phytopathology - Türkiye Fitopatoloji Derneği

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THE JOURNAL OF TURKISH
PHYTOPATHOLOGY
SCIENTIFIC REVIEW BOARD
The Editor-in-Chief and Editorial Boards of The Journal of Turkish Phytopathology would like
to extend their sincere appreciation to all those who have worked as referees for the Journal.
Thank you for sharing your time, effort and professional expertise.
Prof. Dr. Gülay TURHAN
Prof. Dr. Ersin ONOĞUR
Prof. Dr. Nafiz DELEN
Prof. Dr. Figen YILDIZ
Prof. Dr. Filiz ERTUNÇ
Prof. Dr. Savaş KORKMAZ
Prof. Dr. Yeşim AYSAN
Prof. Nuh BOYRAZ
Assoc. Prof. Dr. Himmet TEZCAN
Assoc. Prof. Dr. Seral YÜCEL
Assoc. Prof. Dr. Mustafa GÜMÜŞ
Asist. Prof. Dr. Sibel DERVİŞ
Asist. Prof. Dr. Nedim ÇETİNKAYA
Asist. Prof. Dr. Nazlı Dide Kutluk YILMAZ
Asist. Prof. Dr. Dr. Kubilay K. BAŞTAŞ
Prof. Dr. Semih ERKAN
Prof. Dr. F.Sara DOLAR
Prof. Dr. Berna TUNALI
Prof. Dr. Abuzer SAĞIR
Prof. Dr. Mehmet E. GÜLDÜR
Prof. Dr. Saadettin BALOĞLU
Prof. Dr. Nuray ÖZER
Prof. Dr. Murat H.SİPAHİOĞLU
Prof. Dr. Çiğdem ULUBAŞ SERÇE
Prof. Dr. Semra DEMİR
Assoc. Prof. Mine SOYLU
Assoc. Prof. Dr. Ömer ERİNCİK
Asist. Prof. Dr. Dr. Hülya ÖZGENEN
Dr. Aydan KAYA
Dr. Üftade GUNER
All rights of articles published in this journal are reserved by The Turkish Phytopathological
Society. Any use of the material, including reproduction in whole or in part requires permission
in writing from The Turkish Phytopathological Society.
Meta Basım Matbaacılık Hizmetleri
87 Sok. No. 4 / A Bornova
(0.232) 343 64 54 [email protected]
İzmir, 2013
Basım Tarihi: 22.07.2013
ISSN 0378 - 8024
http://www.fitopatoloji.org.tr
THE JOURNAL OF TURKISH
PHYTOPATHOLOGY
TURKISH PHYTOPATHOLOGICAL SOCIETY
VOL. 39
2010, December
NO. 1-3
CONTENTS
Stolbur Phytoplasma Infections in Potato and Tomato Plants from Different Locations in Turkey
Türkiye'nin Değişik Yörelerinden Alinan Patates ve Domates Bitkilerinde Görülen
Stolbur Phytoplasma Enfeksiyonlari
B. K. ÇAĞLAR, T. ELBEAINO, M. KÜSEK, D. PEHLIVAN, H. FIDAN, M. PORTAKALDALI ......... 1
Solanapyrones Produced by Turkish Isolates of Ascochyta rabiei and Their
Phytotoxicity on Chickpeas
Ascochyta rabiei’nin Türk İzolatlarının Solanapyrone Üretimi ve Nohutlar
Üzerindeki Fitotoksiteleri
M. TÜRKKAN, F. S. DOLAR ..................................................................................................................... 9
Reactions of Local Maize Cultivars to Fusarium verticillioides Based on Disease Severity
and Production of Pectolytic Enzymes and Zearalenone Toxin
Hastalik Şiddeti, Pektolitik Enzim ve Zearalenone Üretimi Açisindan Yerel Misir Çeşitlerinin
Fusarium verticillioides É Karşi Reaksiyonlari
O. BÜYÜK, N. ÖZER ............................................................................................................................... 23
Toxin Production and DNA Sequence Analysis of Turkish Isolates of Ascochyta rabiei,
the Causual Agent of Ascochyta Blight in Chickpea
Nohutta Ascochyta Yanıklıklık Etmeni Ascochyta rabiei’nın Türk İzolatlarının Toksin
Üretimi ve Dna Sekans Analizleri
F. Sara DOLAR........................................................................................................................................... 31
Determination of Variety Reaction to Potato Wart Disease (Synchytrium endobioticum)
in Potato Planting Areas of Nevsehir Province, Turkey
Nevşehir İli Patates Ekiliş Alanlarında Patates Sığıl Hastalığı (Synchytrıum endobıotıcum)’Na
Karşı Çeşit Reaksiyonlarının Belirlenmesi
H. GÜNAÇTI, A. ERKILIÇ ....................................................................................................................... 39
The Effects of Various Inactivation Treatments on Seed Germination Characteristics in
Vegetable Seeds Infected with the Viruses
Viral Etmenler İle Enfekteli Sebze Tohumlarına Yapılan Değişik İnaktifleştirme
Uygulamalarının Çimlenme Özellikleri Üzerine Etkileri
I. C. PAYLAN, S. ERKAN, N. ÇETINKAYA, M. ERGUN, A. CANDAR ............................................. 45
J. Turk. Phytopath., Vol. 39 No. 1-3, 1-8, 2010
ISSN 0378 - 8024
Stolbur Phytoplasma Infections in Potato and
Tomato Plants from Different Locations in Turkey
Behçet Kemal ÇAĞLAR* Toufic ELBEAINO** Mustafa KÜSEK*** Deniz PEHLIVAN*
Hakan FIDAN**** Mustafa PORTAKALDALI****
*
Cukurova University, Faculty of Agriculture, Department of Plant Protection, 01330, Adana- Turkey. [email protected]
Istituto Agronomico Mediterraneo di Bari, Via Ceglie 9, 70010 Valenzano (BA), Italy
***
Kahramanmaraş Sütçü İmam University, Faculty of Agriculture, Department of Plant Protection, Kahramanmaraş, 46100,
Turkey. [email protected]
****
Biological Control Research Station, 01320, Adana, Turkey
**
Accepted for publication February 02, 2013
ABSTRACT
During August 2012, a survey and identification of phytoplasmas associated with diseased potato (Solanum
tuberosum L.) and tomato (Solanum lycopersycum L.) plants were conducted in four regions, in Turkey. Potato
samples with reddish or purplish discoloration and rolling of leaves symptoms were gathered from “Kayseri and
Sivas” provinces, whilst tomato samples were collected form plants exhibiting floral abnormalities, sepal
hypertrophy, virescence and phyllody symptoms, from Kahramanmaraş and Adana provinces. All symptomatic
plants of both species reacted positively when assayed by direct polymerase chain reactions (PCR) using universal
primer pair R16F1/R16R0 and nested PCR using R16F2n/R16R2 primers. Phytoplasmas were detected in 32
symptomatic plants, out of 40 samples collected. However, no PCR amplicon products were obtained from the
asymptomatic ones (8 plants). BLAST sequence analysis of the 16SrDNA amplicons (1250 bp) showed that the
phytoplasma found in potato and tomato samples resembled “Candidatus phytoplama solani” (16SrXII-A ribosomal
subgroup member) and shared with this last 99.8% sequence identity. Similar PCR and sequence results were
obtained from Cicadula inornata (Cicadellidae), insects collected from affected tomato plants in surveyed fields,
when were assayed by PCR and 16SrDNA-sequenced. The RFLP profile of the 1250 bp PCR fragments, restricted
with 7 different endonucleases (EcoRI, TaqI, HhaI, AluI, MseI, RsaI and HpaII) usually used for phytoplasma
subgroups differentiation, showed identical patterns to “Candidatus phytoplasma solani”. RFLP results were in
harmony with the phylogenetic tree constructed with the sequences obtained, which grouped in one cluster all
stolbur phytoplasma from Turkey and those of 16SrXII-A ribosomal subgroup members. To our knowledge, all
phytoplasma diseases were detected from potatoes and tomatoes in Kayseri, Sivas, Adana and Kahramanmaraş
provinces are same phytoplasma. Further investigations are needed to determine whether Cicadula inornata insect,
trapped from affected plants is a potential vector responsible for the transmission of this phytoplasma in that area,
where tomatoes are grown in Turkey.
Key words: 16S rRNA, PCR, RFLP, sequencing and phylogenetic analysis
1
STOLBUR PHYTOPLASMA INFECTIONS IN POTATO AND TOMATO PLANTS FROM DIFFERENT
LOCATIONS IN TURKEY
INTRODUCTION
Phytoplasma of the stolbur group (16SrXII) are phloem limited, insect-transmitted pathogens and they infect
a great number of plants species (Ember et al., 2011; Lee et al. 2000), and depending on the affected species this
phytoplasma induce various systemic symptoms ranging from yellowing, shoot proliferation, witches’-broom
growth to phyllody and virescence. In the last ten years, increasing incidence of stolbur phytoplasma was registered
in different crops (grapevine, maize, sugar beet, potato, tomato, vegetable crops), suggesting its’ progressive spread.
In the vegetable crops, severe yield losses caused by stolbur phytoplasma have been recorded in solanaceous crops
(tomato, potato, pepper) and celery (Carraro et al., 2008; Navràtil et al., 2009; Fialova et al., 2009, Ember et al.,
2011). Stolbur phytoplasma also has a wide host range that includes weeds from the families Asteraceae
(Taraxacum officinale, Cirsium arvense), Convolvulaceae (Convolvulus arvensis) and Urticaceae (Urtica dioica),
which can serve as pathogen reservoirs (Berger et al. 2009; Navràtil et al. 2009; Langer and Maixner 2004).
Potatoes and tomatoes are considered among the most important crops in Turkey, as their total productions
reach high levels (4,648,081 and 11.003.433 tons, respectively) (Anonymous, 2011). The presence of stolbur
phytoplasma disease in potato and tomato fields of Turkey has been recorded (Özdemir et al., 2009), however not
much is known regarding the molecular information of its’ genome.
Accordingly, the first aim of this study is to investigate the etiology of diseased potato and tomato plants in
four provinces in Turkey, Kayseri, Sivas, Kahramanmaraş, and Adana. Surveyed fields showed plants with leaf
yellowing and rolling, reddish and purplish discoloration and presence of aerial tubers, whislt tomato plants
exhibited flower malformations, phyllody and big bud, all symtpoms suspected to be of phytoplasmal origin.
The second aim is to characterize at the molecula level the identified pathogen reponsible of the encountered
diseases, for which the results are hereafter reported.
MATERIALS AND METHODS
Plant Material and Insects Sampling
During August 2012, two lots of 20 samples each were collected from four locations, Kayseri/Sivas and
Kahramanmaraş/Adana, dedicated to potato and tomato plantations, respectively. Samples consisted of leaves
collected from plants showing typical symptoms of phytoplasma (Fig. 1 and 2) and from plants with apparent
symptoms. The survey has also interested the collection of Cicadula inornata (Cicadellidae) insects that were found
nourishing on infected plants. Insects were trapped with a D-Vac apparatus in tomato plots at Kahramanmaraş
(Elbistan) province. Samples gathered from potatoes fields at Kayseri province were denoted “PoSKa2” and
“PoSKa4”, from Sivas province as “PoSSi1” and “PoSSi5”, while samples gathered from tomatoes fields from
Adana provinces were denoted “ToSAd1” and “ToSAd2”. Tomato samples and Cicadula inornata from tomato in
Kahramanmaraş (Elbistan) were denoted “ToSEL1” and “ToSCi” respectively. All samples originated from plants
and insects were subjected to molecular assays.
DNA Extraction and Polymerase Chain Reaction Amplification (PCR)
DNA was extracted from fresh leaves of symptomatic and asymptomatic tomato and potato plants as
described by Ahrens and Seemüller (1992), with some modifications. Tissue samples (1 g) were homogenized in 4
ml of CTAB buffer (2% w/v cetyltrimethylammonium bromide, 1.4 M NaCl, 0.2% 2-β-mercaptoethanol, 20 mM
EDTA, 100 mM Tris-HCl, 2% polyvinylpyrrolydone, pH 8.0) and 1.5 ml aliquots of the extract were incubated at
65°C for 30 min. DNA was further purified by phenol and chloroform-isoamyl alcohol (24:1) extraction and
afterward precipitated. Eluted DNA template was used for direct PCR amplification. DNA was also extracted from
cixiids according to Doyle and Doyle (1990).
2
B. K. ÇAĞLAR, T. ELBEAINO, M. KÜSEK, D. PEHLIVAN,
H. FIDAN, M. PORTAKALDALI
a
b
Figure 1. (a) Potato plants infected with stolbur phytoplasma and showing aerial tubers with severe deformation symptoms. (b) Tomato plants
infected with stolbur phytoplasma and showing sepal hypertrophy symptoms.
1
2
3
4
5
6
7
8
9
10
11
1250 bp
1000 bp
Figure 2. Electropherogram showing 16SrDNA nested-PCR products amplified with R16F2n/R16R2 from potato and tomato phytoplasmaaffected plants. Lane 1: 1 kb DNA marker; lane 2: PoSKa2; lane 3: PoSKa4; lane 4: PoSSi1; lane 5: PoSSi5; lane 6: ToSEL1; lane 7:
ToSCi; lane 8: ToSAd1; lane 9: ToSAd2; lane 10: healthy potato plant; lane 11: healthy tomato plant.
The universal phytoplasma primer pair R16F1/R16R0 (5'-AAGACGAGGATAACAGTTGG-3'/5'GGATACCTTGTTACGACTTAACCCC-3') (Lee et al., 1994) was used in one step PCR for amplifying a 1.8 kbp
fragment of ribosomal operon consisting of the 16SrRNA gene, the 16S-23S intergenic spacer region (SR) and a
portion of the 5’ region of 23SrRNA gene. A 1:100 dilution of the single step PCR product was used as template for
3
LOCATIONS IN TURKEY
the nested PCR round, utilizing the primer pair R16F2n/R16R2 (5'-ACGACTGCTAAGACTGG-3'/5'TGACGGGCGGTGTGTACAAACCCCG-3'), which amplify an internal DNA fragment of 1,250 bp from the
16SrRNA gene (Gunderson and Lee, 1996).
For first step PCR, amplification was performed in 50 reaction mixtures, each containing 100 ng of extracted
DNA, 1.25 μl dNTPs (10 mM), 1 μl forward and reverse primers (10 pmol), 10 μl of 5X Crimson Taq reaction
buffer, 3 μl MgCl2 (25 mM) and 0.25 μl Crimson Taq DNA polymerase (5U/μl ) (BioLabs, USA). PCR was
conducted in a Techne TC 4000 apparatus using the following parameters: 35 cycles of 1 min at 94 °C, 2 min at
50 °C and 3 min at 72 °C. PCR conditions for the second round (nested PCR) were the same, except for the
annealing temperature that was at 58 °C. An extension cycle consisting of 10 min at 72 °C was used for both PCRs.
10 μl of PCR products primed with R16F2n/R16R2 were electrophoresed in 1% agarose gel in 1xTBE buffer (67
mM Tris-HCl, 22 mM boric acid, 10 mM EDTA, pH 0.8) together with 1 kb DNA marker (Fermentas, Milan, Italy),
stained with ethidium bromide and photographed on a UV transilluminator.
Restriction Fragment Length Polymorphism (RFLP) Analyses
Restriction fragment length polymorphism (RFLP) analysis was performed using 100 ng of purified
R16F2n/R16R2-primed nested-PCR amplicons products obtained from potato and tomato samples and reference
stolbur strain. First RFLP was performed using EcoRI to distinguish between R16F2n/R16R2 PCR amplicon
product of phytoplasma and chloroplast DNA of plants (Nejat et. al 2009).
Amplicons were digested separately with 2 µl each of following restriction enzymes: TaqI at 65°C and MseI,
AluI, HpaII, HhaI, RsaI at 37°C, according to manufacturer’s instruction (Fermentas, Milan, Italy). Fragments
patterns were compared after electrophoresis on a 5% polyacrylamide gel followed by ethidium bromide staining,
and photographed under UV at 312 nm using a transilluminator.
Cloning, Sequencing and Phylogenetic Analysis
The R16F2n/R16R2 primed-16S rDNA PCR products obtained from stolbur phytoplasma infected plants
were excised from agarose gel, washed and eluted by centrifugation through siliconized glass wool, as described by
Gromadka (1995). The eluted DNAs were sequenced from both directions using M13 forward and reverse
sequencing-primers. DNA fragments were subjected to automated sequencing (ABI 3130xl Genetic Analyzer,
Applied Bio. REFGEN Gen Araştırmaları ve Biyoteknoloji Ltd. Şti., Ankara, Turkey).
Computer-assisted analysis of nucleotide sequences was assembled using the Strider 1.1 program (Marck,
1988). 16S-23S rDNA sequences of stolbur phytoplasma isolates with similar reference phytoplasmas were
separately aligned using Clustal X 1.81 (Thompson et al., 1997). Phylogenetic tree was constructed using the NJ
plot and Boostrap analysis with 1000 replicates using the NEIGHBOR methods of the PHYLIP package
(Felsenstein, 1989).
RESULTS
Detection of Phytoplasma and Sequence Analysis
The presence of phytoplasma was detected in 32, out of 40 tested, symptomatic potato and tomato plants
(Fig. 1) resulting with an amplification of 1,250 bp DNA fragments using nested-PCR (Fig. 3). No PCR positive
reactions were obtained from the eight asymptomatic plants tested. BLAST sequence analyses conducted on 20
different recombinant DNA clones, originated from 10 different infected plants of each species, showed that the
16SrDNA fragments amplified from potatoes and tomatoes plants share the highest identity (99.8%) with stolbur
phytoplasma and in particular with the “Candidatus phytoplasma solani”, 16SrXII-A subgroup member, from
Serbia (accession number: JQ730750) and Romania (accession number: HQ108388). All 16SrDNA phytoplasmal
sequences of stolbur found in potato and tomato in Turkey were identical (100% homology) when compared
between them.
4
RFLP Analysis
The presence of a single EcoRI restriction site in the 16F2n/R16R2- primed PCR products (1250 bp)
generated a RFLP pattern made of two DNA fragments (750 bp and 500 bp), thus ascertaining the phytoplasmal
nature of the nested-PCR amplicon (Figure 3). Performing separate digestions of PCR products with different
endonucleases (EcoRI, TaqI, HhaI, AluI, MseI, RsaI and HpaII), all samples showed identical restriction profiles
(Figure 3), thus indicating that a single phytoplasma infection has occurred in all affected plants. The RFLP profiles
from stolbur phytoplasma isolates of Turkey and of that found in Cicadula inornata insects, were all similar to that
of the reference strain, “Candidatus phytoplasma solani”, showing that there is no genetic variability within the
Turkish isolates.
EcoR I
1
2
3
4
5
6
Taq I
7
8
9
1
2
3
4
2
3
4
1
2
3
4
5
6
7
8
9
1
2
3
4
6
7
8
9
1
2
3
4
Mse I
5
6
7
8
9
6
7
8
9
6
7
8
9
Alu I
Hha I
1
5
5
Rsa I
5
Hpa II
1
2
3
4
5
6
7
8
9
Figure 3. Electropherogram showing Restriction Fragment Length Polymorphisms of 16SrDNA, amplified by nested-PCR from diseased potato
and tomato plants, using seven restriction enzymes (indicated above gels). Lane 1: 1 kb DNA marker; lane 2: PoSKa2; lane 3:
PoSKa4; lane 4: PoSSi1; lane 5: PoSSi5; lane 6: ToSEL1; lane 7: ToSCi; lane 8: ToSAd1; lane 9: Tomato plant infected with
“Candidatus phytoplasma solani” (16SrXII-A) used as reference control.
