DOI: 10.2478/s11535-006-0027-1 Research article CEJB 1(3) 2006 399–411

Distribution and characterization of Aegilops and Triticum species from the Bulgarian Black Sea coast

Penko Spetsov1∗, Dragomir Plamenov2, Vanya Kiryakova1

1 Dobroudja Agricultural Institute, 9520 General Toshevo, Bulgaria 2 Pedagogical College – Dobrich 9300, Bs. Konstantin Preslavsky University, Shumen, Bulgaria

Received 30 May 2006; accepted 24 July 2006

Abstract: A total of 158 Aegilops-Triticum samples representing six Aegilops species (one diploid, four tetraploid and one hexaploid) and one diploid Triticum were collected along the Bulgarian Black Sea coast, and their distribution on the 350 km long coastal line was reported. The region south of Kamchia river, accepted as the middle point of the coast, was characterized by the greatest diversity of these wild relatives of wheat. The most widely distributed species in this area was Ae. geniculata. Ae. cylindrica was distributed only in north (Durankulak), while Ae. biuncialis and Ae. triuncialis were collected both north and south of Kamchia river. All samples of Ae. neglecta were hexaploid. Natural hybrids of goatgrass and wheat were found in Ae. cylindrica populations. Triticum monococcum ssp. aegilopoides had limited distribution in the south region. Aegilops uniaristata was recorded as a new species for the Bulgarian flora. Most of the samples expressed resistance to powdery mildew in seedling and adult stage, but all of them were polymorphic regarding the resistance to leaf rust (cultures 73760 and 43763). The study revealed additional data for the distribution of Aegilops and Triticum species in Bulgaria and their potential value as genetic resources in wheat improvement. c Versita Warsaw and Springer-Verlag Berlin Heidelberg. All rights reserved.

Keywords: Aegilops, Triticum monococcum, distribution, Bulgarian Black Sea coast, leaf rust, powdery mildew, resistance

1 Introduction

The Aegilops-Triticum group involves diploid, tetraploid and hexaploid species [1]. All cultivated forms of this group belong to the genus Triticum. Aegilops species are closely re-

∗ E-mail: [email protected] 400 P. Spetsov et al. / Central European Journal of Biology 1(3) 2006 399–411 lated to wheats, and include forms with different ploidy levels. The wild wheats T. mono- coccum L. ssp. aegilopoides (Link) Thell., T. urartu Tumanian ex Gandilyan (diploids), T. turgidum L. ssp. dicoccoides (K¨orn. ex Asch. & Graebn.) Thell., T. timopheevii (Zhuk.) Zhuk. ssp. armeniacum (Jakubz.) van Slageren (tetraploids) and their domes- ticated forms constitute the primary gene pool, while the species of Aegilops form the secondary gene pool of wheat [2, 3]. There have been large efforts to evaluate, document and store many Triticeae lan- draces and wild relatives for breeding purposes [4, 5]. As there is concern among breeders that the narrow genetic base of wheat might limit future breeding advances, there is an urgent need to explore the genetic diversity in natural populations of wild species and use those with valuable traits for wheat breeding. Nine Aegilops and one Triticum species are distributed in Bulgaria [6, 7]. Four of the Aegilops species (Ae. geniculata Roth, Ae. triuncialis L., Ae. cylindrica Host and Ae. neglecta Req. ex Bertol.) and T. monococcum ssp. aegilopoides are widespread in the country, while the remaining, Ae. speltoides Tausch, Ae. markgrafii (Greuter) Hammer, Ae. umbellulata Zhuk., Ae. columnaris Zhuk. and Ae. biuncialis Vis. are distributed in southern Bulgaria only, mainly in the Trakyiska lowland and in the Tundjanska hilly plain [8]. Bulgaria is one of the six countries bordering the Black Sea. The climate around the sea is mostly continental, with pronounced seasons, ranging from dry in the west to humid on the eastern end. The lake effect also influences the climate. The Bulgarian Black Sea coast is multiform (heterogeneous) due to many estuaries (five river mouths pour into the Black Sea) and small lakes (four lakes and one marshland are very close to the coast). Soil around the coastal line varies from chernozem to grey and limy. Wild plants can acclimate to such conditions by escape, avoidance, tolerance and other strategies [9, 10]. The information for the frequency and distribution of wild plant populations in the coastal areas is limited at present. The aim of this study was to investigate the wild relatives of wheat (Aegilops and Triticum) along the Bulgarian Black Sea coast: species diversity, distribution, natural wild x cultivated wheat hybrids, and disease resistance with emphasis on powdery mildew and leaf rust. Relationships among the species collected were also analysed.

