Proposed Tomato Tospovirus Initiative for South and Southeast Asia First Asian Solanaceous Roundtable, Bengaluru India 9-10 September, 2014 Peter Hanson, Presenter AVRDC- The World Vegetable Center R&D Advisory Committee APSA Vegetable SIG 1

Tomato Tospoviruses in SE/S Asia: an Established Threat Outbreaks common in South Asia, SE Asia, south China Unlikely to go away Kunkalikar, S. R., Poojari, S., Arun, B. M., Rajagopalan, P. A., Chen, T.-C., Yeh, S.-D., Naidu, R. A., Zehr, U. B. & Ravi, K. S. (2011). Importance and Genetic Diversity of Vegetable-Infecting Tospoviruses in India. Phytopathology 101, 367-376. Courtesy: Lawrence Kenyon

Courtesy: Naidu Rayapati Tospovirus Diversity: S/SE Asia South Asia PBNV WBNV PYSV IYSV CaCV Southeast Asia CaCV WSMV MYSV PCFV TNRV Courtesy: Naidu Rayapati

Tospovirus Resistance Trials: 2007-2008 Entries Tomato line TSW-10: homozygous for TSWV resistance gene Sw-5 Tomato line CK12: homozygous for Sw-7 Checks CLN2498D, TLB182 14 “best-bet” S. peruvianum accessions Seed company trials in India and Thailand Natural field epidemics, mechanical inoculation TSW-10 (Sw-5) S. peruvianum

Tospovirus Trials in India and Thailand Manager Leader Period Tospovirus Inoculation Mahyco-India K.S. Ravi Jan-Feb 2007 PBNV Mechanical 45 DAS 2008 Mechanical 20 DAS JK Agriseed A. Deshpande June-Oct 2008 Natural-field East-West H. Pascha ‘Thailand’ Mechanical 14 DAS CaCV

Tospovirus Screening Results AVRDC Mahyco1 Mahyco2 JK EW-Thai EW CaCV Code No. infected/ total No. infected\total No. sympt./ total Resistant (%) L00689 0/10 12\20 9\21 33 13 L00887 1/10 14\20 9\14 NT L00673 2/10 10\20 8\26 57 90 L00738 12\23 L00889 1\10 15\20 6\26 85 78 L00882 0\10 9\20 10\24 NT  L00640 2\9 L00737 14\24 L00688 10\23 47 55 L06138 3\29 100 L00687 13\25 L00671 2\10 11\20 5\28 60 83 L00678 9\25 L00890 7\25 Tomato ck 4\4 CK12 (Sw-7) 26\27 TSW-10 (Sw-5) 26\26

Outcomes and Challenges: Breeding for Tospo Resistance Available tospo resistance genes Sw-5, Sw-7 ineffective against PBNV, CaCV Promising resistance in some S. peruvianum accessions Challenges Difficult to predict tospo epidemics <100% symptoms on susceptible checks common Symptoms may vary by plant age, environmental conditions, and expression often delayed Various tospo mechanical inoculation methods designed but not easily available.

Tospo Resistance Breeding: What Happened After 2008? Some companies took up resistance breeding using S. peruvianum. Other sources also? Interspecific crosses and backcrosses Some select in the field under natural tospo pressure and some companies developing mechanical, other inoculation methods Good that seed companies are investing in tospo resistance but could these efforts be more efficient through collaboration?

Collaboration on a tospo screening technique makes sense Field selection for tospo resistance is unreliable A reliable, efficient mechanical tospo screening protocol would remove a major bottleneck and allow us to: Identify resistance sources Select and introgress resistance Avoid loss of resistance during backcrossing Map resistance genes, develop markers Standard to evaluate progress Benefits participating companies, reduces development costs

Elements of a Tospo Screening Protocol Correct diagnosis and virus isolation Host plant that maintains virus and long-term virus infectivity for long-term and permits sufficient inoculum for large-scale screening Optimize inoculum concentration, buffers for efficient inoculation (90-100% infection of susceptible check) ELISA and visual symptom scale Technique repeatability under different environmental conditions Simple protocol that does not require heavy investment in facilities, equipment Accomplishing the above is a great technical challenge

Response of the APSA R&D Advisory Committee Solicited proposals from regional universities and national programs targeting design of a tomato tospo screening protocol Two proposals considered: National Center for Genetic Engineering (BIOTEC) Tamil Nadu Agricultural University Consider funding one or both proposals

BIOTEC Proposal Outline Title: Development of an efficient inoculation protocol for TNRV and CaCV resistance in tomato Principal Investigator Dr. Channarong Seepiban Project Duration Two years Budget: USD 73,000 Objective Develop an efficient inoculation protocol for TNRV and CaCV resistance screening in tomato Deliverables Mechanical inoculation protocol Thrips inoculation Graft inoculation

BIOTEC Workplan Approach Confirm tospo species (ELISA, RT-PCR, N gene sequencing) Maintain TNRV, CaCV isolates in host plants Isolation & Maintenance Isolate TNRV, CaCV from infected plants Method Design Disease rating scale (Symptoms + ELISA) Inoculation design Mechanical inoculation Graft inoculation Thrips inoculation Protocol Design Establish standard screening protocol for TNRV, CaCV Assess Mechanical inoculation alone Mechanical inoculation + grafting/thrips inoculations Test methods with different tomato varieties

TNAU Proposal Outline Title: Development of a reliable Inoculation Protocol to screen for resistance against Tospoviruses viz. Groundnut bud necrosis virus (GBNV) and Capsicum chlorosis virus (CaCV) Principal Investigator Dr. V.G. Malathi Project Duration Two years Budget: Under discussion Objective Develop a robust protocol for maintenance, multiplication, inoculation of GBNV and CaCV isolates on tomato and capsicum Deliverables Protocols for isolation and maintenance of cultures of a repertoire of GBNV/CaCV isolates Standardized sap/graft inoculation method on tomato/ capsicum genotypes Descriptors for resistance

TNAU: Work plan Summary GBNV, CaCV isolation, culture, identification by RT-PCR and sequence the nucleocapsid gene Identify indicator hosts and propagate isolate propagation Develop and standardize mechanical inoculation protocol Standardize buffer, reducing agents, pH Standardize inoculum application: vacuum infiltration, air facilitated inoculation, etc. Evaluate effects of temperature/RH, plant age, dosage, number of inoculations, on symptom expression Fine-tune until 80-100% disease expression in susceptible varieties

Building a Tospo Consortium R&D Committee intends to fund the BIOTEC proposal immediately and fund both proposals if enough interested companies join Equal cost sharing among companies: more companies reduces cost Access to results available only to participating companies Expected 2nd phase project applying the mechanical to select and develop resistant breeding material Participation in Phase 2 requires subscribing to Phase 1 Discussion in Break-out Group 1 meeting

“Working Together Works”-Rob Gilbert Committed Companies H.M Clause East-West Seeds Rijk Zwaan Syngenta Enza Zaden Indus Seeds Ankur Seeds BHN Seeds Namdhari Seeds PT BISI International SeedWorks India YOUR NAME HERE “Working Together Works”-Rob Gilbert

Thanks for your Attention There is substantial evidence worldwide to show that vegetables can increase farmer income and can provide moreover a rapid rate of return to investment far exceeding traditional cereal production.