ISSN 1313 - 8820Volume 7, Number 2

June 2015

2015

Scope and policy of the journalAgricultural Science and Technology /AST/ – an International Scientific Journal of Agricultural and Technology Sciences is published in English in one volume of 4 issues per year, as a printed journal and in electronic form. The policy of the journal is to publish original papers, reviews and short communications covering the aspects of agriculture related with life sciences and modern technologies. It will offer opportunities to address the global needs relating to food and environment, health, exploit the technology to provide innovative products and sustainable development. Papers will be considered in aspects of both fundamental and applied science in the areas of Genetics and Breeding, Nutrition and Physiology, Production Systems, Agriculture and Environment and Product Quality and Safety. Other categories closely related to the above topics could be considered by the editors. The detailed information of the journal is available at the website. Proceedings of scientific meetings and conference reports will be considered for special issues.

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Editor-in-Chief

Tsanko YablanskiFaculty of AgricultureTrakia University, Stara ZagoraBulgaria

Co-Editor-in-Chief

Radoslav SlavovFaculty of AgricultureTrakia University, Stara ZagoraBulgaria

Editors and Sections

Genetics and Breeding

Atanas Atanasov (Bulgaria)Nikolay Tsenov (Bulgaria)Max Rothschild (USA)Ihsan Soysal (Turkey)Horia Grosu (Romania)Bojin Bojinov (Bulgaria)Stoicho Metodiev (Bulgaria)

Nutrition and Physiology

Nikolai Todorov (Bulgaria)Peter Surai (UK)Zervas Georgios (Greece)Ivan Varlyakov (Bulgaria)

Production Systems

Dimitar Pavlov (Bulgaria)Bogdan Szostak (Poland)Dimitar Panaiotov (Bulgaria)Banko Banev (Bulgaria)Georgy Zhelyazkov (Bulgaria)

Agriculture and Environment

Georgi Petkov (Bulgaria)Ramesh Kanwar (USA)Martin Banov (Bulgaria)

Product Quality and Safety

Marin Kabakchiev (Bulgaria)Stefan Denev (Bulgaria)Vasil Atanasov (Bulgaria)

English Editor

Yanka Ivanova (Bulgaria)

2015

ISSN 1313 - 8820 Volume 7, Number 2June 2015

Genetics and Breeding

Characterization of as ara t c a isolates from Bulgaria with microsatellite markers

1 2 3 1. osev , I. Simeonov , G. Djakova , Ts. Hvarleva *

1Agrobioinstitute, Agricultural Academy, 8 Dragan Tsankov, 1164 Sofia, Bulgaria2Instiute of iticulture and Enology, Agricultural Academy, 5800 Pleven, Bulgaria3Institute of Agriculture and Seed Science, Agricultural Academy, 7007 Rousse, Bulgaria

Abstract. The fungus Plasmopara viticola is the causal agent of downy mildew, one of the most severe grapevine diseases. The genetic diversity of this pathogen in Bulgaria has not yet been investigated. In order to assess the polymorphism among P. viticola isolates in Bulgaria six samples collected in different regions of the country were genotyped with 7 SSRs. The genetic polymorphism revealed by the used microsatelite markers allowed discrimination of P. viticola isolates originating from the different areas of Bulgaria. All samples were found to have a unique microsatellite profile. One sample was found to have a multiallelic profile at three out of seven loci and was excluded from the calculation of genetic diversity parameters. The number of alleles per locus varied between 2 and 6 with a mean value of 3.5 alleles per locus. The obtained values of observed heterozygosity ranged from 0.2 to 0.8 with an estimated average value of 0.46±0.13, which is a bit lower than the mean expected heterozygosity – 0.53±0.07. With this study the characterization of population structure of P. viticola pathogen in Bulgaria was initiated.

Keywords: grapevine, downy mildew, Plasmopara viticola isolates, SSRs

AGRICULTURAL SCIENCE AND TECHN L G , L. 7, No 2, pp , 2015159 - 161

Introduction different locations in Bulgaria emen, Galabnik, Dobromirka, Ruse, Pleven and Sofia ( igure 1). The samples were extracted from the leaves using filter tips and vacuum pump. Extraction of DNA was Downy mildew, caused by the omycete Plasmopara viticola carried out with Analytic jena Uniprep plant DNA kit. The following 9 (Berk. and Curt.) Berl. and de Toni., is one of the most devastating microsatellite loci were used for the microsatellite profiling: Pv65, diseases in grapevine in temperate climate zones in the whole world. Pv101, Pv137, Pv140, Pv143, Pv144 and Pv147 (Rouxel et al., The pathogen spores overwinter in fallen leaves and initiate infection 2012). PCR reaction was carried out in 15 l final volume including in spring when temperature is 18 20 C and humidity is around 90 MyTaq HS Mix 2x, Bioline, 0.1 M of a dye-labeled forward primer (Lafon and Clerjeau, 1988 Gessler et al., 2011). Downy mildew can and an unlabeled reverse primer. PCR cycles were performed in cause severe defoliation and crop loss (Lafon and Clerjeau, 1988

