Mycotoxins, food security and climate change

Mycotoxins, climate change and food security: do we know enough?

Prof Naresh Magan DSc

Dr Angel Medina and Dra Alicia Rodriguez

Applied Mycology Group

AgriFood Institute, Cranfield, Bedford MK43 0AL, U.K.

n.magan@cranfield.ac.uk

Contents of my talk

Background to climate change issues –context for spoilage moulds/diseases and mycotoxins in staple food crops

Impact of water stress x temperature on mycotoxigenic fungi

Effect of water stress x temp x CO2 on growth and toxin production

Modelling climate change on a global scale to predict impacts on mycotoxins

Future perspectives

Food security is a global issue: prices of somestaple grains have increased in the last 5 years

Climate change: will put pressure on foodsupply, quality, and sustainability world-wide

What will changes in rainfall patterns, drought,temperature and CO2 have on staple foodproduction systems pre- and post-harvest?

Interaction between plant stress andfungal/pest infections will impact on fungalspoilage and mycotoxin contamination

Estimates: +25% losses due to fungal spoilage

Background

Predicted temperature change models in relation to industrial activity

0

100

200

300

400

500

600

700

1920 1940 1960 1980 2000 2020 2040 2060 2080

CO2 (ppmv)

CO2…

Predictions of CO2 based on the available models

Drought /rainfall patterns: total annual rainfall patterns may shift and interact with temp. & CO2 changes

Bebber et al. (2013; Nature Climate Change) have predicted: plant pathogens and pests are moving at about 3-5 km/year towards

the poles.

Expected additional no. of pests per country. Predicted from their model relative to

current levels (Bebber et al. 2014. New Phytologist)

−200 −150 −100 −50 0 50 100 150 200 250 300 350 400 450 500 550

Fischer & Knuttl (2015). Anthropogenic contributions to global

occurrence of heavy precipitation and temp. extremes. Nature

Climate Change: reduction in extremes from 1000 to 200 days

Climate change threatens food

security

Effects of climate change on crops?

Maize: Increasing need for irrigation; otherwise

>5-10% yield penalty; no maximum CO2 levels

determined

Rice: needs 12-38oC for canopy development;

Doubling CO2 promotes photosynthetic rate by

30-40%; also increases rice biomass by 20-25%

Soybeans: water stress would increase

vulnerability; higher temps adversely affect yield

by 40-50%

CO2 % change % change oC % change % change

> ambient biomass yield increase biomass yield

Yr

1 700 +12 +15 +4 -14 -18

2 700 +26 +26 +4 -16 -35

3 700 +17 +16 +4 -1 -6

Mean: +18 +19 -10 -20

Using “Climatron” environmental growth chambers (David Lawlor,

2000; Rothamsted Research)

Effects of increased CO2 (x 2) and temperature on biomass and grain production of winter wheat

[controls at 350 µl/l]

Different predictions by different studies

Climate change can affect crop diseases:

what impacts on mycotoxin production?

Crop(resistance)

Pathogen(changing population)

Environment(changing

climate)

Pre- and Post-

harvest spoilage &

Mycotoxin severity

Our focus has been in mycotoxins – why?

Mycotoxins - naturally occurring toxic secondary metabolites

heat stable/difficult to destroy even during processing

Produced by Aspergillus, Penicillium, Fusarium and Alternaria genera

Contaminated at any time: field, harvesting, drying, transport and storage, milling and in finished products

Low mould counts do not mean that food is free of mycotoxins, as moulds can die but the toxins will remain

Comparison of potency of carcinogens in test animals

Compound Dose * Relative potency

trichloroethylene 3 1

carbon tetrachloride 0.02 150

benzidine 0.005 600

dimethyl nitrosamine 0.0005 6000

Sterigmatocystin 0.00003 100,000

aflatoxin B1 0.000001 3,000,000

* = g/kg/day to produce tumours in 50% of test animals over a life time

ochratoxins

FumonisinsAflatoxinsTrichothecenes

Raw materials, moulds and mycotoxins

Commodities for which EU legislative limits existAflatoxin OTA Patulin Fusarium toxins

_______ ______ _______ ____

Groundnuts + +

Nuts + +

Dried fruit + +

Cereals + + +

Maize + + +

Spices + +

Baby foods + + + +

Coffee +

Cocoa +

Grape juice +

Fruit + + + (apple)

Milk, egg +

Wine +

Climate change impacts on mycotoxins: directly and indirectly? Increased plant diseases/pest reproduction and

interaction with plants? YES Effects on biodiversity of microbiota on plant

surfaces? YES Will this lead to more mycotoxin contamination

of staple crops??? Will different mycotoxins become important on

a regional basis??? Will this make existing legislation out of step

with potential problems??? EU borders: RASFF 30% rejections-mycotoxins

Maize silkingdates in (a) 2016 as reference and (b) + 5 °C scenario in 2050.

