Detection of Citrus Tristeza Virus (CTV) on Lime Leaves Using Enzyme Linked Immunosorebent Assay (ELISA).
A. A. Al-Badi1
1Department of crop sciences, college of Agricultural and marine Sciences , Sultan Qaboos University ,P.O Boox34,Al-KHOD 123,Oman.
Abstract
Six sample of lime leaves were tested in enzyme linked immunosorbent assay
(ELISA) to investigate whether it has Citrus Tristeza Virus or not .the sample were collected from deferent parts of Oman. This test will give an idea about the distribution of the virus among the tested area. After the analyses the disease find on .. and other area were clean
Keywords: Citrus Tristeza Virus ,lime, ELISA,Oman.
Introduction
Citrus Tristeza Virus (CTV) of citrus is worldwide problem. It effects all kind of citrus tree. There are millions of lime tree destroy since 1910 worldwide(Agrios , 2005) The disease has also affected citrus in the Mediterranean area including Spain (Moreno et al., 1955) Israel (Raccah et al., 1976), Egypt (Nour-Eldin and Bishay, 1955), Turkey (Norman, 1963) and Cyprus (Kyriakou et al., 1993) .In Oman there was 39% of the CTV ; zero % in Al-Batinah, 29.7% and 71.5% in Muscat, 42.4% and zero% in Dhofar. No incidence of CTV was found in Al-Dhakhliah and Al-Sharqiah. CTV spread in grafted citrus trees as well as lime trees produced from seeds indicated that the vector was involved in the transmission (Ministry of Agriculture2009).
The CTV cause huge loses of fruit quality and quantity and eventually the death of the infected tree by decline. Sometimes the decline fast and in other times the developed slowly. Other symptom of CTV is stem pitting ,and dwarfing and chlorosis (Agrios ,2005).
To detect the Citrus Tristeza Virus a useful technique were use called the enzyme linked immunosorbent assay (ELISA) (Bar. Joseph et al., 1979).The main idea of ELISA is to get yellow color for the infected sample while free-disease sample remain colorless the color form because the engagement of the virus and the specific antibody and the specific enzyme.
The main objective of this experiment was (i)to learn how to imply ELISA on lime leave, and (ii)to get an idea of distribution of CTV on Oman. The result of this two objective will help us in the future in ELISA application and knowing lime CTV.
Material and methods
First at all, the samples of lime leave were collected from different part of Oman. Then the samples were prepared; one gram of midribs was segregated and grinded with 10ml of extraction buffer. The second step were pipette 100 ul of the samples into duplicated wells and incubated it at 37C for 1 hour and also tow known control one as positive and one as negative were added to the wells. Next the plate was removed from the incubator and washed by 200ul of wash buffer .the previous step was repeated tow time. After that 100ul of conjugate (enzyme) were added to the duplicated wells and then was leaved one hour on the incubator at 37 C. After one hour the duplicated wells were filled by 200ul wash buffer to wash. The previous step was repeated three times to make stainless sample i.e. unwonted residue like unconnected conjugate. Then 100ul of substrate were added and the substrate is a substance reacts with the conjugate to get yellow to green color. Then the wells were warp and incubated at room temperatures in dark place for one hour. Finally the samples were entered in the spectrophotometer at 405nm.Only one sample show clear evidence of infection.
Result
The wells that turn yellow contain the virus against the colorless one, however it is difficult to detect the change by naked eyes. All ridings was multiply by 2 to make clear indication of the difference between the control and the sample. So the spectrophotometer was used and the following readings were shown:
Table 1. spectrophotometer result of ELISA test of CTV.
The sample well #
|
location
|
Reding 1
|
Reading 2
|
Average
|
X 2
|
Negative (control)
|
-
|
0.093
|
0.094
|
0.0935
|
0.187
|
Positive (control)
|
-
|
0.153
|
0.148
|
0.1505
|
0.301
|
A(W152 y-6)
|
Qaraiaat
|
0.096
|
0.106
|
0.101
|
0.202
|
B(W152 y-1)
|
Qaraiaat
|
0.873
|
0.889
|
0.881
|
1.762
|
C(W154 y-5)
|
Qaraiaat
|
0.110
|
0.116
|
0.113
|
0.226
|
D (W154 y-4)
|
Qaraiaat
|
0.089
|
0.093
|
0.091
|
0.182
|
E(w210 0-1)
|
Al khod
|
0.086
|
0.092
|
0.089
|
0.178
|
F(w210 0-3)
|
Al khod
|
0.087
|
0.090
|
0.0885
|
0.177
|
Discussion
One out of six sample which were tested by ELISA test show clear infection with 1.762 comparing with 0.301 the positive control. This sample was from Qaraiat and the author 5 were from Alkod and also Qaraiaat. The sample (W154 y-4) show negative reading which mean that the lime tree is not infected by CTV.It seems clearly that Alkod has no CTV .From the above dada it is clearly that Oman in general has the CTV .However it is not separated all over the country. It is recommended to destroy plant B because it will spared the disease before it die ,and also it is recommended to study the tree E and F for potential use like preparative .
