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Biodiversity of Toxigenic Fungi and its Implications in Disease Management
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Biodiversity of Toxigenic Fungi and its Implications in Disease Management

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Food systems,Fusarium diversity,Aspergillus diversity,Aflatoxin risk in cereals,Mycotoxin Management

Food systems,Fusarium diversity,Aspergillus diversity,Aflatoxin risk in cereals,Mycotoxin Management

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  • 1. Biodiversity of Toxigenic Fungi and itsImplications in Disease Management Ranajit Bandyopadhyay IITA, Ibadan International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 2. Outline• Food systems• Fusarium diversity• Aspergillus diversity• Aflatoxin risk in cereals• Management• Conclusions International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 3. Food Systems Large Scale and Regulated – Developed countries – Trade based – Advanced infrastructure – Capital intensive Small Scale and Unregulated – Developing countries – Informal markets – Subsistence – High food insecurity Tim Williams, Peanut CRSP International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 4. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 5. Major Classes of Mycotoxins• Aflatoxins: Aspergillus flavus, A. parasiticus• Fumonisins: F. verticillioides etc.• Trichothecenes: Fusarium spp, Stachybotrys• Zearalenone: F. graminearum• Ochratoxins: Penicillium verrucosum, A. ochraceous International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 6. Mycotoxin and Trade• Protect consumers from undue exposure• Promote regional and international trade • Stringent regulatory standards in importing countries (e.g., EU) • Rapid Alert Reporting System globally reports food safety issues (on internet) – poor country image• To promote regional and international trade• To encourage national development of agro-based economies• To protect consumers from economic exploitation• Impractical in local systems International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 7. Mycotoxins and Health Death Contributes to 40% of DALYs Impairs growth and development of children Suppress immune system Aflatoxin interacts with HBV • 30 times more potent in HBV+ people • 5-60 times higher cancer risk May impede uptake and utilization of micronutrients in human systems Associated with Kwashiorkor in children, oesophageal cancer in humans, neural tube defect Also affect animal health and productivity International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 8. Confounding Toxicological Effects• Poisoning modes – Acute poisoning • Clear symptoms – Chronic exposure • Indirect symptoms usually attributed to other agents – Immune suppression -> infectious diseases – Nutritional interference -> vitamin deficiencies – Developmental interference – Cumulative exposure • Genetic and carcinogenic• Medical professionals need better information International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 9. Wrong Emphasis on Aflatoxin and Human Disease (Gong et al) Possible Relative public Health effect deaths (No.) attentionBiological weapon 0 (?) Very highAcute aflatoxicosis 100’s HighHepatocellular 10,000’s MediumcarcinomaGrowth impairment/ 100,000’s (?) Low/Noneimmunosuppression International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 10. Aflatoxin (ng/g) Contamination in West Africa Food products • Peanut paste: 3278 – GhanaPrimary products • Peanut sauce: 943 – Ghana • Leaf sauce: 775 – Gambia• Maize: 4000 – Benin • Maize dough: 313 – Ghana• Peanut: 216 – Ghana • Kenkey: 524 – Ghana• Sorghum: 80 – Ghana • Cashew paste: 366 – Ghana• Millet: 200 – Nigeria • Peanut oil: 500 – Nigeria• Tiger nuts: 120 – Nigeria • Yam flour: 7600 – Nigeria MTL: 20 ng/g • Local beer: 135 - Nigeria International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 11. Aflatoxin Exposure in Africa, Europe & USA 100 Gambia (n = 950) Benin (n = 479) USA (n = 48) Europe (n = 74)Number of individuals (%) 80 60 40 20 0 <5 5-25 26-100 >100 Aflatoxin-albumin adducts (pg AFB1-lysine eq./mg albumin) International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 12. Fusarium speciesCourtesy: Leslie & Summerell International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 13. Fusarium – A General Division• Three broad groups• “Dark red” pigment producers – F. graminearum, F. culmorum, F. poae, F. sporotrichioides, etc. – Trichothecenes and zearalenone – Not common in Africa• F. solani species complex – Not strictly toxin producers, but can turn plant host defense compounds into toxins – Most important cause of direct human infections• Gibberella fujikuroi species complex important in Africa – Fumonisins are the most prominent toxin International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 14. Fusarium Diversity and Toxins• Several Fusarium species • An understanding of and types of toxins occur species diversity can help on cereals. in toxin risk management• Risk of toxin based on • Species and diversity Fusarium species present depend on crops, but least understood in Africa Fusarium spp. FB1 FB2 MON DON ZEA T2 NIV F. verticillioides FB1 FB2 F. proliferatum FB1 FB2 MON F. thapsinum MON F. graminearum DON ZEA NIV F. sporotrichioides T2 International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 15. Gibberella fujikuroi Species Complex• Most common problem in Africa, especially if F. oxysporum included here• May be problematic on maize, millet and sorghum• Most prominent toxin is fumonisins, but other potential problems include moniliformin, fusaproliferin and beauvericin• At one time all were called “F. moniliforme”• May produce a compound that can be confused with zearalenone in TLC analyses International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 16. Fusarium spp. from Sorghum• Common F. thapsinum dominates followed by F. andiyazi and F. proliferatum with occasional other species• Egypt F. proliferatum – 52%; F. thapsinum – 26%; F. verticillioides – 17%; F. sp. nov – 5%• West Africa F. thapsinum dominates followed by F. andiyazi and numerous unidentified species• Dominant species varies by location• Many unidentified species with undescribed toxigenic potential• Presence of fumonisins and moniliformin confirmed International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 17. Fusarium Diversity on Maize in Ghana• Data from two AEZ; more than 650 isolates• All grains yielded at least one isolate• 95% were F. verticillioides• F. proliferatum 3%, a third species 1%, and six other species represented by a single isolate each• 51 clones containing 2-4 isolates each of F. verticillioides recovered representing ~ 20% of the total population• Both sexual and asexual reproduction important in this fungal population• Fumonisin contamination most prevalent and risk is high (100+ ppm) Leslie and Bandyopadhyay (2005) Phytopathology 95:S58 International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 18. Fumonisin (µg/g) in Selected Elite Lines Artificially inoculated with F. verticillioidesInbred lines 2003 2004(1368/S.A. Pub Lines36/1368)-2-2-2-B 2.5 0.8(CIM 116 x TZMi 302 x CIM 116)-2-2-B 3.3 3.7KU1414xICAL 36-1xKU1414-6-1-B 4.7 3.5Obantapa-9-3-1-1-B 5.9 6.5Obantapa-33-5-1-B 5.9 4.7Obantapa-31-1-1-B 5.9 0.3PIONEER SEEDS-26-2-1-B 32.4 37.1((KU1414 x 9450) x 9450)-24-2-1-B 43.5 25.4P43SRC9FS100-1-1-8-#1-B1-4-B 55.0 21.61368xINV 534-1x1368-7-1-B 62.0 58.64205 63.5 99.21368xICAL 224-1x1368-2-2-B 87.2 25.0Mean 28.9 17.1SE 4.2 3.3 Afolabi et al. (2007) Plant Disease 91:279. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 19. Status Report• Most populations are diverse and are outbred• Sexual stages not commonly encountered in the field• Many plants are multiply infected• Fusarium spp. from sorghum and millets in Africa are unique and largely undescribed• Basic fungal taxonomy and mycotoxin profiling remains to be done• Fusarium toxins are a clear problem on maize, less so on other crops• Best plant pathogens are not the best toxin producers• The African picture often is different from from Europe or North America• Subsistence farm scenario may be of critical scientific & practical importance International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 20. AFLATOXIN International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 21. Biological Control of Aflatoxin Toxigenic  Ability to produce aflatoxin in A. flavus strains varies  Some strains produce a lot (toxigenic), and others no aflatoxin (atoxigenic)  Competitive exclusion (one strain competing to exclude another) as biocontrol principle in practice in the US  Diversity studies in