Kazakh National Agrarian University

UDC: 632.4(632.7) as a manuscript

AMANGELDI NURGUL

Survey of root diseases, screening of some wheat genotypes on resistance to (Fusarium Spp. and Heterodera filipjevis Hum.) and the protection measures for them in Kazakhstan

6D081100 –Plant protection and quarantine

Thesis is submitted in fulfillment of the requirements for the degree of Doctor of philosophy (PhD)

Scientific consultants: Candidate of biological science, preofessor Agibaev A.J, Candidate of agricultural science Kochorov A.S., Doctor of agricultural science Abdtelfattah Amer Dababat Turkey, CIMMYT

Republic Kazakhstan Almaty, 2018 CONTENT

NORMATIVE REFERENCES 3 DEFINITIONS 4 SYMBOLS AND ABBREVIATIONS 5 INTRODUCTION 6 1 MAIN PART 9 1.1 Diversity of wheat gene pool………………………………………….. 9 1.2 Study of root rot in Kazakhstan ...... 10 1.3 The status of nematode in Kazakhstan till now ...... 12 1.4 Interactions between Heterodera filipjevis and Fusarium culmorum……… 13 1.5 Spreading of Pratylenchus species ...... 15 2 CHARACTERISTIC OF NATURAL-CLIMATIC CONDITIONS OF 17 THE RESEARCH AREA ZONE…………………………………………. 3 OBJECTS AND METHODS OF RESEARCH ...... 25 3.1 Objects of research ...... 25 3.2 Research Methods for root rot ...... 29 3.3 Research Methods for nematode…………………………………………. 39 4 RESULTS OF THE RESEARCH ...... 50 4.1 Microbiologycal identification of Fusarium solanoi...... 50 4.1.1 Assessment of screening methods to identify resistant to root rot (Fusarium colmorum) in wheat...... 53 4.1.2 Assessment of screening methods to identify resistant to Heterodera filipjevis in wheat...... 60 4.2 Survey and determination of cyst nematodes (Heterodera spp) in some cereal growing regions of Kazakhstan...... 64 4.3 Identification and spreading of plant parasite nematodes in wheat growing areas of west and south – east part of Kazakhstan...... 71 4.4 Survey and identification of cyst nematodes from destination Astana to Kostanay ...... 78 4.5 Effectiveness of biologics application against root rot of grain crops...... 82 5 ECONOMIC EFFECTIVENESS 92 CONCLUSIONS 94 LIST OF USED SOURCES 96 ANNEXES 107

2 NORMATIVE REFERENCES

In this dissertation, references are made to the following standards: The Law of the Republic of Kazakhstan "On Science" of 18.02.2011 № 407-IV LRK. State Educational Establishment of the Republic of Kazakhstan 5.04.034-2011: The state compulsory standard of education of the Republic of Kazakhstan. Post-graduate education. Doctoral studies. Basic provisions (changes as of August 23, 2012 No. 1080). The rules for awarding academic degrees from March 31, 2011 № 127. GOST 2.105-95 Single system of construction documentation. Common use for text documents. GOST 2.11-68 Single system of construction documentation. Monitoring time. GOST 6.38-90 Unified docummentation systems. System of organization- documulation. Tractions to the documents. GOST 7.32-2001. Otchet o nauchno-issledovatelskoy rabote. Structure and rules of the mission. GOST 7.1-2003. Bibliographic record. Bibliographic description. Terms of use and rules of conduct. GOST 21507-81. It protects the growth.

3 DIVISIONS

The following terms are used in this thesis with the relevant definitions: Variety – a group of cultivated plants obtained as a result of selection within the framework of the lowest known botanical taxa and possessing a certain set of characteristics (useful or decorative) that distinguish this group of plants from other plants of the same species. Resistance to disease – This is when plants of some sort (sometimes of a species) are not affected by the disease, or are less affected than other varieties (or species). Phytopathological evaluation – revealing the infestation of plants by phytopathogens, suggests timely combating of harmful diseases. Yield - the amount of crop production, obtained from a unit area. Yields are calculated in centners or tons per 1 hectare. Attribute – any feature of the structure, any property of the organism. The development of a sign depends both on the presence of other genes and on environmental conditions, the formation of signs occurs in the course of individual development of individuals. Gene – functionally indivisible unit of genetic material, the site of the DNA molecule, which determines the possibility of developing a separate elementary feature. Diagnosis - the doctrine of methods for the recognition of plant diseases. Pathogenicity - the ability of a parasite to cause a host disease, to harm it. Isolate - pure culture of the microorganism, allocated to a nutrient medium. Isolation - isolation of a pure microorganism culture on a nutrient medium. Sequencing - Determination of the sequence of nucleotides in the DNA molecule. Ekstrasol - a microbiological preparation used in agriculture, possesses a growth- stimulating and protective effect. Extrasol is one of the recent developments of the All- Russian Scientific Research Institute of Agricultural Microbiology and received state registration as a microbiological fertilizer in 1999. The extrasolver successfully combines the best qualities of biological and chemical preparations. The basis of the preparation is a strain of rhizosphere bacteria Bacillus subtilis Ch-13, isolated from the rhizosphere of healthy plants. Vial - two-component systemic fungicide for preseeding processing of seeds of cereals and sunflower from a complex of diseases. Nematode - microscopic worms belonging to the class Nematoda type Roundworms. The body of phytonematodes is threadlike or spindle-shaped, less often of another form, 0.5-3.5 mm long, covered with a coat (cuticle).

