№ 1 2015 End.Indd

Contents

Quinoa as a promising pseudocereal crop for Ukraine S. L. Mosyakin, V. V. Schwartau 3 Breeding and usage of sugar beet cultivars and hybrids resistant to sugar beet nematode Heterodera schachtii L. A. Pylypenko, K. A. Kalatur 12 The importance of agroecology in the process of well-balanced agrosphere formation O. І. Furdуchko, O. S. Demyanуuk 23 Recent data on the causative agent of pale green dwarf (Acholeplasma laidlawii var. granulum incertae sedis) in Ukraine: pathogenicityand virulence factors and host reactions K. S. Korobkova, V. P. Patyka 30 Regulation of nitrogen-carbon interactions in agroecosystems in the forest-steppe zone of Ukraine V. A. Velichko, О. V. Demidenko 35 Soil Spatial Heterogeneity and Systems of Agriculture V. V. Medvedev 50 Detection of antibiotics, active against Bacillus subtilis, in grain and feed O. V. Trufanov, А. M. Kotyk, V. A. Trufanova, О. V. Tereshchenko, О. M. Zhukorskiy 60 Transforming growth factor β1, pituitary-speciﬁ c transcriptional factor 1 and insulin-like growth factor I gene polymorphisms in the population of the Poltava clay chicken breed: association with productive traits R. A. Kulibaba, A. V. Tereshchenko 67 Inﬂ uence of humus acids on mobility and biological availability of iron, zinc and copper A. I. Fateev, D. O. Semenov, K. B. Smirnova, A. M. Shemet 73

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UDC 582.4 : 582.662 + 581.9 + 581.522.6 + 581.527 QUINOA AS A PROMISING PSEUDOCEREAL CROP FOR UKRAINE S. L. Mosyakin, V. V. Schwartau 1 M. G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine 2, Tereshchenkivska Str., Kyiv, Ukraine, 01601 2 Institute of Plant Physiology and Genetics, National Academy of Sciences of Ukraine 31/17, Vasylkivska Str., Kyiv, Ukraine, 03022 e-mail: [email protected]; [email protected] Received on February 09, 2015

The article provides an assessment of perspectives of quinoa (Chenopodium quinoa L.) cultivation in Ukraine, based on international experience and original ﬁ eld tests, with the aim of ensuring further development and diversiﬁ cation of crop production in Ukraine and expanding modern crop rotation systems. The data on the taxonomic position of quinoa and its relationships with other species of the genus Chenopodium and the history of species domestication are provided. Quinoa is a crop of high nutritional value and can be used in gluten-free diets, which are important components of human ration. The results of test cultivation of quinoa in 2013–2014 under conditions of the experimental agricultural farm of the Institute of Plant Physiology and Genetics, Na- tional Academy of Sciences of Ukraine, located in Vasylkiv District (rayon) of Kyiv Region (oblast), are provided. It is concluded that quinoa is a promising crop for domestic grain producers. The introduction of quinoa into crop rotation systems can improve ecological conditions of agroecosystems and promote restoration of soil fertility in the country without diminishing the revenues of farmers. Key words: Chenopodium quinoa, quinoa, biology, taxonomy, domestication, agricultural technologies.

INTRODUCTION Except for the vegetation season of 2014, during the recent fi ve years Ukraine witnessed numerous long- The Big Three cereals – wheat, rice and corn – are term drought periods, which sharply decreased crop justly considered to be the main cereal crops and feed- yield of spiked cereals, technical and other crops. Each ers of humanity. According to FAO (http://www.fao. year the fi elds of the Steppe zone of Ukraine suffer org), cereals account for about 58 % of the annual crop from droughts of varied intensity. Rapid changes in the areas and provide humans with over 50 % of food calo- structure of Ukrainian agriculture caused by the forma- ries. By 2050, the share of the three mentioned crops tion of agroholdings (agricultural holding companies together is expected to amount up to 80 % of the in- and corporations) and the decline of animal farming crease in cereal consumption [1, 2]. However, it is ac- resulted in reduced crop rotations with the cultivation knowledged that along with the increase in the cultiva- of only immediately profi table and cost-effi cient crops tion of the main crops there is a need for diversifi cation with high nutrient removal from soils. This, in turn, re- in cultivation and consumption of other crops, includ- sulted in considerable worsening of phytosanitary con- ing those currently of lesser signifi cance (so-called mi- ditions of agroecosystems and segetal plant communi- nor crops), but which were in the past, and still are, ties, the increase in weed infestation levels and growing components of traditional agricultural systems [2–5]. threats of emergence of pesticide-resistant pathogens, These crops may be better adjusted to the conditions pests, and weeds, as well as the decline of soil fertility. of specifi c geographic regions of the Globe, where the A considerable share of winter wheat fi elds is occupied cultivation of the main crops is complicated, risky, or by cultivars and hybrids of foreign selection, which economically or ecologically unreasonable. often become frostbitten in vast areas. Among staple

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 3 MOSYAKIN et al. cereals registered in Ukraine, spring wheat does not The taxonomic position of quinoa and match the main domestic crop – winter wheat – in the its relationships to other species of Chenopodium yield level and qualitative indices, while other crops According to modern views, the family Chenopodia- (peas, for instance) also require the application of great ceae Vent. belongs to the order Caryophyllales Juss. ex amounts of pesticides. Bercht. & J. Presl of the unranked group of eudicot, or It is important to amend crop rotations in Ukraine true dicotyledonous angiosperms [8, 9]. According to with highly cost-effi cient spring crops with low level our variant of the system of fl owering plants [9], the of nutrient removal, which are resistant to high tem- taxonomic position of Chenopodiaceae is viewed as peratures and droughts and tolerant to the applica- follows: division Magnoliophyta Cronquist, Takht. & tion of agricultural chemicals. Therefore, so-called W. Zimmerm. ex Reveal (angiosperms, or fl owering pseudocereals nowadays appear to be quite promising plants), class Rosopsida Batsch (= Dicotyledonae, eu- food and technical crops. Pseudocereals are defi ned dicots), subclass Caryophyllidae Takht., order Caryo- as non-grass crops that can be used in much the same phyllales Juss. ex Bercht. & J. Presl., and suborder way as true cereals (grass cereals). It means that their Chenopodiineae J. Presl. seeds (“grain”) are used similarly to cereal grain, e.g., In all three published variants of the APG system, the pseudocereal seeds can be ground into fl our, grits, family Chenopodiaceae [8, 10, 11] was merged with etc. Typical pseudocereals include buckwheat (Fago- the family Amaranthaceae, the latter name having no- pyrum esculentum Moench, family Polygonaceae), menclatural priority and thus being used for the com- grain amaranths (several domesticated species of the bined family. However, it is diffi cult to agree with this genus Amaranthus L., family Amaranthaceae), as decision, especially considering available taxonomic well as quinoa (Chenopodium quinoa L.) and some and nomenclatural evidence [9, 12]. More detailed other species of the genus Chenopodium L. (family justifi cation of the need to preserve the independence Chenopodiaceae). of the family Chenopodiaceae will be published in an- Quinoa, an ancient crop that emerged approximately other article (Mosyakin, in print). 7,000 years BP (before present) in the mountain re- Within the family, the genus Chenopodium be- gions of the central part of the Andes in South America, longs to subfamily Chenopodioideae Burnett, tribe currently enjoys the period of its revival and renewed Chenopodieae Dumort. It has been suggested to popularity. It is caused by unique food (nutrient) char- synonymize the latter with tribe Atripliceae Duby acteristics of this species, its easiness of cultivation, [13], but it seems to be not the best option (Mosya- environmental resistance and tolerance, and a great kin, in print). number of available and diverse cultivars [2, 3, 5, 6]. The modern system of Chenopodium sensu lato and This crop was even used as an experimental object in other genera previously included into this aggregate is the NASA space program [7]. Therefore, the United based on the system of Aellen, a Swiss botanist [14– Nations General Assembly and FAO declared 2013 as 16]; that system was later improved and updated by the International Year of Quinoa (http://www.fao.org/ Scott [17], further on – by one of the authors of this quinoa-2013/en/). article [18–21], in particular, in collaboration with the As for nutritional properties of quinoa, its protein US botanist Clemants [22, 23]. Mosyakin and Clem- content in “grain” varies from 13.81 to 21.9 %, depend- ants [22, 23], among other taxonomic and nomencla- ing on a cultivar. Quinoa is one of a few edible plants tural proposals, fi rst justifi ed the phylogenetic isolation containing all essential amino acids and most closely of so-called glandular chenopods (aromatic species corresponding to human food standards approved by with glandular trichomes) from typical representatives FAO. The balance of essential amino acids in quinoa of Chenopodium sensu stricto (in the strict sense) and protein considerably exceeds the indices of amino acid transferred these species to a re-circumscribed genus content in wheat grain, barley, and soybean, approach- Dysphania R. Br., providing necessary nomenclatural ing in that respect the parameters of milk protein. Qui- combinations. This taxonomic decision was initially noa also has signifi cantly higher calcium, magnesium, accepted in our fl oristic and taxonomic treatments for iron and zinc contents as compared to those values in the Flora of North America North of Mexico and the wheat, corn, rice, barley, oats, rye, and triticale, among Flora of China [24, 25], later confi rmed by molecular others. It is important that quinoa is suitable for gluten- phylogenetic data [13, 26−28], and currently it is al- free diet [5]. most universally accepted.

4 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 QUINOA AS A PROMISING PSEUDOCEREAL CROP FOR UKRAINE In addition, modern molecular phylogenetic studies In pre-Columbian times (i.e. prior to the beginning justifi ed further segregation of several additional gen- of colonization by the Europeans) ancient inhabitants era from Chenopodium sensu lato (in the wide sense) of America managed to domesticate quite a wide range [13, 28, 29]. However, these radical changes do not af- of local wild plants; however, these crops considerably fect the group that includes quinoa and other pseudo- differed taxonomically from the domesticated plants of cereals of the genus, since they all remain in Chenopo- Eurasia. The unique feature of the ancient American dium in the strict sense. domestication experience is the use of exclusively local According to modern taxonomic views [30, 31], qui- resources, especially under very limited contacts even noa belongs to sect. Chenopodium (the typical section, between American geographical and cultural centers of containing the nomenclatural type of the genus), which domestication. Several main centers of plant domes- includes most species of the genus further segregated tication and origin of agriculture, with their peculiar into several subsections. Subsection Favosa (Aellen) sets of crops, are distinguished in the Americas (North, Mosyakin & Clemants (= Chenopodium series Fa- Central, and South) (see reviews for further informa- vosa Aellen) includes species geographically ranging tion [45–53]). from South America (C. quinoa Willd., C. hircinum The following centers of plant domestication are usu- Schrad. etc.) to North America (a group of species re- ally distinguished, with some variants, in the Americas: lated to C. berlandieri Moq.) and Eastern and South ▪ Mesoamerican upland and Mesoamerican lowland Asia (C. fi cifolium Sm. sensu lato). The latter species centers (remarkable domesticates are corn Zea mays L., (the lectotype of subsect. Favosa) was widely natural- grain amaranth Amaranthus cruentus L. = A. panicula- ized in other regions outside its initial natural range, tus L., species of such genera as beans Phaseolus, squash due to which it was sometimes erroneously considered and pumpkin Cucurbita, paprika Capsicum etc.); a species of Mediterranean origin. The phylogenetic integrity of the newly outlined subsect. Favosa was ▪ Andean center (potato Solanum tuberosum L. s.l. conclusively supported by modern molecular and phy- (some taxa), ulluco Ullucus tuberosus Caldas, oca Ox- logenetic evidence [13, 28] and comparative anatomic alis tuberosa Molina; quinoa Chenopodium quinoa and carpological data [32]. Willd., qañiwa C. pallidicaule Aellen, foxtail amaranth Amaranthus caudatus L. etc.); Issues of taxonomy and evolution of several species of Chenopodium, in particular, American cultivated ▪ Arizonian-Sonoran center (formed under the infl u- races of quinoa (C. quinoa), huazontle (C. nuttalliae ence of the Mesoamerican upland center; also includes Safford), and their wild relatives (C. hircinum etc.) specifi c cereals at early stages of domestication, in par- and probable ancestors were considered in some sub- ticular, barnyard millet from the Echinochloa muricata stantial, as of that time, publications by Wilson and his (P. Beauv.) Fern. group, species of millet Panicum s.l., colleagues [35−42]. Even at that time, these integral barley Hordeum s.l., bromegrass Bromus s.l. etc.); studies involved the methods of traditional taxonomy, ▪ Eastern North American, also known as Alaba- palaeoethnobotany (archaeobotany), biochemistry, and man-Illinoian, center (Chenopodium berlandieri Moq. – biosystematics. C. nuttallii aggr., species of genera Helianthus, Cy- Recently the nomenclatural type (lectotype) of the clachaena, Ambrosia, Polygonum s.l., Phalaris etc.); species has been selected for C. quinoa [43]; this her- ▪ Peruvian coastal center (mainly adoption of crops barium specimen (the reference sample of the species) from the Andes); is deposited at the Willdenow Herbarium of the Botani- cal Garden and Museum in Berlin (Botanischer Garten ▪Amazonian, also known as Amazonian-Orinocan or und Botanisches Museum Berlin-Dahlem). Brazilian-Paraguayan, center (manioc Manihot escu- lenta Crantz, sweet potato Ipomoea batatas (L.) Lam., History and peculiarities of domestication and some other crops). of quinoa and some other unique crops of America As we can see, the absolute majority of domesticates The issues of history and peculiarities of domestication of American origin, especially those used at the stage of quinoa and some other unique crops of America has of early agriculture, are remarkable for their ruderal already been considered in detail by one of the authors (explerent) life strategy; by their ecological and phy- of this article [44]; subsequent discussion in the present tosociological features they are somewhat similar to article is partially based on that earlier publication. weeds or plants of marginal habitats [44].

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 5 MOSYAKIN et al. Species of families Chenopodiaceae and Amarantha- jonesianum B.D. Smith (the subspecies considered al- ceae occupy a prominent place among the cultivated ready extinct), and some others. Among the mentioned native plants of America [6, 35, 36, 39−42, 49, 52−59]. species and races, only quinoa still maintains its eco- The independent domestication of ruderal species of nomic importance, while remaining species have been Chenopodiaceae and Amaranthaceae in America can either forgotten (for instance, domesticated North be viewed as a unique domestication experiment. The American large-seeded races of the C. berlandieri documented role of these domesticated plants in Ame- group), or are preserved only locally, as relict crops. rica was much more important than the role of crops The majority of domesticated species and subspe- of these families in the Old World. With the exception cies of Chenopodium demonstrate some morphologi- of beet (Beta vulgaris L.) and spinach (Spinacia ole- cal and physiological changes likely caused by do- racea L.) that originated in the Mediterranean region mestication: disruption of the normal mechanism of [60, 61], chenopod crops did not have signifi cant distri- spontaneous dissemination (however, seeds are easily bution in the Old World, mainly being used there from shed while being thrashed), lost (or at least decreased) time to time as leaf crops (salad plants) or as substitutes dormant period in seeds; the sizes of seeds increase of normal and wholesome food in famine years (so- considerably; the pericarp is easily isolated from the called famine food). testa (seed coat), the testa itself becomes thinner, due The ethnobotanical data on the use of wild repre- to which the “seeds” acquire shades of white, yellow sentatives of Chenopodiaceae and Amaranthaceae by or reddish color. native peoples of North and South America are pres- As we can see, practically all cultivated species of ent in many publications (see above), and similar data Chenopodium were domesticated independently in on North America were critically analyzed and sum- various regions of America. In particular, the fact of in- marized in a thorough monograph by Moerman [63]. dependent origin of Andean cultivars of C. quinoa and Fresh green parts of many representatives of Cheno- Mexican cultivars of C. nuttaliae [42] has been proven podiaceae (species of genera Atriplex and Chenopo- without doubt. Despite numerous studies, the origin of dium) were used as green (salad) vegetables or pot- quinoa is still a matter of dispute. This species is likely herbs. The seeds of many wild species of Cheno- to have originated from one of the local Andean tet- podiaceae served as substitutes of cereals: grit or raploid wild species belonging to subsect. Favosa. At fl our were made of them; they were soaked, ground, present the researchers [64, 65] are inclined to think boiled and prepared as porridge, bread substitute, that tetraploid American species of Chenopodium from and pemmican-type dry mixtures. In particular, Mo- subsect. Favosa (including the cultivated ones) are al- erman [63] described the use of fruits and seeds of lopolyploids that originated from ancient hybridization at least 9 species of Atriplex (orache), 10 species of events between the local American and probably some Chenopodium, species of Cycloloma Moq. (winged Asian (or Eurasian) species. In particular, it is believed pigweed), Corispermum L. (bugseed) etc. as food that one diploid (2n = 18) ancestor of species of the products, but the real number of species actually tetraploid (2n = 36) American complex (C. berlandieri, used must have been much higher, as ethnobotanists C. hircinum, etc.) could be the North American species often did not indicate the precise species-level iden- C. standleyanum Aellen. Eurasian species C. album L. tifi cation of plants, reporting only the genus or an- or C. fi cifolium were suggested for the role of the sec- other supraspecifi c group. ond probable diploid ancestor. In our opinion, typical Archaeological and palaeoethnobotanical data (see C. album, being hexaploid (2n = 54), cannot be consid- references above) indicate wide use of seeds of several ered as a potential parental species of a tetraploid tax- species of Chenopodium, which may be considered true on. Therefore, diploid C. fi cifolium is best suited for the domesticates, in North and South America: C. quinoa role of such an ancestral species. The species C. stand- (quinoa, the Andes), C. pallidicaule (qañiwa or qaña- leyanum was placed by us [24, 30] in a separate sub- wa, the Andes), C. nuttaliae Safford (= C. berlandieri section Standleyana Mosyakin & Clemants, which is subsp. nuttalliae (Safford) H.D. Wilson & C.B. Heiser; extremely interesting in terms of its phytogeography, huazontle, Mexico), C. berlandieri (central and south- since it links North American and East Asian taxa. This western part of South America), C. bushianum Aellen subsection also includes East Asian C. bryoniifolium (= C. berlandieri subsp. bushianum (Aellen) Cronq., Bunge (= C. koraiense Nakai s. str.), C. gracilispicum North American Atlantic region), C. bushianum subsp. H.W. Kung (C. koraiense auct. p.p.), C. atripliciforme

6 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 QUINOA AS A PROMISING PSEUDOCEREAL CROP FOR UKRAINE Murr s. str. (C. badachschanicum auct., non Tzvelev), (2) Salares (southern highlands), (3) Inter Andean Val- North American C. standleyanum (C. boscianum auct. leys, (4) arid zones (in particular, western highlands), non Moq.), and probably also C. missouriense Ael- plants of (5) high altitudes and cool climate, (6) coastal len emend. F. Dvořák. Thus, there is an evident “East regions, (7) jungle and tropical zones, and (8) high Asian trace” in the origin of American tetraploid spe- rainfall and humidity zones. Such a considerable diver- cies of subsect. Favosa (including C. berlandieri, C. sity of cultivars and forms opens excellent prospects hircinum, and C. quinoa). for the search for cultivars best pre-adapted to the conditions of different physiographic and climatic zones As currently generally estimated, quinoa emerged of Ukraine. about 7,000 years BP, but there are some indications to an older age of this crop. The archaeobotanical remains Peculiarities of quinoa cultivation of the fruits similar to those of quinoa in the northern part of Peru on the western slopes of the Andes are dat- The culture of quinoa is new for domestic pro- ed by the Las Pircas period (9,800–7,800 years B.P.); ducers in Ukraine. Under the conditions of south- however, it is possible that these earliest fi nds indicate ern Polissia and northern Forest-Steppe zones of the pre-domestication use of plants from natural habi- Ukraine – at the experimental agricultural farm of tats by tribes of hunters-gatherers rather than complete the Institute of Plant Physiology and Genetics, NAS domestication [43]. of Ukraine (Vasylkiv District, Kyiv Region) – quinoa was sown in well prepared leveled soil in early Contrary to the older Andean complex, the so- May in 2013–2014 at stable positive temperatures called “Eastern complex” of crops was formed in (8−10 °С) and suffi cient humidity of soil. The row North America in the eastern and central parts of the planting was made 1–2 cm deep with the row spac- present-day USA about 5,000 years ago, i.e. much later than the time of quinoa domestication in South ing of 30 cm. The seeds were kindly provided by Dr. America. Besides the races of the C. berlandieri – C. Jamal B. Rakhmetov (M.M. Gryshko National Bo- bushianum group, this complex also included Am- tanical Garden, NAS of Ukraine). Late terms of sow- brosia trifi da L. (giant ragweed), Helianthus annuus ing allow using non-selective herbicides based on N- L. s.l. (sunfl owers) and local species of genera Cy- (phosphonomethyl)glycine (Glyphosate), in spring clachaena (marsh-elder) and Amaranthus. Consider- and relieving the fi elds from wheatgrass, Elytrigia able domestication-caused morphological changes repens (L.) Desv. ex Nevski (Agropyrum repens L.), in the seeds of cultivated species of Chenopodium sow-thistle species (Sonchus spp.), and other weeds. are reliably traced in the archaeological records of Being the predecessor, quinoa allows decreasing the this region for the period of 3,500–2,000 years B.P. weed infestation of the fi eld with thermophylic grass During that period, forms with large (about 2 mm species (in particular, foxtail species, Setaria spp. in diameter) seeds and thinner testa (10–15 μm as etc.) on condition of introducing graminicides of the compared to 40–78 μm in wild species) appeared, classes of aryloxyphenoxypropionic acid, cyclohex- which may testify to the emergence of true domes- anediones etc. A considerable drawback is a high ticated cultivars. The cultivation of local Chenopo- level of infestation of quinoa fi elds with common dium domesticates (as well as many other species) goosefoot (Chenopodium album L., a species very in North America gradually ceased starting with the widespread in Ukraine) and other related species of 2nd–3rd centuries C.E. due to the adaptation of more the genus, as well as species of the genus Atriplex L. productive Mesoamerican (Central American) crops, (orache). However, if quinoa is used as a predecessor in particular, corn, several species of beans, squash of winter wheat or corn, on the fi elds of which the and pumpkin [53]. However, the ancient tradition of use of herbicides to control Chenopodium album is cultivation of South American species of Chenopo- effi cient, high levels of weed biomass accumulation dium (especially quinoa) continued and still contin- may be considered as application of green manure. ues until now. Quinoa responds very well to soil enrichment with Quinoa has many native landraces and other cultivars nitrogen [66]. To cultivate the organic crop, quinoa adapted to extremely diverse natural and climatic con- should be sown after the application of organic fertil- ditions of South America. In particular, the following izers, green manure, nitrogen-fi xing grain legumes, or groups of cultivars, landraces, and forms are distin- either nitrogen-fi xing species of plants should be used guished [6]: (1) Altiplano (northern Andean highlands), as green manure.

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 7 MOSYAKIN et al. Quinoa demonstrates good response to the foliar ap- mal for the regions in the Steppe, Forest-Steppe, and plication of macro- and microelements. However, the Polissia zones, the control of dicotyledonous species damage of quinoa leaves with agrochemicals may be of weeds in the fi elds, and some aspects of introduc- observed as early as after the introduction of 1–3 % ing the mechanical aids into crop production. working solutions of fertilizers. Foliar biofortifi cation Кіноа – перспективна для України of quinoa yield is effi cient using fertilizers containing «зернова» культура nutrition elements and amino acids (Megafol, Megafol С. Л. Мосякін 1, В. В. Швартау 2 Protein, Isabion). e-mail: [email protected]; Due to the fact that quinoa seeds have high content e-mail: [email protected] of proteins and amino acids, it was demonstrated that 1 it may condition the increase in selenium accumulation Інститут ботаніки ім. М. Г. Холодного НАН України Вул. Терещенківська, 4, Київ, Україна, 01004 in the yield in the forms of selenomethionine, selenate 2 Інститут фізіології рослин і генетики НАН України (Se(VI)), and in non-protein compounds. On condition Вул. Васильківська, 31/17, Київ, Україна, 03022 of the application of barium selenate and barium sel- enite during the vegetation period, quinoa seeds accu- Для забезпечення зростання й диверсифікації рос- mulate organic selenium [67]. линництва України і розширення сучасних сівозмін на основі узагальнення світового досвіду та проведених Quinoa is a known photoperiod-responsive crop. польових експериментів досліджено можливість виро- For instance, cultivars from Ecuador need at least 15 щування в країні кіноа (Chenopodium quinoa L.). Наве- days with short illumination period for 10 h to transfer дено дані з систематичного положення кіноа та спо- to the fl owering phase. The duration of the vegetation рідненості культури з іншими видами роду Chenopo- period may vary in different years, being from 108 to dium, з історії доместикації виду. Кіноа має високу хар- 148 days [68]. чову цінність, її можна використовувати у безглюте- нових дієтах, що є важливим компонентом раціону лю- Quinoa is uniquely adapted to the cultivation in dif- дини. Представлено результати вирощування кіноа у ferent agroecological regions; it grows at relative hu- 2013−2014 рр. за умов дослідного сільськогосподар- midity from 40 to 88 % and can endure the tempera- ського виробництва Інституту фізіології рослин і гене- tures from −4 °С to +38 °С. This crop uses water with тики НАН України (Васильківський район Київської об- high effi ciency and forms the yield even at 100–200 ласті). Таким чином, культура кіноа є перспективною mm of rainfall during the vegetation season [5]. How- для вітчизняних зерновиробників. Введення кіноа до ever, the physiological mechanisms of stress resistance сівозмін може покращити екологічний стан агрофіто- of this crop are studied insuffi ciently [69]. ценозів та сприяти відновленню родючості ґрунтів країни без зниження прибутків аґраріїв. In the vegetation season of 2014 with high amount Ключові слова: Chenopodium quinoa, кіноа, біологія, сис- of rainfall, the fi elds of quinoa in Kyiv Region were тематика, доместикація, технології вирощування. slightly infested with downy mildew (Peronospora farinosa) and sugarbeet root aphid (Pemphigus fusci- Киноа – перспективная для Украины cornis). No infestation with these pathogen and pest «зерновая» культура should be expected under dry conditions of the vegeta- С. Л. Мосякин 1, В. В. Швартау 2 tion season. e-mail: [email protected]; In 2014, the yield of quinoa was 1.05 t/ha, thus mak- e-mail: [email protected] ing the cultivation of this crop highly cost-effi cient. 1 Институт ботаники им. Н. Г. Холодного НАН Украины According to many authors, the yield may be as high Ул. Терещенковская, 4, Киев, Украина, 01004 as 2.5 t/ha [70]. 2 Институт физиологии растений и генетики НАН Украины Therefore, quinoa is a promising crop for domestic Ул. Васильковская, 31/17, Киев, Украина, 03022 grain producers. The introduction of quinoa into crop Для обеспечения роста и диверсификации растение- rotation systems can improve ecological conditions водства Украины, а также расширения современных of agrophytosystems and promote the restoration of севооборотов на основе обобщения мирового опыта soil fertility in the country without diminishing the и проведенных полевых экспериментов исследована revenues of farmers and grain producers. The issues возможность выращивания в стране киноа (Chenopodi- still to be solved in promoting quinoa cultivation in um quinoa L.). Приведены данные по систематическому Ukraine are the identifi cation of crop genotypes opti- положению киноа и родстве культуры с другими вида-

8 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 QUINOA AS A PROMISING PSEUDOCEREAL CROP FOR UKRAINE ми рода Chenopodium, по истории доместикации вида. 12. Takhtajan A. Flowering plants. 2nd ed. Berlin, Springer. Киноа имеет высокую пищевую ценность, ее можно 2009; xlvi + 871 p. использовать в безглютеновых диетах, что является 13. Fuentes-Bazan S, Uotila P, Borsch T. A novel phylogeny- важным компонентом рациона человека. Представлены based generic classifi cation for Chenopodium sensu lato, результаты выращивания киноа в 2013−2014 гг. в усло- and a tribal rearrangement of Chenopodioideae (Cheno- виях опытного сельскохозяйственного производства Ин- podiaceae). Willdenowia.2012;42:5–24. ститута физиологии растений и генетики НАН Украи- 14. Aellen P. Chenopodium amaranticolor Coste et Reynier, ны (Васильковский район Киевской области). Таким Ch. purpurascens Jacquin, Ch. giganteum Don, Ch. Qui- образом, культура киноа является перспективной для noa Willd., Ch. Moquinianum Aellen und Ch. Reynieri отечественных зернопроизводителей. Введение киноа в Ludwig et Aellen. Eine nomenklatorischen und systema- севообороты может улучшить экологическое состояние tischen Studie. Ber. Schweiz. Bot. Ges.1929;38:5–23. агрофитоценозов и способствовать восстановлению пло- 15. Aellen P, Just T. Key and synopsis of the American дородия почв страны без снижения прибылей аграриев. species of the genus Chenopodium L. Amer Midl Nat. Ключевые слова: Chenopodium quinoa, киноа, биология, 1943;30(1):47–76. систематика, доместикация, технологии выращивания. 16. Aellen P. Chenopodiaceae. G. Hegi. Illustrierte Flora von Mitteleuropa. Berlin & Hamburg: Paul Parey. 1960– REFERENCES 1961; Band 3, Teil 2 (Lief. 2–4):533–762. 1. Rosegrant M, Ringler C, Sinha A, Huang J, Ahammad H, 17. Scott AJ. A review of the classifi cation of Chenopodium Zhu T, Msangi S, Sulser T, Batka M. Exploring alterna- L. and related genera (Chenopodiaceae). Bot Jahrb Syst. tive futures for agricultural knowledge, science and tech- 1978;100:205–20. nology (AKST). ACIAR Project Report ADP/2004/045. 18. Mosyakin SL An outline of a system for Chenopodium L. Canberra, ACIAR.2009;84 p. (species of Europe, North and Central Asia). Ukr Bot Zh. 2. Food Outlook: Biannual report on global food markets. 1993; 50(5):71–7. June 2013. Rome, FAO.2013;132 p. 19. Mosyakin SL, Chenopodium L Flora Europae Orientalis. 3. Neglected crops: 1492 from a different perspective. Eds J. St. Petersburg, Mir i Semya-95.1996;Vol. 9:27–44. E. Hernandes Bermejo, J. Leon. FAO Plant Production and 20. Mosyakin SL The system and phytogeography of Che- Protection Series, no. 26. Rome, FAO.1994; xxii + 341 p. nopodium subgen. Blitum (L.) I. Hiitonen (Chenopodia- 4. Descriptores para quinua (Chenopodium quinoa Willd.) ceae). Ukr Bot Zh.2002;59(6):696–701. y sus parientes silvestres. Roma, Bioversity Internation- 21. Mosyakin SL The system and phytogeography of Cheno- al. 2013; vi + 52 p. podium subgen. Chenopodium (Chenopodiaceae). Ukr 5. Quinoa: An ancient crop to contribute to world food se- Bot Zh.2003;60(1):26–32. curity. FAO, Regional Offi ce for Latin America and the 22. Mosyakin SL, Clemants SE. New nomenclatural combi- Caribbean. 2011; vi + 55 p. nations in Dysphania R. Br. (Chenopodiaceae): taxa oc- 6. Bazile D, Fuentes F, Mujica A. Historical perspectives curring in North America. Ukr Bot Zh.2002;59(4):380– and domestication. Ch. 2. Quinoa: botany, production 85. and uses. Eds A. Bhargava, S. Srivastava. Wallingford, 23. Mosyakin SL, Clemants SE. Further transfers of glan- CABI.2013;16–35. dular-pubescent species from Chenopodium subg. Am- 7. Schlick G, Bubenheim DL. Quinoa: candidate crop for brosia to Dysphania (Chenopodiaceae). J Bot Res Inst NASA’s Controlled Ecological Life Support Systems. Texas.2008;2(1):425–31. Progress in new crops. Ed. J. Janick. Arlington, ASHS 24. Clemants SE, Mosyakin SL. Dysphania R. Brown; Che- Press.1996;632–40. nopodium Linnaeus. Flora of North America north of 8. The Angiosperm Phylogeny Group. An update of the An- Mexico. Ed. by FNA Editorial Committee. New York, giosperm Phylogeny Group classifi cation for the orders Oxford Univ. Press.2003;Vol. 4:267–99. and families of fl owering plants: APG III. Bot J Linnean 25. Zhu Gelin, Mosyakin SL, Clemants SE. Chenopodiaceae. Soc. 2009; 161(2):105–21. Flora of China. Eds Wu Zhengyi, P. H. Raven. Beijing, 9. Mosyakin SL. Families and orders of angiosperms of the Science Press & St. Louis, Missouri Botanical Garden fl ora of Ukraine: a pragmatic classifi cation and placement in Press.2003;Vol. 5:351–414. the phylogenetic system. Ukr Bot Zh.2013;70(3):289–307. 26. Kadereit G, Borsch T, Weising K, Freitag H. Phylogeny of 10. The Angiosperm Phylogeny Group. An ordinal classifi ca- Amaranthaceae and Chenopodiaceae and the evolution of

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UDC 633.63:632.651 BREEDING AND USAGE OF SUGAR BEET CULTIVARS AND HYBRIDS RESISTANT TO SUGAR BEET NEMATODE HETERODERA SCHACHTII L.A. Pylypenko 1, K.A. Kalatur 2 1 Institute of Plant Protection of NAAS 33, Vasylkivska Str., Kyiv-33, 03022 2 Institute of Bioenergy Crops and Sugar Beet of NAAS 25, Klinichna Str., Kyiv-141, 03141 e-mail: [email protected] Received on February 09, 2015

Heterodera schachtii Schmidt, 1871 is one of the most economically important pests of sugar beet (Beta vulgaris L.) worldwide. It is also widespread in most sugar beet growing regions in Ukraine causing serious yield reduction and decreasing sugar content of sugar beet in infested fi elds. An advanced parasitic strategy of H. schachtii is employed to support nematode growth, reproduction and harmfulness. In intensive agriculture systems the nematode control measures heavily rely on nematicides and good agricultural practice (crop rotation in the fi rst place). But alternative strategies based on nematode resistant sugar beet cultivars and hybrids are required as none of nematicides approved for the open fi eld application are registered in Ukraine. Here we review the achievements and problems of breeding process for H. schachtii resistance and provide the results of national traditional breeding program. Since the beginning of 1980s fi ve sugar beet cultivars (Verchnyatskyi 103, Yaltuschkivska 30, Bilotcerkivska 45, BTs-40 and Yuvileynyi) and seventeen lines partly resistant or tolerant to H. schachtii have been obtained throughout targeted crossing and progenies assessment in the infested fi elds. The further directions for better utilization of genetic sources for nematode resistance presented in national gene bank collection are emphasized. There is a need for more accurate identifi cation of resistance genes, broader application of reliable molecular markers (suitable for marker-assisted selection of nematode resistant plants in the breeding process) and methods for genetic transformation of plants. Crop cash value and national production capacity should drive the cooperation in this fi eld. Knowledge as well as germplasm exchange are thereby welcomed that can benefi t breeding progress at national and international level. Key words: sugar beet, Heterodera schachtii, nematode resistance, breeding.

