Mycotoxin Research
by
Belinda Janse van Rensburg, Sonia-Mari Greyling
and Bradley Flett
ARC-Grain Crops Institute, Potchefstroom,2520
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, fungicides
Ms. A. Schoeman - Isolate variation and interactions, molecular, FGSC, etiology
Mr. 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
Fusarium verticillioides, F. proliferatum,
F. subglutinans = fumonisins
Fusarium graminearum species complex =
zearalenone, nivalenol, deoxynivalenol
Aspergillus flavus, A. parasiticus = aflatoxin
Stenocarpella maydis = diplonine?
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)
Why are mycotoxins important?
Techniques
Mycotoxins
ELISA replaced by:
HPLC – fumonisin, aflatoxin, zearalenone, nivalenol, deoxynivalenol
LC-MS – Stellenbosch University
Fungal identification and quantification
Identification 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)
The effect of maize plant stressors on Fusarium
verticillioides and fumonisin production
Dr. B. Janse van Rensburg
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.
1)
2)
3)
4)
AIMS
Determine the effect of drought stress on F. verticillioides
infection and fumonisin contamination (glasshouse).
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.
To investigate the effect of N fertilizer levels on the development
of F. verticillioides infection and fumonisin contamination
(glasshouse).
Investigate the role of PR-proteins during fungal colonisation and
fumonisin production.
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).
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
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
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
RESULTS
1) 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
Means of HPLC
12.00
PAN6P-110 (cv1)
CRN3505 (cv2)
9.00
Mean fumonisins
(ppm)
6.00
3.00
0.00
1
cv
2
RESULTS
1) 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
qPCR mean
(pg/µℓ)
Watering regime
RESULTS
1) Watering regime glasshouse trial:
- Chlorophyll fluorescence data indicated PAN6P-110 to withstand
plant-stress better than CRN3505 at some plant growth stages.
plants/ha
50 000
plants/ha
40 000
plants/ha
30 000
plants/ha
20 000
plants/ha
10 000
cultivar
-
Plant density/
2)
-
RESULTS
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)
fumonisins
fumonisins
fumonisins
fumonisins
fumonisins
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
Mean
a significant relationship between
fungal biomass and fumonisins (R2=0.73 and P=0.00).
2)
-
-
-
RESULTS
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
RESULTS
2) 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%
Performance index parameters of PAN6P-110 and CRN3505
measured during three plant growth stages, applying five
different plant densities.
1)
-
-
DISCUSSION
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.
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.
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.
Prof. B. C. Flett - FlettB@arc.agric.za
Dr. B. Janse van Rensburg - BelindaJ@arc.agric.za
Dr. J. Berner - jacques.berner@nwu.ac.za
Ms. A. Schoeman - BelgroveA@arc.agric.za
Mr. E. Ncube - NcubeE@arc.agric.za
Tel: 018 299 6100