Turkish Journal of Agriculture and Forestry Turk J Agric For (2013) 37: 457-467 http://journals.tubitak.gov.tr/agriculture/ © TÜBİTAK Research Article doi:10.3906/tar-1203-71

PCR detection of Fusarium oxysporum f. sp. radicis-lycopersici and races of F. oxysporum f. sp. lycopersici of tomato in protected tomato-growing areas of the eastern Mediterranean region of

1, 2 Ayşegül ÇOLAK *, Mehmet BİÇİCİ 1 Biological Control Research Station, Adana, Turkey 2 Department of Plant Protection, Faculty of Agriculture, Çukurova University, Adana, Turkey

Received: 30.03.2012 Accepted: 22.12.2012 Published Online: 16.07.2013 Printed: 02.08.2013

Abstract: Fusarium crown and root rot (caused by Fusarium oxysporum f. sp. radicis-lycopersici, FORL) and Fusarium wilt (caused by Fusarium oxysporum f. sp. lycopersici, FOL) are the most important diseases to affect tomatoes in protected growing conditions in the eastern Mediterranean region of Turkey. These diseases cause significant yield losses in this region. Polymerase chain reaction (PCR) studies were used to characterize 87 isolates from Adana and provinces, representative of different locations. Among them, 60% and 40% of the plants were determined to have FORL and FOL, respectively. FORL and FOL race 3 are the dominant pathogens in this region. Some differences between morphological identification and molecular detection (PCR) were observed.

Key words: Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporum f. sp. radicis-lycopersici, PCR detection, tomato

