ISSN Volume 7, Number 4 December 2015

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1 ISSN Volume 7, Number 4 December

2 Editor-in-Chief Tsanko Yablanski Faculty of Agriculture Trakia University, Stara Zagora Bulgaria Co-Editor-in-Chief Radoslav Slavov Faculty of Agriculture Trakia University, Stara Zagora Bulgaria 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) Scope and policy of the journal Agricultural 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. Submission of Manuscripts All manuscripts written in English should be submitted as MS-Word file attachments via to editoffice@agriscitech.eu. Manuscripts must be prepared strictly in accordance with the detailed instructions for authors at the website and the instructions on the last page of the journal. For each manuscript the signatures of all authors are needed confirming their consent to publish it and to nominate on author for correspondence. They have to be presented by a submission letter signed by all authors. The form of the submission letter is available upon from request from the Technical Assistance or could be downloaded from the website of the journal. Manuscripts submitted to this journal are considered if they have submitted only to it, they have not been published already, nor are they under consideration for publication in press elsewhere. All manuscripts are subject to editorial review and the editors reserve the right to improve style and return the paper for rewriting to the authors, if necessary. The editorial board reserves rights to reject manuscripts based on priorities and space availability in the journal. The journal is committed to respect high standards of ethics in the editing and reviewing process and malpractice statement. Commitments of authors related to authorship are also very important for a high standard of ethics and publishing. We follow closely the Committee on Publication Ethics (COPE), elines The articles appearing in this journal are indexed and abstracted in: EBSCO Publishing, Inc. and AGRIS (FAO). The journal is accepted to be indexed with the support of a project BG051PO Science and business financed by Operational Programme Human Resources Development of EU. The title has been suggested to be included in SCOPUS (Elsevier) and Electronic Journals Submission Form (Thomson Reuters). Address of Editorial office: Agricultural Science and Technology Faculty of Agriculture, Trakia University Student's campus, 6000 Stara Zagora Bulgaria Telephone.: Technical Assistance: Nely Tsvetanova Telephone.: editoffice@agriscitech.eu

3 Volume 7, Number 4 December 2015 ISSN

4 AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 7, No 4, pp , 2015 Phosphorus fractions in alluvial meadow soil after long-term organic-mineral fertilization S. Todorova*, K. Trendafilov, M. Almaliev Department of Agro-chemistry and Soil Science, Faculty of Agronomy, Agricultural University, 12 Mendeleev, 4000 Plovdiv, Bulgaria Abstract. The aim of our study was to investigate phosphorus fractions, their content and distribution in alluvial meadow soil after continuous fertilization. Our research was focused on a long-term field experiment with different variants of fertilization set in 1959 in the Experimental field of Agricultural University of Plovdiv. Since 2006 the fertilization has been discontinued. In 2011, we collected soil samples from two depths (0 30 and cm) and from the following variants: variant 1 (control, without fertilization), variant 2 (N50P30K20), variant 3 (N25P15K0 + 6t/da manure), variant 4 (N50P0K20). The studied soil is characterized by relatively good natural phosphate regime. The amount of available phosphates determined by the Egner-Rheem method is high average 16.76% of the total phosphorus. Over 50% of the mineral phosphates in the soil are in available (in varying degree) form. Relatively high is the amount of organic phosphates, which during the mineralization can also be involved in the plant nutrition. The amount of available phosphates (according to Egner-Rheem) is determined to the highest degree by the total phosphorus in soil, which in turn is highly correlated with the content of hardly soluble calcium phosphates. Keywords: alluvial meadow soils, phosphorus fractions Introduction In soil phosphorus is in the form of inorganic and organic compounds. Plant roots and mycorrhizal hyphae absorb soil solution P as orthophosphate ions in either the H2PO4- or HPO42- form, depending on soil ph. The dominant solution inorganic P below ph 7.2 is the monovalent ion, while HPO42- dominates at ph values between 7.2 and Plant uptake of the divalent ion has been shown to be slower than the monovalent ion (Cresser et al.1993). However, these ions can be adsorbed on the surface or absorbed into the solid particles of the soil and then become unavailable for plants. Organic soil phosphorus represents, on average, one-half of the total soil P and varies between 15 and 80% in most soils (Stevenson, 1986). The amount of soil organic P depends on soil organic matter content, therefore it tends to be high in the surface horizon and decreases with soil depth. Soil organic P compounds are synthesized by soil microorganisms as esters of orthophosphoric acid (Anderson, 1980). The most stable and abundant compounds are inositol phosphates which make up 10 to 50% of the total soil organic P. Phosphorus is an element with very complex behavior in the soil, which is determined by its ability to enter into chemical and sorption reactions with soil colloids and mono and polyvalent ions of metals. In a short period of time, these transformations are determined by the biochemical processes associated with the formation and decomposition of soil organic matter. In longer term, however, the behavior of phosphorus in soil is determined by the geochemical transformations specific to soil type (Wandruszka, 2006). As a result of these metamorphoses the applied assimilable phosphorus becomes over time less available for plants. In other researches concerning the studied soil (Todorova et al., 2012) it was found that the soil is silty loam according to the International Texture Classification, and light to medium clay according to the classification developed by Kachinskii (1965). Despite the small amount of total carbonates (2 3%), the soil reaction is slightly alkaline (ph(h2o) ). The same authors found high amount of exchangeable bases (Ca2++ Mg meq/100 g soil) in the soil sorption complex. The aim of our study is to determine soil phosphorus forms after different regimes of fertilizations. Material and methods The object of our study is alluvial meadow soil (Mollic fluvisol, FAO 2006) on which in 1959 a long-term field experiment was set with different variants of fertilization in the Experimental field of Agricultural University of Plovdiv. Since 2006 the fertilization has been discontinued. In the spring of 2011, we collected soil samples from two depths (0 30 and cm, from the second and fifth field and from the following variants: variant 1 (control without fertilization), variant 2 (N50P30K20), variant 3 (N25P15K0 + 6 t/da manure), variant 4 (N50P0K20). To determine phosphorus forms, soil samples were analyzed according to the method for fractionation of soil phosphates by Nikolov (1985). This method allows avoiding or highly reducing some disadvantages of fractionation of inorganic phosphorus in calcareous soils according to the original methodology of Chang and Jackson (1957). In this modification first are extracted the easily and moderately soluble phosphates with malic acid and then phosphates associated with soil carbonates by acetate buffer. After removal of these forms and CaCO3, extraction with NH4F and NaOH + H2SO4, is applied as in the original method. The preliminary extraction of easy and moderately soluble phosphates before application of fluoride extraction makes it possible to eliminate the positive error due to their dilution in the extract with NH4F, noted by Kaila A On the other hand, the extraction of phosphorus associated with CaCO3 and removing from the system the carbonates allows avoiding the negative error in fluoride extraction, resulting from the precipitation of phosphorus and fluorine on the surface of calcium carbonate found by a number of authors * stefi_k.todorova@abv.bg 431