4 SYMBOLS AND ABBREVIATIONS

KSRI PPQ - Kazakh Scientific Research Institute of Plant Protection and Quarantine. FAO - The Food and Agriculture Organization of the United Nations ha - hectare  С - degree centigrade m - metr gr - gram psc. - piece AES - Agricultural experimental station CIMMYT - International Center of Wheat and Corn Improvement LP - Limited partnership R - resistant S - susceptible MR - moderately resistant MS - moderately susceptible CCN - cereal cyst nematode PPN - plant parasite nematode 0.1 (rate of extrasol - 10 times concentrated strains of bacillus subtilis consumption) (0.1 * 10 = 1)

5 INTRODUCTION

Relevance of the research topic “President of the Republic of Kazakhstan N. Nazarbayev”, the people of Kazakhstan, "New Development Opportunities in the Conditions of the Fourth Industrial Revolution", the agro – industrial complex faces the task of increasing labor productivity and exporting processed agricultural products 2.5 times. January 10, 2018 [1]. The Republic of Kazakhstan is among the ten largest grain exporters. Annually, up to 6 million tons of wheat is exported to Central Asia countries and up to 2 million tons to European Union countries. It is common knowledge that the increase in crop yields in developed countries is more than 40% dependent on the introduction of new varieties and hybrids. In developing countries, the renewal of varieties is slow and therefore they have a low yield of cultivated plants. In developed grain countries (Canada, Australia, etc.), there have national wheat selection programs, which analysis plant of resistance genetics, population structure of pathogens, identification of effective genes and donors. In Kazakhstan, the most dangerous disease of cereal crops is root rot. Therefore, resistance of wheat varieties to this disease is very important for protection. Due to the fact that the structure of populations of pathogens in root rot is constantly changing, it is important to replenish wheat genetic resources which is the most dangerous diseases in our country, new donors from international nurseries that have a resistance group. Recently, parasitic nematodes significantly affect the productivity of wheat. As a result of the research work of scientific organization (CIMMYT-Turkey), various varieties of spring wheat (17 CBHL) were obtained by crossing different genotypes. In Kazakhstan, it is necessary to continue scientific research on the study of genotypes of spring wheat which is resistant to soil diseases, and to select the best samples. All the predetermined urgency of chosen topic also the goals and tasks of this dissertational work Purpose of the research Screening some genotypes of wheat for resistant to root rot (Fusarium spp. and Heterodera filipjevis Hum.), Determination of phytoparasitic nematodes in various wheat cultivation zones and effectiveness of biologics application against root rot of grain crops. Research objectives To achieve the goal, the following tasks were set: - sequencing of the pathogen Fusarium spp, root rot of wheat; -identification of effective resistant donors for spring wheat samples to root rot;

6 -selection of spring wheat samples resistant to root rot, adapted to different soil and climatic zones of Kazakhstan and transferring them to breeders for reproduction and obtaining new resistant varieties and hybrids; -phytosanitary monitoring to determine the presence of cyst and parasitic wheat nematodes in Kazakhstan; - determination of the effectiveness of biologics application against root rot of grain crops. Objects of research Spring wheat, winter wheat, cyst and parasitic nematodes, root rot, seed treatment, extrasol, vial-TT. Subject of study Biopreparation "Extrasol", structure productivity whaet. Scientific novelty - phytosanitary monitoring for the presence of cyst (Heterodera spp.) and parasitic nematodes (Aphelenchus spp., Aphelenchoides spp., Tylenchus spp., Filenchus spp., Pratylenchus spp., Parapratylenchus spp., Ditylenchus spp.) was conducted for the first time in the conditional sowing regions of Kazakhstan; - proposed an effective method of using strains of Bacillus subtilis, which is increases the yield of cereals; - carried out the screening of resistant to root diseases (Fusarium spp. and Heterodera filipjevis Hum) for some wheat genotypes from the Siberian-Kazakhstan line. The main provisions to be defended the following questions are raised for defense - screening materials of some wheat genotypes on resistant to root rot (Fusarium spp. and Heterodera filipjevis Hum.); - results of studies of 17 CBHL hybrids containing the dominant donors which is resistant to root rot; - monitoring results in some grain growing areas of Kazakhstan for the presence of cyst and parasitic nematodes of wheat; - results of molecular level of the pathogen Fusarium solani; - information of the effectiveness of biologics application against root rot of grain crops. The author's personal contribution is to determine the purpose, objectives and conduct of scientific research. Field researches were carried out on stationary areas of wheat fields in various regions of Kazakhstan, surveys were conducted to identify cyst and parasitic nematodes. Some experiments were conducted in Turkey base on the International Centre for the Improvement of Wheat and Corn (CIMMYT) and Netherlands at the company «Base clear». Practical significance of the results results of the experimental data possible to use in the selection of 14 samples of spring wheat from the Siberian-Kazakhstan lines (EKADA148, LUTESCENS916, 7

GRECUM1003, LUTESCENS89-06, TULAIKOVSK 100, CIMMYT, SY TYRA, SY ROWYN, SY INGMAR, SELECT, MUCHMORE, URALOSYBIRSKAYA, LYUTESTSENS 12, LYUTESTSENS 6-04-4, containing resistant donors to root rot and recommendations for the use of seed treatment. Research works have been tested and introduced in the wheat fields of Bayserke-Agro LLP, Talgar District, Almaty region. Collaboration of the dissertation topic with the state programs Dissertational work was carried out in 2014-2017. within Kazakh National Agrarian University under the program of granting PhD doctoral students on the specialty 6D081100 - Plant Protection and Quarantine, and scientific research within the framework of the Scientific and Technical Program on the PCF of the MES RK "Innovative Scientific and Technical Support of Phytosanitary Security in the Republic of Kazakhstan" for 2015-2017., where the main performer is the Kazakh Scientific Research Institute of Plant Protection and Quarantine. named after Zh. Zhiembaeva. Approbation of the research work The main provisions, conclusions and results of the thesis are reported at international scientific and practical conferences: 1. "Environmental Topical Problems of the 21st Century" (Kazakh-Turkish International University named after K. Yassaui, Turkestan, 2015); 2. "Agriculture and selection of agricultural plants at the present stage", dedicated to the 60th anniversary of the NPC of the grain farm named after. A.I. Baraev (Astana- Shortlandy, 2016) 3. "Agrarian science for agriculture" (Altai State University, Barnaul, Russian Federation, 2017); 4. "Sixth International Symposium of Cereal Nematodes" (Agadir, Morocco, 2017); 5. "Modern directions of development of education and science in the field of chemistry, biology, ecology and geography" (Kazakh State Women's Pedagogical University, Almaty, 2017). Publications of works by the topic of the thesis 9 scientific articles were published: 3 - in scientific publications recommended by the Committee to Control the Education and Science of the Ministry of Education and Science of the Republic of Kazakhstan, 5 - in the materials of the International Scientific and Practical Conferences, 1 - in the journal included the database of Scopus. Structure and scope of the dissertation The dissertation work consists of an introduction, five chapters, a conclusion and a list of sources used from 149 titles and annexes, sentences which contains 106 pages of computer text, illustrated with 31 figures and 23 tables.