1. Introduction in one cropping season (Rekah et al. 2001). FORL causes In covered tomato-growing areas in the eastern 90% of crop losses with repeated infections that result in Mediterranean region, it was previously known that the microconidia spreading, especially in the same growing wilt disease caused by Fusarium oxysporum f. sp. lycopersici season in greenhouses (Hajlaoui et al. 2001; Hibar 2002). (Sacc.) W.C. Snyder & H.N. Hans (FOL) was of prime Since disinfected greenhouse soil can be recolonized with importance. Later, crown and root-rot diseases caused by FORL, crown and root rot disease control is difficult. F. oxysporum f. sp. radicis-lycopersici Jarvis & Shoemaker On the other hand, while FOL only affects solanaceous (FORL) were encountered more frequently (Yücel and plants, FORL is able to infect a total of 37 plant species Çınar 1989; Can et al. 2004). and varieties belonging to Solanaceae, Leguminosae, F. oxysporum has spread worldwide and is Cucurbitaceae, and Chenopodiaceae (Rowe 1980; Menzies phylogenetically diverse. The species is well known as a et al. 1990). Therefore, some weeds, as well as the tomato mycotoxin producer, and so its precise identification is of crops, should be considered in crown and root rot control. prime concern (Irzykowska et al. 2012). The 2 important Finally, it is essential to make a clear distinction between formae speciales of F. oxysporum, FOL and FORL, these 2 formae speciales, because their control relies on display epidemiological, genetic, and symptomatological their significant differences in terms of the sensitivity of differences (Rowe et al. 1977; Menzies et al. 1990). host tomato cultivars and their epidemiological behavior. Since FORL and FOL can coexist in tomato-growing Differentiation of Fusarium pathogens is not easy. greenhouses, mixed infections in 1 greenhouse, or even in Traditionally, a morphological and physiological features 1 tomato plant, are very probable (Rosewich et al. 1999; assessment was used as a basic identification method. Balmas et al. 2005). The Fusarium wilt caused by FOL can However, the formae speciales and races of F. oxysporum be prevented through growing resistant tomato cultivars are very difficult to discriminate this way (Nelson et al. and soil disinfection by some chemicals. However, 1983; Burgess et al. 1994). As a result, the complexity of commercial tomato cultivars resistant to the crown identification of these 2 formae speciales causes difficulties and root rot caused by FORL have not been developed in controlling the diseases caused by them (Katan et al. yet (Jones et al. 1991; Ozbay et al. 2004). FORL causes 1997; Rosewich et al. 1999; Gale et al. 2003). Although monocyclic and polycyclic infections on tomato plants tester plants have been widely used for the discrimination * Correspondence: [email protected] 457 ÇOLAK and BİÇİCİ / Turk J Agric For of the special forms and races of F. oxysporum, molecular estimate the frequency and severity of FORL and FOL in techniques for rapid and reliable identification have come tomato greenhouses. to the fore in recent years (Garibaldi 1975; Woo et al. 1996). Many molecular techniques are used as practical and 2. Materials and methods reliable methods for identification of fungi. Molecular 2.1. Fungi isolation and culture maintenance markers have recently become technically easy to use, Samples of tomato plants displaying disease symptoms readily yielding informative and conclusive results in were collected from tomato-growing greenhouses of some the identification of the species and subspecies of fungi. districts in Adana (Yüreğir, Seyhan, Karataş, Ceyhan) and Essentially, there are 2 techniques for DNA marking. Mersin (, Erdemli, Adanalıoğlu, Kazanlı, Tarsus), The first is restriction fragment length polymorphism provinces in the eastern Mediterranean region of Turkey, (RFLP), based on DNA hybridization, and the other, between November 2007 and April 2008. Some causal based on polymerase chain reaction (PCR), includes agent isolates of the diseases from tomato plants that random amplified polymorphic DNA, amplified fragment showed typical wilting or crown and root rot symptoms length polymorphism, simple sequence repeats, sequence were thus obtained through sample collections in tomato related amplified polymorphism, translation elongation greenhouses (Ozbay et al. 2004; Hibar et al. 2007). factor 1-α, and b-tubulin gene sequencing (Geiser et Symptoms caused by FORL include crown and root al. 2004; O’Donnell et al. 2009; Irzykowska et al. 2012). rot, with chocolate-brown discoloration of the vascular Although each technique provides a valuable view into system 25–30 cm above the soil line, brown rot of the the species, using PCR with primers specifically chosen cortex and xylem, and stunted and quickly wilted plants. for the taxonomy level is the most widely used approach Pinkish sporulation of the pathogen on exposed necrotic (Sambrook et al. 1989; Assigbetse et al. 1994; Darling and Brickell 1994; Kistler 1997; Summerell et al. 2001; Kuramae lesions on stem can be intensive (Figure 1). Symptoms of and Souza 2002; Hernandez 2004; Mulè et al. 2004). FOL include the yellowing of older leaves, often on only For this reason, the causal agents of the disease isolates one side of the plant. The leaflets on one side of a petiole were obtained for field investigation from tomato plants frequently turn yellow before those on the other side, and showing typical wilting or the symptoms of crown and browning of the vascular system is characteristic for the root rot from the protected tomato-growing areas of the disease, which can be used for its identification (Figure 2). eastern Mediterranean region of Turkey. The present 2.2. Identification based on morphological features study is focused on FORL and FOL distribution in tomato The 87 isolates of Fusarium oxysporum used in this greenhouses. The principal aims of the study were to study, each from different greenhouses, were recovered check specific primer sets for formae speciales and races from tomato plants showing typical FORL or FOL and of F. oxysporum discrimination, to compare the accuracy are listed in Table 3. The roots of the tomato plants were of morphological and molecular identification, and to washed thoroughly with tap water to remove adhering

a b c d

Figure 1. Disease symptoms of F. oxysporum f. sp. radicis-lycopersici: a, b) chocolate-brown discoloration of the vascular system and crown-root rot (Adana/AY-5 isolate, 2008); c, d) light orange-pink sporulation on stem of severely diseased tomato plant (Mersin/ KZ-3 isolate, 2007).

