RPF-III

PERFORMA FOR SUBMISSION OF FINAL REPORT OF RESEARCH PROJECTS Part- I : General Information

800 Project Code : Path. XII (813) 8001 Institute Project Code No. : ICAR-CP2 8002 ICAR Project Code No. 801 Name of the Institute and Division 8011 Name and address of Institute : Indian Institute of Spices Research, Calicut �673 012, Kerala 8012 Name of Division / Section : Division of Crop Protection 8013 Location of the Project : IISR, Calicut, Kerala, India 802 Project Title : Identification and development of diagnostics for the viruses causing stunted disease in black pepper 803 Priority Area : Crop Protection 8031 Research Approach : 804 Specific Area : Identification & development of diagnostics against pathogen 805 Duration of Project : 3 years 8051 Date of start : 1st July 2003 8052 Date of Completion : 30th June 2006 806 Total cost /Expenditure Incurred : : Rs 17,10,502 (Give reasons for variation, if any from original estimated cost) 807 Executive Summary

Random survey of black pepper plantations at the major black pepper growing regions of Karnataka and Kerala revealed that incidence of the disease was highest in Wyanad district (45.4%) followed by Idukki district (29.4%) in Kerala. In Karnataka, Kodagu district (14.9%) had the highest incidence of the disease

Applied Res.

Basic Res. Process or Tech. Dev.

Transfer of Tech.

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followed by Hassan district (5.2%). In general, disease incidence and severity were more in Kerala than in Karnataka. Among the areas surveyed incidence and severity were more in the black pepper plantations situated at higher altitudes such as Idukki and Wyanad. Mosaic, reduction in leaf size and internodal length leading to the stunting and bright yellow mottling along the veins were the important symptoms observed on diseased vines. All cultivars and improved varieties including hybrids were susceptible to the disease under natural conditions. Vines of all ages were found affected by the disease. A few of the weeds found in and around black pepper plantations showed typical virus symptoms, which might act as potential virus source. When isolates were subjected to immunological analysis through enzyme linked immunosorbent assay (ELISA) using antisera to different viruses, majority of the isolates reacted either to Cucumber mosaic virus (CMV) (Genus: Cucumovirus) or Banana streak virus (BSV) (Genus: Badnavirus) indicating the involvement of at least two viruses with the stunted disease of black pepper.

CMV isolated from naturally infected black pepper was propagated on Nicotiana benthamiana and N. glutinosa. CMV particles were purified from these hosts by differential centrifugation and sucrose density gradient centrifugation. Electron microscopy of negatively stained purified preparations showed the presence of isometric particles of about 28 nm in diameter. The antiserum against CMV was produced in New Zealand white rabbit. Immunoglobulin G (IgG) was purified from the crude polyclonal antiserum and coupled with the enzyme alkaline phosphatase. Double antibody sandwich (DAS) ELISA method was standardized for the detection of CMV in diseased black pepper leaves collected from different regions of Karnataka and Kerala.

The badnavirus infecting black pepper was identified as a strain of Piper yellow mottle virus (PYMV). PYMVparticles were purified from infected black pepper leaves. The purification procedure included extraction of sap, clarification and precipitation of virus particles by polyethylene glycol followed by differential and sucrose density gradient centrifugation. Electron microscopy of negatively stained purified preparation showed the presence of bacilliform particles of about 120 x 30 nm in diameter. The antiserum against badnavirus was raised in New Zealand white rabbit by injecting the purified virus particles. Immunoglobulin G (IgG) was purified from the crude polyclonal antiserum and coupled with the enzyme alkaline phosphatase. Double antibody sandwich (DAS) ELISA method was standardized for the detection of badnavirus in diseased black pepper leaves collected from different regions of Karnataka and Kerala.

DAS-ELISA procedure was used to index mother vines of black pepper. Of the 2186 black pepper mother vines belonging to eleven different varieties indexed for the presence of PYMV and CMV, 714 plants were tested positive for either one or both the viruses. More than 50% of DAS-ELISA positive black pepper mother vines did not exhibit any external symptoms indicating the need of sensitive technique like ELISA to identify virus-free plants. To know the variation in virus titre, virus infected black pepper plants belonging to five varieties were tested by DAS-ELISA for PYMV and CMV at monthly intervals from April 2004 to March 2005. Results indicated that the concentration of both the viruses varied during different months of the year. The concentration of both the viruses was found to be higher during October to January. Three varieties of infected black pepper vines were selected to study the distribution of PYMV and CMV in

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different parts of the plant through DAS-ELISA. Results indicated that concentration of PYMV was more in young leaf, old leaf and stem followed by spike and root. The concentration of CMV was found to be more in young leaf and old leaf followed by stem, spike and root.

An effective and reproducible protocol for meristem culture in black pepper was established. It was also established that the same technique can be employed successfully in elimination of CMV and PYMV infecting black pepper. The use of TDZ-containing medium to induce the initial growth of dissected meristem and subsequent transfer of the enlarged meristem to MS containing 1mg/l BAP and 1mg/l IAA was found to be promising. The fully developed shoots were then transferred to Woody Plant Medium (WPM) containing 0.5% charcoal without any growth regulator for rooting. The rooted plantlets were hardened under green house conditions and screened for the presence or absence of PYMV and CMV through ELISA and Polymerase Chain Reaction (PCR).

808 Key words : black pepper, stunted disease, identification,

Cucumber mosaic virus, Piper yellow mottle virus, ELISA, detection, indexing.

Part-II : Investigator Profile (Please identify clearly changes, if any in Project personnel)

810 Principal Investigator : 8101 Name : Dr. A.Ishwara Bhat 8102 Designation : Senior Scientist 8103 Division/ Section : Crop Protection 8104 Location : Indian Institute of Spices Research 8105 Institute Address : Indian Institute of Spices Research Calicut-673 012, Kerala, India 811 Co- Investigator: 8111 Name : Dr. R. Suseela Bhai 8112 Designation : Senior Scientist 8113 Division/ Section : Crop Protection 8114 Location : Indian Institute of Spices Research 8115 Institute Address : Calicut-673 012, Kerala, India

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Par-III: Technical Details 820 Introduction and objectives 8201 Project Objectives:

i) Identification of the causal viruses associated with the stunted disease of black

pepper.

ii).Production of polyclonal antisera against each of the viruses.

iii) Production of sero-diagnostic methodology for detection of viruses

iv) Development of virus-free planting material certification program.

8202 Background information and importance of the projects Stunted disease of black pepper (Piper nigrum L.) was first noticed in black

pepper nursery at district Agricultural Farm, Neriamangalam, Idukki, Kerala during 1975 (Paily et al., 1981). Subsequently stunted disease was recorded from almost all pepper growing regions in South India (Sarma et al., 2001). Disease of a similar nature was also reported from Brazil, Indonesia, Malaysia, Sri Lanka and Thailand. In India, this disease is fast spreading and expected to become a major constraint as the disease is transmitted through stem cuttings used for planting. In Brazil, it is suspected to be caused by cucumber mosaic virus (CMV) (Duarte et al., 1991) while in Malaysia, symptoms were caused by a complex of viruses involving a badnavirus, a spherical virus and a clostero- like virus. In India, Sarma et al. (2001) reported CMV as one of the causal viruses associated with stunted disease. In addition Bhat et al. (2003) reported the association of a Badnavirus serologically related to Banana streak virus and Sugarcane bacilliform virus with stunted disease of black pepper based on electron microscopy, mealybug transmission and serological relationships. Use of healthy planting material and correct identification of the pathogens are essential requirements for integrated management of plant diseases. Although these requirements were recognized for a long time, but these have not been implemented effectively for the stunted disease of black pepper due to the non-availability of diagnostic kits. Sero-diagnosis is currently the method of choice for confirming and identifying viral infections in plants and vectors. Under the proposed project, viruses associated with the stunted disease of black pepper will be identified, purified and sero-diagnostic methods will be developed which will be utilized for the production of virus-free planting materials for black pepper. This will help for the development of virus-free planting material certification programme in black pepper. Besides, protocols will also be established for production of virus-free plants through meristem tip culture. This is essential for minimizing the losses caused by the stunted disease in the form of yield reduction and quality improvement. 821 Project Technical Profile 8211 Technical programme