5
LOCATIONS IN TURKEY
0.02
795
927
794
Acheloplasma laidlawii [M23932]
PosKa2
Ca. phytplasma solani (XII-A) [JQ730750]
ToSEL1
Ca. phytplasma solani (XII-A) [HQ108388]
Ca. Phytoplasma solani (XII-A) [AF248959]
ToSCi
Strawberry lethal yellows phytoplasma (XII-C) [AJ243045]
Ca. Phytoplasma australiense (VII-B) [L76865]
720
Ca. Phytoplasma caricae (XVII-A) [AY725234]
Ca. Phytoplasma graminis (XVI-A) [AY725228]
653
Ca. Phytoplasma americanum (XVII-A) [Dq174122]
Mexican periwinkle virescence phytoplasma (XIII-A) [AF248960]
1000
Clover phyllody phytoplasma (I-C) [AF189288]
Aster yellows phytoplasma (I-F) [AY265211]
625 Blueberry stunt phytoplasma (I-E) [AY265213]
Aster yellows phytoplasma (I-D) [AY265206]
Derbid phytoplasma (XXVIII-A) [AY744945]
979
Buckland valley grapevine yellows phytoplasma (XXII-A) [AY083605]
Sugarcane phytoplasma D3T2 (XXVII-A) [AJ539180]
Sugarcane phytoplasma D3T1 (XXVI-A) [AJ539179]
932
1000
Ca. Phytoplasma cynodontis (XiV-A) [AJ550984)
Ca. Phytoplasma oryzae (XI-A) [AB052873]
827
Ca. Phytoplasma pini (XXI-A) [AJ632155]
Ca. Phytoplasma castaneae (XIX-A) [AB054986]
Ca. Phytoplasma phoenicium (IX-D) [AF515636]
Pigeonpea witches'-broom phytoplasma (IX-A) [AF248957]
1000
Loofah witches'-broom phytoplasma (VIII-A) [AF353090]
Ca. Phytoplasma fraxini (VII-A) [AF092209]
Ca. Phytoplasma trifolii (VI-A) [AY390261]
635 730
Alder yellows phytoplasma (V-C) [AY197642]
589
Ca. Phytoplasma ziziphi’-related strain JWB-Kor1 (V-G) [AB052879]
950 Ca. Phytoplasma ziziphi’-related strain JWB-G1 (V-B) [AB052876]
Phytoplasma sp. Strain LDN (XXII-A) [Y14175]
Sorghum bunchy shoot phytoplasma (XXIV-A) [AF509322]
Coconut lethal yellowing phytoplasma (LYJ-C8) (IV-A) [AF498307]
804
Carludovica palmata leaf yellowing phytoplasma (IV-D) [AF237615]
1000
Phytoplasma sp. LfY5 (E65)-Oaxaca (IV-A) [AF500334]
Weeping tea tree phytoplasma (XXV-A) [AF251672]
Clover yellow edge phytoplasma (III-B) [AF189288]
Western X phytoplasma (III-A) [L04682]
1000
993 Canadian peach X phytoplasma (III-A) [L33733]
Ca. Phytoplasma brasiliense (XV-A) [AF147708]
991 Cactus witches'-broom phytoplasms (II-C) [AJ293216]
Ca. Phytoplasma aurantifolia (II-B) [U15442]
1000
Ca. Phytoplasma australiense (II-D) [L76865]
983
Peanut witches'-broom phytoplasma (II-A) [L33765]
Picris hieracioides phytoplasma (II-E) [JF799094]
Picris echioides phyllody phytoplasma (II-E) [Y16393]_
Ca. Phytoplasma rhamni (XX-A) [X76431]
Ca. Phytoplasma spartii (X-D) [X92869]
1000
Ca. Phytoplasma prunorum (X-F) [AJ542544]
929
995 Ca. Phytoplasma pyri (X-C) [AJ542543]
886 Ca. Phytoplasma mali (X-A) [AJ542541]
Figure 4. Dendrogram, constructed by the neighbour-joining method, showing the phylogenetic relationship of stolbur phytoplasma from
potato (Poska2), tomato (ToSEL1) and cixiid (ToSCi) of Turkey (in bold) and those present in database, based on 16S rRNA gene
sequences. Groups and subgroups of phytoplasmas are reported between brackets ( ). GenBank accession numbers for sequences are
reported between two braces [ ]. The reliability of the analysis was subjected to a bootstrap test with 1000 repeats. Bar, 0.01
nucleotide substitutions per site. Acheloplasma laidlawii was used as an outgroup species to root the tree.
Phylogenetic Tree
The phylogenetic tree constructed with 16SrDNA sequences of the stolbur phytoplasma of potato and
tomato, together with members of 16SrXII-related subgroups, confirmed the RFLP results, hence placed the stolbur
phytoplasma from Turkey in one subclade together with 16SrXII subgroups members (Figure 4).
6
DISCUSSION
In recent years, emerging phytoplasma diseases have increasingly become important in Turkey, due to the
increment of plant material exchanging between agricultural districts and to the moderate weather that favors the
multiplication of these pathogens and their insects-vectors. The molecular investigation carried out on samples
collected from diseased potato and tomato plants, and originated from four different regions particularly dedicated to
their production, showed the presence of a high level of phytoplasma infections; specifically with stolbur
phytoplasma “Candidtus phytoplasma solani”, 16SrXII-A ribosomal subgroup member. The incidence of this
phytoplasma in the surveyed field was somehow significant (80%), considering that 32 samples were PCR-positive
out of 40 tested. The “Candidtus phytoplasma solani”, was previously reported in solanaceous crops in Turkey,
however with a lower incidence (Sertkaya et al., 2007). Accordingly, quarantine measures should be taken in order
to prevent the further expansion of this pathogen where potatoes and tomatoes are grown in the country.
The molecular analysis conducted in this study showed that there is no notable sequence variation in the
stolbur phytoplama found in both species. This result was also confirmed with the RFLP, sequencing and phylogeny
analyses.
An interesting outcome of this study was the identification of stolbur phytoplasma in Cicadula inornata
insects (Cicadellidae); however, further experiments are needed to ascertain whether this phytoplasma is transmitted
in nature by this novel insect.
ÖZET
TÜRKIYE'NIN DEĞIŞIK YÖRELERINDEN ALINAN PATATES VE DOMATES
BITKILERINDE GÖRÜLEN STOLBUR PHYTOPLASMA ENFEKSIYONLARI
Türkiye’nin dört farklı ilinde 2012 Ağustos ayı boyunca yapılan bir survey süresince patates (Solanum
tuberosum L.) ve domates (Solanum lycopersycum L.) bitkilerinde fitoplasma kaynaklı hastalığın tanılanması ile
ilgili bir çalışma yürütülmüştür. Kayseri ve Sivas illerinden kızarıklık, morarma gibi renk değişikliği ve yaprak
kıvrılması simptomu gösteren patates bitki örnekleri, Kahramanmaraş ve Adana illerinden çiçek anormallikleri,
çanak yaprak irileşmesi ve şişmesi simptomu gösteren domates bitki örnekleri toplanmıştır. Toplanan örnekler
Polimeraz Zincir Reaksiyonu (PCR) yöntemi ile R16F1/R16R0 (universal) ve R16F2n/R16R2 (nested) primerler
kullanılarak testlendiğinde, simptom gösteren bütün bitkiler pozitif reaksiyon vermiştir. Araştırmaya dahil edilen
toplam 40 bitki örneğinden 32 simptomlu bitkide fitoplazma saptanırken, 8 simptomsuz bitkide fitoplazma
saptanmamıştır. Domates ve patateslerde saptanan fitoplazmanın 16S rDNA’i üzerinden yapılan PCR sonucunda
elde edilen PCR ürününün (1250 bp) baz dizilimi, “Candidatus phytoplama solani” (16SrXII-A ribosomal altgurub)
ile %99.8 oranında benzerlik göstermiştir. Domates üretim alanlarından toplanan Cicadula inornata
(Cicadellidae)’nın bünyesindeki fitoplazmanın, 16S rDNA’i üzerinde yapılan PCR ve genetik analizlerden de aynı
sonuç elde edilmiştir. İzolatlar arasında genetik farklılıkların olup olmadığını kontrol etmek amacıyla PCR
sonucunda elde edilen 1250 bp PCR ürünü EcoRI TaqI, HhaI, AluI, MseI, RsaI HpaII enzimleri kullanılarak RFLP
çalışmasına dahil edilmiştir. RFLP sonuçlarında Candidatus phytoplasma solani ile aynı sonucu vermiştir. RFLP
sonuçlarının ve genetik dizilimle elde edilmiş filogenetik ağacın uyum içerinde olduğu ve Türkiye’deki araştırmaya
konu olan stolburun 16SrXII-A ribosomal altgruba ait olduğu ortaya konulmuştur. Çalışmada Kayseri, Sivas, Adana
ve Kahramanmaraş illerindeki patates ve domateslerden saptanan fitoplazmaların tamamı benzer olarak
bulunmuştur. Türkiye’deki üretim alanlarında Cicadula inornata’nın fitoplazma vektörü olup olmadığı konusunda
da daha fazla araştırma gerekmektedir.
Anahtar Kelimeler: 16S rRNA, PCR, RFLP, genetik dizilim ve filogenetik analiz
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group-specific oligonucleotide primers for nested-PCR assays to detect mixed- MLO infections in a single
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vegetable crops in South Moravia (Czech Republic) and consequences of yield losses. Crop Prot 28:898–904.
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‘Candidatus phytoplasma cynodontis’ group phytoplasma associated with coconut yellow decline in
Malaysia. Plant Pathol., 58: 389.
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characterization of a phytoplasma causing tomato stolbur disease in Turkey. Proc. IInd Intl. Symposium on
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infecting sesame and solanaceous crops in Turkey. Bulletin of Insectology 60 (2): 141-142, 2007.
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4876–4882.
8
ISSN 0378 - 8024
Solanapyrones Produced by Turkish Isolates of Ascochyta rabiei and
Their Phytotoxicity on Chickpeas
Muharrem TÜRKKAN*, Fatma Sara DOLAR**
* Department of Plant Protection, Faculty of Agriculture, University of Ordu, 52200, Ordu, Turkey,
** Department of Plant Protection, Faculty of Agriculture, University of Ankara, 06110, Ankara, Turkey,
ABSTRACT
Randomly selected four isolates were grown on Czapek Dox liquid medium supplemented with metal cations for
7, 14 and 21 days in order to determine kinetics of solanapyrones production during in vitro growth of Turkish isolates of
Ascochyta rabiei. After culture filtrates were passed through the C18 cartridge, the solanapyrones were eluted with 2 ml
acetonitrile. Quantitation of solanapyrones was determined with LC/MS analyses. Maximum solanapyrones production of
the isolates was observed on 14th day of incubation. Therefore, quantitation of solanapyrones of the rest 63 isolates of A.
rabiei was also determined on the 14th day. Of the 67 A. rabiei isolates used in the present study, it was determined that 66
(98.5 %) isolates produced solanapyrone A, 18 (26.9 %) isolates produced solanapyrone B and 64 (95.5 %) isolates
produced solanapyrone C. Toxicity of solanapyrones on both sensitive (ILC 1929) and resistant (ILC 3279) chickpea
cultivars were demonstrated by the living cell bioassay. The LD50 concentrations for solanapyrone A, B and C in the
bioassay for the sensitive cultivars were respectively 18.6, 23.2 and 96.8 µg/ml while those for the resistant cultivars were
respectively 34.5, 36.2 and 109.3 µg/ml. The LD50 concentrations of the mix of solanapyrones in the sensitive and
resistant cultivars were respectively 42.4 and 45.4 µg/ml.
Keywords: Ascochyta rabiei, Pathotype, Chickpea, Solanapyrones, Bioassay
INTRODUCTION
Chickpea (Cicer arietinum L.) is one of the most extensively grown legume crops in Turkey. According to
FAO records in 2010, total plantation of chickpea is 446218 hectares, production is 530634 tones and rate of yield
is 1189 kg hectare-1 in Turkey (FAOSTAT, 2012). Chickpea production and quality are negatively affected by a
number of biotic and abiotic stresses (Singh et al., 1994). One of the greatest biotic stresses reducing potential yield
in chickpea is Ascochyta blight caused by Ascochyta rabiei (Pass.) Lab. (Singh and Reddy, 1996). A. rabiei is a
heterotallic Ascomycete with two mating types, and both mating types are present in most chickpea production areas
(Trapero-Casas and Kaiser, 1992; Kaiser and Kusmenoğlu, 1997). Pathogenic variability of A. rabiei is enhanced by
the presence of the teleomorphic stage (Kaiser, 1997), which has been reported from almost all chickpea producing
countries in the world (Udupa and Weigand, 1997; Jamil et al., 2000; Maden et al., 2004; Chen et al., 2004; Chongo
et al., 2004; Türkkan and Dolar, 2009). A. rabiei attacks all above-ground parts of plants and causes necrotic lesions
which are circular on leaves and pods and elongate on petioles and stems. When stems and petioles are girdled, they
usually break (Nene and Reddy, 1987). The disease may cause total yield loss if the enviromental conditions are
favorable (Singh and Reddy, 1990).
Phytotoxins are known to be secreted by a number of phytopathogenic fungi (Durbin, 1981). It is thought that
these toxins are playing an important role in plant diseases, because they can induce most or all of the disease
symptoms (Yoder, 1980; Strobel, 1982). It is known that early symptoms of A. rabiei causes epinasty and loss of
turgor in petioles and young branches (Alam et al. 1989). Later, the whole aerial part of the plant may dry out and
die. Solanapyrone toxins (A and C) in culture filtrates of A. rabiei firstly determined by Alam et al. (1989). After
9
SOLANAPYRONES PRODUCED BY TURKISH ISOLATES OF ASCOCHYTA RABIEI AND THEIR
PHYTOTOXICITY ON CHICKPEAS
Chen et al. (1991) optimized solvent system for seperation of solanapyrones, solanapyrone B was also found in
culture filtrate of the fungus. In further studies, the workers reported that production of solanapyrone toxins varied
according to content of the liquid culture medium on which the fungus was grown (Chen and Strange, 1991; Höhl et
al., 1991; Latif et al., 1993; Kaur, 1995; Bahti and Strange, 2004). Application of solanapyrone toxins were
reported to led to morphological changes in chickpea leaves and caused brekage in chickpea stem (Höhl et al., 1991;
Hamid and Strange, 2000). Moreover, Höhl et al. (1991) observed that 100 and 200 µM concentrations of
solanapyrone toxins caused a pronounced bleaching of the chlorophyll in the area of droplet application on leaves.
Therefore, it was suggested that such symptoms could result from solanapyrones which were synthesized by A.
rabiei. Zerroug et al. (2007) determined that 250 μM (75.5 μg ml-1) concentration of solanapyrone A inhibited the
root growth of chickpea seedlings by 50%. Hamid and Strange (2000) reported that bleaching of the stem occurred
and shoots of chickpea broke just below the uppermost leaf after application of 45.3 µg solanapyrone A. Moreover,
they determined that LD50 values of solanapyrone toxins varied widely depending on cultivar and solanapyrone A
was the most toxic of the three solanapyrones.
The present study was carried out to determine the presence of the solanapyrone toxins in Turkish isolates of
A. rabiei and toxicity of solanapyrones on both sensitive (ILC 1929) and resistant (ILC 3279) chickpea cultivars.
Growth, spore production and storage of Ascochyta rabiei
Totally sixty-seven isolates of A. rabiei, sixty-four of which were obtained from the culture collection of the
Department of Plant Protection, Faculty of Agriculture, University of Ankara, were used in this study. The rest of
the isolates representing the pathotypes of A. rabiei were provided by Dr. Bassam Bayaa from ICARDA. The
isolates maintained on microbank were grown on CSMDA (Chikpea Seed Meal Dextrose Agar: chickpea meal 40 g,
dextrose 20 g, agar 20 g, distilled water 1L) for 14 days at 22 ± 1 oC with a 12 h light-photoperiod. The cultures
were flooded with sterile distilled water (SDW) and spores were scraped with sterile glass spatula. After the spores
were filtered through filter paper to remove mycelial fragments, they were centrifuged at 10000xg/rpm for 10 min.
They were resuspended in SDW and centrifuged twice more before finally resuspending them at 1×107 spores/ml.
They were stored as a final suspension in 10% glycerol at -80 oC for use in the future experiments.
Toxin Production
A. rabiei isolates were grown on Czapek Dox medium supplemented with metal cations (ZnSO4.7H2O, 0.05 g l-1,
CaCI2.2H2O, 0.1 g l-1, CuCI2.2H2O, 0.02 g l-1, CoCI2.6H2O, 0.02 g l-1, MnCI2.4H2O, 0.02 g l-1) (Hamid and Strange,
2000). The medium was dispensed in 250 ml Erlenmeyer flasks containing 30 ml Czapek Dox medium. Each flask was
inoculated with 30 µl spor suspension of A. rabiei (107 spores ml-1). Flasks were incubated at 20 ± 1 oC without
shaking. Fungal mycelia and spores were removed by filtration through Whatman No. 1 filtrate paper, 7, 14 and 21
days after incubation. Mycelial mass was dried at 70 oC until constant weight (UNB 500 Oven – 108 lt, “Memmert
GmbH + Co. KG”, Germany). Solid phase extaction cartridges (SPE; 1 g C18, end-capped Isolute, Alltech
Chromatography, USA) were conditioned with methanol (MeOH) (HPLC grade) (5 ml) and then distilled water (5 ml).
Culture filtrates (10 ml) were passed through the column, and after washing with distilled water (5 ml), the toxins were
eluted with 2 ml acetonitrile (ACN) (HPLC grade) and stored at -20 oC until required (Bahti and Strange, 2004).
LC/MS Analysis
The LC analyses for the screening and quantitation of solanapyrone A, B, and C were performed by an
Agilent 1100 HPLC system (Waldbronn, Germany) with Agilent 1100 MS detector equipped with ESI interface.
The analytical separation was performed on a ACE 5 C18 (150 x 4.6 mm, 5 µm) using the isocratic mixture of 0.01
mM acetic acid in 0.2% aqueous solution of formic acid [A] and acetonitril [B], [A:B] (50:50; v:v) at a flow rate of
0.8 ml min-1. MS data acquisition was obtained with positive and negative-ion detection in selected ion monitoring
(SIM) mode.
10
M. TÜRKKAN, F. S. DOLAR
Growth of chickpeas for bioassay study
Chickpea plants consisting of ILC 1929 (susceptible) and ILC 3279 (resistant) were used in bioassay studies.
The seeds were surface sterilized with sodium hypochloride (1%) for 3 min, then washed with sterile distilled water
(SDW) for 3 times and, they were sown in 14 cm pots containing sterilized mixtures of soil, sand and fertilizer
(1:1:0.5, v/v/v). Plants were grown at 22 ± 1 °C with a 14 h light-photoperiod (light intensity, 260 µmoles sec-1 m-2)
for 14 day.
Bioassay
Solanapyrone A, B, C and the mix of solanapyrones (1:0.1:1) were predissolved in MeOH and then diluted in
Czapek Dox containing 5% dimethyl sulfoxide (DMSO) to yield concentration of 250, 125, 62.5, 31.3, 15.6, 7.8 and
3.9 µM.
