Towards improved maize withresistance to Fusarium verticillioides

The Safemaize-projectresults & future possibilities

The Safemaize-projectGenetic improvement of maize to enhance food safety by introducing resistance to Fusarium verticillioides

EU-INCO Project January 2001 - June 2004

Why Safemaize-project?

Maize is the single most important staple food in countries of the Southern African Development CommunityOften, maize produced in SADC countries such as Zambia, Zimbabwe and South Africa, contains too high toxin levels due to infection with F. verticillioides

The Safemaize Project Partners

University of Pretoria (co-ordinator)CSIR, PretoriaARC-Roodeplaat, PretoriaUniversity of ZambiaUniversity of Rome

Maize breeding, Bergamo

PRI

General aims of the Safemaize project

To characterize Fusarium verticillioides isolates from Africa. To develop screening methods to evaluate maize genotypes for resistance to the fungus. To produce transgenic maize with stable inheritance of selected plant anti-fungal genes, and evaluate resistance in the field.

Southern Africa: isolates collection

Fusarium verticillioides isolates from Africa

Collection of Fusarium isolates 60 isolates from Zambia23 isolates from MRC (mainly from Transkei)

Characterisationspecies specific primers (Waalwijk) AFLP

Species specific primers

Acknowledgements: C. Waalwijk (PRI), A. Logrieco & G. Mulè (ITEM)

Species specific primers Origin n Fvert Fsub FprolUNZA 58 43 1 1MRC 23 22Fvert=F. verticillioides , Fsub=F. subglutinans , Fprol=F. proliferatum

Species specific primers

Ackn: C. Waalwijk (PRI), A. Logrieco & G. Mulè (ITEM) , J. Rheeder (MRC)

Species specific primers Fumonisin productionOrigin n Fvert Fsub Fprol +++ ++- -++ -+- --+ ---UNZA 43 43 32 1 4 5* 1

13 1 1 1 10*2 1 1 2

MRC 23 22 22 1Fvert=F. verticillioides , Fsub=F. subglutinans , Fprol=F. proliferatum ,

Fum prod=fumonisin producer (FUMF/R, VERTF-1/2, PKS-exon5F6R)* F. longipes , F. equiseti , F. graminearum , unknown, ** F. sambucinum , F. subglutinans

