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Orchids
Detection, Characterization, and Management of Pineapple
Mealybug Wilt-Associated Viruses
John Hu
University of Hawaii
Pineapple in Hawaii
Hawaii’s number one agricultural commodityHawaii Agricultural Statistics Service (2002)
Symptoms of 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
Healthy
MWP
Association of Mealybugswith 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 Antswith 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 mealybugtransmissibility, 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 Technomyrmexalbipes
• Diazinon– Use in pre-planting dips has been
eliminated
Research Areas
• Detection• Epidemiology • Etiology• Management
Detection Assays
1. dsRNA analyses2. EM & ISEM3. ELISA4. Tissue blot Immunoassay*5. RT-PCR*
Multiple Closterovirusesin 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
Epidemiology1. Virus diversity*
2. Mealybug transmission*3. Interactions between PMWaV and
other stress factors4. Host range
Multiple Closterovirusesin Pineapple?
M 1 2
23.1
2.32.0
9.46.64.4
kb• A doublet of dsRNA was
often resolved by agarosegel electrophoresis
• May represent the replicative forms of two viruses with different genome sizes
Lane 1 - dsRNAs extracted from 100 g of TBIA-positive pineapple tissue2 - dsRNAs extracted from 5 g of citrus bark infected with Citrus tristeza virus
Multiple Closterovirusesin Pineapple?
hsp70hClone
ntHomology • Initial cloning and
sequencing revealed two distinct hsp70h genotypes in viral dsRNA
• PMWaV-1• PMWaV-2
pC15100%
pC16100%
pC1847%
pC12
PMWaV-2 Monoclonal Antibody Selection
PMWaV
2
1 and 2
1
None
dsRNA Analysis of PMWaV-1-and PMWaV-2-Infected Plants
M 1 2
9.4
6.6
4.4
23.1
3kb Lane
1 dsRNAs isolated fromPMWaV-1-infected plants
2 dsRNAs isolated fromPMWaV-2-infected plants
3 dsRNAs isolated fromPMWaV-free plants
Genome Organization ofPMWaV-1 and PMWaV-2
P-PRO MTR HELRdRp
p5/6HSP70
p46/61CP
CPdp20
p215’ 3’
16 kb0 4 8 12
p5
PMWaV-2
PMWaV-1??
% Sequence HomologyBetween PMWaV-1 & -2
Amino AcidGene
NucleotideIdentity Similarity Identity
Helicase 47 59 33Polymerase 66 49 23p5/p6 70 61 25HSP70h 62 56 37p46/p61 36 51 20Coat 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 67 752 38 - 48 393 75 37 - 664 88 37 76 -
% nucleotideidentity
% amino acid identity
PMWaV-Specific RT-PCR Assays
603872
bp
310
M - H2O1 2 3PMWaV
4
10781353
0 2 4 6 8 10 12 14 16 18kb
p59p21
p20 p20
p46
p22p20
protease domain(polyprotein processing)methyltransferase domain(replication)helicase domain(replication)RNA polymerase(replication)
hydrophobic protein(movement)heat shock 70 homolog(structure, movement)see above(structure, movement)major coat protein(structure, movement)
minor coat protein(structure, movement)see above(unknown function)see above(unknown function)see above(unknown function)
p61p24
p61 p23
GLRaV-3
PMWaV-2
5’ 3’+1+2+3
p4p7
p6?
?PMWaV-1 ?
?PMWaV-3 ?
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.
Little cherry virus 2 (LChV-2)
Pineapple mealybug wilt-associated virus 2 (PMWaV-2)
Grapevine leafroll-associated virus 3 (GLRaV-3)
Cucurbit yellow stunt disorder virus (CYSDV)
Sweet potato chloroticstunt virus (SPCSV)
Citrus tristeza virus (CTV)
Beet yellows virus (BYV)
Beet yellow stunt virus (BYSV)
Lettuce infectious yellows virus (LIYV)
Genus Ampelovirus(mealybug transmissible)
Genus Crinivirus(whitefly transmissible)
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 Closterovirus(aphid transmissible)
53
9152
9776
9597
Genus Major Coat Protein (kDa)
Closterovirus
Crinivirus
Ampelovirus
PMWaV-1, -3
Q Q C Vcag cag ugc guuuccg cag cgg guuuP Q R V
NaacagcS
PMWaV-1,-3BYV
S A L Fugc gcg uua uuucgcu ggu ugc uuucA G C F
EgaggagE
PMWaV-2GLRaV-3
22-25
28-31
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.