8 1 MAIN PART

1.1 Diversity of wheat gene pool According to the experts of the international food organization, the general indicator of the national security of any country is the production of wheat grain. Sown area in the world in 2010-2011 amounted to 218 million hectares, and gross harvest - 653 million tons [2]. Currently, products from wheat (Triticum aestivum), rice (Oryza sativa), and maize (Zea mays) provide about two thirds of the world's main food. 35% of them are wheat. Wheat provides 21% of calories and 20% of protein for approximately 4.5 billion people in 94 developing countries from Norway, Russia to Argentina [3]. According to FAO [4], world wheat losses due to pest amount to 34%, in addition to 12% due to diseases. In Kazakhstan, winter and spring wheat is affected by several types of rust (brown, yellow,stem).The spread and development of wheat diseases varies considerably depending on the weather conditions, cultivation zones and varietal characteristics of the culture [5]. The production of grain was and remains an important strategic resource of Kazakhstan, the basic branch of agricultural production. The republic produces grain not only to meet domestic needs of the country, but also for exporting to foreign countries. Winter wheat is mainly cultivated in the southern and south-eastern regions of the republic on an area of 1.5-2 million hectares, including 140.5-170.4 thousand hectares in irrigated lands. At the end of the 1980s wheat was cultivated on 35-36% of the area according to intensive technology, its average yield was 11.2-11.5 c / ha, and on irrigated land it reached 32.5-35 c / ha [6]. Mineral fertilizers were used up to 1189-1212 thousand tons. In food grains, Kazakhstan covered the need not only for itself, but also the countries of Central Asia. Strong and hard wheat, grown in Kazakhstan, is exported for processing into pasta, as well as to produce bread in the Baltic and other countries. Kazakhstan, as one of the world's major grain producers, with a yield level of 15-20 c / ha, annually loses from 3.5 to 9.5 c / ha of wheat [3, 7]. One of the main causes of such damage are mass outbreaks of fungal diseases, among which the most common and harmful are brown leaf rust (causative agent Puccinia triticina Erikss., Puccinia recóndita Rob.) And yellow rust (causative agent Puccinia striiformis West.). The most important element of the integrated protection of wheat from rust diseases are resistant varieties [3]. To successfully manage the host-pathogen pathosystem, it is necessary to maintain diversity in agroecosystems on the basis of resistance, both in time and in space, taking into account the intrapopulation structures of the pathogen [8]. Soft wheat (Triticum aestivum L.) is one of the main food crops in the world. The genetic potential of its productivity is now almost exhausted, and yields largely depend on the resistance of cultivated varieties to unfavorable abiotic and biotic factors of the environment. At the same time, production of ecologically clean grain is becoming more urgent. Great damage to productivity and quality of grain is caused by wheat diseases.

9 Chemical protection against diseases is an expensive and not always justified measure. In addition, their use causes significant harm to the environment [9]. Therefore, in the solution of the problem of growth in the production of ecologically pure grain, a special attention is given to the selection of wheat resistant to harmful diseases. The genetic diversity of the softest wheat is not enough to solve this problem. A significant reserve of effective resistance genes is concentrated in the gene pool of numerous wild-type relatives of soft wheat [9].

1.2 Study of root rot in Kazakhstan Diseases of cereal crops with soil infection include root rot and helminthosporium leaf spots. On winter and spring wheat, there are helminthosporium, fusarial, ophoid, cercosporous and pyotic root rot. On virgin and fallow lands of Northern Kazakhstan and Western Siberia, helminthosporium and helminthosporium-fusarial root decay are the most common and harmful diseases of spring wheat and barley that occur annually. The main causative agent of the disease is the imperfect fungus Bipolaris sorokiniana Shoem. (synonym for Helminthosporium sativum P. R. and Bakke) fungi of the genus Fusarium [10-13]. The disease manifests itself in the form of darkening and rotting rootlets, underground interstice stems and lower leaves. Zh. T. Djimebaev [14] points out that the fungus often affects the base of the stem and rarely the root system. Spores of the fungus that remain on plant residues are able to germinate only in the upper layers of the soil and can be introduced into young plant tissues into the base of the stem during full sprouting and before the formation of the secondary root system. In addition, the disease adversely affects the physiological and biochemical processes occurring in plants during their growing season and the quality of the grain. On barley, root rot was studied in the South-East [15, 16], Northern [11] and North- West Kazakhstan [17], and in Western Siberia [18-19]. Natural cereal deposits are free from the causative agent of helminthosporium root rot [18, 82 p]. According to VA. Chulkina [19, 42 p], on the arable land of the forest steppe of Transuralia and Western Siberia, the contamination of the soil with fungus is 4-17 more than in virgin and fallow their analogs. The role of plant residues of individual crops on which the infection is reserved in the field is different. E. Chumakov [20] M.V. Gorlenko [21] point out that N. sativum is better preserved on fresh, slightly decomposed remains of plants of spring wheat and barley and in soil, sometimes transmitted by seeds. J.T. Djiembaev and Zh.Sh. Alzhanov [22, 23] believe that the main sources of root rot infection in Northern Kazakhstan are post-harvest residues of wheat and wild cereals, in particular the cedar grass. Many scientists of Russia, Ukraine, Byelorussia and other countries point to the phytosanitary significance of agrotechnical methods [24-25]. In Western Siberia, long- term studies were conducted by V.A. Chulkina and her school [26. 27-28] to develop an integrated system for the protection of cereals from helminthosporous root decay. They