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region isolates, which were then stored at 4 °C for use in ab PCR studies (Booth 1971; Nelson et al. 1983). F. oxysporum isolates were also used for laboratory studies to determine the development of the colony, color, phialide, chlamydospore, and micro- and macroconidia in terms of microscopic features. Mycelium features were assessed on a PDA medium and carnation leaf agar (CLA) using light microscopy. For this purpose, isolates belonging to FORL and FOL race 3, found in 5 different regions as dominant, were identified by PCR. Each isolate was then planted in different PDA and CLA media, 10 dishes each, and incubated for 10 days at 25 °C. Following cd the incubation period, the isolates were identified based on their morphological characteristics and microscopic features (Booth 1971; Nelson et al. 1983; Burgess et al. 1994; Leslie and Summerell 2006). 2.3. DNA extraction and PCR assay Genomic DNA of F. oxysporum was isolated from single- spore fungal cultures growing on PDA medium using a Sigma DNA (G2N70-70 prep) miniprep extraction kit. The effectiveness of DNA isolation was checked in 0.8% agarose gel electrophoresis. To determine the formae speciales of FOL (race 1, 2, or 3) or FORL, PCR was Figure 2. Isolates obtained from tomato greenhouses in Mersin/ performed with the specific primer sets uni, sp13, sp23, Yenitaşkent province: a, b) symptoms of FORL, with strong and sprl as described by Hirano and Arie (2006). Primers wilting and brown discoloration at the soil line (YT-4 isolate); c, were synthesized by İontek (Adana, Turkey) (Table 1). The d) symptoms of FOL, with yellowing of the older leaves on one uni primer set amplifies 670–672 bp fragments from all side of the plant and the leaflets (YT-10 isolate). isolates (FOL and FORL). The sp13 primer set amplifies 445 bp fragments for FOL races 1 and 3, while the sp23 soil particles. To isolate the fungus, infected plant tissues primer set amplifies 518 bp fragments for FOL races 2 were first given a surface disinfection with 1.0% sodium and 3. With the sprl primer set, a fragment of 947 bp was hypochloride for 2–3 min. Next, disinfected tissues of amplified specifically from FORL but not from the FOL 3–4 mm in length were placed on petri dishes including isolates (Table 2). 2.0% potato dextrose agar (PDA) and incubated at 25 The PCR reaction mixture (25 µL) contained 18 µL °C for 5 days (Katan et al. 1991). Identification based of Master Mix (1.25 µL of 0.2 mM each dNTP mix, 5 µL on the morphological and cultural characterization of Taq (10X), 2 µL (2.5 mM) MgCl2, 0.2 µL (1 U) Taq DNA F. oxysporum isolates was conducted according to some polymerase, 9.55 µL distilled water), 1 µL (0.5 mM) each of characteristics of macroconidia, microconidia, phialides, the primers, and 5 µL of genomic DNA (10 ng). A GeneAmp existence of chlamydospore, and colony growth traits. A 9700 thermocycler (Eppendorf Mastercycler Gradient single-spore isolation on PDA was then made from all the PCR Thermocycler) was used for PCR amplifications.

Table 1. Specific primer sets of PCR analyses (Hirano and Arie, 2006).

Primer Sequence (5’3’) Fragment (bp) uni-f ATCATCTTGTGCCAACTTCAG 670–672 bp (FO) uni-r GTTTGTGATCTTTGAGTTGCCA sp13-f GTCAGTCCATTGGCTCTCTC 445 bp (FOL races 1 and 3) sp13-r TCCTTGACACCATCACAGAG sp23-f CCTCTTGTCTTTGTCTCACGA 518 bp (FOL races 2 and 3) sp23-r GCAACAGGTCGTGGGGAAAA sprl-f GATGGTGGAACGGTATGACC 947 bp (FORL) sprl-r CCATCACACAAGAACACAGGA

