Tài liệu Bệnh khô đầu lá dứa docx

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
• 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)
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?
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
Epidemiology
1. Virus diversity*
2. Mealybug transmission*
3. Interactions between PMWaV and
other stress factors
4. Host range
Multiple Closteroviruses
in Pineapple?
M1 2
23.1
2.3
2.0
9.4
6.6
4.4
kb
• A doublet of dsRNA was
often resolved by agarose

gel electrophoresis
• 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
Multiple Closteroviruses
in Pineapple?
hsp70h
Clone
nt
Homology
• Initial cloning and
sequencing revealed two
distinct hsp70h genotypes
in viral dsRNA
• PMWaV-1
• PMWaV-2
pC15
100%
pC16
100%
pC18
47%
pC12
PMWaV-2 Monoclonal Antibody Selection
PMWaV
2
1 and 2

1
None
dsRNA Analysis of PMWaV-1-
and PMWaV-2-Infected Plants
M1 2
9.4
6.6
4.4
23.1
3
kb
Lane
1 dsRNAs isolated from
PMWaV-1-infected plants
2 dsRNAs isolated from
PMWaV-2-infected plants
3 dsRNAs isolated from
PMWaV-free plants