Aspergillus section Flavi conducted in Nigeria to identify atoxigenic strains for biocontrolBandyopadhyay et al. (2006) Tropentag Atoxigenic International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 22. Selection Criteria for Biocontrol Agent• Atoxigenic strains evaluated for a set of selection criteria: – Should not produce aflatoxin – Ensure that the candidate strains belong to unique groups that are unable to produce toxigenic progenies in the natural environment. – Propensity to multiply, colonize and survive so that few reapplications will be required once the atoxigenic strains are introduced. – Environmental safety – Field efficacy• Select appropriate strains for further tests• Protocols for mass multiplication International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 23. Areas SampledJ. Atehnkeng Wet Dry M. Donner  Derived Savanna & Southern Guinea Savanna – 5 districts  Northern Guinea Savanna – 2 locations  5 locations / state; 50 to 124 isolates / location; n = 4414 International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 24. Atoxigenic Strain Identification Toxin assay - UnknownStrain characterization cnx nia-D VCGField Field release UnknownCompetition assays cnx nia-D Lab + International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 25. Distribution of Aspergillus in Nigerian Soils & Grains Soil Grain L-strain S-strain A. tamarii A. parasiticus 1% 0.1% 88% 97.9% 5% 0.6% 1.4% 6% L-strains more predominant than S-strains Low frequency of A. parasiticus Atehnkeng et al. (in press) Int. J. Food Microbiol. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 26. Aspergillus Strains in Grain 600 500 L-strain S-strain A. parasiticus 400 A. tamariiStrains / location 300 200 100 0 o ti a ja di a a a ga a f ia a h j ki un id ri w n ko bu ur os La Za an in E ok B ad ak Lo A M om do kw M M K A A gb O Districts International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 27. Proportion of Toxigenic and Atoxigenic Strains in Grain 90 80 70 60Percentage 50 Toxigenic 40 Atoxigenic 30 20 10 0 M fia na Ka ia ga da Ak uja ho wa Ad oja na it i Lo i d r Ek ur La Za an Bi du in os Ab ok k ak M w om o M gb O Districts International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 28. Atoxigenic Performance in Mixtures Grain Inoculation StudiesStrains AfB (ng/g) Reduction (%)La3279 17 99.8Og0222 110 98.7La3303 26 99.7Ab2216 303 96.5Lo4216 147 98.3La3304 44 99.5Ak3020 294 96.6La3108 153 98.2La3228 8567 --(Control)LSD (α = 0.05) 462.6 -- International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 29. Field Testing of Atoxigenics• 24 atoxigenic from 6 locations plus 4 toxigenic strains field- tested in Ibadan• Inoculation of atoxigenics alone and in combination with a toxigenic strain• Toxin production in cobs and competition between toxigenic and atoxigenics evaluated. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 30. Atoxigenic Performance in Mixtures Cob Inoculation Studies AfB1 (ng/g) in AfB1 (ng/g) in inner ring outer ring Strains Alone Mixture Alone Mixture La3279 0.0 9.4 0.2 1.1 Inner ring La3303 0.0 12.1 0.5 2.9 Ab2216 6.6 126.5 9.7 92.4 Lo4216 0.3 141.9 9.7 3.3 La3304 0.8 233.5 0.0 7.2 x Ak3020 0.6 264.4 0.0 15.9 La3108 0.0 272.1 0.0 1.9 La3228 5054.0 253.5 (Control) Outer ring LSD ---- 656.7 ---- 120.1 X = inoculation siteAtehnkeng et al. (submitted) Food Add. Cont. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 31. Field Release of Atoxigenic Strains• One toxigenic and four atoxigenic strains released together in field; with controls• Grain harvested after maturity• Recovery of released strains tested by VCG complementation• Atoxigenic La3279 most frequently recovered (up to 97%)• Further analysis is in progress International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 32. Frequency of Isolate Recovery from Maize after Field Release100 - Unknown 80 cnx nia-D 60 VCG 40 20 0 Ab2216 La3228 La3279 La3303 Ka16127 + Isolate International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 33. On-Station Field Release Trials 2007 • 4 strains • 0.75 ha • Zaria • Mokwa • Ibadan • Ikenne International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 34. Outlook for Aflatoxin Biocontrol• Research to develop atoxigenic strains is resource intensive• Need 4-6 years for large-scale use of biocontrol agents• Linkage needed with other organizations