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Association of Mealybugswith the Disease Dysmicoccus brevipes pink Dysmicoccus neobrevipes gray • In 1931 Illingworth directly associated mealybugs with wilting pineapple plants • Psued

Orchids

Detection, Characterization, and

Management of Pineapple

Mealybug Wilt-Associated Viruses

John Hu University of Hawaii

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 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)

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

• At least two serotypes exist

Tissue blot immunoassay:

PMWaV-Specific RT-PCR Assays

PMWaV-1

PMWaV-2

• 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

sequencing revealed two

PMWaV-2 Monoclonal Antibody Selection

PMWaV

2

1 and 2

1 None

dsRNA Analysis of and PMWaV-2-Infected Plants

% Sequence Homology Between PMWaV-1 & -2

Amino Acid Gene

Nucleotide Identity Similarity Identity

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

% amino acid identity

0 2 4 6 8 10 12 14 16 18kb

p59 p21

p20 p20 p46

p22 p20

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)

p61 p24p61 p23

GLRaV-3

PMWaV-2

+1 +2 +3

p4 p7

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 chlorotic stunt 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 Major Coat Protein (kDa)

P Q R V

N aac agc S

PMWaV-1,-3

BYV

S A L F ugc gcg uua uuuc gcu ggu ugc uuuc

A G C F

E gag gag E

PMWaV-2

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) 100

100

PMWaV-1 PMWaV-3

100 100 100

LChV-2 GLRaV-1

GLRaV-3

PMWaV-2 100 100

68

MVBaV OLYaV

CTV

GLRaV-2 BYSV

BYV

86 100 100

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)

79.2 (84.7)

72.0 (78.2)

63.2 (71.5)

63.7 (70.2)

(38.1)

12.8 (31.9)

44.0 (51.0)

34.9 (43.3)

21.1 (29.8)

25.8 (37.7)

(70.5)

Open reading frame Amino acid identity (similarity)

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)

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)

Amino acid identity and (similarity) of PMWaV-3 with

other Closteroviridae members

USDA National Clonal Germplasm Repository

Pineapple mealybug wilt associated virus

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

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

What is the role of the pineapple

mealybugs in PMWaV dissemination

Dysmicoccus brevipes D neobrevipes

Effect of Mealybug Densities

# of PMWaV infected plants/ total # exposed

Effect of Mealybug Age

# of PMWaV infected plants/ total # exposed Days Prelarvaposition period Larvaposition Post-

after 1st 2nd 3rd young old larvapos.

_

• 1 month after transmission, virus infection

can be detected by tissue blotting.

• Instars are better vectors than adults

1 Symptom induction

2 Mealybug transmission of PMWaVs*

Symptom Induction

Mealybugs

- no MWP no MWP + no MWP YES !

PMWaV

PMWaV-free

PMWaV 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

Infection incidence Symptom incidence Acquisition source Virus

Dysm icoccus 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 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

Synergistic?

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.

Based on 200 amino acids

Optimized alignment using ClustalX.

Neighbor joining using PAUP.

Badnavirus Detection

Polymerase chain reaction assays (PCR)

• Nucleic acid extraction (DNeasy® kit)

Badnavirus Primer sets Amplicon

Purify, purify, purify……….

100 nm

100 nm

100 nm

Badnavirus incidence (Mean percentage)Source

10 50

100 100

100 100

100 100

No of plants sampled

Badnavirus incidence

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

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

Pineapple Transformation and

Regeneration Systems

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

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

USDA-ARS USDA-CSREES Hawaii Department of Agriculture

Pineapple Growers Association of Hawaii

Banana bunchy top virus

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

(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

Stem-pitting