Acknowledgement
I would like to thank Dr.Abdullh Al-Sadi for his protocol of dealing with CTV and ELISA test and for his valuable comments on detecting Citrus Tristeza Virus.
References
-Agrios G. N (2005).plant pathology.fifth edition,Elsvier Academic press,USA.
-Minstry of Agricultur Oman ,(2009). Agriculture and livestock Research Annual Report, p. 97-162
- Bar-Joseph, M., Garnsey, SM and Gonsalves, D. (1979). Adv. Virus Res. 25: 93.
-Kyriakou A., Polycarpou D., Efstathiou A. and A. Handjinicoli 1993. Citrus tristeza virus in Cyprus. In : Proc. 12th conf.. of IOCV, India 1992: 69-72.
-Raccah, B., Loebenstein, M., Bar-Joseph, M. and Oren, Y. (1976). Transmission of tristeza by aphids
prevalent on citrus and operation of the tristeza suppression programme in Israel. In Proc. 7' Conf of IOCV, Greece, pp. 47-49.
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Detection Witch broom disease of lime (Phaytoplasma) using Nested PCR
Ali. A. Al-Badi1
1Department of crop sciences, college of Agricultural and marine Sciences , Sultan Qaboos University ,P.O Boox34,Al-Khod 123,Oman.
Abstract
Witches broom disease is serious diseases that attract the citrus tree. This disease starts in Oman and it's now separate inside and outside the country. This experiment deal with 16 sample of citrus tree from different part of Oman .the sample were tested by normal PCR and nested PCR to get clear result of infection. The result show huge infection of the citrus tree that was tested.
Keywords:
Witch brome disease, PCR, gel electrophoresis, lime, Oman.
Introduction
There are more than 1600 spices of citrus in the world .however, there are 5 species most important, which are C.sinensis ,C,reticulate,C.paradisi ,C,limon and C.aurantiflia (Rieger 2006). In Oman citrus are important fruit crops. Its cover about 4% of the fruit area about 1,691 Ha. (Ministry of Agriculture 2005).
Witches broom disease (WBDL) is an important disease that effect Oman lime .the WBDL appeared in the Northern coastal plain of the Sultanate of Oman, near the border with the United Arab Emirates (UAE)probablyin the 1970s. Since then, the disease has spread within Oman and extends all along the coastal plain from the border with the UAE to Muscat (Joseph. M et al).the WBDL caused by MLO or phytoplasma called Candidatus Phytoplasma aurantifolia (M. M. Faghihi et al , April 2011)
The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular sequence. The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (Bartlett & Stirling 2003).
The nested PCR is a type of PCR.However this type is more specific to the DNA and it use the product of the (normal)PCR,also use the primers R16R2 and R16F2n ,buffer and the nuclutaids. This method will be used during this experiment and the result will be negative /positive infection.
The main objective of this experiment was to get an idea of distribution of phytoplasma across the country using PCR,and to get more knowledge about the daisies because there is lake of knowledge about this disease .This will help in future managements or maybe in reduce spread the of disease in Oman
Material and methods
DNA extraction
First at all, the samples of lime leave were collected from different part of Oman. Then the samples were prepared; one gram of midribs was segregated and grinded by liquid nitrogen.Nexet 100mg were weight and placed on eppendorf tube. Then 400 ul of AP1 buffer were added to lyses the cells. Then the eppendorf tube was vortexes. After that the eppendorf tube which contain the sample were incubated at 65Co for 20 minutes and inverted 2-3 time in the incubators .The next step was by added 130ul of AP2 buffer which will precipitated the debris ,protein and polysaccharide .them the mixture were incubate for 5 minutes on ice. Then the sample was centrifuged for 5 minutes at 14,000 rpm. The next step, the sample were transfer to Qmsc tube and centrifuged for 5 minutes at 14,000 rpm.Then the supernatant were transferred to new eppendorf tube then 1.5 time supernatant of AP3 buffer were added .then the mixture were pipetted to Dmsc tube in two dose and then centrifuged for 1 minutes at 10000 rpm .After that the liquid were discard and the basket was transferred to new collection tube.
Direct PCR
First ,new .5 ml eppendorf tube were labeled .next ,1 ul of P1 and 1 ul of P7 primers were added to the tube. The next step, 2ul of DNA sample was added to the mix. Then 21 ul of Milli-Q water was added. After that the tubes were placed in the thermal cycler and the device was run at the following siting:
Denaturation 94 oC for 2 minutes
35 cycles consisting of :
Denaturation 94 oC for 30 sec.
