AAnnnnuuaall RReeppoorrtt 2012/13 ¦ÉÉ®úiÉÒªÉ ¨ÉºÉɱÉÉ ¡ òºÉ±É +xÉÖºÉÆvÉÉxÉ ºÉƺlÉÉxÉ Indian Institute of Spices Research (Indian Council of Agricultural Research) Kozhikode - 673012, Kerala, India. Indian Institute of Spices Research ANNUAL REPORT 2012/13 +É<Ç +É<Ç BºÉ +É®ú ´ÉÉ̹ÉEò Ê®ú{ÉÉä]Çõ 2012/13 IISR Annual Report 2012/13 ½þ® Eònù¨É, ½þ®ú b÷MÉ®ú ÊEòºÉÉxÉÉå EòÉ ½þ¨ÉºÉ¡ò®ú ¦ÉÉ®úiÉÒªÉ EÞòÊ¹É +xÉÖºÉÆvÉÉxÉ {ÉÊ®ú¹Énù Agr search with a uman touch ¦ÉÉ®úiÉÒªÉ ¨ÉºÉɱÉÉ ¡òºÉ±É Indian Institute of +xÉÖºÉÆvÉÉxÉ ºÉƺlÉÉxÉ Spices Research EòÉäʹÉCEòÉäb÷ Kozhikode 1 Two times winner of Sardar Patel Outstanding ICAR Institution Award Indian Institute of Spices Research ANNUAL REPORT 2012/13 Editors V. Srinivasan R. Dinesh D. Prasath T.E. Sheeja Cover design A. Sudhakaran Citation Indian Institute of Spices Research, Annual Report 2012/13, Kozhikode Published by Director, Indian Institute of Spices Research Kozhikode - 673 012, Kerala, India. Phone: 0495-2731410, Fax: 0495-2731187 Website: www.spices.res.in, E-mail: [email protected] ISBN: 13 - 978 - 81 - 86872 - 43 - 7 June 2013 Printed at GK Printers, Kochi-17. 2 Indian Institute of Spices Research ANNUAL REPORT 2012/13 CONTENTS Preface ....................................................... 4 Bioinformatics ............................................. 69 Executive summary ..................................... 5 Agricultural knowledge management unit ..... 70 Introduction ................................................. 15 Library ......................................................... 71 Past achievements ...................................... 19 Agricultural technology information centre .... 72 Black pepper ............................................... 25 Krishi vigyan kendra .................................... 74 Cardamom .................................................. 42 Research publications .................................. 77 Ginger ......................................................... 49 Education and training ................................. 79 Turmeric ..................................................... 54 Institute technology management unit .......... 80 Vanilla ......................................................... 59 Hindi cell activities ....................................... 81 Tree spices ................................................. 60 Recognition ................................................. 83 Post harvest technology, value addition Institute management committee ................. 84 and high value compounds .......................... 61 List of projects ............................................ 85 Extension and impact assessment ............... 64 Personnel .................................................... 89 All India coordinated research Weather data 2012 ...................................... 92 project on spices ......................................... 67 3 Two times winner of Sardar Patel Outstanding ICAR Institution Award PREFACE I have great pleasure in presenting the Annual Report 2012/13. During this year, the institute enriched the germplasm collections in black pepper from Kannur, Malappuram and Idukki districts of Kerala and Kodagu district of Karnataka. Farmer selected cultivars, drought tolerant and long spike types were among the unique collections. Evaluation of new generation chemicals and actinomycetes against Phytophthora and nematodes of black pepper and cardamom thrips have given good leads. Liq- uid formulation for biocontrol agents have been standardized. Technology for control of anthracnose disease in black pepper nursery was developed and recommended. A quick detection assay based on loop mediated isothermal amplification (LAMP) was devel- oped for detection of Pepper yellow mottle virus (PYMoV) and Cucumber mosaic virus (CMV) infect- ing black pepper and Banana bract mosaic virus (BBrMV) infecting cardamom. A SYBR Green based method for real-time quantitative RT-PCR (qRT-PCR) for detection of Cardamom mosaic virus (CdMV) and BBrMV infecting