AsiaBlight: the beginnings Louise R Cooke Beijing, 25-27 October 2019 History of AsiaBlight • Nepal, November 2014: experts from 10 Asian nations propose creation of AsiaBlight History of AsiaBlight • Nepal, November 2014: experts from 10 Asian nations propose creation of AsiaBlight History of AsiaBlight • China, July 2015: first AsiaBlight meeting at World Potato Conference, population mapping proposed History of AsiaBlight • November 2015: Greg Forbes invited me to assist him with developing AsiaBlight, specifically with the coarse-scale mapping project • 2016: coarse-scale mapping project initiated to: • provide information on the current Asian Phytophthora infestans population • demonstrate the value of Public-Private-Partnerships • develop collaboration among Asian partners • Aarhus, May 2017: report on progress at EuroBlight Recent developments: late blight in Asia AsiaBlight Louise R Cooke, Ruofang Zhang, Lei Wu, Si Hui Chen, Greg Forbes History of AsiaBlight • 2014-2018: initial AsiaBlight activities conducted on a voluntary basis with the generous involvement of many collaborators; not sustainable in the long-term • September 2018: CIP–China Center for Asia-Pacific (CCCAP) took on the challenge of implementing a self-sustaining AsiaBlight network • York, May 2019: report on progress and future plans at EuroBlight Why map the Asian Phytophthora infestans population? • The implications of changes in the population affect the region as a whole, impacting on blight control via: • Initial inoculum source (oospores or tubers) • Cultivar behaviour • Forecasting/decision support systems • Fungicide performance Courtesy of Asko Hannukkala & Ari Lehtinen, MTT, Finland Coarse-scale mapping project • 500 FTA cards funded by Bayer (Regions APAC 1 & APAC 2) • Distribution of FTA cards and genotyping offered by Ruofang Zhang and her team, Inner Mongolia Potato E & T Research Center, Inner Mongolia University, Hohhot, China • Genotyping standards provided by David Cooke, James Hutton Institute (JHI) • Contacts established in Asian countries via CIP, previous collaborations and Bayer Progress to date • Sampling already occurring in Asia from ongoing projects and other collectors; some genotyped and submitted to the database: • Bangladesh (Geert Kessel, Bangladesh Agricultural University, Bangladesh Agricultural Research Institute) • China (Guo Mei) • India (Geert Kessel, Sanjoy Guha Roy, Sanjeev Sharma) • Indonesia (Chris Ursell, Huub Schepers, Phill Wharton) • Republic of Korea (Geert Kessel) • Myanmar (Geert Kessel) • The Philippines (Geert Kessel) Progress to date • FTA cards and sampling instructions and forms were sent to: • Bangladesh (2016) AsiaBlight Protocol for sampling Phytophthora infestans DNA using FTA cards 2019 The process takes longer to explain than to do - it’s really simple! 1. Use 1 card with 4 sampling areas (circles) per site (field). • Georgia (2016, 2017) 2. Sample 4 lesions per infected site. If only one plant is infected, collect 4 separate lesions from the same plant. If there are numerous infected plants, collect 1 lesion from each of 4 separate plants distributed across the site. 3. Correct sampling is critical. Select leaflets with single, fresh, nicely sporulating young • India (2016) lesions for each sample (Figure 1), otherwise we won’t get good results (maybe no results at all). Avoid older lesions, dead leaves, old or dry lesions, leaflets with many lesions, wet, water soaked (bacterially infected) looking leaves and those with small restricted lesions. 4. Label the FTA card with a reference number. Provide the rest of the information on the sample form. • Indonesia (2016, 2017) 5. Cut out a piece of sporulating lesion approx. 1-2 cm square at the edge of the lesion (Figure 2) using sterilised scissors or scalpel (use an alcohol wipe). 6. Place the lesion sample on one clean circular sampling area on the FTA card sporulating side down. Do not touch the FTA card sampling area except with the lesion sample! • Japan (2016) 7. Replace the FTA card’s cover sheet over the lesion sample and apply moderate rubbing pressure to the sample through the cover sheet using a blunt object (e.g. the blunt end of a marker pen or pestle). Don’t rub too hard or you will damage card. When green leaf extract is visible on the back of the FTA card, the collection process is complete. • Kyrgyzstan (2017) 8. Remove excess plant residue from the FTA card, ensuring that no large pieces of plant tissue remain stuck to the card (Figure 3). It doesn’t matter if some small bits remain. 9. Air-dry the FTA cards for at least 1 hour at room temperature. 