for safety, registration, mass production, marketing & other downstream Collaboration: dissemination activities • USDA-ARS• Scope for impact • Univ. of Bonn • Univ. of Ibadan International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 35. NigeriaInternational Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 36. Relative Aflatoxin Risk of Cereals in AfricaM. Kumar • Sorghum and pearl millet traditional cereals • Nigerian maize area – 1980: 465,000 ha; 2004: 4.5 million ha • Maize introduced and replacing in marginal areas • Mycotoxin risk higher in marginal areas • Objective: Compare Aspergillus contamination and aflatoxin levels in maize, sorghum and pearl millet grown side-by-side by subsistence farmers Bandyopadhyay et al. 2007. Food Add. Cont. 24:1109. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 37. Method• 14 farmers’ fields in Northern and Southern Guinea Savanna• Maize, sorghum and pearl millet grown by farmers in adjacent plots• Harvested at maturity by farmers• Samples brought to lab in Ibadan• Analysis: aflatoxin by ELISA, frequency of S (more toxic) and L (less toxic) strains of A. flavus.• Exposure calculated based on historic cereal consumption data International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 38. Fungal Genus (%) on Grains Crop Sample size Aspergillus Fusarium Maize 23 17.7 a 47.3 a Pearl millet 7 1.9 b 26.1 b Sorghum 40 4.2 b 26.4 b• Maize 4 & 9 fold more likely to be contaminated with Aspergillus than sorghum & pearl millet• Maize 1.8 fold more likely to be contaminated with Fusarium than sorghum & pearl millet International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 39. Aspergillus in Cereal Grains CFU per Aspergillus -- % of total (range)Crop* g grain L-strain S-strain parasiticusMaize 9869 83-100 0-17 0-15Sorghum 2390 83-100 0-12 0-4 *Sample size: 13 of each crop grown in same location International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 40. Aflatoxin Exposure from Cereals • Maize could have up to 24-fold MTL, sorghum 4.5-fold • Risk from sorghum is 4-fold less, and pearl millet 8-fold less than maize (consumption: 138 kg/year; BW: 60 Kg) Aflatoxin (ng/g) Samples Exposure > 20 ppb (ng/kg aflatoxin Crop Mean SD Median Range bw/day) (%) Maize 36 100 4.2 1.1 – 480 17 226.8 Sorghum 8.8 14 5.0 1.6 – 90 5 55.5Pearl millet 4.6 1.8 4.4 2.6 – 8.1 0 29.0 Afla-Safe 20 - - - 126.0 International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 41. Mycotoxin Management Strategies• Awareness• Host plant resistance• Biological control• Time of harvest• Grain drying method• Storage structure• Storage form• Sorting and processing• Insect control International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 42. Mycotoxin Management R-4-D at IITA Development and dissemination of mycotoxin management strategies (BMZ) Breeding for resistance (US-FAS; USDA) Biocontrol of aflatoxin through competitive exclusion (BMZ; collaboration with USDA, Univ. of Bonn) Awareness campaign to sensitize the population on aflatoxin risk (Rotary International) Intervention study for the reduction of aflatoxin and impact of nutrition on the toxin (BMZ) Fusarium species diversity and mycotoxin profile in food baskets (cereals/legumes) in Burkina Faso, Ghana, Nigeria and Cameroon (USAID; KSU, CNR, IRAD, SARI, INERA) Training & information exchange (USAID, BMZ, EU) International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 43. Summary• Mycotoxins in food and feed pervasive in Africa• Negative impact overlooked – chronic, unseen• Fungal diversity studies can help in targeting intervention strategies – technical and diet based• Maize riskier than sorghum and pearl millet for aflatoxin contamination• Institutions related to food safety very weak.• Aflatoxin received most attention; studies needed on other mycotoxins as well.• More work required on mycology to explain toxin data International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 44. Aflatoxin Tested Pet Food in NairobiInternational Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 45. P. Cotty, R. Sikora, S. Kiewnick J. Leslie P E O P L E C. AfolabiA. Menkir K. Hell P. Ojiambo International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 46. TeamInternational Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
  • 47. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org