Detection & Detection & Control Of Banana Control Of Banana Bunchy Top VirusBunchy Top Virus

Mazhar UllahMazhar Ullah

Class # 08Class # 08

IntroductionIntroduction

BananaBanana is the common name for is the common name for herbaceous plants of the genus herbaceous plants of the genus MusaMusa and for the fruit they produce. and for the fruit they produce.

Bananas come in a variety of sizes Bananas come in a variety of sizes and colors when ripe, including and colors when ripe, including yellow, purple, and red. yellow, purple, and red.

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Bananas are a staple starch for many Bananas are a staple starch for many tropical populations. tropical populations.

Depending upon cultivar and ripeness, the Depending upon cultivar and ripeness, the flesh can vary in taste from starchy to flesh can vary in taste from starchy to sweet, and texture from firm to mushy. sweet, and texture from firm to mushy.

Both skin and inner part can be eaten raw Both skin and inner part can be eaten raw or cooked. or cooked.

Bananas' flavor is due, amongst other Bananas' flavor is due, amongst other chemicals, to isoamyl acetate which is one chemicals, to isoamyl acetate which is one of the main constituents of banana oil.of the main constituents of banana oil.

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Banana bunchy top virus (BBTV) is one Banana bunchy top virus (BBTV) is one of the most serious diseases of banana.of the most serious diseases of banana.

Once established, it is extremely Once established, it is extremely difficult to eradicate or manage. difficult to eradicate or manage.

BBTV is widespread in Southeast Asia, BBTV is widespread in Southeast Asia, the Philippines, Taiwan, most of the the Philippines, Taiwan, most of the South Pacific islands, and parts of South Pacific islands, and parts of Pakistan, India and Africa. (Saleem et Pakistan, India and Africa. (Saleem et al., 2005)al., 2005)

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Banana bunchy top virus is spread by the banana Banana bunchy top virus is spread by the banana aphid, which acquires the virus after at least four aphid, which acquires the virus after at least four (but usually about 18) hours of feeding on an (but usually about 18) hours of feeding on an infected plant (Naseem et al., 2010).infected plant (Naseem et al., 2010).

The aphid can retain the virus through its adult life, The aphid can retain the virus through its adult life, for a period of 15–20 days. for a period of 15–20 days.

During this time, the aphid can transmit the virus During this time, the aphid can transmit the virus to a healthy banana plant by feeding on it, possibly to a healthy banana plant by feeding on it, possibly for as little as 15 minutes but more typically for for as little as 15 minutes but more typically for about two hours. about two hours.

Disease symptoms usually appear about a month Disease symptoms usually appear about a month after infectionafter infection..

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The initial symptoms of banana bunchy top The initial symptoms of banana bunchy top virus consist of dark green streaks in the virus consist of dark green streaks in the veins of lower portions of the leaf midrib and veins of lower portions of the leaf midrib and the leaf stem.the leaf stem.

Also, dark green, hook-like extensions of Also, dark green, hook-like extensions of theleaf lamina veins can be seen in the theleaf lamina veins can be seen in the narrow, light-green zone between the midrib narrow, light-green zone between the midrib and the lamina.and the lamina.

The short hooks point down along the midrib The short hooks point down along the midrib toward the petiole. They can best be seen toward the petiole. They can best be seen by back-lighting the leaf against the sky.by back-lighting the leaf against the sky.

Annual Losses In Annual Losses In PakistanPakistan

In Pakistan Sindh is the main producer of In Pakistan Sindh is the main producer of bananabanana

Annual loses of banana in Pakistan Because of Annual loses of banana in Pakistan Because of BBTV:BBTV:

Sindh 19 Million RupeesSindh 19 Million Rupees

KPK 8 Million RupeesKPK 8 Million Rupees

Punjab 13 Million RupeesPunjab 13 Million Rupees

Balochistan 5 Million RupeesBalochistan 5 Million Rupees

Review Of LiteratureReview Of Literature

Mazhar & Waheed (1988) isolated & Mazhar & Waheed (1988) isolated & characterized BBTV from different banana characterized BBTV from different banana species using ELISAspecies using ELISA

Humayoun et al (1999) confirmed that BBTV is Humayoun et al (1999) confirmed that BBTV is an RNA virus.an RNA virus.

Dmtzgen & Franeki (1988) reported that BBTV Dmtzgen & Franeki (1988) reported that BBTV can be isolated & detected through ELISA.can be isolated & detected through ELISA.

ObjectivesObjectives

To detect BBT virus.To detect BBT virus.

To control BBT virus.To control BBT virus.

To characterize BBT virus.To characterize BBT virus.

MethodologyMethodology

The present research study will be The present research study will be conducted at Institute Of conducted at Institute Of Biotechnology And Genetic Biotechnology And Genetic Engineering (IBGE) Khyber Engineering (IBGE) Khyber Pakhtunkhwa Agricultural University Pakhtunkhwa Agricultural University Peshawar during 2011-12.Peshawar during 2011-12.

ELISA kit will be purchased from KPL ELISA kit will be purchased from KPL companies Lahore.companies Lahore.

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This research will be carried out on This research will be carried out on the virus isolated banana Xthe virus isolated banana X2 2 specie.specie.

Samples will be collected from Samples will be collected from different areas of the country.different areas of the country.

Sample PreparationSample Preparation

Leaf samples of about 1g will be Leaf samples of about 1g will be taken from the infected banana taken from the infected banana tree.tree.

The sample will be crushed in 5ml of The sample will be crushed in 5ml of extraction buffer using fine extraction buffer using fine abrasive.abrasive.

