Detection of Citrus Greening Organism (Liberobacter Asiaticum) by Polymerase Chain Reaction
Citrus greening disease caused by greening organism (GO; Liberobacter asiaticum) is one of the most destructive diseases of citrus in Malaysia and Indonesia. To detect the GO in infected plant tissues, Polymerase Chain Reaction (PCR), an accurate, rapid and reliable detection method was applied...
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| 格式: | Thesis |
| 语言: | English English |
| 出版: |
2001
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| 主题: | |
| 在线阅读: | http://psasir.upm.edu.my/id/eprint/10548/1/FP_2001_8_.pdf |
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| 总结: | Citrus greening disease caused by greening organism (GO; Liberobacter
asiaticum) is one of the most destructive diseases of citrus in Malaysia and
Indonesia. To detect the GO in infected plant tissues, Polymerase Chain
Reaction (PCR), an accurate, rapid and reliable detection method was applied
to detect the 165 rONA fragments of the GO in leaves showing one of several
typical symptoms of greening collected from GO-infected mandarin trees in
Malaysia and Indonesia.
In GO-infected mandarin trees, four typical symptoms of greening on leaves
were observed , namely mottling (type I), mild chlorosis with green veins (type
II), severe chlorosis with green veins (type III) and vein yellowing (type IV).
Types II and III symptoms were mostly found in GO-infected mandarin trees in
the field, followed by type I symptom, while type IV symptom was rare. Before PCR was used for the detection of GO in infected plant tissues, several
experiments relating to the optimization of the PCR condition were conducted.
Results indicated that the best sample of citrus tissues for DNA extraction was
the midrib plus the petiole. This can be shown by more intense band observed
after agarose gel electrophoresis. A positive amplification was still visible when
the reaction mixture contained 10 ng of total DNA was used. Results of the
optimization of the PCR condition indicated that the optimal PCR buffer for
amplification of GO's DNA was the standard buffer containing 78 mM Tris-HCI
(pH 8.8), 17 mM (NH4hS04, 10 mM ()-mercaptoethanol and 200)1g of Bovine
Serum Albumin (BSA). The optimal concentrations of MgClz, d NTP, primer and
Taq DNA polymerase to be used in reaction mixture were 1. 5 mM, 0.2 mM,
0.4uM, and 1 Unit, respectively. The optimal annealing temperature and
num ber of cycles of PCR condition were 55°C and 40 cycles, respectively.
The 16S rONA fragments of the GO in expected size of 1160 bp were detected
in each typical symptoms. These fragments were amplified from DNA extracted
from mandarin cultivars infected with the GO and were not amplified from DNA
extracted from healthy trees. These fragments were also detected in insect
vector (Diaphorina citn) collected from GO-infected mandarin trees and were
not amplified from DNA extracted from healthy vector collected from Murayya
paniculata using cetyl trimethyl ammonium bromide (CTAB) method for DNA
extraction. |
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