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Table 2. Assessment of F. oxysporum ID (FORL or FOL races) based on symptomatological estimation and PCR analyses based on the results of PCR with specific primer sets (Hirano is shown in Table 3. and Arie, 2006). The electrophoresis of PCR products obtained for isolates belonging to Erdemli (EM-2, EKY-3, EKR-3), Primer Sets Adanalıoğlu (AO-2, AO-22), Kazanlı (KZ-4), Tarsus (Tarsus-1), and Silifke (S-17) in Mersin and isolates from uni sprl sp13 sp23 Karataş (AK-1) and Yüreğir (AY-5) in Adana where FORL + + - - intensive tomato growing is practiced are given in Figures FOL race 1 + - + - 3–7. The PCR analyses of the EM-2 and EKR-3 isolates FOL race 2 + - - + belonging to the greenhouses in Erdemli indicated that the FOL race 3 + - + + EM-2 gave fragments with uni (672 bp) and sp23 (518 bp) specific primer sets for FOL race 2. EKY-3 isolates were The PCR conditions for all primers were set at the initial determined as FOL race 1 with specific primer sets uni denaturation temperature of 94 °C for 5 min, followed by (672 bp) and sp13 (445 bp) (Figure 4). 45 cycles of 94 °C for 1 min, annealing at 61 °C for 1 min, The tomato isolates from AO-2 greenhouses in and elongation at 72 °C for 2 min, with a final extension at Adanalıoğlu were observed to amplify fragments with uni 72 °C for 10 min (Hirano and Arie 2006). All PCR reaction (672 bp), sp13 (445 bp), and sp23 (518 bp), indicating FOL products were electrophoresed in a 2% agarose gel, stained race 3, and AO-22 isolates were identified as FORL based with ethidium bromide, and visualized under UV light. on PCR products obtained with uni and sprl primers (Figure 4). The KZ-4 isolate belonging to a Kazanlı tomato 3. Results greenhouse in the province of Mersin formed specific The PCR analyses of the formae speciales and races of 87 fragments with uni, sp13, and sp23 specific primers, Fusarium oxysporum isolates obtained from the Adana indicating FOL race 3. The Tarsus-1 isolate obtained from and Mersin greenhouse tomatoes were found to be the greenhouses in Tarsus gave PCR products with uni and accurate and reliable. The isolates of the whole region were sprl primers and was identified as FORL (Figure 5). analyzed with the uni primer set, confirming the presence The S-17 isolate of Silifke formed fragments on uni, of F. oxysporum, and the sprl primer set, confirming the sp13, and sp23 primers and was identified as FOL race presence of FORL (Figure 3). Isolates for which specific 3. The AY-5 isolate of the Adana/Yüreğir greenhouse and fragments did not amplify with the sprl primer were the EKH-23 isolate of the Mersin-Erdemli Kocahasanlı amplified by sp13 (FOL races 1 and 3) and sp23 (FOL races greenhouse were detected to be FORL, forming fragments 2 and 3) primer sets. Identification of regional isolates with the uni and sprl primers (Figure 6). The EÜ-1 isolate from Üçtepe, Erdemli, was observed to form fragments on M 1 2 3 4 5 6 M the uni, sp13, and sp23 primers and was identified as FOL race 3; the S-14 (from Silifke) isolate was observed to form fragments on the uni and sprl primers and was identified 1000 bp as FORL; and the S-28 (from Silifke) isolate amplified 947 bp 672 bp fragments with uni and sp23 primers and was identified as 500 bp FOL race 2 (Figure 7). As a result of this study, among the 87 isolates, 60% (52 isolates) were determined to be FORL and 40% (35 isolates) to be FOL. The isolates of FOL were divided as follows: 34% (30 isolates) FOL race 3, 5% (4 isolates) FOL race 2, and 1% (1 isolate) FOL race 1 (Table 3). According to PCR results, among the 13 isolates from Adana (15%), 7 were identified as FORL and 6 as FOL race 3 (Figures 5 and 6). On the other hand, among the 74 isolates from Mersin (85%), 45 isolates were determined to be FORL, 24 isolates Figure 3. Electrophoretic separation of PCR products using FOL race 3, 4 isolates FOL race 2, and 1 isolate FOL race the uni and sprl primers to determine F. oxysporum isolates in 1 (Table 1; Figure 8). FORL and FOL race 3 are thus the tomato plants greenhouses in Adana/Karataş (AK-1 isolate), dominant pathogens in this region. The most infestations Erdemli/Kocahasanlı (EKH-2 isolate), and Erdemli/Türbe (ET-3 of FOL race 3 were found in Erdemli (10 isolates), Silifke isolate). M: molecular marker (100 bp); 1, 2: EKH-2 isolate; 3, 4: (5 isolates), and Yenitaşkent and Ceyhan (3 isolates each). ET-3 isolate; 5, 6: AK-1 isolate; 1, 3, 5: uni primer (672 bp); 2, 4, In contrast, FORL was found most often in Erdemli (12 6: sprl primer (947 bp).