2 Experimental procedures

2.1 Collecting sites

Sample collections in 2000-2001 followed the land road along the Black Sea coast (Fig. 1). The widest distances up to 2 km far from the coast were in the areas to the north of Kamchia river, while the collecting sites to the south followed the coastal line except for Burgas district. Sample collections spanned 350 km, from the north point Durankulak (43 ◦45’N, 28 ◦35’E) to the south point Rezovo (41 ◦59’N, 28 ◦02’E). The locations were between altitudes 0 m to 386 m, with the highest elevation being the region between P. Spetsov et al. / Central European Journal of Biology 1(3) 2006 399–411 401

Kamchia river and Burgas. Collecting was done in habitats as roadsides including dry grassy and stony places, pastures, meadows and wasteland.

2.2 Plant growth and measurements

The plant material studied represents 158 samples of Aegilops and Triticum species col- lected from 94 locations along the Black Sea coast (Table 1). Seeds were collected from each sample. Twenty seedlings per original sample were vernalised in a greenhouse from January to March. Six plants per sample were randomly selected and transplanted in 14 × 20-cm pots with soil (2 plants in a pot). They were included in a randomized de- sign involving 2 plants in a replication. Plants were grown in a non-heated greenhouse (61 samples in 2001 and 97 samples in 2002). Morphological characterization of the samples was based on seven quantitative char- acters: spike emergence (days to heading, from 1 May), plant height (cm), number of tillers per plant, spike length (cm), number of spikelets per spike, number of seeds per spike, and seeds weight per spike (g). STATISTICA cluster analysis module (version 5.0 for Windows 95) was used to construct a dendrogram using UPGMA (unweighted pair group method with arithmetic average).

2.3 Powdery mildew resistance

The resistance to powdery mildew, caused by Blumeria graminis f.sp. tritici was analysed at seedling and adult plant stages. Plants in the two and three leaf stages were evaluated in the greenhouse under natural infection of the pathogen. Common wheat variety Sadovska ranozreika-4 as spreader and control for high susceptibility to powdery mildew, was placed at several sites in the greenhouse. Status of pathogen infection was recorded twice, once at the two-leaf stage, and once at the three-leaf stage. The ratings on a scale 0-9 [11] were applied to record mildew coverage of each plant, as follows: 0 = immune, no visible sign of infection; 1-3 = resistant, increasing from no necrosis to large necrotic areas, and from no mycelium to little mycelium; 4-6 = moderately resistant, necrotic areas changing to chlorotic areas, increasing in amount of mycelium and conidiospores production; and 7-9 = susceptible, decreasing from chlorotic areas to no chlorosis, increasing in amount of mycelium and conidiospore production to complete susceptibility. Disease ratings were grouped into three classes. Class R (resistant) included ratings of 0-3, class I (moderately resistant) included ratings of 4-6, and class S (susceptible) included ratings of 7-9. Resistance of the mature plant stage was also evaluated in the greenhouse under natural disease epiphytotics. Common wheat variety Sadovska ranozreika-4 was also used as susceptible standard for comparison. The infection type (IT, 0-4) and the disease infection severity (DIS) were estimated according to Stakman et al. (1962) [12]. DIS is given in percentage from 0 to 100, where 0 indicates lack of infection, and 100 indicates maximum degree of infection. Disease was assessed as a visual estimate of the percentage leaf area covered by colonies of Blumeria graminis f.sp. tritici. The leaf areas analysed 402 P. Spetsov et al. / Central European Journal of Biology 1(3) 2006 399–411 involved the top three leaves of adult plants, on the upper surface at 10-day intervals between growth stages 51 and 61. Plants were designated as resistant (R) with IT=0-1 and DIS=0-20, intermediate (I) with IT=2-3 and DIS=25-50, and susceptible (S) with IT=4 and DIS=55-100.