Living cell bioassay
Living cell bioassay was performed using the method developed by Shohet and Strange (1989). Chickpea
plants were watered 30 min prior to cell isolation. Leaflets excised from chickpea plants were cut into small pieces
and vacum infiltreted with an enzyme cocktail solution. The enzyme cocktail solution consisted of Cellulase R10,
200 mg (Sigma Aldrich Co., USA); Macerozyme R10, 30 mg (Sigma Aldrich Chemie, Germany) and bovine serum
albumin, 5 mg (BSA, Sigma Aldrich Chemie, Germany) in 10 ml of a holding buffer consisting of citric acid
monohydrate, 10.5 g l-1; CaCl2.2H2O, 5 mM; K2HPO4, 1 mM; Mg(SO4)2.7H2O, 1 mM; glucose, 100 g l-1; NaOH,
6.2 g l-1, adjusted to pH 5.8 with HCl 0.1 M). The suspension of leaflets in the digestion solution was stirred on a
magnetic stirrer at 120 rpm for 20 min. The suspension was filtered through four layers of muslin and then washed
three times by centrifuging at 750 rpm for 5 min in the holding buffer. Cell viability was checked by vital staining
with fluorescein diacetate (FDA). Solutions at different concentrations of solanapyrones were placed in wells of
microtest plates (50 µl/well), and 50 µl of cell suspension was transferred into each well. After cells were incubated
at 25 °C for 3 h in the dark, the cells were stained with FDA. Then, 30 µl of cell suspension was transferred onto a
microscope slide, and the viability of 50 cells was assessed under a fluorescence microscope (cells with intact
plasma membranes fluoresced yellowgreen, while dead cells remained unstained). The LD50 values extracted from
graphs of probit percent cell death corrected for control values. The experiment was carried out with three replicates.
RESULTS AND DISCUSSION
A number of pathogenic fungi produce one or more toxic metobolites which are injurious to plants (Durbin,
1981). It is thought that these toxins are playing an important role in plant diseases, because they can induce most or
all of the disease symptoms (Yoder, 1980; Strobel, 1982). Ascochyta rabiei which produces solanapyrone A, B and
C as well as chytochalasin D in liquid culture medium causes epinasty and loss of turgor in petioles and young
branches on chickpea (Alam et al., 1989; Höhl et al., 1991; Latif et al., 1993). Conventionally, solanapyrone toxins
have been determined by HPLC which is based on C18 silica column separation with ACN/water (1:1) mobile phase
with UV detection (Chen et al., 1991). As has been reported by other researchers, solanapyrone A was also well
separated with UV detection in our study (Chen et al., 1991; Kaur, 1995). However, solanapyone B and C was
coeluted so that they had the same retention time (Figure 1a). This method was not sufficiently separated
solanapyrones for quantitative analysis and need optimization (Chen et al., 1991). Therefore, solanapyrone A and C
toxins were firstly reported from liquid culture filtrate of A. rabiei, but solanapyrone B was not determined in
culture filtrate of the fungus (Alam et al., 1989). After application of solvent optimization to the separation of
solanapyrones toxins, all three solanapyrone A, B and C was isolated from A. rabiei (Chen et al., 1991). However,
solvent optimization is still limited owing to the difficulty of monitoring the crossing over of peaks in different
solvents and the large number of variables which affect the separation (Synder and Kirkland, 1979). The
determination and quantification of solanapyrones in the culture filtrate of A. rabiei was carried out with
chromatography (TLC, HPLC, MS and 1H-NMR) (Höhl et al., 1991). In our study, therefore, the amounts of
solanapyrone A, B and C in the culture filtrates of sixty-seven A. rabiei isolates were determined by HPLC\MS
11
analyses. After chromatographing toxins by HPLC using a diode array detector (DAD), UV spectral data of the
solanapyrone standarts were obtained (Table 1; Fig. 1). The retention time of solanapyrone A, B and C standards
were approximately 11.39, 10.06 and 10.11 min, respectively. Detection was carried out with ESI-MS in SIM mode.
Molecular ions selected for quantification of toxins in samples were 303 (positive ion), 287 (positive ion) and 331
(negative ion) for solanapyrone A, B and C, respectively (Fig. 1b, c and d).
Figure 1. Shows UV and LCMS separation of solanapyrones a) separation of solanapyrone A,B and C by DAD; separation of solanapyrone A
(b), B (c) and C (d) by MS
12
In order to determine kinetics of the solanapyrones production during in vitro growth of Turkish isolates of A.
rabiei, randomly selected four isolates were grown on Czapek Dox liquid medium supplemented with metal cations
for 7, 14 and 21 days under continuous light. It is observed that solanapyrones production of the isolates varied
considerably during three different incubation periods (Table 2). In the 7th day, production of each three
solanapyrones was in very low quantity. The amount of solanapyrone C and B in liquid culture was higher than that
of solanapyrone A on this incubation period. Similarly, Höhl et al. (1991) reported that the major toxin in fluids of
germinating spores was solanapyrone C, and solanapyrone B was detected in trace amount on the 4th day
along with solanapyrone C. In the same period, they did not found solanapyrone A in culture filtrate and it was
observed after 6th day of incubation. Results of our study showed that the concentration of solanapyrone A and C in
culture filtrates of four isolates (except for solanapyrone C production of isolate Ank-3) reached the highest quantity
on the 14th day. This result is in agreement with previous studies reporting that the concentration of solanapyrone A
in the culture filtrates peaked at 14-16 day (Bahti and Strange, 2004; Zerroug et al., 2007). In the present study,
however, solanapyrone B could not be detected in the culture filtrate of the isolates on the 14th day. In following
incubation period, solanapyrone toxins in the fungal culture rapidly decreased. As mentioned by previous
researchers, these results show that production of solanapyrone toxins are limited to a certain stage of growth cycle
and can imply that the increase of mycelial mass of the fungi is closely related with the quantity of solanapyrones
(Höhl et al., 1991; Chen and Strange, 1991; Kaur, 1995). In our main study, therefore, 14th day of incubation was
used for extraction of the rest 63 isolates of A. rabiei. The amounts of solanapyrone A, B and C of all 67 isolates
were shown in Table 3. Although solanapyrone B in culture filtrates of all four isolates in previous study limited to
7th day of the incubation periods, we determined that 18 A. rabiei isolates out of 67 produced solanapyrone B on 14th
day in the following study. Except for isolate Ank-9, Solanapyrone B quantity of these isolates was also very low
compared to the other solanapyrones. Höhl et al. (1991) detected that the concentration of sugar in liquid culture
medium especially affected the amount of solanapyrone B whereas solanapyrone A and C remained unaffected. The
amount of solanapyrone A and C also varied according to isolates and 64 A. rabiei isolates produced both
solanapyrone A and C. These toxins weren’t detected in culture filtrate of isolate Kay-2. All other isolates produced
solanapyrone A and solanapyrone C and they were determined in culture filtrates of 64 A. rabiei isolates, except
Kay-2, Ams-2 and PII.
Table 1. UV data for solanapyrone A, B and C
Solanapyrone A
λmax: 231, 326 nm
Solanapyrone B
λmax: 200, 302 nm
Solanapyrone C
λmax: 237, 317 nm
Table 2. The amounts of solanapyrone A, B and C in the mycelial dry weight (g) of four Ascochyta rabiei isolates
Names of the
isolates
Afy-1
Dez-5
Ank-3
Kmar-1
Incubation
periods (day)
7
14
21
7
14
21
7
14
21
7
14
21
A
0.09
15.79
0.13
7.16
0.35
0.08
38.8
0.90
0.12
6.22
1.73
Solanapyrone (µg/g)
B
C
1.47
1.29
9.41
0.82
0.28
0.78
19.72
9.78
0.4
1.12
0.05
4.27
0.93
1.50
14.96
1.79
13
Table 3. The solanapyrone production of sixty seven isolates of Ascochyta rabiei
Name of the isolates
14
Solanapyrone (µg/g the mycelial dry weight)
Pathotypes**
A
B
C
Total
*Pathotype I
22.2
-
4.23
26.43
Ady-1
14.43
-
7.39
21.82
I
Ady-2
45.88
-
70.0
115.88
I
I
I
Ady-3
1.03
-
9.50
10.53
Ady-4
28.92
1.08
80.00
110.00
I
Ady-5
86.67
-
102.5
189.17
I
Ady-6
97.84
-
107.3
205.14
I
Afy-1
15.79
-
9.41
25.20
I
Ams-1
3.97
-
18.26
22.23
I
Ams-2
0.13
-
-
0.13
I
Ank-1
0.84
-
0.95
1.79
I
Ank-2
90.94
-
124.06
215.00
I
Ank-3
38.80
-
0.05
38.85
I
Ank-4
11.8
-
40.82
52.62
I
Ank-5
47.44
-
101.86
149.30
I
Ant-1
5.66
0.08
0.23
5.97
I
Ant-2
7.35
-
4.51
11.86
I
Ant-3
57.20
-
39.56
96.76
I
Çor-1
16.29
0.14
22.77
39.20
I
Çor-2
24.40
0.26
21.76
46.42
I
Çor-3
83.13
2.50
186.56
272.19
I
Dez-1
0.510
-
10.02
10.53
I
Dez-2
49.10
0.33
83.08
132.51
I
Diy-1
20.74
-
59.26
80.00
I
Diy-2
5.16
-
58.39
63.55
I
Diy-3
56.47
-
121.18
177.65
I
Esk-1
0.75
-
33.40
34.15
I
Esk-2
93.55
-
91.29
184.84
I
Esk-3
15.81
-
106.13
121.94
I
Esk-4
16.36
1.09
39.45
56.90
I
Kmar-1
6.22
-
14.96
21.18
I
Kay-1
10.17
-
9.87
20.04
I
Kır-1
18.30
5.11
28.3
51.71
I
Kır-2
0.88
-
5.33
6.21
I
Tok-1
46.50
-
63.00
109.5
I
Uşk-1
2.28
0.15
21.02
23.45
I
Uşk-2
11.47
-
13.53
25.00
I
I
Uşk-3
16.74
-
14.91
31.65
Yoz-1
11.61
-
3.94
15.55
I
*Pathotype II
9.84
0.05
-
9.89
II
II
Dez-3
12.61
-
174.78
187.39
Kay-2
-
0.08
-
0.08
II
Urf-1
38.52
-
61.85
100.37
II
*Pathotype III
Ady-7
Ady-8
Ams-3
Ams-4
Ank-7
Ank-6
Ank-8
Ank-9
Bur-1
Çor-4
Dez-4
Dez-5
Dez-6
Dez-7
Diy-4
Diy-5
Diy-6
Diy-7
Siv-1
Uşk-4
Uşk-5
Yoz-2
Yoz-3
27.05
9.38
16.82
0.05
13.34
43.20
23.93
20.57
13.85
26.15
43.19
0.22
7.16
24.36
9.80
52.41
10.52
2.72
5.46
3.92
14.55
4.10
34.68
5.44
0.11
0.10
0.16
41.54
0.11
0.08
0.07
-
12.28
5.71
3.41
0.28
44.84
34.33
3.03
20.53
133.08
361.54
69.86
15.43
19.72
18.05
100.40
66.55
4.16
13.97
12.20
22.94
47.27
22.37
80.96
26.83
39.33
15.20
20.23
0.43
58.18
77.69
26.96
41.1
188.47
387.69
113.05
15.65
26.88
42.41
110.20
118.96
14.79
16.69
17.74
26.86
61.82
26.47
115.71
32.27
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
* Referans pathotypes of A. rabiei were provided by Dr. Bassam Baya.
** Türkkan and Dolar (2009)
These results are in agreement with studies of Chen and Strange (1991), who reported that each three
solanapyrones were produced by A. rabiei when it is grown on Czapek Dox medium supplemented with metal
cations.
Pathogenic variability among isolates of A. rabiei has been reported from almost all chickpea producing
countries in the world including India, USA, Syria, Pakistan, Turkey and Canada. The isolates used in these studies
have been classified into 3 to 17 pathotypes based on their reactions on 3 to 16 host genotypes (Udupa and
Weigand, 1997; Jamil et al., 2000; Maden et al., 2004; Chen et al., 2004; Chongo et al., 2004). Recently, Türkkan
and Dolar (2009) categorized 64 A. rabiei isolates into three pathotypes based on differences in aggressiveness on
three differential chickpea cultivars (ILC 1929, ILC 482 and ILC 3279). Pathotype I was the least agressive
pathotype, whereas pathotype III was the most agressive pathotype. Because the same isolates were used in present
study, we could compare solanapyrone production among all three pathotypes. Although solanapyrones production
of some of pathotype I isolates was very low in liquid culture medium, the others’ was very high. Moreover, higher
amounts of solanapyrone toxins were not isolated from the more aggressive isolates of pathotype II and III.
For example, solanapyrones production of isolates Kay-2 and Ams-3 which are belonging to PII and PIII,
respectively was very low. Results of our study therefore showed that there were not a correlation between
pathotypes of A. rabiei isolates and their solanapyrone production in vitro conditions. Similar results were observed
by Latif et al. (1998), who determined that the isolates produced significant levels of the solanapyrones in vitro
growth and also induced significant levels of phytotoxic compounds in fungus infected plants caused a moderate
degree of disease severity. Therefore, they reported that production of phytotoxic compounds had no bearing on the
disease severity or fungal virulence because of the discrepancy in the levels of production of toxins during in vitro
and in vivo situations. Sugawara and Strobel (1987) reported that a phytotoxin involved in a host–parasite
interaction should be demonstrable in susceptible host plants after infection. Although Shahid and Riazuddin (1998)
suggested that solanapyrone C has been found in field infected plants, other researchers could not detect any of
solanapyrone toxins in chickpea tissue infected by the fungus (Höhl et al., 1991; Hamid and Strange, 2000; Bahti
and Strange, 2004). However, they reported that application of the different toxin concentrations to chickpea plants
led to morphological changes in both leaf and stem structures that are well comparable to those observed in the
tissue of chickpea plants when invaded by the fungus. Therefore, it is argued that solanapyrones isolated from
15
A. rabiei may contribute to virulence or may be necessary for pathogenicity owing to their affects on chickpea
plants (Alam et al., 1989; Höhl et al., 1991; Latif et al., 1993; Hamid and Strange, 2000).
In our study, the effects of solanapyrone toxins on both susceptible and resistant chickpeas were determined
with a living cell bioassay. It was found that solanapyrone A (18.6 and 34.5 µg ml-1 LD50 values for susceptible and
resistant cultivars, respectively) was more toxic than solanapyrone B (23.2 and 36.2 µg ml-1) and C (96.8 and 109.3
µg ml-1) on each two chickpea cells as well as the mix of solanapyrone A, B and C (42.4 and 45.4 µg ml-1). This
result is in agreement with previous studies, reporting that LD50 values of solanapyrone toxins varied widely
depending on cultivar and solanapyrone A was the most toxic of all the three solanapyrones (Hamid and Strange,
2000). Morover, the researchers reported that the effect of solanapyrone A, B and C on cells isolated from chickpea
was additive rather than synergistic (Alam et al., 1989; Höhl et al., 1991; Latif et al., 1993).
ÖZET
ASCOCHYTA RABİEİ’NİN TÜRK İZOLATLARININ SOLANAPYRONE ÜRETİMİ VE NOHUTLAR
ÜZERİNDEKİ FİTOTOKSİTELERİ
Ascochyta rabiei izolatlarının solanapyrone A, B ve C üretim kinetiğini belirlemek amacıyla rastgele seçilmiş
4 izolat 7, 14 ve 21 gün süreyle inorganik tuzlarla zenginleştirilmiş Czapek Dox sıvı besin ortamında geliştirilmiştir.
Kültür fitratları C18 isolute kartijlerinden geçirildikten sonra solanapyrone toksinleri 2 ml acetonitril ile elute
edilmiştir. Toksinlerinin kantitatif miktarları LC/MS ile belirlenmiştir. İzolatların solanapyrone A, B ve C üretiminin
maksimum olduğu inkübasyon periyodunun 14. gün olduğu belirlenmiştir. Bu nedenle geri kalan 63 izolatın
solanapyrone A, B ve C miktarları inkübasyonun 14. günü esas alınarak belirlenmiştir. Çalışmada kullanılan
izolatların 66 (% 98.5)’ sının solanapyrone A, 18 (% 26.9)’ inin solanapyrone B ve 64 (% 95.5)’ ünün solanapyrone
C ürettiği belirlenmiştir. Solanapyrone toksinlerinin toksisitesi hassas ve dayanıklı bitkilerde canlı hücre bioassay
çalışmaları ile belirlenmiştir. Solanapyrone A, B ve C’ nin hassas çeşit ILC 1929’ da LD50 değerleri sırasıyla 18.6,
23.2 ve 96.8 µg ml-1 iken dayanıklı çeşit ILC 3279’ da sırasıyla 34.5, 36.2 ve 109.3 µg ml-1’ dir. Hassas ve dayanıklı
çeşitlerde solanapyrone toksinlerinin karışımının LD50 konsantrasyonları sırasıyla 42.4 ve 45.4 µg ml-1’dir.
Anahtar Sözcükler: Ascochyta rabiei, Pathotype, Chickpea, Solanapyrones, Bioassay
ACKNOWLEDGEMENTS
We are thankful to Dr. Estelle GEWİS (University College of London, UK) and Dr. Hideaki OİKAWA
(University of Hokkaido, Japan) for providing solanapyrone standarts, and Dr. Bassam Bayaa for providing
reference pathotypes of A. rabiei and chickpeas cultivars (ICARDA). We would also like to thank the Scientific &
Technological Research Council of Turkey (Project no: 104O115).
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18
ISSN 0378 - 8024
Reactions of Local Maize Cultivars to Fusarium verticillioides Based on Disease Severity
and Production of Pectolytic Enzymes and Zearalenone Toxin*
Orhan BÜYÜK**
Nuray ÖZER***
* This study is a part of Master thesis, accepted by Institute of Natural and Applied Sciences of Namık Kemal University
** Plant Protection Central Research Institute, Food, Agricultural and Livestock Ministry, Ankara 06172 Turkey
*** Department of Plant Protection, Faculty of Agriculture, Namık Kemal University, Tekirdağ 59030, Turkey
Accepted for publication March 13, 2013
ABSTRACT
Eleven local maize cultivars from the West Black Sea Region of Turkey were analyzed for reactions to
Fusarium verticillioides, the causal agent of ear rot in maize. Tests were based on disease severity, associated
quantitative and qualitative polygalacturonase (PG) and pectate lyase (PL) activities, and zearalenone (ZEA)
concentration after kernel inoculation by pathogen. The kernels of same cultivars were also tested for the presence
of ZEA before inoculation. The pathogen caused low disease severity and exhibited low PG and no PL activity in
the cultivars Bartın22 and Düzce50. ZEA concentration in all cultivars except two of them were below the
recommended limits before inoculation of the pathogen and increased at very low rates in the cultivars Bartın22,
Düzce50 and Düzce97 after inoculation. PG and PL activities were positively correlated with disease development.
This study suggests the importance of pectolytic enzyme activity produced by F. verticillioides in maize kernels at
the early phases of pathogenesis and it can be possible use in monitoring resistance.
Keywords: Fusarium verticillioides, cultivar reactions, pectate lyase, polygalacturonase, zearalenone toxin
INTRODUCTION
Maize (Zea mays L.) is one of the most important agricultural crop in hot and temperate regions around the
world. Turkey is the world's 21st producer with an average production of about 4.5 million tons in the last year
(FAOSTAT 2010). The crop is currently the third cereal being cultivated after wheat and barley in the country.
However ear rot caused by Fusarium verticillioides (Sacc.) Nirenberg (FV) (Synonym F. moniliforme Sheldon),
(Telemorph, Gibberella moniliformis) is a limiting factor in maize production. Typical symptoms of the disease are
individual or groups of infected kernels scattered randomly on the entire ear. Whitish-pink fungal growth can be
seen on infected kernels and/or silks. The pathogen is also known with a “starburst symptom” of white or pink
streaks radiating from silk insertion of the kernel or from the base (Payne 1999). Additionally it is able to grow in
kernels without causing visible symptoms, as a seedborne endophyte (Munkvold et al. 1997)
Pectolytic enzymes produced by this pathogen could play a role in tissue colonization.
Endopolygalacturonase of FV is expressed during maize seedling infection and may be necessary for fungal
penetration even in monocotyledons (Daroda et al. 2001). Inoculation of the roots of tomato and cauliflower with
FV isolated from mangrove plants showed that the pathogen produced PG and PL in hypocotyls and roots of tomato
and cauliflower (Niture and Pant 2007; Niture et al. 2008). PG and PL isoforms of the pathogen were generally
detected under in vitro conditions (Rao et al. 1996; Posada et al. 2001; Niture et al. 2001; Niture and Pant 2004).