10 believe that in order to increase the suppressiveness and self regulation of the phytosanitary state of wheat and barley crops, it is necessary to: optimize the seeding density, seed embankment depth, transition to soil, protection treatment of the soil with a system of moisture accumulation measures, seeding, snow retention, the creation of a mulching layer, and the application of organic and mineral fertilizers. The Austrian Scientist V. Zwats [29] points out that the basis for an integrated program for the protection of crops from root rot is agrotechnical measures, in particular, the timing of sowing, the rate of sowing seeds, and the introduction of mineral and organic fertilizers into the soil. A.V. Malikova [30,31], in the forest steppe zone of the Krasnoyarsk Territory, isolated H. sativum fungus from wheat, millet and oats. In the mycological analysis of wild cereals, the fungus infection contained wheatgrass, a hedgehog, and a ryegrass pasture. L.K. Khatskevich and A.A. Benken [32] found in laboratory conditions that vetch, pea, sunflower, and rape are less favorable substrates for the caoprophytic development of the pathogen in comparison with spring wheat and barley. Helminthosporium root rot significantly reduces the productivity of plants. Polyakov [33] notes that it has become the main disease of spring wheat areas of virgin and fallow lands and reduces its yield to 8-20%. G.A. Kraev [34] points out that in the 1950s the losses from this disease exceeded the reduction in the grain yield from all other diseases combined. According to V.P. Shevchenko [35], the epiphytes of root rot in Western Siberia were observed in 1947, 1955, 1965. The size of losses of wheat grain yield from diseases in the average was 14-15, reaching 45-50%, the quality of grain and seeds deteriorated. According to L.M. Gorodilova [36], in the Akmola region, in the first years of plowing virgin lands, root rot on spring wheat developed insignificantly. Gradually the accumulation of infection in the soil was progressing and the development of the disease was intensified. Even with its weak manifestation, the crop decreased by 11.6%, and with a strong development to 42.5%. Affected plants lag behind in development, there is a white collar. In some varieties, a dump and a stalk fracture are observed at the base of hard wheat [36]. The harmfulness of root rot depends on factors such as precursors, the nature of the damage to plants, the timing of seeding and the conditions of the growing season [11-37, 38-17, 39]. In the 70 years in Kostanay region, V.V. Kotova [38] found that a significant decline in the crop begins with the intensity of the disease in the range of 10-15%. The threshold of disease severity varies depending on the agronomist. It is higher when sowing wheat after steam, corn and below when it is grown monoculture for 2-3 years. The harmfulness of root rot of spring wheat increased sharply when plants were damaged by latent stem pests. With a weak manifestation of root rot, the yield of spring wheat decreased by 11.6%, with a strong yield of 42.5%, and when the plants were

11 damaged by hidden pests, losses reached 65.7% [37]. The disease is most damaging to barley. So, with a strong manifestation of the disease, the number of grains in the ear decreased by 25.3-39.2%, the mass of the grain decreased by 10.6-18.6%. S.F. Bedina [40-41], in the conditions of the meadow steppe zone of the East Kazakhstan region, found that the harmfulness of root rot on spring wheat depends on the type of manifestation of the disease. With the defeat of the underground internodes on the spring wheat cultivar, the Saratov 29 coefficient of severity of the disease was 12.5%, stem base was 15.9%, and both organs was 23%. On grade Kharkivska 46, root rots and a node of tillering with a sickness coefficient of 61-69% were noted. Experiments by V.V. Kotova [38] showed that when sowing seeds obtained from plants affected by root rot, the yield of wheat is reduced by 10-25% or more. A research by R.I. Shchekochikhina [42,43], conducted in Kostanay, Kurgan and Saratov regions, revealed that the condition of spring wheat influences the severity of the disease. In stunted plants, when they were affected by 1 point, the mass of grain from the ear decreased by 24%, and when affected by 2 and 3 points, it decreased by 36- 46%. At the same time, in highly medium sized plants, these indicators decreased significantly. At the Carabinka HoxXOC, in normal humidification mode (GTK 1.18), the loss of yield from root rot in the Bezynchuk 98 variety was 19%, and in the arid (GTK 0.65) was 44.2%. In the conditions of the Toraya Oblast in 1981 and 1985, which were more favorable for the growth of wheat, the disease severity was 1.5-2 times lower in comparison with the period from I982-1984, characterized by acute aridity [11,44]. In the years L.A. Ponomareva and M. Koishibaev [17, 44, 45, 46] determined the harmfulness of root rot on barley in the conditions of the Kostanay region. The loss of this crop from the disease, depending on the conditions of the growing season, predecessors and sowing time, varied from 5.4 to 25.2%. In 2012, in the East Kazakhstan region in the Glubokovsky district at the stations of the East Kazakhstan Institute of High Rust, brown rust appeared in the phase of the beginning of the ear of winter wheat. During the period of milk ripeness, the development of brown rust on winter wheat did not exceed 9.5%, and on spring wheat rust reached 47.8-74.9%. Similar data were obtained for Shimonaykhinsky and Zyryanovsk regions - 38.4-43.9%, and in the arid Ulan region it developed at a weak level - 15.0-19.1% [47-50]

1.3 The status of nematode in Kazakhstan till now Kazakhstan, as the second in terms of area and third in terms of agricultural importance in the Soviet Union republic, is of great interest as a base for nematological research due to the unique combination of diverse soil - climatic and agroecological conditions. On the one hand, Southern Kazakhstan (primarily Semirechie), as one of the ancient agriculture areas, is part of the south - western Asian genocenter of agricultural crops [51]. It is very likely that this region coincides with the genocenter of some

12 nematodes - parasites of cultures (primarily vegetable ones). At the same time, some crops (for example, sugar beet) were introduced to Kazakhstan in the last 40 to 50 years, which suggests that the recent introduction of sugar beet heteroders, now registered in 5 regions of the republic [52]. The development of cereals for virgin lands in Central and Northern Kazakhstan was accompanied by the spread and growth of the harmfulness of the bicarious cereals. The giant sizes of the fields in these regions have no precedent in world practice and are therefore of particular interest to nematologists. On the other hand, finding on grains of the British Gallic nematode is unlikely to be associated with the introduction of this parasite from Western Europe, where it was previously known [96] and also requires a special study. In a sheltered state farm of the Alma-Ata and Pavlodar oblasts, 50 -70% of the vegetable production is often destroyed by root-knot nematodes. Significant losses in the production of iluka potato in the southern and eastern zones of Kazakhstan are caused by dietary diseases [53] Over 80 years, since the first reports of the discovery of parasitic nematodes in Kazakhstan [54], the common nematode fauna is of the most important cultural plants in the republic [55], and the most important species parasitic nematodes were separated to fight them [56]. Together with the topic, the results of these studies were not systematically on a modern basis, and could not answer the questions raised by practice. It was necessary to generalize and supplement the existing information, critically analyse the proposed methods of mathematical calculations and modeling of the dependence of losses of plant products on the density of populations of parasitic nematodes, prediction of the harmfulness of the latter, and develop principles for managing phytosanitary conditions based on the integration of antinematode measures [57]. For the first time in our republic professor A.O Sagitov revealed a new pest of cereal crops - the British root nematode (Meloidogyne britannica) on wheat, barley and oats in the East Kazakhstan and Pavlodar regions [58,59].