2.4 Leaf rust resistance

The resistance to leaf rust, caused by Puccinia triticina was investigated in seedlings. Two cultures were used and characterized against 15 known monogenic seedling resis- tance genes: 73760 (avirulence/virulence Lr gene phenotype: 9, 19, 24, 26, 28/ 1, 2a, 2b, 2c, 3, 11, 15, 17, 21, 23) and 43763 (avirulence/virulence: 1, 2a, 9, 19, 28/ 2b, 2c, 3, 11, 15, 17, 21, 23, 24, 26). Evaluation of young plants (2-3 leaves stage) for seedling resistance was carried out after inoculation with urediospores [13]. The inoculated seedlings were incubated at 20 ± 1 ◦C for 24 h before moving to a greenhouse at 20-25 ◦C. Infection levels were recorded 11-15 days after inoculation according to a 0-4 scale [12] as follows: 0 = immune, no visible infection; 0; = no uredia, hypersensitive flecks present; 1 = resis- tant, minute non-sporulating uredia surrounded by distinct necrotic areas; 2 = resistant to moderately resistant, small uredia with slight sporulation; 3 = moderately resistant to moderately susceptible; 4 = susceptible, large sporulating uredia with no chlorosis or necrosis. Plants were classified as resistant (R) – 0-2 type reaction and susceptible (S) – 3-4 type reaction. The wheat variety Michigan Amber was the susceptible check.

2.5 Cytological analyses

Seeds from Ae. uniaristata and Ae. neglecta were only used in somatic chromosome analysis. Counts were made from squashes of root-tip cells pretreated with mono-bromo- naphtalene and stained by the Feulgen method.

3 Results and discussion

3.1 Distribution

A total of 158 samples of Aegilops and Triticum from 94 locations along the Black Sea coast were collected (Table 1). Six species of Aegilops, and one species of Triticum, T. monococcum ssp. aegilopoides, were identified. The most widely distributed species were Ae. triuncialis, Ae. cylindrica and Ae. bi- uncialis. These species were found in habitats on the north and south of Kamchia river estuary, accepted as a middle point of the coastal line (43 ◦02’N, 27 ◦53’E; Fig. 1). Only Ae. cylindrica was found at the northernmost point at Durankulak. All the seven species were distributed south of Kamchia river (Table 1). Of these species, Ae. uniaris- tata was found in two locations only, in the area between Rezovo and Burgas. This species is reported here for the first time as a new plant for the Bulgarian flora. The P. Spetsov et al. / Central European Journal of Biology 1(3) 2006 399–411 403

Species Number of samples Total

North South Ae.cylindrica 16 16 32 Ae.triuncialis 15 28 43 Ae.biuncialis 3811 Ae.geniculata 33 33 Ae.neglecta 29 29 Ae.uniaristata 22 T.monococcum 88 ssp. aegilopoides Total 34 124 158

Table 1 Number of samples collected to the north and south of Kamchia river along the Bulgarian Black Sea coast.

o 27 28o 29o

Romania o 44

Durankulak

Varna

Kamchia 43o

Black Sea Burgas

Rezovo 42o Turkey

Fig. 1 Main collecting sites on the Bulgarian Black Sea coast. cytological observation confirmed that its genome possessed 14 chromosomes (Fig. 2a). Aegilops uniaristata has been previously reported in Turkey, Albania, Croatia, Greece, Italy and (former) Yugoslavia [1]. We collected the spikes of Ae. uniaristata together with spikes of Ae. geniculata and Ae. neglecta at both locations, south of Burgas. The species was identified in 2002 when 12 plants were grown in the greenhouse. Aegilops uniaristata is diploid, but very similar to tetraploid Ae. biuncialis and hexaploid Ae. ne- glecta in spike morphology (Table 2, see means for spike length, number of spikelets per spike and number of seeds per spike). Spike features of Ae. uniaristata include rudimen- 404 P. Spetsov et al. / Central European Journal of Biology 1(3) 2006 399–411 tary spikelets, lateral glume apex with a broad, triangular tooth and awn, and apical spikelet glumes with a flat, 3-5 cm long awn. The two samples of Ae. uniaristata were nearly identical regarding the quantitative characters. The most variable character was the number of tillers per plant (SD=12.21; Table 2). Dispite this, the differences between samples were not significant. The low variability within Ae. uniaristata could be due to the short distance between the two locations, Rezovo and Ahtopol. The latter place is 75 km south of Burgas. Aegilops uniaristata is characterised as Mediterranean element with limited distribution. Triticum monococcum ssp. aegilopoides had a restricted distribution in the southern half of the coastal line, between Kamchia river and Burgas.