UPGMA tree

Nei and Li distance

Bootstrap values above 50%

U N Z A 0 0 1 2

F . V M 1 6

U N Z A 0 0 2 9 U N Z A 0 0 2 6 U N Z A 0 0 1 1

M R C 7 6 5 9

M R C 0 4 3 8

M R C 0 0 8 2

U N Z A 0 0 0 4

U N Z A 0 0 4 1

M R C 2 5 1 1

U N Z A 0 0 5 6

F c I P O 3 9

M R C 0 9 8 1

U N Z A 0 0 2 1

M R C 0 8 2 6

U N Z A 0 0 5 1

U N Z A 0 0 4 4

U N Z A 0 0 4 2

M R C 7 9 3 2

U N Z A 0 0 5 5

U N Z A 0 0 4 7

U N Z A 0 0 4 5

M R C 0 6 1 1

U N Z A 0 0 1 9

M R C 0 9 2 9

U N Z A 0 0 3 0

U N Z A 0 0 1 0

U N Z A 0 0 0 7

M R C 3 2 3 5

M R C 0 6 2 7 M R C 1 0 4 8

M R C 0 8 1 5 M R C 5 6 2 2 M R C 7 8 9 4 M R C 7 8 9 6

M R C 7 8 9 5 M R C 7 9 3 1

U N Z A 0 0 3 4 U N Z A 0 0 2 7

U N Z A C U N Z A 0 0 4 8

U N Z A E U N Z A 0 0 4 6

U N Z A 0 0 4 3 U N Z A 0 0 5 7 M R C 0 1 3 7 U N Z A 0 0 0 6

M R C 0 1 3 8 U N Z A 0 0 1 6

I T E M 0 1 2 6 I T E M 2 2 8 2

M R C 0 6 0 2 U N Z A 0 0 4 0 U N Z A 0 0 1 5 M R C 5 9 0 3

U N Z A 0 0 1 8 U N Z A 0 0 5 0

U N Z A 0 0 2 2 U N Z A 0 0 2 3 U N Z A 0 0 5 3 U N Z A 0 0 2 4 U N Z A 0 0 0 5 U N Z A 0 0 0 9

U N Z A 0 0 3 8 U N Z A 0 0 3 9 U N Z A 0 0 0 8 U N Z A 0 0 1 4 I T E M 1 4 9 6 U N Z A D

F g 8 2 0 U N Z A 0 0 3 7

U N Z A 0 0 0 1 U N Z A 0 0 0 2

F . f u j i U N Z A 0 0 5 4

0 . 10 . 20 . 30 . 40 . 50 . 60 . 70 . 8

5 3

7 8

8 9

9 0

5 89 9

5 6

6 2

5 3

6 2

6 3

8 4

9 1

9 4

9 8

1 0 0

1 0 0

7 6

1 0 0

6 6

1 0 0

MRC+++

UNZA+++

UNZAvariation

others

Screening methods

Greenhouse/in vitro test in the Netherlands Field test in Zambia Aims:

to screen germplasm for resistanceto screen transgenic maize

Greenhouse test in the Netherlands

Good correlation

between years (r2=0.77)

Seedling assay: aggressiveness

control Fv MRC826

Fc IPO-39

Fv M16

Fg ITEM126

Field experiment in Zambia

Carried out at the Golden Valley Research TrustMaize growing season Nov- April

2001/2002 very dry2002/2003 good2003/2004 good

Field test in Zambia

• Silk channel inoculation method

Field test in Zambia 203/2004Blotter PDA

CO255 Netherlands 7.5 10.0LO 1096 Italy 15.0 15.06705-60 Mexico 10.0 15.0A722-2 Zimbabwe 12.5 17.51725-29 Mexico 15.0 25.0LO1010 Italy 11.7 20.0A722-4 Zimbabwe 20.0 23.36705-56 Mexico 16.7 23.3PAN 6363 Zambia 13.3 26.7MRI 455 Zambia 20.0 30.0MRI 624 Zambia 16.7 33.3LO1124 Italy 30.0 33.3F113 Netherlands 21.7 35.0GV 412 Zambia 20.0 36.7MRI 514 Zambia 20.0 36.7MM 502 Zambia 16.7 40.0MRI 734 Zambia 20.0 43.3

Genes available for transformation

Pvpgip2 gene (polygalacturonase inhibitor gene)maize b-32 gene (ribosome inhibiting protein)chitinase gene terpene synthase genes M. Jongsma PRI

Nerolidol Experiment - day 3

0.000

0.050

0.100

0.150

0.200

0.250

1 2 3 4 5 6 7 8 9 10Concentrations

Abso

rban

ce

ITEM2282

MRC3235MRC826

MRC826-2MRC826 wc

Plant transformation: B32 and bean Pgip2Transgenes are inserted into maize genome: PCR, Southern blot

Transgenes are expressed: Northern blot, Western blot

Plant transformation: achieved

Fungal Infection Score of Germinating seedsU

ntr.

Hi-I

I(N

ysta

tin)

Unt

r. H

i-II

Unt

r. H

i-II

(Sap

orin

)

A1E

F1A

F1F

H8E

0

1

2

3

4

5

6

7

Plant Seedlings

Infe

ctio

n Sc

ore

Transgenic maize shows fungal resistance: laboratory test

Putative T0 transgenics in pots under greenhouse conditions [plants are dwarfed due to tissue culture stress]

Plant transformation: achieved

T1 plants in greenhouse T3 plants in larger greenhouse, 3 m old

Plant transformation: achieved

Field Experiment 2003/2004 GMO Field experiment has been carried out in South Africa instead of Zambia, becauseit is not allowed to grow GMO maize in ZambiaResults: look promissing, wait for plating results

Safemaize project training

Guest workers from UNZA to PRIfrom University of Pretoria & ARC to University of Rome

Training includedinvolvement in plant pathological aspects molecular biology

Safemaize conclusions

Collection of Fusarium isolates Screening assays have been developedVariation in resistance levels is present > breeding! GMO-plants with additional plant defense genes have been obtainedLab-assays show improved levels of resistanceTraining

How to improve the situation?

Further research

Plant GMO/non GMO

resistance genesselectable markerscultural measures

Fungus and toxininfection processisolates/species aggressiveness

mixtures of toxins

Medical researchinfluence of other food types

relationship between diet and health

toxin mixtures

Social impactawareness of toxins

acceptance new varietiesand other food