A few studies reported that the pathogen produced PG isoforms during infection of tomato (Niture and Pant 2004)
and maize seedling (Daroda et al. 2001); but the role of PGs and PLs in determining pathogenesis in maize kernels
remains unclear.
19
REACTIONS OF LOCAL MAIZE CULTIVARS TO FUSARIUM VERTICILLIOIDES BASED ON DISEASE
SEVERITY AND PRODUCTION OF PECTOLYTIC ENZYMES AND ZEARALENONE TOXIN
FV is known as a producer of fumonisins (FUM) and studies have focused on this toxin in the pathogen
(Presello et al. 2007; Presello et al. 2008; Blandino et al. 2009; Löffler et al. 2010a; Miedaner et al. 2010; Mukanga et
al. 2010). However, deoxynivalenol (DON), nivenol (NIV) and zearalenone (ZEA) were also detected in the ears and
kernels of maize from which FV was commonly isolated (Cvetnić et al. 2005; Adejumo et al. 2007a; Adejumo et al.
2007b). Previously Büyük (Büyük O., 2009, unpublished data) analyzed the presence of Fusarium spp and their
mycotoxins in the kernels of local cultivars from growers' fields in the West Black Sea Region of Turkey. He
determined that FV was the most commonly isolated fungus (71%). Samples were analyzed for the mycotoxins
FUMB1, DON and ZEA using the technique of LC/MS/MS. Thirty-eight per cent of these samples exceeded the
recommended limit for FUM and sixty-one for ZEA. DON was not detected in the samples. It is well known that ZEA
is dangerous for animals and humans. It causes reproductive disorders of farm animals and may cause premature
thelarche in humans (Pitt 2000; Zöllner et al. 2002). To minimize the risk of human exposure to this mycotoxin, the
European Union and Turkey released limits for ZEA (300 ppb) in unprocessed maize for use indirectly as human food
in 2007 (EU Commission 2007). Results from the Büyük study showed that the kernels of 11 local cultivars did not
contain any Fusarium species, thus providing a possibility for their natural resistance to FV.
The mechanism of maize resistance to FV is complex (Presello et al. 2004). Evaluations associated with
kernel resistance to this pathogen are based on pericarp thickness and wax content (Hoenisch and Davis 1994;
Sampietro et al. 2009), presence of dominant resistance genes, defence related genes and enzymes (Clement et al.
2004; Presello et al. 2004; Lanubile et al. 2010; Lanubile et al. 2012), disease severity level and FUM concentration
(Presello et al. 2007; Presello et al. 2008; Schjøth et al. 2008; Löffler et al. 2010 a; Löffler et al. 2011).
Production of pectolytic enzymes and toxins by the pathogen during the early stages of kernel germination
may represent a reliable indicator of resistance. This study was conducted to assess the resistance of local cultivars,
previously found to be free of any Fusarium species (Büyük O., 2009, unpublished data), to infection by FV based
on production of pectolytic enzymes and ZEA during kernel germination after artificial inoculation of the pathogen.
Plant Material
Eleven local maize cultivars (Bartın22, Bartın43, Bartın47, Bolu74, Bolu84, Düzce50, Düzce72, Düzce97,
Zonguldak3, Zonguldak8 and Zonguldak14) collected from the West Black Sea Region of Turkey were assayed.
Ears of each cultivar were dried at room temperature for one week and shelled. These cultivars were previously
characterized for the absence of any Fusarium spp. placing the kernels of each cultivar on different agar media
(Potato Dextrose Agar, Synthetic Nutrient Agar, Pepton PCNB Agar, Water Agar) and sterile filter paper (Blotter
method) moistened with sterile distilled water in 9 cm petri dishes. Kernel samples were stored at 4oC until used in
assays.
Culture of the fungus
Isolate FV61* was obtained from naturally infected maize growing in the West Black Sea Region of Turkey
and used to produce the inoculum. This isolate was selected for its high level of aggressiveness in preliminary tests
(data not shown) and it was grown in Potato Dextrose Agar (PDA, Oxoid; Unipath Ltd., Basingtone, UK).
For enzyme preparation, the isolate was surface cultured in Czapek's liquid medium (pH 5.0) containing
NaNO3 (2 g/l), KH2PO4 (1 g/l), MgSO4.7H2O (0.5 g/l), KCl (0.5 g/l), FeSO4.7H2O (0.01g/l), ZnSO4.7H2O (0.01g/l)
and 10 g/l citrus pectin as the sole carbon source. The inoculum was one agar disc (6 mm diameter) cut from the
edge of a 7-day-old-culture on PDA. Cultures were grown for 7 days at 25oC in 250-ml Erlenmeyer flasks
containing 50 ml of medium in an incubator (Binder KB240; GmbH, Tutttingen, Germany).
*
This isolate was identified by Prof. Dr. B. Tunalı (Department of Plant Protection, Faculty of Agriculture, Ondokuz Mayıs
University)
20
O. BÜYÜK, N. ÖZER
Kernel inoculation
Kernels from each cultivar were surface sterilized by immersing them in a 1% solution of sodium
hypochlorite for 5 min, rinsing in sterile distilled water and air drying on sterile filter paper. Five kernels were
placed in a petri dish containing PDA. A total of forty petri dishes were prepared from each cultivar. Agar discs (1
cm) from the edges of 7-day-old cultures on PDA were placed on each kernel (Sneh and Ichielevich-Auster 1998).
The same number of kernels without inoculation was prepared as control for each cultivar. All petri dishes were
incubated in the dark at 25oC for 7 days.
Disease assessment
Germinating kernels were rated according to the following 0 to 3 scale: 0, no damage; 1, undeveloped root
and infected root-tips; 3, no germination, kernels completely colonized. The percentage of disease severity (DS) was
calculated from the following equation (Unterstenhöfer 1963).
DS=
Σ ratings of each germinating kernel
X 100
Total number of germinating kernels rated X the high score
Enzyme extraction from fungus culture and inoculated maize kernels
After 7 d of growth on Czapek liquid medium, FV isolate mycelial mats from three flasks were gently
removed. The culture filtrates were centrifuged at 15.000xg/rpm for 15 min at 4oC. Supernatants were dialysed
against several changes of distilled water at 4oC. Enzyme preparations from kernels were obtained by grinding
infected tissues in an ice-cooled mortar in 0.05 M Tris-HCL buffer pH 7.8 (1 g tissue/ml buffer) containing 0.1 M
KCl, 0.5% (mass/v) cysteine, and 1% (mass/v) insoluble polyvinylpolypyrrolidone (Sigma Chemical Co., St. Louis,
MO, USA). The mixture was then strained through three layers of cheesecloth, centrifuged at 15.000xg/rpm for 20
min at 4oC, and dialysed several times against distilled water. The same procedures were applied to control kernels.
Pectolytic enzyme assays
PG activity was determined as the increase of reducing end-groups over time according to Nelson's method
(Nelson 1944), slightly modified, using D-galacturonic acid (Sigma Chemical Co.) as a standard. Activity was
expressed as reducing units (RU). One RU was defined as the amount of enzyme producing 1 µmol/min of reducing
groups from 0.25% (mass/v) polygalacturonic acid (Sigma Chemical Co.) in sodium acetate buffer (0.1 M, pH 5.0)
at 35oC. PL activity was assayed spectrophotometrically by measuring the increase in absorbance at 235 nm. An
increase in absorbance of 1.73 indicated the formation of 1 µmol of unsaturated uronide (Zucker and Hankin 1970).
One unit of enzyme activity catalyzed the formation of 1 µmol/min of unsaturated uronide from 0.25% (mass/v)
polygalacturonic acid in Tris-HCl buffer (0.1 M, pH 8) at 35oC. The experiments were conducted twice.
Isoenzyme identification
Isoenzyme separation by isoelectric focusing (IEF) was realized horizontally with a Mini IEF cell apparatus
(Bio Rad, Milano, Italy) using 0.4 mm thick polyacrylamide gels containing 5% (v/v) ampholyte (Sigma Chemical
Co.) and covering a pH range of 3.5-10.0. The gels were run at 200 V, 450 V, 600 V and 950 V for 15, 30, 20 and
25 minutes, respectively. After IEF, the gels were overlaid with ultrathin (0.4 mm) agarose gels for PL and PG
isoenzyme detection, prepared as described by Ried and Collmer (1985). For PL isoenzyme detection, a 1%
(mass/v) agarose (Sigma Chemical Co.) gel contained 0.1% (mass/v) polygalacturonic acid buffered at pH 8.0 with
50 mM Tris-HCl; for PG isoenzyme detection, a 1% (mass/v) agarose gel contained 0.1% (mass/v) polygalacturonic
acid in 50 mM sodium acetate buffer, pH 5.0. The runs were conducted twice.
21
IEF polyacrylamide gels overlaid with ultrathin agarose gels were incubated at 100% relative humidity for
120 min at 35oC. Activity bands were visualized by staining the agarose overlay for 30 min in 0.05% (mass/v)
ruthenium red (Sigma Chemical Co.), followed by rinsing in distilled water. PL and PG isoenzymes appeared as
white bands. The isoelectric point (pI) values of pectolytic isoenzymes were estimated from a regression equation of
standard protein vs. the distance migrated.
Zearalenone Analysis
Samples (50 g) from each local cultivar were ground to fine powder in a mill (Retsch ZM 200 GmBH Co.
Kg., Haan, Germany) at 230V, 50/60Hz, 1100W,12A with 20 mm mesh screen; 25 g were taken for toxin analysis.
The toxin was extracted in 100 ml methanol:water (80:20 v/v) containing 4 g sodium chloride using a reciprocating
shaker (Johann Otto, GmBH, Germany) at 230V, 50/60 Hz, 0,16A for 1 hour. The mixture was separated through
Whatman filter no.4 paper.
ZEA toxin standard was from Sigma-Aldrich (Germany). The analysis of ZEA was carried out using HPLC
after sample clean up by immunoaffinity column following the method described by Visconti and Girolamo (2005).
The filtrate (10 ml) was mixed with 40 ml distilled water and filtered through glass microfiber. The mixture (20 ml)
was rapidly passed through the immunoaffinity column at a flow rate of one or two drops per sec. The column was
washed with 20 ml of distilled water (two drops per sec) and dried by flushing air through the column. The
Zearalenone was eluted by passing 1.5 ml HPLC grade acetonitrile through the column. The eluate was mixed with
deionized water (1.5 ml) and passed through a filter (0.2 µm), then transferred to an autosampler vial. HPLC
equipment of the Agilent 1100 (Agilent, USA) series was used. The stationary phase was ZORBAX EclipseXDB,
C18 column (4.6X150 mmX5 µm, Agilent) and the eluent was acetonitrile:water (1:1, v/v); the flow rate was
adjusted to 1.0 ml/min. For detection a Fluorescence Detector was used with wavelengths set at λ ex 232 nm and λ em
440 nm. Detection limits of the method ranged between 50 and 800 ppb. The linearity (R) of the standard curve was
0.99927. The recovery rates ranged from 83% to 104 %. The limit of quantification (LOQ) for the toxin was 300
ppb on maize.
Data analysis
Quantitative data on PG, PL, and DS (%) were statistically evaluated by analysis of variance (one-way
ANOVA). Statistical significance of mean differences was estimated according to the Duncan multiple range test
(P=0.05). The increase of ZEA after inoculation was calculated as increase % [= (ZEA concentration of kernel after
inoculation (Z1) − that of kernel before inoculation)/Z1 X 100]. The Pearson coefficient of simple correlation (r)
was calculated between DS and PG, PL, and ZEA production for various infected cultivars. Data statistics were
performed using SPSS 15.0 for Windows (Statistical Package for Social Sciences, Inc., 2001, Chicago).
RESULTS
Disease severity of the cultivars
Kernels of eleven local maize cultivars inoculated with FV isolate were monitored 7 days after inoculation
along with uninoculated kernels for the development of visible symptoms (Figure 1). Disease severity differed
significantly among the cultivars. The cultivars Bartın22 and Düzce50 showed very low disease severity. Maximum
disease (91.5%) was observed in the kernels of cv. Zonguldak14 followed by Bartın47, Bolu84 and Bartın43, and
they were statistically different from other cultivars.
22
Figure 1. Disease severity (± SE) caused by Fusarium verticillioides on the kernels of different cultivars. Ba: Bartın, Bo: Bolu, Dz: Düzce, Zg:
Zonguldak. Bars topped by the same letters do not differ significantly, according to the Duncan Multiple Range Test (p<0.05).
Pectolytic enzymes of FV in liquid culture and during kernel colonization of different cultivars
During the 7-day growth period in liquid culture, FV61 produced a higher amount of PG and PL enzyme
activities than infected kernels (Table 1). When FV61 was able to colonize maize kernels of different cultivars,
production of two pectolytic enzymes (PG and PL) were found depending on the cultivar. The pathogen produced
PG in all cultivars whereas PL was found in only three of them. Significant differences in PG and PL production
were found among the cultivars tested, being significantly low RU and absent in cultivars Düzce50, Bartın22 and
Bolu74, respectively; medium RU and absent in Bartın 43, Düzce97 and Zonguldak8, respectively. However, PG
production by pathogen was significantly high in Bolu84, Zonguldak3 and Zonguldak14, compared with other
cultivars.
Table 1. Polygalacturonase (PG) and pectate lyase (PL) activity from liquid culture and from kernels of local maize cultivars inoculated with
Fusarium verticillioides
Local Cultivars
Bartın22
Bartın43
Bartın47
Bolu74
Bolu84
Düzce50
Düzce72
Düzce97
Zonguldak3
Zonguldak8
Zonguldak14
Liquid culture
PG (RU)
0.46±0.03 ef
0.63±0.06 de
0.83±0.06 bc
0.47±0.01 e
1.37±0.08 a
0.42±0.02 f
0.73±0.03 cd
0.60±0.06 de
0.94±0.06 b
0.58±0.05 def
0.95±0.08 b
2.30±0.05
PL (U)
0.00±0 c
0.00±0 c
0.00±0 c
0.00±0 c
0.13±0.003 a
0.00±0 c
0.10±0.006 b
0.00±0 c
0.00±0 c
0.00±0 c
0.12±0.003 a
0.35±0.01
PG activity is expressed as reducing units (RU), PL activity as units (U). Means (±SE) in each column followed by the same letters do not differ
significantly, according to the Duncan Multiple Range Test (p<0.05).
Enzyme extracts of the isolate from culture filtrates and infected kernel tissues were separated by IEF on thin
layer polyacrylamide gels and evaluated for their PG and PL isoenzyme patterns. Three PG isoenzyme forms, PG1
(pI 4.7), PG4 (pI 6.6), PG5 (pI 7.2) were observed from liquid culture (Figure 2). No PG bands were resolved from
extracts of cultivars Bartın22, Bartın43, Düzce50, Düzce72, Zonguldak97 and Zonguldak8 inoculated with the
pathogen. Cultivars Bartın47, Zonguldak3 and Zonguldak14 were characterized by the presence of PG4; Bartın47
by one acidic and two basic extra-bands at pI 5.5 (PG2-very faint), pI 8.1 (PG6-faint), and pI 8.5 (PG7); Bolu84 by
one acidic extra-band at pI 5.9 (PG3-faint), and PG6 and PG7; and Bolu74 by PG3.
23
One acidic and five alkaline PL isoenzyme patterns, PL1 (pI 6.6), PL2 (pI 7.2), PL3 (pI 7.4), PL4 (pI 8.1),
PL5 (pI 8.5) and PL7 (pI 9.3) were detected from the culture filtrate of the pathogen (Fig. 3). PL3 and PL4 were
observed from the cultivars Bolu84 and Zonguldak14, respectively. Extra bands PL6 (faint-pI 8.7) and PL7 (very
faint) were expressed in the cultivars Bolu84, Düzce72 and Zonguldak14. The pathogen did not exhibit any PL
isoenzyme pattern in kernels of cultivars Bartın22, Bartın43, Bartın47, Bolu74, Düzce50, Düzce97, Zonguldak3 and
Zonguldak8.
Figure 2.Polygalacturonase isoenzyme patterns (white bands) from liquid culture and from kernels of different local maize cultivars inoculated
with Fusarium verticillioides. Ba: Bartın, Bo: Bolu, Dz: Düzce, Zg: Zonguldak. Estimated pIs are indicated on the left, pIs of standard
proteins (S.P) on the right.
Figure 3. Pectate lyase isoenzyme patterns (white bands) from liquid culture and from kernels of different local maize cultivars inoculated with
Fusarium verticillioides. Ba: Bartın, Bo: Bolu, Dz: Düzce, Zg: Zonguldak. Estimated pIs are indicated on the left, pIs of standard
proteins (S.P) on the right.
24
ZEA concentration
Kernels of the local cultivars, which did not contain any Fusarium spp., were analyzed for ZEA before and
after inoculation of the pathogen (Table 2). Among the cultivars, Bolu84 and Zonguldak3 had ZEA concentrations
higher than EU and Turkey limits before and after pathogen inoculation, although the increase in concentration was
low in cultivar Bolu84 after inoculation. ZEA concentrations in other cultivars did not exceed the maximum limits
in either treatment. The lowest increase was detected in the local cultivar Düzce97, followed by Düzce72, Düzce50
and Bartın22.
Table 2. Zearalenone (ZEA) concentration in local maize cultivars (Fusarium spp-free) before and after inoculation with Fusarium verticillioides,
and increase in toxin concentration
Local Cultivar
Bartın22
Bartın43
Bartın47
Bolu74
Bolu84
Düzce50
Düzce72
Düzce97
Zonguldak3
Zonguldak8
Zonguldak14
ZEA concentration (ppb)
After
Before inoculation
inoculation
286.40
293.10
76.40
108.20
129.10
163.70
124.30
136.70
343.40
356.40
106.27
107.63
250.70
253.00
276.30
278.40
307.60
376.30
272.81
286.40
220.10
236.70
Increase in toxin
concentration (%)
2.28
23.39
21.13
9.07
3.64
1.26
0.90
0.75
18.25
4.74
7.01
Correlations between disease severity and pectolytic enzymes and ZEA production
A linear correlation existed between disease severity of the cultivars tested and PG (r=0.68, p<0.05) and PL
(r=0.61, p<0.05) activities detected from diseased tissues upon inoculation with the pathogen. There was no
correlation between disease severity and ZEA production.
DISCUSSION
Some ear and kernel characteristics of specific cultivars provide chemical and mechanical barriers to ear rot
infection caused by FV (Hoenisch and Davis 1994; Munkvold, 2003; Sampietro et al. 2009; Lanubile et al. 2010;
Lanubile et al. 2012). Maize genotypes or cultivars from different countries are currently characterized for their
resistance to infection by FV and also to FUM accumulation in kernels using silk channel inoculation of spore
suspension (Presello et al. 2007; Presello et al. 2008; Miedaner et al. 2010; Löffler et al. 2010 a; Löffler et al. 2010b;
Löffler et al. 2011) and side needle inoculation (Lanubile et al. 2010) under field conditions. Ears and kernels of
maize infected with FV can contain other mycotoxins such as DON, NIV and ZEA (Cvetnić et al. 2005; Adejumo et
al. 2007b). In one of the project (Büyük, O., 2009, unpublished data), it was determined that the rate of ZEAcontaminated kernels exceeding the recommended limit was higher than FUM-contaminated kernels. However
information on the effect of ZEA on resistance to FV in maize kernels is lacking. In addition, Fusarium spp.
perforates the seed coat and invades the endosperm producing pectolytic enzymes as well as mycotoxins (Kikot et
al. 2009). No studies have addressed the role of pectolytic enzymes in resistance to the same disease. However,
these enzymes are thought to play a role in the infection by FV of tomato, cauliflower and maize seedling (Daroda et
al. 2001; Niture and Pant 2004; Niture and Pant 2007; Niture et al. 2008). In this study we evaluated disease severity
as well as production of pectolytic enzymes and ZEA by FV to determine resistance to ear rot of maize cultivars,
and we examined the relationships among them. Eleven local pathogen-free cultivars from the Black Sea Region of
Turkey were used in the study.
