molecular studies on - izbis.com · alignment b1 : e3 : af 3 6 8 3 2 9 : f 4 : ay1 4 1 8 5 5 cr :...

Molecular studies on

Cryphonectria parasitica isolates from

different Central-European plots

Görcsös G. – Irinyi L. –Tarcali G. – Radócz L.

Beograd

September 25-28

Cryphonectria parasitica

The casual agent of chestnut blight

One of the most important fungal pathogens of

chestnut in Europe and North America

Heterothallic ascomycetous fungus

The disease kills the infected tree branches

and cause the rapid death of the entire tree

Trees infected by Cryphonectria

parasitica

Symptom of C. parasitica

The aim of our study

• was to infer phylogenetic relationships and

diversity among Cryphonectria parasitica

isolates collecting from different Central-

European plots.

Materials and Methods

In this study, we analyzed almost 100

Cryphonectria parasitica strains isolated from

different part of Central-Europe but mainly

from the Carpathian-Basin.

Bark samples were collected from chestnut

trees located in geographically determined

(GPS) areas.

Collection sites of Cryphonectria

parasitica isolates

Culture morphology and growth

Isolates were grown on PDA

incubated at 25 C in the dark for one week

then exposed to diffuse daylight at room

temperature

culture morphology was recorded once a week

for a period of 4 weeks

For molecular works, cultures were grown in

malt broth for 2 days

Molecular studies

DNA extraction

Amplification of nuclear ribosomal Internal

Transcribed Spacer (ITS) and translation

elongation factor 1-alpha (tef1) by PCR

DNA sequencing

Phylogenetic analyses

Schematic structure of tef1 gene

and location of primers

Primers

Schematic structure of ITS region

and location of primers

Primers

Phylogenetic analyses

ClustalX

GeneDoc

Paup (parsimony analyses)

Alignment

B1 :

E3 :

AF368329 :

F4 :

AY141855Cr :

DQ368749 :

AY697928 :

AF452123 :

EU442647 :

EU442646 :

DQ368750 :

AY697930 :

AY141857 :

AY141858 :

AY141856 :

AY141863 :

N2 :

EF545115 :

AY141873 :

AY141859 :

AF292042 :

Bob2-2T_IT :

Bob2-4T_IT :

Bob1-3_ITS :

Bob3-3_ITS :

RO4_ITS_F :

024_ITS_F :

C0722ITSF :

C0008ITSF :

C0009ITSF :

C0006ITSF :

EU442645 :

C0722_ITS_ :

Bob1-1ITSR :

Bob3-1ITSR :

ERSEK3ITSR :

VROT3ITSR :

KOBA4TITSR :

PET1ITSR :

RAD5ITSR :

TET9ITSR :

TG4ITSR :

KAS1_ITS_F :

VEVI2_ITS :

VOL4_ITS_F :

ARK14ITSF :

BRAT3ITSR :

KEK6_ITSRu :

KEK8ITSR :

PAR4_ITSRu :

PAR5_ITSR :

SVE3_ITSR :

ERSEK2_ITS :

FEL1T_ITSR :

FEL4_ITSR :

HOR2_ITS_R :

KAV1_ITSR :

KOBA2_ITSR :

RO6_ITSR :

TG2_ITS_R :

MV1/4_ITS_ :

MV1/6_ITS_ :

P5-1_ITS_R :

P5-2_ITS_R :

P73_ITS_R :

PAR9_ITS_R :

PIR1_ITS_R :

BRAT1_ITSR :

KEK2ITSR :

KEK4_ITSR :

MOD4_ITS_R :

C1_ITSR :

OSOJ2_ITS_ :

ME48-2_ITS :

ME48-1_ITS :

MOD1_ITS_R :

PET3_ITS_R :

VEVI1_ITS_ :

VROT1_ITS_ :

EU442647.1 :

EU442646. :

EU442645.1 :

AF452121 :

AY697927 :

AY141862 :

GQ290655 :

GQ290654 :

GQ290653 :

EU199182 :

AF368331 :

AY697937 :

EU331074 :

Cryphonect :

AY697942 :

AF452118 :

AY697934 :

AY697933 :

EU442649 :

EU442650 :

EU442651 :

EU442652 :

EU442653 :

EU442654 :

EU442655 :

AF368327 :

AF452117 :

AF548744 :

AF548745 :

AF452113 :

AF368326 :

AF046905 :

AF368323 :

FJ785445.1 :

FJ785439Ph :

|FJ785437 :