cardamom was also developed. The endosymbiont, Wolbachia on cardamom thrips was documented. Source-sink relationship and biochemical characterization of resistance for shoot borer have been worked out in ginger and turmeric. The functionally annotated subtracted tran- scriptome revealed defense/stress related genes like glutathione-s-transferase, leucine rich protein and various enzymes involved in anti-oxidant defense of Curcuma amada against Ralstonia infection. A Kisan Mela and Farmers-Scientists interaction meet was organized at Cardamom Research Center (CRC), Appangala during the third week of December 2012. About 1700 soil samples from Way- anad and 10110 soil samples from Kozhikode have been analyzed for all the essential nutrients and soil health cards with nutrient advisories were given. Front Line Demonstrations (FLDs) on varieties and technologies of black pepper were conducted at Wayanad. The Krishi Vigyan Kendra (KVK) conducted 9 seminars, participated in 8 Kisan Melas and exhibitions and trained more than 5150 beneficiaries. Par- ticipatory seed production on high yielding varieties of ginger and turmeric was taken up in 10 farmer’s plots and public-private participatory seed production of IISR Prathibha turmeric has yielded dividends to farmers in Andhra Pradesh and Kerala. The institute has participated in 3 national and 4 state level exhibitions, organized training programmes to about 100 horticultural officers and farmers of Uttar Pradesh, Andhra Pradesh, Arunachal Pradesh, Karnataka and Kerala. Media visits to progressive farm- er’s plots were organized and technologies were popularized through video films, AIR programmes and print media. I consider it a privilege to place on record the encouragement given by Dr. S. Ayyappan, Di- rector General, ICAR during his visit to CRC, Appangala. But for the strong support and guidance we received from Dr. N.K. Krishnakumar, Deputy Director General (Horticulture) and Dr. Umesh Srivastava, former ADG (Hort.II) and Dr S.K. Malhothra, the present ADG (Hort.II) we would not have made these achievements. I am equally thankful to the Research Advisory Committee and the Quinquennial Review Team for their suggestions. I appreciate the efforts shown by the staff of this Institute for their zeal in executing the programmes. I also appreciate the editors for having compiled and brought out this An- nual Report. Kozhikode M. Anandaraj Date: 15.06.2013 Director 4 Indian Institute of Spices Research ANNUAL REPORT 2012/13 EXECUTIVE SUMMARY BLACK PEPPER Observations on growth parameters taken after 8 months of application revealed little or no signifi- cant differences among the treatments including Genetic resources control. Available nutrient levels including organic Diverse cultivars of black pepper were col- C were markedly high (5.4-6.0%) in all the treat- lected from farmers’ plots in Kannur, Kozhikode, ments. Dehydrogenase level was lowest in the Malappuram and Idukki districts of Kerala. A to- control and silver oak, but markedly higher in soils tal of 184 collections were made including 7 wild applied with other tree extracts. Microbial bio- Piper species. Spike proliferating cultivar Thekkan, mass C was lowest with silver oak and ailanthus other farmer selected cultivars such as Kumbakkal extracts but greatest with garuga extract. and Ponmani were some of the unique accessions Microbial consortium for growth collected from Idukki district. A drought tolerant type was collected from Malappuram district. IC promotion numbers were obtained for 191 cultivars and 169 wild accessions from NBPGR, New Delhi. Germ- A consortium of three rhizobacterial iso- plasm accessions were planted at the alternate lates (BRB3, BRB13 and BRB23) markedly pro- black pepper germplasm conservation center at moted the growth of black pepper. However, the CHES, Chettalli. PGPR applied in combination with various rates of chemical fertilizers differed in their effects on Organic production package growth parameters. Shoot weight (fresh) was