Spike Plant Number Spike Number Number Seeds Species emergence height of tillers length of spikelets of seeds weight per plant per spike per spike per spike

Ae. geniculata Mean 26.51 36.3 34.17 2.91 3.98 6.61 0.08 Min 8.00 25.00 3.00 2.20 3.00 4.00 0.01 Max 42.00 47.00 72.00 4.00 5.00 10.00 0.15 SD 6.22 4.45 12.42 0.31 0.22 1.19 0.03 Ae. triuncialis Mean 33.74 47.69 30.04 5.81 5.77 8.22 0.09 Min 26.00 28.00 1.00 4.50 5.00 2.00 0.01 Max 47.00 62.00 57.00 7.30 8.00 15.00 0.16 SD 4.48 4.86 8.62 0.51 0.59 1.56 0.03 Ae. neglecta Mean 37.14 43.62 25.82 3.35 4.71 4.44 0.09 Min 26.00 22.00 1.00 2.20 4.00 2.00 0.02 Max 57.00 60.00 49.00 4.30 6.00 8.00 0.15 SD 5.04 7.33 9.84 0.40 0.47 0.96 0.03 Ae. cylindrica Mean 40.94 45.68 19.76 9.67 8.66 12.44 0.10 Min 26.00 22.00 2.00 7.40 7.00 1.00 0.005 Max 68.00 66.00 38.00 13.00 11.00 18.00 0.18 SD 6.50 7.15 7.64 0.92 0.80 3.02 0.03 Ae. biuncialis Mean 31.77 41.95 37.98 3.35 3.19 5.07 0.06 Min 22.00 24.00 6.00 2.60 3.00 3.00 0.02 Max 56.00 55.00 85.00 4.10 4.00 7.00 0.10 SD 7.53 6.23 14.07 0.29 0.39 1.03 0.02 Ae. uniaristata Mean 40.92 40.25 32.50 3.44 4.25 5.50 0.06 Min 38.00 34.00 13.00 3.00 4.00 4.00 0.04 Max 45.00 47.00 49.00 3.80 5.00 6.00 0.08 SD 1.73 4.39 12.21 0.26 0.45 0.80 0.01 T. monococcum Mean 40.58 105.19 6.83 6.20 20.63 20.50 0.25 ssp. aegilopoides Min 35.00 78.00 4.00 4.00 13.00 7.00 0.05 Max 49.00 133.00 15.00 8.80 30.00 34.00 0.45 SD 3.20 11.89 2.13 1.26 4.35 5.72 0.10

Table 2 Results of morphological characterization (mean values, range and SD) for seven species

Among the Aegilops species, Ae. neglecta is remarkable in that it includes tetraploid and hexaploid forms. No morphological trait is known to distinguish the two cytotypes. Aryavand et al.[14] revealed that the increased ploidy in this species has resulted in fewer but larger stomata per unit leaf area. Leaf stomatal and/or its frequency can be used to distinguish tetraploid from hexaploid types. We found Ae. neglecta distributed on the south area of the coastal line and all samples were hexaploid according to our cytological observations (Fig. 2b). Our finding is, therefore, different from previous research reporting tetraploid forms of Ae. neglecta [7]. From the nine Aegilops species distributed in Bulgaria [7], four of them, Ae. speltoides, Ae. markgrafii, Ae. umbellulata and Ae. columnaris, were not found along the Black Sea P. Spetsov et al. / Central European Journal of Biology 1(3) 2006 399–411 405

a b

Fig. 2 a) Chromosome complement of Ae. uniaristata (2n=14); b) Chromosome com- plement of Ae. neglecta (2n=42) coast. This study revealed the region south of the Kamchiya river was characterized by the greatest diversity of wild wheat relatives. Seven species, six from Aegilops and one from Triticum, were found as wild plants, and among them, one diploid was recorded for the first time. This region along the Black Sea coast comprising about 200 km2 can be considered as an ‘in situ conservation area’ in Bulgaria. The most widely distributed species in this region was Ae. geniculata, followed by Ae. neglecta and Ae. triuncialis. These data support the information given by Zaharieva et al.[7]thatAe. cylindrica was the species more frequently found in northern Bulgaria, while Ae. geniculata and neglecta were widely distributed in the south.