(Indicate briefly plan of procedure, techniques, instruments and special materials, organisms, special environments etc.) Survey, collection and maintenance of isolates

Identification of causal viruses Characterization and development of diagnostics against

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Cucumber mosaic virus (CMV) Characterization and development of diagnostics against Piper yellow mottle virus (PYMV) Indexing of mother vines for viruses Distribution of CMV and PYMV in black pepper Production of virus-free black pepper by meristem culture

8212 Total man months involvement of component project workers

a) Scientific 18 b) Technical 72 c) Supporting 12

822 Final Report on the Project

Detailed report containing all relevant data with a summary of results (not exceeding 2-5 pages)

8221 Achievements in terms of targets fixed for each activity

1. Survey, collection and maintenance of isolates 1.1. Survey

Random survey of black pepper plantations at the major black pepper growing regions of Karnataka and Kerala states were taken up. Five districts of Karnataka and five districts of Kerala were surveyed for occurrence of stunted disease (Table 1). Of the two taluks of Dakshina Kannada surveyed, no incidence of stunted disease was noticed. In the Hassan district, two locations of Aloor taluk surveyed were free from the disease. However, other two taluks surveyed (Belur and Sakleshpur) showed the incidence of stunted disease rangning from 0-33% in different gardens (Table 1). All the three taluks of Madikeri (Madikeri, Somvarpet and Virajpet) showed presence of stunted disease with incidence ranging from 0-100% in different gardens. Maximum incidence was noticed at Suntikoppa where two out of three gardens showed 100% incidence of the disease (Table 1). All the three gardens at Idu under Udupi district showed incidence of the disease. Of the 15 gardens surveyed at Uttara Kannada, only one garden at Neernahalli had the disease.

In Kerala, all the five districts surveyed showed incidence of the disease. Of

the four taluks of Idukki, high incidence of the disease was noticed at Peermade and Udumbanchola taluks while no disease was observed at three gardens of Muttom under Thodupuzha taluk (Table 1). Majority of the gardens surveyed at northern districts of Kerala such as Kannur and Kasargod also showed good distribution of the disease. All the gardens surveyed at Wynad district of Kerala showed incidence of the disease ranging from 13-93%.

In general, disease incidence and severity were more in Kerala than in

Karnataka. Among the areas surveyed in Kerala, incidence and severity were more in the black pepper plantations situated at higher altitudes such as Idukki and Wynad (Table 1).

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1.2. Symptomatology Two major kinds of disease symptoms were noticed in the locations surveyed. Typical symptoms of stunting such as mosaic and mottling on leaves, reduced leaf size, brittle leaves and reduced internodal length leading to stunting of the vine were the most prominent symptoms observed in a majority of the black pepper plantations in Idukki and Wyanad Districts of Kerala (Figure 1A). The other symptoms, which were observed mainly in black pepper plantations grown at coastal plains, included chlorotic flecking, bright yellow mottling along the veins coupled with characteristic curling of leaves (Figure 1B). Reduction in spike length and poor filling of spikes were observed in most of the diseased vines. Masking of symptoms (especially in older leaves) during monsoon and winter months were also seen in many of the affected vines. The symptoms were prominently exhibited in the affected vines during March to May.

1.3. Maintenance of isolates Several isolates of black pepper showing distinct symptoms of disease

representing various geographical areas were collected. In addition, Piper longum showing mosaic mottling symptoms coupled with stunting of entire plants were also collected. Few isolates of Piper colubrinum showing mild mottle symptoms were also collected. All the collected isolates are being maintained under insect proof glass house through vegetative propagation.

A B

Fig. 1: Symptoms of viral disease affected black pepper vines: (A) Close-up view of a lateral branch of affected vine showing mottling, curling and reduction in leaf size and internode length (B) Close-up view of leaves of affected vine showing bright yellow mottling along the veins.

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Table 1. Distribution and incidence of viral disease of black pepper in Karnataka and Kerala

Incidence in different plantations (%)

State / District

Taluk Location

A B C

Mean incid-ence (%)

Adyanadka 0 0 0 0 Alangar 0 0 0 0

Bantwal

Peramogaru 0 0 0 0 Sediyappu 0 0 0 0

Karnataka Dakshina Kannada Puttur

Shibara 0 0 0 0 Chennalli 0 0 0 0 Alur Rayarakoppalu 0 7 7 5 Ankehalli 0 0 0 0 Bantenahalli 33 27 0 20

Belur

Biccodu 0 0 7 2 Bagge 0 0 0 0 Belagodu 20 13 20 18 Fatimapura 7 0 0 2

Hassan

Sakleshpur

Rajendrapura 0 0 0 0 Boikeri 33 53 0 29 Hakkathur 0 20 0 7 Heravanad 0 0 0 0 Napokulu 0 0 0 0

Madikeri

Siddapur 0 0 0 0 Almatti 0 13 7 7 Horoor 0 0 0 0 Suntikoppa 100 100 33 78

Somvarpet

Valagunda 13 27 - 20 Balale 0 20 7 9 Margoli 0 0 0 0 Mayamudi 53 27 13 31

Kodagu

Virajpet

Polibetta 7 20 13 13 Balegadde 0 0 0 0 Gadakai 0 0 0 0 Kambara kattige 0 0 0 0 Malenahalli 0 0 0 0

Uttara Kannada

Sirsi

Neernahalli 7 0 0 2 Adimali 20 7 27 18 Devikulam Vellathooval 13 20 0 11 Chottupara 33 27 13 24 Kumily 60 100 73 78

Peermade

Vandiperiyar 20 20 67 36 Thodupuzha Muttom 0 0 0 0

Chakkupallam 47 47 40 45 Kalthoty 20 13 13 15

Kerala Idukki

Udumbanchola

Meppara 0 0 27 9

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Myladumpara 20 53 7 27 Nedumkandam 67 73 53 64 Puliyamala 33 20 27 27 Rajakkad 53 20 13 29 Santhapara 0 0 0 0 Thookkupalam 37 60 67 58 Mathamangalam 0 27 20 16 Nareekamvalli 13 27 33 24

Kannur

Olayambadi 60 53 13 42 Kalikadavu 53 47 13 38 Kuramathur 7 0 7 5

Taliparamba

Taliparamba 7 27 47 27 Ayipuzha 27 20 7 18 Chalodu 0 7 0 2

Kannur

Tellicherry

Paduvilai 13 0 0 4 Balal 33 33 0 22 Kanhangad 40 60 60 53 Plachikara 40 0 0 13

Hosdurg

Vellarikundu 7 0 0 2 Kuntar 7 7 7 7 Kundankuzhi 33 27 20 27 Kuttikole 20 7 0 9 Mulleria 0 0 0 0

Kasaragod

Kasaragod

Poinachi 60 47 - 37 Chembukadavu 33 0 13 15 Kattumunda 7 0 20 9 Kodenchery 53 20 13 29 Koodaranji 67 0 33 33 Koodathai 0 0 0 0 Maranjathi 20 7 0 9 Mykavu 0 7 7 5 Nellipoyil 7 0 20 9 Pulloorampara 13 0 7 7 Shanthinagar 7 33 20 20

Kozhikode

Thiruvambadi 20 27 27 25 Chakkittapara 0 7 0 2 Chembanoda 7 0 20 9 Koorachundu 7 13 7 9 Narinada 13 13 0 9

Quilandi

Poozhithode 20 0 0 7 Kakkat 0 20 0 7 Mokeri 0 0 7 2 Naripetta 0 13 7 7

Kozhikode

Vadakara

Thottilpalam 0 0 7 2 Mananthavady Koileri 67 67 53 62

Ambalavayal 7 67 - 37 Areapally 87 13 - 50

Wyanad Sultan Batheri

Chainthakunnu 67 53 - 60

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Kenichira 67 60 - 64 Kuppadi 27 13 - 20 Nadavayal 73 93 - 83 Pulpally 13 40 - 27 Karumannam 33 33 - 33 Vengapalli 73 27 - 50

Vythiri

Vythiri 13 13 - 13 -, not surveyed

2. Identification of causal viruses The isolates were subjected to immunological analysis through enzyme linked

immunosorbent assay (ELISA) using antisera to different viruses. Results of ELISA showed that majority of the isolates reacted either to Cucumber mosaic virus (CMV) (Genus: Cucumovirus) or Banana streak virus (BSV) (Genus: Badnavirus). Thus results clearly indicate the involvement at least two viruses with the stunted disease of black pepper.