JN942335 :

JN942331 :

EF026147 :

* 140 * 160 * 180 * 200 * 220 * 240

G-------------AAGGCAGATTTTCTTCCTTCTCCCCTCCCT------CCCCCCCCTCTTCCCCCGTGCAAACGGTTGTTGGGGAGCAGGCCCGCCGGCGGCCCACTAAACTCTTTGTTTTT

G-------------AAGGCAGATTTTCTTCCTTCTCCCCTCCCT------CCCCCCCCTCTTCCACCGTGCAAACGGTTGTTGGGGAGCAGGCCCGCCGGCGGCCCACTAAACTCTTTGTTTTT









G-------------AAGGCANATTTTCTTCCTTCTCCCCTCCCT------CCCCCCCCTCTTCCACCGTGCAAACGGTTGTTGGGGAGCAGGCCCGCCGGCGGCCCACTAAACTCTTTGTTTTT


G-------------AAGGCAAATTTTCTTCCTTCTCCCCTCCCT------CCCCCCCCTCTTCCACCGTGCAAACGGTTGTTGGGGAGCAGGCCCGCCGGCGGCCCACTAAACTCTTTGTTTTT





G-------------AAGGCAGATTTTCTTCCTTCTCCCCTCCCTT---CCCCCCCCCCTCTTCCACCGTGCAAACGGTTGTTGGGGAGCAGGCCCGCCGGCGGCCCACTAAACTCTTTGTTTTT


G-------------AAGGCAGATTTTCTTCCTTCTCCCCTCCCT-----CCCCCCCCCTCTTCCACCGTGCAAACGGTTGTTGGGGAGCAGGCCCGCCGGCGGCCCACTAAACTCTTTGTTTTT






G-------------AAGGCAGATTTTCTTCCTTCTCCCCTCCCT------CCCCCCCCTCTTCCACCGTGCAAACGGTTGTTGGGGAGCACGCCCGCCGGCGGCCCACTAAACTCTTTGTTTTT


G-------------AAGGCAGATTTTCTTCCTTTTCCCCTCCCT------CCCCCCCCTTTTCCACCGTGCAAACGGTTGTTGGGGAGCAGGCCCGCCGGCGGCCCACTAAACTCTTTGTTTTT

G-------------AAGGCAGATTTTCTTCCTTCTCCCCTCCCT------CCCCCCCCTTTTCCACCGTGCAAACGGTTGTTGGGGAGCAGGCCCGCCGGCGGCCCACTAAACTCTTTGTTTTT














G-------------AAGGCAGATTTTCTTCCTTCTCCCCTCCCT------CCCCCCCCTCTTCCACCGTGCAAACGGTTGTTGGGGAGCAGGCCCGCCGGCGGCCCCCTAAACTCTTTGTTTTT

G-------------AAGGCAGATTTTCTTCCTTCTCCCCTCCCT-----TCCCCCCCCTCTTCCACCGTGCAAACGGTTGTTGGGGAGCAGGCCCGCCGGCGGCCCACTAAACTCTTTGTTTTT


G-------------AAGGCAGATTTTCTTCCTTCTCCCCTCCCT------CCCCCCCCTTTTTCACCGTGCAAACGGTTGTTGGGGAGCAGGCCCGCCGGCGGCCCACTAAACTCTTTGTTTTT


A-------------AAGGCAGATTTTCTTCCTTCCCCCCCCCCC------CCCCCCCCTCTTCCACCGGGCAAACGGTTGTTGGGGAGCAGGCCCGCCGGCGGCCCACTAAACTCTTTGTTTTT
























G-------------AAGGCAGATTTTCTTCCTTCTCCCCTCCCT--CCCCCCCCCCCCTCTTCCACCGGGCAAACGGTTGTTGGGGAGCAGGCCCGCCGGCGGCCCACTAAACTCTTTGTTTTT

G-------------AAGGCAGATTTTCTTCCTTCTCCCCTCCCT--CCCCCCCCCCCCTCTTCCCCCGGGCAAACGGTTGTTGGGGAGCAGGCCCCCCGGCGGCCCACTAAACTCTTTGTTTTT













ATTGAAAGAGAAGAAAAAAATACTTTTTTCTCTCTCTCTTCCCT----CCCCCCCCCGCCTTCTACCGTGCAAACGGTTGTT--GGAGCAGGCCCGCCGGCGGCCCACCAAACTCTTTGTTTTT