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Erdeml Erdeml Adanalıoğlu Adanalıoğlu EKY-3 EM-2 AO-2 AO-22 FOL 1 FOL 2 FOL 3 FORL

M uni sp13 sp23 sprl uni sp13 sp23 sprl uni sp13 sp23 sprl uni sp13 sp23 sprl M 947 bp 672 bp 518 bp 445 bp

Figure 4. Agarose gel electrophoresis PCR products using 4 specific primer sets to detect F. oxysporum isolates. M: molecular marker (100 bp); EKY-3: FOL race 1; EM-2: FOL race 2; AO-2: FOL race 3; AO-22: FORL; uni primer: 672 bp; sp13 primer: 445 bp; sp23 primer: 518 bp; sprl primer: 947 bp. isolates), Adanalıoğlu (11 isolates), and Silifke (9 isolates). the lower surface, a prominent dark violet was especially Some differences were observed between the observed (Figure 10). FOL race 3 in PDA media was identification results based on the symptomatological determined to be white in the upper surface of colonies assessment and the results from PCR. In the investigations and mostly white and slight pale pink in the lower surface based on symptomatological and morphological features, (Figure 11). Colonies of FORL isolates in CLA media had 61 of the 87 isolates (70%) were identified as FORL and 26 macroconidia lengths of 32.5–57.5 µm and microconidia (30%) as FOL, while PCR results indicated that 52 (60%) lengths of 12–20 µm, while those belonging to race FOL of the isolates were FORL and 35 (40%) were FOL (Figure 3 isolates had macroconidia of 27.5–50 µm long and 9). The macroscopic and microscopic identification of microconidia of 10.5–15 µm long (Booth 1971; Nelson FORL and FOL race 3, belonging to 5 different locations et al. 1983; Windels 1992; Summerell et al. 2001; Balali in the region as the dominant species, were made in PDA and Iranpoor 2006; Leslie and Summerell 2006; Ciampi et and CLA media. The colony colors of the FORL isolates al. 2008). Two forms of F. oxysporum displayed the same were identified on the upper surface as white and light morphological features in the attempt to identify their to dark violet colors in PDA. However, in the center of macroscopic and microscopic properties (Table 4).

M 1 2 3 4 5 6 7 8 M M 1 2 3 4 5 6 7

947 bp 947 bp 672 bp 672 bp 518 bp 445 bp 518 bp 445 bp

Figure 5. Agarose gel electrophoresis PCR products in region of Figure 6. Agarose gel electrophoresis PCR products in region of Mersin/Kazanlı (KZ-4), Mersin/Erdemli (EKR-3), and Mersin/ Silifke (S-17), Adana/Yüreğir (AY-5), and Kocahasanlı/Erdemli Tarsus (Tarsus-1). M: molecular marker (100 bp); 1, 2, 3: KZ-4 (EKH-23). M: Molecular marker (100 bp); 1, 2, 3: S-17 isolate in isolate in FOL race 3 (1: uni, 2: sp13, 3: sp23); 4, 5, 6: EKR-3 FOL race 3; 4, 5: AY-5 isolate in FORL; 6, 7: EKH-23 isolate in isolate in FOL race 2 (4: uni, 5: sp13, 6: sp23); 7, 8: Tarsus-1 FORL; 1, 4, 6: uni primer (672 bp); 2: sp13 primer (445 bp); 3: isolate in FORL (7: uni, 8: sprl). sp23 primer (518 bp); 5, 7: sprl primer (947 bp).