B-96, L B PCR system according to Rouxel et al. (2012) with the Gessler et al., 2011). Currently, fungal disease is controlled by the following conditions: an initial denaturation at 94°C for 4 min and 38 application of fungicides, where the fungicidal treatments can reach cycles of 30 s at 94°C, 30 s at the appropriate annealing up to 14 per year. The application of agrochemicals for plant temperature, and 35 s at 72°C, ending with a 5-min extension at protection in viticulture causes substantial increase of the production 72°C. ragment analysis was carried out in ABI prism 3130 Genetic costs, ecological problems and reduction of the quality of grape and Analyzer. Alleles were scored with Genemapper v.4 software from wine. Recently it was shown that P. viticola can overcome resistance

in some resistant cultivars (Peressotti et al., 2010). In order to keep track of P. viticola populations and genetic diversity, a strategy utilizing Simple Sequence Repeats (SSR) markers was proposed (Gobin et al., 2003a, b Gobin et al., 2005 ). urther development of the SSR markers for P. viticola was done by Delmotte et al., 2006 a, b Rouxel et al., 2012). The populations of P. viticola in Bulgaria and their virulence have not yet been studied.

In this paper microsatellite analysis was used for characterization of 6 samples of P. viticola collected from six regions of Bulgaria emen, Galabnik, Dobromirka, Ruse, Pleven and Sofia. These six samples were genotyped with a set of seven SSR.

ur aim is to assess the genetic diversity of the pathogen in the country.

Material and methods

Infected grapevine leaves were collected from the field in six

159

*e-mail: hvarleva@abi.bg

Figure 1. Physical map of Bulgaria showing regions where P. viticola isolates were collected

Dobromirka

Ruse

Pleven

Galabnik

Sofiaemen

160

Applied Biosystems. For Calculation of allele frequencies, expected profile.and observed heterozygosity, probability of null alleles and The selected SSR markers revealed a high degree of probability of identity software GenAlex (Peakall and Smouse, 2006) polymorphism among the tested isolates, which is in consistence was used. with the high genetic variability of P. viticola pathogen determined in

a number of studies (Gobin et al., 2003a, b; Delmotte et al., 2006 a, b; Rouxel et al., 2012). The results from the calculated genetic diversity parameters are shown in Table 2. One sample from Results and discussionGalabnik was found to have a multiallelic profile at three out of seven loci and was excluded from the calculation of genetic diversity Samples of P. viticola collected from six regions of Bulgaria parameters. were genotyped with seven SSR markers. The obtained

The number of alleles per locus varied between 2 and 6 with a microsatellite profiles of the six studied isolates are presented in mean value of 3.5 alleles per locus. Three loci, PV65, PV101 and Table 1. All samples were found to have a unique microsatellite

Table 2. Genetic diversity parameters in the group of 5 isolates of P. viticola: N – number of samples, Na – number of alleles, Ne – number of effective alleles, Ho – observed heterozygosity, He – expected heterozygosity, PI – probability of identity

Na Ne Ho He PIN

PV137

PV140

PV144

PV65

PV143

Pv147

Pv101

Total

Mean

5

5

5

5

5

5

5

5

5

2

3

6

2

4

3

2

22

3.143±0.6

1.923

2.941

4.167

1.471

2.778

1.515

2.000

16.794

2.399±0.4

0.400

0.800

0.800

0.000

0.800

0.200

0.200

0.457±0.1

0.480

0.660

0.760

0.320

0.640

0.340

0.500

0.529±0.1

-13.9x10 -11.9x10 -28.8x10 -15.1x10 -11.9x10 -14.6x10 -13.8x10 -41.1x10

Table1. Microsatellite profiles of six P. viticola isolates. Allele sizes are given in bp.

Origin \ Nameof primerse PV137 PV137 PV140 PV140 PV144 PV144 PV65 PV65 PV143 PV143 PV147 PV147 PV101 PV101