1.4 Interactions between Heterodera filipjevi and Fusarium culmorum Soil-borne pathogens represent an important, but often over looked, biotic constraint to cereal production worldwide. Inparticular, over the past ten decades, the changing spectrumof soil borne pathogens in wheat has created challenges forcrop rotation practices that were previously able to provideadequate disease control. Heterodera filipjevi, a species of cyst nematode that is parasitic to cereals, is now widely recognized as a major nematode pest of wheat in cereal production areas especially intemperate climates and semiarid areas in Europe and WestAsia [60, 61]. This species is also recorded in East Asia and Pacific Northwest USA [62,60,63]. In addition, it has been found in association withwild host grasses such as Avena ludoviciana, A. fatua,Bromus tectorum, Hordeum distichum and H. spontaneum [64]. H. filipjevi is a sedentary endoparasite that feeds on root vascular tissues and causes severedamage on crops. The nematode has only one cycle pergrowing season and

13 completes its life cycle within 155 daysin winter wheat [65]. For exemple, in Iran, H. filipjeviis the prevailing species of the cereal cyst nematode, whereit is widespread in wheat growing areas in eighteen provinces [66,64]. At thehighest initial population density (20 eggs and juveniles perg soil) in field microplots it caused 48% yield loss in winterwheat [67]. Yield losses caused by soil borne fungal pathogens havebeen reported as a serious problem for small grain growersthroughout the world. Pathogens such as Fusarium culmorum, F. pseudograminearum, F. avenaceum, Bipolaris sorokiniana, Gaeumannomyces graminis and Rhizoctonia solaniare responsible for reducing wheat yields [68,69,70]. The crown rot fungus F. culmorum limits wheat yields intemperate and semitropical areas of the world [71,72]. It infects roots andsub crown internodes causing dry rot of crown and roots, browning of the base of the plant. When infected plantsare subjected to water stress, severe damage appears as whiteheads shortly before crop maturity[69,70]. Fusarium culmorum and F. Pseudograminearum are generally considered to be the most economically important species of crown rot fungi on wheat. Yield losses from each species have been reported for wheat inmany countries and reach up to 30% in the USA [73,70]and 43% in Turkey. However, the damage depends on the virulence ofthe fungal population, environmental conditions and fieldmanagement practices [70]. Common root rot caused by B. sorokiniana also causesyield reductions in wheat [74,75]. Infected seedlings develop dark brown necroticlesions on the crown, sub-crown internode and lower leafsheath. Under suitable environmental conditions, spikeletsmay be affected, causing grain shrivelling [75]. Losses of wheat yield due to common root rot wereestimated as high as 7% in Canada [76], 24% in Australia [77] and 20% in India [78]. Fungal pathogens are known to interact with plantparasitic nematodes causing increased disease severity [79,80]. Riedel (1988) mentioned that plant- parasiticnematodes may also predispose plants or modify plantexpression of resistance to plant pathogens by altering hostphysiology. Interactions of cyst nematodes and fungalpathogens have not been investigated as extensively as forthe root knot nematodes and fungal pathogens. However,there are some reports available for interactions of H. glycines and Macrophomina phaseolina on soybean [81], H. glycines and F. solani on soybean [82], H. cajani and F. udum on pigeonpea [83], Globodera pallida and Verticillium dahliae on potato [84], G. rostochiensis, G. pallida and R. solani on potato [85,86], and H. schachtii and R. solani on sugar beet [87]. Yield losses for disease complexes involvingH. avenae and several soil borne plant pathogenic fungihave also been described [88]. Meagher etal. (1978) found both H. avenae and R. solani on wheat rootsin South Australia and assumed a negative interactionbetween those two pathogens. In semiarid areas of Syria,possible interactive effects between H. latipons and B. sorokiniana on barley yield were postulated by Scholz (2001) and Abidou et al. (2005).

14 Since both root rot fungi and cereal cyst nematodes are found together especially under cereal monoculturing [89,90], an interaction that increases root damage is possible, and a synergistic interaction might cause a higher yield loss than when the fungus or nematode are found individually. Such information was lacking for H. avenae and the root rot fungus F. culmorum on wheat. The interactions between Heterodera avenae and Fusarium culmorum on growth and yield components of durum wheat cv. Sham 3, H. avenae reproduction and crown rot severity were studied in a plastic house experiment. Grain yield reduction caused by the treatment H. avenae alone and F. culmorum alone was 12.3 and 25.5% respectively. The simultaneous inoculation of H. avenae and F. culmorum resulted in 38.4% reduction, indicating an additive effect of yield losses due to the two pathogens. A synergistic interaction was found when the nematode was added one month before the fungus, where the grain yield reduction (43.8%) exceeded the sum of individual loss caused by the nematode and fungus alone. The interaction was antagonistic when the fungus was added one month before the nematode, which caused a reduction in grain yield (33.3%) less than the sum of individual loss caused by the nematode and fungus alone. The interaction of nematode and fungus resulted in decreasing final population densities for the nematode especially when the fungus was added one month before the nematode. The highest fungus infection severity (2.8) and disease index (91.7%) was observed when the nematode was added one month before the fungus [91].

1.5 Spreading of Pratylenchus species Individuals of Pratylenchus species, commonly known as the root lesion nematodes, are obligate migratory endoparasites with a wide variety of host plants. These nematodes parasitize roots and bulbs of host plants roots intracellularly and cause necrotic areas. The aboveground symptoms are difficult to identify but generally are represented by stunted growth, poor vigor and chlorotic plants, and an infected field shows irregular growth and infection occurs in patches [92]. Up to date a total of 89 species are valid species of Pratylenchus worldwide [93,94]. Several species of RLN were recorded to be associated with wheat worldwide, however, P. neglectus and P. thornei are the most damaging and widely distributed species worldwide. Other RLN that also affect wheat production are: P. crenatus, P. fallax, P. mediterraneus, P. penetrans and P. zeae [95-104]. The maize root lesion nematode, P. zeae, was recorded to attack wheat in Europe; Northern Africa; Western Asia [105,106]. The RLN, P. fallax was reported to cause damage on cereals in Europe [107]. On the other hand, the RLN P. mediterraneus is recorded from Algeria, Tunisia, Morocco, Palestine, Syria and Turkey [108,109].While the RLN P. penetrans was recorded to be associated with wheat grown in Canada, Egypt and Iran [95]. Both P. thornei and P. neglectus were reported to be the most common species of Pratylenchus on cereals in several countries [110,111]. Ahmadi et al. (2015) reported that RLN were recovered from 35% of the 200 wheat fields sampled in Khuzestan

