MEALYBUG WILT OF PINEAPPLE: PINEAPPLE VIRUSES johnhu/pineapple.pdf · Pineapple mealybug wilt associated…

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Orchids

Detection, Characterization, and Management of Pineapple

Mealybug Wilt-Associated Viruses

John Hu

University of Hawaii

Pineapple in Hawaii

Hawaiis 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 100s 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

http://images.google.com/imgres?imgurl=http://gemini.biosci.arizona.edu/whitefly/images/nav/images/whiteflyFrontpage4_r2_c5.jpg&imgrefurl=http://gemini.biosci.arizona.edu/whitefly/&h=285&w=495&sz=17&tbnid=otAKZUhstR9WxM:&tbnh=73&tbnw=127&hl=en&start=16&prev=/i

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

X

X

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

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

1 428 1711 18 24 86 34 22

2 3110 165 3 43 56 34 02Costa Rica

Mean 36 10 1610 10 3 4 66 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 crawlersintroduction 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

Identificatio...

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