The Use of an Alternative Differential Set for Determination of Pyrenophora teres f. maculata Pathotypes

Pyrenophora teres f. maculata incites spot form of barley net blotch disease. For determination of Pyrenophora teres f. maculata pathotypes, a differential set consisted of 22 international cultivars and genotypes and a susceptible Turkish barley variety Bülbül 89 were tested using 45 isolates obtained from different regions of Turkey. Nineteen pathotypes were determined out of 45 isolates used. It appears that this differential set could be useful for determination of P. teres f. maculata pathotypes.


Introduction
Barley net blotch disease caused by the fungus Pyrenophora teres (anamorph: Drechslera teres) is a common and important disease which lowers the yield and quality of the barley in the world (Mathre 1982;McLean et al 2009;Karakaya et al 2014). Pyrenophora teres has two biotypes. P. teres f. maculata and P. teres f. teres incite spot and net forms of net blotch disease, respectively (Liu et al 2011). Resistant cultivars are preferred in disease control. However, pathotypes of the fungus complicate the resistance studies. In order to control the pathogen, information about the pathotypes of the fungus is necessary. For pathotype determination studies, different researchers used different cultivars and genotypes. However, most of the time, comparison of these pathotypes were difficult (Wu et al 2003;Grewal et al 2008;Boungab et al 2012;McLean et al 2014a;McLean et al 2014b). This study aimed at contributing to development of an international set for determination of Pyrenophora teres f. maculata (Ptm) pathotypes. each location were considered. In surveys, systematic sampling method was used (Aktaş 2001). Samples were obtained from a diverse set of provinces. Leaves showing characteristic spot form of net blotch symptoms were selected. These leaves were subjected to surface sterilization using 1% NaOCl for 1 minute and they were kept in blotter for 4-5 days. Under a stereomicroscope, single spores were taken and transferred to Potato Dextrose Agar plates. From diseased barley and wild barley (Hordeum spontaneum) plants 45 Ptm single spore isolates were obtained. Typical Pyrenophora teres f. maculata conidia were observed in a light microscope. Symptom morphologies of these isolates were verified using the susceptible barley cultivar Bülbül 89 (Mathre 1982;Çelik Oğuz & Karakaya 2017 Under greenhouse conditions, differential set genotypes were planted in plastic pots, 7 cm in diameter, containing topsoil. Each pot contained 5-10 seeds. There were three replications arranged in a completely randomized fashion. Ten days old cultures grown on Potato Dextrose Agar were used as inoculum. Fungal cultures were scraped using a paintbrush and washed through cheesecloth with water. Inoculum which consisted of mycelium pieces, was adjusted to 1.5-2.0x10 5 mycelium parts per mL. For each 100 mL of inoculum suspension, one drop of Tween 20 was added (Aktaş 1995). Inoculation of the barley seedlings were performed at the two to three leaf stages (Z12-13; Zadoks et al 1974). Fungal suspensions were sprayed onto barley differential set seedlings. Inoculated plants were kept in closed transparent lid boxes covered by transparent nylon covers for 72 h in a greenhouse at high humidity. The nylons were then removed and ventilation lids were opened for another 24 h. The temperature of the greenhouse was 18±1-23±1 o C during night and day with a 14h/10h light/dark period. Following this period, box lids were opened. Seven days after inoculation, barley seedlings were assessed for disease severity using the spot form scale described by Tekauz (1985).
For pathotype determination, methods outlined in Wu et al (2003) and Çelik Oğuz & Karakaya (2017) were used. Seven days later following inoculation, plants were evaluated using a 1-9 scale developed by Tekauz (1985). For evaluation, second leaves were used. Scale values between 1-5 and 6-9 were considered as resistant and susceptible, respectively. Differential test genotypes were numbered 1 through 23 and pathotypes were determined according to their responses to these differential set genotypes. For example, isolate PTM 42 from Yozgat province showed susceptible reactions (>5) on genotypes 13 (Galleon), 18 (Steptoe) and 19 (Stirling) and showed resistant reactions (≤5) on the other differential set genotypes. Therefore, this pathotype was named as 13-18-19. Isolates exhibiting resistant reactions (≤5) to all differential test genotypes were termed as pathotype 0.
In Turkey, Çelik Oğuz & Karakaya (2017) used 25 differential set genotypes. From a total of 50 isolates, 26 Ptm pathotypes were determined. In our current study, we used 23 differential set genotypes and from a total of 45 isolates, 19 pathotypes were distinguished. Karki & Sharp (1986) used a differential set which consisted of 20 genotypes. In their study, 6 groups were evident among the 14 isolates used. Gupta et al (2012) used a differential set which consisted of 26 genotypes. In their study, 7 groups were found among the 49 isolates used.
In our current study, differential genotypes Chebec, CI5286, CI7584, CI9819 and CI16150 exhibited resistant reactions to 43 isolates (95.5%) and susceptible reactions to 2 isolates. Karki & Sharp (1986)        Genotypes CI3576, CI9214, CI9776 and Torrens exhibited resistant reactions to 91% of the isolates in our current study. These genotypes showed susceptible reactions to 4 isolates. Akhavan et al (2016) reported that CI9214 genotype was resistant to all Ptm isolates except two. In another study, genotypes CI9214 and CI9776 showed a resistant reaction to all isolates used (Karki & Sharp 1986) In our current study, Arimont, CI5791, Skiff and TR250 genotypes exhibited resistant reactions to 42 isolates (93%). These genotypes showed susceptible reactions to 3 isolates. Akhavan et al (2016) reported the virulence of 19 (70.4%) Ptm isolates on genotype CI5791. Cultivar Arimont was reported as susceptible in a previous study (Karki & Sharp 1986). Cultivar Skiff was reported as generally moderately resistant and genotype TR250 was reported as moderately susceptible (McLean et al 2012).
In our current study, cultivar Steptoe was susceptible to 40% of the isolates. In Akhavan et al (2016) study, this cultivar was susceptible to 81.5% of the isolates.
In the current study, cultivars Steptoe and Bülbül 89 exhibited susceptible reactions to 18 and 19 isolates, respectively. These cultivars were the most susceptible cultivars. Cultivar Bülbül 89 could be used as universal susceptible genotype in an international Ptm differential set. The genotypes used in this study were useful in differentiating Ptm pathotypes.

Conclusions
For determination of Pyrenophora teres f. maculata pathotypes, a differential set consisted of 22 international cultivars and genotypes and a susceptible Turkish barley variety Bülbül 89 were tested using 45 isolates obtained from different regions of Turkey. Nineteen pathotypes were determined out of 45 isolates used. Cultivar Bülbül 89 could be used as universal susceptible genotype in an international Ptm differential set. The genotypes used in this study were useful in differentiating Ptm pathotypes.