15 Province in south west of Iran. Imren et al. (2015) in the other hand reported that RLN were detected in 73% of the total samples of the collected 75 soil samples from cereal fields in Bolu province in north western Turkey. It was recorded that P. thornei is one of the most important RLN that limits the productivity of wheat in the Pacific Northwest in the United States of America [113, 42 p]. Strausbaugh et al. (2004) reported that both RLN species, P. thornei and P. neglectus, reduced the profitability of wheat fields in Idaho, Oregon and Washington by about $51 million annually [114]. Those two RLN species can reduce the wheat production in Pacific Northwest in the United States of America by 60% of dry land wheat fields [115]. Both P. thornei and P. neglectus were reported to be present in high population in wheat fields [112, 24 p]. While Ahmadi et al. (2015) reported that the number of RLN in wheat root samples taken from wheat fields ranged from 1 to 365 nematodes/g root. Both RLN species were also able to reduce the yield of wheat up to 60% without causing any obvious symptoms on the wheat plants whether foliage or roots (Bridge et al., 2005). Several reports showed that these two species are adapted to dry environment. In these dry conditions these two RLN affect the wheat root ability to extract water from the soil [113]. Wheat yield is negatively correlated with preplant population inoculums level [112, 100 p]. Accurate understanding and identification of the RLN is of a great importance for further epidemiological studies and management issues. The identification of RLN based on morphological characteristics is usually performed using original descriptions of the species as well as diagnostic keys [116-119]. However, it is very difficult to differentiate between too closely related species and require expert taxonomists. For this reason, nematologists use molecular tools to confirm the species identity. Such tools include the amplification of regions in the rDNA using nematode universal primers and species specific primers [120,121, 53 p]. The distribution of RLN is considered an essential step for management of RLN. Such management is the use of resistant cultivars. Several reports indicated that the need for reliable information on the susceptibility and resistance against RLN is an essential issue to assist the growers in making a proper decision on varietal selection and in planning their cropping rotations [122].

16 2 CHARACTERISTICS OF THE NATURAL CLIMATE CONDITIONS OF THE RESEARCH AREA ZONE

The soil distribution and climatic parameters of the experimental region are subordinate to vertical law and are located in the northern foothills of Trans-Ili Alatau. The climate of the region is very continental, with a daily and annual fluctuation of air temperature. In addition, the winter is cold, summer is hot and long, early and late in the spring frosts and atmospheric droughts, the evaporation process is intense, with an atmospheric precipitation deficiency. The annual air temperature is 9,10С. The maximum rainfall at temperatures above 100С will be 250-350 mm. The evaporation rate in these stages is 1450-1700 mm. The peculiarities of natural and climatic conditions vary considerably during each season of the year. For example: the winter is quite warm in the area surveyed. The daily average fluctuation of air temperature passes from the optimum to the cold period from the first decade of November to the second decade of December. The continental snow cover will begin in early December. Snow cover is not uniform, sometimes melts quickly in the winter. Snow cover thickness varies from 20 to 25 cm. The absolute temperature in winter is -30-350С. In winter, there is frequent weather warming. The amount of rainfall in winter is twice as low as in warmer seasons. Spring begins in the region. The spring will last 30-50 days. The air temperature rises rapidly and the daily temperature fluctuations are high, the line is intense and, due to this, the dry surface of the soil is rapidly dried. In addition, the spring season is unstable. The reason is that in some cases, the temperature drops rapidly, and snowfall frequently closes. The cool days in March fluctuate between 10 and 30 days, in April to 3-7 days. According to the long-term research, spring frosts will be completely completed by the end of April. The total number of frosty days is 165-167 days. And the lengthy days of long-term indices range from 151-181 days. One of the most wet periods of the region is the first table and the first figure. In May and April, a third of the annual precipitation will fall. The number of rainy days in these months is 10-15 days. The highest temperature in April reaches 28-300С. And in May it will be between 33-340С. However, due to frequent and frequent precipitation, it is not noticeable. Summer It is the longest, hotest and most dry season in the year. The duration is 125-185 days. The beginning of the hot season begins in April, and in the vicinity of the mountains in May. At the end of September and the beginning of October the air temperature will drop to 13-150С. The average air temperature varies from summer to summer, ie 20-250 C in June, 25-300 C in July, and 27-240 C in August. The amount of precipitation in summer is 2-3 times lower than in spring. Due to the high heat consumption in the summer, it is perfectly suited for the cultivation of greenhouses in the region. And in the hot season the air humidity will drop to 3035%. Daily amplitude of day and night temperatures on the average 19-200 ° C. The range of optimum temperatures in summer varies between 3500-37000 C.

17 Autumn. The duration of the year is 2-2.5 months. The cold days begin in October. Daily fluctuation fluctuates in September at 21-250С, in October - 17-230С, in November - 12-150С. The frosty days in autumn will take place in late September and late October. The amplitude of the daytime and night temperatures varies between 24- 300С. One of the climatic factors necessary for the growth and development of agricultural crops is the distribution of rainfall and their seasonal distribution. One of the main peculiarities of the study area is that the amount of precipitation decreases in spring and declines in summer. The 3-year climatic data are shown in Table 1. According to the data from February to July 2014, the rainfall was 341.9 mm, and in 2016 it was 298.3 mm, which is 43.6 mm lower than in 2014. In 2016, the amount of precipitation in the growing season is 250.8 mm, which is 43.9 mm (206.9 mm) more than the annual amount. According to the weather data, in 2016, the high volumes of precipitation are high. Most of the mountaineers also came from the winter and early spring.