INTRODUCTION The distribution and harmfulness of sugar beet Sedentary cyst-forming nematodes of family Heterode- nematode in sugar beet ﬁ elds ridae are the most dangerous obligate parasites, damag- Sugar beet nematode is known for its spreading in 18 ing the root system of many cultivated crops and causing European countries where sugar beet is cultivated: in their diseases and serious yield reduction. Several species the Netherlands and Poland (on 25 % ﬁ elds), on the ter- are considered to be the most harmful, including grass ritory of former Czechoslovakia (20 %) and Yugosla- (Punctodera punctata), oat (Heterodera avenae), soybean via (10−12 %), Germany (20 %), Italy (19 %), Sweden (Н. glycines), potato golden (Globodera rostochiensis) (10−15 %), Spain, Great Britain (10 %), and France and pale (G. pallida), hop (H. humuli), clover (H. trifolii), (3 %) [3]. In addition, it was found in Belgium, Den- alfalfa (H. medicaginis), pea (H. goettingiana) and sugar mark, Finland, Ireland, Switzerland, Austria and Bul- beet (Н. schachtii) cyst nematodes. The latter is the agent garia [1−3]. Sugar beet nematode was also registered of heteroderosis – a sugar beet disease, wide-spread in in the USA, Canada, Romania, Latin America, Japan, many countries of the world [1, 2]. Australia, India, Morocco, Algeria, Turkey, Israel, Tu-

12 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 BREEDING AND USAGE OF SUGAR BEET CULTIVARS AND HYBRIDS nisia, Iran and on the African continent (Senegal and The reason of high harmfulness of sugar beet nem- Gambia) [1–3]. atode is the impairment of the recommended shift of In the former USSR sugar beet nematode was fi rst crops in the crop rotation and the reduction of the terms discovered by Professor Korab in Ukraine in August of of returning the plants – nematode hosts – to the fi eld 1923 on the fi elds of the Pii sugar beet farm, Kyiv re- (all species of beet, cabbage, radish, mustard, ripe, gion [4] (at present it is spread in 18 regions of Ukraine) spinach, rutabaga, turnip), disrespect of scientifi cally [5–7]. Later (in 1932) it was found in Lithuania [8] and grounded systems of the soil cultivation and the sys- Kazakhstan (in 1939) [9]. Much later – in 1964 – the tems of fertilization, insuffi cient application with plant outbreak of heteroderosis was registered in Moldova protection products. The build up of nematode popula- [10], and in 1966 – in Kirghizia [11]. Similar outbreaks tions in soil is also promoted by weeds of Chenopodia- were reported in Belarus, Estonia, Tajikistan [10], Uz- ceae, Brassicaceae, Caryophyllaceae, Labiateae, and bekistan [12], Georgia [13] and Russia [14]. Polygonaceae families [1, 2, 4, 15, 16, 18–22]. Apart from Н. schachtii distribution rate, external Negative changes, occurring in a plant organism un- symptoms of plant damage have been studied and de- der the impact of H. schachtii invasion, lead to a re- scribed, which are rigorous in the fi eld with a high level markable reduction in the average weight of sugar beet of soil infestation with this pest (over 300 eggs and lar- roots and their sugar content, which eventually ends up vae of nematode (e + l) in 100 ccm of soil). Due to such in the crop yield reduction and sometimes in the death nematode population density the majority of plants are of plants [1−5, 15, 16, 18−22]. It is discovered that the repressed in their growth and development, their leaves degree of such losses depends on the pre-sowing den- become pale green, later the outer leaves discolor to sity of sugar beet nematode population in soil, plant- yellow and die back. Crop damage by heteroderosis predecessor, planting time, soil-climatic conditions of usually appears as patches of poorly growing plants the region, weather conditions of the vegetation period (late June – early July). If the plants are lifted out in etc. Sugar beet seed crop is the most susceptible to het- this period, the beet-roots look “bearded” due to a high eroderosis, because of these plants root system, which number of side roots bearing white bodies of nematode is not as deep as that of plants of the fi rst vegetation year females. The most severe symptom of the disease is [1, 2, 15, 16, 18–21]. For instance, there was a noted complete plant death [1, 2]. sharp reduction in the sugar beet productivity with the At the low (1–100 e + l/100 ccm of soil) and medium pre-sowing density of sugar beet nematode population (101–300 e + l/100 ccm of soil) sugar beet nematode in the range of 210–2600 e + l/100 ccm of soil [19]. In population densities in soil the infested plants do not particular, if 100 ccm of soil contains 210–280 e + l of differ from the healthy ones, however in the afternoon, nematodes, sugar beet yield reduction is 5–10 %, for when the air temperature reaches 20 °С and above, 500 e + l/100 ccm of soil – 20 %, and for 850 e + l/100 their leaves wilt and fall to the ground [1, 2]. ccm of soil – 30 %. The reduction of sugar beet seeds for the abovementioned infestation levels of nematode Sugar beet nematode parasitizing in the root system in soil will be 7–14, 29 and 42 %, respectively. Further leads to the impairment of its main function and the increase in nematode population densities from 1,550 plant does not receive necessary mineral substances to 2,600 e + l/100 ccm of soil will promote the reduc- and water from the soil. At the same time there are tion in the root weight by 40–50 %, and the seeds – by pathological changes of a whole number of physiologi- 57–70 %. The reduction in the sugar content of roots is cal processes: reduction in the total number and area of statistically reliable only at a high level of soil infes- plant leaves, in the content of green pigments, caroti- noids, phosphor, nitrogen compounds and potassium, tation with sugar beet nematode and may range from as well as decrease in photosynthesis intensity; growth 0.8 to 2 % [19]. In the developed countries, the losses regulation is impaired and the breathing process is of sugar beet root weight and reduction in their sugar slowed down [1, 2, 15]. Stunted and wilted plants are content from heteroderosis at the level of 25–30 % are not capable of fi ghting the other ptytopathogens of estimated as 600 USD per 1 ha [23]. the root system and leaves completely. Therefore, the It is noteworthy that most Ukrainian fi elds have the fi elds, infested with sugar beet nematode, are in danger medium level of soil infestation with sugar beet nema- of even wider infestation of sugar beet with Pythium tode of 600 e + l/100 ccm of soil [5, 6], but on some disease, cercosporosis and root rot during the vegeta- farms this level reaches up to 7,000 e + l/100 ccm of tion season [16–18]. soil and above, which leads to the death of plants [5,

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 13 PYLYPENKO et al. 6, 24]. Usually the fi elds of the greatest outbreaks of infested with this pest, is the most ecologically safe and heteroderosis are located on the farms in old areas of economically justifi ed measure [28, 29]. The fi rst se- beet growing; the farms, associated with sugar refi n- lection work in breeding nematode-resistant sugar beet eries and factories, specialized in breeding beet seeds cultivars was started by Moltz in Germany in 1917 [3]. production. In particular, the areas of the highest sugar In 1936 the studies were continued by Hulsenberg [30] beet nematode population densities in soil are located and since 1954 this task was also pursued by Rietberg in Kyiv, Vinnytsia, Cherkasy, Sumy, Chernihiv, and [31] and Filutowicz, Kuzdowicz [32]. However, the Kharkiv regions, and the beet yield reduction due to most relevant scientifi c achievements in solving this heteroderosis in these regions reaches 70–80 % [5, 6, problem were made by Savitsky, who had been work- 19, 20, 21, 24–26]. Due to high density of nematode ing at breeding nematode-resistant sugar beet lines in population in soil there were cases of death of sugar the USA for over 25 years [33–40]. beet seed plants in Cherkasy, Kyiv, Zhytomyr, and In 1950s the genes of resistance to H. schachtii were Ivano-Frankivsk regions [20]. Thus, the usage of the discovered in three wild beet species of the section nematode infested fi elds should be planned consider- Рatellarеs Tran.: Beta procumbens Chr. Sm. (1815), B. ing economic threshold which does not exceed 200 e patellaris Moq. (1849), B. webbiana Moq. (1840) [41]. +l/100 ccm of soil in the forest-steppe of Ukraine [19]. At the same time there was the fi rst crossing of culti- The analysis of the data obtained testify that sugar vated В. vulgaris with the wild species B. procumbens beet nematode is one of the most wide-spread and [42]. The nature of resistance of these beet cultivars to harmful pathogens of sugar beet in many countries heteroderosis is the inability of sugar beet nematode of the world and the losses, incurred from it, are esti- to develop and reproduce in their roots. It was also de- mated as over USD 95 million [27]. Therefore, starting termined that nematode is capable of penetrating roots from the moment of its discovery the researchers tried of both resistant and susceptible sugar beet cultivars. to determine the factors, which would allow limiting However, the nutrition of its larvae in the roots is pos- the population densities and harmfulness of nematode sible only in presence of special gigantic cells or syn- in the soil, and to elaborate the recommendations on cytia, formed due to the dissolution of cellular walls reducing the losses of sugar beet yield and seeds from under the impact of enzymes of these larvae [1, 2]. The heteroderosis. The modern and widely used system of degradation of the syncytia formed takes place only in integrated crop protection from sugar beet nematode resistant plants, leading to the death of nematode larvae involves preventive, agrotechnical, organizational, eco- of the 2nd and 3rd stages of development [3]. nomic and chemical measures. In particular, the system foresees the next measures: prevention of nematode In addition to species from the section Рatellarеs, introduction into the fi elds along with the equipment, other subspecies and cultivars of В. vulgaris (namely, tools for soil tillage etc.; following the recommended diploid and tetraploid sugar beet [31, 32, 34, 43–47], crop rotation, use of better predecessors; timely and fodder beet [48], red beet [49], mangold [50] and qualitative main and pre-sowing soil tillage; introduc- B. maritima cultivar [51]) were studied for the most tion of organo-mineral and microfertilizers, balanced compatible interspecies crossing. Such species as for the needs of the fi eld; high quality of the seeds and B. macrocarpa and B. atriplicifolia, were used as ma- its pre-sowing treatment with protective and stimulat- ternal plants for the crossing with B. procumbens, B. pa- ing substances; optimal terms of sowing; weed control tellaris, B. webbiana, as well as tetraploids, obtained as on all the crop rotation fi elds; timely nutrition of plants a result of hybridization of B. procumbens and B. web- in the vegetation period, etc. [1–7, 14–22, 24–26]. biana [52]. The positive results were achieved in Po- land while crossing tetraploid beet with three wild spe- The history and problems of breeding cies from the section Рatellarеs. However, the revealed sugar beet cultivars and hybrids, resistance against nematodes was lost in backcrosses resistant to sugar beet nematode В1 of the subsequent generations [53]. Similar results In addition to the abovementioned measures a rel- were obtained while crossing the cultivated forms of evant place in the modern system of protection from beet and B. webbiana. The fi rst and second generations heteroderosis is attributed to breeding and use of resis- of the hybrids obtained were remarkable for some resistant and tolerant sugar beet cultivars and hybrids. The tance to H. schachtii which was lost in the subsequent scientists of many countries of the world proved that generations [44]. In addition, it was discovered that cultivation of nematode-resistant plants on the fi elds, the sugar beet nematode resistance breeding, based on

14 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 BREEDING AND USAGE OF SUGAR BEET CULTIVARS AND HYBRIDS interspecies hybrids, faces other diffi culties as well – Savitsky’s work on the transmittance of the trait of it yields either inviable progeny or hybrids with low resistance to В. vulgaris was supported and contin- fertility [30, 40, 54, 55]. Summing up the researches ued by other scientists, in particular, by Speckmann, conducted prior to 1970, Savitsky presented the data De Bock, De Jong [56, 57], Heijbroek et al. [58, 59], about low viability of hybrid progeny: only 53 out of Loptien [60, 61], Jung et al. [62, 63], Lange et al. 3,000 obtained plants proved to be viable [40]. Out of [64–66]. 618 inviable sprouts, grafted to fl oriferous shoots of In the former USSR the breeding of sugar beet culti- sugar beet, only 57 plants reached the stage of fl our- vars and hybrids for resistance and tolerance to sugar ishing, while most hybrids of the fi rst generation did beet nematode started in Ukraine and Russia [14, 54, not form seeds or formed very little thereof [40]. In- 55, 67, 68]. Scientists worked in several directions, in- viability and low fertility of hybrids, no resistance to volving different methods of investigation, genetic in nematode and the doubts regarding the possibility of particular, to obtain hybrids of cultivated plants with gene exchange between В. vulgaris and cultivars of the wild beet species of the section Рatellarеs, bearing re- section Рatellarеs led to the termination of any work in sistance genes to sugar beet nematode, as well as the this direction in almost all the countries [40]. method of classic breeding with the search for new Regardless of some problems which occurred while sources of resistance among different beet cultivars, breeding nematode-resistant sugar beet hybrids, some which had already been obtained. However, due to the scientists continued their work to solve these issues formation of inviable forms or hybrids with low fertil- anyway with Savitsky gaining the highest progress. Af- ity the desired results were not received [54, 55]. In ter crossing tetraploid В. vulgaris with diploid B. pro- addition, a number of undesired traits were transmitted cumbens, out of 6,750 plants four nematode-resistant to the gene pool of sugar beet along with the resistance trisomics were selected that had 18 В. vulgaris chromo- to nematode. Therefore, the main efforts of researchers somes and one B. procumbens chromosome responsible were directed towards the work in estimating and se- for resistance. This B. procumbens chromosome and the lecting nematode-resistant forms among the cultivars, nematode-resistant trait were transferred to the eighth lines and selection numbers of beet of different genetic backcross generation. As a result of the crossing-over origin. During 1925–1927 in Ukraine, on the fi elds of between chromosomes of В. vulgaris and B. procum- Pii and Nizov sugar refi neries, Professor Korab tested bens in trisome plants, two diploid nematode-resistant 11 sugar beet cultivars for resistance to H. schachtii plants were obtained in the progenies of trisomics and [67]. In Russia the determination of the nematode resis- resistance was transferred from both of these plants to tance degree of sugar beet and some wild beet species the fi rst generation of sugar beet hybrids. Due to these was pursued by Skarbilovich in 1940 [14] and Briush- researches the chromosome segment of the wild spe- kova in 1971 [68]. However, these investigations did cies of B. procumbens bearing the gene for nematode not yield any positive results – all the selective materi- resistance, was transferred to a sugar beet chromosome als of sugar beet were found susceptible to sugar beet [40]. Savitsky explains the genetic basis of resistance to nematode. sugar beet nematode as follows: “Nematode resistance Later (1982–1983) at the Institute of Sugar Beet is most likely controlled by a single gene, because it is (currently the Institute of Bioenergy Crops and Sug- transferred by one chromosome. In the nematode-re- ar Beet, NAAS) naturally infested fi elds (the level of sistant species B. procumbens, the genes for nematode sugar beet nematode in soil was 1,000 e + l/100 ccm resistance early bolting, long petioles, and elongated of soil) were used to test 166 samples of mono- and dark-green leaves belong to the same linkage group. polyspermic beet cultivars for resistance to nematode. Nematode resistance is a dominant character” [39]. The results of these experiments highlighted cultivars Since the 1960s, Pawelska joined the work of breed- Verchnyatskyi 103, Yaltuschkivska monosperm 30 and ing nematode-resistant sugar beet hybrids [30, 53]. She six lines of sugar beet, the roots of which had 2–4 times used the material of trisomics, obtained by Savitsky in less females of sugar beet nematode than other culti- the USA, in her studies. In 1977 Pawelska isolated two vars did. The researchers also noted that after growing diploid plants with resistance to sugar beet nematode of Verchnyatskyi 103 cultivar the number of nematode out of 60 plants, obtained from the progeny of resistant cysts in soil decreased by 7 % which also testifi es to trisomics. Later these plants were used in the selection less susceptibility of this cultivar to the infestation with work regarding resistance to nematode [30]. the pest [55].

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 15 PYLYPENKO et al. During the subsequent years (1984–1990) about VII and VIII (Table). It is noteworthy that the chromo- 300 samples, cultivars and lines of sugar beet were somes with genes of resistance of cultivars B. webbi- analyzed for resistance and tolerance to heteroderosis. ana and B. procumbens are homologous [74, 75]. Five lines, obtained from pair mating of the progeny The genes Hs1pro–1, Hs2pro–7, Hs1web–1, Hs1web–7 and of Yaltuschkivska and Yaltuschkivska monosperm 30 Hs1pat–1 are dominant and provide absolute resistance (12175, 12177, 12179, 12181, 12222), were isolated of the corresponding wild beet species to sugar beet out of the selective material of Ivaniv experimental nematode, contrary to partial resistance, ensured by the breeding station, and the degree of nematode infesta- gene Hs1web–8 [63, 72, 76]. tion for them was estimated as the lowest (single cysts pro–1 were found on the roots) attributed to 1 score (out of The gene Hs1 has been studied the most and it 1-5 score scale). Sugar beet cultivars (Bilotcerkivska was proven that it encodes the plant disease resistance 45, BTs-40 and Yuvileynyi) and six selective numbers NBS-LRR proteins, which contain an amino-terminal (108-64, 108-6, 108, 59, 88, 126), tolerant to sugar domain, nucleotide-binding site (NBS) and leucine- beet nematode, were isolated [19, 54, 55]. The results, rich repeats (LRR) [71, 77, 78]. Still an unusual ami- obtained by Ukrainian scientists, confi rmed the possi- noacid composition of these proteins allows referring bility of breeding sugar beet forms, less susceptible to gene Hs1pro–1 to a specifi c independent class of resis- sugar beet nematode, in the course of screening of indi- tance genes, encoding cytoplasmatic proteins [79, 80, vidual plants from relatively resistant (tolerant) forms 81]. on naturally infested fi elds with subsequent in-family The manifestation of resistance mechanism for plants reproduction of selected lines [54]. This work is still with gene Hs1pro–1is evident in syncytia (the system of going on, although the classic breeding for resistance nematode–induced cells used as specifi c feeding struc- to nematode is known to last up to 15 years [69]. How- ture) degradation, which is observed with delay – on ever, this approach to control H. schachtii spread and the 14th day after the nematode larvae penetration into harmfulness is considered to be the most promising as the root of plants, but actually – after the period of de- there are no nematicides, accepted for use in fi eld con- termining the sex of nematodes, which explains the for- ditions in Ukraine. mation of a great number of females that do not mature Modern state of researches in breeding [71, 82, 83]. nematode-resistant sugar beet cultivars and hybrids Although the introgression of gene Hs1pro–1 from The development of genetic engineering and bio- wild species B. procumbens to cultivated В. vulgaris technology methods launched a new trend of work in was generally successful, it was accompanied with the breeding nematode-resistant sugar beet cultivars and transmittance of undesired agronomic traits, in particu- hybrids. At present such methods allowed mapping the lar, low productivity of plants on nematode-free soil genes, controlling resistance to sugar beet nematode, [69]. In addition, it was noted that sugar beet plants, namely, Hs1pro–1, Hs2pro–7, Hs1web-1, Hs1web–7, Hs1web–8 carrying the locus of gene Hs1pro–1, often suffer from and Hs1pat–1 , where Hs − H. schachtii; pro − B. pro- the formation of tumors on leaves and root system and cumbens; web – B. webbiana; pat – B. patellaris [70, the occurrence of so called multi-top phenotype. To 71, 72, 73]; the fi gure at the end of the code indicates prevent this phenomenon, there was an attempt to de- the chromosome of locating the mentioned locus – І, crease the introgression segment down to 35 and 17 % from the initial state. The plants obtained demonstrated The characteristics of mapped genes of resistance to Het- resistance to sugar beet nematode though the molecu- erodera schachtii lar analysis testifi ed to the loss of gene Hs1pro–1, which here may indicate the presence of another gene of nem- Chromosome Gene Origin Literature atode-resistance in the introgression segment [84]. of mapping The discovery of the mentioned genes of resistance Hs1pro–1 B. procumbens І [71]; [72] allowed accelerating the selection process of breeding Hs1pat–1 B. patellaris І [72] sugar beet cultivars and hybrids, resistant to heteroder- web–1 Hs1 B. webbiana І [73] osis. The crossing of sugar beet cultivars and wild spe- web–7 Hs2 B. webbiana VII [73] cies with resistance genes leads to the formation of one Hs2pro–7 B. procumbens VII [72] of three genotypes: with the addition of the chromo- Hs3web–8 B. webbiana VIII [73] some from wild species (monosomy phenomenon, 2n =

16 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 BREEDING AND USAGE OF SUGAR BEET CULTIVARS AND HYBRIDS = 18 + 1) [57, 62, 85], with the addition of the chro- ing resistant sugar beet cultivars on nematode-infested mosome fragment of wild species (2n = 18 + F) [86, fi elds the level of soil infestation did not exceed 216 e + 87] and the introgression of the chromosome fragment + l/100 ccm of soil, whereas after cultivating suscep- of wild species to the genome of the cultivated cultivar tible cultivars the density of the nematode population (2n = 18) [84]. It is noteworthy that three genes of re- increased up to 7,690 (2,260–14,100) e + l/100 ccm of sistance, descending from wild species B. рrocumbens soil [95]. In addition, it was determined that using the (Hs1pro–1), B. webbiana (Hs1web–1, Hs2web–7) and located susceptible sugar beet cultivar the multiplication rate on the fi rst and seventh chromosomes respectively, for sugar beet nematode was 1.8–8.9 (with the infes- were mapped on the fourth chromosome of В. vulgaris tation level of 5,600–17,560 e + l/100 ccm of soil), after the introgression [88]. whereas with the resistant cultivar this index decreased With any type of transferring resistance genes to a by 30–50 % [96]. new genotype its nematode-resistance is usually lower During last decade continuous breeding work re- than of the paternal resistant form. So the resistance sulted in over 15 nematode-resistant cultivars. At trait may be lost in the subsequent generations. It may present foreign companies recommend cultivating the indicate the presence of other, undetected yet, resis- following resistant sugar beet cultivars: Nemata, Pau- tance genes in wild species, the combination of the letta, Hella, Kleist, Brix, Belladonna KWS, Adrianna, former is the basis of absolute resistance of the initial Kristallina KWS, BTS 440, Vasco, Lisanna KWS, paternal form [89], which was evidently demonstrated Finola KWS, Corvetta KWS, Theresa KWS and Kuhn for gene Hs1pro–1, already mentioned above [84]. [97]. Several nematode-resistant sugar beet hybrids, However, regardless of these specifi cities the breeding including Korrida KWS, Slawa KWS and Bison, were and use of resistant cultivars allowed both obtaining a registered in Ukraine. Contrary to the fi rst sugar beet considerable surplus of beet yield on the infested fi elds, nematode-resistant cultivars, the crop yield of modern and decreasing the number of nematode in soil down to cultivars increased by 10 % on average. At the same 73 %, while the nematode population densities in soil time their quality was improved. In particular, the con- under the susceptible plants increased by 35 % [90, 91]. tent of treacle-forming substances in the cellular fl uid decreased by almost 10 % compared to the best stan- The fi rst nematode-resistant cultivars of sugar beet dard cultivars. It is also noteworthy that while the fi rst Evasion and Nemakill were registered in France in bred cultivars had almost 60 % resistance to nematode, 1996. Later (in 1998) another sugar beet nematode-re- this index for modern cultivars may sometimes reach sistant cultivar – Nematop – was included to the exist- as high as 100 % [98]. However, the scientists note that ing list in Germany [92]. However, they did not have it is not reasonable to rely only on resistant cultivars on high crop yield and their cultivation was proven more the fi elds with high level of infestation with sugar beet economically effi cient only on the fi elds, where the nematode. An integrated crop protection system should density of nematode population was up to 800–1,000 e be applied on such fi elds including the cultivation of + l/100 ccm of soil [93]. According to the observations resistant intermediate crops [1–7, 14–22, 24–26]. of Belgian scientists nematode-resistant sugar beet cultivars were somewhat less productive than susceptible CONCLUSIONS cultivars, but they had higher crop yield on the soils Sugar beet remains a strategically important crop in with the infestation rate of over 1,500 e + l/100 ccm of Ukraine and, taking into account a wide distribution soil [28]. In the opinion of German researchers, resis- of sugar beet nematode in the country, the problem of tant cultivars should be cultivated only on the fi elds, breeding domestic nematode-resistant cultivars and hy- densely infested with nematode [94]. However, recent brids of this crop is still urgent. Further development of investigations proved the recommendation to cultivate selection programs for sugar beet regarding resistance nematode-resistant sugar beet cultivars even on the to nematode is related to clearer identifi cation of resis- fi elds, where the nematode population denstity does tance genes and products of their expression, reliable not exceed 250 e + l/100 ml of soil. If the pre-sowing estimation of genetic potential of initial and selection number of sugar beet nematode reaches 750 e + l/100 material using genetic markers and to the development ml of soil and above, the use of the resistant cultivar and introduction of the methods of genetic transforma- will, fi rst and foremost, promote the decrease in its den- tion of plants. Crop cash value and production capac- sity in soil which is more important than obtaining high ity are good motivation to cooperate in solving these crop yield [95]. Thus, it was proven that after cultivat- issues, whereas the exchange of fresh knowledge and

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 17 PYLYPENKO et al. germplasm will be beneﬁ cial both on the domestic and Создание и внедрение в производство сортов и international levels. гибридов сахарной свеклы, устойчивых к свекловичной нематоде Heterodera schachtii Cтворення та впровадження у виробництво сортів 1 2 і гібридів цукрових буряків, стійких до бурякової Л. А. Пилипенко , Е. А. Калатур нематоди Heterodera schachtii e-mail: [email protected] Л. А. Пилипенко 1, К. А. Калатур 2 1 Институт защиты растений НААН Ул. Васильковская, 33, Киев, Украина, 03022 e-mail: [email protected] 2 Институт биоэнергетических культур и сахарной 1 Інститут захисту рослин НААН свеклы НААН Вул. Васильківська, 33, Київ, Україна, 03022 Ул. Клиническая, 25, Киев, Украина, 03141 2 Інститут біоенергетичних культур і цукрових буряків Heterodera schachtii Schmidt, 1871 является одним из НААН наиболее экономически убыточных паразитов сахарной Вул. Клінічна, 25, Київ, Україна, 03141 свеклы (Beta vulgaris L.) во всем мире. Свекловичная Heterodera schachtii Schmidt, 1871 є одним з економічно нематода широко распространена в большинстве свекло- найзбитковіших паразитів цукрових буряків (Beta vul- сеющих регионов Украины, вызывая существенные по- garis L.) в усьому світі. Бурякова нематода широко роз- тери урожайности и снижение содержания сахара в повсюджена у більшості бурякосіючих регіонів України, сахарной свекле с зараженных угодий. Уникальные па- спричиняючи істотні втрати врожайності та зниження разитарные свойства свекловичной нематоды способ- вмісту цукру у цукрових буряках із заражених угідь. ствуют ее росту, размножению и вредоносности. В ин- Унікальні паразитарні властивості бурякової нематоди тенсивных системах земледелия меры контроля свекло- сприяють її росту, розмноженню та шкодочинності. В вичной нематоды заключаются преимущественно в ис- інтенсивних системах землеробства заходи контролю пользовании нематицидов и соблюдении надлежащей бурякової нематоди полягають переважно у викорис- сельскохозяйственной практики (в первую очередь сево- танні нематицидів та дотриманні належної сільсько- оборотов). Поскольку ни один из нематицидов, разре- господарської практики (у першу чергу сівозмін). Ос- шенных к использованию в полевых условиях, не за- кільки жоден з нематицидів, дозволених до застосуван- регистрирован в Украине, актуальной остается аль- ня за польових умов, не зареєстрований в Україні, ак- тернативная стратегия, основанная на создании и ис- туальною лишається альтернативна стратегія, в основі пользовании устойчивых сортов и гибридов сахарной якої лежить створення і використання стійких сортів і свеклы. Сделан анализ достижений и проблем про- гібридів цукрового буряку. Зроблено аналіз досягнень цесса селекции на устойчивость к H. schachtii и при- і проблем процесу селекції на стійкість до H. schachtii ведены результаты национальной селекционной про- та наведено результативність національної селекційної граммы, основанной на традиционных подходах. С програми, що базується на традиційних підходах. З начала 1980-х годов пять сортов сахарной свеклы (Верх- початку 1980-х років п’ять сортів цукрового буряку няцкий 103, Ялтушковская односемянная 30, Белоцер- (Верхняцький 103, Ялтушківський однонасінний 30, ковская 45, БЦ-40 и Юбилейный) и 17 линий, час- Білоцерківська 45, БЦ-40 і Ювілейний) та 17 ліній, тично устойчивых или толерантных к H. schachtii, по- частково стійких або толерантних до H. schachtii, одер- лучены целенаправленным скрещиванием и отбором жано внаслідок цілеспрямованого схрещування та від- устойчивых форм на природных инвазионных фонах. бору стійких форм на природних інвазійних фонах. По- Показаны перспективные направления дальнейшей ра- казано перспективні напрями подальшої роботи для боты для более эффективного использования источни- ефективнішого використання джерел і донорів немато- ков и доноров нематодостойкости, имеющихся в на- достійкості, наявних в національному банку генетичних циональном банке генетических ресурсов растений. ресурсів рослин. Існує нагальна потреба в точнішій Существует настоятельная потребность в более точной ідентифікації генів нематодостійкості, широкому засто- идентификации генов нематодостойкости, широком при- суванні надійних молекулярних маркерів (для маркер- менении надежных молекулярных маркеров (для мар- ної селекції) і впровадженні методів генетичної транс- керной селекции) и внедрении методов генетической формації рослин. Ринкова цінність культури та потуж- трансформации растений. Рыночная ценность культуры ності з її виробництва є добрим підґрунтям для коопе- и мощности по ее производству являются хорошим рації з вирішення цих завдань, тоді як обмін новими основанием для кооперации по решению этих задач, в знаннями та генетичною плазмою стануть корисними як то время как обмен новыми знаниями и генетической на національному, так і міжнародному рівні. плазмой будут полезными как на национальном, так и международном уровне. Ключові слова: буряки цукрові, Heterodera schachtii, Ключевые слова: сахарная свекла, Heterodera schachtii, стійкість до нематод, селекція. устойчивость к нематоде, селекция.

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22 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 ISSN: 2312-3370, Agricultural Science and Practice, 2015, Vol. 2, No. 1

UDC 631.95:001.891(477) THE IMPORTANCE OF AGROECOLOGY IN THE PROCESS OF WELL-BALANCED AGROSPHERE FORMATION O. І. Furdуchko, O. S. Demyanуuk Institute of Agroecology and Environmental Management of NAAS 12, Metrologichna Str., Kyiv, Ukraine, 03143 e-mail: [email protected], [email protected] Received on August 7, 2014

Contemporary realities demonstrate actual features of the environmental crisis in agrosphere which is considered to be both the result of technological impact on its components, and also sharp decline of public morals and the lack of foresight regarding further consequences of the collision of the long-established lifestyle. In this respect the foreground of overcoming environmental problems in the agrarian sphere is taken by the main trends in agroecology. The article discloses the signifi cance of agroecology as a fundamental of well-balanced agrosphere formation, environmental protection, rational use and renewal of natural resources and ensuring ecological safety. It is substantiated that in current complicated environmental and economic conditions agroecology should defi ne the development strategy of agricultural production with the obligatory account of environmental, social and economic factors. The place of agroecology in the system of agrarian sciences and a number of priority tasks of agroecology at the current stage of agricultural science development and production in Ukraine are defi ned. The main aim of agroecology is to ensure sustainable production of quality and safe products, storage and renewal of natural resource potential of the agricultural sector, which means ecological safety of all branches of agricultural production with their economic feasibility. Agrosphere management calls for urgent development of new scientifi cally grounded approaches, based on the main principles of the Sus- tainable Development Concept. Thoughtful management of environmental processes in agrosphere, balanced needs of economic development and opportunities for the renewal of natural resources, comprehensive realiza- tion of environmental measures and technologies in АIC are the basis of the sustainable development of the country, life duration, strong health and well-being of present and future generations. Key words: аgroecology science, agrosphere, sustainable development, environment, natural resources.

