Case study – Pepper mild mosaic virus (PMMoV), description of methods used for seed health test and result interpretation Kai-Shu Ling USDA-ARS, U. S. Vegetable Laboratory Charleston, SC, USA (NAPPO Seed Health Workshop-2015, Riverdale, MD) U. S. Vegetable Laboratory The mission of the U.S. Vegetable laboratory is to improve genetic populations of vegetable crops on yield, quality and pest resistance; to generate knowledge on the etiology, ecology, epidemiology, & pathogenicity of plant pests, thus to develop new management strategies. Broccoli Collard green Cowpea Cabbage Pepper Sweetpotato Cauliflower Tomato Watermelon Major vegetable crops that we are working on: Brassica (Broccoli, Cabbage, Cauliflower, and Collared green), Solanaceous crops (Pepper and Tomato), Southern pea, Sweet potato and Watermelon. Today, I will focus my presentation on the emerging tomato diseases. Virus structure, composition and the strategies in virus detection Detection: RNA or DNA Molecular (PCR) Coat protein Serological (ELISA) (From Agrios’ Plant Pathology, 3rd ed.) Common Methods for Plant Virus Detection Indirect Tests (infectious + dead viruses) Direct Tests (infectious) (quick, sensitive and specific, could result in false positive) (slow, less sensitive, non-specific) Tobamovirus Coat protein RNA or DNA (Local lesions) SqMV (Growout) Major seed-borne viruses and viroids on Solanaceous crops and seed health tests Indirect test Direct test Genus Species Host qRT- ELISA Bioassay PCR PMMoV Pepper + Pepper/ TMV + Local lesion Tobamovirus tomato ? on tobacco Pepper/ ToMV + tomato Systemic on Potexvirus PepMV Tomato + + N. benthamiana PSTVd/P Pepper/ Pospiviroids - + Growout CFVd tomato Pepper mild mottle virus • Symptoms: leaf chlorosis (mild), plant stunting, and distorted fruits (severe). • Spread: – Long distance: Seed-borne (outer seed coat). – Short distance: mechanical transmission (hands, tools, clothing). Particular in greenhouse production. – no insect vector. But in soil and water. Humans as a vector (stool and fecal pollution)? • Distribution: Worldwide. Pepper Seed-borne Tobamoviruses (PMMoV + TMV) • Family: Virgaviridae • Genus: Tobamovirus • Species: – Pepper mild mottle virus (PMMoV) – Tobacco mosaic virus (TMV) – Tomato mosaic virus (ToMV) PMMoV-pepper seed Seed health test by serological method (enzyme- linked immunosorbent assay -ELISA) for three tobamoviruses in pepper ISTA/ISHI/NSHS: 3,000 seeds/lot in 250 An absorbent value at OD405 nm seeds/subsample (12 subsamples in that is 2x above the healthy control duplicate wells), tests for 3 viruses could should be considered positive. be carried out using the same seed extract. Rep1 Rep2 Negative sample -CK Positive sample +CK (yellow color) Bioassay with a local lesion test for tobamoviruses on tobacco plant • ISTA/ISHI/NSHS: minimum 3,000 seeds in 250-500 seeds/subsamples (6- 12 reps) through mechanical inoculation on tobacco, Nicotiana tabacum Xanthi NN to induce necrotic local lesions. • When such sample is inoculated on to a healthy pepper plant, a new disease would likely occur. • Local lesion test is quicker (5-7 days) than 4-5 weeks, quantification of virus titer by counting local lesions, local lesion is more obvious than mild mottle. Risk assessment: Interpretation of seed health test results If following the standard method, a negative test result is used to certify a seed lot. Planting such certified seeds will likely not introduce that virus to a region or a country. Even if a seed-borne disease has established in a country, planting a certified virus-tested seed lot is still important to reduce initial virus inoculum, slow disease progression and minimize yield loss to an economic threshold level. Risk assessment (cont.): Interpretation of seed health test results A right balance in seed health test is to prevent an infected seed lot to be traded in the market. A false negative result may occur due to the poor sensitivity, specificity, or inhibition of a test. A false positive resulting from detection of dead or non- infectious viruses could result in an unnecessary rejection of a valuable seed lot. Under this situation, particularly with a treated seed lot, the positive result from an indirect test (like ELISA, PCR) should be validated with a direct test to determine virus infectivity, i.e., seedling growout or bioassay, as appropriate. Real-time RT-PCR for specific detection of tobamoviruses, an alternative method or a future for seed health test? • RNA extraction of seed samples using PMMoV 2800 Trizol reagent. 2700 2600 2500 2400 2300 2200 • Conserved sequences for primer and 2100 2000 1900 1800 1700 1600 probe design. 1500 1400 1300 1200 Fluorescence(norm) 1100 1000 • Using an internal amplification control 900 800 700 600 500 400 (i.e., 18S rRNA). 300 200 100 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 • At least 10 x higher sensitivity than Cycle Threshold: 199 (Adjusted manually) ELISA. Baseline settings: automatic, Drift correction OFF ToMV TMV 580 3400 560 3200 540 520 3000 500 2800 480 460 2600 440 2400 420 400 2200 380 2000 360 340 1800 320 1600 300 280 1400 260 1200 240 1000 Fluorescence(norm) 220 Fluorescence(norm) 200 800 180 160 600 140 400 120 100 200 80 0 60 40 -200 20 -400 0 -20 -600 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Cycle Cycle Threshold: 99 (Adjusted manually) Threshold: 251 (Adjusted manually) Baseline settings: automatic, Drift correction OFF Baseline settings: automatic, Drift correction OFF Management of PMMoV Host resistance (L1, L2, L3, and L4 R genes) Avoidance (clean seed), seed health test. Hygiene, sanitation and disinfection to limit secondary mechanical transmission. Rotation to a non-host crop, like tomato, eggplant. Most Effective Disinfectants for ToMV/TMV (likely effective for PMMoV too) + - + - + - 10% Bleach 20% NFDM 50% Lysol Most effective disinfectants against major greenhouse tomato viruses/viroid (should be useful to manage pepper tobamoviruses) Disinfectants PepMV ToMV TMV PSTVd Clorox (10%) Virkon S (2%) NFD Milk (20%) Lysol (50%) Partial Summary Planting a certified virus-tested seed lot is an important preventative measure against seed-borne viruses. A reliable seed health test should be sensitive in detecting the presence of virus particles, but also need to determine the virus infectivity through appropriate bioassay, particularly on those treated seed lots. An integrated disease management should be employed, from planting a virus-resistant cultivar, to the use of effective disinfectants, and crop rotation to non-host plants. Acknowledgments USDA-ARS, Charleston -Rugang Li • USDA-NIFA SCRI project on -Andrea Gilliard greenhouse tomato disease management, (PI: Sally Miller) • USDA-NIFA SCRI project on tomato viral disease detection and resistance (PI: Kai Ling) • Chet Kurowski, Monsanto (Photos on PMMoV). .