1

بسم الله الرحمن الرحیمبسم الله الرحمن الرحیم

Virulence and Molecular diversity in

Puccinia striiformis f.sp. tritici from IRAN

Hojjat Rabbani

General Manager of North khorassan Agricultural and Natural Resources Research Center

1,3PST-31964

1,3,11PST-38

1987

1,3,12 PST-22

1981

1,3,11,12PST-421990

PST-591998 1,3,11,12,16

1,3,8,11,12,16,17,18,19,20 PST-80

2000

PST-972002

1,3,10,11,12,16,17,18,19,20

PST-98

2002

1,3,8,10,11,12,16,17,18,19,20

New2003 1,3,8,9,10,11,12,16,17,18,19,20

Stepwise Evolution of PST Races Stepwise Evolution of PST Races

12345678

R 47-51

Lemhi Chinese 166

Heines VIIMoroPaha

Druchamp

Produra9

10

1112131415

YamhillStephens

Lee Fielder Tyee Tres Hyak

16 Express 17 Yr818 Yr9 19 Clement

PST-782000 1,3,11,12,16,17,18,19,20

20 Compair

1,3,9,11,12,16,17,18,19,20 PST-99

2002

Start of Race studing

20A & 25A

1960 Neiman et al.,1968

Bamdadian 1972

14/8، 14/8A،19, 2D 20A and 25A

96E16 on Falat and Qods cultivars

Bamdadian 1984

YrSD, YrA, Yr25,Yr24, Yr10, Yr9, Yr7, Yr6, Yr4 and Yr2

Torabi et al., 1997

Yr 3 5 4 10 sp nd cv

Torabi et al., 1999

YrSD, Yr24,Yr23, Yr22, Yr19,

Yr9, Yr8, Yr7, Yr2, YrA and Yr1

Nazari et al., 1998

Yr24,Yr23, Yr22, Yr19, Yr9, Yr8, Yr7,Yr6, Yr24,YrA , YrSU,YrND,YrCV

Torabi 2000, Afshari 2003 and 2006

Yr24, Yr9, Yr7, Yr6, Yr2 and YrA

5

Special Greenhouse for Yellow Rust Research

Seed and Plant

Improvement Institute

Glass house

7

Sample Collection

8

9

Multiplication of the Fungus7-days-old seedlings of cultivar BulaniMaleic hydrazide acid (0.25 g·liter−1) per pot

10

11

12

After 10 days, latent lesions could be observed for each successful infection

13

Collecting Urediniospores

Collecting Urediniospores

14

Dried in a Desiccator at 4°C for 3 days, and stored in microtubes

15

Microtubes stored in Liquid Nitrogen

Race Determination

according to

Johnson et al., 1972

McNeal et al., 1990

two-leaf-stage onto seedlings of each variety.Incubation: dark climatic room for 24 h at 8°C and 100% relative

humidity greenhouse at 20°C for 10 days

17

Molecular Research

18

DNA extraction

Digestion

Adapter Ligation

Preamplification PCR

Selective amplication

Polyacryl -Amid gel Electrophoresis

Data Analysis

AFLP Analysis

19

DNA Extraction1- Grinding ( 15 mg Urediniospores + Mercapto-Ethanol)65C 1h

2-Phenol-Chloroform- Isoamylalchohol 14000rpm 3min 4C

3-Chloroform 14000 3min 4C4-Chloroform 14000 3min 4C

5- IsoPropanple -20C 30min17000rpm 4C 30min

Drying TE BufferRnase 37C 1h

Nanodrop Spectrophotometer

20

Selective amplification PCR EcoRI+AC / AG/ GC/ GT MseI+ AA/ AC/ AG/ GT13 cycles at 94°C for 30 s, 65°C for 30 s with a 0.7°C decrement per cycle, and 72°C for 1 min, and then 23 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min.

DigestionGenomic DNA (100 ng) EcoRI and MseI Restriction Enzyme (5 U each; Biolabs) at 37°C in 40 μl of RL buffer 1 h

Adapter Ligation 5 pmol EcoRI adapter, 50 pmol MseI adapter, 1 pmol ATP 1 U ligase (Fermentas), incubated 3 h more at 37°C diluted Three times

Preamplification PCR

EcoRI+0(5-GACTGCGTACCAATC-3) MseI+0(5-GATGAGTCCTGAGTAA-3)

30 cycles at 94°C for 30 s, 56°C for 1 min, and 72°C for 1 min.