18 Table 1 - Monthly averages of precipitation and air temperature in 2015-2017

Indicators І ІІ ІІІ IV V VI VII VIII IX X XI XII yearly

Precipitation settling, mm 2015 29,8 26,4 93,8 113,2 49,6 91,3 6,1 54,8 27,9 115,1 59,2 32,0 669,6 2016 55 14,1 66,2 152,1 230,9 132,6 108,2 6,4 24,0 56,5 97,8 30,9 947,9 2017 48,3 42,6 46,2 221,3 115,7 54,6 54,5 15,2 0 90,7 76,2 82,0 847,3 The average long-term 44,3 27,7 68,7 162,2 93,8 92,8 56,2 25,3 17,3 87,4 77,7 48,3 801,7 Average air temperature , 0С 2015 -3,0 -2,0 4,0 14,1 19,8 23,1 24,2 24,6 16,3 9,3 1,3 -3,1 10,7 2016 -2,0 -1,7 9,2 13,6 17,0 23,1 24,8 23,8 22,1 12,6 3,8 0,2 12,2 2017 -8,0 -3,0 3,0 12,7 14,2 22,0 22,1 23,6 20,5 4,1 0 -1,6 9,1 The average long-term -4,3 -2,2 5,4 13,4 17,0 22,7 23,7 24,0 19,6 8,6 1,7 -1,5 10,6 air temperature, 0С Humidity of air , % 2015 41 42 50 66 49 39 57 52 49 60 57 68 52,5 2016 68 57 58 69 79 69 66 40 50 58 56 69 61,5 2017 54 50 56 65 58 58 57 51 52 62 60 70 57,7 The average long-term 54,3 49,6 54,6 66,6 62,0 55,3 60,0 47,6 50,3 60,0 57,6 69,0 57,2 of air humidity, %

19

The climate of the Ural region is marked by a sharp continentality, which increases from the north-west to the south-east. It manifests itself in sharp temperature contrasts of day and night, winter and summer, in a rapid transition from winter to summer. The entire region is characterized by instability and deficiency of precipitation, low snow and heavy deflation of snow from fields, great dryness of the air and soil, the intensity of evaporation processes and an abundance of direct sunlight throughout the growing season. Winter is cold, mostly overcast, but not long, and the summer is hot and quite long. The first zone is the most moisture-rich area of the region. But even here, the humidification conditions are very severe and in most years moisture is not enough. The annual precipitation is 280-320 mm, and during the warm period 125-135 mm falls. Steady snow cover is usually remains 120-130 days, its height reaches 25-30 cm, water reserves in snow are 75-95 mm. The hydrothermal coefficient (GTK) during the vegetation period of grain crops is characterized by a value of 0.5-0.6, the sum of positive average daily air temperatures above 10 ° C is about 2800 ° C. The period of active plant vegetation is 150-155, frost-free 130-135 days. The productive reserves of moisture in the soil at the beginning of spring field work are rather limited: in the arable layer - they are in the average for Uralsk 34 mm, for Chingirlau - 31 mm; in the meter layer of the soil, the total moisture reserves in the Uralsk area are 174 mm, and in Chinhgirlau are 136 mm. Heat is sufficient here for the ripening of early grain crops, millet, potatoes, early ripening, mid-ripening varieties and maize hybrids and most vegetable crops. The second zone is more arid than the first (SCC = 0.5-0.3). The sum of positive air temperatures above 10 ° C varies between 2800-3000 ° C; duration of the period with a temperature above 10 ° C is 155-160 days. During this period, 100-130 mm of precipitation falls, 240-260 mm falls for a year. The frost-free period is 145-155 days. Winter with a stable mainly snow cover lasts about four months. The duration of the period with a stable snow cover is 110-120 days, the average height of the snow cover is 20-25 cm, the water reserves in the snow are 75-90 mm. Reserves of moisture in a meter layer of soil by the beginning of spring are no more than 80 mm. Thermal resources of the region ensure the maturation of most agricultural crops. The third zone is a zone of sharply arid, hot desert steppes, semi-deserts and deserts. The SCC varies between 0.3 and 0.2. The sum of positive air temperatures above 10 °C is 3000-3400 °C. During this period 100-120 mm of precipitation falls, for a year 190 to 230 mm falls. The frost-free period lasts 160-180 days. The period with a stable snow cover is 80-105 days, the average heights of the snow cover is 10-15 cm, the water reserves in the snow are 40-50 mm. In the extreme south, the snow cover at a low altitude lies 1.5-2 months. In this zone, summer precipitation is very unstable. The number of them sharply fluctuates over the years, often within two to three months in a row there is no more than 5 mm of precipitation. The abundance of heat and the lack of rainfall limit the possibility of farming.

20 In the first zone of Uralsk region, dark chestnut and chestnut soils predominate. In the extreme north of the region, an insignificant area, about 6.5 thousand hectares, is occupied by southern chernozem. The texture of the soil is mainly heavy loamy, and in terms of the amount of hydrolyzable nitrogen and mobile potassium are classified as medium-income. Dark chestnut soils constitute the main agricultural fund and occupy an area of 2295 thousand hectares. Among them, dark-chestnut carbonate, residual-carbonate and alkaline ones are distinguished. Their mechanical composition is from clay to sandy loams and even sandy ones. Dark chestnut soils have sufficient natural potential fertility for cultivating any crops. The humus content in them varies from 1.7 to 4.7%. The thickness of the humus horizon (A + B1) is 36-53 cm. With high potassium reserves (1- 1.5%) and gross nitrogen (0.1-0.2%), a low content of phosphorus (0.06- 0.15%). A significant area of dark chestnut soils (359,000 ha) in arable land is dangerous for water erosion and requires anti-erosion treatment. In the first zone, and partly in the second zone, there are 655 thousand hectares of soils in arable land, prone to wind erosion. They should be used anti-deflation measures. There are also solonets complexes with a solonetz content of 10 to 50% or more. Dark chestnut soils in combination with solonetzes up to 30% do not require complex measures and can be used in arable land, and complexes containing 50% or more of solonetses need radical melioration. Chestnut soils are characteristic of the second zone, the area of which is 491.6 thousand hectares. They are somewhat inferior in their fertility to dark chestnut. The structure of their profiles is similar to that of dark chestnut trees, and is distinguished from them by a lower thickness of the humus horizon and closer to the surface of calcium carbonate salts. The thickness of the humus horizon (A + B1) is 32-43 cm, the humus content is 2.1-3%. Provision of phosphorus is low, nitrogen is medium, and potassium is high. Chestnut soils are used for arable land without any measures to improve them, but they need replenishment with moisture, which can be achieved by accumulation of snow in the fields. The soil cover of the third zone (182 thousand ha) is light chestnut and brown soils (3.1 thousand hectares). Almost all the light-chestnut soils of this zone have clear signs of solonetsousness, and sometimes solonchakiness, which is due to the close occurrence of water-soluble salts on the surface. Light chestnut soils are characterized by low natural fertility. The humus content varies between 1.3-1.6% with a humus horizon (A + B1) of 35-45 cm. Light chestnut soils and mobile forms of nutrients are poor. The main obstacle to the development of agriculture on light chestnut soils is a lack of moisture. Therefore, they are most often used as pastures. Stable yields on these soils can be obtained only under condition of artificial irrigation. On non-complex