Considering the implementation of the provisions of the as the crisis of existence philosophy, the crisis of spiri- Sustainable Development Concept and the transformation tuality. processes in the human perception about the re-estimation The degradation of environment results both from the of the relevance of environmental quality and safety, as technological impact and sharp decline of public mor- well as the use of natural resources, it is impossible not to als, lack of foresight regarding further consequences of recognize the priority signiﬁ cance of agroecology at the the collision of the long-established lifestyle. current stage of the agriculture development. Taking the abovementioned into account, the fore- The benchmarks, primarily targeted at economic re- ground of overcoming the ecological problems in the sults and the implementation of scientiﬁ c achievements agriculture is gradually taken by the main trends of and novel technologies without any consideration for agroecology. the priority of the development of ecological and social factors, gradually pass into history. In current complicated ecological and economic conditions agroecology determines the strategy of de- Many scientists believe that Ukraine has got all the veloping the agricultural production, which should be features of the ecological crisis, which is now viewed aimed at the maintenance and renewal of soil, water

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 23 FURDУCHKO et al. and biological resources fi rst and foremost, the envi- Agroecological investigations are a specifi c synthesis ronment protection and the provision of high-quality of environmentology (environmental science) and eco- food products in suffi cient amounts for people. sozology (science of natural environment protection). Agroecology is formed as an independent branch of Agroecology acts both as a sectoral agricultural sci- science at the interface of many disciplines. On the one ence that studies the agrosphere to meet the needs of hand, it is based on the set of natural sciences, includ- humanity and also explores general agroecological ing general ecology, physiology, chemistry, morphol- problems related to nature preservation, which is an ogy, physics, meteorology, hydrology, biochemistry, important component of sustainable environment de- mathematics, etc., and on the other hand it is grounded velopment. on the industrial sciences on crop cultivation and live- The main aim of agroecology is to ensure sustainable stock production, including agriculture, plant cultiva- production of quality and safe products, storage and re- tion, agrochemistry, soil science, agroforestry, forestry, newal of natural resource potential of the agrarian sec- melioration, animal husbandry, animal science, bio- tor, i.e. the ecological safety of all the branches of agri- technologies, biosafety, environmental management, culture along with their economic feasibility. It studies etc. (Figure). human interaction with the environment in the process In addition, agroecology is closely related to envi- of agricultural production, namely, the impact of agri- ronment safety and social ecology. Agroecology is the culture on natural systems, the interaction between the science, which focuses on the study of agrosphere in components of agroecosystems, energy transfer, and general, investigates the foundations of the sustainable the specifi cities of functioning of agroecosystems in use of agricultural land for obtaining crop and live- conditions of technological impact [4]. stock products and their processing with simultaneous Agroecology and environmental management are two preservation of natural resources (biota, soil, water, at- related and interconnected sciences, designed to create mospheric air, etc.), biotic diversity and the protection the new philosophy of agrosphere knowledge and re- of human environment and manufactured goods from source management of animate and inanimate nature, contamination [2]. primarily renewable man-made resources, created in Although agroecology is the interdisciplinary sci- the process of proper production, with the purpose of ence, yet it belongs to agricultural sciences with the preserving a dynamically sustainable state of environ- dominating emphasis on the elaboration and scientifi c ment as well as ensuring better life for current and fu- substantiation of the measures, required for obtaining ture generations, and providing for social adaptation of high-quality and safe agricultural products, preventive society to regular changes in the environment. It means estimation of undesired consequences of the negative that agroecology is the scientifi c study of agrosphere impact of human activity on agroecosystems, biogeo- state and dynamics, while environment management cenoses and landscapes in general, and the elimination presupposes practical measures for the use of natural of the mentioned consequences. objects and recommendations regarding the technologies of using natural resources, monitoring of their The founder of the domestic agroecological science, condition and searching for the ways of optimizing the full-member of NAS of Ukraine, NAAS and RAAS, ecological and economic indicators of activities of the Sozinov O. O. emphasized that modern agroecology acting agents. Therefore, agroecology should become is complex science, based on the synthesis of many a leading force in solving the problems of sustainable sciences and grounded on the systemic approach, pre- nature management and ensure obtaining high quality supposing the use of political, economic and other and environmentally safe agricultural products [1]. factors. The object of agroecology research is agrosphere, The implementation of agroecology achievements in and its subject is human relations with the environment the Ukrainian context does not require immense addi- in the process of agricultural production, the impact of tional expenses; on the contrary, it would ensure more agriculture on natural systems, the relations between effi cient utilization of our natural potential. The only the components of agroecosystems and the specifi cities requirement is the political will of state authorities to of circulation of substances, energy and information implement the principles of agriculture biologization therein under the anthropogenic impact. Agroecology and the formation of stable agricultural landscapes and as a science considers agricultural systems and tech- agroecosystems [3]. nologies of crop cultivation and livestock production

24 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 THE IMPORTANCE OF AGROECOLOGY IN THE PROCESS OF WELL-BALANCED AGROSPHERE

Agroecology in the system of agrarian sciences [1]

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 25 FURDУCHKO et al. in the terms of the utilization and renewal of natural The evolution of agroecology depends on the devel- resources; it also assesses the validity of ecological so- opment of natural processes in biosphere, human re- lutions. It should develop theoretical foundations for lations with the environment, as well as political and ecologically disposable and harmless crop cultivation economic processes in the society. The retrospective and livestock production, and design the formation of studies suggest that thousands of years ago spontane- agricultural landscapes, ensuring harmonious balance ous human activities led to signifi cant environmental (homeostasis) with biosphere. changes, which sometimes compromised the existence Agrosphere is both the main source of providing the of mankind [7]. During the long history of humanity human population with food and raw materials for food their relations with environment were unequal and con- and light industry (mostly due to solar energy and other stantly changing. natural resources – soil, water, climatic factors, etc.), Agriculture has got the longest standing and remains and also the habitat of the majority of the population. the most powerful factor of terrestrial ecosystems and It is remarkable for specifi c fundamental regularities of biosphere transformation in general. The development internal development, which result from the interaction of settled agriculture caused the fi rst serious anthropo- of various natural and socio-economic factors [5]. So genic biosphere shock. Crises, cataclysms and other agroecology, the main purpose of which is the harmo- disorders of environmental conditions within civili- nization of relations of agrosphere and natural environ- zation were not uncommon. In the past centuries, the ment, is the one to determine the ways of sustainable resolution of such crises was quite simple: the center of development of the agrarian sector [6]. economic development shifted to another area or a per- Therefore, agroecology should be understood as a son changed the way of management. At the end of the comprehensive agricultural science that studies all ХХ century humanity felt the beginning of one more contemporary environmental issues related to the agro- environmental crisis, which had qualitatively different industrial production and the ways of applying the en- origin compared to all previous ones. This crisis was vironmental protection principles in all the branches caused by technological and production factors, includ- of AIC. Methodologically, it is important to ensure the ing agricultural production. General degradation of ecological direction in the agricultural technologies global environment was started. The elements of civili- taking into account the trends of scientifi c and tech- zation pressure on environment were the technologies nological progress, specialization characteristics and with signifi cant fi nancial expenses and large hazardous concentrations according to natural and commercial wastes, used in industry and agriculture. zones. Nature-viability concept should be built into the Agrosphere in Ukraine covers more than 70% of industrial system, and in order to evaluate productivity the total area. Its fi rst “islands” arose as a result of it should be considered as the ratio of manufactured the Neolithic revolution about 8−10 thousand years products and quantities of used resources and received B.C. (Trypil culture). Its signifi cant development oc- waste. The requirements of rational nature manage- curred in the ХІХ century. The main contradictions ment should be considered in all the subsystems of between agrosphere and environment in those days modern agriculture (sphere of manufacturing the farm was the expansion of the former due to forests de- production supplies, sphere of material and technical struction as well as steppe ecosystem damages be- supplies service for agriculture, agricultural industry, cause of substantial increase in the number of sheep harvesting, storage, primary processing and marketing in these areas. However, in general the effect of an- of agricultural products). thropogenic factors in those times did not lead to The main and perhaps ultimate aim of agroecol- homeostasis disorders in global environment. Nev- ogy is to fi nd the formula of optimal ratio, i.e. the ertheless, even on the verge of the last century such balance in cultivating plants and producing livestock outstanding scientists as Vernadskyi V., Dokucha- under certain environmental conditions. The mea- ev V., Kostychev P., Vysotskyi G., and Izmailskyi O. sure of this ratio is the productivity of agricultural warned about possible environmental crisis due to plants and animals, which, in addition to quantita- the growing anthropogenic pressure on agrosphere. tive indicators, should be characterized by high qual- They substantiated the need for purposeful actions ity products and safety for environment; and this is for conservation and restoration of natural resour- largely determined by the characteristics of ecologi- ces, including agricultural and forest lands, water cal processes in agrosphere. and forest ecosystems etc.

26 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 THE IMPORTANCE OF AGROECOLOGY IN THE PROCESS OF WELL-BALANCED AGROSPHERE In the second half of the ХХ century, the situation be- and global, and require fast optimization of agricultural gan to change rapidly as a result of active industrializa- production. tion of agriculture and the increase in the negative in- Modern agroecology, based on the integrated and fl uence of industry and urbanized areas on agrosphere. systematic approach, determines the ways of agro- The area of arable lands increased dramatically, the ecosystem transition to the foundations of sustainable intensity of their cultivation enhanced, the processes development. This means that permanent obtaining of of soil erosion accelerated, and soil degradation and the required amount of high quality and competitive contamination by xenobiotics deepened. Small rivers products should be carried out with limiting the anthro- gradually disappeared, in many areas the hydrologi- pogenic energy, the restoration of natural resources, the cal regime was broken, which was partially caused by formation of well-balanced agroecosystems and mini- gross mistakes in water melioration. mum environmental contamination, based on the cri- However, in this period there was also consider- teria of rational use of natural resources and bioethics able work, carried out on land management, forest principles. melioration, and introduction of crop rotation, the Agrosphere is man-made and continuously man- application of mineral and organic fertilizers was in- maintained; it is inertial in its essence. Its manage- creased, resulting in greatly improved crop produc- ment requires systematic approach and scientifi cally tivity, and the increase in the number of animals. The substantiated strategy. Regardless of the fact that agro- 80s witnessed the beginning of the implementation sphere is mostly an anthropogenic system, according of the soil-protecting and profi le melioration system, to its fundamental essence it is a part of biosphere, developed by domestic scientists. Nevertheless, the and the basic mechanisms, remarkable for the latter, ecological crisis in the agrosphere of Ukraine deep- operate therein. This means the presence of photoau- ened, which was particularly acute after the Cher- totrophs and hemoheterotrophs (including humans); nobyl nuclear disaster. this involves the circulation of biogenic elements and The reasons of current complicated environmental energy, and the balance of pathogenic factors interac- situation in the agrosphere include the following fac- tion (viruses, microorganisms, insects) with plants and tors: the ineffi ciency of public administration, unsatis- animals. The violation of this balance may have cata- factory use of economic instruments for the implemen- strophic consequences [8]. tation of environmentally-friendly technologies, low It is well known that the foundation and basis of ex- level of ecological culture of both manufacturers and istence and balanced state of the biosphere is biodiver- population, low activity and effi ciency of environmen- sity. With its deterioration the whole system becomes tal organizations and social movements. unstable, which can lead to its complete collapse. This Special attention should be paid to the issue of im- is especially true for agroecosystems. Unfortunately, plementing and setting up the system of national agro- the problems of biodiversity preservation in the agroecological monitoring, using modern information and sphere have not been considered properly. Due to space technologies, assessing the degree of contami- agricultural production intensifi cation, humans have nation of all components of agricultural landscapes mistakenly thought about the dominance of anthropo- by pathogenic organisms (viruses, bacteria, micromy- genically-controlled factors in solving all the problems ceta), organic xenobiotics and heavy metals, the study in agrosphere, especially related to equipment, fertil- of migration and transformation of toxicants in the soil izers, crop protection chemicals and animals. How- and in the soil – plant – animal – human system etc. ever, humans almost forgot about the fundamentals of Another relevant trend is also the development of the biological system functioning, about the mandatory methods and techniques of contaminated soil remedia- existence of the corresponding biodiversity in agroeco- tion and their return into the agricultural production. systems. The underestimation of this factor, the lack of In most countries the peculiarities of agricultural pro- studies to identify the ways of biodiversity preservation duction are determined by the priority of the consumer- endangers the possibility to achieve sustainable devel- related function. Providing people with food and raw opment of agroecosystems and consequently the well- materials requires intensifi cation of all the agricultural being of the population [9]. sectors, which has caused degradation processes in Undoubtedly, the function of the main man’s bread- agrosphere. While in the beginning of the XX century winner will always be performed by agrosphere. How- they were local, now they have become wide-spread ever, this goal must be achieved on the basis of the prior-

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 27 FURDУCHKO et al. ity of natural resources preservation, the improvement gies, suitable for obtaining of high quality agricultur- of production quality, a signifi cant increase of effi cien- al production with sustainability of natural resources cy of solar energy using, especially by green plants, the (land, water, biological resources) with the minimum intensifi cation of microbiological processes in soil as environmental impact. an important link of substances circulation in agroeco- Finally, it should be mentioned that only the aware- systems, in particular biological nitrogen fi xation and ness of the importance of agroecology in modern agri- phosphorus mobilization. This requires not only new cultural production, the thoughtful management of en- ways of solving problems in social and economic rela- vironmental processes in agrosphere, balanced needs tions in the sphere of agricultural production, but also of economic development and opportunities for repro- new relations between agrosphere, technosphere and duction of natural resources, comprehensive realiza- urbosphere, the application of high energy saving and tion of agri-environmental measures and technologies environmental protection technologies. in AIC are the basis of the sustainable development of The problem of forming a new sustainable agro- the country, life duration, strong health and well-being sphere is of special importance for Ukraine. There is of present and future generations [1]. an urgent need to defi ne a new development strategy Значення науки агроекології for both agricultural production and agrosphere in gen- у формуванні збалансованої агросфери eral. We need decisive action and support at the state level for the implementation of the main provisions of O. І. Фурдичко, O. С. Дем’янюк the Sustainable Development Concept, the agrosphere e-mail: [email protected], [email protected] formation based on its principles, and the biospheric- noospheric approach, founded on the ideas of Verna- Інститут агроекології і природокористування НААН dskyi V. First of all, it requires the development of the Вул. Метрологічна, 12, м. Київ, Україна, 03143 Agrosphere Model of Ukraine for the XXI century that Реалії сьогодення свідчать про наявні ознаки екологіч- would be based on the well-established principles of ної кризи в агросфері, що розглядається не лише як agroecology and economic science, taking into account наслідок техногенного тиску на її складові, а й зубо- the mechanisms, active in the agrosphere as a part of жіння моральності суспільства, недалекоглядності щодо the biosphere. It is necessary to take into account the майбутніх наслідків колізій усталеного рівня життя. З qualitative changes in environment, which resulted огляду на це на передній план подолання екологічних from signifi cant increase in the anthropogenic impact проблем в аграрній сфері виходять основні напрями on environment in the XX century and modern tenden- науки агроекології. В огляді розкрито значення науки агроекології як фундаментальної основи формування cies of global climate change etc. збалансованої агросфери, охорони довкілля, раціональ- There is still no economic incentive of environmen- ного використання та відтворення природних ресурсів, tally friendly technologies introduction in Ukraine. The а також гарантії екологічної безпеки. Обґрунтовано, що application of innovative, resource-saving and envi- наука агроекологія за сучасних складних екологічних ronmental protection technologies, including the tech- та економічних умов повинна визначати стратегію роз- nologies of agricultural waste disposal and utilization, витку аграрного виробництва з обов’язковим урахуван- is on the low level. ням екологічних, соціальних і економічних чинників. Визначено місце науки агроекології у системі аграрних Negative processes occur especially rapidly in the re- наук та низку пріоритетних завдань на сучасному cent decades, when there are all the features of global етапі розвитку аграрної науки і виробництва України. climate changes, the number of technological disas- Головною метою агроекології є наукове забезпечення ters increases, and industrial technologies usually do збалансованого виробництва якісної і безпечної продук- not meet current requirements of environmental safety. ції, збереження і відтворення природно-ресурсного по- Therefore, the issue of creating sustainable agricultural тенціалу аграрного сектора, тобто екологічна безпека landscapes, increasing the area of environmentally sta- всіх галузей сільського виробництва за економічної до- цільності. Зазначено, що управління агросферою потре- bilizing lands and optimizing the area of forest plan- бує розроблення нових науково обґрунтованих підходів, tations in the structure of agricultural landscapes, the які базуються на основних принципах Концепції стало- preservation and restoration of natural resources in го розвитку. Продумане управління екологічними проце- general, is still urgent. As for the fi eld of crop cultiva- сами в агросфері, збалансованість потреб економічного tion and livestock production, it is necessary to focus розвитку і можливостей відтворення природних ресурсів, on the elaboration of environmentally safe technolo- комплексна реалізація екологічних заходів і технологій

28 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 THE IMPORTANCE OF AGROECOLOGY IN THE PROCESS OF WELL-BALANCED AGROSPHERE в АПК – це основа стабільного розвитку держави, научно обоснованных подходов, базирующихся на основ- тривалості життя і міцного здоров’я та благополуччя ных принципах Концепции устойчивого развития. Про- нинішніх і прийдешніх поколінь. думанное управление экологическими процессами в агро- сфере, сбалансированность потребностей экономичес- Ключові слова: наука агроекологія, агросфера, збалан- кого развития и возможностей воспроизводства при- сований розвиток, навколишнє природне середовище, родных ресурсов, комплексная реализация экологичес- природні ресурси. ких мероприятий и технологий в АПК – это основа Значение науки агроэкологии стабильного развития государства, продолжительности в формировании устойчивой агросферы жизни и крепкого здоровья и благополучия нынешних и грядущих поколений. О. И. Фурдычко, Е. С. Демьянюк Ключевые слова: наука агроэкология, агросфера, cба- e-mail: [email protected], [email protected] лансированное развитие, окружающая среда, природ- Институт агроэкологии и природопользования НААН ные ресурсы. Ул. Метрологическая, 12, Киев, Украина, 03143 REFERENCES Реалии сегодняшнего дня свидетельствуют о наличии признаков экологического кризиса в агросфере, что 1. Furdychko O. I. Agroecology: monograph. – Kуіv: DIA, рассматривается не только как следствие техногенного 2014. – 400 p. прессинга на ее составляющие, но и обнищания нрав- 2. Glossary of agroecology and nature management / Ed. ственности общества, недальновидности относительно Furdychko O. I. – Kyiv: DIA, 2012. – 336 p. будущих коллизий в устоявшемся уровне жизни. В 3. Sozinov O. O. Agroecology – the philosophy of этой связи на первый план при преодолении эколо- agriculture of the ХХІ century // Visnyk agrarnoi nauky. – гических проблем выходят именно основные направ- 1997. – N 9. – P. 61–67. ления науки агроэкологии. В обзоре раскрыто значе- 4. Agroecology / Eds V. A. Chernikov, A. I. Chekeresa. – ние науки агроэкологии как фундаментальной основы Moscow: Kolos, 2000. – 536 p. формирования сбалансированной агросферы, охраны 5. Sozinov O. O. Agrosphere of Ukraine in the ХХI centu- окружающей природной среды, рационального использо- ry // Visnyk NAN Ukrai›ny. – 2001. – N 10. вания и воспроизводства природных ресурсов, а также 6. Furdychko O. I. Agrosphere as the object of agroecology гарантии экологической безопасности. Обосновано, что research // Agroekologichnyj Zh. – 2008. – P. 12–14. – наука агроэкология в современных сложных экологи- (Special Is.) ческих и экономических условиях должна определять 7. Chalavan V. V. Evolution of agroecological science and стратегию развития аграрного производства с обяза- the strategy of modern agricultural production // Agro- тельным учетом экологических, социальных и экономи- ekologichnyj Zh. – 2008. – P. 259–269. – (Special Is.) ческих факторов. Определено место науки агроэкологии 8. Sozinov O. O. On the most relevant indices and quan- в системе аграрных наук, а также приоритетные зада- titative and qualitative properties of mega-agroecosystem чи на современном этапе развития аграрной науки и (agrosphere) of Ukraine / Sozinov O. O., Prydatko V. I., производства Украины. Главной целью агроэкологии Burda R. I. et alt. / Agrobioriznomanittja Ukrai›ny: является научное обеспечение сбалансированного произ- teorija, metodologija, indykatory, pryklady. – Kn. 2. / водства качественной и безопасной продукции, сохра- Eds. Sozinov O. O., Prydatko V. I., Lysenko O. I. – Kyiv: нения и воспроизводства природно-ресурсного потен- ZAT «Nichlava», 2005. – P. 17–30. циала аграрного сектора, то есть экологическая безо- 9. Agrobiodiversity of Ukraine: theory, methodology, пасность всех отраслей сельского производства при indices, examples / Eds. Sozinov O. O., Prydatko V. I., экономической целесообразности. Определено также, Lysenko O. I. –Kyiv: ZAT «Nichlava», 2005. – V. 1. – что управление агросферой требует разработки новых 384 p.

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UDC 579.887 + 577.1 + 577.15 RECENT DATA ON THE CAUSATIVE AGENT OF PALE GREEN DWARF (ACHOLEPLASMA LAIDLAWII VAR. GRANULUM INCERTAE SEDIS) IN UKRAINE: PATHOGENICITY AND VIRULENCE FACTORS AND HOST REACTIONS K. S. Korobkova, V. P. Patyka D. K. Zabolotny Institute of Microbiology and Virology, NAS of Ukraine 154, Zabolotnoho Str, Kyiv, Ukraine 03680 e-mail: [email protected] Received on December 3, 2014

Contemporary state of the distribution of mycoplasma diseases of cultivated crops in Ukraine was analyzed. The changes of the physiological state of plant cells under the impact of mollicutes were investigated. It was demonstrated that there is temporary increase in the activity of peroxidase, catalase, polyphenoloxidase, phenylalanine-ammonia-lyase at the early stages of interaction. The adhesive properties are changed in the mollicutes under the impact of plant lectin; there is synthesis of new polypeptides. It was determined that the phytopathogenic acholeplasma is capable of producing a complex of proteolytic enzymes into the culture medium. It was concluded that when plant cells are infected with acholeplasma, a number of signaling interactions and metabolic transformations condition the recognition of pathogenesis and ensure the aggregate response of a plant to stress in the form of defense reactions. It was assumed that some speciﬁ cities of the biology of phytopathogenic acholeplasma determine their avoiding the immune mechanisms of plants and promote long-term persistence of mollicutes. Key words: cultivated crops, mycoplasmosis, phytopathogenic acholeplasma, persistence, enzymes.

The elaboration of balanced agroecosystems and the [3]. All this increases the danger of the microorgan- maintenance of conditions for their stable development isms, not registered before, penetrating the territory is rather a complicated process, related to a wide scope of Ukraine and spreading therein. It is noteworthy that of issues. These include the determination of physical, in these conditions mycoplasma, plant disease agents, chemical and biological processes in soil, the elabora- enhance their aggression and harmfulness, accelerating tion of modern agrotechnologies, the improvement of the penetration of phytomycoplasma, regulated by the the specialization of agrarian production systems, the European and Mediterranean Plant Protection Organi- optimization of the structure of agricultural landscapes zation, into Ukraine. and the organization of the territory of land-utilization [1, 2]. Ukraine has witnessed some movement in eco- The apple tree proliferation disease, grape yellow- nomic relations with Western countries which has a ing, stolburs, wheat light green dwarf disease are quite positive impact on the general state of agrarian produc- common in the territory of Europe and conditioned tion; at the same time there are some objective reasons, by plant damage with phytopathogenic mycoplasma. like privatization of land resources, restructuring of The harmfulness of mycoplasma diseases is a common agricultural enterprises, which often impair the techno- problem, as the infections, caused by mycoplasma, are logical foundation of crop cultivation. related to catastrophic diseases, which often become epiphytoties [4]. In addition, there is a change in climatic conditions, remarkable for speciﬁ c zones of cultivating crops which Mycoplasmas (mollicutes) are the smallest prokaryo- conditions the ﬂ uctuation of biodiversity in ecosystems tic microorganisms, deprived of a cellular wall and

30 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 MYCOPLASMA DISEASES IN MODERN STRATEGY OF CROP PRODUCTION capable of independent existence and restoration [5]. these mollicutes, A. laidlawii var. granulum st. 118 is It was described that the mollicutes, parasitizing on the agent of wheat mycoplasmosis which is capable of plants, are capable of avoiding the non-specifi c defense causing the outbreaks of the disease and the decrease in reaction of plants – oxidative stress, which leads to the the yield of infected fi elds [12, 13]. persistence of mollicutes. The scientists even assumed Among famous biologically active wheat substances that this reaction of plants may somehow promote the a relevant role is attributed to wheat germ agglutinins viability of mycoplasma, but the mechanisms of my- (WGA) – the lectin, which may perform the function of a coplasmosis emergence are yet to be elucidated [5–7]. plant signaling molecule [14–18]. It promotes the growth According to recent notions, the symptomatic mani- and development of wheat with simultaneous change in festation of infectious processes depends considerably the metabolism of symbiotic bacteria as a communication both on the infection agent and the specifi cities of the factor in the plant-microbe symbiosis. At the same time interaction of genetically determined signaling path- there are data on the increase in WGA level in Triticum ways in the parasite-host system [8–10]. As for mol- vulgare L. plants after the abiogenous stresses under the licutes, a close connection to the membrane of the eu- impact of drought, osmotic and heat shock. Taking this karyotic cell may result in masking their antigens and into consideration one of the tasks of our work was to in direct biochemical effects which cause local damage study the impact of this lectin on the agent of wheat light of the target cell structures. The inhibition and control green dwarf disease – A. laidlawii var. granulum st. 118. of mycoplasma infections of plants are related to the It was demonstrated that the introduction of WGA to the study of molecular mechanisms of mycoplasma inter- culture medium of acholeplasma causes the pleiotropic action with plant cells and the persistence principles effect: there is the activation of growth processes, the in- for these microorganisms and the phytopathogenesis, crease in the total amount of protein compared to the con- conditioned by latter. trol, the decrease in hemagglutinating activity which leads to the attenuation of adhesive properties of the pathogen. Fine mechanisms of mycoplasmosis emergence are We believe that after the direct contact of mollicute glyco- yet to be studied, as the interaction of cells is a compli- polymers and a wheat germ lectin the signal is transmit- cated and multi-component process. Therefore, our ef- ted inside the cell, due to which metabolism is enhanced, forts have been directed at the investigation of transfor- there is expression of new proteins, and the biomass of mations in the living parasite-host complex on the model the microorganism is increased. On the other hand, under of plant cells, infected with phytopathogenic strains of the impact of WGA the investigated microorganism dem- acholeplasma [11]. The dominant of these investigations onstrates a change in adhesive properties, which may be is the analysis of specifi cities of signaling and metabol- a manifestation of non-specifi c plant protection due to the ic relations in the network of molecular interactions of intrusion of the pathogen. It was assumed that it promotes mollicutes and cells of the host macroorganism. the transition of the infection into the latent state and long- Specifi c virulence factors have not been determined term persistence of acholeplasma in wheat plants. for plant mycoplasma, which are the agents of cata- Cell cultures are widely used as a model to study strophic epiphytoties, and the phytopathogenesis is many physiological and biochemical processes in caused by mycoplasma persistence and related phyto- plants as it allows having adequate estimation of ex- immunity reactions. The damage of plants with phy- change processes in plant cells and their response to toplasmosis may be manifested in the form of single various irritations of the environment in controlled symptoms of mycoplasmosis, and not as epiphytoty, conditions [9, 11, 19–21]. In our opinion the system of for a long time [4–7, 12, 13]. Therefore, it is extremely joint cultivation of the pathogen and cultures of target important and urgent to conduct a detailed study of the plant cells is a convenient model in the study of speci- agents of such dangerous diseases and the mechanisms fi cities of plant cell response to the infection with phy- of manifestation of their phytopathogenic properties topathogenic mycoplasma, thus it was further used in to prevent the occurrence of mycoplasma diseases of the investigations of stress condition of plants in condi- plants, new to Ukraine. tions of developing artifi cial mycoplasmosis. Acholeplasma laidlawii is a mollicute, widely spread The infestation of plants with obligate pathogens is in the environment, which is found in soil, manure, accompanied with the increase in the energy content of wastewater, cell cultures, tissues of humans, animals, cellular processes, in particular, there is the increase in and plants [5]. The phytopathogenic representative of breathing intensity which is related to the activation of

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 31 KOROBKOVA et al. enzymatic systems of peroxidase, catalase, polyphenol- in different organisms may be considerably different oxidase which perform the functions of terminal oxidases. [8, 22–26]. Thus, as seen from the abovementioned, The protection of biological molecules and cell organelles the impact of mollicutes on the plant organism may is carried out by enzymes that catalyze the biotransforma- be considered to be a biotic stress, as its consequence tion of primary reactive oxygen species (ROS) and low is a considerable disbalance of antioxidants and pro- molecular weight compounds of different chemical nature oxidants to the benefi t of the latter. The oxidative that react with these substances [8, 10, 19, 22–25]. stress results in the accumulation of reactive oxygen There are numerous data in the study of oxidation- species, which may act as inducers of corresponding reduction enzymes in conditions of oxidative stress in defense mechanisms in plant cells. plants, infected with different fungal or bacterial patho- Therefore, immediately after being infected with a gens [19, 22–26]. Taking into account the insuffi cient phytopathogenic mollicute the plant tissues are in condi- amount of publications on the issues of investigating tions of severe stress, total metabolism is inhibited. On these enzymes while infecting plants with mollicutes, the other hand, the stress-induced activation of the en- we analyzed the impact of the phytopathogenic mol- zyme indicates its specifi c role in overcoming this stress. licute A. laidlawii var. granulum st. 118 on the change The dynamics in the activity of enzymes of phenolic in the activity of peroxidase and catalase of plant cells plant metabolism, in particular, phenylalanine-ammo- in standard in vitro conditions (unpublished data). nia-lyase (PAL), when they are infected with the repre- For this reason we used callus cultures of sugar beet to sentatives of Mollicutes class, has not been studied yet infect them with the mollicute A. laidlawii var. granulum and is necessary to disclose the specifi cities of patho- st. 118. During the initial contacts of the plant and the genesis of plant mycoplasmosis. It was determined that pathogen the activity of peroxidase and catalase relative to PAL activity of a plant culture under the impact of A. the control is increased. Several hours after the infection laidlawii var. granulum st. 118 starts increasing one hour there was a decrease in enzymatic activity, after which the after the infection, and in two hours its level increases indices were stable. The changes, revealed in the activity 4-fold compared to the control. With further cultivation of oxidative enzymes, are related to the induction of de- of calluses in the presence of the pathogen, the level of fense mechanisms of plant cells as a response to the stress. PAL activity decreases and 6 hours after the infection it It is assumed that polyphenoloxidase (PPO) is activat- is only 50 % higher than the indices of the control non- ed in case of infesting plant cells with phytopathogenic infected samples. After 24 h of cultivation the PAL ac- organisms. Its function is to oxidize phenols to highly tivity decreases to the initial level and remains the same toxic quinones, inactivating foreign exoenzymes, de- during the whole experiment. Compared to other micro- stroying the cells of pathogens and the host-plant, i.e. organisms, this “outbreak” of activity is non-permanent it is involved in the reaction of plant hypersensitivity [27, 28]. Acholeplasma may be capable of overcoming [8, 22]. The analysis of PPO activity in callus tissues of the defense activity of non-specifi c mechanisms of plant sugar beet demonstrated that A. laidlawii var. granu- cells and/or avoiding hypersensitivity reactions, which is lum strain 118 has a considerable impact on the change a likely constituent of the adaptation of phytopathogenic in enzyme activity which may be compared to the ef- mollicutes to long-term persistence. The increase in PAL fect of hydrogen peroxide activity. activity during the infection of sugar beet calluses with A. laidlawii var. granulum st. 118 may also be viewed Thus, it may be concluded that the infection of sugar as a manifestation of defense reactions of plant cells in beet calluses with the cells of phytopathogenic acho- response to the activity of the pathogen. leplasma leads to the stimulation of defense reactions of plant cells – temporary increase at the early stages Along with other enzymes the phytopathogenic mi- of the activity of peroxidase and catalase enzymes, croorganisms produce extracellular proteases. In many participating in the development of systemic acquired cases there is some correlation between the activity of sensitivity as well as polyphenoloxidase enzymes. It extracellular proteases of the phytopathogen and the confi rms the literature data, according to which the intensity of the plant disease. The proteases of phyto- ratio in the activity of different oxidative systems has pathogenic microorganisms are capable of cleaving the adaptive meaning and may be considered as a nec- antimicrobial proteins of plants and playing a relevant essary condition for the manifestation of plant resis- role in the destruction of cellular wall proteins [29]. tance and the content of enzymes of the system of an- It was assumed that the ability of producing extracel- tioxidative protection and low molecular antioxidants lular enzymes may be one of pathogenicity factors for

32 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 MYCOPLASMA DISEASES IN MODERN STRATEGY OF CROP PRODUCTION phytopathogenic mollicutes. It was determined that the нілаланін-аміак-ліази. У молікутів під дією рослинного mollicute A. laidlawii var. granulum st. 118 is capable лектину змінюються адгезивні властивості, відбувається of producing proteolytic enzymes along with nucleases синтез нових поліпептидів. Встановлено здатність фіто- and some hydrolases into the cultivation medium. It патогенної ахолеплазми продукувати у культуральне was also demonstrated that the complex of proteolytic середовище комплекс протеолітичних ферментів. Зроб- enzymes, which are a part of the preparation, obtained лено висновок, що низка сигнальних взаємодій і мета- болічних перетворень за інфікування клітин рослин by us, is rather manifold and capable of cleaving such ахолеплазмою обумовлюють розпізнавання патогену і protein substrates as casein, hemoglobin, and fi brin. забезпечують сумарну відповідь рослини на стрес у ви- The comparative analysis of structural and function- гляді реакцій захисту. Висунуто припущення, що певні al specifi cities of proteases of phytopathogenic mol- особливості біології фітопатогенних ахолеплазм визна- licutes and related representatives of bacilli allowed чають уникання дії механізмів імунітету рослин і спри- determining a certain level of affi nity of their physical яють тривалій персистенції молікутів. and chemical properties and substrate specifi city. It is Ключові слова: мікоплазмоз, фітопатогенні ахолеплазми, anticipated that in this case the purpose of proteolytic персистенція, ферменти. enzymes is overcoming defense barriers of plants rather Новые данные о возбудителе бледно-зеленой than providing mollicutes with peptides and aminoac- карликовости зерновых (Acholeplasma laidlawiivar. ids, required for the growth and development. Further granulum incertae sedis) в Украине: studies will allow defi ning the activation mechanisms факторы патогенности, вирулентности and determining the source of inhibition of the activity и реакции растения-хозяина of proteolytic enzymes of phytopathogenic mollicutes. Е. С. Коробкова, В. Ф. Патыка e-mail: [email protected] Thus, it may be concluded that when plant cells are infected with acholeplasma, a number of signaling in- Институт микробиологии и вирусологии teractions and metabolic transformations condition the им. Д. К. Заболотного НАН Украины recognition of the pathogen and ensure the aggregate Ул. Академика Заболотного,154, Киев, Украина, 03680 response of a plant to stress in the form of defense Проанализировано современное состояние распростра- reactions. Some specifi cities of the biology of phyto- нения микоплазменных болезней сельскохозяйственных pathogenic acholeplasma determine their avoiding the культур в Украине. Исследованы изменения физиологи- immune mechanisms of plants and promote long-term ческого состояния клеток растений под влиянием мол- persistence of mollicutes. ликутной инфекции. Показано, что на ранних этапах взаимодействия временно увеличивается активность пе- The results of our work may be used as a founda- роксидазы, каталазы, полифенолоксидазы, фенилаланин- tion for the elaboration of the concept of decreasing аммиак-лиазы, у молликутов под действием раститель- the harmfulness of mycoplasma diseases of plants to ного лектина изменяются адгезивные свойства, проис- maintain ecological balance in agroecosystems, which ходит синтез новых полипептидов. Установлена спо- is in good agreement with modern trends of developing собность фитопатогенной ахолеплазмы продуцировать sustainable agriculture in Ukraine. в культуральную среду комплекс протеолитических ферментов. Сделан вывод о том, что ряд сигнальных Нові дані про збудника блiдо-зеленої карликовостi взаимодействий и метаболических превращений при зернових (Acholeplasma laidlawiivar.granulum incertae инфицировании клеток растений ахолеплазмой опре- sedis) в Україні: фактори патогенності, вірулентності деляют распознавание патогена и обеспечивают суммар- та реакції рослини-господаря ный ответ растения на стресс в виде реакций защиты. К. С. Коробкова, В. П. Патика Выдвинуто предположение, что определенные особен- e-mail: [email protected] ности биологии фитопатогенных ахолеплазм обусловли- Інститут мікробіології і вірусології вают избежание действия механизмов иммунитета рас- ім. Д. К.Заболотного НАН України тений и способствуют длительной персистенции мол- ликутов. Вул. Академіка Заболотного,154, Київ, Україна, 03680 Ключевые слова: микоплазмоз, фитопатогенные ахоле- Зроблено аналіз сучасного стану розповсюдження міко- плазмы, персистенция, ферменты. плазмових хвороб сільськогосподарських культур в Ук- раїні. Досліджено зміни фізіологічного стану клітин REFERENCES рослин під впливом молікутної інфекції. Показано, що 1. Patyka VP, Tihonovich ІA, Fіlіp’ev ІD, Gamajunova VV, на ранніх етапах взаємодії тимчасово збільшується ак- Andrusenko ІІ. Microorganisms and alternative agri- тивність пероксидази, каталази, поліфенолоксидази, фе- culture. Kyiv, Urozhaj.1993;176 p.