25
Virulence and production of PG and PL pectolytic enzymes by the pathogen varied greatly depending on
cultivar. Our results demonstrated different degrees of susceptibility among the cultivars; “Düzce50” was the most
resistant based on the disease severity, the production of PG and PL by pathogen; “Bolu84” was a susceptible
cultivar in which the pathogen exhibited the highest PG and PL activity; another susceptible cultivar,
“Zonguldak14”, had the highest disease severity and the pathogen produced high PG and PL enzyme activity. The
high resolving capacity and sensitivity of the staining technique for pectolytic activity after IEF permitted the
detection of a number of isoenzymes of PG and PL. Three PG isoenzyme forms (PG1, PG4 and PG5) were
expressed in the extracts from liquid culture; among them one PG form (PG4, pI 6.6) appeared during pathogenesis
in susceptible maize cultivars. This suggests that this form may be important for colonizing the pathogen into the
kernel tissues. Daroda et al (2001) determined that a commercial preparation of FV produced four endo PG isoforms
(38.0, 41.5, 45.0 and 48.5 kDa) in vitro and in infected maize seedlings. In our experiments extra PG acidic and
alkaline isoforms, which were not expressed in liquid culture, were detected in the extracts from infected kernels of
some cultivars. Among those, an alkaline PG isoenzyme focusing at pI 8.1 during infection of kernels of sensitive
cultivars Bartın47 and Bolu84 was also previously reported in liquid culture of the pathogen and during infection of
tomato tissue (Niture et al. 2001; Niture and Pant 2004).
This study showed that during growth on pectin as a sole carbon source, FV produced PL activity and six PL
isoforms, although Rao et al. (1996) detected the presence of a PL isoenzyme form (pI 9.1) in the extracts from
liquid culture. Low levels of PL activity and four isoenzyme forms as faint bands were identified during infection of
local cultivars Bolu84, Zonguldak14 and Düzce72. Studies reporting PL isoforms produced by FV during maize
kernel or seedling infection are not available in the literature.
It has been shown in various plant-fungal pathogen interactions that the ability to produce symptoms parallels
the level of PG isolated from infected tissues (Baayen et al., 1997; Le Cam et al. 1997; García-Maceira et al. 2001;
Roncero et al. 2003). Positive correlations between disease development and PG and PL activity by the pathogen in
the infected maize kernels are suggestive of a possible causal relationship. The IEF analysis did not show isoenzyme
forms of PG and PL from local cultivars Bartın22, Bartın43, Düzce50, Düzce97, which also had low disease
severity.
FV can produce FUM in kernels without visible disease symptoms (Munkvold et al. 1997). Our results also
demonstrated the presence of ZEA in symptomless kernels of different local cultivars. Interestingly, no Fusarium
spp. could be found in these cultivars. It is well known that sporulation and mycotoxin production in Fusarium are
both regulated by G protein signaling pathways which commonly regulate fungal development, stress response and
expression of virulence. However fungal development is also influenced by external factors such as lipids and, in
particular, oxylipin signals in maize kernels which have the potential to elicit profound changes in sporulation in the
fungus (Brodhagen and Keller 2006). Although amounts of oxylipin in maize kernels were not determined in the
current study, the results based on presence of ZEA in Fusarium spp-free kernels indicates that sporulation of
Fusarium spp, including FV, seems to be affected by oxylipin content of the kernels. This study clearly showed that
laboratory inspection of maize kernels alone for the presence of FV is not a reliable predictor for ZEA.
ZEA concentrations tested in nine cultivars before and after inoculation were under the recommended
maximum limits. The least increase in concentration after inoculation was recorded in cultivar Düzce97, followed
by Düzce72, Düzce50 and Bartın22. These cultivars showed low disease severity, but ZEA concentration was not
significantly correlated with disease severity. In previous studies, no correlation or a negative association between
disease severity and FUM concentration for FV was observed (Presello et al. 2007; Presello et al. 2008; Covarelli et
al. 2012). Conversely, in another study a positive correlation between disease severity and FUM concentration was
recorded (Löffler et al. 2011). Therefore we hypothesize that ZEA production depends on the given Fusarium
isolate, the substrate and the environment.
In conclusion, for this set of cultivars and experimental conditions, local cultivars Bartın22 and Düzce50 were
determined as resistant to FV. Cultivar Düzce 97 showed low disease severity and had also the lowest increase in
ZEA concentration after inoculation with FV. The enzyme activity tests described in this study can be used as
26
additional tools to evaluate resistance mechanisms to FV as well as contributing to a better understanding of the
infection ability of this pathogen.
ACKNOWLEDGMENTS
This research was supported by the General Directorate of Agricultural Researches and Policies of Food,
Agricultural and Livestock Ministry, Turkey (Project No: TAGEM-BS-09/07-03/05-/02). We are grateful to Dr.
Martha Rowe (University of Nebraska-Lincoln) for improving the language and for useful remarks.
ÖZET
HASTALIK ŞİDDETİ, PEKTOLİTİK ENZİM VE ZEARALENONE ÜRETİMİ AÇISINDAN YEREL
MISIR ÇEŞİTLERİNİN Fusarium verticillioides É KARŞI REAKSİYONLARI
Türkiyeńin Batı Karadeniz Bölgesinden toplanan 11 yerel mısır çeşidinin mısırda koçan çürüklüğü etmeni
Fusarium verticillioidesé karşı reaksiyonları belirlenmiştir. Testlerde, etmenin mısır tanelerine inokulasyonundan
sonra oluşan hastalık şiddeti, kalitatif ve kantitatif poligalakturonaz (PG) ve pektat liyaz (PL) enzim aktiviteleri, ve
zearalenone (ZEA) üretimi dikkate alınmıştır. Aynı çeşitlerin tanelerinde inokulasyondan önceki ZEA miktarı da
tespit edilmiştir. Patojen Bartın22, Düzce50 çeşitlerinde düşük hastalık şiddetine neden olmuş, az miktarda PG
sergilemiş, PL üretmemiştir. İki çeşit hariç, tüm çeşitlerde inokulasyondan önceki ZEA konsantrasyonu sınırların
altında olmuş, inokulasyondan sonra ise Bartın22, Düzce50 ve Düzce97 çeşitlerinde çok düşük oranlarda artış
göstermiştir. PG ve PL aktiviteleri ve hastalık oluşumu arasında pozitif bir ilişki olduğu belirlenmiştir. Bu çalışma
sonuçlarına göre mısır tanelerinde hastalık oluşumunun başlangıcında F. verticillioides tarafından üretilen pektolitik
enzimlerin önemli olduğu ve dayanıklılığı kontrol etmek için kullanılabileceği ileri sürülebilir.
Anahtar kelimeler: Fusarium verticillioides, çeşit reaksiyonu, pektat liyaz, poligalak-turonaz, zearalenone toksini
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ISSN 0378 - 8024
Toxin Production and DNA Sequence Analysis of Turkish Isolates of Ascochyta rabiei,
the Causual Agent of Ascochyta Blight in Chickpea
F. Sara DOLAR
University of Ankara, Faculty of Agriculture, Department of Plant Protection, 06110, Ankara, (Turkey)
Accepted for publication March 10, 2013
ABSTRACT
In this study, twenty isolates of Ascochyta rabiei were isolated from disease-enfecteol chickpea plants which
were collected from chickpea growing areas in Turkey. In order to determine solanapyrone production of these
isolates, the fungus was grown on Czapek Dox liquid culture medium (CDLCM) for 12 day at two different
temperatures. Quantitation of solanapyrones was determined with HPLC analyses. The results demonstrated that all
isolates produced solanapyrone A in CDLCM at 200C but not at 300C.
Confirmation of the identity of the pathogen was sought by sequence analysis of rDNA. These experiments
showed that the sequences of the internal transcribed spacers (ITS) and 5.8 S gene of the seven isolates, which were
identical to each other, were also identical to that of a Pakistan isolate of A.rabiei. rDNA sequences of the PCR
products of isolates of A.rabiei which were produced different amount of toxin were same.
Keywords: Solanapyrone, Ascochyta rabiei, Chickpea, DNA Sequence
INTRODUCTION
Chickpea (Cicer arietinum L.) is an important legume crop in several parts of the world, such as West Asia,
North Africa, Central and South America (Nene 1982; Singh and Reddy 1990). It is the most produced legume crop
of Turkey (Anonymous 2008). The most important disease of chickpea is Ascochyta blight caused by Ascochyta
rabiei (Pass.) Labr. Serious crop losses occur when environmental conditions, especially cool, wet weather, favour
disease development and spread (Singh and Reddy 1990). This fungus, which is known to be seed-borne, causes
characteristic dark necrotic lesions on the stems, leaves and pods of the host, and severe infection can kill the plants
(Maden et al. 1975; Nene 1982).
The taxonomy of Ascochyta species have been based on morphology and host plant association.
Classification systems based upon data from morphological studies have been mostly successful. However, in some
cases, morphology has been unsuccessful in characterizing fungi. Recently, significant advances in fungal taxonomy
and identification have come about through DNA analysis. A wide range of molecular techniques are available for
the identification of fungi, including Restriction Fragment Length Polymorphism (RFLP) and Random Amplified
Polymorphic DNA (RAPD). Fungi of the genus Ascochyta have not been extensively studied by molecular tools.
Some publications describe DNA fingerprinting of A. rabiei, by means of RFLPs (Weising et al. 1991), RAPD
(Fischer et al. 1995) and the RAPD-like PCR technique (DAF)-DNA amplification fingerprinting (Kaemmer et al.
1992). RFLP has been used to solve systematic problems in the genus Phytophthora (Förster et al., 1990), to
characterize Pythium species (Wang and White, 1997) and to determine genetic polymorphism among isolates of A.
rabiei (Morjane et al., 1994). A large group of A. rabiei isolates were analysed by RAPD but analysis of the
combined data failed to reveal any correlation between amplification patterns and pathotype clasification (Fisher et
31
TOXIN PRODUCTION AND DNA SEQUENCE ANALYSIS OF TURKISH ISOLATES OF
ASCOCHYTA RABIEI, THE CAUSUAL AGENT OFASCOCHYTA BLIGHT IN CHICKPEA
al. 1995). Fatehi and Bridge (1998) detected multiple rRNA-ITS regions within nine cultures of Ascochyta and
Khan et al.(1999) distinguished A.rabiei from Phoma medicaginis var pinodella both of which were found in lesions
on chickpea in Australia.
Sequence data of an appropriate part of the fungal genome provides unequivocal identification of fungal
species. Analysis of DNA sequences, particularly those of the ribosomal repeat unit, has proved to be a definitive
and rapid method for the identification and taxonomic studies of fungi as well as for the studies of evolution and
speciation (White et al. 1990). This technique has been used to identify fungi and to delineate species (Sherriff et
al. 1995: Kusaba and Tsuga 1995).
An organism may damage plants by secreting one or more toxins. Early symptoms of Ascochyta blight
include epinasty, loss of turgor and cellular disintegration. It has been suggested that such symptoms could result
from toxin production by the pathogen (Höhl et al. 1991, Hamid and Strange 2000). In liquid culture, isolate of
A.rabiei have been reported to synthesizses a total of four toxic compounds; solanapyrone A, B, C and cytochalasin
D (Alam et al. 1989; Höhl et al. 1991; Latif et al. 1993) which are released in the culture medium. Solanapyrones
A, B and C were first found in culture filtrates of Alternaria solani, the causal agent of early blight of tomato and
potato (Matern et al. 1978). The chemical structure of these toxins were elucidated by NMR-spectroscopy and
mass- spectrometry (Ichihara et al.,1983). Phoma exiqua var. heteromorpha, which was formerly known as
Ascochyta heteromorpha, produced cytochalasins A, B, U and V when grown on a semi-synthetic medium (Capasso
et al. 1991)
The objective of this research was to determine production of toxin by Turkish isolates of A.rabiei and
demonstrate DNA sequence of A.rabiei isolates which produced different amount of toxin or not.
MATERIALS and METHODS
Ascochyta rabiei Isolates
Isolates of Ascochyta rabiei, designated T 1-22, were isolated from diseased leaves, stems and pods of
chickpea collected from chickpea growing areas in different regions (Central Anatolia, South Eastern Anatolia and
Aegean) of Turkey. These cultures were cultivated on the CSMDA medium (Chickpea Seed Meal Dextrose Agar:
chickpea meal 40 g, dextrose 20 g, agar 20 g, distilled water 1 l) at 20±20C with a 12 h period of near UV light for
10 days. From these cultures, single spore isolates were obtained. Inoculum was further multiplied on chickpea
seeds according to Alam et al. (1987). After incubation at 200C for 7-10 days the inoculated seeds were agitated
with sterile distilled water and the spores suspended in 10% glycerol (107 spore ml-1 )..The suspension was distributed
to 1.8 ml Nunc tubes in 1 ml aliquots and were stored in liquid nitrogen.
Toxin Production and HPLC Analysis
The fungus was grown on Czapek Dox liquid culture medium, consisting of Czapek Dox nutrients
supplemented with zinc sulphate (50 mg/l), manganese chloride (20mg/l), calcium chloride (100mg/l), cobalt
chloride (20mg/l), cupric chloride (20mg/l) per litre (CDLCM; Chen and Strange, 1991). After distribution to 250
ml Erlenmeyer flasks (30 ml per flask) and autoclaving, each flask was inoculated with 30µl spore suspension (107
spores ml-1) of A.rabiei and incubated without shaking at 200C and 300C in continuous light for 12 days. The fungus
was removed by filtration through 6 layer of muslin and filtered using Whatman No.1 filter paper. Mycelial mats
and the relatively few spores were discharged and dried at 800C for 48 h to give a measure of fungal growth.
Samples of the filtrates (30 ml) were passed through an Isolute 1g C18 cartridge (International Sorbent Technology
Ltd., Mid Glamorgan, UK) and after washing with water (5.0 ml), toxin was eluted with acetonitrile (100%:2.0ml).
Samples of the acetonitrile eluate (20µl) were injected onto a Philips HPLC consisting of a PU4100 quaternary
pump, PU4021 diode array detector and computer equipped with PU6003 diode array software for data handling.
The stationary phase was a Jones Chromatography C18 column ODS (5µm particle size: 4.6 x 150 mm: Jones
Chromatography Ltd., Mid Glamorgan, UK). The column was developed with a mobile phase consisting of
32
F. S. DOLAR
tetrahydrofuran 20.6%, methanol 23.1% and bidistilled water 56.3% at a flow rate 1.0 ml/min. The solanapyrones
were recognized by their retention times and their characteristic UV spectra, which were compared with authentic
samples. Chromatograms were abstracted from the chromascans at λ=327nm and solanapyrone A quantified by
reference to an external standard of the compound.
Growth of Ascochyta rabiei and DNA Extraction
Isolates (T-4, 5, 9, 10, 14, 15 from Turkey and P-8 from Pakistan) were grown in a medium consisting of
Czapek-Dox Nutrients (45.5 g: Oxoid, Unipath Ltd., UK), bacto peptone (1g), yeast extract (1g), casein hydrolysate
(1g), dissolved in 1 L water and supplemented with 200 ml of clarified V-8 juice (Campbell Grocery Products Ltd.,
UK) prepared by filtering the juice through four layers of muslin and centrifuging at 20009g for 5 min. The medium
was distributed to 250 ml Erlenmeyer flasks (100 ml per flask) and autoclaved at 1210C for 20 minutes. After
cooling to room temperature, 0.01g of Streptomycin sulphate was added to each flask. Spore suspension (100 μl:
1x107 spore/ml) of isolates, which were stored in liquid nitrogen, were used to inoculate the medium. After
incubation for 3 days at 250C on an orbital shaker the mycelia were harvested. DNA was extracted using a
commercial kit (Nucleon Phytopure Plant DNA Extraction Kit, Scotlab, UK) according to the manufacturer`s
instructions. Precipitated DNA was resuspended in 180 μl of TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0) and
20μl RNase (1mg/ml) was added. After incubation at 370C for 1 hour on a shaker, DNA concentration and purity
was ascertained by monitoring UV absorption at 260 and 280 nm and by electrophoresis in 1% agarose gel
containing ethidium bromide (0.2 μg ml-1 gel).
DNA Amplification
The two internal transcribed spacers (ITS1 and ITS2) and the 5.8S rDNA were amplified with the primer
pairs ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) (Gardes and Bruns, 1993) and ITS4 (5′TCCTCCGCTTATTGATATGC-3′) (White et al. 1990). Each DNA sample (50ng) and the two primers (10pmol of
each) were added to a “Ready to Go” PCR bead (Amersham Pharmacia Biotech Inc., Sweden) which contained 1.5
units of Taq DNA polymerase, 10 mM Tris-HCl (pH 9.0), 50mM KCl, 1.5mM MgCl2 and 200 μM of each dNTP
when brought to a final volume of 25 μl. The PCR conditions were 40 cycles of 1 min at 940C, 45 s at 500C, 2 min
at 720C, followed by a final extension step of 5 min at 720C (Fatehi and Bridge 1998) PCR products were
electrophoresed in 1.7 % agarose gel.
DNA Sequencing and Analysis
DNA sequencing of PCR products were performed using the ABI PRISM Big Dye Terminator Cycle
Sequencing Ready Reaction Kit (Protocol number 4303152) with AmpliTaq DNA polymerase (Perkin Elmer
Corporation) using an ABI PRISM 377 DNA Sequencer according to the manufacturer`s instructions. Primers
ITS1F and ITS4 (Gardes and Bruns, 1993; White et al. 1990) were used to sequence the PCR products.
The sequences of the PCR products were aligned by the clustal method using the programme MAGI
(Multiple Alignment General Interface) at the HGMP-RC (Human Genome Mapping Project Resource Centre;
www.hgmp.mrc.ac.uk) according to the service providers instructions.
A reference A.rabiei sequence was obtained from CABI and compared with sequences from the Turkish
isolates using the cluster method of MAGI.
Production of Solanapyrone A by Ascochyta rabiei Isolates
The variation in solanapyrone A concentrations of the culture filtrates from 21 isolates of A.rabiei is shown
in Fig.1. Toxin production varied from 0.89 to 11.0 μg/g dry weight of mycelium after 12 days on Czapek-Dox
33
Liquid Cation Medium (CDLCM) at 200C. Isolate T-21 produced the highest concentrations of the solanapyrone A
with values of 11.0 μg/g dry weight of mycelium. T-17, T-15 and T-12 produced 8.79, 8.04 and 7.73 solanapyrone
A μg/g dry weight of mycelium on the same medium, respectively. Three isolates (T-1, T-22 and T-11) produced
small amounts of sol A. All of 20 Turkish isolates produced solanapyrone A on CDLCM but not sol B and sol C.
No solanapyrones were detected in culture filtrates of any isolates incubated at 300C.
Figure 1. Production of solanapyrone A (μg/g dry weight mycelium) of isolates Ascochyta rabiei grown on Czapek-Dox Liquid Cation Medium
The solanapyrones were first described as products of Alternaria solani, the causal agent of early blight of
potato and tomato (Ichihara et al. 1983). Alam et al. (1989) isolated two toxins from culture filtrates of Ascochyta
rabiei and identified them as solanapyrones A and C. Further work in which the fungus was grown on chickpea
seed extract with glucose or Richard’s medium (Höhl et al 1991) or a defined medium (Chen and Strange 1991)
allowed additionally the production of solanapyrone B. Latif et al. (1993) found one of nine strains of the A.rabiei
produced a cytochasin which was identified as cytochalasin D. The results reported in this paper demonstrate the
capacity of twenty one pathogenic isolates of the fungus to synthesize phytotoxic compounds in vitro. All of the
isolates produced solanapyrone A in CDLCM at 200C but not at 300C.