5 (Garbuchev, 1978; Nejkova-Bocheva, 1978; Williams et al., 1967). Then dithionite reduction is applied in the presence of sodium acetate with which only reductively soluble phosphorus bounded with the iron is extracted, and phosphorus bound to aluminum does not pass into the extract, which is determined in the dithionite-citrate extraction, applied in the original method. The amount of available phosphates by Egner-Rheem (GOST ), organic phosphorus Metha et al. (1954) and total phosphorus in the soil after combustion with 60% HClO4 (Bray and Kurtz, 1945) were also determined. In the extracts for determining fractions of inorganic phosphates, available phosphates by EgnerRheem and organic phosphates, phosphorus was determined with ascorbic acid reducer by the method of Murphy-Riley (Murphy and Riley, 1962) and total phosphorus was determined by the molybdate-vanadate method (Agrochemical methods for soil investigation, 1975). Results and discussion The results for the amount and composition of the forms of phosphate compounds in the studied alluvial-meadow soil are presented in Tables 1 and 2. We searched correlations between the content of available phosphates determined by Egner-Rheem and fractions of phosphorus determined by the method of Nikolov (1985) organic phosphorus and total phosphorus. Data show that the content of easily soluble phosphates is not high and varies from 0.31 to 1.66 mgp/100g soil, which by itself is not enough to meet the requirements of plants. It is considered that the minimum amount of available phosphorus to ensure the normal phosphorus diet for most of the crops is 5 mgp/100g soil. The amount of this mostly available for plants fraction represents from 0.70 to 3.65% of total phosphorus in the soil. The content of moderately soluble phosphates extracted from the soil with malic acid is a little higher 0.49 to 3.51 mgp/100g. These phosphates, which play a significant role in the nutrition of plants, comprise from 1.04 to 7.72% of total phosphorus. The main part of mineral phosphorus in the studied soil is presented by fractions of phosphates associated with CaCO3 average 6.00 mgp/100g (14.22% of total phosphorus) and hardly soluble calcium phosphates average mgp/100g (26.18% of total phosphorus). The reason for this distribution of phosphorus forms in the soil lies in its genesis. Applied during fertilization, monocalcium phosphate from the superphosphate reacts with the carbonate soils and at lower concentrations of phosphorus at soil solution, phosphate anions are mainly adsorbed on the surface of CaCO3, while at higher concentrations along with the adsorption, precipitation of dicalcium phosphate takes place, octacalcium phosphate and, in some cases it is possible to form harder soluble calcium phosphates such as apatite (Nikolov, 1985; Cole et al., 1953). Reductively soluble iron phosphates are formed under conditions of poor aeration, excessive compaction of the profile as a result of high content physical clay, and during gleization of soils i.e. in soils where reduction processes dominate. Since such conditions are not observed in the studied soil, then the content of this fraction is low mgp/100g, which is an average of 2.98% of the total phosphorus. Table1. Content of mineralforms of phosphorus, organic phosphorus and total phosphorus, mgp/100 g soil Available phosphorus by Egner-Rheem Insoluble residue Total Р Mineral Р Occluded Fe - Р Organic Р Occluded Al - Р Hardly soluble Ca - Р Reductively soluble Fe - Р 4 Hardly soluble Fe - Р 3 Hardly soluble Al - Р 2 Second 1 Phosphorus associated with CaCO3 4 Moderately soluble Р 3 Easily soluble Р 2 Fifth 1 Depth Field Variants Fractions