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UDC 631.8:631.45:631.559 REGULATION OF NITROGEN-CARBON INTERACTIONS IN AGROECOSYSTEMS IN THE FOREST-STEPPE ZONE OF UKRAINE V. A. Velichko 1, О. V. Demidenko 2 1 National scientiﬁ c center “Institute for Soil Science and Agricultural Chemistry named after O. N. Sokolovsky” NAAS of Ukraine 4, Chaikovskoho Str., Kharkiv, Ukraine, 61024 2 Cherkasy State Agricultural Experimental Station, NSC “Institute of Agriculture”, NAAS of Ukraine 13, Dokuchaiev Str., Kholodnianske village, Smila District, Cherkasy Region, Ukraine, 20731 e-mail: [email protected] Received on January 26, 2015

Aim. To determine the speciﬁ ed parameters of the complex model of nitrogen-carbon circulation while using different types of crop rotation, kinds of organic fertilizers and ways of soil cultivation in agroecosystems of the forest-steppe zone of Ukraine. Methods. Field, laboratory, computational, mathematical and statistical.

Results. Speciﬁ c types of organic fertilizers affect the emission of СО2 into the lowest atmospheric layer: in case of humus the typical emission interval is 25–85 t/ha, while in case of secondary products it is 70–160 t/ ha. The impact of the way of chernozem preparation on nitrogen-carbon circulation is manifested in the fact that in case of subsurface tillage the carbon balance in soil was positively increasing compared to ploughing.

The interval of СО2 emission into the lowest atmospheric layer due to the mineralization of humus and organic fertilizers with ploughing changes in a wider range compared against subsurface tillage. Conclusions. The nitrogen-carbon interactions are impaired due to the introduction of humus and removal of secondary products beyond the boundaries of the agroecosystem in the course of ploughing. The application of ground secondary products of crop production as organic fertilizers, wrapped up into the surface layer of chernozem during the subsurface tillage of soil, simulates the natural course of nitrogen-carbon circulation in agroecosystems of different types. Natural soil formation process is simulated due to the activation of photosynthetic activity of

cultivated crops with СО2 saturation in the lowest atmospheric layer, which provides for extensive restoration of chernozem fertility in the forest-steppe zone of Ukraine.

Key words: carbon, nitrogen, СО2, subsurface tillage, deep ploughing, agroecosystem.

INTRODUCTION climate changes [2, 6, 7]. The isolation of the nitrogen cycles from the consideration of the consequenc- The circulation of nitrogen and carbon are the es of climate changes leads to incomplete estimation main biochemical cycles, taking place in terrestrial of the response of ecosystems and agroecosystems, eco- and agroecosystems [1–3]. Recent studies dem- where mineral forms of nitrogen in soil are the limit- onstrate that, on the one hand, it is common to in- ing factor for the development of terrestrial plants in clude the carbon cycle of the ecosystem into climatic nature [8–10] and agroecosystems [11]. models as an integral characteristic of a carbon cycle

– СО2 concentration in the atmosphere [4, 5]; on the There is considerable impact of the nitrogen circula- other hand, the deﬁ ciency of mineral forms of terres- tion on the inverse relation between the change in the trial nitrogen has negative effect on the development climate characteristics and the carbon cycle: the higher of autotrophs both in natural systems and in agroeco- the dependence of the performance of agro- and natural systems, inﬂ uencing the depositing of atmospheric systems on the amount of assimilated nitrogen in soil, carbon which enhances the consequences of global the faster the emission of СО2 is accumulated by ter-

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 35 VELICHKO et al. restrial plant aggregations [12], thus the complex quan- Therefore, the elaboration of a complex model of the titative estimate of the interaction between the carbon interaction of nitrogen circulation (using different types and nitrogen cycles has considerable impact on the of crop rotation and kinds of organic fertilizers as well process of increasing or decreasing the carbon stocks as different methods of chernozem cultivation) and of terrestrial eco- and agroecosystems [13, 14]. The carbon circulation allows estimating carbon circulation interdependence and intensity of nitrogen and carbon with expected climatic changes reliably, determining cycles result in changes in the content of nitrogen and the main regularities of the direction of nitrogen-carbon carbon in plants, detritus layer and soil organic matter circulation, nature and mechanisms of restoring natural [1, 15–17] and are the foundation of the natural soil soil-formation using soil-restoring adaptive measures formation in agrosystems using soil-restorative adap- in the agroecosystems of the modern climatic system tive systems of soil preparation [16]. of the forest-steppe of Ukraine. As a rule, climate warming leads to the decrease in This work was aimed at determining the specifi ed parameters of the complex model of the interaction be- СО2 depositing in eco- and agroecosystems [18] which is related to the increase in the intensity of both productive tween nitrogen circulation and carbon circulation using and destructive processes: the rate of organic matter de- different types of crop rotation and alternative types of composition in soil is enhanced, soil breathing is intensi- organic fertilizers with different methods of soil culti- fi ed which results in higher dependence of the perfor- vationas well as determining the main regularities of mance of different plant aggregations on soil humidity the direction of nitrogen-carbon circulation, nature and and air temperature. In case of excessive manifestation mechanisms of restoring natural soil formation using of the abovementioned processes, the intensity of soil soil-restoring adaptive measures in the agroecosystems in the modern climatic system of the forest-steppe of breathing starts exceeding the rate of atmospheric СО2 accumulation by plants, and eco- and agroecosystems Ukraine. transform into the sources of emission of carbon dioxide MATERIALS AND METHODS [17] and nitrous oxide [3, 19] into the atmosphere. The study was conducted in the long-term fi eld sta- The failure to take the interaction of nitrogen and tionary experiment of the Cherkasy State Agricultur- carbon circulation in the conditions of climate warm- al Experimental station on typical heavy loam, light ing into consideration results in the reduction of stocks clay, low humus chernozem with the humus content of terrestrial carbon in plant aggregations (including of 3.8–4.2 %, the content of mobile phosphorus of agroecosystems) and in soil due to the intensifi cation 12–14 mg per 100 g of soil and mobile potassium – 8– of autotrophic breathing and the rate of the decom- 10 mg per 100 g of soil, рНQ = 6.8–7.0. The area position of organic matter of detritus and soil, i.e. the of the seeding bed was 162 m2, the reporting area – carbon-climate interaction gets positive direction. The 100 m2, the experiment was laid down in triplicate. course of the nitrogen cycle refl ects on the increase in The results for 35 years were rated. Two fi ve-year crop the terrestrial carbon content due to the increase in the rotations were studied. Crop rotation 1: peas–winter air temperature which is conditioned by the increase in wheat–sugar beets–corn–corn. Crop rotation structure:

СО2 concentration in the atmosphere and the intensiﬁ - 60 % of grain crops, 20 % grain legumes, 20 % tech- cation of mineralization processes in soil, which results nical crops. Crop rotation 2: perennial grass–winter in terrestrial accumulation of available mineral nitro- wheat–sugar beets–corn–barley with grass. Crop rota- gen, stimulating the performance of eco- and agroeco- tion structure: 60 % grain crops, 20 % technical crops, systems and intensifying the productivity of photosyn- 20 % perennial grass. The system of fertilization: with- thesis [11, 20, 21]. In case of a sufﬁ cient level of the out fertilizers, single dose (one dose – N33Р31К41) and mentioned processes the need for atmospheric carbon double dose (two doses − N66Р62К82) of mineral fertil- in plant aggregations starts exceeding the emission of izers. Until 2000 the mentioned doses of mineral fertil- carbon in soil, while terrestrial eco- and agroecosys- izers were introduced on the background of 6 t/ha of tems get transformed into systems – accumulators of humus with the extraction of secondary products, and atmospheric organic matter, i.e. the carbon-climate in- in 2001–2010 humus was substituted with 6 t/ha of sec- teraction becomes an inverse correlation model [2, 14] ondary products. The ways of soil cultivation: different which diminishes negative manifestations of the green- deep ploughing; subsurface tillage, surface tillage. The house effect of the climate changes in the forest-steppe calculation of carbon balance in the agroecosystems of Ukraine. of different types and forecast of the humus condition

36 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 REGULATION OF NITROGEN-CARBON INTERACTIONS IN AGROECOSYSTEMS

Fig. 1. The general model of nitrogen-carbon circulation in agroecosystems of short-term crop rotations for the forest-steppe of Ukraine

of chernozem as per weight of released СО2 was per- RESULTS AND DISCUSSION formed using the following fl ows: The calculations were used to determine the specifi ed average parameters of nitrogen-carbon circulation Сγ – weight of СО2, released due to humus mineralization, t; for the forest-steppe zone (Fig. 1). The average removal N with the harvest (30.6 t of feed units (f.u.) per Ср – weight of СО2, released due to the mineralization of secondary products, crop and root remains, t; one crop rotation or 6.12 kg/ha per year) was 596 kg (119 kg/ha) per one crop rotation with typical interval С(γ + р) – total weight of СО2, released due to mineralization, t; values of 426–718 kg. The total removal of nitrogen from the agroecosystem is 744 kg, and the typical in- Сj – weight of СО2, released via the breathing of soil organisms, t. terval removal is 525–937 kg. The input of N due to post-harvest, root, post-mowing remains and humus The level of ensuring potential bioproductivity of was 478 kg on average, and 342–634 kg in the interval crops with СО2 resources was determined using the measurement. The total input of N with the consider- balance of this resource, while the balance of organic ation of mineral fertilizers, nitrogen-fi xation, N of seed carbon was estimated as the difference between the material and rainfall amounted to 715 kg or 424–935 input of Сorg to the agroecosystem and its introduc- kg in the interval measurement with increasing remov- tion from the secondary products or pus into humus. al. The balance of N in agroecosystems amounted to 30

The expenditure item involves the removal of Сorg kg (–6 kg/ha) and was −203…+163 kg in the interval with the harvest and the weight of Сorg of secondary measurement. The intensity of N balance was 101 %, products, which passed to СО2 due to mineralization. which was 77–122 % in the interval measurement. The

For clarity of calculations Сj was taken as a constant interaction between nitrogen circulation and carbon for all the variants. The carbon balance was calcu- circulation was established: there is 1.12 t of negative lated using the common method taking into account carbon balance per one unit of negative value of ni- the removal of nitrogen (N) by the level of the per- trogen balance. The negativity of nitrogen balance in formance of crops; it was used as a basis to calculate agroecosystems ensures the positivity of carbon bal- the balance (N) [22] in the agroecosystems of crop ance in soil (BС(s)) on the level of simple replacement rotation with further elaboration of the recommenda- (BС(s) = +0.5 t or BС(s) = 0.1 t/ha, which in the interval tions for the ofﬁ cial publishing of the 6th National measurement is BС(s) = −1.7…−2.9 t. On average the Communication of Ukraine on the Issue of Climate intensity of N balance (Іb) was 101 % or 77–122 %, Change, 2012 [18]. and Іb (Сagr) = 21.5–86 %; in soil Іb (Сsoil) = 69.5–116 %.

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 37 VELICHKO et al. While introducing humus and removing the secondary In the crop rotation with peas (Fig. 2, Table 1) us- products from the agroecosystems, the parameters of ing the secondary products the removal of N with the nitrogen circulation as per the removal of yield were yield increases by 120 %, and the total removal of N decreased, compared to the average values, down to from the agroecosystem decreases by 180 kg (36.0 kg/ 551 kg (110 kg/ha) and the interval values were 383– ha) against the average value. The interval value of the 680 kg. Here the total removal of N increased up to removal of N tapers with the use of the secondary prod- 821 kg (164 kg/ha) and in the interval estimates it was ucts, and extends with the introduction of humus with 528–1031 kg (106–206 kg/ha). The input of N with the typical interval range of 1.68 (Table 1). With the afterharvest and root remains decreased by 129 kg use of humus the input of N with the secondary prod- (253.8 kg/ha) and the interval value of nitrogen input ucts decreases 2.02 times, which is a 3-fold decrease corresponded to 300–389 kg (60–78 kg/ha). by the typical interval range. The total input of N in- The total input of N into the agroecosystem was 629 creases 1.42-fold, which is a 1.27-fold increase by the kg (125.8 kg/ha), which is 352–846 kg (70–169 kg/ha) typical range. In case of applying secondary products in the interval values. With humus introduction the ni- the balance of N in the agroecosystem becomes posi- trogen balance in agroecosystems was −203 kg (−41 tive (+97.8 kg), while in case of humus the balance of kg/ha), which was −403…−64 kg or −80.6…−12.8 kg/ N was negative (–345 kg), which was +32.6 and −55 ha in the interval estimates. The intensity of N balance kg by the typical interval range respectively. The inten- was 37–91 %, or 82.1 % on average. The increase in sity of N balance was 109 and 66.7 % respectively with the defi ciency of N balance with systematic introduc- the 1.62-fold decrease of the typical range using the tion of humus is related to the increase in the defi ciency secondary products. The removal of N with the yield increased by 62 kg or by 113 % in the crop rotation of carbon balance both in agroecosystems (BС(а)) and in soil: B = −28 t (B = −31.5…−23.0 t, B = −1.3 with perennial grass using the secondary products than С(а) С(а) С(s) in case of humus introduction, while the total removal t, BС(s) = −4.5…−1.1 t). Here Іb of carbon in the agroecosystem and soil were on the level of 59.4−80.8 %. of nitrogen decreased by 129 kg (25.8 kg/ha). With the systematic introduction of secondary prod- The input of N due to the secondary products in the ucts the total removal of N with the harvest compared crop rotation with grass (Fig. 2) was 116 and 20 kg against the average value increased by 45 t (9 t/ha), and lower compared to the crop rotation with peas in case as per interval estimates – by 102–93 t (20.4–18.6 t/ha). of humus introduction, but in the framework of the The input of N from the secondary products increased crop rotation with the application of humus the input by 127 and 174 % against the average value and the of N decreased by 183 kg (36.6 kg/ha), which is a 1.58- systematic introduction of humus, thus it amounted to fold decrease by the typical interval range. In case of 607 kg (121 kg/ha) or 425–702 kg (85–140 kg/ha). The introducing the secondary products, the total input of N total input of N amounted to 802 kg which exceeded in the crop rotation with grass decreased 1.14 times and the average value and the introduction of humus by increased 1.13 times in case of humus application com- 113 and 128 % respectively, and the interval value pared to the crop rotation with peas, while in the crop amounted to 432–1052 kg or 86.4–210 kg/ha. The N rotation with peas the application of secondary prod- balance in agroecosystems was positive (+143 kg or ucts ensured 1.15 times higher input of N compared to +28.6 kg/ha) which was +44...+272 kg (8.8–54.4 kg/ humus application. ha) in the interval estimate. The intensity of the balance In the fi rst case the balance of N was +189 kg (37.8 changed in the range of values from 112 to 130 %. In kg/ha), and in the second one it was –61.4 kg (–12.3kg/ case of applying secondary products the positivity of N ha). Thus, in case of applying the secondary products Іb balance in the agroecosystem was accompanied with was 1.34 times higher. the increase in the defi ciency of carbon balance (B (а) С The positivity of N balance in case of applying the = −38.8 t) and the positivity of carbon balance in soil secondary products does not ensure the positivity of (B = +2.3 t). Thus, І in the fi rst case was 62.1 %, and С(s) b the balance of organic carbon (–38.8 t) in the agroeco- in the second one − 110 %. systems of crop rotations with peas and grass, whereas Due to the mineralization processes on average 101 t the negative balance of carbon was formed in the men-

СО2 were released into the atmosphere per one crop rota- tioned crop rotations with the humus introduction and tion, while in case of humus introduction it was 2.43 times it was 1.30–1.5 times less deﬁ cient. However, in case less, and it was 1.42 times less against the average value. of applying the secondary products the positivity of N

38 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 REGULATION OF NITROGEN-CARBON INTERACTIONS IN AGROECOSYSTEMS

Fig. 2. The general model of nitrogen-carbon circulation in agroecosystems of crop rotations for the forest-steppe of Ukraine: А − crop rotation with grass; B – crop rotation with peas

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 39 VELICHKO et al. balance affected the balance of organic carbon in soil yield is 645 kg; with subsurface tillage (20–25 cm)– (+2.0…−2.7 t or +0.40…−0.54 t/ha) in the crop rota- 628 kg, and with surface tillage (10–12 cm) – 457 kg. tion with peas and perennial grass. With the humus According to the typical interval range the removal of introduction the negativity of N balance determined the N was more stable with the subsurface tillage, whereas negativity of carbon balance in soil; −3.12 t (−0.63 t/ha) in case of deep ploughing and surface tillage the re- in the crop rotation with peas and −0.93 t (−0.06 t/ha) moval of N was 1.24 and 1.23 times higher (Table 2). in the crop rotation with perennial grass. Here Іb of car- The total removal of N from the agroecosystem was bon in the agroecosystems of crop rotations regardless the highest in case of ploughing (813 kg), while it was of the kind of organic fertilizers amounted to 56.6−62.2 17 and 252 kg or 3.4 and 50.4 kg/ha less for subsurface

%, and Іb of carbon balance in soil for the application tillage and surface tillage, respectively. The introduc- of the secondary products in the crop rotation with peas tion of N with the secondary products was the same for was 114.2 %, and in the crop rotation with perennial deep ploughing and subsurface tillage (488 kg) and it grass – 107.6 %. In case of humus introduction in the was 38 kg lower for surface tillage. The total introduc- former case Іb = 59.4 %, and in the latter – 102.6 %, tion of N into the agroecosystem for deep deep plough- which testifi es to the effi ciency of applying humus and ing was 717–742 kg (143–148 kg/ha), and it decreased secondary products specifi cally with the saturation of by 105 and 80 kg respectively for surface tillage. The crop rotations with perennial grass. balance of N for ploughing and subsurface tillage was The calculations demonstrate that alongside with the negative: −68.4 and −80.4 kg, whereas it proved to be effect of the kind of organic fertilizers and type of crop positive for surface tillage – +103 kg (+20.6 kg/ha). rotation the kind of soil preparation has its impact on The balance intensity for deep ploughing was 94.1− the circulation of N (Fig. 3, Table 2). Thus, with the 94.6 %, and for surface tillage – 122 %. The intensity systematic deep ploughing the removal of N with the of carbon balance in the agroecosystem and soil was

Table 1. The impact of a crop rotation and a kind of organic fertilizers on the speciﬁ ed parameters of nitrogen-carbon circulation in the agroecosystems of the forest-steppe of Ukraine

Crop rotation with peas Crop rotation with grass Parameter Humus, Secondary products, Humus, Secondary products, of circulation 6 t/ha 6 t/ha 6 t/ha 6 t/ha

Xaver Interval Xaver Interval Xaver Interval Xaver Interval Yield of feed units, t 29.0 24.8−33.3 33.0 25.3–41.0 29.3 25.8–32.4 30.7 27.8–35.3 Nitrogen removal with 601 501–721 718 551–895 502 356–644 564 370–718 the yield, kg Total removal of nitro- 92.5 634–1204 745 563–921 717 450–981 588 384–749 gen, kg Input of N, kg: secondary products 330 265–389 665 473–849 366 314–387 549 379–669 N in the agroeco- 591 291–824 837 505–1096 667 387–875 767 441–1038 system, kg Nitrogen balance, kg (±) −345 −535…−215 +97.8 −64−188 −61.4 −176…−406 189 55.5–305

Іb in the agroeco- 66.5 47.0–76.0 109 89–124 97.0 79.0–95.5 130 115–137 system, %

Balance of Сorg of −25.8 −29.0…−22.5 −38.7 −38.0…−25.0 −30.2 −33.5…−23.8 −38.8 −46.3…−33.0 the agroecosystem, t

Balance of Сorg of soil, t −3.12 −5.88…−3.31 +2.7 +0.9…+4.0 –0.93 –0.63…–1.62 2.00 0.4–2.7

Іb of Сorg of the agro- 62.2 14.0–92.0 62.0 26.0–85.0 56.6 18.5-85.0 62.2 23.0–85.5 ecosystem, %

Іb of Сorg of soil, % 59.4 54.0–65.0 114.2 109–129 102.2 94.5–112.5 107.6 103–117

Сorg (СО2), t 30.8 26.0–35.0 111 77.0–142 52.3 42.6–58.9 91.5 86.0–102

Note. Хaver − average value.

40 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 REGULATION OF NITROGEN-CARBON INTERACTIONS IN AGROECOSYSTEMS

Fig. 3. The general model of nitrogen-carbon circulation in agroecosystems of crop rotations for the forest-steppe of Ukraine: А – deep ploughing; B – subsurface tillage

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 41 VELICHKO et al. under 100 % regardless of the way of soil cultivation: the input of nitrogen from the secondary products was 55.3−62.7 % for carbon in the agroecosystems and 2.13 times higher than in case of humus application. 93.5−98.2 % for carbon in soil. Here СО emission 2 In case of humus introduction the relationship bet- due to the mineralization of the organic matter demon- ween СО emissions due to mineralization and the strates a stable tendency towards the decrease, caused 2 input of nitrogen corresponded to the level of weak by ploughing (СО – 77.5 t or 15.5 t/ha), compared to 2 correlation (R = 0.28−0.30 ± 0.05), whereas in case of subsurface tillage (67.9−68.5 t or 13.5 t/ha) and 60.2 t introducing the secondary products it was on the level (12.4 t/ha) for surface tillage. There was an ambiguous of direct strong correlative dependence (R = 0.79− effect of the way of soil cultivation on the amount of 0.85 ± 0.02), while the unit of СО released from the NО emission: 21−23 kg (4.2−4.6 t/ha). 2 2 mineralization of the secondary products had 5.59 and The estimates demonstrated (Table 3) that the kind 8.44 kg/ha of N of secondary products and nitrogen of of organic fertilizers affects the correlation level be- general input. tween the yield of feed units and the components of the nitrogen balance. In case of humus introduction In case of applying the secondary products the bal- there was a direct correlative relationship between the ance of Сorg in soil was positive in the typed interval performance of crop rotations, the total removal of of CО2 emission, whereas with the humus introduction nitrogen, its input from the secondary products and the positivity of carbon balance was accompanied with the total input of N: R = 0.69 ± 0.02; R = 0.57 ± 0.03, the deﬁ ciency of nitrogen balance in the typed interval R = 0.87 ± 0.02 and R = 0.82 ± 0.02 respectively, of СО2 emission due to mineralization. Direct correla- whereas in case of introducing the secondary prod- tion was established between the performance of a crop ucts the relationship enhanced up to the level of di- rotation with peas, the total removal of nitrogen, the rect strong correlation: R = (0.82−0.88) ± 0.03. Here introduction of nitrogen from the secondary products, with the increase in the performance of crop rotations total nitrogen introduction and the nitrogen removal on using humus introduction the balance of organic car- the level of R = 0.57 ± 0.03, with nitrogen input − on bon in the soil was negative, whereas in case of intro- the level of R = (0.79−0.91) ± 0.03. In a crop rotation ducing the secondary products it was positive in the with grass the relationship between the performance whole interval of the typed performance. In the latter and components of nitrogen balance weakens down to case one unit of performance increase had 2.1 times the average level by the removal and input of nitrogen smaller amount of the removal of total nitrogen, and from the secondary products R = (0.58−0.61) ± 0.03,

Table 2. The impact of the ways of soil preparation on the speciﬁ ed parameters of nitrogen-carbon circulation in the agroecosystems of the forest-steppe of Ukraine

The way of soil cultivation Parameter Deep ploughing Subsurface tillage Surface tillage of circulation

Xaver Interval Xaver Interval Xaver Interval

Yield of feed units, t 31.7 27.4−34.2 31.0 27.0–34.6 28.1 23.5–31.6 Nitrogen removal with the yield, kg 645 646–732 628 514–723 47.5 371–518 Total removal of nitrogen, kg 813 593–1005 796 650–979 561 452–648 Input of N, kg: secondary products 488 339–638 488 340–657 450 371–525 N in the agroecosystem, kg 742 461–966 717 452–907 637 376–966 Nitrogen balance, kg (±) –68.0 −215…+115 –80.4 –245…+149 103 –93.0…+334

Іb in the agroecosystem, % 94.6 77.5–117 94.1 71.0–122 122 83.0–102

Balance of Сorg of the agroecosystem, t –39.8 –42.1…–26.9 –29.5 –34.1…–22.1 –27.3 –33.0…–22.0

Balance of Сorg of soil, t –0.75 –3.7…+1.2 1.4 –0.5…+3.8 0.7 –0.6…+2.8

Іb of Сorg of the agroecosystem, % 62.7 21.5–87.5 55.3 21.0–85.0 57.8 22.0–84.0

Іb of Сorg of soil, % 93.9 71.0–110 97.3 69.0–127 93.6 59.0–116

Сorg (СО2), t 77.5 41.5–103 67.9 35.0–69.9 60.2 33.0–86.0

42 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 REGULATION OF NITROGEN-CARBON INTERACTIONS IN AGROECOSYSTEMS and there is an established direct strong correlation be- is accounted for by the unit of increasing the crop per- tween the total input of nitrogen and performance of formance in the crop rotations regardless of the way of crops (R = (0.73−0.83) ± 0.02); this dependence weak- soil preparation. In both cases the balance of Сorg in soil ens to the weak level with the nitrogen balance (R = was positive, and the balance of Сorg in agroecosystems = (0.82−0.87) ± 0.03). The same amount of nitrogen got impaired and, thus, defi cient. Table 3. The values of correlation coeffi cients and regression coeffi cients of linear equations of nitrogen-carbon circulation depending on the components of the cultivation system in the agroecosystems of the forest-steppe of Ukraine N balance, kg Type of crop rotation, kind of organic fertilizers, way Total Entered the agroecosystem of soil preparation removal from the agroecosystem with secondary products total

Yield of feed units in a crop rotation, t Introduction of 6 t/ha of secondary products Crop rotation with peas 0.85/12.9* 0.88/48.4 −0.01/−0.33 Crop rotation with perennial grass 0.37/7.90 0.86/53.6 0.13/5.9 Introduction of 6 t/ha of humus Crop rotation with peas 0.94/24.5 0.94/37.4 0.77/15.3 Crop rotation with perennial grass 0.29/2.60 0.26/4.80 –0.06/–0.56

СО2 emission in a crop rotation, t Introduction of 6 t/ha of secondary products Crop rotation with peas 0.78/10.7 0.79/40.7 –0.17/–8.93 Crop rotation with perennial grass 0.03/0.21 0.13/2.91 –0.11/–1.82 Introduction of 6 t/ha of humus Crop rotation with peas 0.91/5.55 0.93/8.80 0.83/3.88 Crop rotation with perennial grass 0.66/5.59 0.62/10.6 0.35/2.78 Yield of feed units in a crop rotation, t Ploughing for 22–25 cm 0.73/23.2 0.83/40.7 0.32/14.8 Subsurface tillage for 22–25 cm 0.75/22.0 0.81/39.5 0.37/17.8 Surface tillage for 10–12 cm 0.72/18.7 0.79/22.4 0.82/46.6

СО2 emission in a crop rotation, t Ploughing for 22–25 cm 0.87/4.48 0.59/4.65 0.66/4.94 Subsurface tillage for 22–25 cm 0.85/4.08 0.53/4.26 0.58/4.55 Surface tillage for 10–12 cm 0.76/3.61 0.52/4.90 0.32/2.60 Yield of feed units in a crop rotation, t (general model) Crop rotation with peas 0.57/21.8 0.79/26.9 0.91/42.2 Crop rotation with perennial grass 0.61/28.5 0.58/16.6 0.74/39.1

СО2 emission in a crop rotation, t (general model) Crop rotation with peas –0.04/–0.19 0.94/4.53 0.72/4.7 Crop rotation with perennial grass 0.03/0.34 0.71/4.35 0.39/4.36

Yield of feed units in a crop rotation, t (general model) 6 t/ha of humus 0.69/47.1 0.57/10.7 0.87/51.2 6 t/ha of secondary products 0.82/23.2 0.88/22.7 0.82/35.1

СО2 emission in a crop rotation, t (general model) 6 t/ha of humus 0.28/1.61 0.30/5.56 0.48/6.51 6 t/ha of secondary products 0.85/5.59 0.79/8.44 0.48/2.64

*Correlation coefﬁ cients (R)/regression coefﬁ cients (b) in linear equations of dependencies between the parameters.