3. Characterization and production of diagnostics against Cucumber mosaic virus

3.1. Host range studies

Attempts were made to transmit CMV mechanically onto indicator hosts such as cowpea, Chenopodium album, Nicotiana benthamiana, N.glutinosa, N. tabacum cv. White Burley and Physalis floridana. Mechanical inoculations were done by extracting sap in chilled 0.1M phosphate buffer (pH 7.2) containing 0.1% 2 mercaptoethanol in a mortar kept in an ice tray. The extracted sap was rubbed on the leaves of test plants pre-dusted with celite or carborandum powder and kept for observation under insect proof conditions. Results showed that CMV could be efficiently sap transmitted to the indicator hosts (C.album, N. benthamiana, N.glutinosa, N.tabacum cv. White Burley and P. floridana). Chlorotic local lesions appeared on C. album within 3-4 days after inoculation. The symptoms appeared 7-10 days after inoculation on other hosts. Symptoms on N. benthamiana included severe puckering, mosaic mottling and downward curling of leaves while in N. glutinosa, vein clearing followed by mosaic and yellow mottling with slight curling at leaf margins were the prominent symptoms. Sap inoculated N. tabacum cv. White Burley and P. floridana plants showed dark green mosaic followed by dark green blister like symptoms on the leaves. Further in order to know the host range of CMV, plants belonging to six plant families (Table 2) were grown in pots raised under insect proof glass house and were rub-inoculated. A minimum of five plants of each species were inoculated and kept under observation for four weeks. Results showed that in addition to the above mentioned indicator hosts, CMV was also transmitted to other cultivated hosts such as Benincasa hispada, Capsicum annuum, Cucurbita pepo, Cucumis sativus, Lycopersicon esculentum and Trichosanthes anguina which showed systemic, mosaic and other related symptoms (Table 2).

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Table 2. Reaction of different plant species to Cucumber mosaic virus infecting Black pepper. Family /Plant species Symptoms Days required for

symptom expression

Chenopodiaceae Chenopodium album

CLL, NLL

3-4

Cucurbitaceae Benincasa hispada (Thunb.) cogn. Cucumis sativus L. Cucurbita pepo L. Trichosanthes anguina L.

Mo M SL CS

7-10 7-10 - 3-7

Fabaceae Cajanus cajan (L.) Millsp. Cicer arietinum Linn. Glycine max (L.) Merr. Vigna radiata (L.) Wilczek Vigna unguiculata (L.) Walp

SL SL SL SL SL

- - - - -

Malvaceae Abelmoschus esculentus L.

SL

-

Poaceae Zea mays L.

SL

-

Solanaceae Capsicum annuum L. Lycopersicon esculentum L. Nicotiana benthamiana W. Nicotiana glutinosa L. Nicotiana tabacum L.

M, LC D, M M, Mo, LC B, M, Mo, LC B, M, Mo, LC

5-7 5-7 7-10 7-10 7-10

B-blisters on leaves; CLL-chlorotic local lesion; CS-chlorotic spot; D-deformation of leaves; LC-curling of leaves; M-mosaic; Mo-mottling; NLL-necrotic local lesion; SL-symptomless.

3.2. Purification of CMV

Virus isolated from naturally infected black pepper was propagated on Nicotiana benthamiana and Nicotiana glutinosa, which were used as the source for virus purification. Purification was carried out using the procedure of Lot et al. (1972) with some modifications. Young infected leaves collected 2-3 weeks after inoculation were triturated at 1:2 dilution (w/v) in 0.5M sodium acetate buffer pH 6.4 containing 5 mM ethylenediamine tetraacetic acid (EDTA) di-sodium salt and 0.5% thioglycolic acid. The sap was expressed through muslin cloth and emulsified with an equal volume of chloroform by stirring for 30 min at 4oC followed by centrifugation at 12,000g for 10 min. The buffer layer was removed and the virus was precipitated by adding polyethylene glycol (PEG 6000, 10%) in the presence of sodium chloride

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(1.75%, w/v) with continuous stirring for 1h to precipitate the virus. The virus was pelleted down by centrifugation at 12,000g for 15 min. The resulting pellet was suspended overnight in one-fifth original volume of 5 mM sodium borate buffer, 0.5 mM EDTA, pH 9.0. Triton X 100 was added to a final concentration of 2% (v/v) in the suspension, stirred for 30 min and centrifuged at 18,000g for 25 min. The supernatant obtained was subjected to centrifugation at 45,000g for 3h in an ultracentrifuge (Beckman Optima LE 80K). The pellet was dissolved in about 3 ml of 5 mM borate buffer containing 0.5mM EDTA, pH 9.0. Further purification was carried out by layering partially purified preparation on a linear 10-40% (w/v) preformed sucrose density gradient (prepared by layering 5 ml of 10% and 10 ml each of 20%, 30% and 40% sucrose in 5 mM borate buffer containing 0.5mM EDTA, pH 9.0 and incubating overnight at 4oC). Gradients were centrifuged at 55,000g for 2h using SW 28 rotor (Beckman). The virus-containing band was located with the help of light vertically passing through the tube. The band was collected, dialyzed and concentrated by centrifugation at 45,000g for 3h. A 260/280 ratio of the virus preparation was measured using spectrophotometer (Carry 50 Bio-spectrophotometer, Australia).

The purification procedure resulted in a clean virus preparation. A single opalescent band was seen in the sucrose density gradient centrifugation. Electron microscopy of negatively stained purified preparations revealed the presence of isometric particles of about 28 nm in diameter (Fig. 2).

3.3. Antiserum production

The antiserum against CMV was produced in New Zealand white rabbit by injecting purified virus preparations intramuscularly six times at 10-day intervals. On each occasion, 500 µl purified virus-containing 0.5 mg of virus emulsified with incomplete Freund�s adjuvant (1:1, v/v) was injected. The animal was bled 15 days after the last injection and antiserum collected.

Figure 2: Electron micrograph of purified preparations of

Cucumber mosaic virus

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3.4. Isolation of IgG and preparation of enzyme conjugate Immunoglobulin G (IgG) was purified from the crude polyclonal antiserum by

affinity chromatography. Affinity column contained protein A coupled to cyanogen

bromide activated agarose (Genei, Bangalore). 5 ml of polyclonal antiserum was

passed through the column and the column was washed with 25 ml of wash buffer to

remove all unbound materials. The IgG bound to the column was later eluted by

adding 5 ml of elution buffer and quantified by taking O.D values at 280 nm (1.4 O.D

= 1mg/ml of IgG). One mg of this IgG was used for conjugate preparation. One step

glutaraldehyde method described by Avrameas (1969) was followed for the

preparation of IgG-alkaline phosphatase conjugate.

3.4. Standardization of Double antibody sandwich (DAS) ELISA for detection DAS-ELISA was done on polystyrene plate (Co-Star) using the protocol

described by Clark et al. (1986). Wells were initially coated with CMV IgG at 1µg

per ml in coating buffer. Antigen preparation included grinding leaf tissues in five

volumes of PBS-T containing 2% polyvinyl pyrrolidone (PVP) and 0.2% BSA

followed by centrifugation at 8000 rpm for 1 min. Supernatant obtained was used to

load onto ELISA plates. CMV specific alkaline phosphatase conjugate was used at

1:500 dilution. The reactions of ELISA were read at 405 nm, 1 h after addition of

substrate (p-nitro phenyl phosphate, Genei, Bangalore) by using an ELISA reader

(µQuant, Bio Tek Instruments Inc., USA).