3.2 Natural hybrids

Cultivated wheat is an autogamous plant propagated through seeds by self-pollination. The floral architecture and the prevailing environmental conditions are main factors which affect the outcrossing rate in wheat. Interspecific hybridization between jointed goatgrass and wheat can occur naturally when the two species come into contact [15]. We found many hybrid plants in an Ae. cylindrica population north of Varna. This region is the north-eastern part of fertile land in Dobroudja, a wheat producing area of the country. Hybrid spikes were collected in July before harvest. Hybrid plants were identified by their distinctive traits: a spreading plant habit and height, large tiller number and intermediate spike morphology (Fig. 3). It is characterized by long spike (6-18 cm), narrowly cylindri- cal shape and two glume awns (2-4 cm) terminating at the spike apex. Hybrid spikes were usually darker brown to black in colour as compared to both parents. Hybrids averaged 10-20 tillers per plant. The capacity for high tiller production was inherited from the jointed goatgrass parent, as well as the spike disarticulation. At maturity, hybrid spikes disarticulated at the base, falling to the ground as a whole spike dispersal unit. Each individual hybrid spike was checked by hand for seed. Hybrid spikes were scored as ‘fer- tile’ when at least one seed was found. Jointed goatgrass population was located around the field exterior and neither wild nor hybrid plants were observed in the field interior. 406 P. Spetsov et al. / Central European Journal of Biology 1(3) 2006 399–411

The jointed goatgrass population was dense and spread extensively along the field, just between the wheat stand and the steep coast. Among the wild plants, the hybrid plant community can be evaluated as large, with greater than 50 hybrid plants.

Fig. 3 Spike morphologies: right, two spikes of hybrid plants; left, cultivated wheat; center, Ae. cylindrica.

On the basis of spike morphology, hybrid plants appeared to be F1 or early genera- tion backcross plants that resembled the F1-hybrid phenotype. Such hybrid spikes were not identified in the wheat field adjacent to the goatgrass plants. These analyses sug- gest hybridization dynamics in which jointed goatgrass served as the predominant F1 female parent and wheat as the predominant backcross male parent. From the 15 hybrid spikes collected, 4 seeds were found, of which only one germinated and grew in the green- house. The plant was vigorous and showed resistance to powdery mildew at seedling and adult plant stages. It was also fertile (according to the term accepted by Morrison et al.[15]). Seven spikes were selfed and set only one seed. Five other spikes were arti- ficially backcrossed using wheat pollen to continue the next generation. Our data were in accordance with the results published [1, 16]thatF1 hybrids were common in jointed goatgrass-infested wheat fields, and that goatgrass was most often the female parent.

3.3 Disease resistance

All samples of Ae. geniculata, Ae. neglecta, Ae. uniaristata, Ae. biuncialis (with the ex- ception of two samples in the second year) and T. monococcum ssp. aegilopoides showed complete resistance to powdery mildew at seedling and adult plant stages (Table 3). Such high levels of resistance have been described for Ae. geniculata and Ae. neglecta [17]. Six- teen of the 19 Ae. triuncialis samples collected in 2000 were resistant to powdery mildew at seedling stage, but all 24 samples expressed resistance in the second year. Eleven P. Spetsov et al. / Central European Journal of Biology 1(3) 2006 399–411 407 samples were found to segregate for this trait. Most samples showed resistance in the adult plant stage. Eight samples from the first year expressed resistance at both stages compared to 17 resistant samples in the second year. Aegilops cylindrica showed the low- est resistance to powdery mildew; from all 32 samples collected, 27 were susceptible to the pathogen in the adult plant stage (Table 3). Only one sample segregated. Promising samples were No.73 from the first year and No.111 from the second year, showing resis- tance at both seedling and adult stages. Most of the samples collected in the second year were resistant at the seedling stage, but only three samples showed resistance at the adult stage. The results correspond with previous evaluations of Ae. cylindrica resistance to powdery mildew [8, 18, 19].

