it contain the detail of banana viruses in weed plants using DAC-ELISA. This is more use full for your study and project work

1. PRESENTED BY
SHONE THANKACHAN SHAJI
REG.NO:140021101373
M.M.N.S.S.COLLEGE
KONNI,PATHANAMTHITTA
Undertaken at
KERALA AGRICULTURAL
UNIVERSITY
BANANA RESEARCH STATION
KANNARA.,THRISSUR

2. INTRODUCTION
 Banana is the one of the world most important tropical fruit crop.
 Banana grown in south India can be broadly grouped into three type
like deserts , culinary and dual purpose varieties.
 About 23.2millions tons banana production in India
 So banana cultivation has high socio-economic and agricultural impact
in India.
 But some phytopathogens are the major setback to banana cultivation.
 There are mainly four viruses known to naturally infect banana widely
in different countries are BBTV, CMV, BBrMV, BSV.

3. Sl.no Disease Causal virus
1 Bunchy top Banana Bunchy Top Virus (BBTV)
2 Infectious chlorosis Cucumber mosaic virus (CMV)
3 Bract mosaic Banana bract mosaic virus (BBrmv)
4 Streak Banana streak virus (BSV)
5 Mosaic Tobacco mosaic virus (TMV)
6 Die-back Nepovirus
7 Potexvirus
8 Abaca mosaic Abaca mosaic virus (AbaMV)

4. Cucumber mosaicvirus
 Cucumber mosaic virus (CMV) is a plant pathogenic virus in the family
Bromoviridae.
 It is the type member of the plant
virus genus, Cucumovirus.
 CMV is found worldwide, is very
easily spread and causes damage
to the host.
 CMV can be transmitted
by more than 60 different
aphid species and other vector.

5. Bananabunchy top virus
 Banana bunchy top is a viral disease caused by a single-stranded
DNA virus called the Banana Bunchy Top Virus (BBTV).
 BBTV is the type species of
the genus Babuvirus in the
family Nanoviridae.
 Genome of BBTV is composed
of at least six DNA components.
 It was first identified in Fiji in 1891

6. Bananabract mosaic virus
 Banana bract mosiac virus (BBrMV) is a plant pathogenic virus of the
family potyviridae.
 Banana bract mosaic virus
(BBrMV) was first isolation
from banana in the Philippines
in 1979
 The virions lack an envelope and
are flexuous, filamentous rods
720 to 850 nm long and 12-15 nm
in diameter

7. Direct antigencoating(DAC)-ELISA
 This is the simplest of ELISA, and also referred as Antigen Coated Plate
(ACP)-ELISA.
 Antigen is bound to the plate surface.
 In the second step, polyclonal antiserum or IgGs are used to detect the
trapped homologous antigen.
 The main advantage with DAC-ELISA is one secondary antibody can be
utilized with several systems.
 Enzyme substrate , p-nitro phenyl phosphate (PNPP) is added into the
ELISA wells to develop positive reactions.

8. Aim
 To detection of banana viruses in weed plant through DAC-ELISA
from the field of banana research station, Kannara.
 This will help to disease resistance of banana plant.

9. MATERIALS
 96 well microtitret plate
 Micro centrifuge tube 1.5 ml
 Micropipette and microtips
ELISAplatereader(BIO-RAD)
Centrifuge
Incubator

10. REAGENTS REQUIRED
1. Coating buffer pH 9.2 (10x)
Sodium carbonate – 15.9g
Sodium bicarbonate -29.3g
Sodium nitrate -2.8g
Distilled water -1000ml
2. Phosphate buffered saline (PBS) pH 7.4 (10x)
Sodium chloride -80g
Potassium dihydrogen phosphate -2g
Disodium hydrogen phosphate -11.6g
Potassium chloride -2g
Distilled water -1000ml
3. Wash buffer i.e, PBS-Tween(PBST)
For 1 litre of PBST add 0.5ml of tween-20
4. Blocking buffer
5g of spray dried milk to 100ml of PBST

11. 5. Antibody Diluent Buffer/Enzyme Conjugate Diluent
Buffer(PBS-TPo)
Add 20g of polyvinyl pyrrolidine PVP and 2g of Bovine serum
albumin to 1 litre of PBST
i. Primary antibody concentration rate 1:500 dilution
ii. Seconday antibody concentration rate 1:1000 dilution
6. Substrate buffer pH 9.8
Diethanolamine -9.7ml
Distilled water –made upto100ml
Store in brown colored bottle
7. Substrate buffer solution
1mg of para nitro phenyl phosphate (PNPP) per 1ml of
substrate buffer.