Initial experiments using various concentrations of coating antibody (IgG) and

conjugate with an aim to standardize DAS-ELISA was achieved at 1µg per ml of IgG

coating and 1:500 dilutions of CMV IgG-alkaline phosphatase conjugate. The DAS-

ELISA procedure thus standardized detected the CMV in dilutions of extracts from

diseased black pepper plants from different regions of Karnataka, Kerala and Tamil

Nadu (Table 3). The varying O.D values seen with diseased black pepper samples

from different regions indicate varying virus concentration in these samples.

4. Characterization and development of diagnostics against Piper yellow mottle virus (PYMV)

4.1. Host range studies Attempts were made to transmit PYMV mechanically onto indicator hosts

such as cowpea, Chenopodium album, Nicotiana benthamiana, N.glutinosa, N. tabacum cv. White Burley and Physalis floridanan as explained in section 3.1.

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Results showed that none of the hosts inoculated with badnavirus took up the infection. However virus could be transmitted from infected to healthy black pepper through grafting.

4.2 Purification of PYMV

Black pepper vines exhibiting distinct symptoms collected from Indian Institute of Spices Research (IISR) Farm, Peruvannamuzhi, were used as the source for virus purification The virus was purified using the protocol of de Silva et al., (2002), with few modifications. The infected leaves with distinct symptoms were homogenized with extraction buffer in 1:10 (leaf: buffer) ratio. The extraction buffer included 0.25 M Tris-HCl pH 7.4, containing 0.5% (w/v) sodium sulphite, 4% (w/v) polyvinyl pyrrolidone (PVP), 0.5% (v/v) β-mercapto ethanol and 0.25% (w/v) diethyl dithiocarbamic acid (DIECA). After grinding, the extracts were filtered through muslin and to the filtrate Triton X�100 (20 ml. L-1 conc.) was added and stirred for a period of 35 min at room temperature. The extract was centrifuged at 12000 g for 20 min at 10ºC and supernatant was collected. To this supernatant 4% polyethylene glycol (PEG-6000) and 0.2 M NaCl were added and stirred for 4.5 h at 4ºC in order to precipitate the virus particles. The solution was centrifuged at 12000 g for 20 min at 10ºC. The resulting pellet was resuspended for overnight in suspension buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) at 4ºC, which was 1/5 of the volume of the extraction buffer taken. The next day this suspension was subjected to centrifugation at 10000 g for 10 min and the supernatant was overlaid onto 6 ml of 30% sucrose solution prepared in suspension buffer and centrifuged for 3 h at 51000 g at 10ºC (Beckman Optima LE-80 K). The pellet was collected and suspended in small volume of suspension buffer. Further purification was attempted by using sucrose gradient (10-40%) where 2 ml of the virus sample was overlaid onto the preformed gradient and centrifuged at 35000 g for 3 h using SW-28 rotor (Beckman). Alternatively, the pellet obtained from 30% sucrose step was also subjected to purification by cesium chloride (CsCl) step gradient (0-40%) at 35000 g for 5 h using SW-41 rotor.

After the run was over the gradient tubes were checked for the presence of light scattering zone containing the virus particles. Final purification using sucrose density gradient (10-40%) or cesium chloride step gradient (0-40 %) did not reveal the presence of light scattering zone of virus in the gradient tube. Reducing the duration of centrifugation and varying the sucrose gradient (20-60%) also did not give any light scattering zone. But the pellet in the gradient tube revealed the presence of bacilliform particles when observed under Electron Microscope. This was used for the production of antiserum against the virus.

4.3. Electron microscopy

Electron microscopy of negatively stained purified preparations revealed the presence of bacilliform particles of about 120 x 30 nm in diameter (Fig 2). This purified virus preparation was used for raising antibodies.

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Table 3. Detection of Cucumber mosaic virus (CMV) by double antibody sandwich

(DAS) ELISA* in black pepper and other Piper species from different regions

Isolate A 405 value $ Isolate

A 405 value$

Karnataka state Hassan District Bantana halli Belur Madikeri District Balale Kodlepet Mayamudi Polibetta Udupi District Idu Kerala state Idukki District Adimali Chakkuppalam Chottupara Muttom Thookupalam Vandiperiyar Wyanad District Kenichera Kappadi Pulpally Vengapalli Calicut District Peruvannamuzhi Tamil Nadu state Walayar Healthy black pepper

0.43 0.57 0.32 0.78 0.37 0.41 0.68 0.75 0.22 0.19 0.15 0.19 0.15 0.49 0.57 2.01 0.78 0.61 1.16 0.00

P. chaba Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 P. colubrinum Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Sample 12 Sample 13 Piper longum Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 N.benthamiana N.glutinosa Healthy black pepper

0.33 0.31 0.30 0.21 0.20 3.92 3.89 3.98 3.76 2.00 0.76 0.71 0.57 0.51 0.35 0.33 0.30 0.21 0.53 0.46 1.33 1.65 1.16 0.22 2.23 1.98 0.04

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4.4. Antiserum production, isolation of IgG and preparation of enzyme conjugate The antiserum against PYMV was produced in New Zealand white rabbit.

500µl of the virus preparation was mixed with 500µl of Freund�s incomplete adjuvant (1:1 v/v) was injected intramuscularly seven times at weekly intervals. Fourteen days after the last dose of injection, the rabbit was bled and the antiserum was collected. Immunoglobulin G (IgG) was purified from the crude polyclonal antiserum and IgG-alkaline phosphatase conjugate was prepared as explained in sections 3.5 and 3.6 above.

Fig.2: Purified badnavirus particles as detected by electron microscopy with dimensions of 120 x 30 nm.

4.5. Detection of badnavirus through double antibody sandwich (DAS) ELISA DAS-ELISA was done on polystyrene plate (Tarsons) using the protocol

described by Clark et al. (1986). Wells were initially coated with badnavirus IgG at 1µg per ml in coating buffer. Antigen preparation included grinding leaf tissues in ten volumes of PBS-T containing 2% polyvinyl pyrrolidone (PVP) and 0.2% BSA followed by centrifugation at 8000 rpm for 1 min. Supernatant obtained was used to load onto ELISA plates. Badnavirus specific alkaline phosphatase conjugate was used at 1:2000 dilutions. The reactions of ELISA were read at 405 nm, 90 min after the addition of the substrate (p-nitro phenyl phosphate, Genei, Bangalore) by using an ELISA reader (µQuant, Bio Tek Instruments Inc., USA).

Initial experiments using various concentrations of coating antibody (IgG) and conjugate with an aim to standardize DAS-ELISA was achieved at 1µg ml-1 of IgG coating and 1:2000 dilutions of badnavirus IgG-alkaline phosphatase conjugate. The DAS-ELISA procedure thus standardized detected the virus in dilutions of extracts from diseased black pepper plants from different regions of Karnataka, Kerala and Tamil Nadu (Table 4). The varying O.D values seen with diseased black pepper samples from different regions indicate varying virus concentration in these samples.

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Table 4: Detection of badnavirus by Double Antibody Sandwich (DAS) ELISA in different Piper nigrum isolates and Piper species.

