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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.