Vegetative Compatibility Groups and Pathogenicity of Verticillium dahliae Isolates from Potato Plants in Erzurum Province ı n ı n Vejetatif Uyum Gruplar ı ve Patojeniteleri

One hundred eleven isolates of Verticillium dahliae were obtained from potato plants in Erzurum province, Turkey. The pathogen was isolated from 7.6% of the stems collected. All isol ates were assigned to vegetative compatibility groups (VCGs) using nitrate-nonutilizing ( nit ) mutants. In total, 240 nit mutants were obtained from V. dahliae isolates, and classified as nit 1 (71%) and nit M (29%). Two VCGs were found and identified as VCG 2B (34 isolates) and VCG 4B (77 isolates) by using tester isolates of known VCGs. Pathogenicity of V. dahliae isolates was tested on potato (cv. Marfona) by the root-dip method. Both VCG 2B and VCG 4B isolates showed similar aggressiveness on potato. This is the first study of VCGs of V. dahliae isolates from potato plants in Turkey.


Introduction
Verticillium dahliae Kleb. is a soilborne plant pathogen responsible for severe damage on many crop species including potato (Pegg & Brady 2002). Potato is an important crop in Erzurum province, where more than 3,000 ha of potatoes are planted. Verticillium wilt on potato caused by V. dahliae has been a serious problem in this area recently. On potato, this fungus causes early senescence of plants and a light brown discoloration in TARIM BİLİMLERİ DERGİSİ  JOURNAL OF AGRICULTURAL SCIENCES 18 (2012) 110-114 18 (2012) [110][111][112][113][114] 111 the vascular ring of tubers (Rich 1986), and it can cause a reduction of both yield and quality. Many weed species also have been reported as hosts of V. dahliae (Pegg & Brady 2002;Ligoxigakis et al 2002). The pathogen has been isolated from five weed species in potato fields in Erzurum (Demirci & Genc 2009).
The aim of this research was to determine the VCGs of V. dahliae isolates from potato plants in Erzurum, Turkey and to investigate the pathogenicity of these isolates on potato.

Isolation of V. dahliae from potato plants
Potato plants generally showing wilt symptoms were collected from 9 locations (Table 1) in Erzurum province between August and September in 2003-2005 growing seasons. Plants were washed with tap water, and then stem sections 1 cm long were excised from potato plants. The tissue sections were surface disinfected with 0.5% sodium hypochlorite solution for 1 min, rinsed with sterile distilled water, dried on sterile filter paper and placed on water agar (WA, 2%) amended with 100 mg L -1 streptomycin sulfate in Petri plates. Plates were incubated at 24 °C in the dark for 7 days until verticillately branched conidiophores formed around the stem sections. Emerging fungi were subcultured on potato dextrose agar (PDA). Singlespore isolates of V. dahliae were obtained, identified as described previously (Hawksworth & Talboys 1970;Goud et al 2003), and maintained on PDA medium in tubes at 5 °C.

Generation and characterization of nit mutants
Nit mutants of V. dahliae were generated on cornmeal agar with 0.02% glucose amended with 3% potassium chlorate (CMC) as described previously (Korolev & Katan 1997). Mycelial discs (5 mm diam.) of V. dahliae isolates were removed from the margin of each actively growing colony on PDA and placed on CMC at six separate points in 9 cm diameter Petri plates. Plates were incubated in the dark at 24 ºC for 2-4 weeks. Chlorate-resistant sectors were transferred to Czapex-Dox Agar (CDA) plates. Sectors that grew on CDA as thin expansive colonies with no aerial mycelium were considered nit mutants.
CDA amended with sodium nitrite (0.5 g L -1 ) or hypoxanthine (0.2 g L -1 ) was used for partial phenotyping of the nit mutants (Correll et al 1987). Mutants that grew profusely on sodium nitrite and hypoxanthine were classified as nit1, whereas mutants that grew profusely on sodium nitrite but sparsely on hypoxanthine were classified as nitM. Phenotypically distinct mutants were placed 1.5 cm apart on CDA in 9 cm diameter Petri plates and incubated at 24 ºC for 2-4 weeks. Complementation was evident by the development of prototrophic growth where two mutant colonies met and formed a stable heterokaryon (Bao et al 1998). The degree of complementation was ranked as follows: (+) = dense prototrophic growth, (+/-) = small microsclerotial dots with or without a little aerial mycelium, (-) = prototrophic growth absent or inconspicuous (Korolev et al 2000). Each pairing was repeated at least twice. When mutants of two isolates formed a heterokaryon, their parents were assigned to the same VCG.

Pathogenicity of V. dahliae isolates on potato
Pathogenicity of 10 isolates of each VCG, selected at random on the basis of geographical origin, was determined on potato plants (cv. Marfona) by the rootdip method. This cultivar has been grown in Erzurum for a long time. Surface-disinfected potato tubers (1 min in 2% formaldehyde and rinsed in sterile distilled water) were planted in 15 cm diameter pots containing a sterile soil mix of topsoil and sand (1:1, v/v) in a growth chamber. After 5 weeks, 10 to 20 cm tall plants were selected for inoculation (Joaquim & Rowe 1991). The isolates were grown on PDA (9-cm plates) at 24°C in the dark for 10 days. Conidia were washed off the agar surface with sterile distilled water, and the inoculum density adjusted to 10 6 conidia mL -1 with a hemacytometer and sterile distilled water (Strausbaugh 1993

Isolates of V. dahliae from potato plants
Stem samples were collected from potato fields in Erzurum province. The number of potato stems excised from plants onto culture media in the laboratory totaled 1460 during the 3 years, and V. dahliae was isolated from 7.6% of the stems examined. Totally, 111 isolates of V. dahliae were obtained from potato stems from 9 locations ( Table 1). Most of these isolates were collected from Pasinler.