PBNSPaV (p)
GLRaV-6 (p)GLRaV-9GLRaV-5 (p)GLRaV-4 (p)
100100100
PMWaV-1PMWaV-3
100
100
100
LChV-2GLRaV-1
GLRaV-3
PMWaV-2 100100
68
MVBaV
OLYaV
CTV
GLRaV-2 BYSVBYV
86100
100
100
LChV-1
LIYV
SPCSV
CYSDV
100100
100
65
Ampelovirus
Crinivirus
Closterovirus
100
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 HSP70complete
P46 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)
Open reading frame -- Amino acid identity (similarity)
GLRaV-9 CA 59.3 (67.5)
Virus RdRp Hydro HSP70 P46 Coat
GLRaV-1 Australia
34.5 (45.4) 15.7 (23.5)
35.0 (43.0)
19.5 (26.4)
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)
LChV-2USA6b 32.2 (45.0) 16.3 (34.7)
34.2 (43.8)
23.9 (33.6)
27.5 (33.3)
Amino acid identity (similarity) of PMWaV-3 with other Ampeloviruses
Open reading frame -- Amino acid identity (similarity)
RdRp Hydro HSP70 P46 Coat
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)
Closterovirus
GLRaV-2 Italy 34.8 (45.8) 28.5 (49.0) 33.7 (40.6) 22.2 (50.0) 17.9 (23.4) CPd22.6 (33.9) CP
Unassigned
Tentative Ampeloviruses
PBNSPaV 46.6 (55.2)
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)
Amino acid identity and (similarity) of PMWaV-3 with other Closteroviridae members
USDA National ClonalGermplasm Repository
Of 35 TestedOf 35 TestedBy TBIA and RTBy TBIA and RT--PCRPCR
φX
φX
20 (57%)
2 (6%)
4 (11%)
2 (6%)
12 (34%)
Total
PMWaV-1, -2, -3
PMWaV-2 and -3
PMWaV-1 and –3
PMWaV-3 Only
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 0Hybrid 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 8 0 0 0 0 0 0
Hybrid 9 31 ± 7 5 ± 2 9 ± 1 3 ± 3 5 ± 1 5 ± 1
Hybrid 7 <1 ± 1 <1 ± 1 0 0 0 0
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
2 31±10 16±5 3 4±3 5±6 3±4 0±2Costa 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 PMWaVNo. of PMWaV infected plants/ total no. exposed
Experimental Initial Days after initial mealybug introductionConditions status 44 75 125 175________________________________________________________Without mealybugs
PMWaV “-” 0/40 0/40 0/40 0/40 0/40PMWaV “+” 20/20 20/20 20/20 20/20 20/20
Virus-free mealybugsPMWaV “-” 0/40 0/40 0/40 0/40 0/40
Viruliferous mealybugsPMWaV “-” 0/40 7/40 21/40 31/40 40/40PMWaV “+” 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 5/1530 0/45 1/15 6/14 7/15 8/1550 1/45 3/15 10/14 14/15 13/1575 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 0/1580 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+ no MWP YES !PMWaV
PMWaV-free
PMWaVinfected
PMWaV-free
PMWaV-infected
Mealybug-free Mealybug-inoculated
MWP SusceptibilityPineapple X/X V/X V/MSelection 1 0/10 0/10 17/20Selection 2 0/10 0/10 20/20Selection 3 0/10 0/10 18/20Selection 4 0/10 0/10 18/20Selection 5 0/10 0/10 10/10
Mealybugs AcquisitionSource
No. infected/No. exposed
MWP
D. brevipes PMWaV-2 54/72 20/20D. neobrevipes PMWaV-2 28/30 20/20D. brevipes PMWaV-1 7/10 0/10D. neobrevipes PMWaV-1 10/10 0/10D. brevipes PMWaV-free 0/10 0/10D. neobrevipes PMWaV-free 0/10 0/10
Transmission of PMWaVsand Symptom Induction
Infection incidence Symptom incidenceAcquisition source Virus combination S2 H5 S2 HY5
D. brevipesAccession 100 1 and 3 4/5 5/5Accession 111 2 and 3 4/5 5/5 4/5 5/5Accession 126 2 and 3 3/5 4/5 3/5 4/5Hybrid 9 3 4/5 5/5 0/5 0/5
D. neobrevipesAccession 100 1 and 3 5/5 5/5 0/5 0/5
Accession 126 2 and 3 5/5 5/5 5/5 5/5
Hybrid 9 3 5/5 5/5 0/5 0/5
Accession 111 2 and 3 5/5 5/5 5/5 5/5
Selection 1 - 0/5 0/5 0/5 0/5
0/5
0/5 0/5
Selection 1 - 0/5 0/5 0/5
Vector Transmission and MWP
Dysmicoccus brevipes D. neobrevipes
PMWaV-3 can be acquired and transmitted by pink and grey pineapple mealybugs.