highest with 75%N + 100%P + 100%K + BRB3 + Organic production package involving 10 BRB13 + BRB23, while greatest root weight (fresh) kg FYM + 500g neem cake + 500g ash+ 2kg ver- and tallest plants were registered in the treatment micompost with biofertilizers – Azospirillum and P 100%N + 100%P + 75%K + BRB3 + BRB23. Based solubilizing bacteria (20g) and Trichoderma (50g) on a series of experiments, microbial formulation and Pseudomonas (IISR 6) (50g) per vine and spray involving a consortium of PGPR viz., IISR Biomix of 1% Bordeaux mixture (BM) and neem oil (5mL was developed for licensing and commercializa- L-1 of water) for disease and pest control has been tion. developed, tested and demonstrated. Results showed that organic management system yielded Drought tolerance on par with integrated system, whereas oleoresin content was found to be superior under organic Fifty germplasm accessions were screened system. for drought tolerance and the accession 6707 was identified as relatively tolerant based on its high Black pepper- tree species interac- relative water content (RWC) and low membrane tion leakage (ML) values even at 8.0-8.5% soil mois- ture content. The extracts of trees like garuga, eryth- rina, gliricidia, ailanthus, jack and silver oak, the New chemicals against foot rot most commonly used support trees of black pep- The antioomycete activity of RIL-070/FI per at 100% concentration was applied to bush (72WP) and Ergon 44.3% (w/w) were tested against pepper @100mL per plant twice every month. 5 Two times winner of Sardar Patel Outstanding ICAR Institution Award different developmental stages of Phytophthora Characterization of Phytophthora capsici and Colletotrichum gloeosporioides infect- isolates using multi-gene approach ing black pepper. RIL-070/FI (72WP) when tested in vitro showed 100% inhibition against mycelia A multi-gene analysis was done for the at 50ppm, sporulation at 100ppm and zoospore characterization of Phytophthora isolates from germination at 200ppm concentrations. The av- black pepper. Nine loci were selected viz., 28S Ri- erage ED50 value of RIL-070/FI (72WP) against P. bosomal DNA, 60S Ribosomal protein L10, Beta- capsici was 30ppm. Ergon 44.3% (w/w) showed tubulin, Elongation factor 1α, Enolase, Heat shock 100% inhibition of mycelial growth and sporula- protein 90, TigA gene fusion protein, Mitochon- tion at 6000ppm with an ED50 value of 210.6ppm drial genome region between gene COX2 and for mycelia inhibition and 1480.4ppm for sporula- tion inhibition. Inhibition of zoospore germination gene COX1 and Ras-related protein (Ypt1) gene. was 100% at 1000ppm. All the loci were amplified using the correspond- ing primers, which revealed 2 diverse groups viz., Evaluation of new chemicals against where Group 1 showed similarity to P. capsici and Group II showed similarity to both P. capsici and Radopholus similis P. tropicalis. Nematicidal activity of eight chemicals Identification of virulence-associat- viz., Fipronil, Thiamethoxan, Acephate, Car- tap hydrochloride, Quinalphos, Flubendamide, ed Phytophthora gene Carbosulfan and Chloropyriphos was evaluated against R. similis in the green house. Out of these, Gene expression studies using qPCR un- 5 chemicals viz., Fipronil, Thiamethoxan, Cartap dertaken in P. colubrinum challenge inoculated hydrochloride, and Carbosulfan showed good ne- with Phytophthora revealed highest level of ex- maticidal activity. pression of polygalacturonase inhibitor protein (PGIP), indicating its importance in disease resist- Greenhouse evaluation of actino- ance. mycetes against P. capsici Evaluation of Trichoderma isolates Nine actinomycetes were evaluated un- der green house conditions for disease suppres- Fifteen different isolates of Trichoderma sion and growth promotion. The results revealed isolated as efficient for various crops were tested the efficacy of 3 isolates (Act5, Act2, and Act9) in against P. capsici. PhytoFuRa10 was found to be reducing foot rot incidence due to P. capsici under