It is known that many toxins are responsible for pathogenicity of fungus (Wheeler and Luke 1955, Nadel and
Spiegel-Roy 1988, Vidhyasekaran et al. 1990). Whereas it is not difficult to show the relevance of host-selective toxins
to pathogenicity since isolates that lose their ability to produce toxin are non-pathogenic, it is more difficult to
demonstrate the role of non-selective toxins in disease (Strange 1998). Solanapyrone compounds by the fungus
A.rabiei are not selectively toxic but the symptoms caused by the solanopyrone A, epinasty, chlorosis and necrosis, are
consistent with the disease (Strange 1997). Furthermore cuttings allowed to take up solanopyrone A lodged, a symptom
typical of the disease (Hamid and Strange 2000). Solanapyrone produced by A.rabiei may therefore be related to the
virulence of the pathogen.
Sequence Data
DNA was successfully extracted from six Turkish isolates of A.rabiei which produced different amounts
solA and a Pakistan isolate of fungus using the commercial kit .An amplicon of about 600 bp was obtained with the
primers ITS1F and ITS4 from all isolates. Sequencing of the amplicons which contained the ITS1 region (139bp),
the 5,8 S gene (158bp) and ITS2 region (143bp) showed that they were identical (Fig2).
Confirmation of the identity of the pathogen was sought by sequence analysis of rDNA. These experiments
showed that the sequences of the internal transcribed spacers and 5.8 S gene of the six Turkish isolates, which were
identical to each other and, were also identical to that of a Pakistan isolate of A.rabiei. Thus, the causal agent of
blight of chickpea in Turkey was additionally confirmed to be A.rabiei at the molecular level. Two interpretations of
34
F. S. DOLAR
the perfect match of the DNA sequences of the Turkish and the Pakistan isolates are that either these regions are
particularly conserved within in A.rabiei or that the Pakistan and Turkish isolates are of common origin. A.rabiei
isolates obtained from Paul Bridge (CABI) contained one more base pair in ITS1 region (140bp) than Turkish and
Pakistan isolates of A.rabiei.
ITS
1CCTAGAGTTTGTGGGCTTTGCCCGCTACCTCTTACCCATGTCTTTTGAGTACTTACGTTTCCTCGGCGGGTCCGCCCG
CCGATTGGACAAAATCAAACCCTTTGCAGTTGCAATCAGCGTCTGAAAAACATAATAGTTA
5.8 S
CAACTTTCAACAACG GATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAGTGTGAATTGCAGAA
TTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCATGGGGCATGCCTGTTCGAGCGTCATTT
ITS 2
GTACCTTCAAGCTTTGCTTGGTGTTGGGTGTTTGTCTCGCCTCTGCGTGTAGACTCGCCTTAAAACAATTGGCAGCCGGCG
TATTGATTTCGGAGCGCAGTACATCTCGCGCTTTGCACTCATAACGACGACGTCCAAAAGTA
Figure 2. Sequences of the internal transcribed spacer (ITS) 1, the 5.8 S and the ITS2 regions (440bp in total) of
the Turkish isolates of Ascochyta rabiei.
Ribosomal DNA (rDNA) sequences have been aligned and compared in a number of living organism. Studies
of rDNA sequences have been used to infer phylogenetic history across a very broad spectrum, from studies among
the basal lineages of life to relationships among closely related species and populations (Hillis and Dixon 1991).
In this study, all isolates were confirmed as A.rabiei by rDNA sequencing and they all produced solanapyrone
A although the amounts were variable. These data provide additional evidence for the importance of solanopyrone A
to the pathogen since if its production were gratuitous some isolates would be expected not to produce the
compound, particularly as the fungus has a sexual stage, Didymella rabiei.
ACKNOWLEDGEMENTS
I am grateful to Dr. Richards Strange for his great help and supervision. I also wish to acknowledge Miss Laura
Winskill for sequencing the PCR products. and Dr. Paul Bridge (CABI)) for providing A.rabiei isolate. This project
was funded by British Council.
ÖZET
NOHUTTA ASCOCHYTA YANIKLIKLIK ETMENİ ASCOCHYTA RABIEI’NIN TÜRK
İZOLATLARININ TOKSİN ÜRETİMİ VE DNA SEKANS ANALİZLERİ
Bu çalışmada 20 adet Ascochyta rabiei izolatı Türkiye’nin nohut üretimi yapılan alanlarından toplanan
hastalıklı nohut bitkilerinden izole edilmiştir. İzolatlar solanapyrone üretimlerinin belirlenmesi için 12 gün süreyle
iki farklı sıcaklıkta Czapek Dox sıvı kültür ortamında (CDLCM) geliştirilmişlerdir. Solanapyronların kantitatif
ölçümleri HPLC analizi ile yapılmıştır. Tüm izolatların sıvı ortamda (CDLCM) 200C de solanapyrone A ürettikleri
buna karşın 300C de üretmedikleri tespit edilmiştir.
Patojenin teşhisinin doğrulanması rDNA sekans analizi ile yapılmıştır. Bu çalışmada yedi izolatın ITS ve 5.8
S gen bölgelerinin sekanslarının hem birbirleri ile hem de A.rabiei’nin Pakistan izolatı ile aynı olduğu görülmüştür.
Farklı miktarlarda toksin üreten A.rabiei izolatlarının PCR ürünlerinin rDNA sekansları aynı bulunmuştur.
Anahtar kelimeler: Solanapyrone, Ascochyta rabiei, Nohut, DNA Sekans.
35
LITERATURE CITED
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copious inoculum production. The Mycologist, 21: 20.
convenient method for
Alam, S.S., Bilton, J.N., Slawin, A.M.Z., Williams, D.J., Sheppard, R.N. and Strange, R.N. 1989. Chickpea blight:
production of the phytotoxins solanapyrones A and C by Ascochyta rabiei. Phytochemistry, 28:2627-2630.
Anonymous, 2008. F.A.O. http://www.fao.org. (Date accessed: May 2008)
Capasso, R., Evidente, A. and Vurro, M.,1991. Cytochalasins from Phoma exiqua var heteromorpha.
Phytochemistry, 30:3945-3950.
Chen, Y.M. and Strange, R.N., 1991. Synthesis of the solanapyrone phytotoxins by Ascochyta rabiei in response to
metal cations and development of a defined medium for toxin production. Plant Pathology, 40:401-407.
Chen, Y.M., Peh, E.K. and Strange, R.N., 1991. Application of solvent optimisation to the separation of the
phytotoxins, solanopyrone A,B and C from culture filtrates of Ascochyta rabiei. Bioseparation,2:107-113.
Fatehi, J. and Bridge, P., 1998. Detection of multiple rRNA-ITS regions in isolates of Ascochyta. Mycological
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Förster, H., Oudemans, P. and Coffey, M.D., 1990. Mitochondrial and nuclear DNA diversity within six species of
Phytophthora. Experimental Mycology, 14:18-31.
Hamid K and Strange RN. 2000. Phytotoxicity of solanopyrone A and B produced by the chickpea pathogen
Ascochyta rabiei (Pass.)Labr. and apparent metabolism of solanopyrone A by chickpea tissues. Physiol. and
Mol. Plant Pathol. 56:235-244.
Hillis, D.M. and Dixon, M.T., 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. The
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Höhl, B., Weidemann, C., Höhl, U. and Barz, W. 1991. Isolation of solanapyrone A, B and C from culture filtrates
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Ichihara, A., Tazaki, H. and Sakamura, S., 1983. Solanapyrones A, B and C, phytotoxic metabolites from the fungus
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Khan, M.S.A, Ramsey, M,D,,Corbiere,R., Infantino, A., Porta-Puglia, A., Bouznad, Z. and Scott, E.S.1999.
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Kusaba, M. and Tsuge, T. 1995. Phylogeny of Alternaria fungi known to produce host-specific toxins on the basis of
variation in internal transcribed spacer of ribosomal DNA. Current Genetics. 28: 491-498.
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and cytochalasin D among nine isolates of Ascochyta rabiei. Plant Pathology, 42:172-180.
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38
ISSN 0378 - 8024
Determination of Variety Reaction to Potato Wart Disease (Synchytrium endobioticum) in
Potato Planting Areas of Nevsehir Province, Turkey
Hale GÜNAÇTI*
Ali ERKILIÇ**
* Biological Control Research Station,01321, Adana
** Department of Plant Protection, Faculty of Agriculture, University of Cukurova, 01330 Adana, TURKEY.
ABSTRACT
Potato wart disease is caused by the soil borne fungus Synchytrium endobioticum (Schilberszky) Percival
belong to Chytridiomycetes class as an obligate fungus. In order to determine the varietal reactions against the
pathogen, an experiment was conducted in Derinkuyu district in Nevşehir, in 2008. Thirty varieties including
industrial and table ones, were used in the trial. The lowest disease intensity ratio (11.1%) was recorded on Jelly
variety. In addition, 15 varieties showing much tolerant reaction than Jelly variety were placed in the same group,
statistically. Binella was found as the most susceptible variety with the disease intensity in rate of 26.2 %. In
general, the most resistant variety was Jelly (11.1 %) and the most susceptible variety was Binella (26.2 %). Nearly
half of the varieties showed disease intensity ratios between 11.1–15.4 %, and the rest showed 16.0-26.2 % disease
reaction.
Key Words: Potato, Synchytrium endobioticum, susceptibility, resistance
INTRODUCTION
Potato (Solanum tuberosum L.) as a member of Solanaceae family, originated Peru and the Andean region of
Bolivia. Potato was introduced to our country at the end of 19th Century, first of all in Eastern Black Sea region, and
then the west of Thrace (Simsek, 2002).
According to 2009 data, potatoes had 18.326.242 hectares of plantation area, 329.556.911 tons of production
and 1798,3 kg of yield around the world. According to 2009 data, the planting area was 142.684 hectares,
production 4.397.711 tons and the yield 3082,1 kg in Turkey (FAO, 2009). Turkey ranked number seven after
China, Russia, India, Poland, USA and Germany in terms of the amount of planting area and production
(Anonymus, 2002).
The potato wart disease spread at the end of the nineteenth century from its original range in the Andes in
South America to parts of North America and Europe and then other potato growing countries like in Asia, Africa,
and Oceania continents (EPPO/CABI, 1997). Potato Wart Disease is caused by the soil borne fungus Synchytrium
endobioticum (Schilberszky) Percival belong to Chytridiomycetes class as an obligate fungus (Langerfeld, 1984).
Resting spores of the fungus in soil are extremely long lived, in the range of 10-40 years or more (Langerfeld, 1984;
Laidlaw, 1985; Hampson, 1996; EPPO/CABI, 1997; Strachewicz ve Langerfeld, 1998).
The pathogen usually preferring cool climates is known to exist in 43 countries today (Baayen et al. 2006).
Losses due to the disease have been changing between 50 and 100 percent worldwide (Hampson, 1993; Melnik,
1998). The first determination of disease was taken place in 1992 in Ordu (Aybastı), Niğde (Ağcaşar) and Nevşehir
(Kaymaklı, Derinkuyu) in Turkey.
39
DETERMINATION OF VARIETY REACTION TO POTATO WART DISEASE (SYNCHYTRIUM
ENDOBIOTICUM) IN POTATO PLANTING AREAS OF NEVSEHIR PROVINCE, TURKEY
A typical symptom of the disease occurring on tubers is cauliflower-like warts or tumors of different sizes.
Initially the size of warts changes from pea size to hand punch size with white to green color. Above-ground warts
are green but later become black and subterranean warts are white to brown, becoming black on decayed area
(Hampson, 1981). The disease can cause symptoms to the underground components of potato plants including
crown, stolons and tubers except roots (Hampson ve Haard, 1980).
Phytosanitary regulations, national and international, have been applied throughout the world for nearly a
century to prevent spread of the fungus. The time that infected fields are identified, strict phytosanitary control and
prohibition of cultivation of susceptible cultivars have been the main components of official control. Because the
serious nature of the disease and the fact that spores can remain viable in the soil for many years it has to be controlled
in the European Union by a Wart Disease Directive (EU, 1969). According to the legislation, potatoes cultivation does
not allowed on the area which an outbreak has occurred. Therefore, only the resistant cultivars may be grown in a
safety zone around the infected sites.
The purpose of this study was to find out the efficacy of some applications to provide a base for Potato Wart
Disease control, since there is any control measures exist, apart from quarantine legislations in Turkey. For this
purpose, susceptibility of potato varieties was tested to the pathogen in the soil.
In this study, 30 potato varieties were used which are commercially grown in Turkey, named as; Agata,
Agria, Almera, Ambition, Anuckha, Armada, Binella, Cosmos, Elfe, Elodi, Esprit, Faluca, Floris, Hermes, Jelly,
Consul, Latona, Madeleine, Marabel, Maranca, Marfona, Markies, Matador, Milva, Presto, Provento, Safran, Sante,
Van Gogh, Zafira.
In order to determine the variety reactions against the pathogen, a study was conducted in Derinkuyu district in
Nevşehir, in 2008. A total of 30 varieties including industrial and table ones were used in the trials. The experiment
was established according to completely randomized design with three replications. The study was conducted on
infested soil in the pots (25 cm) in the open air. The pots were filled with S. endobioticum infected soil from Derinkuyu
and two potatoes from each potato varieties were planted. And then, 150g of inoculum from the pathogen compost was
prepared like the method of Spieckermann and Kothoff (1924) added in the pots and covered with infected soil. At the
end of the vegetation period of 120 days, the plants were pulled out and all the underground plant parts, root,
underground housing, stolons and tubers were evaluated (Figure 1). After harvest, the tubers’ wart development was
evaluated for the stem base, stolons and tubers by rating on the scale of 1-9 (EPPO, 2004) where 1=Not affected,
2=Single proliferation (<5 mm), 3= 2 or 3 proliferations (<5 mm) or a single larger one (5-10 mm), 4=Several small
warts (5-10 mm), 5=Several medium-sized warts (>10mm), 6=Several large warts, at last one of these being > 10
mm, and beginning deformation of the tuber, 7=Large warts with a diameter of > 10 mm and disruption of tuber
formation, 8=Very large warts, but individual tubers still recognizable, 9=Very large warts, no normal tubers
present.
Preparation of Synchytrium endobioticum Inoculum and Compost Production
S. endobioticum inoculum and compost production were prepared using the fresh warts on potato tubers
according to the method of Spieckermann and Kothoff, (1924). Compost inoculums were obtained from the
Nevşehir province. The warts were cut approx. 1cm. in size and added to sand by one portion of wart in three
portions of sand (1W:3S). The mixture was incubated at 18-25°C in dark for six months and the experiments were
conducted with two replicates. The production of compost was completed in three steps:1) one-third of the wart and
sand mixture in the clean trays were wetted with tap water and stirred every day during a period of two months. 2)
wetting and stirring processes were carried out in weekly intervals for two months 3) the compost left to dry without
any wetting and stirring process and kept at +4°C for further use.
40
H. GÜNAÇTI, A. ERKILIÇ
Figure 1. Wart on potato stolons, stem base and tuber
Disease severity scale of values was calculated and variance analysis was applied to these values. Later, by
LSD multiple comparison tests with the varieties of S. endobioticum been in their sensitivity showed differences
between (Karman, 1971).
In this work, 30 potato varieties which grown commercially in Turkey were tested for the variety reactions
against the pathogen. Sensitivity tests were carried out in the field on all the varieties. In the pot tests, all plants were
removed from the soil at the end of the ripening period, and then evaluated according to the scale of 1-9 in terms of
formation of the potato wart.
The cultivars were classified into resistance categories as given in Table 1. The lowest disease development
was recorded from Jelly variety as 11.1 %. In addition, 15 varieties showing much tolerant reaction than the Jelly
were placed in the same group, statistically. Binella was found as the most susceptible variety with the disease
development in a rate of 26.2 %.
In general, the most resistant and most susceptible varieties were Jelly (11.1 %) and Binella (26.2 %),
respectively. However, the average disease incidence was resulted in between the range of 11.1–15.4 % in the half
of the varieties and 16.0-26.2 % in the rest (Figure 2).
41
Table 1. Sensivity of Potato varieties to Synchytrium endobioticum
Variete
Jelly
Matador
Madeleine
Almera
Van Gogh
Safran
Ambition
Anuckha
Elfe
Faluka
Disease
severity(%)
11,1
a*
11,4
ab
11,7
ab
12,0
ab
12,6
ab
13,1
ab
13,2
ab
13,3
ab
14,0
ab
14,1
ab
Variete
Floris
Marabel
Sante
Maranca
Zafira
Hermes
Agata
Milva
Markies
Latona
Disease
severity(%)
14,2
ab
14,2
ab
14,3
ab
15,4
ab
15,4
ab
16,0
ab
17,3
ab
17,3
ab
17,4
b
17,4
b
Variete
Cosmos
Marfona
Agria
Esprit
Armada
Presto
Provento
Consult
Elodi
Binella
Disease
severity(%)
18,7
b
18,3
b
19,8
bc
20,0
bcd
20,2
bcd
20,7
bcd
22,2
bcd
23,5
bcd
25,1
cd
26,2
d
* Different letters of means are statistically different by LSD(0.05) test
Figure 2. Levels of sensivity potato varieties to Synchytrium endobioticum
Agria commonly grown in the region is being referred as susceptible to some sources. In this study it also
showed that the severity of disease rate was 19.8% in this variety. The varieties of Van Gogh, Latona and Provento
referred as resistant in some literature, however in our study it showed a disease severity of 12.6 %, 17.4 % and
22.2% respectively. The variety Provento is similar to the most susceptible variety Binella. Van Gogh has given
similar results like Jelly which is the most resistant.
The study done for determining the resistance and susceptibility reactions of potato varieties against potato
wart disease showed that the severity of disease was between 11.1% (in Jelly, the most tolerant) and 26.2% (in
Binella, the most susceptible). According to the studies in the region and observations and manufacturers'
statements, the varietiy of Van Gogh is resistant to the disease. Van Gogh and 16 potato varieties which were
statistically in the same group, they may likely to be tolerant against S. endobioticum in the region. However, a
comprehensive study may clarify this issue with determining distribution and density of all the races of pathogen in
the region.
Because of the intense pressure of inoculum, both the land intensive sporangium and wart compost added to
soil in pot trials, the infection was ranged between 11.1-26.2% in some potato varieties. It may not sufficient to say
42
H. GÜNAÇTI, A. ERKILIÇ
these cultivars susceptible or resistant under those extreme conditions. There were some studies done in different
regions in Turkey, but they were not on isolates of pathogen and their densities in the location. Only one study was
carried out on reactions of potato cultivars with 6 isolates of the pathogen collected from Nevsehir and Ordu. They
found that the cultivar reactions and isolates of pathogen gave different reactions in 2005 and 2007 years trials. The
cultivar Miriam was resistant against Nevşehir 1 isolate in 2005 and susceptible in 2007 (Cakir et al., 2009).
The observations at the harvest time for 3 years and with growers' statements in potato fields in Nigde and
Nevsehir provinces showed that the Van Gogh variety is disease resistant one. In this case, the type of Van Gogh
and statistically in the same group, which include the 16 potato cultivars in some regions of there may be likely to be
tolerant against to S. endobioticum. However, the result may be much clear on this issue with a study on the
distribution and density of all the isolates of pathogen can reveal by a comprehensive study.
ÖZET
NEVŞEHIR İLI PATATES EKILIŞ ALANLARINDA PATATES SİĞİL HASTALIĞI (SYNCHYTRIUM
ENDOBIOTICUM)’NA KARŞI ÇEŞİT REAKSİYONLARININ BELİRLENMESİ
Patatesin en önemli yumru hastalığı olan Patates Siğil Hastalığı’na Synchytrium endobioticum (Schilb)
Percival neden olur. Etmen, Chytridiomycetes sınıfına ait, toprak kökenli ve obligat bir fungustur.