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 43 VELICHKO et al. The estimation of the effect of the way of soil prepa- ± 0.02). The regression coeffi cients in the linear equa- ration on the relationship between the crop perfor- tions decreased 1.21–1.79 times between the total re- mance in the agroecosystems of short-time rotations moval and input of nitrogen, and increased 3.15 times and the components of nitrogen balance demonstrates with the total input. The amount of СО2 released due that regardless of the way of soil cultivation there to the mineralization with the surface tillage correlates are relationships between the total removal and input with the components of the nitrogen balance in the of nitrogen from the secondary products on the level way, similar to that of deep ploughing, but according to of direct strong correlation (R = (0.73−0.83) ± 0.02), the regression coeffi cients of the dependence equations whereas the dependence on the total removal of nitro- one unit of СО2 emission is accounted for by a 1.25 gen weakens down to the weak correlative relationship times smaller amount of nitrogen from the secondary (R = (0.32−0.37) ± 0.02). products and a 1.9 times smaller change in the nitrogen The direct strong correlation (R = (0.85−0.87) ± balance. The regression coeffi cients were similar in the ± 0.03) was established between nitrogen input from values in the regression equations of the dependence of crop rotation performance and СО2 emissions on the the secondary products and the amount of СО2 regardless of the way of soil cultivation, and with the total in- components of nitrogen balance, which testifi es to the put of nitrogen and its balance the relationship weakens uniformity of the impact of the way of soil cultivation down to R = (0.55−0.65) ± 0.03, which testifi es to the in the mentioned processes of biogenic element circu- decrease in the effect of the ways of chernozem prepa- lation. In case of systematic surface tillage the yield of ration on the relationships between the components of feed units per 1 ha in the agroecosystems of crop rota- nitrogen-carbon cycle in the agroecosystems of differ- tions (similar to deep ploughing) is related to the total ent crop rotations. The types of organic fertilizers as removal and input of nitrogen via strong direct correla- well as a crop rotation have the greatest effect on the tion (R = (0.72−0.79) ± 0.02). Contrary to deep plough- nitrogen-carbon circulation, while the way of soil cul- ing the level of a correlative relationship between the tivation is a subordinate factor in the agroecosystems. performance and the total removal of N increased up to the strong direct correlation. The production of CО2 depending on the type of a crop rotation is related to the total input of nitrogen (R = The use of secondary products as organic fertilizers (0.71−0.94) ± 0.03), and the balance of nitrogen in the with the localization of the former at the subsurface and crop rotation with peas correlates with the performance systematic surface tillage simulates natural circulation on the level of a direct strong correlation (R = 0.72 ± of the organic carbon in agroecosystems, which in the 0.03), whereas this relationship weakens in the crop ro- long run promotes the restoration of the resources of tation with grass (R = 0.45 ± 0.03). Сorg both in the agroecosystem and in soil, bringing The determination of the effect of the crop rotation its content closer to the natural status [19, 23]. It was also demonstrated in the works [19, 23] that the total factor on the balance of Сorg in soil demonstrates that the crop rotation with grass has the positivity of the bal- emission of СО2 decreases in the following order: wild land–fallow–tillage. In spring and summer the emis- ance in the typed interval of СО2 emission; there was an sion of СО on the wild land exceeds the emission of increasing tendency of the balance of Сorg in the crop 2 rotation with peas, and the positivity of the balance of carbon dioxide for tillage 10–23 times, also in spring the emission of СО is 10–25 times higher than in au- Сorg in soil is achieved due to the maximal input of ni- 2 trogen from the secondary products and the total input, tumn with the maintenance of wild land and fallow and 4–5 times higher – in case of systematic tillage in the which simultaneously decreases the defi ciency of Сorg of the agroecosystem down to −30 t/ha. There is an in- long run. crease in the defi ciency of С balance in the agroeco- org The interaction between carbon and nitrogen circula- system due to the removal of the weight of perennial tion in the agroecosystems has a considerable impact grass in the crop rotation with grass. on the content of nitrogen and carbon in the cultivated

The amount of СО2 released from the mineralization crops, detritus and organic matter of soil, and the inten- of the organic matter is a direct function of the input from sity of nitrogen and carbon circulation is determined the secondary products (R = (0.85−0.87) ± 0.02), while by the C to N ratio both in soil and agroecosystems the relationship with the total input of nitrogen to the in general [18]. The use of different types of organic agroecosystem and the balance of N weakens to the di- fertilizers tells both on the removal of nitrogen and car- rect correlation of the medium level (R = (0.53−0.66) ± bon. With humus introduction the removal of nitrogen

44 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 REGULATION OF NITROGEN-CARBON INTERACTIONS IN AGROECOSYSTEMS beyond the boundaries of agroecosystems exceeds its in the total circulation – 1.15 times higher compared input by 154 kg, whereas, vice versa, in case of apply- to the crop rotation with grass. ing the secondary products the input of nitrogen into In the crop rotation with peas the removal of Сorg the agroecosystem exceeds its removal by 145 kg due from the agroecosystems, regardless of their type, was to the input of nitrogen from the secondary products, 36.0−39.0 t, and the input of Сorg was 1.23 times higher which increases 1.75 times with humus introduction. though the total amount of С in the agroecosystems Thus, the removal of С with the introduction of the org org with peas and grass was in the range of 59.4−60.8 t. The secondary products increases by 7.6 t, and the input of C to N ratio in terms of removal from the crop rotation С increases 2.1 times compared to the application of org with grass was 1.53 times higher. The input of nitrogen humus (Table 4). and carbon was registered in the range of 39.5–41.7 Regardless of the kind of organic fertilizers, the t regardless of the type of agroecosystem, though the amount of nitrogen from 1424 to 1468 kg is involved volume of the total circulation of C and N in the crop in the agroecosystems of crop rotations with the in- rotation with grass was 1.24 times higher compared to crease in case of introducing secondary products, and the crop rotation with peas. The impact of the way of in the latter case there is 1.48 times more carbon than soil cultivation on the circulation of N was manifested for humus introduction. The C to N ratio for the to- in the fact that with subsurface/surface tillage the total tal removal using the secondary products was 97 to 1 removal, input and volume of N in the agroecosystems against 45 to 1 on average with humus introduction, decreased 1.05, 1.03 and 1.09 times respectively, while and by the articles of input the C to N ratio was 53 the input and total amount of Сorg had a stable tendency to 1 and 28 to 1. In the general circulation it was 54 towards decreasing 1.14, 1.12 times. It affected the C to 1 and 37 to 1, respectively. The impact of the type to N ratio, the value of which for subsurface/surface til- of crop rotation on the C to N ratio has some speci- lage decreased 1.05, 1.08 times against tillage, respec- ﬁ cities: the total removal of nitrogen was 1.19 times tively, compared to the application of humus and straw higher in the crop rotation with peas compared to the with tillage, which testiﬁ es to the decrease in the inten- crop rotation with grass. The input of nitrogen from sity of C and N circulation with surface tillage both in the secondary products was 1.1 times higher; the total soil and agroecosystem in general. A higher amount of input – 1.05 times higher, and the amount of nitrogen reliable correlative inverse relationships between the C Table 4. The ratio of carbon and nitrogen in agroecosystems of different types using different types of organic fertilizers and ways of chernozem preparation

Parameter X * Interval * X * Interval * of circulation aver aver

Types of organic fertilizers Humus, 6 t/ha Secondary products**

Сorg (removal) to N (removal) 44.7 34.0–49.0 67.4 52.0–75.0

Сorg (input) to N (input) 28.0 17.5–39.5 53.2 37.0–64.0

Сorg (agroecosystem) to N (agroecosystem) 36.9 27.5–40.5 54.2 38.0–57.0 Crop rotation with Peas Grass

Сorg (removal) to N (removal) 44.3 34.0–53.5 67.8 45.0–76.5

Сorg (input) to N (input) 41.7 18.0–58.5 39.5 28.5–44.5

Сorg (agroecosystem) to N (agroecosystem) 40.6 27.5–52.5 50.5 34.5–63.5 The way of soil cultivation Ploughing for 22–25 cm Subsurface for 22–25 cm

Сorg (removal) to N (removal) 57.7 41.0–56.5 55.1 41.0–60.0

Сorg (input) to N (input) 42.4 33.5–54.0 39.0 23.5–57.0

Сorg (agroecosystem) to N (agroecosystem) 47.7 35.5–54.0 44.3 32.5–53.5

*Сorg, t per 1 kg of N, **7 t/ha of secondary products.

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 45 VELICHKO et al. to N ratio of the abovementioned articles of nitrogen fertilizers which improves the balance of the organic and carbon articles was revealed using different kinds carbon in soil and causes the reduction of its defi ciency of organic fertilizers: R = (−0.55…−0.81) ± 0.02 for in the agroecosystem in general. humus introduction and R = (−0.56…−0.73) ± 0.02 for The types of organic fertilizers affects the emission the introduction of secondary products. of СО2 due to mineralization into the atmosphere: ac- For different ways of soil cultivation the level of cor- cording to the typical interval estimate the amount of relative relationships decreased down to a weak inverse emission in case of humus application corresponds to correlation with the removal and input of nitrogen with 25–85 t, whereas in case of introducing the secondary tillage, and there was a considerable strengthening of products the weight of emission reaches up to 80–160 the correlative relationship between the removal and t which is accompanied with the increased removal of total circulation of C and N as well as their ratio: R = nitrogen compared to its input into the agroecosystems (−0.65…−0.73) ± 0.02. In case of surface tillage the in the former case, and vice versa, linearly to the in- inverse reliable correlative relationships were revealed creasing input of the total nitrogen relative to its re- relative to the total circulation of C and N and their moval due to mineralization of the secondary products ratio, and the strengthening of the relationship to the – in the latter. At the same time there is increasing de- values of the medium level – relative to the total refi ciency in the carbon balance in soil with the humus moval of nitrogen. In the crop rotation with peas the application and the increase in its positivity in case of relationships between the mentioned parameters of the applying secondary products, i.e. there is extensive res- nitrogen-carbon circulation were on the level of a weak toration of chernozem fertility due to surface tillage. inverse correlation, and in the crop rotation with grass The impact of the type of agroecosystem (crop ro- they approximated the level of values of R = (−0.55… tation) on nitrogen-carbon circulation manifests itself −0.77) ± 0.02. The emission of СО due to mineral- 2 in the fact that the saturation of crop rotations using ization of С in the agroecosystems was 10.9–14.9 t org crops with a high yield of secondary products (crop according to the normalized interval of values with the rotation with peas) ensures linear increase in the nitro- increase up to 16.7 t with the introduction of second- gen input in the typical interval measurements of in- ary products. The emission of СО due to mineraliza- 2 creasing the performance due to the mineralization of tion of secondary products was 3.85−6.83 times higher secondary products and nitrogen removal from them. compared to humus mineralization and amounted to The rate of removal and input of nitrogen gets leveled 41.4−102 t, increasing to 139 t by the maximally typi- with the removal of the weight of perennial grass, and cal value. The share of СО emission due to mineraliza- 2 with the maximal performance they are not compen- tion of humus and secondary products was from 74 to sated with remaining secondary products and nitrogen- 85 %, and reached 89−90 % in its maximal values. fi xing, which results in the increase in the defi ciency of CONCLUSIONS the carbon balance with the inverse linear dependence The circulation of nitrogen, which is one of the main whereas in the crop rotation with peas the increase in limiting factors of the manifestation of performance the carbon defi ciency changes parabolically in the typi- and intensity of the destructive processes in the agro- cal interval of performance increase. In the latter case ecosystems of different crop rotations, has a consider- the increased emission of СО2 is accompanied with the able impact on the circulation of organic carbon with increasing carbon balance of soil and the increased per- the application of different types of organic fertilizers formance of the agroecosystem. and ways of chernozem preparation in the climatic sys- The effect of the way of chernozem preparation on tem of the forest-steppe of Ukraine. the nitrogen-carbon circulation manifests itself in the The use of humus as an organic fertilizer creates the fact that regardless of the type of organic fertilizers and conditions when the total removal of nitrogen from the that of agroecosystem in case of surface tillage the car- agroecosystems exceeds its input in the typical inter- bon balance in the interval of increasing performance val of the performance increase which is related to the is positively increasing compared to ploughing, where increasing defi ciency of the organic matter of humus the increase in the defi ciency is parabolic. The interval with excessive mineralization and removal of nitrogen. of СО2 emission into the atmosphere due to mineraliza- In case of applying the secondary products the total tion after ploughing was wider relative to surface till- removal of nitrogen from the agroecosystems is less age, which testifi es to the increase in the mineralization than its input from the secondary products and mineral processes in soil with the uniformity of nitrogen-car-

46 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 REGULATION OF NITROGEN-CARBON INTERACTIONS IN AGROECOSYSTEMS bon circulation and determines the way of soil cultiva- ity is ensured by the activation of the photosynthetic tion as a subordinate regulatory subsystem of the crop activity of cultivated crops due to the restoration of rotation type and the kind of organic fertilizers in the drain mechanisms of carbon with the increase in the general circulation. СО2 content in the atmosphere and the accumulation The introduction of humus and removal of secondary of heat resources in the agroecosystems in general products from the agroecosystem while deep plough- which should be the foundation of extensive restora- ing leads to the balance between nitrogen-carbon cir- tion of the fertility of typical chernozem of the forest- culation which is accompanied with the decrease in the steppe of Ukraine. stocks of terrestrial carbon in the agroecosystem and Управління азотно-вуглецевим обігом its components (plants, soil, pool of microorganisms). в агроценозах Лісостепу України It leads to the intensifi cation of autotrophic breathing В. А. Величко 1, О. В. Демиденко 2 and the acceleration of the rate of detritus and humus decomposition i.e. the relationship between carbon cir- e-mail: demo06@yandex culation and climate becomes positive. In case of this 1 Національний науковий центр model of agroecosystem maintenance the replacement «Інститут ґрунтознавства і агрохімії of ploughing with surface tillage only increases the імені О. Н. Соколовського» НААН України processes of humifi cation of the organic matter in soil Вул. Чайковського, 4, Харків, Україна, 61024 2 and somewhat improves the impact of the nitrogen- Черкаська державна сільськогосподарська дослідна carbon circulation on the year to year variations in the станція ННЦ «Інститут землеробства НААН України» Вул. Докучаєва, 13, с. Холоднянське, Смілянський р-н, surface heat resource due to the emission of СО from 2 Черкаська обл., Україна, 20731 the agroecosystem into the atmosphere. Мета. Встановити нормативні параметри комплексної The application of secondary products as an organic моделі обігу азоту і вуглецю при застосуванні різних fertilizer enhances the process of optimizing the nitro- типів сівозмін, видів органічних добрив і різних спо- gen-carbon circulation in agroecosystems of differ- собів обробітку ґрунту в агроценозах Лісостепу Украї- ent types towards natural organization which leads to ни. Методи. Польовий, лабораторний, розрахунковий, the increase in the stocks of surface carbon with the математико-статистичний. Результати. Вид органічного enlargement of heat resources, conditioned by the in- добрива впливає на емісію СО2 в приземний шар ат- мосфери локалізації стеблостою культур: за використання creased emission of СО2 into the atmosphere due to mineralization of the excess of the secondary products гною типовий інтервал викидів вуглекислоти становить and the release of mineral forms of nitrogen into soil. 25−85 т/га, тоді як при застосуванні побічної продукції − The latter increases the performance of agroecosys- 70−160 т/га. Вплив способу обробітку чорнозему на азото-вуглецевий обіг зводиться до того, що за безпо- tems due to the “stimulating effect of N” and enhanced лицевого обробітку баланс вуглецю у ґрунті був додатно- adsorption of СО2 of the atmosphere. In these condi- зростаючим порівняно з оранкою. Інтервал емісії СО2 tions the growth of the greenhouse effect promotes the в приземний шар атмосфери від мінералізації гумусу і accumulation of organic carbon in agroecosystems and органічних добрив за оранки змінюється у більш широкому farming ecosystems which get the properties of waste- діапазоні відносно безполицевого обробітку. Висновки. water systems, i.e. the higher dependence of the perfor- За внесення гною та вилучення побічної продукції за mance of agroecosystems on the intensity of nitrogen межі агроценозу за оранки порушується виваженість в assimilation which entered the system of a farming азотно-вуглецевому обігу. Використання як органічного добрива подрібненої побічної продукції рослинництва, ecosystem, the faster the emission of СО2 due to the mineralization is consumed. яка за безполицевого обробітку загортається у поверх- невий шар чорнозему, моделює природний характер The maintenance and application of ground second- азотно-вуглецевого обігу в агроценозах сівозмін різного ary products of crop production, suffi ciently compen- типу. При цьому посилюється природне ґрунтоутво- sated in terms of nitrogen using mineral fertilizers, as рення за рахунок активізації фотосинтетичної актив- organic fertilizers, and wrapped up into the surface ності сільськогосподарських культур при насиченні СО2 layer of chernozem during the surface tillage, simu- приземного шару атмосфери локалізації стеблостою у lates the natural course of nitrogen-carbon circulation період вегетації, що забезпечує розширене відтворення in agroecosystems of different short-term crop rota- родючості чорноземів Лісостепу України. tions. Here the simulation of natural soil formation in Ключові слова: вуглець, азот, СО2, безполицевий обро- agroecosystems alongside the microbiological activ- біток, оранка, агроценоз.

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forest-steppe zone of Ukraine. Int J Agric Res Rev. land model response to CO2 fertilization and climate 2014;2(8):092-098. variability.Global BiogeochemCycles.2007;21:GB4018. 17. Zaehle S, Dalmonech D. Carbon−nitrogen interactions 22. Baliuk SA, Grekov VO, Lisovyy MV, Komarysta AV. on land at global scales: current understanding in Estimation of the balance of humus and nutrients in modelling climate biosphere feedbacks.Curr Opin Envi- agriculture of Ukraine on different levels of management. ron Sustain.2011;3(5):311–20. Kharkiv,Mis’ka drukarnia.2011;30 p. 18. The 6th National Communication of Ukraine on the 23. Poluektov RA. Description of ammoniﬁ cation process in Issues of Climate Change.Kyiv.2012;342 p. the framework of transformation of carbon and nitrogen in soil.Problemy agrokhimii i ekologii.2011;(4):25−8. 19. Avksentiev АА. Emission of greenhouse gases (СО2, N2O,

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 49 ISSN: 2312-3370, Agricultural Science and Practice, 2015, Vol. 2, No. 1

UDC 631.45 SOIL SPATIAL HETEROGENEITY AND SYSTEMS OF AGRICULTURE V. V. Medvedev NSC «O. N. Sokolovsky Institute for Soil Science and Agrochemistry» NAAS 4, Chaikovskoho Str., Kharkiv, Ukraine, 61024 e-mail: [email protected] Received on February 4, 2015

Aim. To consider soil continuality and discreteness as features of heterogeneity manifestation in a soil cover, important for construction of agriculture systems. Methods. Geostatistical research of soil spatial heterogeneity, revealing the contours of a fi eld with various parameters of fertility. Results. The use of principles of precise agriculture and inspection of indicative properties of fi eld soils using a regular grid allowed to divide a fi eld into contours with three levels of fertility: the fi rst one is characterized by optimal or close to optimum properties which allows refusing from (or reducing substantially) tillage, introduction of fertilizers or chemical ameliorates; the second one has average parameters of fertility corresponding to zonal soils and demands the application of zonal technologies; the third one (with the worst parameters of fertility) presupposes regular use of the improved technologies. Conclusions. The introduction of precise agriculture will allow replacing a traditional zonal system with thenew which is soil-protecting and resource-saving one. Key words: soil continuality and discreteness, zonal and precise agriculture.

INTRODUCTION in discussing this issue. Armand D. L. adhered to the There have been studies on different manifestations continuality principle, Stepanov I. N. – to the discrete- of heterogeneity, namely, continual heterogeneity, ness principle, Fridland V. M., Voronin A. D., Dmitriev when spatial soil properties change gradually (simul- E. A. believed that a soil cover is both a discrete and taneously with soil formation factors), and discrete, in- continual body at the same time (quoted from [1]). In termittent heterogeneity, when soil properties change in recent years the interest to this issue has risen consid- the framework of small areas. Here the main factors of erably, because there is more qualitative information, soil formation do not change. The interest to heteroge- obtained by remote sensing methods, which are the neity is quite reasonable as it is the actual basis for the most correct ones in estimating these properties of a agriculture systems – zonal, restricted to some zones, soil cover. Any regular network of dots, however thick and precise agriculture, the specifi cities of which are it is, does not measure up to a real map, obtained by a formed regardless of the heterogeneity of the crop rota- remote sensing method. For this reason the application tion fi eld. Here the main attention is focused on precise of some remote spectrometric means allows interpret- agriculture as it corresponds to the spirit of the age the ing a soil cover as mainly a continual object, where the most – it allows protecting soil from degradation and properties (heterogeneity) gradually change in space saving resources (due to the abolition of activities in [2, 3]. At the same time the radar probing in the verti- the part of the fi eld where they are not required). cal direction always reveals heterogeneity in the profi le In order to understand the essence of the notions of composition, which may be explained by the changes continuality and discreteness of a soil cover, it is rea- in soil texture, density and humidity [4, 5]. sonable to recall some recent live discussions of this Therefore, it is reasonable to state that the soil topic. In the 70–80s of the last century many outstand- cover in a 2-D format is mainly a continual forma- ing scientists, geographers and soil scientists took part tion, the continuality of which is disrupted only

50 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 SOIL SPATIAL HETEROGENEITY AND SYSTEMS OF AGRICULTURE due to a complex composition of valleys and bot- two – in the Forest-Steppe (Korotych and Kommunar) tom land, high-relief terrain in submountain regions and one – in the Steppe (Donetsk). and mountains, due to alteration of different types Romaniv (Volyn’ Region). Gray podzolic, sod-pod- of vegetation and other reasons. At the same time in zol and gleyed black soils prevail in the soil cover. a 3-D format the soil cover is an evident continual- The terrain is fl attened. The soil texture is light clay discrete formation due to genetic horizons, different loam. The size of the fi eld is 63 ha, the number of in their composition and structure (especially in the elementary patches is 35. Cereal and forage crops are differentiated soils). Most probably, soil is discrete cultivated. The agrotechnical methods of crop cultiva- in a 4-D format as well, when the abovementioned tion are not differentiated, regardless of evident mot- formats are added time as a factor of the soil cover tling of the fi eld. transformation. It is well known that in the course of long-term use the soil loses humus, there are new Kolky (Volyn’ Region). The soil cover is a complex features in its morphology, properties and regimes. of sod-podzol gleyed, sod-gley and meadow-bog soils. All the abovementioned facts allowed defi ning a new The terrain is fl attened. The soil texture is argil sand. type of soil – agrozem, this term appeared in soil The size of the fi eld is 11 ha. The number of patches classifi cations of Russia, Belarus, Ukraine and other is 27. Forage crops are cultivated (on the non-boggy countries. These changes in the soil cover enhance part). The fi eld has been dewatered by an open network its heterogeneity in space and promote the occur- of channels, which function only partially, unfortu- rence of new boundaries [6]. Thus, time becomes a nately. The studies have been conducted only on the factor of discreteness of the soil cover. non-boggy part. The agrotechnical methods are not differentiated in the fi eld. Unfortunately, it should be admitted that the soil cover has been mainly studied as a continual and very Vediltsy (Chernihiv Region). Sod-mesopodzol loamy little – as a discrete body, especially if viewed at a pe- soils. The terrain is fl attened. The size of the fi eld is culiar medium hierarchy level (in terms of spatial het- 105 ha, some part of the fi eld is grassy, the number of erogeneity of many soil properties in the framework of patches on the non-grassy part is 117. Cereal and for- a polypedon or, in the context of this article – a crop age crops are cultivated. rotation fi eld). Certainly, it inhibits successful develop- Korotych (Kharkiv Region). Dark grey heavy loamy ment of the notions of a horizontal profi le of soils and, podzolic soil prevails. The terrain is slightly sloping. as a consequence, practical applications of heterogene- The size of the fi eld is 31 ha. The number of patches is ity to precise agriculture. 35. Cereal and forage crops are cultivated by the meth- The aim of the article is to consider continuality and od, traditional for the forest-steppe. discreteness as specifi cities of the manifestation of het- Kommunar (Kharkiv Region). Typical, low humus, erogeneity in the soil cover, relevant for the establish- leached, heavy loamy chernozem. The terrain is fl at- ment of an agriculture system, new boundaries in the tened. The size of the fi eld is 30 ha. The number of soil cover, formed due to the investigation of indicative patches is 26. Cereal and forage crops are cultivated. properties of soils in the regular network of dots as well Donetsk (Donetsk Region). Typical heavy loamy as the value of new boundaries for precise agriculture. chernozem. The terrain is fl attened. The size of the fi eld MATERIALS AND METHODS is 105 ha, the network of patches (51) is established on the part of the fi eld of 50 ha. Cereal and forage crops We made an attempt to use the traditional notions are cultivated. about spatial specifi cities of soil properties with the purpose of improving modern agriculture practice, es- RESULTS AND DISCUSSION pecially precise agriculture, including the ways of fi nd- Modern agriculture systems and their drawbacks. ing the boundaries of contours with different fertility The solution to a large complex of issues, related to the indices. The materials of long-term studies of spatial enhancement of soil fertility, its protection, rational use heterogeneity of soils in Polyssia, Forest-Steppe and (with the consideration of the terrain, climate, econom- Steppe, summarized in monographs, were used in the ic and social demand, ecologic requirements) depends work [6, 7]. on the agriculture system. It includes the issues of ra- Six fi elds were used as objects, three of which are tional balance of the fi elds, the structure of planting and located in Polyssia (Romaniv, Kolky and Vediltsy), crop rotations, introduction of fertilizers, application of

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 51 MEDVEDEV chemical and other kinds of amelioration, agrotechni- soils of Forest-Steppe are characterized by the process- cal methods. The role of zonality, microzonality and es of dehumifi cation and loss of calcium, due to which landscapes in the location of crops is defi ned in the the soils are inclined to the destruction of aggregates agriculture systems rather objectively and comprehen- and acidifi cation of soil; these processes are not global, sively – it is a very wide scope of problems, solving they are manifested only in specifi c parts of the fi eld. which is focused both on meeting the needs of popula- As shown in multiple 2-D and 3-D diagrams, the soil tion in food products, and the need of production in raw cover of the fi eld in the Steppe is also heterogeneous, materials, but also on the stable character of agriculture the morphological, physical and physical-chemical at present and in the long run. However, there are many properties of the fi eld soil vary in space which actual- claims regarding modern agriculture systems as the lat- izes the problem of differentiating the agriculture sys- ter have been the reason of multiple manifestations of tems in this zone as well. degradation and worsening of soil quality. Their main The abovementioned is of exclusive importance for drawback, considered in this article, is a too general- the differentiation of agrotechnologies in accordance to ized content, corresponding to the conditions of the actual parameters of the fi eld soil. If the fi eld proper- natural zone (which is why they were called zonal agri- ties – the ones, determining the content of agrotech- culture systems), and ignoring spatial specifi cities of a nologies (structure density, content of nutrients, etc.) – specifi c fi eld of a crop rotation. did not have any expressed mottling, the latter could be For instance, the zonal agriculture system in Polys- neglected and the fi eld could be cultivated with even sia may be considered the most intensive. Sod-podzolic introduction of fertilizers on the whole space. Unfortu- soils, prevailing in this zone, have an acid reaction of nately, it does not correspond to reality. There are more the soil solution, sandy and loamy soil texture, it quick- and more data on the heterogeneity of fi elds regardless ly restores its increased initial density after the tillage, of genesis and the level of previous tillage of soils [7]. and easily forms a superfi cial crust due to the intensive The heterogeneity is manifested even in the fi eld where increase in the temperature in spring. Due to the in- the elements of high farming standards were applied creased amount of rainfall, sloping in the terrain and for almost 150 years [8] which allowed for the con- often shallow compacted illuvial horizons these soils clusion on the heterogeneity as a property, immanently have the signs of being gleyed. In addition, Polyssia inherent to soils. soils are mainly defi cient in nutrients. Due to the above At present some fi elds of crop rotations are not sub- mentioned specifi cities the agriculture system in this ject to detailed study using a regular network of dots. zone requires multiple tillage, introduction of fertiliz- Even in case of agrochemical certifi cation, when it ers and lime. Still, it is just a general scheme. As shown could have been done without the increase in expenses, below using the example of investigated fi elds, their the preference is given to the reconnaissance survey, morphological, physical and physical-chemical prop- performing which does not provide any adequate no- erties, this universal approach based on the averaged tion on the spatial heterogeneity of the fi eld [9]. For zonal characteristics requires considerable corrections this reason in any natural zone the agriculture system regarding practically all the components of the agricul- is based on the averaged indices of soils and climate ture system. without the consideration of specifi c fi eld specifi cities. The same is true regarding the soils of the Forest- Certainly, the changes in landscape are considered for Steppe. Typical chernozem, podzolic and dark grey signifi cant sizes of natural zones. In this case the agri- soils of medium and heavy loamy soil texture with culture system refl ects the peculiarities of accumula- good structure and introduction of humus, and mod- tive, transit and automorphous territories. The same is erately dense structure dominate in the soil cover; they true for climate – the share of moisture-retaining tech- are mainly provided with nutrients and have the me- nologies increases in more arid conditions. However, dium reaction, close to optimum. At the same time they the application of the zonal agriculture system presup- are not water-resistant enough, often too compacted in poses gradual change in the soil cover in the bound- the furrow bottom and in the underplanting layer after aries of the natural zone, refl ecting only the continual the spring cycle of tillage, and they are inclined to the constituent of heterogeneity and ignoring the heteroge- formation of lumps, crust and cracks. The drawbacks neity in the framework of the crop rotation fi eld. of these soils are not manifested on the whole fi eld, The discreteness of the fi eld in precise agriculture us- they are remarkable for its boundaries or sloping. The ing fertility norms. The notions of the soil cover as a

52 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 SOIL SPATIAL HETEROGENEITY AND SYSTEMS OF AGRICULTURE discrete formation are being formed in precise agricul- one (Table 1, 2). These norms have been obtained by ture, as the purpose of the latter is to detect the patches the authors on the basis of literature data and adapta- with different fertility in the fi eld and then to make the tion of recommendations to the specifi cities of precise technology of cultivating crops more discrete. The dis- agriculture, indicated therein [6]. creteness in these notions is a compulsory measure, as Precise introduction of organic fertilizers comes fi rst. it is a more convenient and easier way of implementing The parts of the fi eld, where stable increased humus the principles of precise agriculture. At the same time content was registered in the course of several evalua- these notions are nothing but an artifi cial transformation courses, were used to prove the possibility of re- tion of the continual soil cover into the discrete one. fusing from the introduction of manure. The average How can one interpret regular interchange of struc- norm of manure, based on the possibilities of the farm, tures with specifi c properties, revealed in actual soil should be introduced in the parts of the fi eld, where the cover? Is it discreteness, intermittency, or continu- deviation from the previous content reaches –5...–10 ality, a mistake? In the work [6] this interchange is %. Finally, the increased norm of manure should be ap- viewed as a manifestation of continuality and gradu- plied in the parts of the fi eld, where the content is 10 % ality in the change of properties. Here the discrete- less than the increased level, or even more (Table 1). ness is introduced via the application of interpolatory Traditionally the data on crop yield is nearly the only (somewhat formal) kriging-methodology, when the source of information for planning precise agriculture graduality is artifi cially interrupted with classic sub- and, in particular, for introducing organic and mineral divisions of properties. 2-D diagrams, numerously fertilizers. There is an attempt to use these data in this used by the author (in this case block kriging is not work, although the author has his own and more accu- applied in their creation), demonstrate the graduality rate information on the spatial heterogeneity of practi- of the interchange of properties in the space of all the cally all the main macroelements of nutrition and other investigated fi elds [6]. factors of fertility and yield in the investigated fi elds. Below are the norms, serving as a basis for the trans- As stipulated above, the spatial heterogeneity of the formation of the continual soil cover into the discrete data about the yield of cereal crops and sunfl ower is

Table 1. The rationing of the introduction of fertilizers and lime in precise agriculture

Mineral fertilizers Organic fertilizers Lime application Content, Deviation from Norm of Norm of Index mg/kg the average рН Scenario introduction introduction of soil content, %

Total mineral nitro- > 30 0 Stable increased 0 7.0−5.5 Without melio- gen 30–15 By removal with content for 2–3 ration expected yield courses of agro- < 15 The same + addi- chemical certiﬁ ca- tionnal amount due tion to variant 0 Mobile phosphorus >150 0 −5 … −10 Average estimated 5.5− 5.0 Sustaining me- (by Chirikov) 150–50 By removal with introduction, ba- lioration expected yield sed on the possi- < 50 The same + addi- bilities of a farm tionnal amount due to variant 0 Mobile potassium > 120 0 −10 and above The same + addi- < 5.0 Systematic in- (by Chirikov) 120–40 By removal with tional amount due troduction of li- expected yield to economy in va- me in higher riant 0 doses < 40 The same + addi- tionnal amount due to variant 0

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 53 MEDVEDEV moderate and refl ects some averaged value of spatial the sphere of tillage practice is the data on the ratio heterogeneity of all the investigated indices, includ- of zones with favorable, less favorable and unfavor- ing the cases when the spatial heterogeneity of the able agrophysical conditions on the investigated fi elds. yields was analyzed in the aftereffect (Korotych, 2 Thus, the recommendation on differentiating zero (no years of aftereffect, Kommunar, 1 year of aftereffect). tillage), minimal and traditional tillage on the fi elds Rather coordinated data about the spatial heteroge- becomes clear. The higher the share of patches with neity of the humus content and yields were received favorable parameters of balanced bulk density on the only for the objects of Romaniv and Donetsk. Almost fi eld in the pre-sowing period or prior to the main till- complete coincidence of heterogeneity was revealed age is, the more relevant the precise tillage becomes in these cases. Some differences were present in other (Table 2). objects. Here the data about the spatial heterogeneity According to the data of Table 2, potential possibili- of humus content were used for the delimitation of the ties of reducing the intensity of the pre-sowing tillage fi eld. In general the coincidence of fi elds was proven of soil and even completely refusing from tillage turned satisfactory in the observations of the productivity in out to be surprisingly high even on the soils with unfa- aftereffect as well. vorable agrophysical conditions. It should be noted that If the pH value of the soil is in the subacid/neutral the evidence to this fact can be found in the works of interval, which is favorable for all the crops, cultivated some other authors [10]. in the Forest-Steppe, and almost all the crops of Polys- The boundaries in the soil cover for precise agricul- sia, lime application is not required. From 30 to 80 % ture. Precise agriculture applies new boundaries to di- of such patches were found in the investigated fi elds vide the soil space into separate contours. The latter act which is a relevant fact as usually lime application of as independent working areas, on condition that their acid soils in these natural zones is performed along the sizes provide for economically profi table operations. whole area of the fi elds without any exceptions. Con- Precise agriculture and new technical possibilities of siderable economy of lime materials is evident. The mapping, based on geoinformational technologies and norm and regularity of amelioration increase with the remote means became the tool of obtaining principally rise in acidity. According to the data about the spatial new mapping information. These are electronic digital homogeneity of the soil solution reaction, full-scale maps. As the contours of soil properties on these maps lime application is required for only one fi eld on the almost never coincide with the contours (of kinds and Kolky object. even higher taxonomic units) on the traditional soil An additional argument in favor of the research ac- maps, the connection between these two maps has been tivity and implementation of precise agriculture in lost. Gradually precise agriculture has not had any need

Table 2. The standards of estimating physical properties to substantiate the intensity of tillage practice*

Qualitative estimate Recommendation on intensity Index of the tilled soil layer of pre-sowing tillage

Number of blocks in the sowing layer, %: < 5 Favorable No tillage needed 5–15 Satisfactory Moderate tillage > 15 Unsatisfactory Intense Bulk density in the sowing layer, g/ccm: < 1.2 Favorable No tillage needed 1.2–1.3 Satisfactory Moderate tillage >1.3 Unsatisfactory Intense Penetration resistance in the furrow bottom, kgf/sq.cm: < 20 Favorable No tillage needed 20–40 Satisfactory The same > 40 Unsatisfactory Intense

*The standards are applicable to soils of medium and heavy loamy soil texture.