21

Formammid Temprature UREA

22

Silver staining

1- Fix (10% acetic acid)

2- Rinse ( Ultr pure water)

3- Impregnate (AgNo3 1)

4- Rinse

5- Develop

6- Stop

7- rinse and store

AFLP Finger printing Gel

IGS (intergenic spacers) SEQUENCE RESEARCHIGS (intergenic spacers) SEQUENCE RESEARCH

L318 5-GCTACGATCCACTGAGGTTC-3

5SK 5-CTTCGCAGATCGGACGGGAT-3

PCR Reaction

24

PCR Product

25

Band Cutting

Biospin Gel Extraction Kit

DNA Extraction from the Gel

26

DNA Cloning according to pGEM - T Vector System

R

1- Ligation of the pGEM-T vector and PCR product

Promega Cooporation

T4 DNA Ligase

T4 DNA Ligase 10 X buffer

pGEM-T vector

Insert DNA

At least 3 h. at 15o C

Transformation of Ligated PCR:pGRM-T vectorTransformation of Ligated PCR:pGRM-T vector

27

1- High efficiency Competent Cells( JM1o9)

2- LB/ Ampicillin/IPTG/X-gal plate

6 h 37oC

Incubate 4oC

28

White colones are

selected as Positive

transformed colones

29

+

_

30

ABI PRISM BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA)

31

32

Virulence to resistanse Genes Yr1 ، Yr2 ، Yr4 ،

Yr6 ، Yr7 ، Yr8 ، Yr9 ، Yr10 ، Yr19 ، Yr22 ، Yr23 ، Yr24 ،

Yr SD ، Yr ND ، Yr SU ، Yr CV و Yr SP

But Yr3 and Yr5

35 Phisiologic Races were Detected178E0A+ Khuzestan and Iranshahr64E 241 A+ Mazandaran Fars Khuzestan Hamadan

Virulence for Yr2, Yr24, Yr7 and YrA was detected in all around Iran

33

Safi3 , Ahvz, Ilam and Ghazvin 4.6%

First time Detected 1999 Khorasan

Yr1

Persian Gulf

34

Heines Kolben Yr2 77% Heines PEKO Yr2 and Yr6 37% Kalyansona Yr2

2000 reported

Yr2

35

Yr 4 1997 for the first time34%

36

Yr 6

Torabi et al., 1997Nazari et al., 1998 High F.Torabi et al., 2000 High F.Afshari et al., 2003, 2005 High F.

37

Yr7

Yr 7

In Lee and Riechesberg 42

Torabi et al., 1997Nazari et al., 1998 Common.Torabi et al., 1999Torabi et al., 2000 High F.Afshari et al., 2003, 2005 High F.

38

Yr 8

Nazari et al., 1998 Common.Torabi et al., 1999 Low F.Torabi et al., 2000 High F.Afshari et al., 2003, 2005 High F.

39

Yr9

1993 Falat EpidemyTorabi et al., 1997Nazari et al., 1998 Common.Torabi et al., 1999Torabi et al., 2000 High F.Afshari et al., 2003, 2005 High F.

Yr10Torabi et al., 1997Mardukhi and Torabi., 1998 Shiraz and Moghan Torabi et al., 1999Torabi et al., 2000 High F.Afshari et al., 2003, 2005 High F.

Yr19Compare line ( Yr8)

Torabi et al., 1997Nazari et al., 1998

Yr22

Lee Yr7Yr22 and Yr23( Chen and Line 1995)Torabi et al., 1998Nazari et al., 1998

Yr23

Lee Yr7Yr22 and Yr23( Chen and Line 1995)Torabi et al., 1998Nazari et al., 1998

Yr 24

Meering+24 Isogenic lineTorabi et al., 1997Torabi et al., 1999Nazari et al., 1998 CommonTorabi et al., 2000 High F.Afshari et al., 2003, 2005 High F.

45

A

Golestan and

Mazandadaran

B

Mazandaran

C

Khuzestan

46

D Fars

E Fars

F Fars

G Khorassan

H Ardebil

I Center West and North west

47

D Fars

E Fars

F Fars

G Khorassan

H Ardebil

I Center West and North west

B

D

E

H

A

C

F

G

I

48

High Genetic Variation AND Relationship between Geographical regions and AFLP groups

Why:1- Different Environmental Condition Different cultivars Different History of EvolutionIn a uniform environment selection may favor particular genotypes

This metapopulation view of evolution and diversity of pathogen population differed markedy from models that are based on evolution in a uniform invironment where mutation rate and frequency dependent selection are the most important pressure.

2- In Addition to Different Climatic condition Each Provinces Prefer special Cultivars Special History of Evolution

Individuals respond asynchronously to differences in the local selective environment.

49

Genetic Distance and Identity of Populations

54% Similarity

metapopulation

Golestan

Mazandaran

Khorassan

Khuzestan

West & nwest

Fars

Center

50

D Fars

E Fars

F Fars

G Khorassan

H Ardebil

I Center West and North west

AB

C

F D

E

G

H

I

M & GW& NWKhoKhuF

Mediteranian Wind

Flow

Sudanian Wind Flow

52

Conclusion:However the interplay of selection, Genetic drift, Migration and Mutation has a major effect on the genetic diversity of this pathogen population The relative roles of this factors may change markedly beteween different Pathogen-host association, Between stages in the epidemiological cycle and between association in agricultural and natural ecosystems.

It is inevitable that the layer of selection is imposed on individual pathogen demes by a range of biotic and abiotic factors.

Host related factors: Major resistance genes , Variation in presence, Quantity and relative balance of phenolic compounds , Variation in morphology and .. Are Important However Their effect on population diversity is not predictable.

53

DamavandIRAN

Thanks