Piper nigrum isolates Visual symptoms

A405 value

Piper nigrum isolates Visual symptoms

A405 value

KARNATAKA STATE Kodagu Balale

Kodlipet KERALA STATE Idukki Adimali Thookkupalam Kannur Taliparamba Kasargod Vellarikundu Kozhikode Chelavoor Chembanoda Peruvannamuzhi Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

M, CF M, VC, S M, C, S M, VC, S M, Mo M, CF M, CF M, CF M, CF M, CF M, CF M, CF M, CF

0.26 0.61 0.18 0.40 0.39 0.20 0.19 0.26 0.23 0.24 0.20 0.25 0.21

Peruvnnamuzhi (contd.,) Sample 6 Sample 7 Wyanad

Chainthkunnu WEST BENGAL STATE

Shantiniketan Piper spp. P. colubrinum P. hapnium P. longum P. mullesua Healthy sample

M, CF M, CF, VC Asymptomatic M, VC M, Mo, LD M, CF M, Mo, LD M, Mo, LD Asymptomatic

0.26 0.32 0.20 0.34 0.35 0.26 0.40 0.28 0.14

C: chlorosis; CF: chlorotic flecks; LD: leaf distortion; M: mottle; Mo: mosaic; S: stunting of the plant. 5. Distribution of CMV and PYMV in black pepper 5.1. Variation in concentration of PYMV and CMV during different months

Variation in the concentration of PYMV and CMV in five different varieties of black pepper during different months was studied through double antibody sandwich (DAS) ELISA. The five different varieties included HP-34, HP-813, Karimunda, Panniyur- I and Panchami. Samples were collected from plants of these varieties at monthly intervals (April to March) and subjected to DAS-ELISA. Buffer and healthy black pepper samples served as negative controls for all the ELISA tests. DAS-ELISA procedure standardized by us earlier for PYMV and CMV was used in the study.

Results showed varying concentrations of both PYMV and CMV during different months. In general, the concentration of both the viruses were found to be higher during the months of October to January (Table 5). Virus concentration was found to be lower during remaining months of the year. During the month of March

17

no virus could be detected through ELISA in two cultivars namely HP 34 and HP 813. Among the different varieties tested, the concentration of the viruses was found to be higher in Panniyur I followed by HP-34 and Panchami. The remaining two varieties although had viruses; their concentration was relatively low when compared to the other varieties. 5.2. Distribution of PYMV and CMV in different parts of black pepper plants

Various parts of infected black pepper plants like young leaf, old leaf, stem, spike and root were used to check for the presence of the viruses and their concentrations by DAS-ELISA during different months of the year. Three different varieties (Karimunda, Panchami and Panniyur-I) were selected for the study for a period of one year. Table 5. Variation in the concentration of PYMV and CMV in black pepper plants during different months as detected by DAS-ELISA*

Month HP34 HP813 Karimunda Panniyur-I Panchami PYMV CMV PYMV CMV PYMV CMV PYMV CMV PYMV CMV

April + + + + + ++ ++ ++ + ++ May + + + + ++ + + + + + June + + + + + + ++ + ++++ ++++July ++ ++ + + ++ ++ ++ + ++ ++

August + + + + ++ + + + ++ +++ September + + ++ + + + ++ + + +

October +++ ++ +++ +++ ++ +++ +++++ ++++ ++ +++ November +++++ ++++ + + ++ ++ ++++ +++ ++ ++ December +++ +++ ++ + ++ ++ ++++ +++ +++ ++ January ++++ +++ ++ + + + +++ ++ ++++ + February ++++ +++ ++ ++ ++ ++ +++ ++ ++ ++ March - - - - + + ++ + + +

* The average O.D. values obtained after 1h of substrate addition was deducted from O.D. values obtained with healthy controls. The resulting O.D. values were then ranked as follows: +, 0.0 to 0.20; ++, 0.21 to 0.40; +++, 0.41 to 0.60; ++++, 0.61 to 0.80; +++++, > 0.81.

Different parts of black pepper plants collected separately, were subjected to DAS-ELISA, where buffer and healthy black pepper samples served as negative controls. DAS-ELISA procedure standardized by us earlier for PYMV and CMV was used in the study.

Results of DAS-ELISA clearly showed uneven distribution of both the viruses in different parts of the plant in all the varieties tested (Table 6). Further, the results indicated that portions of plant comprising young leaf, old leaf and stem had higher concentration of PYMV followed by spike. In the case of CMV, young leaf and old leaf had higher virus concentration. Concentration of both the viruses were once again found to be higher during the months of October to January. Panniyur-I had more concentration of the virus than the other two varieties.

18

Table 6. Distribution of PYMV and CMV in various parts of infected black pepper plants as detected during different months by double antibody sandwich (DAS) ELISA*

Month Part of the

plant Panchami

PYMV Karimunda

PYMV Panniyur-I

PYMV Young leaf + ++ + + + +

Old leaf ++ ++++ ++ + ++ + Stem ++ + ++ + ++ ++ Spike ++ + ++ + ++ +

August

Root + ++++ + + ++ + Young leaf + + + + ++ +

Old leaf ++ + + + ++ + Stem ++ + + + ++ + Spike + + + + + +

September

Root + + + + ++ + Young leaf + + +++ + +++++ ++++

Old leaf + + ++ ++ +++++ ++ Stem ++ ++ ++ ++ +++++ ++++ Spike + + - - ++ +++

October

Root ++ + - - +++ + Young leaf + + ++ + ++++ +++++

Old leaf + + ++ + +++ ++++ Stem ++ + + + +++++ ++ Spike + + - - ++ ++

November

Root ++ ++ - - ++++ ++ Young leaf ++ ++ ++ + +++ +++

Old leaf ++ + ++ + ++ ++ Stem ++ + + + ++ ++ Spike + + - - ++ ++

December

Root ++ ++ - - + ++ Young leaf +++++ + + + + +

Old leaf + ++ ++ + ++++ + Stem ++ + ++ + +++ ++ Spike + ++ - - - -

January Root ++ + + + + +

Young leaf +++ ++ ++ ++ ++ ++ Old leaf ++ ++ ++ + +++ +

Stem ++ + ++ + +++ ++Spike ++ ++ - - - -

February

Root + + + + + + Young leaf ++++ + + + ++ +

Old leaf + + ++ + +++ + Stem + + ++ ++ ++ + Spike + + - - - -

March

Root ++ + + + +++ +

19

Young leaf +++ + + + ++ + Old leaf ++ + ++ + +++ +

Stem + + ++ ++ ++ + Spike + + - - - -

April

Root ++ + + + +++ + Young leaf +++ + + + ++ +

Old leaf ++ + ++ + +++ + Stem + + ++ ++ ++ + Spike - - - - - -

May

Root - - - - - - Young leaf ++ + + + ++ +

Old leaf ++ + ++ + +++ + Stem + + ++ ++ ++ + Spike - - - - - -

June

Root + + + + + + Young leaf ++++ + + + ++ +

Old leaf + + ++ + +++ + Stem + + ++ ++ ++ + Spike - - - - - -

July

Root + + + + +++ +

* The average O.D. values obtained after 1h of substrate addition was deducted from O.D. values obtained with healthy controls. The resulting O.D. values were then ranked as follows: +, 0.0 to 0.20; ++, 0.21 to 0.40; +++, 0.41 to 0.60; ++++, 0.61 to 0.80; +++++, > 0.81. 6. Indexing of black pepper mother plants for the presence of PYMV and CMV

6.1. Indexing by DAS-ELISA A total of 2186 black pepper mother plants comprising of eleven varieties

from Indian Institute of Spices Research (IISR) Experimental Farm, Peruvannamuzhi, were indexed for the presence of PYMV and CMV using DAS-ELISA procedure standardized by us earlier. The different varieties included Coll.1041, Panchami, Panniyur-I, Panniyur-II, Panniyur-III, Panniyur-V, Panniyur-VI, Panniyur-VII, Pournami, Sreekara and Subhakara. Of the 2186 mother vines tested, 713 were found infected with viruses (Table 7). Among these varieties, Panniyur-III had maximum number of mother vines infected (51.61%) followed by Subhakara (43.51%) while Panniyur-V had the least number of mother vines infected (14.81%). About 5% of mother vines of Subhakara and 2% of mother vines of Sreekara were infected with both PYMV and CMV. Panchami variety had the highest percent of PYMV infection (30.46%) while Panniyur-III carried highest percent of CMV infection (41.93%).