Powdery mildewb Leaf rust culturesc Species Yearsa Samples Seedling stage Adult plant stage 73760 43763 tested R I S R I S R S R S

Ae. geniculata 1121212 2938 22121 21107154 Ae. triuncialis 11919d 16e 1 2 16 3 9 10 224242112186176 Ae. neglecta 1131313 12210 2161616 511412 Ae. cylindrica 1135f 17 1 12 12210 2 1917 2 3 1 15811710 Ae. biuncialis 144 4 44 277 7g 346 1 Ae. uniaristata 222 2 2 2 T. monococcum 288 8 1762 ssp. aegilopoides

a, 1=2001, 2=2002; b, infection type (IT) and disease infection severity (DIS) were estimated for powdery mildew resistance in adult plant stage; R = resistant (ratings 0-3, IT=0-1, DIS=0-20); I = intermediate (ratings 4-6, IT=2-3, DIS=25-50); S = susceptible (ratings 7-9, IT=4, DIS=55-100); susceptible standard Sadovska ranozreika-4 showed S type of reaction to powdery mildew at both stages; c, leaf rust resistance was estimated in seedlings: R=resistant (ratings 0-2), S=susceptible (ratings 3-4); samples tested do not always correspond to the collected ones due to limited number of seeds; d, three samples segregated; e, eight samples segregated; f , one sample segregated; g,two samples segregated. Table 3 Evaluation of Aegilops and Triticum samples for resistance to powdery mildew and leaf rust in two successive years.

Zaharieva et al.[17] established resistance to the pathogen in Bulgarian samples of Ae. triuncialis, and also indicated that Ae. cylindrica samples were the most susceptible to powdery mildew. B¨orner et al.[4] investigated 489 samples belonging to 20 species of the genus Aegilops and most of them were homogeneous and highly resistant against powdery mildew and leaf rust in adult plant stage. Similarly to powdery mildew resistance, most of T. monococcum ssp. aegilopoides samples gave resistance to P. triticina culture 43763 [20]. The leaf rust infection test showed that approximately half of the samples of Ae. genic- ulata, Ae. triuncialis and Ae. biuncialis expressed resistance to both P. triticina cultures. 408 P. Spetsov et al. / Central European Journal of Biology 1(3) 2006 399–411

The potential of Ae. geniculata as a source of resistance to leaf rust was also indicated by previous research [19, 21, 22]. Harjit-Singh et al. [23], Harjit-Singh and Dhaliwal [24] observed intraspecific diversity within this species for this trait. Our results support these findings. Previous research has also shown resistance of Ae. triuncialis to the rust pathogen [19, 23]. Aegilops biuncialis expressed a higher level of resistance to leaf rust than both Ae. geniculata and Ae. triuncialis. Only one out of six samples of this species, grown in 2002, was susceptible to leaf rust culture 43763. Variation in resistance levels has previously been described for Ae. biuncialis; out of five samples, two were resistant and three were susceptible [19]. The same study also demonstrated that most Ae. cylindrica and Ae. neglecta samples expressed high levels of disease resistance. However, according to our study, the two species showed similar susceptibility levels to the pathogen. Pre- vious screening of Aegilops samples from Bulgaria also showed that Ae. cylindrica was the most susceptible goatgrass species to rusts [17]. The two Ae. uniaristata samples showed susceptibility to both P. triticina cultures. Our data indicate that this species lacks genes for leaf rust resistance, and support the assessment reported by Gill et al.[19] for its susceptibility to the rust pathogen. Most T. monococcum ssp. aegilopoides samples showed resistance to P. triticina culture 43763. Gill et al.[25] evaluated samples of this species from different countries and concluded that greater than 40% of them were resistant to leaf rust.