highly promising with less than 10% disease in- challenge inoculation. The potential isolates were cidence followed by PhytoFuRa8 , PhytoFuRa11, identified as Kitasatospora setae (Act2), Strepto- and PhytoFuRa13 with 10- 30% disease incidence myces sp. (Act5) and S. tauricus (Act9). suggesting the plasticity of isolates. Development of liquid formulation Potential endophytic fungi for biological control agents Endophytic fungi were evaluated for its Mature conidial suspension in sterile de- biocontrol potential against P. capsici in vitro. The ionized water was a promising medium for long inhibition ranged from 70-78% and the isolates term storage and preservation of viable conidia of were identified as species of Diaporthe, Phomop- Trichoderma with consistent biocontrol potency. sis, Annulohypoxylon nitens, Daldinia eschscholzii, An encapsulated bead formulation of Trichoder- Fusarium proliferatum, Fusarium moniliformae ma harzianum and Pochonia chlamydosporia was and Ceriporia lacerata. also formulated. 6 Indian Institute of Spices Research ANNUAL REPORT 2012/13 Radopholus similis diversity Real-time PCR and LAMP for detec- tion of viruses Sixteen isolates of R. similis collected from different parts of Kerala were studied for their ge- A SYBR Green based quantitative real netic diversity through ITS-PCR and sequencing. time polymerase chain reaction (qPCR) for Piper Phylogeographic studies using ITS sequences of yellow mottle virus (PYMoV) and reverse tran- R. similis isolates from different states of India re- scription (RT) qPCR for Cucumber mosaic virus vealed extensive genetic diversity in African and (CMV) infecting black pepper was developed for Indian populations. sensitive and specific detection and quantification of these viruses. qPCR and RT-qPCR were more Phenyl propanoids vis-à-vis Radop- sensitive than conventional PCR and RT-PCR in the detection of viruses. The method was validated holus-black pepper interaction using field samples collected from different re- Quantitative assays to estimate phenyl gions. propanoids indicated that the levels of anthocya- A quick detection assay based on loop nidins and total phenols were significantly high in mediated isothermal amplification (LAMP) was HP39, the nematode resistant hybrid, compared developed for detection of PYMoV and reverse to the susceptible Sreekara variety. Histochemical transcriptase (RT) LAMP for detection of CMV studies proved that lignification was significantly infecting black pepper. The assay successfully de- high in HP39 roots compared to Sreekara. tected both the viruses in infected plants while no cross-reactions were seen with healthy plants. Studies on endophytic bacteria Detection limit of LAMP was up to 100 times high- er than conventional PCR and up to 100 times less Endophytic bacteria such as spontaneous sensitive than real time PCR. The optimized LAMP rifampicin resistant Bacillus megaterium BP17R and RT-LAMP were validated by testing field sam- and Pseudomonas putida BP25R were character- ples of black pepper collected from different re- ized by Biolog, biochemical and antibiotic sensitiv- gions. ity assays. Reaction of the above 2 bacteria to 17 antibiotics indicated that P. putida BP25 was re- sistant to several antibiotics while B. megaterium CARDAMOM BP17 was resistant only to rifampicin. The whole metabolome extracted from BP17R and BP25R on assaying against P. capsici, P. tropicalis, P. myrioty- Genetic resources lum, Rhizoctonia, Fusarium, R. solanacearum and nematodes by in vitro bioassays did not show any A total of 592 accessions have been maintained in the National Active Germplasm activity against any of the tested pathogens, in- Site (NAGS) at Appangala. Five accessions includ- cluding nematodes. However, at a concentration of 20mg mL-1 these extracts inhibited germina- ing high biomass type, deep green capsules and tion of P. capsici zoospores. drought tolerant types were collected from Sulta- nia region of Megamalai area. Appangala-1 vari- Technology for