Patates çeşitlerinin patojene karşı reaksiyonlarının belirlenmesi amacıyla 2008 yılında Nevşehir ili Derinkuyu
ilçesinde bir deneme yürütülmüştür. Denemede 30 adet sanayilik ve sofralık patates çeşidi kullanılmıştır. En düşük
hastalık şiddeti %11.1 oranı ile Jelly çeşidinde görülmüştür. Jelly çeşidinin ardından daha yüksek hastalık şiddeti
gösteren 15 patates çeşidi de istatiksel olarak aynı gurupta yer almıştır. Binella patates çeşidi %26.2’lik hastalık
şiddeti ile en duyarlı çeşit olarak bulunmuştur. Genel olarak değerlendirildiğinde denemedeki çeşitlerin en
dayanıklısı Jelly (%11.1) ve en duyarlısı Binella (%26.2) olup, tüm çeşitlerin yaklaşık yarısı %11.1-15.4 arasında,
diğer yarısı da %16.0-26.2arasında hastalık şiddeti göstermiştir.
Anahtar Kelimeler: Patates, Synchytrium endobioticum, duyarlılık, dayanıklılık
LITERATURE CITED
Anonymus, (2002). http:www.fao⁄statistics.
Baayen, R.P., Cochius, G., Hendriks, H., Meffert, J.P., Bakker, J. and Bekker, M., 2006. History of potato wart
disease in europe a propasol for harmonisation in defining pathotypes. European Journal of Plant Pathology,
116:21–31
Çakir, E., Van Leeuwenn, G.C.M., Flath, K., Meffert, J.P., Lanssen and Maden, S. 2009. Identification of
pathotypes of Synchytrium endobioticum found in infested fields in Turkey. Eppo Bulltein. 39,175-178.
Eppo/Cabi. 1997. Quarantine pest for Europe, 2nd end. CABI International, Wallingford (GB).
EU (1969). Council directive 69/464 of 8 December 1969 on control of potato wart disease. Official Journal of the
European Communities L323, 561–562.
Eppo Bulletin (Europen And Mediterrnean Plant Protection Organization). 2004. Dignostic Protocols for Regulted
Pests: Synchytrium endobioticum. Eppo Bulltein Vol. 34. (2): 213–218.
FAO, 2004. FAO Resmi internet sitesi verileri: http:www.fao⁄org.90.
Hampson M.C. 1981. Potato sprouts and potato wart disease. Can. Agric. 26(3):30–31.
Hampson M.C. 1993. History, biology and control of potato wart disease in Canada. Can. J. Plant Pathol. 15: 223–244.
Hampson M.C. 1991. Agriculture and agri-food Canada atlantic cool climate crop research centre. Minister of
supply and services Canada 1991. Cat No. A22-131/1991E. ISBN0-662-19166-8.
43
Hampson, M.C. 1996. A qulitative assessment of wind dispersal of resting spores of Synchytrium endobioticum the
causal agent of wart disease of potato. Plant Disease, 80(7):779–782.
Hampson M.C. and Haard, N.F. 1980. Pathogenesis of Synchytrium endobioticum: 1. infection responses in potato
and tomato. Can. J. Plant Pathol. 2:143–147.
Karman, M. 1971. Mesleki kitaplar serisi. Bitki koruma araştırmalarında genel bilgiler. denemelerin kuruluşu ve
değerlendirme esasları. Bölge Zirai Araştırma Enst. İzmir-Bornova, 279 s.
Laidlaw, W.M.R. 1985. A method for the detection of the resting sporangia of the potato wart disease(Synchytrium
endobioticum) in the soil of old outbreak sites. Potato Res. 28:223–232.
Langerfeld, E. 1984. Synchytrium endobioticum (Schilb.) Perc. zusammenfassende darstellung des erregers des
kartoffelkrebses anhand von literaturberichten. mitteilungen aus der biologischen bundesanstalt für land-und
forstwirtschaft. Berlin-Dahlem, 219:1–142 (in German).
Melnik, P.A. 1998. Wart disease of potato, Synchytrium endobioticum (Schilb.) Perc..Eppo Technical Documents
No. 1032. Eppo, Paris (Fr).
Spieckermann, A. and Kothoff, P., 1924. Testing potatoes for wart resistance. Deutsche Landwirtschaftliche Presse.
51:114-115.
Stachewicz, H. and Langerfeld, E., 1998. Synchytrium endobioticum (Schilb.) Perc. zur geschichte des
kartoffelkrebses in deutschland. Mitteilungen Aus der Biologischen Bundesanstalt für Land-und
Forstwirtschaft, Berlin-Dahlem 335:38–62.
Stachewicz, H. and De Boer, S. 2002. Pathotype determination of potato wart from Prince Edwart Island, Canada.
Nachrichtenblatt Fur Den Pflanzenschutz İn Der Ddr 54:269.
Şimşek, Y. 2002. Patates tarımı. Ankara.
44
ISSN 0378 - 8024
The Effects of Various Inactivation Treatments on Seed Germination Characteristics in
Vegetable Seeds Infected with the Viruses
Ismail Can PAYLAN, Semih ERKAN, Nedim ÇETINKAYA, Müge ERGUN, Ayşe CANDAR
Ege University, Faculty of Agriculture, Department of Plant Protection, 35100 Bornova, Izmir, Turkey
Accepted by April, 24 2013
ABSTRACT
The effects of different virus inactivation methods on seed germination properties were investigated in the
present study. Certain inactivation treatments applied to essential vegetable seed such as tomato, pepper, melon,
squash, bean and lettuce which are important for seed production in Turkey. For the study, 8 different seed samples
infected by Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Soybean
mosaic virus (SMV) and Lettuce mosaic virus (LMV) were exposed to virus-inactivation treatments such as
chemicals, dry heat, ozone, heat water and UV. As a result of treatments in question, the germination tests were
applied to seed samples and the obtained data were assessed statistically. Germination powers (%) and average
germination periods (day) of seeds in our work were considered for the criteria of assessments. According to test
results, other seed treatments except from HCl and ozone, reduced the germination power of vegetable seeds
generally. On the other hand, germination power in HCl and ozone treatments found at the similar levels with
control values. Theheatwatertreatments (65 °C) reduced the germination power of seedsfrom 94% to 34%.
Consequently, HCl and ozone treatments did not have any negative effects on germination values where these
treatments were also successful in elimination of viral agents.
Keywords: Vegetable, seed, virus inactivation treatments, seed germination.
GİRİŞ
Bitkisel üretimde tohumun rolü ve tohumluğun ticari bir nitelik kazanması kalite unsurunu ön plana çıkarmış
ve bunun sonucunda da ulusal ve uluslararası bazda yeni yapılanmalara gidilmiştir. Bu değişimin ilk sonucu üretilen
tohumluk miktarındaki artış ve bunun dikkati çeken diğer yönü ise sağlanan parasal değerde yükseliş olmasıdır. Bazı
tahminlere göre dünya ölçeğinde günümüzde yılda 127-128 milyon ton tohumluk kullanılmaktadır. Bu üretimin
parasal değeri yaklaşık 50 milyar $’dır. Bunun da yaklaşık 30 milyar $’lık bölümünü ticari tohumluk
oluşturmaktadır. Ticari tohumluk üretimi içinde ilk sırayı ABD yaklaşık 5,7 milyar $ ile almakta onu 4,9 milyar $
toplam değerle diğer AB ülkeleri izlemektedir. Ülkemiz ise tahmini 170 milyon $ olan bir değerle oldukça gerilerde
yer almaktadır. Türkiye 20-25 milyon $’lık tohum ihracatı ve 40-45 milyon $’lık tohum ithalatı değerlerine sahiptir
(Açıkgöz vd.,1997).
Kültür bitkilerinin yetiştirilmesinde tohumun niteliği diğer girdilerin potansiyellerini gerçekleştirebilmeleri
üzerinde etkili olmaktadır. Tohumlarda kalite ve verim gibi özelliklerin yanında sağlık durumu da önem taşıyan bir
konudur. Tohumlar bünyelerinde bulundurdukları hastalık etmenlerinden zarar görebildikleri gibi, bu patojenlerin
yayılmalarında ve taşınmalarında aracılık görevi de yapabilmektedirler. Kültür bitkilerinin üretiminde nicel ve nitel
biçimde ürün kayıplarına neden olan faktörler arasında viral kaynaklı etmenlerin ayrı bir önemi vardır. Bu etmenlere
karşı diğer patojen gruplarının önlenmesi için sıkça başvurulan kimyasal savaş yönteminin uygulanamaması ve
diğer kontrol yöntemlerinin de üretici tarafından yeterli düzeyde bilinmeyişi virüslerden kaynaklanan kayıpların
45
THE EFFECTS OF VARIOUS INACTIVATION TREATMENTS ON SEED GERMINATION
CHARACTERISTICS IN VEGETABLE SEEDS INFECTED WITH THE VIRUSES
artmasına neden olmaktadır. Viral kökenli etmenlerin önemli taşınma ve bulaşma yollarından biride tohumdur. Bitki
virüslerinin belirlenen taksonomik gruplarının 28’nin 21’inde tohumla taşınmagerçekleşmektedir. Tüm bitki
virüslerinin % 18’i tohumla taşınabilmektedir (Antignus, 1999). Özellikle dar konukçu dizisine sahip virüsler için
tohumla taşınma yaşamı devam ettirme ve mevsimler arası geçişte bir araç olarak düşünülmektedir. Tohumla
taşınma, viral hastalıkların böcek vektörler ile yayılabilmesi için ilk enfeksiyon kaynağını oluşturmaları açısından da
önemlidir. Tohumla taşınan viral etmenler ticari yolla uzun mesafelere ulaşabildiği gibi, tohumun doğal özelliği
nedeni ile kısa mesafelere de taşınabilmektedir (Erkan, 1998). Tohumla taşınan bazı virüslerin neden oldukları ürün
kayıplarına ait örnekler Çizelge 1’de görülmektedir.
Çizelge 1. Tohumla taşınan virüslerin neden oldukları ürün kayıpları (%)
Virüs Adı
Bean common mosaic potyvirus
Broad bean stain comovirus
Cucumber mosaic cucumovirus
Lettuce mosaic potyvirus
Pea seed-borne mosaic potyvirus
Soybean mosaic potyvirus
Tomato mosaic tobamovirus
Tobacco mosaic tobamovirus
Zucchini yellow mosaic potyvirus
Ürün
Fasulye
Mercimek
Acı bakla
Marul
Bezelye
Soya Fasülyesi
Domates
Domates
Kabakgil
%
Ürün Kayıp Oranı
35-98a
14-61b
25-42c
≤30a
11-36d
48-99e
5-50f
≤94 f
0-99a
Kaynaklar: a Shukla et al. (1994), Richardson, 1990; b Makkouk and Kumari (1990); c Bwye et al. (1994); d Khetarpal and Maury (1987); e Tu (1989); f
Walkey (1991);
Sebze tohumlarının kalite ve kantitesini bu denli etkileyen tohum kaynaklı virüslerin mücadelesi de oldukça
önem taşıyan konu halini almıştır. Bu nedenle de, sebze tohumlarındaki viral etmenlerin inaktifleştirilmesi
konusunda önceki yıllarda dünyada ve ülkemizde bazı kimyasal maddeler, sıcak su ve sıcak hava uygulaması, ozon
uygulaması ve depolama gibi çeşitli uygulamalar gerçekleştirilmiştir (John and Sova, 1955; Taylor et al., 1961;
Gooding and Suggs, 1976; Erkan, 1983; Yorgancı vd., 1993; Değirmenci vd., 2009). Bahsedilen çalışmaların
bazılarında önemli başarılar elde edilmiş, bazıları ise başarısız olmuştur. İnaktifleştirme uygulamalarında başarılı
olunsa dahi en önemli konu tohumun çimlenme gücünün korunmasıdır. Zira, genelde virüsün inaktif duruma
gelmesine neden olan uygulamalar aynı zamanda tohumun canlılığını da azaltmaktadır (Erkan, 1998).
Bu çalışmada, sebze tohumlarında belirlenen viral etmenlerin elemine edilmesi amacıyla tohum örneklerine
çeşitli kimyasal maddeler ve bazı inaktifleştirme yöntemleri uygulanmıştır. Ayrıca, bahsedilen uygulamaların
tohumun çimlenmesine etkileri araştırılmıştır. Sonuçta; ülkemiz sebze üretiminde sağlıklı tohum kullanmak ve
sağlıklı bitki yetiştirmek amacına hizmet etmek için gerçekleştirilecek sonraki çalışmalara basamak oluşturacak
önemli bulgular elde edilmiştir.
MATERYAL VE METOD
Virüs İnaktifleştirme Çalışmaları ve Çimlendirme Testlerinde Kullanılan Tohum Örnekleri
Gerçekleştirilen DAS-ELISA ve RT-PCR testlerinde enfeksiyon düzeyi diğerlerine oranla daha yüksek
olarak saptanan örneklerden tesadüf ilkesi dikkate alınarak, her uygulama için tohum örnekleri seçilmiş ve virüs ile
enfekteli tohum örnekler iinaktifleştirme uygulamalarının materyalini oluşturmuştur.Virüs inaktifleştirme
uygulamalarında kullanılan tohum örnekleri ayrıca, yapılan değişik uygulamaların tohumların çimlenme güçlerine
olan etkilerini araştırmak için yürütülen çimlenme testlerinin de materyalini oluşturmuştur.
Tanılanan Viral Etmenlerin İnaktifleştirilmeleri
Tohumlardaki virüsleri inaktifleştirme işlemleri yapılacak olan virüslü tohum örnekleri, inaktifleştirme
uygulamaları belirlenmiş ve virüslerle enfekteli olan tohum örneklerinde bu uygulamalar gerçekleştirilmiştir. Daha
46
I. C. PAYLAN, S. ERKAN, N. ÇETINKAYA, M. ERGUN, A. CANDAR
önceden DAS-ELISA ve RT-PCR testleri ile virüs(ler)le enfekteli oldukları belirlenen tohum örnekleri,
inaktifleştirme uygulamalarından önce kontrol amaçlı olarak DAS-ELISA yöntemi ile tekrar testlenmiştir.
Virüs(ler) ile enfekteli tohum örneklerine yapılacak olan inaktifleştirme uygulamaları için değişik virüsler ile
enfekteli olan 8 farklı tohum örneği seçilmiştir. Bunlar arasında;
• ToMVve TMV ile enfekteli domates tohum örnekleri,
• TMV ve CMV ile enfekteli biber tohum örnekleri,
• CMV ile enfekteli kavun tohum örnekleri,
• CMV ile enfekteli kabak tohum örnekleri,
• SMV ile enfekteli fasulye tohum örnekleri ve
• LMV ile enfekteli marul tohum örnekleri bulunmaktadır.
Viral etmenlerin inaktifleştirilmesi uygulamaları 3 tekerrürlü olarak gerçekleştirilmiştir. İlk olarak, yüksek
enfeksiyon düzeyine sahip oldukları belirlenmiş olan tohum örneklerinden tesadüf ilkesi dikkate alınarak, her
uygulama için 600 tohum seçilmiştir. Bu tohumlar tülbent bezi içerisine konularak ağızları lastikle kapatılmıştır.
Daha sonra, hazırlanmış olan bu tohumlar 1000 ml’lik beherler içerisine belirlenen sürelerde daldırılmıştır. HCl
uygulamasından sonra, tohumlar bir kap içerisinde su ile 30 dakika yıkanmıştır. Ozon uygulamalarında özel delikli
kaplara konulan tohumlar belirtilen sürelerde ozon tanklarına daldırılmıştır. Kuru sıcaklık ve UV uygulamaları
tohumlara direkt olarak gerçekleştirilmiştir. Sıcak su uygulaması ise ozon uygulamalarında olduğu gibi özel delikli
kaplar içinde sıcak su banyosuna daldırılarak gerçekleştirilmiştir. Sıcak hava uygulamaları da etüv içerisinde
kontrollü olarak belirli sıcaklık, süre ve nemde gerçekleştirilmiştir. Tohumlarda belirlenen viral etmenleri
inaktifleştirmek amacı ile yapılmış olan uygulamalar Çizelge 2’de görülmektedir.
Çizelge 2. Bazı Sebze Tohumu Örneklerinde Bulunan Viral Etmenlerin İnaktifleştirilmeleri İçin Yapılmış Olan Uygulamalar
Uygulama
Asetik asit (CH3-COOH)
Hidrojen peroksit (H2O2)
Hidroklorik asit (HCl)
Kuru sıcaklık
Ozon (O3)
Ozon (O3)
Ozon (O3)
Sıcak su
Sodyum Hipoklorit (NaOCl)
Triton X 100
UV
Uygulama Oranı / Sıcaklık Derecesi
% 0.8
%4
%2
80 oC (%30-40 nem)
5g/m3
10g/m3
10g/m3
65 oC
% 0.4
% 10
305 nm
Uygulama Süresi
20 dakika
20 dakika
30 dakika – 30 dakika su ile yıkama
1 gün
60 dakika
3 dakika
10 dakika
25 dakika
30 dakika
20 dakika
10 dakika
Çimlenme Testleri
Virüslerle enfekteli olan tohum örneklerine gerçekleştirilen inaktifleştirme uygulamalarının, bazı sebze
tohumlarındaki çimlenme güçlerine olan etkilerini araştırmak için bu tohumlara çimlenme testleri uygulanmıştır.
Çimlenme testleri 12-15 cm petri kaplarında 25’er adet tohum kullanılarak 4 tekerrürlü olarak çift katlı kurutma
kağıtları arasında yapılmıştır. Radikula 2 mm uzunluğuna ulaştığında tohumlar çimlenmiş olarak sayılmıştır ve
çimlenen tohumlar ortamdan uzaklaştırılmıştır. Her gün sayım yapılmış ve 3 gün aynı değer kaydedilince deneme
sonlandırılmıştır. Bu denemelerde çimlenme gücü, standart ISTA çimlenme koşulları (24-28 oC, nemli ortam ve 14
günlük gözlem) ile tespit edilmiştir. (Anderson, 1987; Duman ve Eşiyok, 1998; ISTA, 2007).
İstatistik Analizler
Üç tekerrürlü olarak gerçekleştirilen inaktifleştirme uygulamalarının ardından virüslerle enfekteli
tohumlardaki çimlenme testleri ve çimlenme süreleri dikkate alınarak SPSS 15.0 İstatistik Programında Duncan
47
çoklu karşılaştırma testi uygulanmış ve yapılan uygulamalar arasındaki farklar ortaya konulmuştur. (Karman, 1971;
Durmuşoğlu, 2010; Knezevic et al., 2007 ).
SONUÇLAR VE TARTIŞMA
Viral etmenler ile enfekteli olan tohum örneklerine yapılan inaktifleştirme uygulamaları ve çimlenme testleri
için değişik virüsler ile enfekteli olan 8 farklı tohum örneği seçilmiştir. Seçim sırasında tohum örneklerindeki
virüsler ve bulunma durumları dikkate alınmıştır. Buna göre; inaktifleştirme çalışmaları ve çimlendirme testleri
ToMV ve TMV ile enfekteli olan domates, TMV ve CMV ile enfekteli olarak belirlenen biber, CMV ile enfekteli
bulunan kavun ve kabak, SMV ile enfekteli olan fasulye ve LMV enfeksiyonu belirlenen marul tohum örnekleri ile
yürütülmüş ve elde edilen bulgular Çizelge 3, 4, 5, 6, 7, 8, 9 ve 10’da gösterilmiştir.