6.2. Symptomatology of indexed black pepper plants

To know whether the indexed DAS-ELISA positive mother vines exhibited any visible external symptoms, each ELISA positive vine was examined and the results of the same is summarized in Table 8. The result indicated that more than 50% of ELISA positive mother vines did not exhibit any visible external symptoms and hence the presence of virus and symptoms of the plant may not be correlated at all times as symptomless apparently healthy plants might be carrying the virus(es). Thus

20

a plant cannot be judged healthy solely based on external symptoms. Hence use of detection method such as ELISA is necessary to confirm virus free nature of plants.

Table 7. Indexing of black pepper mother plants for the presence of PYMV and CMV in different varieties

7. Virus elimination through meristem culture It is well known that when a plant is infected with certain virus, the titre of the

virus within the plant is highly variable. Most of the viruses fail to reach the

meristematic tissue. There are several hypotheses which explain this phenomenon.

The long distance movement of plant viruses is mainly through vascular tissue, which

is not developed in meristem. Also, the cell to cell movement of the viruses is too

slow that the virus never reaches the meristematic tissue as it divides and grows very

fast. Apart from these, the active metabolites like auxins may have certain role in

preventing the viruses. It seems that all of these factors play certain role in keeping

the virus out of meristem.

PYMV Infected

CMV infected

Both PYMV and CMV infected

Variety

Total numbe

r of vines tested

No. of vines

infected

Percen

t infecti

on

No. of plants Perce

No. of plants

Percent infection

No. of plants

Percent infecti

Sreekara 878 249 28.35 121 13.78 128 14.57 20 2.27 Subhakara 547 238 43.51 122 22.30 116 21.20 27 4.93 Panniyur-I 68 29 42.64 18 26.47 11 16.17 - - Panniyur -II 81 22 27.16 12 14.81 10 12.34 - - Panniyur -III 31 16 51.61 03 09.67 13 41.93 - - Panniyur -V 81 12 14.81 05 06.17 07 08.64 - - Panniyur -VI 69 24 34.78 13 18.84 11 15.94 - - Panniyur -VII 76 23 30.26 13 17.10 10 13.15 - - Coll. 1041 60 16 26.66 06 10.00 10 16.66 - - Panchami 151 62 41.05 46 30.46 16 10.59 - - Pournami 144 23 15.97 15 10.41 08 5.55 - - TOTAL 2186 714 32.66 374 17.10 370 16.92 47 2.32

21

Table 8. Symptomatology of indexed black pepper plants

7.1. Standardization of black pepper regeneration from meristem

7.1.1. Surface sterilization

The meristem was isolated from green house-grown virus-infected plants. Various methods were tested to optimize the protocol for surface sterilization as given in Table 9. MS basal medium was used through out the experiment.

Table 9. Surface sterilization of black pepper shoot tips

Variety Total no. Plants tested

No. Plants positive in ELISA

Symptomatic Plants

Asymptomatic Plants

Sreekara 878 248 100 148 Subhakara 547 238 109 129 Panniyur-I 68 29 05 24 Panniyur - 81 22 10 12 Panniyur - 31 16 11 05 Panniyur - 81 12 09 03 Panniyur - 69 24 20 04 Panniyur - 76 23 13 10 Coll. 1041 60 16 10 06 Panchami 151 62 18 44 Pournami 144 23 18 5

Surface sterilization Chemical Conc. Duration

(Minutes)

Result % Response after 10 days

Sodium hypochlorite

0.2% 10 Died/ Contaminated, 43/43 0

HgCl2 0.1% 10 Died/ Contaminated, 52/52 0 HgCl2 0.2% 5 Proliferated rooting alone

5/47 13

Ethanol 70% 10 Rooting, 10/43 28 HgCl2 0.1% 5 Rooting and shooting, 12/39 30.76 Ethanol 70% 5 Proliferated rooting alone

5/47 13

HgCl2 0.2% 5 Rooting and shooting, 13/183 7 Ethanol 70% 10 Shooting and rooting, 7/ 78 9

22

7.1.2. Media composition

The dissected meristems were placed in seven different media with varying concentrations of growth factors (Table 10). The isolated meristem swelled and enlarged in size with a diameter of about 3 cm after eight weeks of incubation at 25°C in diffused light, but without any differentiation in MS+ 3mg/l BAP and 1mg/l kinetin. When the meristem was placed in MS medium without any growth regulator followed by incubation at room temperature in diffused light for eight weeks, 2-3 small sprouts came up with a very low frequency (0.23%) and 4.06% swollen meristem remained viable. None of the other media compositions supported the growth for more than four weeks. Out of the 1303 meristem inoculated in P0, 92 (7%) survived upto 4 weeks, indicating the superiority of P0 over other media compositions to support the initial growth. Periodically, the contaminated meristem tips in the plates were removed and the non-contaminated ones were transferred to fresh Petri plates containing the same medium. When the cultures were initially incubated at dark for 3-4 weeks, gradually it enlarged in size, ranging from 1-3 cm in diameter. Three explants were observed to grow in size and regenerated the shoot system without any rooting in the MS basal medium. Such shoots were transferred to the medium containing IAA or NAA for inducing the root system but in vain and subsequently they died. So other media compositions were also tested as followed later in the text.

7.1.3. Addition of antibiotics to the medium

Much of the early death in meristem culture of black pepper occurs during the initial period due to the endophytic bacterial contamination. In order to minimize the problem of bacterial contamination, the different contaminating bacterial colonies were tested for their susceptibility to antibiotics. Antibiotic sensitivity assay

Three antibiotics viz. tetracycline, chloremphenicol and ampicillin were tested at 50, 100 and 150mg/l concentration. The antibiotics were added to the molten MS basal medium when it is sufficiently cooled down. The medium, containing antibiotic was poured into Petri-dishes and allowed to be solidified. The bacterial colonies from the contaminated cultures were transferred to the medium and were observed for bacterial growth at daily intervals. Chloremphenicol and ampicillin were ineffective in suppressing bacterial growth. Tetracycline tested at all concentrations inhibited the bacterial growth. No bacterial growth was seen even up to 13 days in tetracycline treated plates. Table 11 shows the antibiotic sensitivity assay performed. The meristem tips were inoculated in the medium after surface sterilization with 0.1% mercuric chloride for 5 minutes followed by three washings with sterile distilled water. Tetracycline has no significant inhibitory effect on the meristem explants, as it is evident from Table 12.

23

Table 10. Response of black pepper meristem (derived from shoot tip) in different media

Table 11. Antibiotic sensitivity assay for contaminating bacteria in meristem culture of black pepper

Ampicillin (mg/l)

Chloremphenicol(mg/l)

Tetracycline (mg/l)

Control (No antibiotic)

50 100 150 50 100 150 50 100 150 I day + - - - - - - - - - II day + + - - + - - - - - III day + + + + + + + - - - IV day + + + + + + + - - - V day + + + + + + + - - -

-Sensitive (no growth); + Resistant (growth seen)

Table 12. Effect of tetracycline in minimizing bacterial contamination during meristem culture of black pepper

Number of meristem Total Contaminated after

Medium

3 days 6 days 10 daysSurvived after 10 days*

P0 123 42 82 84 3 P0 + 50mg/l Tetracycline 124 3 11 14 18 P0 + 150mg/l Tetracycline 124 3 9 11 22

* The rest were died or contaminated with fungi

Variety No.meriste

ms

Medium 1 week 2 weeks

3 weeks

4 weeks

6 weeks

8 weeks

12 weeks

16 weeks

20 weeks

Karimunda Panniyur

1303 122

MS Basal (P0) 208 49

147 39

102 23

92 16

62 9

53 3

31 3

18 0

3 0

Karimunda 176 MS+1mg/L GA (P2)

28 15 15 12 0 0 0 0 0

Karimunda 31 MS+2mg/L GA (P3)

4 3 3 2 0 0 0 0 0

Karimunda 124 MS+7mg/L BAP (P1)

18 12 9 0 0 0 0 0

Karimunda 175 MS+3mg/L BAP+1mg/L kinetin (M4)

27 18 16 14 0 0 0 0 0

Karimunda 36 ½ MS+3mg/L BAP+1mg/L kinetin (M4)

4 0 0 0 0 0 0 0 0

Karimunda 122 MS+2mg/l IAA+3mg/lBAP+15g/l sucrose+10g/l glucose (R1)

13 0 0 0 0 0 0 0 0

24

7.1.4. Pre-treatment of shoot tips The second reason for early death of the meristems in culture is the inhibitory

effect of polyphenolic exudates from the explant itself, turning it to brown and

subsequently to black. Frequent transfer of the explants to fresh medium with an

interval of one day and two days was tested, but it affected the establishment of the

meristem adversely. For minimizing the inhibitory effect caused by polyphenolic

exudates, the shoot tips were kept under running tap water for two hours and agitated

with 0.7% (w/v) polyvinyl pyrrolidone (PVP) in 2% (w/v) sucrose solution for 30

minutes and gave a rapid rinse with antioxidant solution (ascorbic acid: citric acid 50:

75mg/l in water) followed by surface sterilization with 0.1% mercuric chloride for

five minutes and three washes with sterile distilled water. This method extended the

life of the dissected meristem in culture as 19.6% survived for two weeks after this

treatment opposed to 11.2% that survived for two weeks without this treatment.