Zemen

Galabnik

Dobromirka

Ruse

Pleven

Sofia

259

254/256

259

256

256

256

259

259

259

259

259

256

199

199/203

195

199

195

195

203

206

199

203

195

203

180

172/180

172

172

178

172

201

199

199

199

199

199

201

201

199

199

199

199

130

130

134

134

138

128

134

138

138

138

138

138

217

217

217

217

198

217

217

217

217

217

204

217

267

267

267

270

270

267

270

267

267

270

270

267

186

184

172

182

184

186

PV137 showed low number of alleles, only two, while the highest markers allowed discrimination of P. viticola isolates originating from number of alleles, six, was observed at locus PV144. Calculated different areas of Bulgaria. This result is in agreement with the values of genetic diversity per locus ranged between 0.32 at locus current findings that the spread of different P. viticola genotypes is PV65 and 0.76 at locus PV144 with a mean value of 0.53±0.07, restricted to short distances by spatial barriers (Gobin et al., 2005). which values are similar to those found in other studies (Gobbin et Furthermore, only a few genotypes are responsible for an epidemic al., 2003a; Delmotte et al., 2006a) The most informative marker for through asexual multiplication (Gobin et al., 2003a,b). These the investigated set of P. viticola isolates was found to be PV144 with genotypes are probably favored by natural selection of genotypes

-2 adapted to specific environments. 6 alleles and PI value 8.8x10 . The obtained values of observed Five out of six samples analyzed in this study showed diploid heterozygosity ranged from 0.2 to 0.8 with an estimated average

genetic profile, while the sixth sample had a multiallelic profile with value of 0.46±0.13, which is a bit lower than the mean expected three alleles at three loci. Because P. viticola is diploid, this profile heterozygosity. Heterozygote deficiency was observed for four out could be due to the presence of a triploid or tetraploid individuals or of seven microsatellite markers (PV65, PV101, PV137, PV147), to a mixed infection of two diploid individuals. The persistence of which is probably due to the presence of null alleles. The values of polyploid genotypes in P. viticola populations collected on Greek observed heterozygosity are comparable with the corresponding islands was reported by Rumbou et al. (2006). Authors suppose that values of observed heterozygosity for the same loci reported in these polyploid genotypes could be a result of natural selection in Rouxel et al. (2012). conditions of isolation or chemical treatments rather than mixed The genetic polymorphism revealed by the used microsatellite

161

infection in one lesion. disease management. Phytopathologia Mediterranea, 50, 3-44.With this study the characterization of population structure of Gobbin D, Jermini M, Loskill B, Pertot I, Raynal M and Gessler C,

P.viticola pathogen in Bulgaria was initiated. Understanding the 2005. Importance of secondary inoculum of Plasmopara viticola to genetic structure of the population could help us develop better epidemics of grapevine downy mildew. Plant Pathology, 54, 522-strategies for protection. 534.

Gobbin D, Pertot I and Gessler C, 2003a. Identification of microsatellite markers for Plasmopara viticola and establishment of high throughput method for SSR analysis. European Journal of Plant ConclusionPathology, 109, 153-164. Gobbin D, Pertot I and Gessler C, 2003b. Genetic structure of a This study presented the first characterization of genetic Plasmopara viticola population in an isolated Italian mountain diversity of the fungus P.viticola in Bulgaria through molecular vineyard. Journal of Phytopathology, 151, 636-646. markers. The obtained genetic polymorphism between the Gobbin D, Jermini M, Loskill B, Pertot I, Raynal M and Gessler C, investigated isolates of P.viticola showed that the used set of 2005. Importance of secondary inoculum of Plasmopara viticola to microsatellite markers were proper for discrimination of the epidemics of grapevine downy mildew. Plant Pathology, 54, 522-investigated samples of P. viticola. More samples from the same and 534. different geographical and climate regions of Bulgaria are necessary Lafon R and Clerjeau M, 1988. Downy mildew. In: Pearson RC, to elucidate the existing genetic diversity of this pathogen in the Goheen AC, eds. Compendium of Grape Diseases. St Paul, MN, country. USA: APS Press, 11-3. Peakall R and Smouse PE, 2006. GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. References Molecular Ecology Notes, 6, 288-295. Peressotti E, Wiedemann-Merdinoglu S, Delmotte F, Bellin D, Di Gaspero G, Testolin R, Merdinoglu D and Mestre P, 2010. Breakdown of resistance to grapevine downy mildew upon limited deployment of a resistant variety, BMC. Plant Biology, 10,147.Rouxel M, Papura D, Nogueira M, Machefer V, Dezette D, Richard-Cervera S, Carrere S, Mestre P and Delmotte F, 2012. Delmotte F, Martinez F, Némorin A, Wei-Jen C, Richard-Cervera Microsatellite Markers for Characterization of Native and Introduced S and Corio-Costet MF, 2006. Spatial genetic structure of Populations of Plasmopara viticola, the Causal Agent of Grapevine grapevine downy mildew epidemic. In: Proceedings of the 5th Downy Mildew. Applied Environment Microbiology, 78, 6337-6340. International Workshop on Grapevine Downy and Powdery Mildew, Rumbou A and Gessler C, 2005. Particular Structure of San Michele all'Adige, 18-23 June 2006, 63. Plasmopara viticola Populations Evolved under Greek Island Gessler C, Pertot I and Perazzolli M, 2011. Plasmopara viticola: a Conditions. The American Phytopathological Society, 96, 501-509.review of knowledge on downy mildew of grapevine and effective

Delmotte F, Chen WJ, Richard-Cervera S, Greif C, Papura D, Giresse X, Mondor-Genson G and Corio-Costet MF, 2006. Microsatellite DNA markers for Plasmopara viticola, the causal agent of downy mildew of grapes. Molecular Ecology Notes, 6, 379-381.