54 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 SOIL SPATIAL HETEROGENEITY AND SYSTEMS OF AGRICULTURE in using the soil map to plan agrotechnical operations. It should be noted that previously the same happened to the agrochemical map of available nutrients content in the soil. The boundaries of soil contours and the boundaries of providing soils with nutrients do not coincide on these maps. Usually the agrochemical map contains much fewer contours which is very convenient for production purposes. Instead of the soil map, the agrochemical one was used to plan the introduction of fertilizers on the farms. Precise agriculture has got a new foundation – partial analysis [11] of the soil cover, i.e. spatial (geostatistical) analysis of the heterogeneity of specifi c properties of soils, relevant for planning of agrotechnical operations. These properties were called indicative in our laboratory; their systematization was performed. Fig. 1. The alignment (co-kriging) of the soil map of Roma- In these conditions the soil scientists sounded the niv object with the 2-D diagram of humus content. Soil types alarm, as the very basic concepts of the theory on the are shown in grey shades, humus content (%) – by isopleths soil cover structure as an integral soil unit were chal- lenged. Gorjachkin [11] cites a number of interesting references to the works, published in Russia and other countries. The idea of Kozlovsky [12] about the soil individual as an attempt to unite the traditional [13] and geostatistical approaches, which seemed quite rational to some researchers [14] and to the author of this article at fi rst [7], is likely to require substantial stipulations and modifi cation. The soil individual as an elementary primary unit may actually become a link, connecting two approaches, but its application in precise agriculture as a working area cannot be eligible due to extremely small horizontal sizes – from 0.6 to 12 m [15]. Actu- ally, it cannot as of date only. In future, when there is new equipment for agrotechnological operations with high resolution capacity, this possibility may be real. Fig. 2. The alignment (co-kriging) of the soil map of Ro- Still, in any case, the idea of uniting elementary units maniv object with the 2-D diagram of mobile phosphorus into larger ones, the economic activity on which will be content. Soil types are shown in grey shades, phosphorus economically profi table, should be developed in pre- content (mg/100 g of soil) – by isopleths cise agriculture. Thus, the soil map, obtained on the basis of studies in cording to Fridland and the soil individual, estimated the non-regular network of “typical” sections, mainly, according to Kozlovsky. The result was evident non- the profi le morphology, the boundaries of which are de- coincidence of the contours and their sizes. fi ned using the local topography, cannot coincide with At the same time a relevant unifying moment of the the map, developed on the basis of investigations in the maps, created on the basis of two approaches, differ- regular network of sections and analytical data, pro- ing in their principles, is the terrain. This factor serves cessed using formal kriging-methodology of data inter- as a specifi c moderator in the fi nal determination of polation. To verify this fact, one may compare the soil contours on the traditional map. At the same time it is map of one of the investigated objects (for instance, the reason of forming heterogeneity. Therefore, there Romaniv) and 2-D diagrams (Fig. 1, 2). Previously should be some similarity between the traditional and there were comparisons of the elementary soil area ac- geostatistical maps. However, it is considerably dis-

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 55 MEDVEDEV guised by the anthropogenic activity as the introduction of fertilizers, ameliorants, soil tillage and any other human activity enhances heterogeneity. This is precisely why in the author’s opinion [6] and according to much other data [8, 16] the spatial heterogeneity of the content of mobile nutrients in soil reaches extremely high values. Noteworthy is another relevant reason of non-coincidence of boundaries on the maps, namely, differences of deﬁ ned analytical characteristics. A soil map is usually created without the consideration of physical, physical-mechanical and agrochemical characteristics, whereas these very properties are the basis of the indicative estimates, used to plan the introduction of mineral fertilizers, ways and depth of tillage, other agrotechnological operations in precise agriculture. But the main reason of the non-coincidence of the soil map, obtained on the basis of “typical” sections, and the map, developed on the basis of a regular network of the test run, is the fact that in the former the soil cover is a discrete body, the properties of which change jump-like, while in the latter it is a continual body, the properties of which change gradually. The application of modern software made the creation of the map of any soil property a simple and very productive procedure. Moreover, it is possible to perform various transformations with maps in the computer program. It is easy to change the legend in order to increase or decrease the number of contours, to calculate their areas or even create various spatial models, the samples of which have previously been demonstrated [6]. These and many other transformations are possible without printing and manual operations, without extracting the map from the computer. In its essence it is a virtual map, wide-spread both in precise agriculture and beyond it. Moreover, it should be admitted that the study of the experience of precise agriculture in the USA and Germany, and, according to the literature data, in many other countries, including the experience of the leading researchers of this trend [8, 17] leads to the conclusion that a soil map is not required to plan precise agriculture. More precisely, if the structure of the soil cover was registered in the land tenure projecting accurately, and the studies of the indicative properties of soil were conducted Fig. 3. 2-D diagrams of spatial heterogeneity of the con- using the regular network of the selection points, then tent of blocks (A, %), equilibrium bulk density (B, g/ccm), the search for the most reasonable working places to mobile nitrogen forms (C, mg/kg of soil) in the sowing organize the differentiated introduction of fertilizers layer and the grain yield (D, feed units), sod-podzol soil and performing other precise agrotechnological op- (Vediltsy) erations may be conducted, using the maps of indica-

56 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 SOIL SPATIAL HETEROGENEITY AND SYSTEMS OF AGRICULTURE tive properties of soils instead of the soil map. A soil The reason of non-coincidence of soil maps, the map would be required only if spatial planners make maps of soil cover structures and the maps, used in a mistake while cutting the fi elds, like when it hap- precise agriculture, is explained by the fact that the pened on the Romaniv object, and unite non-compat- contours on the soil maps are isolated using the tradi- ible soils in the framework of one crop rotation fi eld. tional soil-geographic principles, ignoring the indices The subsequent experience of working on this fi eld of soil fertility to some degree. The agricultural pur- demonstrated that its part has to be taken out of active poses require establishing both permanent (regular) use and left for grass. Therefore, this mistake was cor- boundaries between specifi c parts of the fi eld and the rected without the soil map. temporary (random) ones, formed synchronically with weather changes, the technology applied, development Certainly, one may not fl atly refuse from the soil map. It is only a question of the suffi ciency and pos- Table 3. The ratio of areas with the application of different ways sibility of planning precise agriculture, taking into ac- of pre-sowing tillage on the investigated objects depending on count the virtual maps of specifi c indicative proper- the level of equilibrium density of soil structure, % ties of soils. As for the fundamental scientifi c concept Tillage of the soil cover structure, actual soil maps, synthe- Object Standard No tillage Minimal sizing “private” information and forming the notion for the zone on the soil cover as an integral natural formation, the refusal from it or any revision is out of the question. Vediltsy 10 50 40 On the contrary, the new information about the soil Romaniv 60 30 10 cover, which previously was not accounted for in the Kolky 25 40 35 development of the concept of the soil cover struc- Korotych 50 40 10 ture, will allow improving the theory and especially Kommunar 70 25 5 Donetsk 75 22 3 its practical applications.

Table 4. The ratio of areas on the investigated ﬁ elds with different levels of applying mineral and organic fertilizers and scenario of chemical amelioration, %

Scenario of chemical Mineral fertilizers Organic fertilizers amelioration Object No For The No Average The No ame- Stan- Kind ferti- expected same + fertili- amount same + Sustaining lioration dard lizers yield addition zers – addition

Romaniv N 100 0 0 14 65 21 30 26 44 P 21 79 0 − − − − − − K 37 63 0 − − − − − − Kolky N 100 0 0 45 17 38 80 13 7 P 0 22 78 − − − − − − K 0 42 58 − − − − − − Vediltsy N 0 0 100 20 53 27 0 0 100 P 70 26 4 − − − − − − K 24 73 3 − − − − − − Korotych N 0 17 83 9 47 44 45 35 20 P 28 72 0 − − − − − − K 90 10 0 − − − − − − Kommunar N 15 85 0 30 62 8 39 61 0 P 100 0 0 − − − − − − K 100 0 0 − − − − − − Donetsk N 0 42 58 1 87 12 − − − P 10 90 0 − − − − − − K 15 85 0 − − − − − −

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 57 MEDVEDEV and state of the crop. That is why the concept of the Просторова неоднорідність ґрунтів soil cover structure is not suffi cient for the purposes of і системи землеробства precise agriculture. В. В. Медведєв Therefore, it seems reasonable and quite justifi ed to e-mail: [email protected] introduce new boundaries in the soil cover, based on lateral study of soil properties – morphological, physi- Національний науковий центр «Інститут ґрунтознавства і агрохімії cal, physical-mechanical and others. These boundaries імені О. Н. Соколовського» НААН України were used to substantiate the confi guration of produc- Вул. Чайковського, 4, Харків, Україна, 61024 tion working places for differentiated application of agrotechnological operations. Мета. Розглянути континуальність і дискретність як особливості прояву неоднорідності в ґрунтовому по- The ratio of areas of fi eld soils with different level of криві, важливі для побудови систем землеробства. fertility. In conclusion it is logical to demonstrate the ra- Методи. Геостатистичне дослідження просторової не- tio of areas with justifi ed different, zero, in particular, однорідності ґрунтів, виявлення контурів поля з різ- agrotechnology in the investigated fi elds. The 2-D dia- ними параметрами родючості. Результати. Викорис- grams obtained (see samples in Fig. 3) and the above- тання принципів точного землеробства і обстеження mentioned standards may be used for this purpose. It is індикаторних властивостей ґрунтів полів за регулярної noteworthy (Tables 3 and 4) that even in the fi elds of сітки дозволило поділити поле на контури з трьома the most problematic zone of Polyssia their considerable рівнями родючості: перший характеризується оптималь- part does not require intensive tillage, lime application ними або близькими до них властивостями, що дозволяє or even introduction of fertilizers. The system of agri- відмовитися від здійснення обробітку (або істотно його скоротити), внесення добрив або хіммеліорантів; дру- culture on all the other fi elds should be equally precise. гий − із середніми параметрами родючості, які відпо- It is clear that the larger the area of the fi eld, allowing відають зональним ґрунтам, потребує застосування зо- the minimization of agrotechnologies, is, the higher eco- нальних технологій; третій (з найгіршими параметрами nomic and ecological advantage of precise agriculture родючості) – вимагає систематичного використання по- is. In the author’s opinion, the data obtained explain in- ліпшених технологій. Висновки. Впровадження точ- creasing attention to precise agriculture in the world [8, ного землеробства дозволить замінити традиційну зо- 17] and its evident promising future in Ukraine. нальну систему землеробства точною – ґрунтозахисною і ресурсозберігаючою. CONCLUSIONS Ключові слова: континуальність і дискретність ґрунтів, Continuality and discreteness were considered as the зональні і точні системи землеробства. forms of manifestation of heterogeneity of the soil cover, Пространственная неоднородность почв used in the elaboration of zonal and precise systems of и системы земледелия agriculture, respectively. A notion of indicative properties and standards of soils, due to the application of which the В. В. Медведев continual soil cover becomes discrete, was introduced. e-mail: [email protected] Precise agriculture is based on new soil boundaries, Национальный научный центр which form the contours with different levels of fer- «Институт почвоведения и агрохимии tility for the differentiation of the components of the имени А. Н. Соколовского» НААН Украины agriculture system in the crop rotation fi eld. Ул. Чайковского, 4, Харьков, Украина, 61024 The application of principles of precise agriculture Цель. Рассмотреть континуальность и дискретность and the investigation of indicative properties of fi eld как особенности проявления неоднородности в поч- soils according to the regular network allowed dividing венном покрове, важные для построения систем зем- the fi eld into the contours with three levels of fertil- леделия. Методы. Геостатистическое исследование про- ity, one of which is characterized with optimal or close странственной неоднородности почв, выявление кон- to optimum properties, which allows refusing from (or туров поля с различными параметрами плодородия. reducing considerably) the application of tillage, intro- Результаты. Использование принципов точного земле- duction of fertilizers of chemical ameliorants. делия и обследование индикаторных свойств почв полей по регулярной сетке позволили разделить поле на кон- The introduction of precise agriculture will allow re- туры с тремя уровнями плодородия: первый характери- placing the traditional zonal system of agriculture with зуется оптимальными или близкими к ним свойствами, the new – soil-protecting and resource-saving one. что позволяет отказаться (или существенно сократить)

58 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 SOIL SPATIAL HETEROGENEITY AND SYSTEMS OF AGRICULTURE от осуществления обработки, внесения удобрения или JP, Richards T, Blackmore BS, Carver MJ, Knight S, химмелиорантов; второй − со средними параметрами Welti B. “Precision farming” of cereals. Practical gui-de- плодородия, соответствующими зональным почвам, тре- lines and crop rotation. Project Report 267. Home-Grown бует применения зональных технологий; третий (с наи- Cereals Authority. London.2002;8 p. худшими параметрами плодородия) – предполагает сис- 9. Samsonova VP, Meshalkina JL, Dmitriev EA. Structure тематическое использование улучшенных технологий. of spatial variability of agrichemical properties of arable Выводы. Внедрение точного земледелия позволит за- soddy-podsolic soil. Theoretical and methodological менить традиционную зональную систему земледелия problems of soil science. Ed. EA. Dmitriev.Moscow, точной – почвозащитной и ресурсосберегающей. GEOC. 2001;318−31. 10. Shein EV, Ivanov AL, Butylkina MA, Mazirov MA. Spatial Ключевые слова: континуальность и дискретность почв, and temporal variability in agrophysical properties of зональные и точные системы земледелия. gray forest soils. Pochvovedenie.2001;(5):578−586. REFERENCES 11. Gorjachkin SV. Problem of priorities in modern researches of a soil cover: the structurally–functional-in- 1. Stepanov IN. Forms in the world of soils. Moscow, formation approach or the partial analysis. Proceedings Nauka.1986;190 p. “Modern natural and anthropogenous processes in soils 2. Achasov AB, Bidolakh DI. Application of materials of and geosystems”.Moscow.2006;53−80. spectrozonal space photography in investigating the con- 12. Kozlovsky FI. Theory and methods of soil cover study. dition and mapping the soil cover. Agrarna nauka i Moscow, GEOC.2003;536 p. osvita.2006;7(5):65−9. 13. Frydland VM. Structure of a soil cover. Moscow, Idea 3. Truskavetskyy SR, Gychka MM, Byndych TYu. Trends of Publishing house.1984; 236 p. improving remote methods of mapping and monitoring 14. Kuzyakova IF. The concept of the soil of the individual of soils. Naukovyy visnyk NAU.2005;(81):176−80. in the light of modern concepts of soil heterogeneity. 4. Petersen H, Fleige H, Rabbel W, Horn R. Geophysical Modern natural and anthropogenic processes in soil methods for imaging soil compaction and variability of and geosystems.Moscow, Soil Inst. Dokuchaev.2006; soil texture on farm land. Advances in Geoecology 38 Soil 324−44. Managing for Sustainability. Reiskirchen, Catena.2006; 15. Korsunov VM, Kraseha EN, Galdin VP. Methodology 261−72. of soil ecology-geographical researches and soil 5. Gychka MM. Seasonal speciﬁ cities of radar probing of cartography. Ulan-Ude, publ. of Buryat NC of the agrophysical properties of soils. Agrokhimia i grunto- Siberian Branch of the Russian Academy of Science. znavstvo. Kharkiv, NNC “IGA im. О. N. Sokolovsky”. 2002;234 p. 2005; Is.66:59−66. 16. Romanenkov VA, Larin VE, Lukin SM. Research of the 6. Мedvedev VV. Soil heterogeneity and precise agriculture. processes deﬁ ning spatial change of arable soil fertility Pt 2. Results of investigation. Kharkiv,13 Publishing for modelling productivity. Modern natural and anthro- house.2009;260 p. pogenic processes in soil and geosystems.Moscow, Soil 7. Medvedev VV. Soil heterogeneity and precise agriculture. Inst. Dokuchaev.2006;305−23. Pt 1. Introduce in the problem. Kharkiv,13 Publishing 17. Jakushev VP, Poluektov RA, Smoljar EI, Topazh AG. Pre- house.2007;296 p. cise agriculture (state-of-the-art review). Agrochemical 8. Godwin RJ, Earl R, Taylor C, Wood GA, Bradley RI, Welsh bulletin.2001;(5):28−33.

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 59 ISSN: 2312-3370, Agricultural Science and Practice, 2015, Vol. 2, No. 1

UDC 615.33:636.086 DETECTION OF ANTIBIOTICS, ACTIVE AGAINST BACILLUS SUBTILIS, IN GRAIN AND FEED O. V. Trufanov 1, А. M. Kotyk 1, V. A. Trufanova 1, О. V. Tereshchenko 1, О. M. Zhukorskiy 2 1 State Poultry Research Station, NAAS of Ukraine 20, Lenin Str., Birky village, Zmiiv District, Kharkiv Region, Ukraine, 63421 2 National Academy of Agrarian Sciences of Ukraine 37, Vasylkivska Str., Kyiv, Ukraine 03022 e-mail: [email protected] Received on January 14, 2015

Aim. Detection of antibiotic substances in samples of grain, extraction cake, and oilcake. Methods. The bioautography method using strains of Bacillus subtilis as test-microorganisms was used to study 102 samples of feed substrates (corn, maize gluten, barley, wheat, sorghum, chaff, dust middling, sunﬂ ower oilcake and extraction cake, soybean meal, feed yeast and grains). Results. From one to four antibiotic substances, inhibiting the growth of B. subtilis and characterized by a wide range of values of chromatographic mobility index, were detected in 95 % of samples of feed substrates. Average areas of the zones of absent growth of a test- microorganism, corresponding to 2.5 g of the sample, were in the range of 52–217 mm2. Conclusions. It was established that feeder grain and other feed substrates are highly contaminated with antibiotics which indicates the necessity of their identiﬁ cation, search for contamination sources, study of prevalence and estimation of the possible impact on the indices of health, performance and reproduction of farm animals and poultry. Key words: grain, oilcake, extraction cake, antibiotics, bioautography.

INTRODUCTION antibiotics may penetrate the agricultural soils with The list of maximally acceptable levels of unde- the excrements of animals, fed with the fodder, pro- sired substances in animal feed and fodder in Ukraine duced with the addition of antibiotic as growth pro- includes inorganic pollutants, nitrogen compounds, moting agents or veterinary antibiotics, prescribed by mycotoxins, toxins of plant origin, chlororganic com- the veterinary physician [3, 4]. There are published pounds, dioxins and microelements. The content of data proving that antibiotics, penetrating the soil from coccidiostatics in the composition of pharmaceutical organic fertilizers, may be consumed by the root sys- products for veterinary use is speciﬁ ed in case when tem of agricultural crops and accumulated in different they are not meant for animals [1]. At the same time tissues and organs, including grain [5]. It was deter- the European Union and a number of countries regulate mined that microbiocidal agents are capable of being the content of antibiotics in feeds which are prohibited accumulated by plants, cultivated on the soil using for use as growth promoting agents [2]. Ukraine has solid organic wastes, obtained in the process of puri- not got any restrictions on the content of antibiotics in fying wastewater, as fertilizers [6, 7]. Besides, a po- feeds therefore they are usually not indicated in the list tential source of antibiotic substances in feeds of plant of feed ingredients. origin may be metabolites of symbiotic soil [8, 9] and phytopathogenic bacteria [10]. In recent years there have been some communications informing that intentional introduction of vet- This study was aimed at detecting antibiotics, ac- erinary antibiotics into fodder combination is not the tive against sensitive strains of Bacillus subtilis, in the only way of contaminating the latter with the sub- samples of grain and vegetative feed for farm animals stances of antibiotic activity. It was determined that and poultry.

60 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 DETECTION OF ANTIBIOTICS, ACTIVE AGAINST BACILLUS SUBTILIS, IN GRAIN AND FEED MATERIALS AND METHODS twice, shaken each time. After the phase distribution the lower layer (chloroform) was isolated for further During 2010–2014 102 samples of feed substrates analysis. The chloroform extracts were combined, 5 g (corn, maize gluten, barley, wheat, sorghum, chaff, dust of anhydrous sodium sulfate was added for dehydra- middling, sunfl ower oilcake and extraction cake, soy- tion, shaken and kept for 10 min. The solution was fi l- bean meal, feed yeast and grains) from the combined tered, the fi ltrate was evaporated. The dry residue was feed-processing plants, livestock enterprises and poul- dissolved in 1–2 ml of benzene, evaporated, and the try farms of different forms of ownership from eight residue was dissolved in 100 μl of benzene. regions of Ukraine were tested for the presence of antibiotic agents. The Sorbfi l slides for thin-layer chromatography (TLC) (Imid Ltd, Russian Federation) were applied The sensitive strain of bacteria was selected out of 10 μl of the extract of the investigated samples, chro- the strains of B. subtilis from the Museum of the Labo- matographed in the system of such solvents as ethyl ratory of poultry feeding and mycotoxicology of the acetate:toluene (3:1, volume/volume) and dried. State Poultry Research Station NAAS of Ukraine, remarkable for even growth in the cultivation medium To detect the antibiotic agents, the surface of hori- and absence of sensitivity to mycotoxins, namely − af- zontally placed chromatographic slides was applied the melted MPA, which was inoculated with the sus- latoxin В1, Т-2 toxin, НТ-2 toxin, deoxynivalenol, fu- monisin, zearalenone and aurofusarin. For this purpose pension of a sensitive strain of B. subtilis. The slides the sensitivity of strains of B. subtilis ap-2, sn-1, sn-2, were kept in a wet chamber at 32°C for 16–18 h. No sn-3, sn-4 and sn-5 to antibiotics was tested using the growth of a test-microorganism on the slide testifi ed to disk diffusion method. Such antibiotics as polymyxin the presence of an antibiotic agent in the sample. The B, streptomycin, tetracycline, amikacin, carbenicillin, linear sizes of zones and the indices of their chromato- cefepime, cefuroxime, cefalexin, cefalotin, cefotaxime, graphic motility (Rf) were taken into consideration. chloramphemide, ciprofl oxacin, netiline, norfl oxacin, RESULTS AND DISCUSSION ofl oxacin or perfl oxacin were applied on paper discs in the amount of 5 to 300 μg. The cultures of strains were The investigated strains demonstrated different sen- cultivated in Petri dishes in lawns using meat-and-pep- sitivity to antibiotics (Table 1). B. subtilis strains sn-1, tone agar (MPA). The diameters of the delayed growth sn-3 and sn-4 were insensitive to cefepime, and sn-1 – zones around the disks with antibiotics were measured to cefuroxime. B. subtilis strains ap-2, sn-2 and sn-5 and their values were ranged. The selection of the most demonstrated their sensitivity to each of the investigat- sensitive strain involved calculation of the rank sum, ed antibiotics. B. subtilis strain sn-2 was characterized the arithmetic mean value for the diameters of delayed with the the highest sensitivity to antibiotics both in growth zones and the sensitivity to specifi c antibiotics. the average value of the diameter of zones of growth To detect the antibiotic substances, 25 g of ground inhibition, and in the rank sum, thus it was selected as grain or combined fodder was introduced into the coni- a test-microorganism for further studies. cal 500 ml fl ask with the addition of 20 ml of the aque- The antibiotic agents, capable of inhibiting the growth ous NaCl solution (10 %, weight/volume) and mixed. of B. subtilis sn-2, were found in 95 % of the investigat- Then 120 ml methanol was added; the mixture was ed samples of feed substrates (Table 2). Therefore, the shaken for 1 h and fi ltered through a paper fi lter. To pre- bioautographic method of applying the sensitive strain cipitate protein admixtures, 100 ml of the fi ltrate was of B. subtilis, sn-2, allows revealing the antibiotic sub- added to 20 ml of the aqueous solution of lead acetate stances with different chromatographic characteristics, (15 %, weight/volume) and 30 ml of distilled water, separating the mixture of several antibiotics and esti- mixed and kept for 10 min. The mixture was fi ltered; mating the concentration in relative units of activity. 120 ml of the fi ltrate was transfered to the separation The bioautographic method of applying Escherichia funnel. To isolate the triglycerides, the fi ltrate was coli was previously used for the purpose of quantitative added to 40 ml of hexane and shaken; the lower water- determination of tabtoxin, mangotoxin and phaseolo- methanol layer was isolated after the phase distribution. toxin – Pseudomonas syringae metabolites, character- The latter was added to 20 ml of hexane twice, shaken ized with antibiotic activity [11]. It should be noted that each time, and the layer of hexane was isolated after a considerable amount of P. syringae strains are phyto- the phase distribution. Then the water-methanol extract pathogenic endophytic microorganisms, causing bac- in the separation funnel was added 40 ml of chloroform terioses of gramineous plants, due to which they may

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 61 TRUFANOV et al. be the source of grain contamination with antibacterial vestigated sample of barley caused four zones of delayed activity [12]. growth of B. subtilis sn-2 on the slide for TLC. It is note- Most frequently, namely, in 46 % of cases, the samples worthy that the distribution of frequencies of simulta- of grain and vegetable raw materials were simultaneously neous detection of several antibiotic substances in one contaminated with two substances, inhibiting the growth sample corresponds to prevailing schemes of applying the of B. subtilis sn-2 (Fig. 1). Approximately 1.5 times less antibiotic as growth promoting agent individually and in frequently (i.e. in 29 % cases) there were samples, con- synergistic combinations [13, 14]. taminated with only one antibiotic substance; almost 2.5 The investigated samples of grain and feed were re- times less frequently – samples, contaminated with three markable for a wide variety of antibiotic substances antibiotics at the same time. Only the extract of one in- both by the index of chromatographic mobility and

Table 1. The sensitivity of six strains of B. subtilis to antibiotics

Antibiotic, Diameter of zones of growth inhibition for B. subtilis strains, mm μg/slide ap-2 sn-1 sn-2 sn-3 sn-4 sn-5

Polymyxin B, 300 12 17 17 13 12 15 Streptomycin, 10 30 26 26 28 29 21 Tetracycline, 10 32 30 30 33 32 30 Amikacin, 10 31 35 35 31 30 27 Carbenicillin, 100 14 28 28 13 13 42 Cefepime, 30 0 16 16 0 0 27 Cefuroxime, 30 0 17 17 10 12 30 Cefalexin, 30 15 37 37 25 13 30 Cefalotin, 30 15 43 43 14 15 34 Cefotaxime, 10 20 24 24 21 20 34 Chloramphemide, 30 33 33 33 35 33 32 Ciprofl oxacin, 30 33 40 40 35 35 32 Netiline, 30 31 34 34 33 32 33 Norfl oxacin, 10 29 30 30 30 30 25 Ofl oxacin, 5 30 37 37 30 30 27 Perfl oxacin, 5 28 34 34 28 30 25 Arithmetic mean value 29.8 22.1 30.1 23.7 22.9 29 Rank sum 45 59 31 49 55 63

Table 2. Number of feed substrate samples with antibiotic substances in the extract

Samples with different amounts Total number Feed substrate of detected antibiotic agents of samples 01234

Corn 43 1 14 24 4 0 Maize gluten 2 0 0 1 1 0 Barley 11 0 2 6 2 1 Wheat 11 1 3 4 3 0 Sorghum 2 0 2 0 0 0 Chaff 2 0 0 1 1 0 Dust middlings 3 1 1 1 0 0 Sunﬂ ower cattle cake, extraction cake 15 0 3 5 7 0 Soybean meal 9 2 4 2 1 0 Feed yeast, grains 4 0 1 3 0 0 Total 102 5 30 47 19 1 % 100 4.9 29.4 46.1 18.6 0.98

62 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 DETECTION OF ANTIBIOTICS, ACTIVE AGAINST BACILLUS SUBTILIS, IN GRAIN AND FEED the area of delayed growth zones (Fig. 2). Depend- ing on the average area of zones of growth inhibition with different Rf they can be divided for clarity into four groups. It should be noted that the antibiotic substances with the rounded value of chromatographic mobility index of 0.5 are remarkable for the highest average value of the area of growth inhibition zones of the test-microorganism − 217 mm2. Somewhat lower areas (from 152 to 173 mm2) were registered for the zones of antibiotic substances with rounded Rf values of 0.3, 0.4 and 0.6. The third place in terms of sizes of growth inhibition zones is taken by antibiotic Fig. 1. The frequency of simultaneous detection of several substances with Rf 0.1 and 0.2, the fourth – with 0.7 antibiotics in different amounts and 0.8, and the substances, remaining on the start of the TLC slide. The results of the studies testify that the antibiotic substances with the chromatographic mobility index of 0.5 and the substances, remaining on the start line in the applied system of eluents are registered the most frequently (Fig. 3). The occurrence frequency for these antibiotic substances is 24 and 23 % respectively. The antibiotics with Rf value of 0.1, 0.2, 0.3 and 0.4 were registered with the frequency from 9 to 12 %, with Rf

0.6 and 0.7 – 6 %, and the least frequently − with Rf 0.8 (frequency of 0.5 %).