6 Table2. Percentage of phosphorus fractions of the total phosphorus in soil (as % of Р total) The low content of hardly soluble phosphates bounded to Al and Fe is reasonable, respectively 2.16 mgp/100g and 2.80 mgp/100g. The blocking of phosphorus in these soils does not take place by way of its binding with iron and aluminum oxides and hydroxides, as their amount and activity in these soils are small because of slightly expressed processes of destruction of silicate minerals and the absence of the process of acidification. In confirmation to this is the fact that in most variants, in both depths iron phosphates predominate over aluminum The indicated high positive correlation between available phosphates (according to Egner-Rheem) and the fraction of hardly soluble Al-P (Figure 1), may have resulted from slightly alkaline reaction of the medium in which the solubility of these phosphates is increased to some extent. It must be noted that the amount of these phosphates is low. The fraction of hardly soluble Al-P constitutes about 5% of total phosphorus, which leads us to believe that in our case, these phosphates are actually not essential for the phosphorus nutrition of plants Egner-Rheem Egner-Rheem Iavailable phosphorus by Egner-Rheem Hardly soluble Ca - Р Insoluble residue Occluded Fe - Р Organic Р Occluded Al - Р Mineral Р Reductively soluble Fe - Р 4 Hardly soluble Fe - Р 3 Hardly soluble Al - Р 2 Second 1 Phosphorus associated with CaCO3 4 Moderately soluble Р 3 Easily soluble Р 2 Fifth 1 Depth Field Variants Fractions y= *x Hardly soluble Al-P Figure1. Linear regression between hardly soluble Al-P and Egner-Rheem phosphates with intervals of variation y= *x P-CaCo Figure2. Linear regression between phosphorus associated with CaCO3and Egner-Rheem phosphates with intervals of variation 433

