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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Main Author:	Mahmud Sara, Muh. Asaad
Format:	Thesis
Language:	English English
Published:	2001
Subjects:	Organisms - Polymerase chain reaction - Case studies
Online Access:	http://psasir.upm.edu.my/id/eprint/10548/1/FP_2001_8_A.pdf
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spelling	my-upm-ir.105482011-05-04T03:41:06Z Detection of Citrus Greening Organism (Liberobacter Asiaticum) by Polymerase Chain Reaction 2001-09 Mahmud Sara, Muh. Asaad 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. Organisms - Polymerase chain reaction - Case studies 2001-09 Thesis http://psasir.upm.edu.my/id/eprint/10548/ http://psasir.upm.edu.my/id/eprint/10548/1/FP_2001_8_A.pdf application/pdf en public masters Universiti Putra Malaysia Organisms - Polymerase chain reaction - Case studies Faculty of Agriculture English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Organisms - Polymerase chain reaction - Case studies
spellingShingle	Organisms - Polymerase chain reaction - Case studies Mahmud Sara, Muh. Asaad Detection of Citrus Greening Organism (Liberobacter Asiaticum) by Polymerase Chain Reaction
description	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.
format	Thesis
qualification_level	Master's degree
author	Mahmud Sara, Muh. Asaad
author_facet	Mahmud Sara, Muh. Asaad
author_sort	Mahmud Sara, Muh. Asaad
title	Detection of Citrus Greening Organism (Liberobacter Asiaticum) by Polymerase Chain Reaction
title_short	Detection of Citrus Greening Organism (Liberobacter Asiaticum) by Polymerase Chain Reaction
title_full	Detection of Citrus Greening Organism (Liberobacter Asiaticum) by Polymerase Chain Reaction
title_fullStr	Detection of Citrus Greening Organism (Liberobacter Asiaticum) by Polymerase Chain Reaction
title_full_unstemmed	Detection of Citrus Greening Organism (Liberobacter Asiaticum) by Polymerase Chain Reaction
title_sort	detection of citrus greening organism (liberobacter asiaticum) by polymerase chain reaction
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Agriculture
publishDate	2001
url	http://psasir.upm.edu.my/id/eprint/10548/1/FP_2001_8_A.pdf
_version_	1747811148056494080

Detection of Citrus Greening Organism (Liberobacter Asiaticum) by Polymerase Chain Reaction

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