Significant impacts on fungal infection/pests

Battilani et al., 2012. EFSA Report

(a)

(b)

Maize harvest date in (a) 2016 as reference and (b) + 5 °C scenario in 2050.

Impacts on diversity, infection and toxin contaminstion

Battilani et al., 2012. EFSA Report

Maize: Map of predicted risk of aflatoxin B1 contamination in maize in the +2 and + 5°C climate change scenarios. Used predicted 2079 meteorological conditions vs existing ones (Battilani etal., 2012. EFSA Report). No account of CO2

+2oC +5oC

Ecology of mycotoxigenic fungi

Consider different interacting factors which will affect growth and mycotoxin production

Water stress (aw) x temperature changes

Climate change: Aw x temperature x CO2

1.00

0.90

0.80

0.70

0.60

0 10 403020 50

Temperature (oC)

Wat

er

acti

vity

(a w

)

Fusarium/Alternariaspecies

Penicilliumspp

Aspergillus spp.

Diagrammatic profiles of growth/no growth limits for the three key mycotoxigenic genera

Potential changes in mycotoxins due to Temp and water stress interactions

Aw Tmax range T+3 T+5

/temp

Alternariol 0.95 100-500/25 20-40 5-20(A.alternata) 0.90 5-20/25 0 0

Ochratoxin A 0.95 >50/20 >50 30-50(P.verrucosum) 0.90 30-50/20 30-50 30-50

Ochratoxin A 0.95 1500-2000/20 1000 0-500(A.carbonarius) 0.90 500-1000/20 0-500 0-500

(Pen/Asp: ng/g) Magan et al. (2011) Plant Pathol.

Integrating molecular, ecological stress and toxin data: A.flavus and aflatoxin B1

Water x temperature impacts

Systems approach: combining gene

expression, growth and aflatoxin B1 production

Expression of 10 genes in the biosynthetic

pathway: aflF, aflD, aflE, aflM, aflO, aflP,

aflQ, aflX, aflR, aflS

+

Radial Growth +Aflatoxin production

Used a mixed growth model: growth associated product

formation model (Shuler and Kargi, 2007)

Comparison of the observed vs. predicted aflatoxin B1 production.

0

0.5

1

1.5

2

2.5

3

3.5

4

0 1 2 3 4

Alat

oxin

pre

dict

ed (p

pm)

Aflatoxin observed (ppm)

Model validation outside of the experimental area

Factors Conditions

Temperature (oC) 37 37 40

Water activity (aw) 0.90 0.95 0.90

Growth, µ (mm day-1) 0.29 0.59 0.14

Observed AFB1 production

(µg g-1)

3.96±0.20 2.68±0.14 0.00

Predicted AFB1 production

(µg g-1)

4.90±0.00 3.75±0.18 0.00

Table 4. Model validation outside the regions in which the model was developed

Abdel-Hadi et al. (2012), & Medina et al. (2013) Journal of Royal Soc. INTERFACE.

Climate change: important factors to

consider

Water stress (water activity, aw)

Temperature fluctuations (+2 to +5oC)

CO2 (x2 and x3 present value)

Very limited data if any on

mycotoxigenic pathogens

Temperature, water and CO2 interactions on growth, gene expression and aflatoxin B1

production by A. flavus

0

2

4

6

8

10

12

14

350 350 350 650 650 650 1000 1000 1000

0.97 0.95 0.92 0.97 0.95 0.92 0.97 0.95 0.92

Gro

wth

rat

e (

mm

of

rad

ium

/day

)

CO2 concentrationWater activity

A. flavus NRRL3357-YES-34oC

0

2

4

6

8

10

12

14

350 350 350 650 650 650 1000 1000 1000

0.97 0.95 0.92 0.97 0.95 0.92 0.97 0.95 0.92

Gro

wth

rat

e (

mm

of

dia

mte

r/ d

ay)