The detected antibiotic substances do not decrease Fig. 2. The average area of zones of growth inhibition for the wheat grain quality of rye, barley, triticale, corn, B. subtilis sn-2 with different values of Rf oats, millet, not damaged by insects, fungi and bacteria. They are different in their chromatographic mobility, their capability of inhibiting the growth of test-microorganisms and sizes of zones of absent growth of test- microorganisms (Fig. 4). One of the ways of antibiotic substances penetrating into grain and secondary products of oil-seed crops processing (oilcake and extraction cake) is through the soil. Recent articles of different authors contain the data on soil contamination by antibiotics for veterinary purposes which penetrate the soil as a result of fertil- izing the latter with the excrements of animals, who were kept using antibiotic growth promoting agents or using the schemes of treating the bacterial diseases, Fig. 3. The frequencies of detecting antibiotic substances presupposing treatment with veterinary antibiotics. with different values of chromatographic mobility For instance, according to the data of Leal et al. [15], 30 % of samples of chicken excrements and 27 % of cently considerable efforts were taken to elaborate the soil samples from Brazil, Austria, China and Turkey means for puriﬁ cation of soils and surface water from were contaminated with enroﬂ oxacin in average con- contamination with sulfonamide antibiotics to prevent centrations of 6.68 mg/kg and 22.93 μg/kg, respective- the occurrence of resistant strains [16]. ly. The authors believe that the results obtained indicate After veterinary antibiotics penetrate the environ- veterinary antibiotics to be a potential source of envi- ment, their further destiny is determined by three pro- ronment pollution, which has been ignored [15]. Re- cesses: adsorption by soil particles, biotransformation

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 63 TRUFANOV et al. antibacterial activity – triclosan, triclocarban and car- bamazepine, which are a part of hygiene products and household cleaning products and contaminate soils due to the introduction of products of wastewater cleaning as fertilizers [21]. Then there is a question whether veterinary antibiotic preparations are the only potential source of contamination of plant cultivation products with antibiotics. The substances with antibacterial and anti- fungal properties are known to be produced by symbiotic bacteria, present in rhizoplane and rhizosphere of cultivated plants and in the root zone of soil [9]. It is quite possible that such antibiotic substances may Fig. 4. The zones of absent growth of a test-microorganism be absorbed by the root system of plants and get ac- B. subtilis sn-2 in case of biography of extracts of barley cumulated in different tissues and organs, including (А) and wheat (B) fruit and seeds. One more possible source of antibiotic substances under the effect of enzymes of microorganisms and in grain may be phytopathogenic and opportunistic bioaccumulation in plant tissues [17]. It was deter- pathogenic endophytic bacteria. Recently there have mined that the sorption of sulfadiazine, a veterinary been some communications on the aggravation of antibiotic, is described with non-linear equations and the problem of bacterioses of cultivated crops due to depends on the level of acidity and type of soil. The wide-scale application of pesticides with fungicidal study of biotransformation parameters of this antibi- activity to protect cultivated plants from fungal dis- otic testifi ed that its half lifetime is in the range from eases [22]. It was determined that fungicides inhibit one to six months [18]. According to the experiment, the development of both fungi as pathogens and rep- which lasted for 120 days, the periods of half lifetime resentatives of fungal saprotrophic soil microfl ora. of erythromycin, oleandomycin, tylosin, tiamulin and Due to the emptying of ecological niches, previously salinomycin in soil are 20, 27, 8, 16, and 5 days, re- taken by saprotrophic fungi, namely, crop debris, spectively. The concentration of roxithromycin did not there is uncontrolled development of bacteria, a con- change during the whole 120-day-period [4]. The re- siderable part of which belongs to phytopathogenic sults of these studies testify that veterinary antibiotics species. are stable substances which may remain in soil for a Phytopathogenic bacteria are capable of penetrat- long time. ing the transport vessels of plants and getting accu- It should be noted that the experiments, aimed at de- mulated in grain, producing a wide spectrum of anti- tecting antibiotics in soil, were conducted in the 50–60s biotic substances and compounds, toxic to eukaryotic of the previous century to search for new antibiotic organisms, including mammals. For instance, Pseu- substances and their producents. Novel studies, aimed domonas syringae atrofaciens is an agent, causing at detecting antibiotics in the soil, emphasize the detec- bacteriosis of barley endosperm, one of the conse- tion of only veterinary antibiotics which penetrate soils quences of which is the deterioration in quality of due to human economic activity [19]. The problem of grain and fl our [23]. An interseasonal factor of the soil contamination with veterinary antibiotics raises transmission of most agents of bacterial diseases of the question on the possibility of bioaccumulation of gramineous plants is seeds, i.e. bacterial cells get ac- these substances in plant tissues, including agricultural cumulated in grain during its development and infect crops. For instance, it was determined that such anti- the sprouts during the germination. It is quite prob- biotics as gentamicin and streptomycin are accumu- able that the products of bacterial synthesis, includ- lated in the tissues of carrots (Daucus carota), lettuce ing antibiotics, also get accumulated in grain. It is (Lactuca sativa) and radish (Rhaphanus sativus), in known that P. syringae is the producent of antibi- addition, gentamicin, the molecular mass of which is otic substances, such as tabtoxin, phaseolotoxin and lower, has more expressed propensity for accumulation mangotoxin, harmful both for bacteria [12] and for [20]. Soybeans may accumulate the substances with mammals [24, 25].

64 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 DETECTION OF ANTIBIOTICS, ACTIVE AGAINST BACILLUS SUBTILIS, IN GRAIN AND FEED CONCLUSIONS Обнаружение в зерне и кормах антибиотиков, активных в отношении Bacillus subtilis The data obtained testify to high frequency and considerable levels of grain contamination of gramineous А. В. Труфанов 1, А. М. Котик 1, В. А. Труфанова 1, plants and secondary products of seed processing of А. В. Терещенко 1, А. М. Жукорський 2 oil-seed crops with antibiotic substances, active against e-mail: [email protected] B. subtilis strain, rather sensitive to a wide spectrum 1 Государственная исследовательская станция of antibiotics. The differences in the indices of chro- птицеводства НААН Украины matographic mobility testify to a high variety of physi- Ул. Ленина, 20, с. Борки, Змиевской р-н, cal properties, and therefore, chemical structures of the Харьковская обл., Украина, 63421 antibiotic substances revealed. The contamination of 2 Национальная академия аграрных наук Украины grain and vegetative raw material with antibiotics is of Ул. Васильковская, 37, Киев, Украина, 03022 potential risk to the environment, health of farm ani- Цель. Выявление антибиотических субстанций в образ- mals and humans. Further studies are planned for the цах зерна, шрота и жмыха. Методы. Методом биоавто- identifi cation of the detected antibiotic substances, the графии с использованием штаммов Bacillus subtilis в investigation of their impact on the environment and качестве тест-микроорганизмов исследованы 102 образ- living organisms as well as determination of sources of ца кормовых субстратов (кукурузы, глютена кукурузно- their penetration into grain and other tissues and organs го, ячменя, пшеницы, сорго, отрубей, мучки кормовой, of cultivated plants. подсолнечного жмыха и шрота, соевого шрота, дрожжей кормовых и барды пивной). Результаты. В 95 % образ- Виявлення в зерні і кормах антибіотиків, цов кормовых субстратов обнаружены от одной до че- активних відносно Bacillus subtilis тырех антибиотических субстанций, угнетающих рост О. В. Труфанов 1, А. М. Котик 1, В. А. Труфанова 1, B. subtilis и характеризующихся широким диапазоном О. В. Терещенко 1, О. М. Жукорський 2 значений показателя хроматографической подвижности. e-mail: [email protected] Средние площади зон отсутствия роста тест-микроорга- низма, соответствующие 2,5 г образца, были в преде- 1 Державна дослідна станція птахівництва лах 52−217 мм2. Выводы. Обнаружен высокий уровень НААН України загрязненности фуражного зерна и других кормовых Вул. Леніна, 20, с. Бірки, Зміївський р-н, субстратов антибиотиками, что указывает на необходи- Харківська обл., Україна, 63421 мость их идентификации, нахождения источников загряз- 2 Національна академія аграрних наук України нения, изучения распространенности и оценки возмож- Вул. Васильківська, 37, Київ, Україна, 03022 ного влияния на показатели здоровья, продуктивности и Мета. Виявлення антибіотичних субстанцій у зразках репродукции сельскохозяйственных животных и птицы. зерна, шрота і макухи. Методи. Методом біоавтографії Ключевые слова: зерно, жмых, шрот, антибиотики, био- з використанням штамів Bacillus subtilis як тест-мікро- автография. організмів досліджено 102 зразки кормових субстратів (кукурудзи, глютену кукурудзяного, ячменю, пшениці, REFERENCES сорго, висівок, мучки кормової, соняшникової макухи і шроту, соєвого шроту, дріжджів кормових і дробини 1. Nakaz Ministerstva agrarnoi’ polityky ta prodovol’stva пивної). Результати. У 95 % зразків кормових субстратів Ukrai’ny vid 19 bereznja 2012 roku N 131. Perelik виявлено від 1 до 4 антибіотичних субстанцій, які при- maksymal’no dopustymyh rivniv nebazhanyh rechovyn гнічують ріст B. subtilis і характеризуються широким u kormah ta kormovij syrovyni dlja tvaryn. Ofi cijnyj діапазоном значень показника хроматографічної рухли- visnyk Ukrai’ny.2012; (29). вості. Середні площі зон відсутності росту тест-мікро- 2. Regulation (EC) No 1831/2003 of the European Par- організму, що відповідають 2,5 г зразка, були в межах liament and the Council of 22 September 2003 on ad- 52−217 мм2. Висновки. Визначено високий ступінь за- ditives for use in animal nutrition. Offi cial Journal of брудненості фуражного зерна та інших кормових суб- European Union. L 268,2003;29−43. стратів антибіотиками, що вказує на необхідність їх- 3. Velagaleti RR. Behavior of pharmaceutical drugs (human ньої ідентифікації, знаходження джерел забруднення, and animal health) in the environment. Ther Innov Regul вивчення розповсюдженості і оцінки можливого впливу Sci.1997; 31(3):715−22. на показники здоров’я, продуктивності та репродукції 4. Schlusener MP, Bester K. Persistence of antibiotics such сільськогосподарських тварин та птиці. as macrolides, tiamulin and salinomycin in soil. Environ Ключові слова: зерно, макуха, шрот, антибіотики, біо- Pollut.2006; 143(3):565−71. автографія. 5. Dolliver H, Kumar K, Gupta S. Sulfamethazine uptake

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 65 TRUFANOV et al. by plants from manure-amended soil. J Environ Qual. litters and soils from Sao Paulo State, Brazil. Sci Total 2007; 36(4):1224−30. Environ.2012;432:344–9. 6. Pannu MW, Toor GS, O’Connor GA, Wilson PC. Toxicity 16. Martucci A, Braschi I, Marchese L, Quartieri S. Recent and bioaccumulation of biosolids-borne triclosan in food advances in clean-up strategies of waters polluted with crops. Environ Toxicol Chem.2012; 31(9):2130−7. sulfonamide antibiotics: a review of sorbents and rela- 7. Prosser RS, Lissemore L, Topp E, Sibley PK. Bioaccu- ted properties. Mineralogical Magazine. 2014; 78(5): mulation of triclosan and triclocarban in plants grown 1115−40. in soils amended with municipal dewatered biosolids. 17. Sassman SA, Sarmah AK, Lee LS. Sorption of tylosin Environ Toxicol Chem. 2014; 33(5):975−84. A, D, and A-aldol and degradation of tylosin A in soils. 8. Bonsall RF, Thomashow LS, Mavrodi DV, Weller DM. Environ Toxicol Chem.2007; 26(8):1629−35. Extraction and detection of antibiotics in the rhizosphe- 18. Kasteel R, Mboh CM, Unold M, Groeneweg J, Vander- re metabolome. Curr Trends Mass Spectrom. (Suppl. borght J, Vereecken H. Transformation and sorption of Spectrosc.)2007; 11:14–9. the veterinary antibiotic sulfadiazine in two soils: a short- term batch study. Environ Sci Technol. 2010; 44(12): 9. Mavrodi DV, Mavrodi OV, Parejko JA, Bonsall RF, 4651−7. Kwak YS, Paulitz TC, Thomashow LS, Weller DM. Accumulation of the antibiotic phenazine-1-carboxylic 19. Subbiah M, Mitchell SM, Ullman JL, Call DR. β-lactams acid in the rhizosphere of dryland cereals. Appl Environ and fl orfenicol antibiotics remain bioactive in soils while ciprofl oxacin, neomycin, and tetracycline are neutralized. Microbiol. 2012; 78(3):804–12. Appl Environ Microbiol.2011; 77(20):7255−60. 10. Bender CL, Alarcon-Chaidez F, Gross DC. Pseudomonas 20. Bassil RJ, Bashour II, Sleiman FT, Abou-Jawdeh YA. syringae phytotoxins: mode of action, regulation, and Antibiotic uptake by plants from manure-amended soils. biosynthesis by peptide and polyketide synthetases. J Environ Sci Health B.2013; 48(7):570−4. Microbiol Mol Biol Rev.1999;63(2):266–92. 21. Wu C, Spongberg AL, Witter JD, Fang M, Czajkows- 11. Arrebola E, Cazorla FM, Duran VE, Rivera E, Olea F, ki KP. Uptake of pharmaceutical and personal care pro- Codina JC, Perez-Garcia A, de Vicente A. Mangotoxin: a ducts by soybean plants from soils applied with biosolids novel antimetabolite toxin produced by Pseudomonas sy- and irrigated with contaminated water. Environ Sci ringae inhibiting ornithine/arginine biosynthesis. Physiol Technol. 010; 44(16):6157−61. Mol Plant Pathol.2003;63(3):117–27. 22. Gvozdjak RI, Pasichnyk LA, Jakovleva LM. Fitopatogenni 12. Arrebola E, Cazorla FM, Perez-Garcia A, de Vicente A. bakterii’. Bakterial’ni hvoroby roslyn: Monografi ja. Kyiv, Chemical and metabolic aspects of antimetabolites to- TOV «NVP «Interservis».2011; 444 p. xins produced by Pseudomonas syringae pathovars. To- 23. Schober-Butin B, Garbe V, Bartels G. Farbatlas Krank- xins(Basel).2011; 3(9):1089−110. heiten und Schadlinge an Landwirtschaftlichen Kultur- 13. Landers TF, Cohen B, Wittum TE, Larson EL. A review pfl anzen: Kartoffel, Zuckerrübe, Raps, Getreide, Mais, of antibiotic use in food animals: perspective, policy, and Sonnenblume, Hanf. Stuttgart, Eugen Ulmer.1999; 240 S. potential. Public Health Rep.2012; 127(1):4–22. 24. Lamar CJr, Sinden SL, Durbin RD, Uchytil TF. The pro- 14. Agunos A, Leger D, Carson C. Review of antimicrobial duction of convulsions by an exotoxin from Pseudomonas therapy of selected bacterial diseases in broiler chickens tabaci. Toxicol Appl Pharmacol.1969; 14(1):82−8. in Canada. Can Vet J.2012;53(12):1289–300. 25. Sinden SL, Durbin RD. Glutamine synthetase inhibition: 15. Leal RM, Figueira RF, Tornisielo VL, Regitano JB. Oc- possible mode of action of wildfi re toxin from Pseu- currence and sorption of fl uoroquinolones in poultry domonas tabaci. Nature. 1968;219(5152):379−380.

66 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 ISSN: 2312-3370, Agricultural Science and Practice, 2015, Vol. 2, No. 1

UDC 636.52/.58:575.113/.118 TRANSFORMING GROWTH FACTOR Β1, PITUITARY-SPECIFIC TRANSCRIPTIONAL FACTOR 1 AND INSULIN-LIKE GROWTH FACTOR I GENE POLYMORPHISMS IN THE POPULATION OF THE POLTAVA CLAY CHICKEN BREED: ASSOCIATION WITH PRODUCTIVE TRAITS R. A. Kulibaba, A. V. Tereshchenko State Poultry Experimental Station, NAAS of Ukraine 20, Lenin Str., Birky village, Zmiiv District, Kharkiv Region, Ukraine, 63421 e-mail: [email protected] Received on January 29, 2015

Aim. To investigate the gene polymorphisms of transforming growth factor β1 (TGF-β1), pituitary-specifi c transcriptional factor 1 (PIT-1) and insulin-like growth factor I (IGF-I) in the population of Poltava clay chicken breed, used for egg and meat production, and to analyze the association of different genotypes for each locus with productive traits. Methods. Genotyping of the chickens was performed using the polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP). Results. Transforming growth factor TGF-β1, pituitary-specifi c transcriptional factor PIT-1, and insulin-like growth factor IGF-I were shown to be polymorphic in the studied populations. The association between genotypes by the loci TGF-β1 and PIT-1 and the indices of egg and meat production of chickens was demonstrated. Conclusions. The data on the genetic structure of the population of Poltava clay chicken breed by loci TGF-β1 and PIT-1 is recommended for the targeted selection of chickens to produce offspring with desirable genotypes, which will, in addition to classical breeding methods, reveal the productive potential of chickens as effi ciently as possible. Keywords: Poltava clay chicken breed, polymorphism, transforming growth factor β1, pituitary-specifi c transcriptional factor 1, insulin-like growth factor I, productive traits.

INTRODUCTION of their polymorphism in experimental populations. The investigation of polymorphism of candidate genes Successful development of poultry industry is in the studied populations of chickens is the ﬁ rst step, considerably determined by the efﬁ ciency of selection. required for further analysis of the association of Due to recent rapid development of molecular and different genotypes with productive traits which may genetic methods of research there is a new possibility be directly used in the selection [2]. The promising of putting selection into practice on the essentially candidate genes, the association of which to poultry new foundation – applying the data on polymorphism productivity traits was proven in numerous foreign of target genes and their association with productivity investigations, include, in particular, such objects as traits. This approach, called marker-assisted selection the genes of transforming growth factor β1 (TGF-β1), (MAS-selection), is based on the study of polymorphism pituitary transcriptional factor 1 (PIT-1) and insulin- of the selected genes, the association of allele variants like growth factor I (IGF-I). with productive traits, the categorization of individuals according to the complex of genes, and the selection TGF-β1 belongs to one of the most important groups of of individuals with desirable genotypes for further genes (family of transforming growth factors β), partici- breeding [1]. An important stage of research in this pating in the maintenance of tissue homeostasis, growth context is the selection of target genes for the study and differentiation of different types of cells, the forma-

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 67 KULIBABA et al. tion of intercellular matrix, the induction of apoptosis, the biological material (blood) was sampled from each regulation of the immune system, etc [3]. Gene TGF-β1 chicken at the experimental poultry farm “Preservation is localized on the 13th chromosome and contains 17 ex- of State Genetic Pool of Poultry” of the State Poultry ons. It was demonstrated that allele variants of TGF-β1 Experimental Station NAAS of Ukraine. The blood correlate with meat production traits of chickens of dif- sample was taken from the crown using the scarifi cator ferent breeds [4]. and sterile fi lter paper. Each sample was dried, marked PIT-1 participates in the expression regulation of and individually packed to prevent any contamination. growth hormone genes, prolactin, thyreotropin, prolif- DNA was isolated from the experimental samples us- eration and differentiation of hormone-secreting cells, ing the commercial kit of reagents DNA-sorb-B (Am- the control of immune system development [5]. Its pliSens, RF). The effi ciency of DNA isolation was de- functioning is directly related to that of the controlled termined by electrophoresis in 0.7 % agarose gel at 200 genes (growth hormone, prolactin) which defi nes PIT-1 V for 5 min. as a promising candidate for the study of the associa- The following parameters were used for the amplifi - tion of different genotypes and the productivity traits cation: due to the especially important role of prolactin in the TGF-β1 – Forward: 5′-GGGGTCTTCAAGCTGA- regulation of the reproductive cycle of birds. PIT-1 GCGT-3′, Reverse: 5′-TTGGCAATGCTCTGCATG- gene contains 6 exons and 5 introns. Several SNP were TC-3′ [4]; revealed in different gene regions [5]. It was shown ′ that allele variants of PIT-1 gene are associated with PIT-1 – Forward: 5 -GTCAAGGCAAATATTCTGT- ′ ′ the live weight of chickens of different breeds [6]. ACC-3 , Reverse: 5 -TGCATGTTAATTTGGCTCT- G-3′ [5]; IGF-I performs a number of physiological func- ′ tions, related to the growth and differentiation of tis- IGF-I – Forward: 5 -GACTATACAGAAAGAACC- ′ ′ sues, and belongs to the family of insulin-like growth AC-3 , Reverse: 5 -TATCACTCAAGTGGCTCAAG- ′ factors [7]. Its functioning is closely related to the T-3 [9]. growth hormone and it is an active participant of the The amplifi cation was conducted using the reagents growth regulation and development of the embryo. of DreamTaq PCR Master Mix (ThermoScientifi c, IGF-I gene contains 4 exons and 3 introns. High level USA) and programmed thermocycler Tertsik (DNA- of polymorphism was registered for 5′ and 3′ non- technologies, RF) using the corresponding programs – coding regions (5′UTR and 3′UTR), as well as intron 1 cycle: denaturation at 94 °C − 3 min; 35 cycles: dena- and exon parts of IGF-I gene. The association of dif- turation (94 °C) − 1 min, annealing − 1 min (65 °C for ferent allele variants of the gene and productive traits TGF-β1; 58 °C for PIT-1; 53 °C for IGF-I), elongation of chickens was studied [8−10]. (72 °C) − 1 min; 1 cycle: fi nal elongation (72 °C) − Previously we investigated the genetic structure of 10 min. The volume of the fi nal mixture was 20 μl, the the populations of chickens, bred in Ukraine for egg concentration of primers – 0.2 μM in each case. and meat production by loci of prolactin, growth hor- The amplifi ed fragments were treated with restric- mone, growth hormone receptor, family of transform- tion endonucleases MboII (restriction site GAAGA ing growth beta-factors, etc [11, 12]. (8/7)↓) and PstI (restriction site CTGCA↓G) ac- The current work was aimed at the analysis of the cording to the standard methods of the manufacturer polymorphism of genes TGF-β1, PIT-1 and IGF-I in the (ThermoScientifi c). In case of TGF-β1 gene MboII population of Poltava clay chicken breed, bred for egg was used, and for IGF-I – PstI. The restriction analy- and meat production, as well as the determination of sis was not conducted to study polymorphism by lo- the association of different genotypes by each of the cus PIT-1. The products of amplifi cation/restriction loci with productivity traits. were separated in 1.5 % agarose gel at 150 V for 40 min. The visualization was conducted using ethidium MATERIALS AND METHODS bromide in the ultraviolet spectrum. The size of the The study was conducted in the Laboratory of Poultry amplifi ed/restrictional fragments was determined us- Disease Prevention and Molecular Diagnostics of the ing the markers of molecular mass M-50 and M-100 State Poultry Experimental Station NAAS of Ukraine, (Isogen, RF). using the Poltava clay chicken breed, line 14 (n = 100), The genotyping by each locus was conducted using selected in Ukraine for egg and meat production. The the analysis of the electrophoregrams obtained.

68 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 TRANSFORMING GROWTH FACTOR Β1, PITUITARY-SPECIFIC TRANSCRIPTIONAL FACTOR 1 Gene TGF-β1: the size of the amplifi ed fragment – 240 bp. Genotype B/B was presented on the electrophoregram in the form of DNA fragments of 173 and 67 bp; F/F – 240 bp; B/F – 240, 173 and 67 bp respectively. Gene PIT-1: fragments of 387 bp are present on the electrophoregram, which testifi es to genotype I/I, and 330 bp – D/D. Heterozygote chickens I/D are present in the combination of both variants – 387 and 330 bp respectively. Gene IGF-I: the size of the amplifi ed fragment – 621 bp. Genotype C1/C1 on the electrophoregram corresponds to the fragment of 621 bp; C /C – 257 and 364 2 2 Fig. 1. The electrophoregram of restriction products of the bp; C /C – three fragments of 621, 257 and 364 bp 1 2 exon region of TGF-β1 gene: 1 – genotype B/B; 2, 4, 8, 9, respectively. 12 – genotype F/F; 3, 5, 7, 10, 11 – genotype B/F; 6 –– mar- The frequency of alleles of polymorphic loci was ker of molecular mass М-50 determined by the formulas of maximum likelihood. The data obtained was used for the calculation of actual and theoretical distribution of genotypes, the cor- respondence to genetic equilibrium of the population 2 by Hardy-Weinberg, using criterion χ , actual (Ho) and expected (He) heterozygosity, effi cient number of alleles (ne), Wright’s fi xation index (Fis) using the Popgen32 program. The productivity traits of chickens were also taken into consideration: the number of eggs in 12 weeks of productivity (En12); the number of eggs in 40 weeks of productivity (En40); the weight th of eggs on the 30 week of life (Ew30); the weight nd of eggs on the 52 week of life (Ew52); live weight; the weight of eviscerated carcass, pectoral muscles, Fig. 2. The electrophoregram of the amplifi cation products muscles of legs, thighs, gizzard stomach, liver, heart, of the second intron of PIT-1 gene: 1–4, 7, 9, 11 – I/D; 5, 10 – I/I; 8 – D/D; 6 – marker of molecular mass M-50 and visceral fat. RESULTS AND DISCUSSION The use of the molecular and genetic analysis allowed revealing the variability of each gene, studied in the current work – TGF-β1, PIT-1 and IGF-I. The polymorphism of TGF-β1 gene is related to the transversion of cytosine in position 632 – mis- sence mutation, leading to the substitution of glu- tamin for asparagine in the encoded protein, which results in the occurrence of the restriction site for MboII in allele B. The allele variants of TGF-β1 gene, occurring due to MboII-polymorphism of the exon region, are presented Fig. 3. The electrophoregram of restriction products of amplifi ed fragment of IGF-I gene: 1, 3, 7, 10 – C /C ; 2, 4, 5, on the electrophoregram of restriction products (Fig. 1). 2 2 9 – C1/C2; 8, 11 – C1/C1; 6 – marker of molecular mass М-100 The data obtained present evidence to the fact that TGF-β1 gene is polymorphic in the studied population The polymorphism of the gene is related to the of chickens. There are individuals of all the possible presence of the insert of 57 bp in the second intron, genotypes (B/B, B/F, F/F). which leads to the formation of two allele variants –

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 69 KULIBABA et al. I and D. Figure 2 presents the electrophoregram of of chickens. There are chickens of all three possible the ampliﬁ cation products of the second intron of genotypes: I/I, I/D and D/D. PIT-1 gene. The allele variants of IGF-I gene occur due to the By the presence of the insert in the second intron PIT- transition of cytosine into thymine which results in 1 gene is also polymorphic in the studied population the loss of restriction site for PstI in case of alle-

Table 1. The genetic structure of the population of Poltava clay chicken breed by loci TGF-β1, PIT-1, and IGF-I

Locus Genotype frequency Allele frequency χ2

TGF-β1 B/B B/F F/F B F 0.03 0.1 0.43 0.47 0.31 0.69 PIT-1 I/I I/D D/D I D 0.62 0.38 0.5 0.12 0.63 0.37 IGF-I C1/C1 C1/C2 C2/C2 C1 C2 1.17 0.17 0.42 0.41 0.38 0.62

Table 2. The association of different genotypes by loci TGF-β1, PIT-1 and IGF-I and the indices of egg productivity of Pol- tava clay chicken breed

Index Gene Genotype

En12 (it.) En40 (it.) Ew30 (g) Ew52 (g)

TGF-β1 B/B 67.0 ± 2.91a 173.7 ± 8.13a 49.1 ± 1.32a 56.8 ± 2.68a B/F 66.5 ± 1.84a 190.2 ± 4.32a 51.9 ± 0.69a 58.7 ± 0.75a F/F 67.8 ± 1.18a 193.2 ± 2.38b 51.9 ± 0.56a 58.6 ± 0.68a PIT-1 I/I 66.6 ± 1.62a 184.2 ± 5.06a 51.1 ± 0.63a 57.2 ± 0.72a I/D 68.4 ± 1.13a 191.7 ± 3.25a 51.8 ± 0.57a 58.9 ± 0.69a D/D 69.8 ± 2.44a 195.0 ± 7.03a 51.4 ± 1.61a 60.6 ± 2.15a IGF-I C1/C1 65.9 ± 2.55a 186.5 ± 6.32a 53.6 ± 1.28a 58.1 ± 1.59a C1/C2 68.1 ± 1.31a 190.2 ± 5.05a 50.9 ± 0.64a 58.3 ± 0.79a C2/C2 68.4 ± 1.49a 189.6 ± 4.39a 51.1 ± 0.64a 58.5 ± 0.77a

Note. a, bDifferences are reliable (p < 0.05) within the locus.

Table 3. The association of different genotypes by loci TGF-β1, PIT-1 and IGF-I and the indices of meat productivity of Poltava clay chicken breed

Geno- Live Eviscerated Pectoral Thigh Leg muscles, Gizzard Visceral Gene type weight, kg carcass, kg muscles, g muscles, g g stomach, g fat, g

TGF-β1 B/B 2.21 ± 0.070a 1.37 ± 0.065a 112.7 ± 5.17a 77.9 ± 3.46a 59.1 ± 1.89a 32.2 ± 1.08a 54.1 ± 10.97a B/F 2.46 ± 0.051ab 1.44 ± 0.041b 116.2 ± 2.99a 82.3 ± 2.46ab 64.00 ± 1.63ab 35.1 ± 1.07b 43.3 ± 4.27a F/F 2.46 ± 0.046b 1.56 ± 0.039c 120.3 ± 2.77a 88.0 ± 2.03b 66.5 ± 1.44b 35.2 ± 0.91b 52.2 ± 3.32a PIT-1 I/I 2.32 ± 0.049a 1.44 ± 0.037a 116.6 ± 3.19a 83.6 ± 2.52a 64.4 ± 1.60a 34.3 ± 0.98a 45.7 ± 3.95a I/D 2.40 ± 0.044a 1.50 ± 0.037a 117.9 ± 2.44a 83.3 ± 1.97a 63.5 ± 1.33a 35.2 ± 0.87a 48.0 ± 3.52a D/D 2.53 ± 0.165a 1.61 ± 0.140a 120.2 ± 9.20a 96.8 ± 4.43b 73.2 ± 3.52b 36.3 ± 3.34a 60.3 ± 17.48a IGF-I C1/C1 2.44 ± 0.095a 1.55 ± 0.067a 115.7 ± 4.87a 88.9 ± 4.74a 65.5 ± 2.69a 34.9 ± 1.82a 59.8 ± 6.49a C1/C2 2.36 ± 0.048a 1.48 ± 0.040a 119.6 ± 3.05a 83.7 ± 2.19a 64.6 ± 1.64a 34.4 ± 0.96a 44.7 ± 3.72b C2/C2 2.36 ± 0.042a 1.46 ± 0.042a 116.2 ± 2.77a 82.8 ± 2.13a 63.9 ± 1.44a 35.5 ± 0.97a 46.5 ± 4.48b

Note. a, b, cDifferences are reliable (p < 0.05) within the locus.

70 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 TRANSFORMING GROWTH FACTOR Β1, PITUITARY-SPECIFIC TRANSCRIPTIONAL FACTOR 1 le C1. The indication of alleles (C1 and C2) is related to Reliable differences were revealed in the meat produc- the number of cytosine residues in the restriction site tivity of chickens by loci TGF-β1 and PIT-1 (Table 3). for PstI. The electrophoregram of restriction products It was demonstrated that by locus TGF-β1 chickens (PstI) of 5’UTR fragment of IGF-I gene is presented with genotype F/F are characterized by reliably higher in Fig. 3. values of indices of live weight, mass of eviscerated The studies demonstrated that similar to the above- carcass, muscles of thigh, legs and gizzard stomach mentioned cases IGF-I locus is polymorphic in the ex- compared against chickens of genotype B/B (Table 3). perimental population of chickens. There are chickens In their turn some reliable differences between the of all the possible genotypes: C /C , C /C and C /C . 1 1 1 2 2 2 chickens of genotypes D/D and I/I by the indices of the The genetic structure of the experimental population of weight of muscles of thigh and legs were registered for chickens for all three studied loci is presented in Table 1. locus PIT-1. There is also a tendency of prevalence of The values of the actual heterozygosity by different genotype D/D chickens by the indices of live weight loci in the experimental population of chickens were and the mass of eviscerated carcass. At the same time in the range of 0.42–0.5 while the values of expected there were no reliable differences for locus IGF-I by heterozygosity were from 0.428 to 0.471. The high- the studied indices, except for the index of visceral fat est level of H was observed by locus IGF-I (0.471), e mass (chickens of genotype C1/C1 are reliably exceed- the lowest – by TGF-β1 (0.428). Some defi ciency of ing chickens of genotype C1/C2). heterozygous chickens was defi ned by locus IGF-I (F is There were no reliable differences between the val- = 0.108). In its turn some excess of heterozygotes was ues of the weight of heart and liver for any locus. observed by locus PIT-1 (Fis = −0.07). The value of the effi cient number of alleles fl uctuated in the range of CONCLUSIONS 1.75–1.89, and the highest level of polymorphism in The genetic structure of Poltava clay chicken breed was the studied population was noted for locus IGF-I. studied by loci of transforming growth factor β1, pituitary The analysis of actual and theoretical distribution of transcriptional factor 1 and insulin-like growth factor I. It chickens of different genotypes (by all the studied loci) was determined that all the investigated loci in the experi- in the experimental population of chickens did not re- mental population of chickens were polymorphic. The as- veal any disorder of genetic equilibrium which testifi es sociation between genotypes by the loci TGF-β1 and PIT- to the absence of any selection pressure. 1 and the indices of egg and meat production of chickens was demonstrated. The data obtained are recommended The presence of chickens of all the genotypes, pos- for the application in the targeted selection of Poltava clay sible for each locus, in the experimental population al- chicken breed with the purpose of obtaining the offspring lowed investigating the connection of different geno- of desired genotypes which will promote maximum ef- types to the productivity traits of chickens. fi ciency in realizing productive potential of poultry in ad- As for locus TGF-β1, it was demonstrated that the dition to classic selection methods. egg productivity of chickens with genotype F/F in 40 weeks exceeded that of chickens with genotype B/B Поліморфізм генів трансформуючого ростового and amounted to 193.2 ± 2.38 and 173.7 ± 8.13 (p < фактора β1, гіпофізарного фактора транскрипції 1 та інсуліноподібного ростового фактора I у популяції < 0.05) (Table 2). курей породи полтавська глиняста: зв’язок It was also noted that by locus PIT-1 the egg pro- з продуктивними ознаками ductivity of chickens with genotype D/D for the same Р. О. Кулібаба, О. В. Терещенко time period was 195.0 ± 7.03, which was more than e-mail: [email protected] the index for chickens with genotype I/I – 184.2 ± ± 5.06. However, in this case the difference is not reliable Державна дослідна станція птахівництва НААН України which is considerably related to a small number of chick- Вул. Леніна, 20, с. Бірки, Зміївський р-н, Харківська обл., Україна, 63421 ens with genotype D/D. As for the rest of the loci, no signifi cant differences in the indices of egg productivity were Мета. Дослідження поліморфізму генів трансформую- revealed between the chickens of different genotypes. чого ростового фактора β1 (TGF-β1), гіпофізарного фак- тора транскрипції 1 (PIT-1) та інсуліноподібного рос- There are no remarkable differences for each locus тового фактора I (IGF-I) у популяції курей яєчно-м’яс- by the egg weight indices on the 30th and 52nd weeks ного напрямку продуктивності породи полтавська гли- of life. няста та аналіз зв’язку різних генотипів за кожним

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 71 KULIBABA et al. локусом з продуктивними ознаками. Методи. Особин Ключевые слова: порода полтавская глинистая, поли- генотипували з використанням полімеразної ланцюго- морфизм, трансформирующий ростовой фактор β1, гипо- вої реакції та аналізу поліморфізму довжини рестрик- физарный фактор транскрипции 1, инсулиноподобный ційних фрагментів (ПЛР-ПДРФ). Результати. Показа- ростовой фактор I, продуктивные признаки. но, що в популяції курей породи полтавська глиняста локуси TGF-β1, PIT-1 та IGF-I є поліморфними. Вста- СПИСОК ЛИТЕРАТУРЫ новлено зв’язок генотипів за локусами TGF-β1 і PIT- 1. Khlestkina E K. Molecular markers in genetic studies 1 з показниками яєчної та м’ясної продуктивності. and breeding. Russ J Genetics: Appl Res.2014;4(3): 236– Висновки. Отримані дані щодо генетичної структури 44. популяції курей породи полтавська глиняста за локуса- ми TGF-β1 і PIT-1 рекомендовано використовувати при 2. Dodgson J B, Cheng H H, Okimoto R. DNA marker здійсненні спрямованої селекції курей для отримання technology: a revolution in animal genetics. Poult нащадків з бажаними генотипами, що дозволить (як до- Sci.1997;76(8):1108–14. повнення до класичних селекційних методів) максималь- 3. Kim I Y, Kim M M, Kim S J. Transforming growth но ефективно розкрити продуктивний потенціал птиці. factor-β: biology and clinical relevance. J Biochem Mol Biol.2005;38(1):1–8. Ключові слова: порода полтавська глиняста, полімор- фізм, трансформуючий ростовий фактор β1, гіпофізарний 4. Li H, Deeb N, Zhou H, Mitchell A D, Ashwell C M, фактор транскрипції 1, інсуліноподібний ростовий фак- Lamont S J. Chicken quantitative trait loci for growth and тор I, продуктивні ознаки. body composition associated with transforming growth factor-beta genes. Poult Sci.2003;82(3):347–56. Полиморфизм генов трансформирующего ростового 5. Nie Q, Fang M, Xie L, Zhou M, Liang Z, Luo Z, Wang G, фактора β1, гипофизарного фактора транскрипции 1 Bi W, Liang C, Zhang W, Zhang X. The PIT1 gene и инсулиноподобного ростового фактора I polymorphisms were associated with chicken growth в популяции кур породы полтавская глинистая: traits. BMC Genet.2008;9(20):20–9. связь с продуктивными признаками 6. Rodbari Z, Alipanah M, Seyedabadi H R, Amirinia C. Р. А. Кулибаба, А. В. Терещенко Identiﬁ cation of a single nucleotide polymorphism of e-mail: [email protected] the pituitary-speciﬁ c transcriptional factor 1 (Pit 1) Государственная опытная станция птицеводства gene and its association with body composition trait in НААН Украины Iranian commercial broiler line. African J Biotechnol. Ул. Ленина, 20, с. Борки, Змиевской р-н, 2011;10(60):12979–83. Харьковская обл., Украина, 63421 7. Hwa V, Oh Y, Rosenfeld R G. The insulin-like growth Цель. Исследование полиморфизма генов трансформи- factor-binding protein (IGFBP) superfamily. Endocr рующего ростового фактора β1 (TGF-β1), гипофизарно- Rev.1999;20(6):761–87. го фактора транскрипции 1 (PIT-1) и инсулиноподобного 8. Khadem A, Hafezian H, Rahimi-Mianji G. Association ростового фактора I (IGF-I) в популяции кур яично- of single nucleotide polymorphisms in IGF-I, IGF-II мясного направления продуктивности породы полтавс- and IGFBP-II with production traits in breeder hens of кая глинистая и анализ связи различных генотипов по Mazandaran native fowls breeding station. African J каждому из локусов с продуктивными признаками. Biotechnol.2010;9(6):805–10. Методы. Особей генотипировали с использованием по- 9. Li W, Li F, Li D. IGF-1 gene polymorphism and weight- лимеразной цепной реакции и анализа полиморфиз- related analysis. Int J Biol.2009;1(2):113–8. ма длины рестрикционных фрагментов (ПЦР-ПДРФ). 10. Kim M H, Seo D S, Ko Y. Relationship between egg Результаты. Показано, что в популяции кур породы productivity and insulin-like growth factor-I genotypes полтавская глинистая локусы TGF-β1, PIT-1 и IGF-I in Korean native Ogol chickens. Poult Sci.2004;83, являются полиморфными. Установлена связь генотипов (7):1203–8. по локусам TGF-β1 и PIT-1 с показателями яичной и мясной продуктивности кур. Выводы. Полученные дан- 11. Kulibaba R A, Podstreshnyi A P. Prolactin and growth ные о генетической структуре популяции кур породы hormone gene polymorphisms in chicken lines of Uk- полтавская глинистая по локусам TGF-β1 и PIT-1 rainian selection. Cytology and Genetics.2012;46(6): рекомендуется использовать при проведении направ- 390–5. ленной селекции кур для получения потомства с же- 12. Kulibaba R A. Polymorphism of transforming growth лательными генотипами, что позволит (в дополнение к beta-factor family genes in the chicken line of Ukrainian классическим селекционным методам) максимально эф- breeding. Molecular and Applied Genetics: Proceedings. фективно раскрыть продуктивный потенциал птицы. Minsk.2014;Vol. 17:7–103.

