Biotechnological strategies for immunity to aflatoxin contamination in groundnut. Pooja Bhatnagar-Mathur

Transcription

1 Sixth Annual South Asia Biosafety Conference, September 15-17, 2018 Biotechnological strategies for immunity to aflatoxin contamination in groundnut Pooja Bhatnagar-Mathur

2 Outline Aflatoxin problem & regulations Groundnut and aflatoxin Current status of combating aflatoxin strategies Biotechnological interventions Summary & Future prospects

3 WHAT IS ICRISAT? Nonprofit, apolitical, international Agricultural Research Institute Established in 1972 Member of the CGIAR System Organization Sorghum Pearl millet & Finger millet Groundnut Chickpea Pigeonpea

4 THE DREADED STORY OF AFLATOXIN

5 Health economics of aflatoxin Correlation between high liver cancer rates and high risk of chronic exposure to aflatoxin Source: Williams et al. (2004); Wu et al., (2011)

6 Trade Economics Vicious-Link Trade Health Quality reduction Trade Restrictions Aflatoxin contamination in food and feed Malnutrition Liver cancer Reduced ability to cope with diseases, especially HIV/AIDS Liver cirrhosis, immuno-suppression, blocks nutrient absorption, growth abnormalities, etc. Synergistic interaction with Hepatitis-B & C While chronic sub-lethal doses have nutritional and immunologic consequences, large doses can lead to acute intoxication resulting to direct liver damage and death. Both types of exposure have a cumulative effect on the risk of developing liver cancer. Country Product Aflatoxin Maximum allowable limit (µg/kg) China, Japan, Thailand, Groundnuts Total 10 Egypt, Turkey Indonesia, Malaysia, Groundnuts Total 15 Taiwan province of China, Australia EU Groundnuts Total 4 (direct consumption) 15 (further processing) B1 2 Kenya Groundnuts Total 20 Russia Groundnuts B1 5 Canada Nuts and Nut products Total 15 India All food products Total 30 Philippines Nuts and products Total 20 Singapore Nuts Total 5 USA All foods except milk Total 20 Viet Nam Food stuffs Total 10 (CODEX ALIMENTARIUS COMMISSION, 2013)

7 Knowledge on fungal biology genetics & management Efficient preharvest phenotyping/ screening methods HIGS/ RNAi Proteomics Candidate genes/ RAPs/ RIPs Biocontrol strategies Plant breeding strategies Host plant resistance Transgenics Overexpression of antifungal / anti-toxin genes Exploring nonhost resistance? TILLING/ Mutagenesis Molecular markers Marker assisted selection Field level assessments & Implementation across regions & environments

8 Complexity of the problem Cultivars and farming practices Prolonged end-of season moisture stress & insect damage to pods Time of harvest / pod removal Mechanical injury Methods of drying & storage Conditions of packing and distribution In many SAT countries, monitoring and enforcement of food safety standards are rare due to poor awareness and lack of human skills and infrastructure

9 Traditional plant breeding options Types of Resistance required Host and aflatoxin-producing fungi interactions 1. Pod wall: Physical barrier and resistance is attributed to pod shell structure 2. Seed coat: Moisture and heat stress can cause microscopic fissures in seed coat 3. Cotyledons: Provide sustenance to pathogen and aflatoxin is produced Challenges No durable sources of resistance available?? Genetics of resistance mechanisms not clearly elucidated Statistically significant genotype-environment interaction

10 Bio Control-Competitive atoxigenic fungal technology Sporulation on moist soil 3-20 days Spores Wind Soil colonization and displacement of toxigenic fungi Competitive exclusion: atoxigenic strains displace toxigenic strains - Shift strain profile from toxigenic to atoxigenic Broadcast Source: IITA

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12 A 13 year long journey A. flavus (%) T3 T2 Controls Genes used for engineering groundnut i. Chitinases (plant & fungal origin) ii. iii. iv. Lipoxygenases Forisomes Defensins

13 Pre-harvest screening of peanut transgenics (T7) in micro-sick plots

14 Towards combinatorial strategies

15 Groundnut immunity to aflatoxin contamination: a biotechnology in seed Approach 1: Defensin line Fungal growth inhibited Gene for resisting Aspergillus growth taken from alfalfa plant Introduced into groundnut DNA Approach 2: HIGS (Host-Induced Gene Silencing)line Defensin proteins produced Aflatoxin-immune groundnut (<1 ppb 3 days after fungus inoculation) compared to heavily contaminated seeds (over 3000 ppb) Aflatoxin production stopped Small Aspergillus RNA segments introduced in groundnut plant Fungus attacks groundnut Aflatoxin-producing gene silenced in Aspergillus > % reduction in aflatoxin production

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17 Host-Induced Gene Silencing

18 Fungal bioassays

19 Aflatoxin profile (Ultra performanceliquid chromatography- LOD )

20 Consistency across A. flavus strains OE-Def hp-omt WT

21 Host antioxidative machinary

22 Effect on pathway genes and feedback inhibition Morphology of infecting A. flavus after 40 hpi

23 Trait stability

24 2nd generation pipeline ,000 Aflatoxin pathway genes - aflm/ver1 NsdC and vea: Global transcription factors regulate sclerotium formation, conidiation and aflatoxin production aflr gene, involved in transcriptional regulation of genes required for developmental processes of sclerotium morphogenesis and conidiation and also regulates aflatoxin production

25 Candidate resistance associated proteins (RAPs) and genes (RAGs) Partners: ICRISAT, USDA-ARS New Orleans and LSU

26 Silencing regulatory genes Colony growth, pigmentation, and sclerotium production and reduced conidiation in A. flavus ΔnsdC and ΔnsdD mutants Cary et al. (2012). Eukaryotic Cell, 11: Coursey: Jeff Cary, SRRC, ARS-USDA

27 Fungal bioassays & aflatoxin analysis T1/ T2 progenies Construct Genotype Aflatoxin content (ppb) <= pcambia 2300: Nsdc ICGV RNAi 37 4RNAi 44 pcambia 2300: Vea ICGV pcambia 2300: amy ICGV ppzp200:4rnai ICGV RNAi B

28 Evaluation for Pre-harvest A. flavus and aflatoxin contamination Screening transgenic events carrying Rchit gene, Resistant and Susceptible checks and studying A. flavus drought interactions under micro-sick plot conditions

29 Roadmap for translation Event selection trial in the field planned for post rainy season Four lines selected for introgression in regionally adapted germplasm for assessment of resistance in the field. Interdisciplinary team of scientists work together to develop pipeline Regulatory and stewardship aspects are critical to address at the beginning Communication on food safety and the uniqueness of technology will be important. Identification of potentially novel mirnas/ metabolites associated with suppression of pre-harvest infection and aflatoxin production Genome editing holds tremendous promise!!

30 If you want something you ve never had, then you ve got to do something you ve never done. President Thomas Jefferson Principal author of Declaration of Independence and third President of the United States Thank you Dileep Shah- Danforth Research Center Deepak Bhatnagar- SRRC, ARS-USDA Jeff Cary- SRRC, ARS-USDA K. Rajashekaran- SRRC, ARS-USDA ZY Chen- Louisiana State University