3.4 Phenotypic clustering

A phenogram based on the degree of similarity among the samples analyzed showed two basic clusters (Fig. 4). All Aegilops species were included in a separate cluster. The most close related were Ae. biuncialis and Ae. uniaristata at linkage distance 8.1. Closely related to them were Ae. triuncialis and Ae. neglecta. Aegilops geniculata and Ae. cylindrica were divergent from the others. Triticum monococcum ssp. aegilopoides formed the second basic cluster. The largest distance (78.4) was established between T. monococcum ssp. aegilopoides and Ae. geniculata.Hegdeet al.[3] also showed that diploid Aegilops species were genetically more related to each other than to the related polyploidy taxa. A study of allozyme genetic diversity within populations of T. monococ- cum ssp. aegilopoides, T. urartu, T. turgidum ssp. dicoccoides and Ae. speltoides from Syria and Lebanon found that T. monococcum ssp. aegilopoides (syn. T. boeoticum)was more closely related to Ae. speltoides than to T. urartu [26]. The clustering based on Jaccard’s and Dice’s coefficients reflected the phylogenetic classification of T. boeoticum, T. durum and T. aestivum [27]. Further, investigating ten T. boeoticum samples from the Czech Republic, the authors did not find any correlations between the relative genetic distances and the places of their origin. The results from the cluster analysis showed that the closest relationship existed between Ae. biuncialis and Ae. uniaristata, and the most morphologically distinct was T. monococcum ssp. aegilopoides. In conclusion, all samples of Aegilops geniculata, Ae. neglecta, Ae. uniaristata, P. Spetsov et al. / Central European Journal of Biology 1(3) 2006 399–411 409

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Fig. 4 A phenogram based on the degree of similarity between the wild relatives collected.

Ae.biuncialis and T. monococcum ssp. aegilopoides from the Bulgarian Black Sea coast showed resistance to powdery mildew and could be used to improve wheat for this trait. Aegilops cylindrica was the species with least powdery mildew resistance, with exceptions of a few samples. Leaf rust test showed that only particular samples from the collected Aegilops species and T. monococcum ssp. aegilopoides possess genes for leaf resistance to two P. triticina cultures. The resistant samples seemed to be valuable resources for wheat improvement.

References

[1] M.W. van Slageren: Wild wheats: a monograph of Aegilops L. and Amblyopyrum (Jaub. & Spach) Eig (Poaceae), Wageningen Agricultural University Papers 94-7, Wageningen, The Netherlands, 1994. [2] J.R. Harlan and J.M.J. de Wet: “Toward a rational classification of cultivated plants”, Taxon, Vol. 20, (1971), pp. 511–514. [3] S.G. Hegde, J. Valkoun and J.G. Waines: “Genetic diversity in wild and weedy Aegilops, Amblyopirum and Secale species - A Preliminary Survey”, Crop Sci.,Vol. 42, (2002), pp. 608–614. [4] A. B¨orner, U. Freytag and U. Sperling: “Analysis of wheat disease resistance data originating from screenings of Gatersleben genebank accessions during 1933 and 1992”, Genet. Resour. Crop. Ev., Vol. 53, (2006), pp. 453–465. [5] A. Karag¨oz and A. Zencirci: “Variation in wheat (Triticum spp.) landraces from different altitudes of three regions of Turkey”, Genet. Resour. Crop Ev., Vol. 52, (2005), pp. 775–785. 410 P. Spetsov et al. / Central European Journal of Biology 1(3) 2006 399–411

