1. 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

2. 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,
Courtesy: Lawrence Kenyon

3. 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

4. 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

5. 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

6. 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

7. 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.

8. 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?

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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 % disease expression in susceptible varieties

16. 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

17. “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

18. 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.