CO2 concentrationWater activity

A. flavus NNRL 3357-YES-37oC

x 2 CO2 x 3 CO2Air

A.flavus: absolute aflD expression relative to the control (=0.99 aw/30oC)

0.6

0.8

1

1.2

1.4

1.6

1.8

300.91 0

300.91650

300.911000

300.99 0

300.99650

300.991000

370.91 0

370.91650

370.911000

370.99 0

370.99650

370.991000

Calibrator: 30ºC, 0.99 aw, 350 ppm CO2

Rel

ati

ve

exp

ress

ion

Regulatory gene: aflR gene relative expression: A. flavus cultures on Glucose-based medium Calibrator: 30ºC, 0.99 aw, 350 ppm CO2

0.6

1.6

2.6

3.6

4.6

5.6

6.6

30 0.91

350

30 0.91

650

30 0.91

1000

30 0.99

350

30 0.99

650

30 0.99

1000

37 0.91

350

37 0.91

650

37 0.91

1000

37 0.99

350

37 0.99

650

37 0.99

1000

Re

lativ

ea

flR

ge

ne

exp

ress

ion

Temperature, water activity and CO2 concentration

0

200

400

600

800

1000

1200

1400

1600

1800

350 650 1000 350 650 1000 350 650 1000

0.97 0.97 0.97 0.95 0.95 0.95 0.92 0.92 0.92

ng

of

AFB

1/g

CO2 concentrationWater activity

A. flavus NRRL 3357 - AFB1

production at 37oC on YES

Summary impacts: A. flavus NRRL3357-YES

Temp (oC) aw CO2 (ppm) aflD aflR AFB1

34

0.97650 = = =

1000 = = =

0.95650 = = =

1000 = (x3.6) =

0.92650 = (x24.4) (x2.6)

1000 = (x2.0) (x2.0)

37

0.97650 (x4.6) = (x30.7)

1000 (x6.5) = (x23.8)

0.95650 (x6.4) (x14.6) (x79.2)

1000 (x3.2) (x43.9) (x78.5)

0.92650 = (x40.4) (x15.1)

1000 (x22.5) (x1680) (x23.8)

Medina et al. (2014) Frontiers in MicrobiologyMedina, A., Rodríguez, A., Sultan, Y & Magan, N (2015). Climate change factors and A.flavus: effects on gene expression, growth andaflatoxin production- World Mycotoxin Journal.Medina, Rodriguez and magan (2015). Current Genetics. In Press.

Stimulation of relative expression of biosynthetic pathway genes

by these 3-way interacting factors and increasing phenotypic

AFB1

Studies on maize: A. flavus NRRL3357

Comparison of relative gene expression and

aflatoxin production on maize under normal and

elevated conditions of CO2 x temp x aw

Temp (oC) aw CO2 (ppm) aflR AFB1

30

0.99650 = =

1000 = =

0.91650 (x 2) (x2.7)

1000 = =

37

0.99650 (x2) (x3.6)

1000 (x3) (x5.0)

0.91650 (x2) (x1.8)

1000 = (x1.5)

RNA sequencing now completed and being analysed in collaboration with NCSU, USA and USDA,.

Coffee: contamination with ochratoxin A

Contaminated with:

Aspergillus westerdijkiae (A. section Circumdati)

A. carbonarius (A. section Nigri)

0

20

40

60

80

0.95 0.98 0.99

OT

A (

ng

g-1

)

Water activity

A. westerdijkiae (B 2) 30°C

Effect of aw x CO2 on OTA production by A.westerdijkiae grown on a coffee medium at 30

and 35°C (Akbar & Magan, unpublished data)Note: scale ranges are different for OTA production.

0

1000

2000

3000

4000

5000

6000

0.9 0.95 0.97

OT

A (

ng

g-1

)

Water activity

(a) A. westerdijkiae (B2)

30°C

Effect of aw x CO2 x temperature on OTA production by A. westerdijkiae (B2) on coffee beans stored at (a) 30 and (b) 35°C (Akbar & Magan, unpublished data)Note: scale ranges are different for OTA production.

