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