%
pC12
PMWaV-2 Monoclonal Antibody Selection
PMWaV
2
1 and 2

None
 dsRNA Analysis of PMWaV-1-
and PMWaV-2-Infected Plants
M 1 2 3
kb Lane
23.1
1 dsRNAs isolated from
PMWaV-1-infected plants
9.4
2 dsRNAs isolated from
6.6 PMWaV-2-infected plants

3 dsRNAs isolated from

4.4
PMWaV-free plants
 Genome Organization of
PMWaV-1 and PMWaV-2

PMWaV-2

RdRp HSP70 CP p20 p5

5’ P-PRO MTR HEL p5/6 p46/61 CPd p21 3’

? ? PMWaV-1

0 4 8 12 16 kb
% Sequence Homology
Between PMWaV-1 & -2

Nucleotide Amino Acid

Gene Identity Similarity Identity
Helicase 47 59 33
Polymerase 66 49 23
p5/p6 70 61 25
HSP70h 62 56 37
p46/p61 36 51 20
Coat Protein 41 49 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 - 47 67 75
% nucleotide
2 38 - 48 39
identity
3 75 37 - 66
4 88 37 76 -

% amino acid identity

PMWaV-Specific RT-PCR Assays
PMWaV
M 1 2 3 4 - H2O
bp

1353
1078
872
603
310
 0 2 4 6 8 10 12 14 16 18kb

5’ p4 3’
p21 p7 +1
p59 +2
GLRaV-3 p20 p20
+3

p22
? p6
p20
PMWaV-2 p46

PMWaV-1 ? p24
p61 ?

? p61 p23
PMWaV-3 ?

protease domain hydrophobic protein minor coat protein

(polyprotein processing) (movement) (structure, movement)
methyltransferase domain heat shock 70 homolog see above
(replication) (structure, movement) (unknown function)
helicase domain see above see above
(replication) (structure, movement) (unknown function)
RNA polymerase major coat protein see above
(replication) (structure, movement) (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 color-
coordinated.
 Beet yellow stunt
virus (BYSV)
52
91 Genus Closterovirus
Beet yellows virus (BYV) (aphid transmissible)

Citrus tristeza virus (CTV)

53
Sweet potato chlorotic
stunt virus (SPCSV)
76
97 Cucurbit yellow stunt Genus Crinivirus
disorder virus (CYSDV) (whitefly transmissible)
Lettuce infectious
yellows virus (LIYV)

Grapevine leafroll-associated
virus 3 (GLRaV-3)
97
95 Pineapple mealybug wilt-associated Genus Ampelovirus
virus 2 (PMWaV-2) (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 Major Coat Protein (kDa)
S A L F
PMWaV-2 ugc gcg uua uuuc E Closterovirus 22-25
GLRaV-3 gcu ggu ugc uuuc gag
A G C F gag
E Crinivirus 28-31

Q Q C V 35-38
Ampelovirus
PMWaV-1,-3 cag cag ugc guuu N
ccg cag cgg guuu aac
BYV P Q R V 28-29
agc
PMWaV-1, -3
S

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
GLRaV-2 BYSV
BYV

CTV 86
LChV-1
100

Crinivirus
100
OLYaV LIYV
100
100 100
100 SPCSV
65
MVBaV
100
CYSDV
68 100
100
PMWaV-2 100 PBNSPaV (p)
100
100 GLRaV-6 (p)
100 GLRaV-9
GLRaV-3 100 100 GLRaV-5 (p)
GLRaV-4 (p)
GLRaV-1 PMWaV-1
PMWaV-3
LChV-2

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 Coat

complete Protein
PMWaV-1 63.9 72.5 79.2 72.0 63.2 63.7
(70.6) (82.4) (84.7) (78.2) (71.5) (70.2)
PMWaV-2 30.4 12.8 44.0 34.9 21.1 25.8
(38.1) (31.9) (51.0) (43.3) (29.8) (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 34.5 (45.4) 15.7 35.0 19.5 (26.4)