ToMV ile enfekteli domates tohumlarına gerçekleştirilen inaktifleştirme çalışmaları ve çimlenme testlerinin
sonuçlarına göre sıcak su uygulaması çimlenme gücünü kontrol değeri olan %99’dan %36’ya indirirken, ortalama
çimlenme zamanını da 5,14 günden 7,81 güne çıkartmıştır. Sıcak su uygulaması dışındaki diğer inaktifleştirme
uygulamalarının çimlenme değerleri üzerinde önemli bir etkisinin olmadığı istatistiki olarak belirlenmiştir.
İnaktifleştirme uygulamalarının ardından belirlenen çimlenme gücü değerleri; HCl için %90, Ozon (60 dk. 5g/m3)
için %96, Ozon (10 dk. 10 g/m3)için %96, UV için %93, Triton için %94, H2O2 için %93, Ozon (3 dk. 10g/m3) için
%95, NaClO için %94, CH3COOH için %89, Etüv (80oC 24saat) için %86 ve kontrol için %99 olarak belirlenmiştir.
Ortalama çimlenme zamanı değerlerinin sıcak su uygulaması dışındaki uygulamalar için 5.23 ile 6.28 gün arasında
değişmekte olduğu saptanmıştır. ToMV enfekteli domates tohumlarına uygulanan inaktifleştirme çalışmalarına ve
çimlenme testlerine ait bulgular Çizelge 3’de, uygulmaya ait resimler ise Şekil 1’de görülmektedir.
Şekil 1. Domates tohumlarına uygulanan çimlenme testleri sonuçları, (A) ToMV ile enfekteli domates tohumlarında gerçekleştirilen çimlenme
testlerine (kontrol) ait görünümü, (B) Ozon (3 dk. 10g/m3) uygulaması yapılmış olan ToMV ile enfekteli domates tohumlarında
gerçekleştirilen çimlenme testlerine ait görünüm.
Çizelge 3. ToMV ile enfekteli domates tohumlarına yapılan inaktifleştirme uygulamaları ve çimlenme testleri sonuçları
Uygulama
Çimlenme Gücü (%)
HCl
Sıcak Su (65 oC)
Ozon (60 dk. 5g/m3)
Ozon (10 dk. 10g/m3)
UV
Triton
H2O2
Ozon (3 dk. 10g/m3)
NaClO
CH3COOH
Etüv (80oC 24saat)
Kontrol
* Farklı harfler farklı istatistiki grupları ifade etmektedir ( Duncan, p ≤ 0,05).
48
90
36
96
96
93
94
93
95
94
89
86
99
c-e*
f
ab
ab
b-d
a-d
b-d
a-c
a-d
de
e
a
Ortalama
Çimlenme Zamanı (gün)
5,23 a
7,81 c
5,50 ab
5,88 ab
5,61 ab
6,28 b
5,75 ab
5,75 ab
5,67 ab
5,35 ab
5,61 ab
5,14 a
TMV ile enfekteli domates tohumlarına gerçekleştirilen inaktifleştirme ve çimlenme testleri çalışmalarından
elde edilen bulgulara göre çimlenme gücü değerleri; HCl için %89, ozon (10 dk. 10 g/m3)için %92, sıcak su (65oC)
için %36, ozon (60 dk. 5g/m3) için %91, ozon (3 dk. 10g/m3) için %92, CH3COOH için %86, Triton için %92,
NaClO için %90, etüv (80oC 24 saat) için %86, H2O2 için %93, UV için %88 ve kontrol için %95 olarak
belirlenirken, ortalama çimlenme zamanı verileri 4.93 ile 8.83 gün arasında değişmektedir. TMV enfekteli domates
tohumlarına uygulanan inaktifleştirme çalışmalarına ve çimlenme testlerine ait bulgular Çizelge 4’de verilmiştir.
Çizelge 4. TMV ile enfekteli domates tohumlarına yapılan inaktifleştirme uygulamarı ve çimlenme testleri sonuçları
Uygulama
89
92
36
91
92
86
92
90
86
93
88
95
HCl
Ozon (10 dk. 10 g/m3)
Sıcak Su (65 oC)
Ozon (60 dk. 5g/m3)
Ozon (3 dk. 10g/m3)
CH3COOH
Triton
NaClO
Etüv (80oC 24saat)
H2O2
UV
Kontrol
a-c*
a-c
d
a-c
a-c
c
a-c
a-c
c
ab
bc
a
Ortalama
5,34 a
5,26 a
8,83 c
5,25 a
5,36 a
5,21 a
5,41 a
4,93 a
5,43 a
5,22 a
7,05 b
4,97 a
TMV ile enfekteli biber tohumlarına gerçekleştirilen inaktifleştirme ve çimlenme testleri çalışmalarından
elde edilen bulgulara göre çimlenme gücü değerleri; ozon (10 dk. 10 g/m3) için %91, ozon (3 dk. 10g/m3) için %90,
ozon (60 dk. 5g/m3) için %91, NaClO için %90, Triton için %86, etüv (80 O C 24saat) için %66, HCl için %80,
H2O2 için %82, sıcak su (65oC) için %30, CH3COOH için %78, UV için %74 ve kontrol için %95 olarak
belirlenirken, ortalama çimlenme zamanı verileri 9.13 ile 11.12 gün arasında değişmiştir (Çizelge 5).
Çizelge 5. TMV ile enfekteli biber tohumlarına yapılan inaktifleştirme uygulamaları ve çimlenme testleri sonuçları
Uygulama
Ozon (10 dk. 10 g/m3)
Ozon (3 dk. 10g/m3)
Ozon (60 dk. 5g/m3)
NaClO
Triton
Etüv (80oC 24saat)
HCl
H2O2
Sıcak Su (65 oC)
CH3COOH
UV
Kontrol
91
90
91
90
86
66
80
82
30
78
74
95
ab*
ab
ab
ab
bc
f
c-e
cd
g
de
e
a
Ortalama
9,42 bc
9,85 bc
9,64 bc
9,36 bc
9,87 bc
10,01 c
10,83 d
9,78 bc
11,12 d
10,04 c
9,13 ab
8,46 a
CMV ile enfekteli biber tohumlarına gerçekleştirilen inaktifleştirme ve çimlenme testleri çalışmalarından
elde edilen bulgulara göre sıcak su (65oC) uygulamasının çimlenme gücünü %29’a indirdiği görülürken çimlenme
49
değerleri diğer uygulamalar için %70 ile %92 arasında değişen oranlarda veriler oluşturmuştur. Ortalama çimlenme
zamanları sıcak su (65oC) uygulaması için 12,68 gün, HCl uygulaması için 10,66 gün, Ozon (10 dk. 10 g/m3)
uygulamasıiçin 9,95 gün, CH3COOH uygulaması için 10,12 gün, Ozon (60 dk. 5g/m3) uygulaması için 10,65 gün,
Ozon (3 dk. 10g/m3) uygulaması için 10,37 gün, NaClO uygulaması için 9,50 gün, Triton uygulaması için 9,84 gün,
Etüv (80oC 24 saat) uygulaması için 10,40 gün, UV uygulaması için 9,83 gün, H2O2 uygulaması için 10,16 gün ve
kontrol için 8,81 gün olarak belirlenmiştir. Belirtilen uygulamalara ait veriler Çizelge 6’da verilmiştir.
Çizelge 6. CMV ile enfekteli biber tohumlarına yapılan inaktifleştirme uygulamaları ve çimlenme testleri sonuçları
Uygulama
Sıcak Su (65 oC)
HCl
Ozon (10 dk. 10 g/m3)
CH3COOH
Ozon (60 dk. 5g/m3)
Ozon (3 dk. 10g/m3)
NaClO
Triton
Etüv (80oC 24saat)
UV
H2O2
Kontrol
29
82
88
80
87
90
87
83
70
74
82
92
f*
bc
a-c
cd
a-c
ab
a-c
bc
e
de
bc
a
Ortalama
12,68 d
10,66 c
9,95 bc
10,12 bc
10,53 c
10,37 bc
9,50 ab
9,84 bc
10,40 bc
9,83 bc
10,16 bc
8,81 a
CMV ile enfekteli kavun tohumlarına gerçekleştirilen inaktifleştirme ve çimlenme testleri çalışmalarından
elde edilen bulgulara göre inaktifleştirme uygulamalarının ardından çimlenme gücü değerleri; sıcak su (65oC) için
%62, HCl için %99, Ozon (10 dk., 10 g/m3)için %98, Ozon (60 dk., 5g/m3) için %99, CH3COOH için %100, Ozon
(3 dk., 10g/m3) için %96, Triton için %80, etüv (80oC, 24saat) için %92, H2O2 için %92, UV için %94, NaClO için
%96 ve kontrol için %100 olarak belirlenirken ortalama çimlenme zamanı verileri 3,92 ile 5,58 gün arasında
değişmiştir (Çizelge 7).
Çizelge 7. CMV ile enfekteli kavun tohumlarına yapılan inaktifleştirme uygulamaları ve çimlenme testleri sonuçları
Uygulama
Sıcak Su (65 oC)
HCl
Ozon (10 dk. 10 g/m3)
Ozon (60 dk. 5g/m3)
CH3COOH
Ozon (3 dk. 10g/m3)
Triton
Etüv (80oC 24saat)
H2O2
UV
NaClO
Kontrol
62
99
98
99
100
96
80
92
92
94
96
100
d*
a
ab
a
a
ab
c
b
b
ab
ab
a
Ortalama
5,58 e
3,92 ab
4,08 a-c
4,09 bc
3,92 ab
4,15 c
5,01 d
3,94 ab
3,92 ab
4,17 c
3,92 ab
3,88 a
CMV ile enfekteli kabak tohumlarına gerçekleştirilen inaktifleştirme uygulamalarının ardından çimlenme
gücü değerleri; HCl için %82, sıcak su (65oC) için %24, Triton için %69, Ozon (10 dk., 10 g/m3) için %89, Ozon
(3 dk., 10g/m3) için %89, UV için %83, NaClO için %80, Ozon (60 dk., 5g/m3) için %89, H2O2 için %88,
50
CH3COOH için %86 ve kontrol için %90 olarak belirlenirken ortalama çimlenme zamanı verilerinin 3,91 ile 5,36
gün arasında değişmekte olduğu yapılan denemeler sonucunda belirlenmiştir. CMV enfekteli kabak tohumlarına
uygulanan inaktifleştirme çalışmalarına ve çimlenme testlerine ait bulgular Çizelge 8’de verilmiştir.
Çizelge 8. CMV ile enfekteli kabak tohumlarına yapılan inaktifleştirme uygulamaları ve çimlenme testleri sonuçları
Uygulama
Ortalama
3,91 a
HCl
82
bc*
Sıcak Su (65 oC)
24
e
5,36
d
Triton
69
d
4,81
c
Ozon (10 dk. 10 g/m3)
89
ab
4,26
b
Ozon (3 dk. 10g/m3)
89
ab
4,38
b
UV
83
a-c
4,30
b
Etüv (80oC 24saat)
80
c
4,07
ab
NaClO
80
c
4,29
b
Ozon (60 dk. 5g/m3)
89
ab
4,31
b
H2O2
88
ab
4,32
b
CH3COOH
86
a-c
3,92
a
Kontrol
90
a
3,90
a
SMV ile enfekteli fasulye tohumlarına gerçekleştirilen inaktifleştirme uygulamalarının ardından çimlenme
gücü değerleri; HCl için %66, Ozon (60 dk. 5g/m3) için %83, Ozon (10 dk., 10 g/m3) için %83, etüv (80oC 24saat)
için %69, Ozon (3 dk., 10g/m3) için %86, sıcak su (65oC) için %33, Triton için %73, UV için %68, NaClO için
%81, H2O2 için %72, CH3COOH için %59 ve kontrol için %86 olarak belirlenirken, ortalama çimlenme zamanı
verileri 7,50 ile 8,82 gün arasında değişmiştir (Çizelge 9).
Çizelge 9. SMV ile enfekteli fasulye tohumlarına yapılan inaktifleştirme uygulamaları ve çimlenme testleri sonuçları
Uygulama
HCl
Ozon (60 dk. 5g/m3)
Ozon (10 dk. 10 g/m3)
Etüv (80oC 24saat)
Ozon (3 dk. 10g/m3)
Sıcak Su (65 oC)
Triton
UV
NaClO
H2O2
CH3COOH
Kontrol
66
83
83
69
86
33
73
68
81
72
59
86
bc*
a
a
b
a
d
b
b
a
b
c
a
Ortalama
8,17 c-e
7,69 a-c
8,07 cd
7,95 b-d
8,06 cd
8,82 f
8,38 d-f
8,58 ef
7,50 ab
8,28 de
8,06 cd
7,22 a
LMV ile enfekteli olan marul tohumlarına gerçekleştirilen inaktifleştirme çalışmaları ardından yapılan
çimlenme testleri sonucunda sıcak su uygulamalarının marul tohumlarındaki çimlenme gücünü %0’a indirdiği
saptanmıştır. Diğer uygulamaların çimlenme gücü değerleri; Triton için %89.5, CH3COOH için %84, Ozon (10 dk.
10 g/m3) için %89.5, Ozon (3 dk., 10g/m3) için %91, Ozon (60 dk., 5g/m3) için %90, H2O2 için %84, Etüv (80oC 24
51
saat) için %80, HCl için %84.5, UV için %85, NaClO için %90 ve kontrol için %92.5 olarak belirlenirken ortalama
çimlenme zamanı verileri 4,08 ile 4,78 gün arasında değişmektedir (Çizelge 10).
Çizelge 10. LMV ile enfekteli marul tohumlarına yapılan inaktifleştirme uygulamaları ve çimlenme testleri sonuçları
Uygulama
89,5
84,0
89,5
91,0
90,0
0,0
84,0
80,0
84,5
85,0
90,0
92,5
Triton
CH3COOH
Ozon (10 dk. 10 g/m3)
Ozon (3 dk. 10g/m3)
Ozon (60 dk. 5g/m3)
Sıcak Su (65 oC)
H2O2
Etüv (80oC 24saat)
HCl
UV
NaClO
Kontrol
a*
b
a
a
a
d
b
c
b
b
a
a
Ortalama
4,53 de
4,08 ab
4,33 b-d
4,24 a-d
4,53 de
0,00 --4,41 cd
4,78 e
4,11 ab
4,14 a-c
4,44 d
4,03 a
Sonuç olarak, inaktifleştirme uygulamalarının genel olarak çimlenme güçleri üzerine olan etkisine baktığımız
zaman, sıcak su uygulaması dışındaki uygulamaların çoğunlukla kontrol değerlerine yakın değerlerde olduğu
görülmüş ve önemli derecede olumsuz etkilerinin olmadığı kanısına varılmıştır. Sıcak su (65oC) uygulamalarının ise
hemen hemen tüm bitki türleri ve virüslerle yapılan çalışmalarda çimlenme değerleri üzerinde olumsuz etkileri
olmuştur. Sıcak su uygulamaları çimlenme gücünü ortalama %94’ten %31 gibi düşük rakamlara indirmiştir (Şekil
2). Daha önce yürütülen çalışmalarda da benzer sonuçlar elde edilmiştir, Değirmenci vd. (2009) 50oC ve 65oC’lik
termoterapi uygulamalarının mısır tohumlarında Maize dwarf mosaic potyvirus (MDMV) etmeninin
konsantrasyonunu azalttığını, ancak çimlenme zamanı üzerinde olumsuz etkileri olduğunu belirtmişlerdir.
Şekil 2. İnaktifleştirme uygulamalarının çimlenme güçleri üzerine ortalama % etkileri
Bu veriler ışığında gerek viral konsantrasyon üzerine etkileri, gerekse çimlenme özellikleri üzerine herhangi
bir olumsuz etkileri olmaması nedeniyle, HCl ve ozon uygulamaları viral etmenleri elimine etme amacıyla
gerçekleştirilen inaktifleştirme uygulamaları için en etkili ve umutvar uygulamalar olarak göze çarpmaktadır.
Benzer şekilde diğer bir araştırmada da, HCl uygulamalarının diğer uygulamalara göre daha etkili olduğu ve
çimlenme üzerine herhangi bir olumsuz etkisinin olmadığı belirtilirken, sıcak su ve CH3COOH uygulamalarının
çimlenme üzerine olumsuz etki ettikleri vurgulanmıştır (Koyuncu, 1993). CGMMV ile yapılan bir çalışmada 75
52
dakika süre ile 6 g/saat ozon uygulanması ve ToMV ile yapılan başka bir çalışmada ise 20 gr/saat ozonun 60 dakika
muamelesi sonucunda etmenlerin tamamen eradike olduğu belirlenmiştir (Runia, 1995). Ozon bitkilerde gösterdiği
benzer etkileri mikrobiyal yapılarda da göstermektedir. Fungal patojenleri toprak veya hava kaynaklı olmalarına
bakmaksızın öldürdüğü (Yamamoto et al., 1990), bakteriyel membranları etkilediği, enzim yapılarını ve nükleik asit
metabolizmasını bozduğu önceki çalışmalarda belirtilmiştir. Viral etmenlerde ise, modifiye olmasına neden olduğu
veya proteininin parçalanabildiği ifade edilmektedir (EPA, 1999). Bitkilere ozon uygulaması ile virüs
konsantrasyonlarıazalmakla birlikte sinyal iletişim mekanizmaları harekete geçirilerek hastalıklara dayanıklılık
sağlayan faktörlerin uyarılabildiği de belirtilmektedir (Sandermann, 1996; Schubert et al., 1997). Tüm bu veriler
dikkate alınarak, farklı doz ve sürelerde yapılacak yeni çalışmalar ile HCl ve ozon uygulamalarının viral
konsantrasyonu azaltma konusundaki başarısının daha da artabileceği düşünülmektedir.
VİRAL ETMENLER İLE ENFEKTELİ SEBZE TOHUMLARINA YAPILAN DEĞİŞİK
İNAKTİFLEŞTİRME UYGULAMALARININ ÇİMLENME ÖZELLİKLERİ ÜZERİNE ETKİLERİ
ÖZET
Ülkemizde tohumluk üretimi açısından önemli olan domates, biber, kavun, kabak, fasulye ve marul
tohumlarına uygulanan virüs inaktifleştirme yöntemlerinin tohumların çimlenme özellikleri üzerine etkilerinin
araştırılması bu çalışmanın içeriğini oluşturmaktadır. Araştırma için Tomato mosaic virus (ToMV), Tobacco mosaic
virus (TMV), Cucumber mosaic virus (CMV), Soybean mosaic virus (SMV) ve Lettuce mosaic virus (LMV) ile
enfekteli 8 tohum örneğine kimyasal, kuru sıcaklık, ozon, sıcak su ve UV gibi virüs inaktifleştirme uygulamaları
yapılmıştır. Bahsedilen uygulamalar sonucunda tohum örneklerine çimlendirme testleri uygulanmış ve sonuçlar
istatistikî olarak değerlendirilmiştir. Değerlendirme için tohumların çimlenme gücü (%) ve ortalama çimlenme
süresi (gün) dikkate alınmıştır. Buna göre, HCl ve ozon uygulaması dışındaki uygulamalar çimlenme gücünü
oldukça düşürmüştür. Öte yandan, HCl ve ozon uygulamalarında çimlenme gücü kontrole yakın bulunmuştur.
Çimlenme gücünü en çok etkileyen yöntem ise sıcak su (65 °C) uygulaması olmuştur. Sıcak su uygulamaları
çimlenme gücünü ortalama %94’ten %31 gibi düşük rakamlara indirmiştir.Sonuç olarak, HCl ve ozon uygulamaları
viral etmenleri elimine etmedeki başarısının yanısıra çimlenme değerleri üzerine olumsuz etkileri olmaması
nedeniyle, en etkili yöntemler olarak saptanmıştır. Bu veriler ışığında, HCl ve ozon uygulamalarının farklı doz ve
sürelerde tohumlardaki viral konsantrasyonu azaltma konusundaki etkisinin daha da artabileceği düşünülmektedir.
Anahtar Sözcükler: Sebze, tohum, virüs, virüs inaktifleştirme uygulamaları, çimlenme özellikleri
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