Models which have been used to predict mycotoxins in relation to climatic conditions

Predictive models: for Fusarium head blight and DON uses regional weather parameters during ripening (DONCAST)

Geostatistics approaches: to identify spatial hotspots which are high risk areas for mycotoxins (Europe; USA)

Agricultural production system simulators: in AustraliaDeveloped an Aflatoxin Risk Index (ARI) for peanuts/maize

Maize: Related seasonal temp and soil moisture during critical silking period to determine the ARI. Hot/dry conditions. Peanuts: Fractional amounts of available soil water during pod filling determined the ARI. Real time model developed (Chauhan et al., 2008, 2010)

Variable N° of stations EMDa

Mean Temperature 20828 48.36

Mean Minimum

Temperature11550 64.94

Mean Maximum

Temperature11544 64.96

Rainfall 27375 41.71

World meteorological data available in FAOCLIM

Variable Africa Asia America Europe Asia Antarctica World

Mean T 605 1256 2765 765 515 90 5996

Rain 3395 2172 5611 1389 915 48 13530

Inventory of time series (number) of variables considered according to

continents.

Worldwide prediction

Risk map – A. flavus in maize

Relative risk maps for ochratoxin A in grapes

Predictive models which exist at present: are they good enough to predict impact of climate change?

Impact of drought stress x CO2 changes on crop physiology and interaction with mycotoxinproducing fungi – required urgently

Cycling of drought/temperature events - effects on plant /fungus interface and mycotoxin production

Metabolomics: changes in mycotoxins produced by specific species??? Ratios ?? Switch between mycotoxins?

Future perspectives I

New mycotoxins by existing drought tolerant fungal species pre- and post-harvest??

Relationship between climate change factors, especially temp x CO2 increases may stimulate pest reproduction in staple crops

This could have a significant impact on mycotoxincontamination by causing damage

Food security in both developing and developed countries could be profoundly compromised under such scenarios

Future perspectives II

Acknowledgements:

Dr Angel Medina, Dr David Aldred, Dra. Alicia Rodriguez, Esther Baxter (Cranfield); Pof. Rolf

Geisen, Dr. Markus Schmidt-Heydt (Max RubnerInstitute, Germany; Dr. Roberto Parra (Monterey

Tech, Mexico)

Dr Deepak Bhatnagar (USDA) and Prof. Gary Payne (NCSU, USA) help with RNA Seq of A.flavus

(analysis in progress)

Pre-harvest: Wheat fusarium ear blight

• Decreases yield

• Produces mycotoxins (damage human/animal health)

• Important across Europe, North and South America

Climate change – wheat flowering dates

predicted to be earlier: implication for

FHB and mycotoxins?2050s High CO21980s

Madgwick et al., 2011, Eur J Plant Path 130, 117-131

Climate change – predicted increase in %

plants with Fusarium ear blight: what does

this mean for mycotoxins - DON??1980s 2050s High CO2

0

400

800

1200

1600

0.95 0.98 0.99

OT

A (

ng

g-1

)

Water activity

(a) A. carbonarius (ITAL 204) 30°C

0

20

40

60

0.95 0.98 0.99

Water activity

(b) A. carbonarius (ITAL 204) 35°C Air (400ppm)

CO2 (1000ppm)

Growth of F. graminearum: elevated temperature and CO2 (1000 ppm; 3x ambient)

conditions at 2 aw levels (Medina & Magan, unpub. data)

0

5

10

15

20

25

25 30 35 40

Gro

wth

rat

e (

mm

dia

met

er/

day

)

Temperature oC

Fusarium graminearum

aw 0.995+ CO2

aw 0.98+CO2

aw 0.995

aw 0.98

0

0.5

1

1.5

2

2.5

3

GS50 GS60 GS65 GS75

350

700

350+4C

700+4C

Growth stage

Log

10

CF

Us

cm2

Biodiversity: fungal populations affected by climate change on flag

leaves/ears of wheat

Magan & Baxter, 2000

Different sub-arrays shown in different colours red = ochratoxin A, light brown = aflatoxin, dark blue =

trichothecenes (type A), light blue=trichothecenes; dark green =fumonisins

This corresponds to the colours of the frames surrounding thehybridized spots after microarray analysis

Schmidt-Heydt & Geisen (2007) Int. J. Fd Microbiol.

MYCOCHIP ARRAY

Maize: colonisation by A. flavus NRRL3357 at 30oC

-1

1

3

5

7

9

11

13

15

17

19

350 650 1000 350 650 1000

0.91 0.99Re

lati

ve

afl

R g

en

e e

xp

res

sio

n

Water activity x CO2 concentration

aflR gene (30°C)0

0.2

0.4

0.6

0.8

1

1.2

350 650 1000 350 650 1000

0.91 0.99Rela

tive a

flD

gen

e e

xp

ressio

n

Water activity x CO2 concentration

aflD gene (30°C)

Key: where P is the AFB1 production (ppm) and b1, b2, α, and β are parameter estimates from the model and µ was calculated based on a period of 9 days growth.