461 ÇOLAK and BİÇİCİ / Turk J Agric For

M 1 2 3 4 5 6 7 8 9 10 M

947 bp 672 bp 518 bp 445 bp

Figure 7. Agarose gel electrophoresis PCR products in the region of Erdemli/Üçtepe (EÜ-1) and Silifke (S-14 and S-28). M: molecular marker (100 bp); 1, 2, 3: EÜ-1 isolate in FOL race 3; 3, 4: S-14 isolate in FORL; 6, 7, 8: S-28 isolate in FOL race 2; 9: healthy control; 10: DNA-free; 1, 4, 6: uni primer (672 bp); 2, 7: sp13 primer (445 bp); 3, 8: sp23 primer (518 bp); 5: sprl primer (947 bp).

4. Discussion diagnosis of the disease as a result of this method will be In the present study, the identification of 87 F. oxysporum quicker and easier. isolates originating from tomato greenhouses in Results obtained indicated that all FORL and FOL the provinces of Adana and Mersin in the eastern isolates were highly pathogenic on tomato plants in the Mediterranean region using symptomatological and PCR area. In the present study, of the 13 F. oxysporum isolates methods was undertaken. obtained from the greenhouses in the province of Adana, As a result of this study, FORL and FOL race 3 were, 7 isolates were identified as FORL and the remaining 6 as for the first time, identified through a symptomatological FOL, while of the 74 F. oxysporum isolates obtained from and molecular method (PCR) to be the most common the greenhouses in the province of Mersin, 45 isolates were pathogens in tomato greenhouses in this region. identified as FORL and the remaining 29 as FOL. The fact Differences were observed in diagnosis conducted with that the wastes of harvest are dumped into the irrigating conventional methods and the PCR results. These 2 races canals around the greenhouses in Adanalıoğlu, Yenitaşkent, are difficult to differentiate morphologically. Furthermore, and Kocahasanlı, districts of Mersin where covered since these races may cause disease symptoms on the same tomato growing is intensively practiced, the same diseases host tomato plant at the same time, they are sometimes have frequently been encountered in nearby greenhouses. confused with each other. The previously conducted race For this reason, the probability that F. oxysporum, which and vegetative conformity group (VCG) identification has the capacity to stay alive in soil and in plant wastes has not been sufficient in determining these F. oxysporum in the form of chlamydospores for many years, will cause forms. Particularly in VCG identification, while some problems in these areas and lead to huge economic losses is formae speciales conform to only one VCG, others may high. Poor conditions in greenhouses; problems in heating; belong to many VCGs. For this reason, although VCG the accumulation of water inside greenhouses; the fact that identification may be beneficial, it cannot be utilized plant wastes from the soil in greenhouses are not removed as a universal method in identifying formae speciales efficiently and become a source of inoculum when they or isolates that are not pathogenic (Attitalla et al. 2004). are dumped around greenhouses; growing of the same However, due to the greater accuracy and reliability of type of tomato in the same greenhouse every year; poor PCR for identification purposes, putting into practice the practices in the disinfection of greenhouse construction, which cause FORL microconidia to proliferate; and the 50 occurrence of repeated infections are particularly thought Adana 40 to be the major causes of the increase in FORL infections. 30 Mers n Another reason why FORL was increasing in the region was that other plant families in addition to Solanaceae were 20 observed as hosts (Menzies et al. 1990). Furthermore, a 10 resistant variety of tomato has not been developed against 0 FORL yet and the widely accepted and commercially FOL 1 FOL 2FOL 3FORL resistant varieties of tomato are not sufficiently able to Figure 8. Results of molecular identification of F. oxysporum fight off FORL. For this reason, urgent measures must be isolates originating from Adana and Mersin provinces. taken to prevent the spread of the disease in the provinces

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Table 3. Formae speciales and races of F. oxysporum separation obtained by symptomatological assessment and PCR analyses.