Review

Genetics and Breeding

Nutrition and Physiology

Effect of physical form and protein source of starter feed on growth and development of dairy calves E. Yavuz, G. Ganchev, N. Todorov

Characterization of Plasmopara viticola isolates from Bulgaria with microsatellite markersK. Kosev, I. Simeonov, G. Djakova, T. Hvarleva

Total phenol content, antioxidant activity of hip extracts and genetic diversity in a small population of R. canina L. cv. Plovdiv 1 obtained by seed propagationM. Rusanova, K. Rusanov, S. Stanev, N. Kovacheva, I. Atanassov

Correlation between qualitative-technological traits and grain yield in two-row barley varieties N. Markova Ruzdik, D.Valcheva, D.Vulchev, Lj. Mihajlov, I. Karov, V. Ilieva

Application of path coefficient analysis in assessing the relationship between growth-related traits in indigenous Nigerian sheep (Ovis aries) of Niger State, NigeriaS. Egena, D. Tsado, P Kolo, A. Banjo, M. Adisa-Shehu-Adisa

Effect of height of stem on the productivity of winter common wheatN. Tsenov, T. Gubatov, E. Tsenova

Influence of the direction of crossing on activities of heterosis regarding the height of plants and number of leaves in Burley tobacco hybridsTs. Radoukova, Y. Dyulgerski, L. Dospatliev

Common winter wheat lines with complex resistance to rusts and powdery mildew combined with high biochemical indexV. Ivanova, S. Doneva, Z. Petrova

Study of emmer (Triticum dicoccum (Schrank) Shuebl.) accessions for traits related to spike productivity and grain quality in connection to durum wheat improvementK. Taneva, V. Bozhanova, B. Hadzhiivanova

Phenotypic stability of yield on varieties and lines of durum wheat (Triticum durum Desf.)R. Dragov, D. Dechev

Classification and regression tree analysis in modeling the milk yield and conformation traits for Holstein cows in BulgariaA. Yordanova, S. Gocheva-Ilieva, H. Kulina, L.Yordanova, I. Marinov

Potential N-supplying ability of soil depending on the size of soil units under different soil tillage systems M. Nankova, P. Yankov

CONTENTS 1 / 2

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Production Systems

Agriculture and Environment

Product Quality and Safety

Tolerance and own tolerance of wheat under conditions of permanent and long-term rotationN. Nankov, G. Milev, A. Ivanova, I. Iliev, M. Nankova

Influence of fertilization and sowing density on grain production of Sorghum bicolor L., in the climatic conditions of Central Moldavia, RomaniaS. Pochişcanu, T. Robu, A. Gherasim, M. Zaharia

Effect of locomotor activity of Russian sturgeons (Acipenser Gueldenstaedtii Brandt) on water heat flows in a recirculation system K. Peychev, Y. Staykov, S. Stoyanova

The effect of stocking density on some hydrochemical parameters and growth traits in European perch (Perca fluviatilis L.), cultivated in a recirculation systemG. Zhelyazkov

Agroecological assessment of wastewater from Municipal Wastewater Treatment Plant by physico-chemical parameters G. Kostadinova, D. Dermendzhieva, G. Petkov, I. Taneva

Exploring the yield potential and spike characteristics of Tritordeum (×Tritordeum Ascherson et Graebner) accessions under the conditions of South DobrodzaH. Stoyanov

Effect of amitraz on varroosis in bees (Apis mellifera L.)K. Gurgulova, I. Zhelyazkova, S. Takova, K. Malinova

New data about Crocus olivieri J. Gay on the territory of Sinite Kamani Natural Park, BulgariaN. Grozeva, M. Todorova, M. Gerdzhikova, G. Panayotova, N. Getova, D. Dohchev, K. Tsutsov

Near Infrared Spectroscopy and aquaphotomics for monitoring changes during yellow cheese ripeningS. Atanassova

Investigation on the technological traits of Bulgarian and imported merino wool batchesD. Pamukova

CONTENTS 2 / 2

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thmance in dairy cows,IX International Conference on Production Diseases in Farm Animals, September 11–14, Berlin, Germany.Thesis:Hristova D, 2013. Investigation on genetic diversity in local sheep breeds using DNA markers. Thesis for PhD, Trakia University, Stara Zagora, Bulgaria, (Bg).

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