[g] – linear model for gene expression

Abdel-Hadi et al. (2012) Journal of Royal Soc. Interface (aflatoxins)Medina et al. (2013). Journal of Royal Soc. Interface (fumonisins)

11

0

tRT

ab

eeXgPw

Linked: Gene expression; growth, aflatoxin B1 production

Biosynthetic genes: aflatoxin production: Grouped according to expression profile at temperature x water activity combinations, to the gene cluster. Group 1 – light grey; Group 2 – dark grey (Schmidt-Heydt et al. 2010: Int J Fd Microbiol)

Ratio of aflS/aflR in relation to aflatoxin biosynthesis and

different parameter combinations

Parameter combination Ratio aflS/aflR Aflatoxin [ng/g]

25oC/0.90 0.5 3.7

35oC/0.90 0.3 4.7

25oC/0.95 7.4 830.2

30oC/0.95 7.1 3016.9

25oC/0.99 1.5 1957.3

30oC/0.99 2.7 2758.7

0

50

100

150

200

250

300

350

350 650 1000 350 650 1000

0.91 0.99

Re

lati

ve

afl

D g

en

e e

xp

res

sio

n

Water activity x CO2 concentration

aflD gene (37°C)

0

20

40

60

80

100

120

140

160

180

350 650 1000 350 650 1000

0.91 0.99

Re

lati

ve

afl

R g

en

e e

xp

res

sio

n

Water activity x CO2 concentration

aflR gene (37°C)

Maize: colonisation by A. flavus NRRL3357 at 37oC

Boundaries for growth and mycotoxinproduction

We now have significant knowledge of the impact of environmental stress factors on mycotoxigenic fungi: germination, growth, toxin production

F.culmorum DONF.graminearum DONF.langsethiae T-2/HT-2F.verticillioides FumonisinsP.verrucosum OTAA.flavus AflatoxinsA.carbonarius OTAA.westerdijkiae OTAAlternaria alternata Alternariol, AME, TZA, ALTXII

Magan & Sanchis, 2004; Magan et al., 2011; Magan et al., 2015

Effects of climate change on

maize?

Increasing need for irrigation; otherwise

>5-10% yield penalty; no maximum CO2

levels determined

Climate change Threat for agriculture

Maize is sensitive to water

stress and A.flavus is able to

grow in drought stress

conditions

Crop diseases, food security and

climate change

Need information to guide strategies for

adaptation to impacts of climate change on crop

disease losses

Need publicity to show how improved crop

disease control can contribute to climate change

mitigation

Need vigilance to maintain/improve crop disease

control despite changing pathogen/climate to

ensure global food security

New mycotoxins by existing drought tolerant

fungal species pre- and post-harvest??

Relationship between climate change factors,

especially temp x CO2 increases may stimulate pest

reproduction in staple crops

Could have a significant impact on mycotoxin

contamination by causing damage

Food security in both developing and developed

countries could be profoundly compromised under

such scenarios

Aflatoxin biosynthesis gene cluster

The genes involved in aflatoxin production are clustered together

Yu et al. (2004)

Re

gu

lato

ryg

en

es

Sst

ruc

tura

lg

en

es Schmidt-Heydt et al. (2010)

- Some genes of this cluster act

together in different groups

- Ratio aflR/aflS & aflatoxin

production (under different

environmental conditions)

Abdel-Hadi et al. (2012)

- A.flavus can grow over a wide

range temperature x aw, butaflatoxin production is over a

narrower range.

- Relationship between activity of

aflD gene with regulatory genes

(T/aw)Sst

ruc

tura

lg

en

es

F. verticillioides CO2

0

2

4

6

8

10

12

14

16

24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Gro

wth

rat

e (

mm

dia

met

er/

day

)

Temperature oC

Fusarium verticillioidesaw 0.995+ CO2

aw 0.98+CO2

aw 0.995

aw 0.98

Growth of F.verticillioides under increased temperature and CO2 (1000 ppm; 3x ambient) conditions at 2 aw levels (Medina & Magan, unpub. data)