[6] N. Andreev, M. Anchev, S. Kojuharov, M. Markova, D. Peev and A. Petrova: A guide for higher plants in Bulgaria, Science and Art, Sofia, 1992 (in Bulg.). [7] M. Zaharieva, J.-M. Prosperi and P. Monneveux: “Ecological distribution and species diversity of Aegilops L. genus in Bulgaria”, Biodivers Conserv, Vol. 13, (2004), pp. 2319–2337. [8] M. Zaharieva: “Bulgarian Aegilops germplasm collection. Diversity and potential use in wheat improvement”, In: Proc 5 th Int Wheat Conference, 10-14 June 1996, Ankara, Turkey, Kluwer Academic Publishers, the Netherlands, pp. 450–451. [9] A. Blum: Plant breeding for stress environments, CRC Press, Boca Raton, FL, 1988. [10] M. Zaharieva, E. Gaulin, M. Havaux, E. Acevedo and P. Monneveux: “Drought and heat responses in the wild wheat relative Aegilops geniculata Roth: Potential interest for wheat improvement”, Crop Sci., Vol. 41, (2001), pp. 1321–1329. [11] J.R. Tomerlin, M.A. El-Morshidy, J.G. Moseman, P.S. Baenziger and G. Kimber: “Resistance to Erysiphe graminis f.sp. tritici, Puccinia recondita f.sp. tritici,and Septoria nodorum in wild Triticum species”, Plant Dis., Vol. 68, (1984), pp. 10–13. [12] E.C. Stakman, D.M. Stewart and W.Q. Loegering: “Identification of physiologic races of Puccinia graminis var. tritici”, Agric. Res. Serv. E617, United States Department of Agriculture, Washington DC., 1962. [13] L.F. Browder: “Pathogenic specialization in cereal rust fungi, especially Puccinia recondita f.sp. tritici: concepts, methods of study, and application”, In: Technical Bulletin 1432, Agricultural Research Service, 1971, p. 45. [14] A. Aryavand, B. Ehdaie, B. Tran and J.G. Waines: “Stomatal frequency and size differentiate ploidy levels in Aegilops neglecta”, Genet. Resour. Crop. Ev., Vol. 50, (2003), pp. 175–182. [15] L.A. Morrison, L.C. Cremieux and C. Mallory-Smith: “Infestations of jointed goat- grass (Aegilops cylindrica) and its hybrids with wheat in Oregon wheat fields”, Weed Sci., Vol. 50, (2002), pp. 737–747. [16] J. Snyder, C. Mallory-Smith, S. Balter, J.L. Hansen and R.S. Zemetra: “Seed pro- duction on Triticum aestivum by Aegilops cylindrica hybrids in the field”, Weed Sci., Vol. 48, (2000), pp. 588–593. [17] M. Zaharieva, A. Dimov, P. Stankova, J. David and P. Monneveux: “Morphological diversity and potential interest for wheat improvement of three Aegilops L. species from Bulgaria”, Genet. Resour. Crop Ev., Vol. 50, (2003), pp. 507–517. [18] A.B. Damania, J.P. Srivastava and L. Pecetti: “Evaluation of wild relatives of wheat and identification of useful traits”, In: Cereal Improvement Program annual report, Aleppo, Syria, ICARDA, 1989, pp. 149–152. [19] B.S. Gill, H.C. Sharma, W.J. Raupp, L.E. Browder, J.H. Hatchett, T.L. Harvey, J.G. Moseman and J.G. Waines: “Evaluation of Aegilops species for resistance to wheat powdery mildew, wheat leaf rust, hessian fly and greenbug”, Plant Dis., Vol. 69, (1985), pp. 314–316. P. Spetsov et al. / Central European Journal of Biology 1(3) 2006 399–411 411

[20] T.S. Grewal, H.S. Dhaliwal, Harjit-Singh and K. Datta: “Identification and transfer of powdery mildew resistance from related species into cultivated wheats (Triticum aestivum)”, Indian J. Agr. Sci., Vol. 69, (1999), pp. 707–711. [21] Y. Anikster, J. Manisterski, D.L. Long and K.J. Leonard: “Resistance to leaf rust, stripe rust, and stem rust in Aegilops spp. in Israel”, Plant Dis., Vol. 89, (2005), pp. 303–308. [22] J. Manisterski, A. Segal, A.A. Levy and M. Feldman: “Evaluation of Israeli Aegilops and Agropyron species for resistance to wheat leaf rust”, Plant Dis., Vol. 72, (1988), pp. 941–944. [23] Harjit-Singh, H.S. Dhaliwal, J. Kaur and K.S. Gill: “Rust resistance and chromosome pairing in Triticum x Aegilops crosses” Wheat Inf. Serv., Vol. 76, (1993), pp. 23–26. [24] Harjit-Singh and H.S. Dhaliwal: “Intraspecific genetic diversity for resistance to wheat rusts in wild Triticum and Aegilops species”, Wheat Inf. Serv., Vol. 90, (2000), pp. 21–30. [25] B.S. Gill, L.E. Browder, H. Hatchett, T.L. Harvey, T.J. Martin, W.J. Raupp, H.C. Sharma and J.G. Waines: “Desease and insect resistance in wild wheats”, In: Proc. 6 th Int. Wheat Genet. Symp., Kyoto, Japan, 1983, pp. 785–792. [26] S.G. Hegde, J. Valkoun and J.G. Waines: “Genetic diversity in wild wheats and goat grass”, Theor. Appl. Genet., Vol. 101, (2000), pp. 309–316. [27] J. Ovesn´a, L. Kuˇcera, R. Bockov´a and V. Holubec: “Characterisation of powdery mildew resistance donors within Triticum boeoticum accessions using RAPDs”, Czech J. Genet. Plant Breed, Vol. 38, (2002), pp. 117–124.