10. Store dry FTA cards separately in paper envelopes. Do not place several cards in the same • Nepal (2016) envelope as this can result in cross-contamination. Figure 3 • Pakistan (2017, 2019) • The Philippines (2017) • Island of Taiwan (2016) Figure 1 • Tajikistan (2016, 2017) Figure 2 • Uzbekistan (2016) Figure 3. FTA card with samples after they have been used for SSR analysis (DNA has been extracted from the 2 mm disks punched out of each sample) and close-up of card with lesion debris. • Vietnam (2016) Progress to date • FTA card samples were collected or are to be collected in: • Bangladesh (2017) • Georgia (2016, 2018) • Indonesia (2016, 2017) • Japan (2016) • Kyrgyzstan (2020) • Nepal (2016, 2017) • Pakistan (2018, 2019-20) • The Philippines (2017) • Island of Taiwan (2014-2016) • Vietnam (2017, 2018, 2019) • Indian biosecurity legislation does not permit FTA cards with pathogen samples to be sent out of the country for genotyping • Re-organisations in Central Asian Countries meant that Armenia could not participate and resulted in loss of contacts and cards in Tajikistan and Uzbekistan AsiaBlight: success • Voluntary collection of blight samples by many contacts across the region • Genotyping using the 12-plex SSR in Hohhot (implemented by Sihui Chen and Yu Lan) proved difficult to maintain, but the current student Zhang Zhibin has produced results (mainly for China) running each SSR marker separately Sihui Chen and Yu Lan Zhang Zhibin • Half FTA cards sent to the James Hutton Institute, November 2018; thanks to David Cooke and his staff we now have samples genotyped and mapped Sampling progress Country/ Year and crops sampled Comments on Region genotyping in JHI 2016 2017 2018 Bangladesh 25 potato 24 samples genotyped Georgia 10 potato 10 potato No results 2016, 8 of 2018 samples genotyped Indonesia 10 potato 16 potato 17 samples genotyped, plus 9 genotyped in Hohhot Japan 10 potato 8 samples genotyped Kyrgyzstan No late blight 2018, samples collected September 2019, not yet genotyped Nepal 9 potato 15 potato 23 samples genotyped 10 tomato Pakistan 15 potato 2018 samples no results, 5 tomato hope to sample 2019-20 Philippines 10 potato 10 samples genotyped Taiwan 12 tomato Plus 8 tomato isolates 2014- 15; 4 genotyped Vietnam 4 potato 3 potato Plus 4 tomato samples 2019; 1 tomato 11 genotyped AsiaBlight: challenges • Obtaining samples across a large, politically diverse region • Difficulty in maintaining stable communication channels, leading to loss of contacts and of FTA cards • National biosecurity legislation restricted opportunities for direct participation in some countries • Genotyping: technical issues, difficulty in standardising allele calling despite provision of web workshop by David in October 2018 • But we have PROGRESS!! AsiaBlight: genotype map 2017 AsiaBlight: genotype frequency map 2016-2019 AsiaBlight: genotype frequency chart 2008-2019 AsiaBlight: MSN tree genotypes 2009-2019 13_A2 Run using all uploaded Asia samples: 2_A1 421, 2009-2019 US_1_A1, AU_1 MSN = Minimum Spanning Network AsiaBlight: MSN tree countries 2009-2019 Indonesia Philippines Run using all uploaded Asia samples: 421, 2009-2019 Bangladesh, India, MSN = Minimum Spanning Network Myanmar, Nepal AsiaBlight: PCA analysis Very marked and distinct groups PCA = Principal Component Analysis Findings: • Population highly clonal • 13_A2 (Blue 13), aggressive, phenylamide-resistant, is widespread across mainland Asia (Bangladesh, China, Georgia, India, Myanmar, Nepal, Vietnam) • US_1 still persists particularly on tomato (Nepal, Vietnam) • Island populations: Indonesia, Japan, the Philippines, Taiwan are distinct and disparate, 13_A2 not so far found Next steps: • Planning to write up for publication during 2019-2020 • Need to ascertain from collectors/owners whether genotyping data from their non-AsiaBlight samples can be included • Hoping for samples from Kyrgyzstan and Pakistan in 2019-2020 • Need more samples from Japan, Taiwan • How to progress genotyping in Asia? • Fungicide sensitivity data: vital to allow appropriate management advice (particularly for the genotypes in Indonesia, the Philippines), need isolates for this • Future: CCCAP’s plans to implement a self-sustaining AsiaBlight network will be described in the following presentations AsiaBlight: Acknowledgements • David Cooke • Jens Hansen and Poul Lassen • Jonathan Yuen • Ruofang Zhang, her staff and students • Bayer (Regions APAC 1 & APAC 2) • Country contacts • Bangladesh: Abdullah-Al-Mahmud, Monower Hossain, Ebna Habib Md. Shofiur Rahaman • CACs: Zurab Khidesheli, Rusudan Mdivani and colleagues • Indonesia: Ineu Sulastrini, Koko Tjintokohadi, Silviya Wiltin • Japan: Seishi Akino • Nepal: Buddhi Sharma • Pakistan: Muhammad Taufique Siddiqui, Waqas Raza • The Philippines: Robert Babaan • Taiwan: Rishi Burlakoti, Lawrence Kenyon, Wallace Chen • Vietnam: René van Rensen, Ho Ngoc Anh • Thanks particularly to Greg Forbes for inviting me to be part of CIP is a research-for-development organization with a focus on potato, sweetpotato and Andean roots and tubers. It delivers innovative science-based solutions to enhance access to affordable nutritious food, foster inclusive sustainable business and employment growth, and drive the climate resilience of root and tuber agri-food systems. Headquartered in Lima, Peru, CIP has a research presence in more than 20 countries in Africa, Asia and Latin America. www.cipotato.org CIP is a CGIAR research center CGIAR is a global research partnership for a food-secure future. 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