Australia (23.5) (43.0)
GLRaV-3 NY1 37.6 (47.1) 25.6 36.6 20.7 26.0 (32.0)
(39.5) (45.8) (30.2)
GLRaV-5 58.1 21.4 59.3 (70.1)
(67.0) (29.9)
GLRaV-9 CA 59.3
(67.5)
LChV-2USA6b 32.2 (45.0) 16.3 34.2 23.9 27.5 (33.3)
(34.7) (43.8) (33.6)
 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)
PBNSPaV 46.6 (55.2)
Closterovirus
GLRaV-2 Italy 34.8 (45.8) 28.5 (49.0) 33.7 (40.6) 22.2 (50.0) 17.9 (23.4) CPd
22.6 (33.9) CP
Unassigned
GLRaV-7 VAA42 35.3 (43.9)
LChV-1 30.0 (42.9) 23.3 (33.3) 26.0 (37.0) 27.3 (29.5)
OLYaV 30.9 (39.7)
OLYaV Sicilian 32.6 (46.6) 22.7 (38.6) 27.3 (37.5)
 USDA National Clonal
Germplasm Repository
Of 35 Tested
By TBIA and RT-PCR
φX

PMWaV-3 Only 12 (34%)

PMWaV-1 and –3 2 (6%)

PMWaV-2 and -3 4 (11%)

φX
PMWaV-1, -2, -3 2 (6%)

Total 20 (57%)
 Pineapple mealybug wilt associated virus

Clone 1 only 2 only 3 only 1 and 3 2 and 3 1, 2, and 3

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

 PMWaV incidence, Hybrid 1, Oahu island

PMWaV incidence (Mean ± S.E. )

Source Loc
+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

Costa
2 31±10 16±5 3 4±3 5±6 3±4 0±2
Rica

Mean 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 Post-
after 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
- +

- no MWP no MWP
PMWaV
+ no MWP YES !
 Mealybug-free Mealybug-inoculated
PMWaV- PMWaV PMWaV- PMWaV-
free infected free infected
 MWP Susceptibility
Pineapple X/X V/X V/M
Selection 1 0/10 0/10 17/20
Selection 2 0/10 0/10 20/20
Selection 3 0/10 0/10 18/20
Selection 4 0/10 0/10 18/20
Selection 5 0/10 0/10 10/10
 Transmission of PMWaVs
and Symptom Induction
Mealybugs Acquisition No. infected/ MWP
Source No. exposed
D. brevipes PMWaV-2 54/72 20/20
D. neobrevipes PMWaV-2 28/30 20/20
D. brevipes PMWaV-1 7/10 0/10
D. neobrevipes PMWaV-1 10/10 0/10
D. brevipes PMWaV-free 0/10 0/10
D. neobrevipes PMWaV-free 0/10 0/10
 Vector Transmission and MWP
Acquisition source Virus Infection incidence Symptom incidence
combination 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 2 and 3 3/5 4/5 3/5 4/5
Hybrid 9 3 4/5 5/5 0/5 0/5
Selection 1 - 0/5 0/5 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? MWP

Badnavirus
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 A B C M

size
500

A 642/573 505 bp
B 654/655 553 bp Agarose gel analysis
C 656/657 563 bp
M 652/653 573 bp
 Purify, purify, purify……………………………….

100 nm

100 nm 100 nm
Badnavirus incidence
No. of Badnavirus incidence
plants (Mean percentage)
Source sampled
+A +B +C +M

Hybrid 1 30 0 100 100 47

(Costa Rica)
Hybrid 1 30 0 100 100 23
(Philippines)
12 100 100 100 100
Hybrid 2

12 10 100 100 100

Hybrid 2
Hybrid 3 12 50 100 100 100
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 silencing

Transient expression assays

A. Assay for suppressors of local silencing
B. Assay for suppressors of systemic 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 Resulting plant
meristem
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

RB pBI121 Backbone LB
1 NOS NPT II NOS-T UBI9 AMV CPS NOS-T

2 NOS NPT II NOS-T UBI9 AMV CPS HSP CPAS NOS-T

pCAMBIA1300 Backbone

3 UBI9 AMV CPS NOS-T 35S HYG 35S

4 UBI9 AMV CPS HSP CPAS NOS-T 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 stem-
pitting, was first reported in
Hawaii in 1952
• Brown citrus aphid
(Toxoptera citricidus), the
most efficient vector of CTV,
has been present in Hawaii Mike Melzer
since 1907 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.