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Mycotoxin Research by Belinda Janse van Rensburg, Sonia-Mari Greyling and Bradley Flett ARC-Grain Crops Institute, Potchefstroom,2520

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Mycotoxin Research. by Belinda Janse van Rensburg , Sonia-Mari Greyling and Bradley Flett ARC-Grain Crops Institute, Potchefstroom,2520. Mycotoxin research team – ARC-GCI. - PowerPoint PPT Presentation

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Page 1: Mycotoxin  Research

Mycotoxin Research

byBelinda Janse van Rensburg, Sonia-Mari Greyling

and Bradley Flett

ARC-Grain Crops Institute, Potchefstroom,2520

Page 2: Mycotoxin  Research

Mycotoxin research team – ARC-GCI

Prof. B. C. Flett - Mycotoxin Research Team Coordinator, resistance, screening, breeding, management practices (CA/conventional)Dr. B. Janse van Rensburg - Epidemiology, stressors, fungicidesMs. A. Schoeman - Isolate variation and interactions, molecular, FGSC, etiologyMr. E. Ncube - Bt technology, stem borers, subsistence farmers, storage facilities

Collaborators:Stellenbosch University, Free State University, North West University, ARC-PPRI, ARC-OVI, PANNAR Seed Company, Cape Peninsula University of Technology, South African Sugar Research Institute, South African Grain Laboratories, University of Nairobi, Kenya, CIMMYT, Kenya, Partnership for Aflatoxin Control in Africa, Ethiopia, IITA, Nigeria and various individuals and institutions from Europe and the USA.

Commodities:Maize, groundnuts, sorghum

Page 3: Mycotoxin  Research

Fusarium verticillioides, F. proliferatum, F. subglutinans = fumonisins

Fusarium graminearum species complex = zearalenone, nivalenol, deoxynivalenol

Page 4: Mycotoxin  Research

Aspergillus flavus, A. parasiticus = aflatoxin

Stenocarpella maydis = diplonine?

Page 5: Mycotoxin  Research

Why are mycotoxins important?

•Affect the entire chain of food and feed production

•Reduction of marketable grain, increased cost of drying, decreased weight gain in animal feeding, fertility problems, and increased costs for animal health

• Toxic to humans and animals• Restrict markets (for developing countries)

Page 6: Mycotoxin  Research

Why are mycotoxins important?

Page 7: Mycotoxin  Research

TechniquesMycotoxins ELISA replaced by: HPLC – fumonisin, aflatoxin, zearalenone, nivalenol, deoxynivalenolLC-MS – Stellenbosch University

Fungal identification and quantificationIdentification and quantification by plating out replaced by:qPCR – probe (mycotoxin specific) , SYBR green (species specific)Plate out method to obtain fungal isolates.

Sequencing, phylogenetics, SSH (population studies), tagging mycotoxins with fluorescent dye.

PR Proteins: spectrophotometer, plate reader, RT-qPCR?

Insect volatiles, insect repulsion/attraction studies

Leaf nutrients (NWU laboratory) , soil analysis (ARC)

Page 8: Mycotoxin  Research

The effect of maize plant stressors on Fusarium verticillioides and fumonisin production

Dr. B. Janse van Rensburg

Page 9: Mycotoxin  Research

Introduction- Substances or conditions that impose stress on the fungus also have an influence on mycotoxin production.- Not enough is known about the effect of plant stressors caused by high plant populations, drought, heat and N depletion on F. verticillioides growth and fumonisin production.- It is important to understand the effect of stressors on F. verticillioides and fumonisin production in order to reduce the risk of fumonisins on human and animal health.

Page 10: Mycotoxin  Research

AIMS1) Determine the effect of drought stress on F. verticillioides infection and fumonisin contamination (glasshouse). 2) To investigate the effect of plant density and nutrient availability on Fusarium ear rot, fumonisin producing Fusarium spp. infection and fumonisin contamination under field conditions. 3) To investigate the effect of N fertilizer levels on the development

of F. verticillioides infection and fumonisin contamination (glasshouse).

4) Investigate the role of PR-proteins during fungal colonisation and fumonisin production.

Page 11: Mycotoxin  Research

MATERIALS AND METHODS

1) Watering regime glasshouse trial: Glasshouse 9, 2012 and 2013 30ℓ-, 25ℓ-, 20ℓ-, 15ℓ- and 10ℓ-water per week, CRN3505 and PAN6P-110 planted in 80 ℓ black bags (x3 reps). Ears inoculated at silking. HPLC (toxin quantification), qPCR (fungal biomass).

Page 12: Mycotoxin  Research

MATERIALS AND METHODS 2) Plant density field trial: ARC-GCI, 2011/12 and 2012/13. 10 000, 20 000, 30 000, 40 000 and 50 000 plants per ha of CRN3505 and PAN6P-110 (x3 reps).

Soil analysis Gradual depletion of N from 2nd season onwards Leaves sampled and analysed for available N,C and S Hand harvested, HPLC, qPCR

Page 13: Mycotoxin  Research

MATERIALS AND METHODS 3) N glasshouse trial: CRN3505 and PAN6P-110 were planted in

80 ℓ bags. KAN and UREA applied at the rates of 0kg-, 25kg-, 50kg-, 75kg- and 100kg-ha.