72 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 ISSN: 2312-3370, Agricultural Science and Practice, 2015, Vol. 2, No. 1

UDC 631.423.4 : 631.453 : 631.417.2 : 631.417.8 INFLUENCE OF HUMUS ACIDS ON MOBILITY AND BIOLOGICAL AVAILABILITY OF IRON, ZINC AND COPPER A. I. Fateev, D. O. Semenov, K. B. Smirnova, A. M. Shemet National Scientiﬁ c Center “Institute for Soil Science and Agrochemistry Research named after O. N. Sokolovsky” 4, Chaikovskoho Str., Kharkiv, Ukraine, 61024 e-mail: [email protected] Received on October 29, 2014

Soil organic matter is known as an important condition for the mobility of trace elements in soils, their geochemical migration and availability to plants. However, various components of soil organic matter have different effect on these processes due to their signifi cant differences in structure and properties. Aim. To establish the role of humic and fulvic acids in the process of formation of microelement mobility in soils and their accumulation in plants. Methods. A model experiment with sand culture was used to investigate the release of trace elements from preparations of humic and fulvic acids and their uptake by oat plants. Results. It was found that among biologically needed elements humic acids are enriched with iron, fulvic acids – with zinc, and copper distribution between these two groups of substances may be characterized as even. These elements have unequal binding power with components of soil organic matter, as evidenced by their release into the cultivation medium and accumulation in plants. In the composition of fulvic acids zink has the most mobility – up to 95 % of this element is in the form, accessible for plants; the lowest mobility was demonstrated by copper in the composition with humic acids, for which no signifi cant changes in the concentration of mobile forms in the substrate and in the introduction to the test culture were registered. Despite signifi cantly higher iron content in humic acids, the application of fulvic acids in the cultivation medium provides a greater increase in the concentration of mobile forms of this element. Conclusions. The results confi rm the important role of organic substances of fulvic nature in the formation of zinc and iron mobility in the soil and their accumulation in plants. Key words: soil, trace elements, humic acids, fulvic acids, mobility, accessibility.

INTRODUCTION erty of soil may act both as a regulator of the mobility The most severe shortage of trace elements in the of trace elements and their source for plants and soil mineral nutrition of cultivated crops is primarily relat- biota. In particular, many trace elements are remark- ed to the properties of soils, affecting the accessibility able for the accumulation of different types of soils in of these trace elements to plants, rather than to their the organic matter, for instance, for Mo, Ni, Cu, V, Co, low concentration in soil. The increase in the concen- Zn, Pb the enrichment factor is in the range from 10 to tration of protons and in the soil humidity as well as 1,000 [2]. Still some components of soil organic matter the decrease in the oxidation-reduction potential (ORP) have a different capacity of binding trace elements – are referred to as the factors, enhancing the mobility of their majority is concentrated in fulvic acids (FA). This most trace elements. On the contrary, the factors, inhib- is a regular phenomenon, taking into consideration iting the mobility of trace elements, include carbonate high dispersibility, hydrophilicity and higher reaction content of soils, increase in ORP and the increase in the capacity of FA which are more saturated with function- − − al groups compared to humic acids (HA). Thus, com- concentration of ОН and РО4 . The organic matter of soils has ambiguous effect on the performance of the pared to HA, FA have a higher capacity of interacting cations of trace elements – the increase in its content with trace elements. enhances the availability of Zn and Mn and inhibits the Notable ways of interacting with trace elements are mobility of Cu [1]. However, it is known that this prop- ion exchange, chelation and adsorption. One kilo of

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 73 FATEEV et al. humic acid is known to adsorb up to 200 g of metals ally soluble. In the anaerobic acid medium these com- and fulvic acids have even higher base exchange ca- pounds of metals are so mobile that they are capable pacity. The carboxyl and phenolic groups are the most of penetrating ground waters and permanent streams in active in the processes of fi xing metals, but due to the large amounts which is primarily notable for rivers of ion exchange heavy metals may leave the composition the boreal belt. It is known that 50–70 % of organic of these compounds rather easily. A separate kind of matter content in the Dnieper’s water is composed of organic and mineral derivatives is complex salts. In the compounds of Fe, Mn, Ni, Cu with humus acids. this case the cations of trace elements displace hydro- The compounds of fulvic and oxy-acids and cations of gen ions and penetrate the inner sphere. Here the che- metals migrate in the geochemical stream and get ac- late structures may be formed due to the interaction of cumulated in depressions. The soils of swamps, fl ood metal with two groups of СООН− or with СООН− and plains and river deltas are usually enriched with these ОН− groups. This is notable for Zn, Cu and Pb that eas- components. On the contrary, humic acids precipitate ily form covalent bonds due to high electronegativity iron, manganese, copper, zinc, nickel and cobalt. This which was proven by infrared spectroscopy [4]. results in the formation of insoluble precipitates of hu- One of the main mechanisms, conditioning the ac- mates which are both salt compounds and the products cessibility of trace elements for living organisms, is of mutual coagulation and adsorption [2]. The interac- the formation of chelates, and in this process FA are tion of heavy metals and HA depends on the availabil- the main chelating agent. Contrary to single salts, the ity of alkaline-earth metals. For instance, in presence of chelate compounds of trace elements maintain their in- Ca and Mn ions the interaction of Cu with the organic creased mobility in a wide range of pH values, while matter decreases 5-fold. However, the mechanism of the concentration of ionic forms of trace elements de- interaction between the cations of heavy metals and creased by tens of times. This phenomenon is of ut- humates and fulvates does not have any considerable most importance to soil-forming processes and to the differences from their binding to HA and FA, where input of nutrients to plant roots. The trace elements in metal displaces hydrogen from the phenolic group [8]: the composition of FA or more low molecular organic Cu2+ + Н-FA-СООСа+ ↔ Cu-FA+-СООСа+ Н+. compounds of soils are more susceptible to microbio- MATERIALS AND METHODS logical destruction and, as a result, more available for plant roots than the ones, accumulated in HA [5]. We have determined reliable increase in the mobility of zinc, manganese and iron along with the increase in The role of HA in the fi xation of trace elements is the share of fulvic acids in the organic matter [9, 10]. revealed not only in the background soils. In case of However, in natural conditions the availability of trace contamination these substances serve as a natural bar- elements in soils is affected by a considerable number rier to fi x the excessive amount of heavy metals. The of factors, the main among them being the granulomet- prevailing signifi cance of fulvic compounds is noted in ric composition of soils, humus content, pH of the me- this process as well – the share of Zn, Pb and Cd in the dium, the presence of СаСО3 (especially in the active composition of fulvic acids of alluvial soils with tech- form) and a number of other abovementioned soil char- nogenic pollution in Czech Republic is 95.7, 82.0 and acteristics. In order to bring out the effect of merely 98.4 % respectively. The signifi cance of this ecologic humus acids we have conducted a model experiment factor is also evidenced in the close interrelation of the with sandy culture and Mitscherlich’s nutrient mixture total content of metals and their concentration in fulvic (without Fe addition), the composition of which and acids. The correlation coeffi cient for Zn, Pb and Cd is the conditions of preparing sand for the experiment are 0.928, 0.855 and 0.873, respectively, р = 0.001 [6]. presented in fi ne detail in [11]. The experiment was However, the complex compounds in the biosphere conducted in three variants with four repeats – with the are not mere regulators of the input of chemical ele- introduction of preparations of humic and fulvic acids ments to plants – FA are the main complexone of nat- and control, respectively. The preparations were ob- ural surface waters and soil solutions. Therefore, the tained by six isolations of the organic matter from the organometallic complexes are the main form of migra- patch of soil using 0.1 m NaOH after its decalcination tion of trace elements and heavy metals in soils which with 0.05 n H2SO4. The fractions of HA and FA were is an integral part of geochemical circulation of these separated by the acidifi cation of the obtained and re- elements [7]. For instance, the fulvates of manganese, fi ltered solution to pH of 1.5–2 with 10 % H2SO4. After aluminum, cobalt, copper and other elements are usu- the corresponding purifi cation and drying-up according

74 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 INFLUENCE OF HUMUS ACIDS ON MOBILITY AND BIOLOGICAL AVAILABILITY to [12] the preparations of HA and FA were analyzed for the content of trace elements (Table 1). The data presented testify that out of biologically required elements the humic acids are enriched with iron and the fulvic acids – with zinc. The distribution of manganese and copper may be characterized as even. The preparations were introduced in the amount of 1.2 g/vessel (0.4 %) which corresponds to the natural content of humic and fulvic acids in soils with sandy texture. The oat plants (Avena sativa L.) were cultivated until the tillering stage. The changes in the concentration of trace elements in the substrate and their accumulation in plants were determined. We studied the changes in the content of trace elements, the concentration of which in the composition of humus acids was found to be the highest, namely – Fe, The impact of humus acids on the content of trace elements Zn, and Cu, for more accurate defi nition of their bal- in the cultivation medium: 1 – copper; 2 – zinc; 3 – iron ance using atomic absorption analysis. The content of bility of this element in the composition of fulvic ac- mobile forms of trace elements was determined using ids, regardless of a considerably smaller total content. 1 N of ammonium-acetate buffer solution (pH 4.8), The form, accessible for plants, was noted for 14.8 % the soil:extractant ratio was 1:5. The concentration of Fe from the composition of exogenous FA, and only trace elements in the preparations of humus acids and 1.9 % – from the composition of HA preparation. herbage of a test-crop was determined after annealing at 550 °С and the dissolution of the ash obtained in The effi ciency of Fe in the complex with FA was 10 % solution of HCl. also determined [14]. Fulvate iron does not concede the EDTA–Fe and citrate–Fe complexes in the stabil- The statistical processing of the data obtained was ity constants; its introduction to the nutrient solution conducted using Dospehov and Statistica 10 programs. in conditions of the sandy crop resulted in more active RESULTS AND DISCUSSION input of this metal into the plants of sunfl ower and Humic and fulvic acids have ambiguous effect on the beans [13]. mobility of trace elements in substrates. It was deter- The effect of zinc in the composition of humus mined that the content of iron in the cultivation medium acids was proven to be somewhat different. Con- after the cultivation of the test-crop in the control vari- trary to Fe, zinc was mostly present in fulvic acids – ant was 0.67 mg/kg. The introduction of preparations 273.5 mg/kg of the preparation against 57.6 mg/kg in of humus acids to the cultivation medium increases humic acids which turned out to be the additional fac- this index up to 1.15 mg/kg for humic acids and up to tor of the release of this trace element from the prepa-

1.47 mg/kg for fulvic acids at the value of LSD05 which rations. The highest content of mobile zinc compounds corresponds to 0.25 mg/kg (Figure). However, the data was observed in the variant with FA introduction – of the analysis of HA and FA preparations testify that 1.40 mg/kg of the substrate with the control value of 1 kg of the mixture was introduced 25.94 and 5.42 mg 0.36 mg/kg. The reliability of the difference was prov- of iron respectively which evidences to high accessi- en by the results of the dispersion analysis − LSD05 Table 1. The content of trace elements in the preparations of humic and fulvic acids from typical chernozem

Content of trace elements, mg/kg of preparation Preparation Fe Mn Cu Zn Co Ni Cr Cd Pb

HA 6485.8 33.8 257.4 57.6 13.4 15.37 22.2 0.72 37.0 FA 1354.9 33.1 255.0 273.5 3.7 4.99 20.8 1.12 26.5

LSD05 411.7 5.07 9.12 22.21 4.62 4.36 5.17 0.33 15.59

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 75 FATEEV et al. corresponds to 0.19 mg/kg. The surplus of accessible components of the organic matter, but this metal has Zn is 1.04 mg/kg with the introduction of 1.09 mg Zn closest relations with humus acids, which is testifi ed to in the composition of fulvic acid preparation, thus the by the absence of reliable changes in the concentration accessibility of this trace element in the composition of its mobile forms. of the preparation of fulvic acids is 95 %. After the Still the most objective index of mobility and bio- introduction of HA preparation the content of mobile logical accessibility of nutrition elements is their ac- forms of zinc was considerably smaller – 0.50 mg/kg, cumulation in plants. The data on the effect of humus so the surplus is 0.14 mg/kg of the substrate which acids on the mobility of Fe, Zn, and Cu in soil are may be considered as a tendency only. If the introduc- partially confi rmed by their accumulation in test- tion of Zn per 1 kg of the medium is 0.23 mg/kg in crops (Table 2). The content of iron in test-crops in this case, the accessibility of zinc in the composition the control variant is 93.1 mg/kg of dry mass. The in- of humic acids is rather high – almost 61 %. The data troduction of the preparations of humus acids to the obtained testify to high mobility of zinc, connected to cultivation medium increases the accumulation of humus acids. The result demonstrates that the presence Fe in plants up to 109.6 mg/kg for humic and up to of FA in soils is a considerable factor of the mobil- 115.5 mg/kg – for fulvic substances. The enhanced ity of this trace element. The introduction of 0.4 % of uptake of iron by plants in these two variants com- the preparation of fulvic acids transfers soil to another pared to the control is statistically proven – LSD is category of provision regarding the content of mobile 05 Zn – over 1 mg/kg according to commonly accepted 9.79 mg/kg. gradations [1]. The data on the accumulation of zinc in plants pro- Quite different results were obtained for copper. Re- vide substantial evidence to the effect of humus sub- gardless of the high amount of the latter in both prepa- stances on the provision of accessible forms of trace rations of humus acids there were no registered reliable elements for soils. The content of Zn in oat plants in changes in mobile forms of this element. The content the control variant is 41.0 mg/kg, calculated per dry of mobile copper is 0.37 mg/kg in the control variant weight. The introduction of the preparation of humic and 0.39 and 0.42 mg/kg – in variants with the intro- acids to the cultivation medium does not induce stable duction of the preparations of humic and fulvic acids changes in the indices of translocation of this trace respectively at the value of the lowest signifi cant dif- element – 40.6 mg/kg. However, the application of FA ference of 0.24 mg/kg of soil. preparation increases zinc accumulation by oat plants more than 1.5-fold – up to 62.3 mg/kg which proves The data obtained brings one to the conclusion that the relevance of the availability of fulvic humus in the the behavior of trace elements, located in humic acids, processes of forming the mobility of this trace ele- depends both on the nature of humus substances and ment in soils. on the properties of metals proper. Iron is primarily located in humic substances, but it is much more ac- The use of preparations of HA and FA has a different cessible in the composition of fulvic acids. The high- effect on the input of copper into plants. The introduc- est mobilization indices are attributed to zinc, which tion of humic acids to the cultivation medium does not is notable for its accumulation in the fulvate humus. induce stable changes in Cu accumulation in the herb- Copper is evenly distributed between the investigated age of oats – 10.5 mg/kg with the control value of 11.7

and LSD05 which equals 7.06 mg/kg. However, the in- Table 2. The accumulation of trace elements in the herbage troduction of 0.4 % of fulvic acids ensures almost twice of oats enhanced introduction of copper into plants compared to the control which is crucially different from their ef- Content of trace elements in plants, fect on the copper release in the cultivation medium. mg/kg of dry weight Variant This effect may be explained by higher susceptibility Fe Zn Cu of plants to the consumption of trace elements i.e. bio- testing using plants is more sensitive to the changes in Control 93.1 41.0 11.7 the mobility of Cu compared to the use of extract of HA 0.4 % 109.6 40.6 10.5 ammonium-acetate buffer solution with pH 4.8. FA 0.4 % 115.5 62.3 21.0 The data obtained testify that the presence of even LSD 9.79 17.05 7.06 05 small amounts of humus substances, of fulvic origin

76 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 INFLUENCE OF HUMUS ACIDS ON MOBILITY AND BIOLOGICAL AVAILABILITY in particular, in the cultivation medium results in the переважну роль фульватних органічних сполук у фор- enhanced accumulation of Fe, Zn, and Cu in plants. муванні рухливості цинку і заліза в ґрунтах та їхньому накопиченні в рослинах. CONCLUSIONS Ключові слова: ґрунт, мікроелементи, гумінові кисло- The results obtained allow the conclusion that humic ти, фульвокислоти, рухливість, доступність. acids are a relevant factor of the mobility of trace elements in soils and their accumulation by plants, but it Влияние гумусовых кислот на подвижность depends both on the nature of humic substances and и биологическую доступность железа, цинка и меди the properties of metals. The predominant role of fulvic А. И. Фатеев, Д. А. Семенов, acids in the processes of the formation of mobility of Е. Б. Смирнова, А. М. Шемет trace elements, the introduction of the preparation of which leads to the increase in the level of provisions for e-mail: [email protected] soils in terms of mobile forms of iron and, especially, Национальный научный центр «Институт почвоведения zinc, has been proven. и агрохимии имени А.Н. Соколовского» Вплив гумусових кислот на рухливість Ул. Чайковская, 4, Харьков, 61024 та біологічну доступність заліза, цинку і міді Известно, что органическое вещество почвы является А. І. Фатєєв, Д. О. Семенов, важным условием подвижности микроэлементов в поч- К. Б. Смірнова, А. М. Шемет вах, их геохимической миграции и доступности расте- ниям. Однако разные компоненты органического вещест- e-mail: [email protected] ва почвы неодинаково влияют на указанные процессы, Національний науковий центр «Інститут ґрунтознавства что обусловлено значительными отличиями в строении та агрохімії імені О. Н. Соколовського» и свойствах. Цель. Установление роли гуминовых и Вул. Чайковська, 4, Харків, 61024 фульвокислот в процессах формирования подвижности микроэлементов в почвах и их накопление в растениях. Відомо, що органічна речовина ґрунтів є важливим чин- Методы. В условиях модельного эксперимента с пес- ником рухливості мікроелементів у ґрунтах, їхньої гео- чаной культурой изучали высвобождение микроэлемен- хімічної міграції та доступності рослинам. Проте різні тов из препаратов гуминовых и фульвокислот и их компоненти органічної речовини ґрунтів неоднаково поглощение растениями овса. Результаты. Установле- впливають на зазначені процеси внаслідок значних від- но, что среди биологически необходимых элементов мінностей у побудові та властивостях. Мета. Вста- гуминовые кислоты обогащены железом, фульвокисло- новлення ролі гумінових та фульвокислот у процесах ты – цинком, а распределение меди между указанными формування рухливості мікроелементів у ґрунтах та группами веществ можно охарактеризовать как равно- їхнє накопичення у рослинах. Методи. За умов мо- мерное. Данные элементы имеют неодинаковую силу дельного досліду з піщаною культурою вивчали ви- связи з компонентами органического вещества почвы, вільнення мікроелементів з препаратів гумінових та о чем свидетельствует их высвобождение в среде роста фульвокислот та їхнє поглинання рослинами вівса. и накопление в растениях. Наибольшая подвижность Результати. Встановлено, що з-поміж біологічно необ- присуща цинку в составе фульвокислот – до 95 % хідних елементів гумінові кислоти збагачені залізом, данного элемента находится в доступной растениям а фульвокислоти – цинком, розподіл міді можна оха- форме; наименьшая – меди в составе гуминовых кис- рактеризувати як рівномірний. Перелічені елементи ма- лот, для которой не зафиксировано достоверных измене- ють неоднакову силу зв’язку з компонентами органічної ний в концентрации подвижных форм в субстрате и в речовини ґрунтів, про що свідчить їхнє вивільнення поступлении в тест-культуру. Несмотря на значительно в середовищі росту та накопичення в рослинах. Най- более высокое содержание железа в гуминовых кисло- більша рухливість притаманна цинку у складі фуль- тах, внесение препарата фульвокислот в среду роста вокислот – до 95 % цього елемента знаходиться у обеспечивает больший прирост концентрации подвиж- доступній рослинам формі; найменша – міді у складі ных форм данного элемента. Выводы. Полученные ре- гумінових кислот, для якої не зафіксовано достовірних зультаты подтверждают важную роль органических ве- змін у концентрації рухливих форм у субстраті та в ществ фульватной природы в формировании подвиж- надходженні до тест-культури. Незважаючи на значно ности цинка и железа в почвах и в их накоплении в вищий вміст заліза в гумінових кислотах, додавання растениях. препарату фульвокислот у середовище росту забезпечує більший приріст концентрації рухливих форм даного Ключевые слова: почва, микроэлементы, гуминовые елемента. Висновки. Отримані результати свідчать про кислоты, фульвокислоты, подвижность, доступность.

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 77 FATEEV et al. REFERENCES 8. Pivovarov S. Physico-chemical modeling of heavy metals (Cd, Zn, Cu) in natural environment. Encyclopedia of 1. Optimization for trace element nutrition of agricultural Surface and Colloid Science. Boca Raton, CRC Press. crops (second edition, revised and enlarged). Ed. A. I. 2004;Vol.6:468–93. Fateev. Kharkiv, “ART-PROEKT” LLC.2012;38 p. 2. Kovda V A. Biogeochemistry of soil cover. Moscow, 9. Fateev A I, Semenov D O, Miroshnychenko M M, Lyko- va O A, Smirnova K B, Shemet A M. Ratio of С /С in Nauka.1985;264 p. HA FA soils of Ukraine as a measure of trace elements mobility. 3. Stepanova M D. Trace elements in soil organic matter. Visnyk agrarnoi’ nauky.2013;(7):16−9. Novosibirsk, Nauka.1976;107 p. 10. Semenov D O. Effect of ratio of humic acids in soil 4. Minkina T M, Motuzova G V, Nazarenko O G. Com- organic matter of soils in Ukraine on content of mobile position of heavy metal compounds in soils. Rostov-on- forms of trace elements. Agrohimija i g’runtoznavstvo. Don, Jeverest.2009;208 p. 2013;(80):116−22. 5. Kabata-Pendias A, Pendias H. Trace elements in soils and plants. Moscow, Mir.1989;439 p. 11. Sokolov A V, Ahromeiko A I, Panﬁ lov V N. Vegetative method. Moscow, Sel’hozgiz.1938;292 p. 6. Boruvka L, Drabek O. Heavy metal distribution between fractions of humic substances in heavily polluted soils. 12. Orlov D S, Grishina L A. Practical work on humus Plant Soil Environ.2004;50(8):339−45. chemistry. Moscow, MGU.1981;272 p. 7. Karpuhin A I. Functions of complex compounds in the 13. Kaurichev I S, Karpuhin A I. Water-soluble iron-organic genesis and soil fertility. Proc. Timiryazev Agricult. compounds in soils of Taiga-Forest zone. Pochvovedenie. Acad. 989; 4):54−61. 1986;(3):66−72.

78 AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 ПРАВИЛА ДЛЯ АВТОРІВ У журналі «Agricultural Science and Practice» публікуються результати фундаментальних і прикладних досліджень з питань грунтознавства, землеробства, рослинництва, ветеринарії, тваринництва, кормовиробництва, генетики, селек- ції та біотехнології, механізації, агроекології, радіології, меліорації, переробки та зберігання сільськогосподарської продукції, економіки, інноваційної діяльності. Друкуються статті, які раніше не видавалися, огляди літератури, короткі повідомлення. Повідомлення публікуються лише англійською мовою; російською та українською – резюме. В електронній версії журналу (http://www.agrisp.com) з 2015 р.розміщуються резюме і список літератури англійською мовою. Комплект документів, необхідних для реєстрації статті 1. На папері подаються (надсилаються): • один примірник рукопису українською або російською мовою (разом з таблицями і рисунками), пронумерований з першої до останньої сторінки і підписаний на останній сторінці тексту всіма авторами; • договір про передачу авторських прав, оформлений окремо кожним із співавторів, наприклад, 4 автори – 4 договори (див. зразок договору). • Звертаємо Вашу увагу на те, що договір про передачу авторських прав набуває чинності після прийняття статті до публікації. У разі відхилення Вашої статті редколегією журналу договір автоматично втрачає силу. Підписання договору автором (авторами) означає, що він (вони) ознайомлені та згодні з умовами договору; • лист − направлення від організації. 2. В електронному вигляді (електронною поштою або на CD/DVD дисках) представляються: • рукопис, ідентичний паперовій версії (прохання називати файл прізвищем першого автора статті англійською мовою, наприклад orlyk.doc); • всі ілюстрації у кольоровому і чорно-білому варіантах в одному зі стандартних графічних форматів − «ppt», «xls» або «psd» (ris1_orlyk.ppt, ris2_orlyk.xls); • інформація про авторів (auth_ orlyk.doc): прізвища, імена, по батькові всіх авторів трьома мовами; при цьому необхідно виокремити одного з них для листування та зазначити його e-mail і номер телефону (з кодом), а також назви і поштові адреси установ, де виконано роботу, англійською, українською і російською мовами. Статті обов’язково супроводжуються українсько(російсько)-англійським словником специфічних термінів (не менше 30), використаних у статті (voc_ orlyk.doc). Оформлення рукопису Матеріали для публікації необхідно подавати у форматі, підтримуваному Microsoft Word, розмір паперу А4, книжкова орієнтація, шрифт Times New Roman – розмір 14, міжрядковий інтервал – 1,5. Повний обсяг (включаючи текст, таблиці, рисунки та підписи до них, резюме трьома мовами, ключові слова і перелік літератури) експериментальної статті не повинен перевищувати 27 000 знаків з пробілами (~ 13 сторінок), оглядової статті – 50 000 знаків (24 сторінки), короткого повідомлення – 12 000 знаків (шість сторінок). Рукопис має містити: індекс УДК; назву статті українською, російською і англійською мовами; прізвища та ініціали усіх авторів трьома мовами; назву і поштову адресу(и) установи(в), де працює(ють) автор(и), трьома мовами; електронну пошту автора для листування. Пропонована структура тексту експериментальної статті: «Вступ», «Матеріали і методи», «Результати і обговорення», «Висновки», «Підтримка». Таблиці повинні мати заголовок і порядковий номер. Примітки до таблиць розміщують безпосередньо під ними. Кількість ілюстрацій не може перевищувати 4 в оглядах, 6 – в експериментальних статтях і 2 – у короткому повідомленні. Всі громіздкі написи на рисунку слід замінити цифровими або літерними позначеннями, а їхнє пояснення перенести в підпис. У пункті «Підтримка» при посиланнях на гранти необхідно вказувати фонд, назву гранту та/або номер. Перелік літератури складається винятково англійською мовою (назви статей з періодичних видань повинні відповідати таким з англомовних резюме, розміщених у зазначених виданнях; заголовки монографій або статей з них також мають бути перекладені англійською мовою, транслітерація допускається лише у разі назв україно(російсько)мовних періодичних видань (Agrarna nauka i osvita), місць видання і видавництв (Kharkiv, NNC «IGA im. О. N. Sokolovsky»))

AGRICULTURAL SCIENCE AND PRACTICE Vol. 2 No. 1 2015 79 у порядку цитування, оформлення джерел слід здійснювати за прийнятим в журналі стандартом (див. приклади). Посилання в переліку нумерують у порядку їхнього цитування в тексті, де їх позначають цифрою у квадратних дужках. Неприпустимо залишати гіперпосилання і посилатися на сайти в інтернеті. Джерела повинні бути загальнодоступними, не можна посилатися на автореферати дисертацій. Приклади оформлення списку літератури: посилання на книгу – 1. Мedvedev VV. Soil heterogeneity and precise agriculture. Pt 2. Results of investigation. Kharkiv, 13 Publishing house. 2009;260 p. на статтю з журналу – 2. Oka Y, Chet I, Spiegel Y. An immunoreactive protein to wheat-germ agglutinin antibody is induced in oat roots following invasion of the cereal cyst nematode Heterodera avenae, and by jasmonate. Mol Plant Microbe Interact. 1997;10(8):961−9. на статтю з книги – 3. Pivovarov S. Physico-chemical modeling of heavy metals (Cd, Zn, Cu) in natural environment. Encyclopedia of Surface and Colloid Science. Boca Raton, CRC Press. 2004;Vol. 6:468–93. Структуровані резюме англійською, російською та українською мовами повинні мати ідентичний зміст (кожне не менше 1500 знаків з пробілами). Орієнтовна структура тексту: Мета. Методи. Результати. Висновки. Ключові сло- ва – не більше 6. Увага! Статті, оформлені не за правилами, повертаються авторам без реєстрації та розгляду. Рецензування статей виконують незалежні експерти, призначені редакційною колегією, після чого автору надсилається примірник рукопису статті із зауваженнями рецензентів. Виправлений авторами варіант статті, погоджений з рецензентами, вважається остаточним і має бути підписаний рецензентами та авторами «До друку», після чого неприпустимими стають заміни тексту, рисунків або таблиць. При публікації статей редакція керується датою надходження останнього варіанту. У разі відхилення рецензентами статті редакція надсилає автору письмове (e-mail) повідомлення.

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