Symptomatological Isolate name Location Results of PCR FOL or FORL AS-1 Seyhan/Adana Forl Forl AS-3 Seyhan/Adana Forl Forl AS-6 Seyhan/Adana Fol Fol 3 AY-2 Yüreğir/Adana Forl* Fol 3* AY-4 Yüreğir/Adana Fol Fol 3 AY-5 Yüreğir/Adana Forl Forl AK-1 Karataş/Adana Forl Forl AC-2 Ceyhan/Adana Forl Forl AC-3 Ceyhan/Adana Fol Fol 3 AC-5 Ceyhan/Adana Fol Fol 3 AC-6 Ceyhan/Adana Forl Forl AC-7 Ceyhan/Adana Forl Forl AC-9 Ceyhan/Adana Fol Fol 3 AO-1 Adanalıoğlu/Mersin Forl Forl AO-2 Adanalıoğlu/Mersin Forl Fol 3 AO-3 Adanalıoğlu/Mersin Forl Forl AO-4 Adanalıoğlu/Mersin Forl Forl AO-5 Adanalıoğlu/Mersin Forl Forl AO-6 Adanalıoğlu/Mersin Forl Forl AO-7 Adanalıoğlu/Mersin Forl Forl AO-8 Adanalıoğlu/Mersin Forl Forl AO-9 Adanalıoğlu/Mersin Forl Forl AO-12 Adanalıoğlu/Mersin Forl Forl AO-13 Adanalıoğlu/Mersin Forl Forl AO-16 Adanalıoğlu/Mersin Fol Fol 3 AO-22 Adanalıoğlu/Mersin Forl Forl KZ-1 Kazanlı/Mersin Forl Forl KZ-2 Kazanlı/Mersin Forl Forl KZ-3 Kazanlı/Mersin Forl Forl KZ-4 Kazanlı/Mersin Forl Fol 3 KZ-5 Kazanlı/Mersin Forl Forl YT-1 Yenitaşkent/Mersin Fol Fol 3 YT-2 Yenitaşkent/Mersin Forl Forl YT-4 Yenitaşkent/Mersin Forl Forl YT-5 Yenitaşkent/Mersin Forl Forl YT-8 Yenitaşkent/Mersin Fol Fol 3 YT-10 Yenitaşkent/Mersin Fol Fol 3 YT-12 Yenitaşkent/Mersin Forl Forl YT-13 Yenitaşkent/Mersin Forl Forl EM-2 Merkez/Erdemli Fol Fol 2 ET-1 Türbe/Erdemli Fol Fol 3 ET-3 Türbe/Erdemli Forl Fol 3 ET-4 Türbe/Erdemli Forl Forl

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Table 3. (continued).