Inoculated at silking Hand harvested HPLC, qPCR

Page 14: Mycotoxin  Research

MATERIALS AND METHODS 4) PR-Proteins collected at: 8 leaf-, silking-, after inoculation , milk- and soft dough-stages. -1,3-glucanase, chitinase, peroxidase activity Chlorophyll fluorescence

Page 15: Mycotoxin  Research

RESULTS1) Watering regime glasshouse trial: - ANOVA – significant cv effect regarding fumonisin (P=0.00)- PAN6P-110 mean fum 4.04 ppm, CRN 3505 mean fum 7.72 ppm

0.00

3.00

6.00

9.00

12.00

1 2

Means of HPLC

cv

PAN6P-110 (cv1) CRN3505 (cv2)

Mean fumonisins(ppm)

Page 16: Mycotoxin  Research

RESULTS1) Watering regime glasshouse trial: - Watering regime (P=0.01) and cv (P=0.00) sign. for fungal biomass- Mean fungal biomass = 25.25 pg at 30 ℓ per week 45.12 pg at 15 ℓ per week- Significant watering regime x cv interaction (P=0.01) for fungal biomass

Watering regime

qPCR mean(pg/µℓ)

Page 17: Mycotoxin  Research

RESULTS1) Watering regime glasshouse trial: - Chlorophyll fluorescence data indicated PAN6P-110 to withstand plant-stress better than CRN3505 at some plant growth stages.

Page 18: Mycotoxin  Research

RESULTS2) Plant density field trial (2011-2012) and 4) PR-Proteins: - Plant density had no effect on fungal biomass- ANOVA indicated significant plant density effect regarding

fumonisin (P=0.03)- Sign. plant density x cv interaction regarding fumonisin (P=0.04)

Plant density/cultivar

10 000 plants/ha              

 

20 000 plants/ha

30 000 plants/ha

40 000 plants/ha

50 000 plants/ha

fumonisins fumonisins fumonisins fumonisins fumonisins Mean

PAN6P-110 1.47 2.79 3.20 3.66 16.65 5.55

CRN3505 0.49 2.93 3.11 3.83 2.40 2.55

Mean 0.98 2.86 3.15 3.75 9.53  

-Regression analysis yielded a significant relationship between fungal biomass and fumonisins (R2=0.73 and P=0.00).

Page 19: Mycotoxin  Research

RESULTS2) Plant density field trial (2011-2012) and 4) PR-Proteins: - Plant density - significant effect on:- Peroxidase - 8 leaf stage (P=0.01) Peroxidase activity 111 273 nmol guaicol. mg-1 protein. min -1

at 10 000 plants/ha Peroxidase activity 156 667 nmol guaicol. mg-1 protein. min -1

at 50 000 plants/ha- Peroxidase - silking stage (P=0.00) Peroxidase activity 18 339 nmol guaicol. mg-1 protein. min -1

at 10 000 plants/ha Peroxidase activity 97 598 nmol guaicol. mg-1 protein. min -1

at 50 000 plants/ha- Chitinase during plant silk stage (P=0.02)- 0.26 A550 nm. mg-1 protein. min-1 at 10 000 plants/ha- 0.29 A550 nm. mg-1 protein. min-1 at 50 000 plants/ha

Page 20: Mycotoxin  Research

RESULTS2) Plant density field trial (2011-2012) and leaf nutrients - Plant density had a significant effect on available N (P=0.00) and C

(P=0.00) in maize leaves: 8 leaf stage, silk stage, milk stage, and soft dough stage.

10 000 plants/ha 50 000 plants/ha

N - 8 leaf stage 3.63% 3.16%

N - silk stage 2.97% 2.28%

N - milk stage 7.28% 2.13%

N - soft dough stage

6.08% 1.23%

C - 8 leaf stage 43.58% 43.46%

C - silk stage 43.14% 43.04%

C - milk stage 40.16% 35.91%

C - soft dough stage 40.12% 35.12%

Page 21: Mycotoxin  Research

Performance index parameters of PAN6P-110 and CRN3505 measured during three plant growth stages, applying five different plant densities.

Page 22: Mycotoxin  Research

DISCUSSION1) Watering regime glasshouse trial: - Fungal biomass is not a direct reflection of fumonisin synthesis, this could explain why watering regime did not significantly effect fumonisin. - Inoculation could have attributed to the significant water regime x cultivar effect regarding fungal biomass. - Trial repeated

2) Plant density field trial:- Plant density had no effect on fungal biomass possibly due to

natural infection of plants. - Significant higher fumonisin levels at increased plant densities

could be attributed to “stress” factors such as competition for water and nutrients.

Page 23: Mycotoxin  Research

DISCUSSION

2) Plant density field trial (continued): - Low temperature and water stress reduce fungal growth.- Increased water stress increases FUM 1 expression.- This could explain significantly higher fumonisin levels in increased plant densities although fungal biomass did not increase. - CRN3505 - lower fungal biomass and fumonisins compared to PAN6P-110.- Potchefstroom, Bethlehem and Ermelo (2013/14) to determine

possible cultivar x environmental conditions. - Peroxidase and chitinase (silking) – important stages for fungal

infection and fumonisin synthesis.- More N available at lower plant densities.

Page 24: Mycotoxin  Research

Relevance and importance to the maize industry

- Management of Fusarium spp. and mycotoxins is reliant on an integrated system.

- One of the components in such an integrated system is to limit plant stressors.

- Literature available, but paucity of work to determine effect of stressors on fungal infection and mycotoxin synthesis under local conditions.

- For example: increase in fumonisins at higher plant populations with an added cultivar effect.

- Assist with management decisions.- Applied by small scale and commercial producers.

Page 25: Mycotoxin  Research

Prof. B. C. Flett - [email protected]. B. Janse van Rensburg - [email protected]. J. Berner - [email protected]. A. Schoeman - [email protected]. E. Ncube - [email protected]

Tel: 018 299 6100