A considerable number of meristems (31.3%) in culture were also lost by

fungal contamination, as it is true with many of the woody plants. Incorporation of

pentachloronitrobenzene (PCNB) at the rate of 1% in the culture medium reduced the

fungal contamination frequency. But later it was discontinued due to its inhibitory

effect on the explants as none of the meristen survived beyond seven days.

7.1.5. Effect of TDZ in induction of meristem growth

The in vitro response and viability of the meristem could further be improved

by using the plant growth regulator Thidiazuron (TDZ) at a concentration of 0.1 mg/l

in MS medium. TDZ enhanced the growth rate of meristem, but again without any

differentiation. When these meristems were transferred to MS medium supplemented

with 1mg/l BAP and 1mg/l kinetin, shooting was induced at a frequency of 53%. The

use of TDZ-containing medium to induce the initial growth of dissected meristem and

subsequent transfer of the enlarged meristem to MS containing 1mg/l BAP and 1mg/l

IAA was found to be promising. The cultures responded well and the induction of

shooting was consistent with a frequency of 53%. Since rooting was not observed in

this medium, all shooted cultures were transferred to woody plant medium (WPM)

supplemented with 0.5% activated charcoal. Activated charcoal absorbs the

polyphenolic exudates and extends the life of explants. Hundred percent of such

cultures developed rooting and successfully transferred to sterilized potting mixture

25

(soil: sand: cowdung::1:1:1) in medium-sized pots under green house conditions.

After a series of trials as discussed above, a finalized protocol was developed for

establishing meristem cultured black pepper plants.

7.2. Production of virus-free black pepper plants by meristem culture An effective and reproducible protocol for meristem culture in black pepper is

established. It is also established that the same technique can be employed

successfully in elimination of the major viruses (Cucumber mosaic virus and Piper

yellow mottle virus) infecting black pepper. Two cultivars of black pepper (Panniyur I

and Karimunda) that showed the viral symptoms were vegetatively propagated in

green house through stem cuttings. These plants served as mother plant and source of

meristem during the period of study. Shoot tips were collected from these plants and

kept under running tap water for one hour followed by frequent agitation with 0.7%

(w/v) polyvinyl pyrrolidone (PVP) in 2% (w/v) sucrose solution for 30 minutes. This

was then subjected to rapid rinse with antioxidant solution (ascorbic acid: citric acid

50: 75mg/l in water) followed by surface sterilization with 0.1% mercuric chloride for

five minutes and three washes with sterile distilled water. Then the meristem was

dissected out under aseptic conditions in a laminar airflow and placed in MS medium

supplemented with 0.1% thidiazuron (TDZ) and 50 mg/l tetracycline. Tetracycline

was used to suppress the endophytic bacterial contamination in the medium based on

the previous experiments. TDZ induces the meristem growth but without any

differentiation. Even though pentachloronitrobenzene (PCNB) was used in the

medium to minimize the fungal contamination, later it was discontinued due to its

inhibitory effect on the explants.

Out of 1328 meristem that were incubated in the above medium, 613 were lost

due to fungal contamination, 137 due to bacterial contamination and 288 were dried

up. After 30 days of culture, only 290 (21.83%) survived. After 45 days, 114

meristem that swell and enlarged in size were transferred to MS medium

supplemented with 1mg/l benzyl amino purine (BAP) and 1 mg/l naphthalene acetic

acid (NAA) for shoot induction. Out of 114 meristem transferred to this medium, 86

started shooting at 90 days of culture and the frequency of shoot induction was found

to be 75.43% in this medium. At 110 to 120 days, the fully developed shoots were

transferred to Woody Plant Medium (WPM) containing 0.5% charcoal without any

26

growth regulator for rooting. At 140 to 150 days, all such cultures developed well-

established root system and these cultures were transferred to sterilized potting

mixture (soil: sand: cowdung::1:1:1) in medium-sized pots under green house

conditions at 180 days of culture. Table 13 shows the frequency of regeneration and

Figure 2 shows the completely regenerated meristem into plantlet and hardening in

green house.

Table 13. Survival and regeneration frequency of black pepper meristem

Total number of meristem in MS+TDZ+Tetracycline 1328 Percentage

Fungal contamination (till 30 days) 613 46.15 Bacterial contamination (till 30 days) 137 10.31 Death (till 30 days) 288 21.68 Surviving (till 30 days) in MS+TDZ+Tetracycline 290 21.83 Surviving till 45 days and transferred to MS+BAP+NAA 114 8.58 60 days: started differentiation 86 6.47 90 days: differentiated shoots transferred to WPM for rooting

56 4.21

110-120 days: Rooting initiated 56 4.21 140-150 days: Transferred to soil for hardening 56 4.21

27

Figure 2. Different stages of meristem culture in black pepper. (a) Dissected meristem; (b) after 45 days in MS+TDZ; (c) after 60 days in MS+BAP+NAA; (d) after 90 days in MS+BAP+NAA+charcoal; (e). after 120 days in MS+BAP+NAA+charcoal; (f) after 140 days in WPM+charcoal; (g) after 150 days in WPM+charcoal; (h) after 180 days in WPM+charcoal; plantlet transferred to potting mixtire at 180 days.

chain reaction (mRT-PCR). As PYMV infecting black pepper is a DNA virus

while CMV is an RNA virus, total nucleic acid (DNA and RNA) extracted from the

28

black pepper plants was used as template in mRT-PCR. Young, but fully developed

leaves of plants were brought to the laboratory in ice and twenty-five milligram of

tissue was homogenized using pre-chilled sterile pestle and mortar in 250 µl TRI

reagent. The homogenate was incubated at room temperature for five minutes and

mixed with 100 µl chloroform: iso-amyl alcohol (49:1). The mixture was shaken

vigorously for 15 seconds and again incubated at rooom temperature for five minutes

followed by centrifugation at 12,000g for 15 minutes at 4°C. The aqueous phase

containing RNA was transferred to a new tube and equal volume of iso-propanol was

added, incubated at room temperature for five minutes after a brief vortex and finally

incubated at -80°C for one hour*. The inter-phase (from the above step) containing

DNA along with the organic phase was used for DNA purification. This was mixed

with 85 µl Tris base (100mM, pH 10.5), vortexed for 10 seconds and incubated in ice

for 30 minutes followed by centrifugation at 2,000g for 15 minutes at 4°C. The

aqueous phase from this step was transferred to a new tube and mixed well with equal

volume of chloroform: iso-amyl alcohol (49:1) prior to centrifugation at 12,000g for

10 minuets at 4°C. The aqueous phase was mixed with 0.1 volume 3M sodium acetate

(pH 4.0) and one volume of iso propanol. Then it was mixed with RNA precipitation

tube above (step indicated by *) and incubated at -80°C for one hour followed by

centrifugation at 12,000g for 15 minutes at 4°C. The pellet containing both DNA and

RNA was washed with 70% ethanol, centrifuged at 12,000g for five minutes at 4°C

and air-dried before dissolving in 40µl sterile de-ionized water. One microlitre of this

preparation was used as template in multiplex PCR.