ET-7 Türbe/Erdemli Forl Forl EKY-1 Koyuncu/Erdemli Forl Forl EKY-3 Koyuncu/Erdemli Fol Fol 1 EKY-4 Koyuncu/Erdemli Forl Forl EKY-5 Koyuncu/Erdemli Forl Forl EKY-6 Koyuncu/Erdemli Fol Fol 3 EKH-2 Kocahasanlı/Erdemli Forl Fol 3 EKH-3 Kocahasanlı/Erdemli Fol Fol 3 EKH-3 Kocahasanlı/Erdemli Fol Fol 3 EKH-4 Kocahasanlı/Erdemli Forl Forl EKH-12 Kocahasanlı/Erdemli Fol Fol 3 EKH-14 Kocahasanlı/Erdemli Fol Fol 3 EKH-18 Kocahasanlı/Erdemli Fol Fol 3 EKH-19 Kocahasanlı/Erdemli Forl Fol 3 EKH-22 Kocahasanlı/Erdemli Forl Forl EKH-23 Kocahasanlı/Erdemli Forl Forl EKH-24 Kocahasanlı/Erdemli Forl Forl EY-1 Yüksek/Erdemli Forl Forl EY-2 Yüksek/Erdemli Forl Forl EY-3 Yüksek/Erdemli Forl Forl EÜ-1 Üçtepe/Erdemli Fol Fol 3 EÇ-1 Çiriş/Erdemli Forl Fol 3 EÇ-2 Çiriş/Erdemli Forl Forl EKR-1 Karakeşli/Erdemli Fol Fol 3 EKR-3 Karakeşli/Erdemli Fol Fol 2 Tarsus-0 Tarsus/Mersin Forl Forl Tarsus-1 Tarsus/Mersin Forl Forl Tarsus-4 Tarsus/Mersin Fol Fol3 Tarsus-6 Tarsus/Mersin Forl Forl S-2 Atik/Silifke Forl Forl S-4 Atik/Silifke Forl Forl S-6 /Silifke Forl Forl S-7 Arkum/Silifke Forl Forl S-9 Arkum/Silifke Forl Forl S-10 Arkum/Silifke Forl Forl S-13 Arkum/Silifke Forl Forl S-14 Atayurt/Silifke Forl Forl S-17 Atayurt/Silifke Forl Fol 3 S-20 Atayurt/Silifke Forl Forl S-22 Atayurt/Silifke Forl Fol 3 S-24 Kabasakallı/Silifke Fol Fol 2 S-28 Kurtuluş/Silifke Fol Fol 2 S-29 Kurtuluş/Silifke Fol Fol 3 S-34 Karadereli/Silifke Fol Fol 3 S-35 Karadereli/Silifke Fol Fol 3

*Isolates identified differently by symptomatological and PCR results are given in bold.

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70 Symptomatolog cal 61 60 52 PCR 50 40 35 30 26 20 10 a b 0 FOL FORL Figure 10. F. oxysporum f. sp. radicis-lycopersici (AK-1 isolate) Figure 9. The results of symptomatological and PCR colony growth in PDA medium: a) colony growth of upper identification of isolates from screened region of Turkey. surface in PDA, b) colony growth of lower surface in PDA.

Table 4. Morphological characteristic features of FORL and FOL race 3 in PDA and CLA media.

Parameters FORL FOL race 3

Upper surface: white – light to dark violet Upper surface: white Color of colony (PDA) Lower surface: dark violet Lower surface: white and slightly pale pink Abundant, More than macroconidia, Features of microconidia (CLA) oval-ellipsoid, nonseptate, oval-ellipsoid, nonseptate, 12–20 µm 10.5–15 µm Abundant, Less than microconidia, 4–5 septa 3–4 septa, rarely 5 septa, Features of macroconidia (CLA) 32.5–57.5 µm 27.5–50 µm, Apical cell: slightly curved-hooked apical cell and basal cell the same as in FORL Basal cell: foot-shaped Features of chlamydospore (CLA) Globose, abundant, single or pairs Globose, abundant, single or pairs Features of phialide (CLA) Short monophialide Short monophialide

a b c

Figure 11. Colony growth of F. oxysporum f. sp. lycopersici races in PDA medium: a) FOL race 1 (EKY-3 isolate); b) FOL race 2 (EKR-3 isolate); c) FOL race 3 (S-17 isolate). of Adana and Mersin. Dealing with the wilt disease caused Consequently, the development and utilization of the by FOL could be possible through developing resistant tomato varieties resistant to this specific form and race varieties of tomato and improving disinfection practices identified in this region will be more appropriate. Today, of the soil. Moreover, if irrigation, fertilization, and similar breeding programs are mostly focused on developing cultivation practices were improved, the disease could resistant varieties, since this method is the most effective in be kept within tolerable limits (Yücel and Çınar 1989; dealing with the diseases that cause huge economic losses. Takahashi et al. 2005). However, as mentioned in the In this respect, the identification of formae speciales and present study, since the FOL 3 race is dominant in the races of F. oxysporum in the greenhouses in the eastern region, growing the varieties of tomato that are resistant to Mediterranean area will promote the development of the this disease will particularly decrease the rate of infection. varieties of tomato that are resistant to these diseases.

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