managing anthrac- ety was registered with Protection of Plant Variety & Farmers Rights Authority, New Delhi. nose disease in nursery Drought tolerance Pre-planting treatment of 3 node cuttings from the runner shoots with the combination of Twelve elite lines with 3 checks were eval- carbendazim + mancozeb (0.1%) was found to be uated for drought tolerance under field condition. superior in delaying disease initiation and devel- Significant variations were recorded for growth opment compared to other treatments and was with reduction in yield characters under stress. validated and recommended for the manage- Two genotypes with bold capsule and early matu- ment of the disease in the nursery. rity maintained yield under moisture stress. 7 Two times winner of Sardar Patel Outstanding ICAR Institution Award Mycelial compatibility among iso- virulent F. oxysporum isolates from Kerala, Kar- nataka and Tamil Nadu led to the identification lates of C. gloeosporioides of WYD T6, RT 7B and RT 2A, respectively as the Mycelial compatibility studies among the most effective isolates. selected isolates of C. gloeosporioides (3 each RT-PCR and RT-LAMP for detection from Kerala and Tamil Nadu and 8 isolates from Karnataka) revealed that majority of the isolates of viruses showed incompatible reaction when isolates from A SYBR Green based real-time quantita- different geographical locations were paired. Iso- tive RT-PCR (qRT-PCR) for detection of Cardamom lates from same geographical region exhibited mosaic virus (CdMV) and Banana bract mosaic vi- compatible reaction. rus (BBrMV) was developed. RT-qPCR was more Characterization of endophytes sensitive than conventional RT-PCR. Reverse tran- scriptase (RT) LAMP method was also developed Endophytic fungi and bacteria were iso- for quick and sensitive detection of BBrMV infect- lated from three ecotypes viz., Malabar, Mysore ing cardamom. Detection limit of RT-LAMP was and Vazhukka. Isolations made from different higher than conventional RT-PCR. The optimized plant parts including leaves, petioles, pseu- techniques were validated by testing field samples dostem, roots and rhizomes of Amomum sp. and of cardamom collected from different regions. Alpinia sp. yielded 50 fungal isolates and 5 bacte- rial isolates. Among the plant parts, the isolates Screening for thrips resistance had more preference for pseudostem followed by rhizomes. Two hundred and ninety six accessions/ lines were field screened against thrips during the IPM for leaf spot disease year. Fifty three accessions recorded capsule dam- Evaluation of fungicides, a neem based age below 20%. IC 349441 recorded the lowest product and an isolate of Trichoderma harzianum capsule damage of 3%. Five Accessions recorded revealed that spraying the combination product of below 10% capsule damage. Nine accessions re- carbendazim + mancozeb (0.1%) in combination corded more than 70% capsule damage with IC with soil application of T. harzianum was promis- 349520 exhibiting the highest damage of 96.2%. ing in managing leaf spot disease under nursery The accessions with less than 10% capsule dam- conditions. age belonged to Malabar type except IC 349416 which belonged to Vazhukka. All the 9 highly sus- Diversity of rhizome- root rot path- ceptible accessions belonged to either Mysore or ogens Vazhukka types. Surveys were repeated in Wayanad and Bacterial endosymbiont and en- Idukki districts of Kerala, Hassan and Kodagu dis- tomopathogen tricts of Karnataka to study the seasonal variation of rhizome and root rot diseases. Eighty fungal The endosymbiont Wolbachia was identi- isolates were isolated from rhizome and root rot fied in thrips populations collected from Kodagu disease affected samples. Among the isolates, (Karnataka), Wayanad, Palakkad, Idukki (Kerala), Rhizoctonia solani, Pythium vexans and Fusarium Yercaud, Ooty and Dindigul districts (Tamil Nadu). species were found to be dominant. Artificial in- Both larvae and adults (male and female) were oculation studies proved that