Annealing 60 oC for 40s
Extension 72 oC for 1.5 min
Nested PCR:
First at all the product of direct PCR were diluted with deionized water by use 1ul of the product of the direct PCR and diluted it with 22 ul Mili-Q water on .5 ml eppendorf tube. Next 1 ul of R16R2 and 1 ul of R16F2n premier were added to the mixture. After that the tubes were placed in the thermal cycler and the device was run at the following siting:
35 cycles consisting of:
Denaturation 94 oC for 1 min
Annealing 60 oC for 1 min
Extension 72 oC for 1.5 min
And the final Extension at 72 oC for 7.5 min
Electrophoresis
First, 1.5% agarose gale was prepared by using 1.5g agarose and 75 ml TBE buffer and 1.5ul Ethidium bromide. Second, 2ul of the PCR product and 5 ul of nested PCR products were added and mixed with 2ul dye and then were loaded to the 1.5% agarose gale. Then the Electrophoresis was run for 40 minutes at 120V and 110 mA. Finally the gale was observed under the UV light.
Result
Table1: The infection that cause by witch broom disease in defiant pat of Oman
Code
|
The sample type
|
Place of collection
|
Direct PCR result
|
Nested PCR result
|
W0135-04
|
Lime
|
Nizwa
|
-
|
-
|
2
|
Lime
|
Shnas
|
+
|
+
|
W0135-05
|
Lime
|
Nizwa
|
-
|
-
|
4
|
Lime
|
?
|
-
|
+
|
5
|
Lime
|
Al kod
|
-
|
-
|
W0132-y1
|
Lime
|
Nizwa
|
-
|
-
|
7
|
Lime
|
Nizwa
|
+
|
+
|
8
|
Lime
|
?
|
-
|
+
|
9
|
Lime
|
?
|
-
|
+
|
W340
|
Lime
|
Al wasta
|
-
|
+
|
W0131
|
Lime
|
Nizwa
|
+
|
+
|
12
|
Lime
|
Bahla
|
+
|
+
|
13
|
Lime
|
?
|
-
|
-
|
14
|
Lime
|
Nizwa
|
-
|
-
|
15
|
Lime
|
Al kod
|
-
|
+
|
16
|
Lime
|
Restaq
|
-
|
-
|
Figure 1: Ethidium bromide-stained of PCR products after gel electrophoresis. 16 different lime samples were amplified.to get positive /negative infliction by phytoplasma (WBDL).
|
Figer 2: lime tree in wlayt bahla has WBDL
|
Discussion
It is clearly seen that phytoplasma which cause witch broom disease of lime (WBDL) still attack lime tree in Oman ,the result were approved by using direct and nested polymerase chain reaction (PCR).The sample that come from Shinas ,Nizwa ,AlKode and Alwasta represent presence of the disease there. However in Alrustaq and in undefined place the diseases not recorded yet. The above data confirm with ( K.-R. Chung et al) research, that say " It is estimated that over 98% of limes currently grown in Oman are infected with WBDL".
Now the diseases (WBDL) spread heavily in Oman and in other country ,to stop or reduce its expansion ,its recommended to(i) develop resistant to disease tree, and (ii) control ,or modify the transmission agent which is leafhopper (Hishimonus phycitis) (Joseph. M et al) and made it inactive.
Acknowledgement
I would like to thank Dr.Abdullh Al-Sadi and Mr. Isaa Al Mahmoli for their priceless assistance and valuable information.
References
- Mark.R (2006).Introduction to Fruit Crops .First edition,Food production press,New York ,USA.
-Agrios G. N (2005).plant pathology.Fifth edition,Elsvier Academic press,USA.
-M. M. Faghihi, A. N. Bagheri, H. R. Bahrami, H. Hasanzadeh, and R. Rezazadeh, Hormozgan ,(2011). Witches'-Broom Disease of Lime Affects Seed Germination and Seedling Growth But Is Not Seed Transmissible, 419-422 pp.
-Minstry of Agricultur Oman, (2009). Agriculture and livestock Research Annual Report, 97-162 pp
- Bar-Joseph, M., Garnsey, SM and Gonsalves, D. (1979). Adv. Virus Res. 25: 93.
-Joseph M. BovC, Leyla Zreik, Jean-Luc Danet, Jacques Bonfils, A. M. M. Mjeni
and Monique Garnier. Witches' Broom Disease of Lime Trees: Monoclonal Antibody and DNA Probes for the Detection of the Associated MLO and the Identification of a Possible Vector ,342-348 pp.
-Minstry of Agricultur Oman, (2005). Agriculture and livestock Research Annual Report
- Bartlett & Stirling (2003)—A Short History of the Polymerase Chain Reaction. In: Methods Mol Biol. 226:3-6
- K.-R. Chung, I. A. Khan, and R. H. Brlansky(2006). Citrus Diseases Exotic to Florida: Witches' Broom Disease of Lime (WBDL) PP-228