Generation and characterization of nit mutants
In total, 240 nit mutants were obtained from 111 isolates of V. dahliae, ranging from 1 to 7 mutants per isolate. Nit mutants were identified based on their phenotype; 171 mutants were classified as nit1 and the remainder as nitM (

Vegetative compatibility grouping
The genetic diversity among one hundred eleven V. dahliae isolates was determined. After complementation with the tester isolates of known VCGs, 34 isolates were assigned to VCG 2B, and 77 to VCG 4B (Table 1). Isolates assigned to VCG 2 showed strong complementation only with tester isolates of VCG 2B. Cross-reactions occurred between isolates VCG 4 from potato plants and tester isolates of VCG 4 (subgroups A and B), VCG 4 isolates showed strong complementation (+) with the tester isolates of VCG 4B, but all were also weakly compatible (+/-) with the tester isolates of VCG 4A. Unfortunately a mistake was made on the determination of the VCG of a number of isolates of V. dahliae from potato plants (Dane 2007). In this thesis, isolates of V. dahliae were typed as VCG 4A due to problems with some tester isolates. After we were aware of this problem, new VCG tester isolates [VCG 4A (BB, P103) and VCG 4B (S-39, MT)] of V. dahliae were provided by Dr. M. M. Jimenez-Gasco. The vegetative compatibility of isolates previously typed as VCG 4A was re-evaluated. All nit mutants of VCG 4 obtained in this study were paired with nit mutants of new tester isolates of VCG 4A and 4B. Eventually, all VCG 4 isolates from potato plants in Erzurum were re-classified as VCG 4B in this article.
Both VCG 2B and VCG 4B isolates were identified from Center, A ş kale, Köprüköy and Pasinler. Only VCG 2B isolates were identified from İspir and 18 (2012) 110-114 113 Narman, and only VCG 4B isolates were identified from Horasan, Ilıca and Tortum. Both VCG 2B (Zeise & Tiedemann Von 2002) and VCG 4B (Joaquim & Rowe 1991;Strausbaugh 1993;Korolev et al 2000;Tsror et al 2001;Zeise & Tiedemann Von 2002) isolates have been reported before on potato. In a study from Erzurum, V. dahliae isolates from some of the common weeds in potato fields also were assigned to VCG 2B and VCG 4B (Demirci & Genc 2009;2011). The results suggest that the population of V. dahliae isolates from potato plants and weeds in potato fields is the same. This is the first study of vegetative compatibility of V. dahliae isolates from potato plants in Turkey.

Pathogenicity of V. dahliae isolates on potato
The pathogenicity of twenty isolates representing VCG 2B and VCG 4B was determined on potato plants by the root-dip method. Potato plants all exhibited typical Verticillium wilt symptoms in response to inoculation with the tested V. dahliae isolates. Disease symptoms were visible 5-6 weeks after inoculation and developed over time from chlorosis to necrosis and wilting. When stem sections of these plants were dissected, they all showed vascular discoloration. Based on the results of pathogenicity tests, all isolates were pathogenic on potato at various levels of aggressiveness (Table 2). Disease severity ranged from 1.5 to 2.8 for VCG 2B isolates, and from 2.0 to 2.5 for VCG 4B isolates. No significant differences ( F 4,80 =0.66, P=0.42) were observed among the tested VCG 2B and 4B for disease severity. Control plants showed no disease symptoms. V. dahliae was recovered from all the inoculated plants but not from control plants.
The present study showed that most isolates of VCG 2B and VCG 4B from potato plants were highly aggressive on potato cv. "Marfona", and there was no difference between the isolates for disease severity.
Disease severity of potato plants infected with V. dahliae isolates from weed species in Erzurum ranged from 1.6 to 2.3 for both VCG 2B and VCG 4B isolates (Demirci & Genc, 2009& 2011. However, symptom severity was significantly higher in potato plantlets inoculated with VCG 4B than VCG 2A and VCG 2B (Tsror et al 2001). In other researches, VCG 4A isolates were more virulent on potato than VCGs 2 and 4B (Joaquim & Rowe 1991) or VCGs 4B and 4A/B isolates (Strausbaugh 1993). Rotation is one of the major components of Verticillium wilt management. However, many field crops, vegetables and weeds are susceptible to the pathogens causing this disease. In addition, microsclerotia produced by V. dahliae in the dying tissues of the infected plant can survive in the soil for many years (Pegg & Brady 2002). Moreover, one study has reported that formation of microsclerotia in senescent tissues of infected weeds could be an important factor in the failure of rotation programs to control V. dahliae effectively (Johnson et al 1980). Another study showed that some of the common weeds in potato fields can act as potential hosts of V. dahliae and potentially play important role in the survival of this pathogen (Demirci & Genc 2009). Therefore, weed control in potato fields is also very important.

Conclusion
The results of this study show that V. dahliae isolates from potato in Erzurum were classified as VCG 2B and VCG 4B. There are no statistically significant differences between VCG 2B and 4B isolates on disease severity.