among the different observed to harbour the endosymbiont. fungi isolated, R. solani, P. vexans and F. oxyspo- rum were pathogenic and F. oxysporum was found Management of thrips to be the predominant species. In vitro screening of respective Trichoderma spp. against the most Ten new insecticides and organic prod- 8 Indian Institute of Spices Research ANNUAL REPORT 2012/13 ucts such as neem soap, Spinosad, Vertemec, and quality, especially in soils deficient in Zn and Thiamethoxam, Thiacloprid, Imidacloprid, L-cy- B. halothrin, Zolone, Fipronil and Quinalphos were evaluated in the field for the management of Organic production package thrips. Among the treatments, Fipronil (1.0mL L-1) Organic package consisting of 20t FYM was the most effective and was on par with Imida- cloprid (0.5mL L-1), Quinalphos (2mL L-1), Thiaclo- ha-1 + 2t ha-1 neem cake + 1t ha-1 ash + 4t ha-1 prid (0.5mL L-1), and Thiamethoxam (0.3mL L-1). vermicompost, Azospirillum and P solubilising Neem soap was not effective and was at par with bacteria (20g bed-1) and PGPR (GRB35) as seed control. treatment and as drench at 45 and 90 DAP has been standardized. Application of BM spray (1%) to contain foliar diseases and neem oil (5ml L-1 of water) in combination with the cultural control is TURMERIC recommended for shoot borer management. Re- sults showed that organic management system Genetic resources yielded on par with integrated system, while cur- cumin content was greater under organic system. One thousand three hundred and forty two Curcuma accessions are being maintained. Chemoprofiling of varieties Germplasm conservatory was enriched with 92 accessions received from RAU, Dholi, Bihar and Essential oil profile of rhizomes of 7 varie- TNAU, Coimbatore, Tamil Nadu as part of NAGS. ties indicated turmerone (5.6-25.8%), ar- turmer- Promising nematode tolerant accessions viz., Acc. one (3.5-20.8%) and curlone (5.4-15.6%) as the 48 and Acc. 79 have been shortlisted for yield. Six chief components. Among the varieties, ar-tur- novel polymorphic SSR markers were isolated by merone was low in Sobha (3.5%). Turmerone was selective hybridization method using 3’ biotinylat- 20-25% in Rajendra Sonia, Sugandham, Narendra ed microsatellite probe mix [(ACT)12, (AAAC)6, Haldi and Co-1, 5-7% in Sobha and Sona and 13% (ACCT)6] for identification of different accessions. in Varna. With regard to curlone, Varna, Rajendra Sonia, Sobha and Sona formed a group with 5.0- Source-sink relationship 6.8% and Sugandham, Narendra Haldi and Co-1 Varieties IISR Prathibha, IISR Alleppey Su- formed another group with 12.5-15.6%. preme and Suguna were sampled at monthly in- Biochemical characterization for re- tervals starting from 50 days after planting (DAP). All the varieties were on par with regard to leaf sistance to shoot borer area and dry weight. Maximum levels of endoge- nous IAA and zeatin riboside levels were recorded In mature leaves of resistant accessions, at 110 DAP which was just prior to rapid rhizome carbohydrates and proteins ranged from 7.56- bulking. Rapid starch accumulation (rhizome bulk- 19.71 and 1.08-7.75mg 100 mg-1 of dried leaves, ing) was seen after 120 DAP. The level of photo- respectively. The lignin content ranged from 26.4- synthesis was observed to be lowest in Suguna at 48.7%. In mature leaves of susceptible accessions; 130 DAP. carbohydrates and proteins ranged from 8.78- 12.76 and 2.82-11.10mg 100 mg-1 of dried leaves. Micronutrients for yield and quality The lignin contents ranged from 23.9-39.1%. The lignin content in immature shoots of both re- Based on the field studies on effect of Zn sistant and susceptible accessions was identical and B on the quality of IISR Prathibha for 3 years, and ranged from 43.0-44.0%. The fiber content in soil application of Zn up to 10kg ha-1 or foliar immature shoots of resistant and susceptible ac- spraying of ZnSO (0.25%) and Borax (0.2%) twice 4 cessions ranged from 24.3-30.3% and 25.7-37.0%, (60 and 90 DAP) is recommended for high yield respectively. 9