7 Egner-Rheem Egner-Rheem y= *x Organic P Total P Figure3. Linear regression between organic phosphorus and Egner-Rheem phosphates with intervals of variation Figure4. Linear regression between total phosphorus and Egner-Rheem phosphates with intervals of variation correlation between available phosphates (according to EgnerRheem) and organically bounded phosphorus (Figure 3). Despite the high degree of mineralization of soil organic matter, only a small part of organically bounded phosphorus is susceptible to decomposition and is involved in the plant nutrition. The insoluble residue of soil phosphorus represents single primary apatite crystals included in other (larger) unweathered aluminosilicate minerals, phosphorus replacing the silicon in the crystal lattice of silicate minerals and some phosphate minerals insoluble in the reagents for fractionation. The amount of this fraction in different variants included in our study varies widely from 0.53 to 20,08% of total phosphorus. From an agrochemical point of view, this insoluble residue cannot be considered as a reserve for plants nutrition. The strongest correlation was found between available phosphates determined by the Egner-Rheem method on one hand, and total phosphorus and hardly soluble Ca-P, on the other side (Figures 4 and 5). The amount of available phosphates (according to Egner-Rheem) is determined to the greatest extent by the total phosphorus in soil which in turn is highly correlated with the content Egner-Rheem In accordance with the small absolute amount of the hardly soluble aluminum and iron phosphates, the relative amount of their occluded forms is low, respectively 1.79 and 2.19% of the total phosphorus, and again the forms bounded with iron predominate. This confirms the statement that in the studied soil there are no reduction processes related to dissolution and deposition of sheaths of iron hydroxides during wetter periods of the year and consequently the formation of occluded forms of phosphorus. From the mineral phosphorus compounds with the highest availability for plants are characterized phosphates of alkali and alkaline earth metals, except for calcium phosphates such as apatite (hardly soluble Ca-P). The reason for Ca-phosphates (fraction of CaCO3-P) to be the best potential food for plants, lies in their good solubility and in the fact that along with phosphorus plants also absorb calcium from these compounds. The great importance of this fraction of mineral phosphates is confirmed by the established high correlation between available according to Egner-Rheem phosphates and phosphates associated with CaCO3 (Figure 2). It is considered that Fe and Al phosphates can also play a certain role in plant nutrition, especially hardly soluble Al-P and Fe-P. Unlike calcium, however, these ions are not absorbed by the plants so intensively and in many cases are toxic, especially Al. In total, inorganic phosphate compounds are from to 84.42% of total phosphorus, while organic phosphates are from to 29.78%. The course of the curve of organically bounded phosphorus down the soil profile, for most of the variants, is similar to the course of soil organic matter, which speaks about close relationship between organic carbon and organic phosphorus. According to Hinov et al. (1967), about 20% of organic phosphorus is bounded to the high-molecular fractions of humic acids and is not susceptible to hydrolysis. This is a residue that cannot be referred to as reserve phosphorus for plants. The remaining 80% of the organic phosphates assign to relatively easily hydrolysable components of soil organic matter, and it is assumed that it can be a potential phosphoric food for plants during the mineralization of humic acids. Compared to control variants there is a tendency for reducing the amount of organic phosphates in variants with prior mineral and organic-mineral fertilization. Application of fertilizers in these variants created more favorable conditions for microbiological activity and thus stimulated the mineralization of soil organic matter. The results of regression analysis showed a weak positive y= *x 7.50 y= *x Hardly soluble Ca-P Figure5. Linear regression between hardly soluble Ca-P and Egner-Rheem phosphates with intervals of variation 14.00

8 of hardly soluble Ca-P. Under conditions of neutral to slightly alkaline medium (as in our case), hardly soluble Ca-P retain their inertness and to the greatest extent determine the reserves of total phosphorus in soil, so a strong correlation between available phosphates and hardly soluble phosphates of Ca is reported. Conclusion In relation to the above mentioned, it can be concluded that the studied soil is characterized with relatively good natural phosphate regime. This is confirmed by the high amount of available phosphates according to the Egner-Rheem method average 16.76% of the total phosphorus is in assimilable for the plants form. Considering all fractions that could potentially be involved in plant nutrition, it can be seen that over 50% of mineral phosphates are in available (to varying degree) form. This assumption is complemented by the relatively high amount of organic phosphates, which during the mineralization can also take part in phosphorus nutrition. References Agrochemical methods for soil investigation, Moscow, Press Nauka (Ru). Anderson G, Assessing organic phosphorus in soils. p In F.E. Khasawneh et al. (ed.) The role of phosphorus in agriculture. ASA, Madison, WI. Bray RH and Kurtz LT, Determination of total, organic and available forms of phosphorus in soils. Soil Sciences, 59, Chang SC and Jackson ML, Fractionation of soil phosphorus. Soil Sciences, 84, Cole CV, Olsen SR and Scott CO, The nature of phosphate sorption by calcium carbonate. Soil Science Society of America Proceedings, 17, Cresser MS, Killham K and Edwards T, Soil Chemistry and its applications. Cambridge University Press, Cambridge, UK. FAO, World Reference base for Soil Resources, Rome, Italy, pp Garbuchev I, Adjusting the phosphate regime of the main soils in Bulgaria. Zemizdat Press, Sofia, (Bg). GOST Soils. Determination of available forms of phosphorus and potassium according Egner-Rheem method (DLMethod) (Ru). Hinov G, Ganev S and Hadjigencheva M, Behavior of phosphorus from humic acids during their acid hydrolysis. Symposium Phosphorus, Sofia-Belgrade (Ru). Kachinskii NA, Soil Physics, Vysshaya Shkola, Moskow, 1 (Ru). Kaila A, Fertilizer phosphorus in some Finnish soils. The Journal of the Scientific Agricultural Society of Finland, 33, Metha NC, Going JO and CA Black, Determination of organic phosphorus in soils Extraction method. Soil Science Society of America Proceedings, 18, Murphy J and Riley JP, A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta, 27, Nejkova-Bocheva E, New aspects of phosphate regime of the soils and possibilities for prediction of phosphorus fertilization. Thesis for PhD, Sofia, (Bg). Nikolov N, Advanced methods for control and regulation of phosphorus in soils. Thesis for PhD, Sofia, (Bg). Stevenson FJ, Cycles of soil; Carbon, nitrogen, phosphorus, sulfur, micronutrients. John Wiley and Sons, New York, NY. Todorova S, Simeonova N, Trendafilov K and Valcheva V, Change of some chemical properties of alluvial-meadow soil (Mollic fluvisols) after long term fertilization. Agricultural Science and Technology, 4, Wandruszka RV, Phosphorus retention in calcareous soils and the effect of organic matter on its mobility. Geochemical Transactions, 7, 6. Williams JDH, Syers JK and Walker TW, Fractionation of Soil Inorganic Phosphate by a Modification of Chang and Jackson`s Procedure. Soil Science Society of America Proceedings, 31,