ATTGAAAGAGAAGAAAAAAATACTTTTTTCTCTCTCTCTTCCCT----CCCCCCCCCGCCTTCTACCGTGCAAACGGTTGTT--GGAGCAGGCCCGCCGGCGGCCCACCAAACTCTTTGTTTTT

ATTGAAAGAGAAGAAAAAAATACTTTTTTCTCTCTCTCTCTTCCCT--CTCCCCCCCGCCTTCTACCGTGCAAACGGTTGTT--GGAGCAGGCCCGCCGGCGGCCCACCAAGCTCTTTGTTTTT

ATTGAAAGAGAATAAAAAAATACTTTTTTCTCTCTCTCTCCCCT-----CTCCCCCCGCCTTCTACCGTGCAAACGGTTGTT--GGAGCAGGCCCGCCGGCGGCCCACCAAACTCTTTGTTTTT

ATTTTTGAGAGAG-------T-C-----TCTCTCTCCTTCCTTC-----------TCGCCTTCTACCGTGCAAACGGTTGTT--GGAGCAGGCCCGCCGGCGGCCCACCAAACTCTTTGTTTTT

ATTGAAAGAGAAGAAAAAAATACTTTTTTCTCTCTCTCTCTTCC---CTCTCCCCCCGCCTTCTACCGTGCAAACGGTTGTT--GGAGCAGGCCCGCCGGCGGCCCACCAAACTCTTTGTTTTT

ATTGAAAGAGAATAAAAAA-TACTTTTTTCTCTCTCTCTCCCCT-----CTCCCCCCGCCTTCTACCGTGCAAACGGTTGTT--GGAGCAGGCCCGCCGGCGGCCCACCAAACTCTTTGTTTTT

ATTGAAAGAGAAGAAAAAAATACTTTTTTCTCTCTCTCTTCCCT-----CTCCCCCCGCCTTCTACCGTGCAAACGGTTGTT--GGAGCAGGCCCGCCGGCGGCCCACCAAACTCTTTGTTTTT

ATTTTTGAGAGAG---------------TCTCTCTCTCCTTCCT---------TCTCGCCTTCTACCGTGCAAACGGTTGTT--GGAGCAGGCCCGCCGGCGGCCCACCAAACTCTTTGTTTTT

ATTTTTGAGAGAG---------------TCTCTCTCTCCTTCCT---------TCTCGCCTTCT--CGTGCAAACGGTTGTT--GGAGCAGGCCCGCCGGCGGCCCACCAAACTCTTTGTTTTT

ATTGAAAGAGAATAAAAAAATAC-TTTTTTCTCTCTCTCTCCCCT----CTCCCCCCGCCTTCTACCGTGCAAACGGTTGTT--GGAGCAGGCCCGCCGGCGGCCCACCAAACTCTTTGTTTTT

ATTGAAAGAGAAGAAAAAAAATACTTTTTTCTCTCTCTCTCTTCCCT--CTCCCCCCGCCTTCTACCGTGCAAACGGTTGTT--GGAGCAGGCCCGCCGGCGGCCCACCAAACTCTCTGTTTTT

AA------GTAAAAGAGGAGAATCTTTTTCTCCTTCTTTTCTTTTTTCCCCCCCTTCCCCTCCATCCGTGTAAGAACGGTTGCTGGAGCAGGCCCGCCGGCGGCCCATCAAACTCTTTGTTTTT









AA--AAAAAAAAAAAGGGGGGAAATTCTGTTTCCCCTTTTTCTTTTTCCCCCCCTTCCCCTTCATCCGGGTAAAATCGGGTGCTGGAGCAGGCCCGCCGGCGGCCCATTAAACTCTTTGTTTTT

AA--AAAAAAAAAAAGGGGGGAAATTCTGTTTCCCCTTTTTCTTTTTCCCCCCCTTCCCCTTCATCCGTGTAAAATCGGGTGCTGGAGCAGGCCCGCCGGCGGCCCATTAAACTCTTTGTTTTT

AA---AAAAAAAAAAGGGGGGAAATTCTGTTTCCCCTTTTTCTTTTTCCCCCCCTTCCCCTTCATCCGTGTAAAATCGGGTGCTGGAGCAGGCCCGCCGGCGGCCCATTAAACTCTTTGTTTTT

GT------------------------------------------------------------GCCCCCCCACCGTGCAAGCGGTGGAGCAGGCCCGCCGGCGGCCCACCAAACTCTTTGTTTTT

