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LOLITA M.DOLORES
University Researcher , CSC-IPB, UPLB
College, Laguna
• Production Constraint
– NMK
- severely reduces yield
Causal Pathogen
CABYV virus particles
- 25-30 nm in diameter
- hexagonal
- no envelope
- icosahedral symmetry
Transmitted efficiently by
-aphids in persistent manner
Objectives
1. To collect and isolate different CABYV isolates
from major ampalaya production areas
Purification of
isolates thru series RT-PCR
of inoculations from
Survey & NMK infected
Symptom
Collection ampalaya to
of virus healthy ampalaya Confirmed
isolates seedlings & vice
versa Transmission:
mechanical or
graft-inoculation ELISA
Host
Biological
range
characterization
Confirmed
Through aphid
transmission Vector
transmissibility
METHODOLOGY: Molecular identification and
characterization
RNA extraction using Trizol
F 5’ GAATACGGTCGCGGCTAGAAATC3’
R 5’ CTATTTGGGGTTCTGGACCTGGC3’
Biological Characterization
c Transmission and Host range
Pangasinan
Tarlac
Pampanga
Bulacan
Laguna
Ilocos sur
RESULTS
Table1. Survey and collection of NMK infected ampalaya:
3. Pangasinan:
* = wilted
RESULTS
Table1. Survey and collection of NMK infected ampalaya:
3. Pangasinan:
3. Pangasinan:
* = wilted
RESULTS
2. Bulacan 2 Baliwag 17 17
3. Pangasinan Tayug 33 50
Cucurbita pepo- Laguna isolate Luffa acutangula (patola ridge) –Laguna isolate
RNA extraction and RT PCR Optimization
1. Total RNA was extracted from 100 mg of symptomatic leaves of
infected ampalaya using Trizol (Gibco, BRL Life Technologies,
England).
2. Briefly, tissue was ground to a fine powder in liquid nitrogen and
homogenized in 1.0 ml of Trizol reagent and 20 µl mercaptoethanol.
3. After vortexing for 15-20 sec, the homogenate was incubated at
56 C for 5 min then centrifuged at 4,000 rpm for 10 min.
4. The aqueous phase was collected and incubated at room
temperature for 2-3 min then centrifuged at 4,000 rpm at 4 C for 15
min.
5. The aqueous phase was collected and added 750 µl isopropanol
and salt mixture (0.8 M sodium citrate, 1.2 M sodium chloride).
6. Total RNA was precipitated by centrifugation at 14,000 rpm at 4
C for 10 min.
7. The RNA pellet was washed with 1.0 ml of 75% ethanol and
dissolved in 50 µl of diethyl pyrocarbonate (DEPC) treated water
(Ambion, Austin, Texas).
RNA EXTRACTION ( Trizol)
The results of PCR showed the expected 600 bp for both NMK
samples 1 and 2 in a 1.5% agarose gel with good DNA fragments
obtained using an annealing temperature of 57C under the PCR
program profile number 2.
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
~600bp
~600bp
~600bp 600bp
2. Terminal Report
DOLORES, L.M. 2008. Molecular and Biological Characterization of Cucurbit
Aphidborne Yellows Virus (CABYV) causing the Namamarako Syndrome in
Ampalaya. Research Terminal Report funded by UPLB Basic Research,
UPLB, College Laguna.
Thank You!