Plants infected with PMWaV-3 and exposed to mealybugs didnot 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: Plantsinfected withPMWaV-3 onlythat wereexposed toDymiscoccusbrevipes
Right: Plantsinfected withPMWaV-3 andPMWaV-2that wereexposed toDymiscoccusbrevipes
Working Hypothesis of the Etiology of MWPPineapple 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 PMWaVinfection and MWP symptoms disappear.
BADNAVIRUSES
•Family CaulimoviridaeGenus Badnavirus
• Circular dsDNA(7.35 kb – 8.3 kb)
• Possible synergistic effects with other viruses
Host plants :
MWP DISEASE COMPLEX
MWP
Vector
PMWaV-2
PMWaV-2 + Mealybugfeeding
Badnavirus
Synergistic?
PCR with degenerate oligonucleotide Badna1a & Badna 4 using total DNA from pineapple plants representing differenthybrids.
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.
Based on 200 amino acidsOptimized alignment using ClustalX.
Neighbor joining using PAUP.
Badnavirus Detection
Polymerase chain reaction assays (PCR)
• Nucleic acid extraction (DNeasy® kit)
Badnavirus Primer sets Ampliconsize
A 642/573 505 bpB 654/655 553 bpC 656/657 563 bpM 652/653 573 bp
Agarose gel analysis
500
A B C M
Purify, purify, purify……………………………….
100 nm
100 nm
100 nm
Badnavirus incidence(Mean percentage)
Source +A +B +C +M
Hybrid 1 (Costa Rica)
Hybrid 1(Philippines)
Hybrid 2
Hybrid 2 Hybrid 3
30 0 100 100 47
30 0 100 100 23
12 100 100 100 100
12
12
10
50
100
100
100
100
100
100
No. of plants
sampled
Badnavirus incidence
Objective 1. Develop universal and specific polymerase chain reactionassays 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 assaysA. Assay for suppressors of local silencingB. 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 MWP1. 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 culturepropagation (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 Methodologiesto 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 incidencesover time
Determine if correlations exist betweenrelative mealybug numbers detected
and virus spread
andmealybug wilt
Strategy 4. Compare and demonstrate IPM tactics
Based on alternative technologies including:
1. Virus incidence 2. Pesticide application methods3. 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
GoalApplication of RNA-mediated virus resistance to this pathosystemwill allow for the development of pineapple plants which are resistant toPMWaV and MWP.
Gene Constructs
RB LBNPT II NOS-T UBI9 AMV CPS NOS-TNOS1
NPT II NOS-T UBI9 AMV CPS CPAS NOS-TNOS
pBI121 Backbone
HSP2
pCAMBIA1300 Backbone
3 UBI9 AMV CPS NOS-T 35S HYG 35S
4 UBI9 AMV CPS CPAS NOS-T 35S HYG 35SHSP
Pineapple Transformation and Regeneration Systems
Conclusions1. 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.
Conclusions5. 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. SipesP. Wood, C. Hubbard, C. Oda, H. Fleisch
Acknowledgments
USDA-ARSUSDA-CSREES
Hawaii Department of AgriculturePineapple Growers Association of Hawaii
Banana bunchy top virus (BBTV) is the most important virus disease in banana worldwide.
Kheng Cheah, Chen YanEden Perez
Impacts
1. BBTV-resistant banana plants2. Resistance to other banana diseases3. Improved quality of bananas4. 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 since 1907
Mike MelzerPh.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.