12. procedure
Collection of weeds
We collect health weeds from outside from the farm and we collect the
disease weeds from the farm. There are various of weed are collected. There are
Kallurukki, Poochavally, Glyricidia, Ageratum, Ponnakanni, Thondi, Makania,
Thamazytham, Cleome, Clitoria, Vayara, Node weed
Coating ELISA plate with antigen
1. Take 1 g of weeds and grind the test weeds in carbonate coating
buffer at 1.5w/v and using a sterile mortar and pestle. Transfer the sample
to 1.5ml micro centrifuge tubes and centrifuge at 5000rpm for 10 minutes.
2. Dispense 100ul of sample into ELISA micro test plate containing 2%
PVP dissolved in 1x coating buffer (100ul) and incubate for 1 hour at
incubate for 1 hour at 37oC
3. Wash the plate with three change of PBS-T buffer, allowing 3 minutes
for each wash. Then add 200ul of blocking buffer. Then plate were kept for
incubation at 37oC for 1 hour.
4. Wash the plate with three change of PBS-T buffer. Then the plate are
load with 200ul of primary antibody solution (1:1000v/v). Cover the plate
and incubate at 4oC for overnight.

13. Delection usingalp system
 On day two,dilute anti-rabbit ALP conjugate to (1:10,000v/v) in PBS-
TPo, wash the plates with three change of PBS-T add 200ul of
secondary antibody solution and incubate at 37oC for 2 hour.
 Wash the plate and add 200ul of pNPP substrate solution into each
well and cover the plates and incubate in dark at room temperature for
30 min (Note: Substrate solution turns yellow when exposed to light for
long time.)
 Take reading of the plate using ELISA plate reader.
 Sample with 405nm values. Twice or Thrice than health sample reading
are considered as virus positive

14. Result
As we take 13
weeds
we get
CMV BBTV BBRMV
Sl.no weed healthy disease healthy disease Healthy Disease
1 kallurukki 0.193 0.1105 0.0815 0.0915 0.212 0.227
2 Poochavally 0.1345 0.244 0.1265 0.165 0.1525 0.293
3 Glyricidia 0.1505 0.136 0.082 0.0835 0.2335 0.2365
4 Ageratum 0.1285 0.333 0.091 0.1595 0.1375 0.335
5 Ponnakanni 0.1105 0.5765 0.077 0.1335 0.1875 0.33
6 Thondi 0.1265 0.1925 0.076 0.1225 0.169 0.2015
7 X2 0.2075 0.1395 0.151 0.1165 0.186 0.1405
8 Makania 0.2075 0.202 0.092 0.0765 0.1085 0.1315
9 Thamazytha
ma
0.216 0.482 0.1915 0.097 0.481 0.254
10 Cleome 0.091 0.1245 0.089 0.5015 0.1105 0.69
11 Clitoria 0.369 0.2985 0.128 0.169 0.4585 0.7155
12 Vayara 0.128 0.1485 0.1 0.13 0.34 0.375
13 Nod weed 0.1525 0.5495 0.091 0.1315 0.1475 0.6515

15. Image of test plate

16.  By taking twice the value of healthy weeds. There are many weed are
disease affect
 They are
CMV
Sl.no Weeds Healthy
Disease
1 Ageratum 0.1285 0.333
2 Poonakanni 0.1105 0.5765
3 Thamazythama 0.216 0.482
4 Nod weed 0.1525 0.5495

17. BBTV
BBrMV
Sl.no Weeds Healthy Disease
1 Cleome 0.089 0.5015
Sl.no Weeds Healthy Disease
1 Ageratum 0.1375 0.335
2 Cleome 0.1105 0.609
3 Nod weed 0.1475 0.6515

18. DISCUSSION
this weed are observed as positive. So that remove this weed from field.
Otherwise the banana get disease infection