Then this viral extract will be Then this viral extract will be filtered with nylon cloth.filtered with nylon cloth.

Detection Of Viral Detection Of Viral PresencePresence

The presence of viral particles will be The presence of viral particles will be detected through ELISA test.detected through ELISA test.

The ELISA plates will be coated with The ELISA plates will be coated with BBTV specific antibodies & will be BBTV specific antibodies & will be incubated at 34C for 2-3 hours.incubated at 34C for 2-3 hours.

Then the plates will be washed.Then the plates will be washed. Then the viral sample (antigen) will be Then the viral sample (antigen) will be

introduced to the wells of ELISA plates.introduced to the wells of ELISA plates.

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The antigen added ELISA plates will be The antigen added ELISA plates will be incubated at 4C overnight.incubated at 4C overnight.

Washing will be done again.Washing will be done again. Then the ELISA plates will be coated with Then the ELISA plates will be coated with

specific antibodies linked with enzymes specific antibodies linked with enzymes that will bind to the antigen.that will bind to the antigen.

Then Phenol phospho nitrate (PNP) will be Then Phenol phospho nitrate (PNP) will be added to break the antibody-antigen added to break the antibody-antigen assembly.assembly.

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It will give yellow colour which It will give yellow colour which indicates the presence of virus.indicates the presence of virus.

The intensity of the yellow colour The intensity of the yellow colour shows the intensity of viral shows the intensity of viral infection.infection.

Control Of BBTV By Control Of BBTV By Pathogen Derived Pathogen Derived Resistance (PDR)Resistance (PDR)

DNA will be extracted from the isolated DNA will be extracted from the isolated viral particles .viral particles .

Core Protein (CP) gene will be isolated Core Protein (CP) gene will be isolated from the viral genome using proper from the viral genome using proper restriction enzymes.restriction enzymes.

The CP gene will be amplified through The CP gene will be amplified through PCR.PCR.

The gene will be loaded on a binary The gene will be loaded on a binary vector.vector.

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Then this gene will be introduced to Then this gene will be introduced to banana by banana by A.tumefaciens.A.tumefaciens.

The gene will be incorporated to The gene will be incorporated to banana’s genome by banana’s genome by A.tumefaciens A.tumefaciens through lateral DNA transfer.through lateral DNA transfer.

This gene will confer banana plant This gene will confer banana plant resistance against BBTV.resistance against BBTV.

Literature CitedLiterature Cited

Naseem, Z., M. Ihsan & M. Rameez. (2010). The Naseem, Z., M. Ihsan & M. Rameez. (2010). The study of BBTV symptoms in different species of study of BBTV symptoms in different species of banana. Journal of Virology 10(15):313-320.banana. Journal of Virology 10(15):313-320.

Dmtzgen, R. G. & Franeki, R. I. B. (1988). Dmtzgen, R. G. & Franeki, R. I. B. (1988). Analysis of lettuce necrotic yellows virus Analysis of lettuce necrotic yellows virus structural proteins with monoclonal antibodies structural proteins with monoclonal antibodies and concanavalin A. Virology 166, 486-494. and concanavalin A. Virology 166, 486-494.

Saleem, A., Q.A.Arbab & H.U.Jan. (2005). Saleem, A., Q.A.Arbab & H.U.Jan. (2005). Prevalence of BBTV in different areas of Nigeria. Prevalence of BBTV in different areas of Nigeria. African journal of virology 9(6):107-110.African journal of virology 9(6):107-110.

Humayoun, F., F.Ali & W. A. Khan. (2006).Isolation Humayoun, F., F.Ali & W. A. Khan. (2006).Isolation & genotyping of BBTV using ELISA. International & genotyping of BBTV using ELISA. International Journal Of Food Microbiology 87(03):109-112. Journal Of Food Microbiology 87(03):109-112.

Budget:Budget:

Fixed Budget:Fixed Budget:S.NoS.No Year 1Year 1 CostCost Year 2Year 2 CostCost TotalTotal

1.1.

2.2.

ELISA ELISA KitKit

IncubatIncubatoror

50,00050,000

50,00050,000

-- -- 1,000001,00000

3.3. -- -- PCRPCR 1,000001,00000 1,000001,00000

Grand Grand totaltotal

200000200000

Recurrent budgetRecurrent budgetS.NoS.No Year 1Year 1 AmountAmount Year 2Year 2 amountamount TotalTotal1.1. ChemicalsChemicals 10,00010,000 ChemicalsChemicals 10,00010,000 20,000 20,000

2.2. GlasswarGlasswareses

5,0005,000 GlasswarGlasswareses

5,0005,000 10,00010,000

3.3. StationaryStationary 10,00010,000 StationaryStationary 10,00010,000 20,00020,000

4.4. ResearcheResearche

r,sr,s salarysalary50,00050,000 ResearcheResearche

r,s salaryr,s salary50,00050,000 1,000001,00000

5.5. Honraria Honraria 30,00030,000 HonrariaHonraria 30,00030,000 60,00060,000

6.6. TravellingTravelling 50,00050,000 TravellingTravelling 50,00050,000 100,000100,000

7.7. seminarsseminars 100,000100,000 seminarsseminars 100,000100,000 200,000200,000

8..8.. MiscellaneoMiscellaneo

usus 200,000200,000 MiscellaneoMiscellaneo

usus200,000200,000 400,000400,000

TotalTotal -- -- - - -- 9,10,0009,10,000

Total BudgetTotal Budget

Total Budget = Fixed Budget + Total Budget = Fixed Budget + Recurrent BudgetRecurrent Budget

= 200,000 +9,10,000= 200,000 +9,10,000

=11,10,000=11,10,000