7.3.2. Multiplex RT-PCR (Reverse transcriptase polymerase chain reaction)

The multiplex RT-PCR that detects both PYMV and CMV simultaneously in

the same reaction tube was carried out in 25 µl reaction volume with the reagents and

respective concentration as given in Table 14.

Just prior to the addition of RNA/ DNA template to the reaction mixture, it was

heated to 75°C for 10 minutes and rapidly cooled down in ice for 3 minutes to make

RNA linear. The PCR tube containing all the above reagents was subjected to the

following temperature profile in Eppendorff�s Master Cycler Gradient. First, it was

held at 42°C for 45 minutes for cDNA synthesis followed by 94°C for 30 seconds to

denature, 50°C for one minute for primer annealing and 72°C for one minute for DNA

29

synthesis by Taq DNA polymerase. The whole process except cDNA synthesis was

repeated for 39 cycles and a final extension was allowed at 72°C for 10 minutes. After

the multiplex RT-PCR, the reaction products were subjected to electrophoresis in

0.8% agarose gel and positive reactions were identified by the presence of 450 bp

product specific for PYMV and 650bp product specific for CMV.

Table 14. Reagents and its concentration in a single multiplex RT-PCR

Reagents Stock Quantity (µl)

Final concentration/ reaction

Assay buffer (Taq DNA polymerase) with 15mM MgCl2

10x 2.5 1x with 1.5mM MgCl2

Dithiothreitol (DTT) 100mM 2.5 10m M DNTP mixture 10 mM 1.875 750µM

Primer AIB 1 (Forward CMV) 0.5 15 pM Primer AIB 2 (Reverse CMV) 0.5 15 pM

Primer AIB 35 (Forward PYMV) 0.5 10 pM

Primer AIB 36 (Reverse PYMV) 0.5 10 pM RNAase Inhibitor 40 U/µl 0.1 4 U MMuLv Reverse transcriptase 10 U/µl 0.125 1.25 U Taq DNA Polymerase 3 U/µl 0.25 0.75 U De-ionized water _ 14.65 _ Template DNA/RNA _ 1 _ Total 25

A total of six black pepper plants were propagated to be used as the source of

meristem, out of which four plants were tested positive for both PYMV and CMV

while the other two were positive for CMV alone. Altogether 32 meristem-

regenerated plants were tested using multiplex RT-PCR. Each time when the

meristem-regenerated plants were tested, the corresponding mother plants were also

included in the tests, which served as positive control while total nucleic acid

extracted from a known healthy plant served as negative control. Out of the 28

meristem-plants that derived from PYMV-infected mother plants, 22 were found to be

negative for the virus while six were positive. Similarly, out of 32 meristem-derived

plants raised from CMV-infected mother plants, 27 were negative for the virus while

30

five were positive for the virus. A representative gel showing the virus elimination is

given in Figure 3.

Figure 3. Elimination of CMV and PYMV from black pepper. Lane 1- molecular size marker, mother plants (lanes 2 and 7) and their meristem-derived plants (lanes 3-6) and (lanes 8-13).

The frequency of CMV elimination was 84.37% while that of PYMV was

78.57%. This may be explained by the differing ability of viral movement and the

actual length of meristem taken for culture; longer the length, higher the chance of

virus subsistence and vice-versa. As a thumb rule, meristem, not longer than 100 µm

is preferred to eliminate the viruses. A few of the badnaviruses are known to be

integrated in the hosts� nuclear genome besides their presence in the episomal form.

However, whether such integration occurs in PYMV-black pepper combination is yet

to be studied.

8222 Questions- Answered The incidence and severity of the stunted disease is high at high altitudes such as Idukki and Wynad Two viruses either singly or in combination can cause the disease A reliable ELISA based detection both the viruses in nursery and field samples. A technique for virus elimination through meristem culture

5000bp 2000 1500 1000 CMV 500 PYMV

1 2 3 4 5 6 7 8 9 10 11 12 13

31

8223 Process/ Product/ Technology/ Developed A reliable DAS-ELISA protocol was developed for efficient detection of CMV and PYMV An efficient protocol for virus elimination through meristem regeneration was developed 8224 Practical Utility (not more than 150 words)

For management of stunted disease use of virus-free planting materials is important. As symptoms can not be reliably used to identify healthy plants, use of sensitive methods based on ELISA is needed to identify virus-free plants. The project developed a reliable and sensitive ELISA methodology for the detection of both the viruses which can be used in certification programme to develop virus-free planting materials. Project also developed protocol for the meristem culture for elimination of viruses from an infected plant. Constraints, if any: Nil 823 Publications and Material Development (One copy each to be supplied with this proforma.) 8231 Research papers Bhadramurthy, V., Retheesh, S.T., Bhat, A.I., Madhubala, R., Hareesh, P.S. and Pant,

R.P. 2005. Development of ELISA based technique for the detection of a putative badnavirus infecting black pepper (Piper nigrum L.). Indian Phytopath. 58: 314-318.

Bhat, A.I., Devasahayam, S., Venugopal, M.N. and Suseela Bhai, R. 2005. Distribution and incidence of viral diseases of black pepper in Karnataka and Kerala, India. J. Plantation Crops 33: 59-64

Bhat, A.I., Faisal, T.H., Madhubala, R., Hareesh, P.S. and Pant, R.P. 2004.

Purification, production of antiserum and development of enzyme linked immunosorbent assay-based diagnosis for Cucumber mosaic virus infecting black pepper. J. Spices & Aromatic Crops, 13: 16-21.

Papers under preparation Retheesh, S.T. and Bhat, A.I. Production of virus-free plants through meristem tip

culture in black pepper Bhadramurthy, V. and Bhat, A.I. Indexing and distribution of Cucumber mosaic virus

and Piper yellow mottle virus in black pepper.

8232 Popular articles Bhat, A.I. 2005. ELISA- a powerful tool for the detection and diagnosis of viruses.

Spice India 18 (1): 36-38.

32

Parthasarathy, U., Bhat, A.I., Devasahayam, S., Jayarajan, K., Shareef, B.H.A and Parthasarathy, V.A. 2005. Pepper plots throw light on stunted disease. Geospatial, September-October 2005: 52-56. Parthasarathy,U., Bhat, A.I., Devasahayam, S. 2005. GIS helps studying viral disease of black pepper. SAIC Newsletter, 15(3): 11. 8233 Reports Nil 8234 Seminars, conferences and workshops (relevant to the project) in which the scientists have participated. (List abstracts forwarded) Nil 824 Infrastructural facilities developed (Details of field, laboratory, note books and final material and their location) A swing out rotor, magnetic stirrer, vortex machine and micropipette were purchased

under this project. Laboratory note books and materials are with P.I.

825 Comments / Suggestions of Project Leader regarding possible future line of work that may be taken up arising out of this Project.

A detailed study on the epidemiology and spread of the disease is needed.

Effect of single virus and combinations of both viruses also should be studied. Efforts

should also be continued to know involvement of any other viruses in the disease.

Studies are also needed to know whether PYMV gets integrated into black pepper

genome.

Part-IV : Project Expenditure (2003-06) (Summary)

Head Grant

allotted Total grant released till date

Grant utilized Balance

Non-recurring 660000 660000 660000 0 Manpower 611446 487246 613200 -125954 Recurring contingencies

360000 300000 360000 -60000

Institutional charges

79056 65328 65328 -13728

Total 1710502 1512574 1698528 -199682 Balance to be released

199682

33

Part-V : Declaration

This is to certify that the final report of the Project has been submitted in full consultation with the Project workers as per the approved objectives and technical programme and the relevant records, note-books, materials are available for the same.

Signature of the Project Investigator: A. Ishwara Bhat

Co-Investigator: Dr. R. Suseela Bhai

Signature & Comments of the Head of the Division/ Section Signature & Comments of the Director