9 AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 7, No 4, 2015 CONTENTS 1/2 Review Effect of feeding program for first two months after birth of female calves on growth, development and 389 first lactation performance G. Ganchev, E. Yavuz, N. Todorov Genetics and Breeding Involvement of the transcriptional variants of histone H3.3 in the development and heat stress response of Arabidopsis thaliana M. Naydenov*, B. Georgieva, V. Baev, G. Yahubyan 402 Study of factors affecting sporophytic development of isolated durum wheat microspores V. Bozhanova, Hlorst Lörz 407 Screening Pisum sp. accessions for resistance to Pseudomonas syringae pv. pisi M. Koleva, I. Kiryakov 411 Investigation on the parthenogenetic response of sunflower lines and hybrids M. Drumeva, P. Yankov 415 Hybridization between cultivated sunflower and wild annual species Helianthus petiolaris Nutt. D. Valkova, G. Georgiev, N. Nenova, V. Encheva, J. Encheva 419 Nutrition and Physiology Ethological and haematological indices in yearling sheep fed various dietary nitrogen sources I. Varlyakov, V. Radev, Т. Slavov, R. Mihaylov 423 Phosphorus fractions in alluvial meadow soil after long-term organic-mineral fertilization S. Todorova, K. Trendafilov, M. Almaliev 431 Energy productivity, fertilization rate and profitability of wheat production after various predecessors 436 I. Energy productivity of wheat Z. Uhr, E. Vasileva Influence of mineral nitrogen and organic fertilization on the productivity of grain sorghum S. Enchev, G. Kikindonov 441 Production Systems Influence of the farm construction, farm regimen and season on the comfort indices of dairy cows D. Dimov, Ch. Miteva, Zh. Gergovska 444 Effect of the way of pre-sowing soil tillage for wheat on the development of its roots P. Yankov, M. Drumeva, D. Plamenov 451

10 AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 7, No 4, 2015 CONTENTS 2/2 Occurrence of grapevine leafroll-associated virus complex in the Republic of Macedonia E. Kostadinovska, S. Mitrev, I. Karov 455 Influence of sowing and fertilization rates on the yield and plant health of einkorn wheat (Triticum Monococcum L.) V. Maneva, D. Atanasova, T. Nedelcheva, M. Stoyanova, V. Stoyanova 460 Effect of stocking density on growth intensity and feed conversion of common carp (Cyprinus caprio L.), reared in a superintensive system S. Stoyanova, Y. Staykov 464 Agriculture and Environment Monitoring of fungal diseases of lavender K. Vasileva 469 Nitrogen mineralization potential of alluvial meadow soil after long-term fertilization V. Valcheva, K. Trendafilov, M. Almaliev 476 Changes in the leaf gas exchange of common winter wheat depending on the date of application of a set of herbicides Z. Petrova, Z. Zlatev 481 Leaves area characteristics of Betonica bulgarica Degen et Neiĉ., during vegetation M. Gerdzhikova1*, N. Grozeva2, D. Pavlov1, G. Panayotova1, M. Todorova1 486 Short communications Design and development of a device for measuring vacuum-pulsation parameters of milking unit G. Dineva, V. Vlashev, L. Tsanov 494

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Todorov N and Mitev J, Effect of level of feeding during dry period, and body condition score on reproductive performance in dairy cows,ixth International Conference on Production Diseases in Farm Animals, September 11 14, Berlin, Germany. Thesis: Hristova D, Investigation on genetic diversity in local sheep breeds using DNA markers. Thesis for PhD, Trakia University, Stara Zagora, Bulgaria, (Bg). The Editorial Board of the Journal is not responsible for incorrect quotes of reference sources and the relevant violations of copyrights. Animal welfare Studies performed on experimental animals should be carried out according to internationally recognized guidelines for animal welfare. That should be clearly described in the respective section Material and methods.

12 Volume 7, Number 4 December