GT------------------------------------------------------------GTCCCCCCAACCTTCAAACGGT-GAGCAGGCCCG-CGGCGGCCCACCAAACTCTTTGTTTTT

GT------------------------------------------------------ATGCCCCCCCCTTACCGTACTCGCGGTGGGAGCAGGCCCGCCGGCGGCCTACCAAACTCTTGGTTTT-

GCACCCAGAAACCCTTTGTGAACTTATACCTATTGTTGCCTCGGCGTAGGCCGGCCTCTTCA--CTGAGGCCCCCTGGAAACAGGGAGCA-GCCCGCCGGCGGCCAACCAAACTCTT-GTTTCT

GCACCCAGAAACCCTTTGTGAACTTATACCTACTGTTGCCTCGGCGCAGGCCGGCCTTTTGTGACAAAGGCCCCCTGGAGACAGGGAGCA-GCCCGCCGGCGGCCAACCAAACTCTT-GTTTCT

GCACCCAGAAACCCTTTGTGAACTTATACCTACTGTTGCCTCGGCGCAGGCCGGCCTTTTGTGACAAAGGCCCCCTGGAGACAGGGAGCA-GCCCGCCGGCGGCCAACCAAACTCTT-GTTTCT

CCCTTTGTGAACTTATACCTTTTTATCGTTGCCTCGGCGCCGAGCCGGGAGTGCTCTTCTGTGCTCCCCCACCGCGCAAGCAGTGGAGCAGGCCCGCCGGCGGCCCACCAAACTCTTTGTTTTT

CCCTTTGTGAACTTATACCTTTTTATCGTTGCCTCGGCGCCGAGCCGGGAGTGCTCTTCTGTGCTCCCCCACCGCGCAAGCAGTGGAGCAGGCCCGCCGGCGGCCCACCAAACTCTTTGTTTTT

TTGTGAACTTATACCTATTTATCGTTGCCTCGGCGCTGAGCTGGGGGCACTCTCTTCTGTGTGCCCCCCCACCGTGCAAGCGGTGGAGCAGGCCCGCCGGCGGCCCATCAAACTCTTGTTTTTT

aagg atttt ttc tctc c tccct cccccccc cttc accgtgcaaacggttgtt gGAGCAgGCCCgcCGGCGGCCcac AAaCTCTttgTTTtt

: 167

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: 178

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: 153

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: 124

: 117

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: 148

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: 150

: 192

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: 192

Green: Hungary

Red: Greece

Blue: Ukraine

Lila: Slovakia

Orange: Romania

Brown: Macedonia

Yellow: Bulgaria

Black: from GeneBank

Results

The phylogenetic tree based on ITS sequences is drawn by

parsimony analysis. Topological robustness in parsimony

analysis was estimated using 1000 bootstrap replicates.

The C. parasitica isolates are grouped in several clades

which indicates the high variability in ITS sequences.

However, the clades do not represent the studied

geographical regions. The isolates originated from the same

regions are not grouped together, they are found mixed in

different groups.

Green: Hungary

Red: Greece

Blue: Ukraine

Lila: Slovakia

Orange: Romania

Brown: Macedonia

Yellow: Bulgaria

Black: from GeneBank

Results

The phylogenetic tree based on partial sequences of tef1

sequences is drawn by parsimony analysis.

Parsimony analysis of the tef1 fragment did not revealed

any estimable values.

Our results show that there is no variability in tef1

sequences among C. parasitica isolates collected from

Europe.

Summary

In this study we employed ITS region and a part of the translation elongation factor (tef1), as a potential genetic markers to infer phylogenetic relationships among Cryphonectria parasitica isolates.

The differences among the Cryphonectria parasitica isolates originated from the Carpathian-Basin were not significant.

Differences were only found in ITS region

The bootstrap values were not enough high either to support real differences among isolates. Significant, well based differences were not found between isolates.

Summary

Little differences were found between Hungarian, Greek

and isolates from database which constitute separate

groups.

It show also that the tef1 and ITS sequences could only be

suitable for studying C. parasitica isolates originated from

geographical places far away from each other.

In our studies, molecular differences were insignificant to

infer well-based phylogenetic relationships.

Summary

In the future, for studying the variety and diversity of C.

parasitica at population level in Central-Europe as well as

in Hungary, it is recommended to choose a more diverse

variable molecular marker like micro-satellites.

Thanks for your attentions!

molecular studies on - izbis.com · alignment b1 : e3 : af 3 6 8 3 2 9 : f 4 : ay1 4 1 8 5 5 cr :...

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