21 massifs, it is possible to irrigate agriculture with growing grain and fodder crops. For these purposes, the lightest in texture of the soil is most favorable. In brown soils, the humus horizon is weak (within 20-40 cm) and with low natural fertility. They contain humus 1-1.5%, and are poor in mobile forms of basic nutrients. Basically brown soils are used as pastures. When irrigation of brown and light chestnut soils, it is necessary to provide drainage, since with the existing salinization of soils and aridity of the climate, processes of secondary salinization of the soil can easily develop here and withdraw them from the agricultural turnover. Solonetzes and solonetzic soils on the territory of the region are widely distributed in the second and third zones. Their total area in the region is 7451.1 thousand hectares, including complexes of zonal soils with solonetzes of 2410.7 thousand hectares (10- 30%), complexes with solonetzes of 1405.2 thousand hectares (30-50% ) and solonetses in a complex with zonal soils of 3635.2 thousand hectares (10-30%). The lands occupied by zonal soils with solonetzes (10-30%), when used in arable land, require selective improvement of solonetz spots by applying manure in doses of at least 30 tons / ha. Complex soils with solonetzes (30-50%) require, during cultivation, complex reclamation with considerable economic costs. Lands containing zonal soils with solonetzes over 50% need radical melioration. Along with the zonal soils in the region, considerable areas are occupied by meadow-chestnut and meadow-brown soils, with a total area of 2625.7 thousand hectares. Their properties are characterized by a high content of humus (2.6-7%). They have a more powerful humus horizon (40-60 cm). Their characteristic feature is that they do not have large integral arrays, but lie in small parts of all possible configurations. This inhibits the productive use of these soils. For the most parts, they are mown pastures and hayfields. The Akmola region is located in the north of the central part of the Republic of Kazakhstan. It borders with Kostanay in the west, with North Kazakhstan in the north, with Pavlodar in the east and with Karaganda region in the south. The region has significant economic potential and natural resources. Prospects for further development are determined by the possibility of positioning the region, not only as an exporter of raw materials, but also as a provider of medium and hightech goods and services. The volume of gross regional product for 2008 was 457.5 billion tenge, with an average annual growth rate over the past three years of more than 39%. Per capita GRP amounted to 614.3 thousand tenge, with growth by 80.7% in comparison with 2006. The terrain of the territory is diverse: the greater part is occupied by steppes, melkosopochniki, lowland, weakly dissected and river valleys, mountains covered with forests.

22 The climate of the region is sharply continental. Summer is short, warm, and winter is long, frosty, with strong winds and snowstorms. The minimum air temperature is above minus 40 ° C, the maximum reaches plus 44 ° C. Vegetation is represented by steppe species of herbage and, correspondingly, to landscapes, especially in the northern part of the region, pine-birch forests, herbage and grass vegetation, which covers the slopes of the mountains. Mountain pine forests are the wealth of the region. The fauna of the region is characterized by considerable wealth and diversity: 55 species of mammals, 180 species of birds, 300 species of waterfowl, etc. On the territory of the region there are State national nature parks "Kokshetau" and "Burabai", Korgalzhyn State Reserve of International Importance. The territory of the region contains explored unique in its composition and scale reserves of gold, silver, uranium, molybdenum, technical diamonds, kaolin and muscovite, as well as iron ore, coal, dolomite, common minerals, mineral waters and therapeutic mud. Soils are represented by ordinary chernozems and chestnut, characterized by heavy mechanical composition, increased solonetsiness and salinity, and low water permeability. Laboratory studies were conducted in Turkey, in the greenhouse CIMMYT (International Center for Wheat and Corn Improvement) located in Eskishehir. The climate of Eskishehir is characterized as moderately continental, semiarid. Summer is hot and long. Winter is relatively cold and snowy. In the summer rains are rare. In winter, precipitation usually falls out in the form of snow. The average duration of the snow cover is 45 days. The average temperature in January is +0.3 C; and 23 C in July. Average annual temperature is 12.1 C. On average, 402 mm of precipitation falls in Eskishehir over a period of approximately 104 rainy days, mostly in the form of snow in winter. In autumn and winter of 2016, the temperature dropped to -30C at night and rose to + 100C during the day. The amount of precipitation that fell in January was 27.0 mm, which is the rainiest month of the year. Thus, in the years of research, the climate is very diverse, which has had a significant impact on the water regime of the soil, the growth and development of summer wheat plants and their productivity. The year 2016 was favorable for plants, yellow and brown rust. In general, in 2016, weather conditions were quite favorable for the formation of the yield of winter wheat. High humidity contributed to the development of spores of yellow and brown rust. It should be noted that the distinctive feature of the current conditions in the years of research is the precipitation and relative humidity of the air during the growing season of the studied crop. In general, the climatic conditions of the study zone are not significantly different from the mean annual indicators. 2015-2017 years were beneficial for the growth and development of summer wheat as the number of fallen offsets, and so on the temperature system. The number of discharges and the temperature of the air contributed to the

23 formation of the winter carnivore. For the characteristics of climatic conditions during the conduct of research, the data of the meteorological station of KazNIIZiR LP were used.

24 3 OBJECTS AND METHODS OF RESEARCH

3.1 Objects of research The main objects of research are: root rot (Fusarium spp), cereal cyst nematode and plant parasite nematode. The tests were carried out at the experimental facility of Kazakh Research Institute of Plant Protection and Quarantine (“Kapal” farm situated in the village of “Akyn Sara” in the Eskeldy District of the Almaty Region). Spring wheat variety “Kazakhstan 10” was the research object in the current study. The experiments were laid out in the Almaty Region, in Talgar District, in the village of “Panfilov”, LLP “Bayserke Agro”, at the Experimental Base of Kazakh Research Institute of Plant Protection and Quarantine. Laboratory, and field research methods were used when conducting experiments. “Baisheshek” barley variety was taken as a research object. Baisheshek - created by the method of individual selection from a hybrid population (Glozir x Unumli-arpa). Resistance to diseases and climatic conditions: Helminthosporium is affected in the middle and above average degree, powdery mildew is above average. For the study from Turkey (CIMMYT), we obtained seeds of spring wheat 17CBHL in the number of 90 samples containing sources of standard, resistant to disease and commercial varieties of spring wheat in the Almaty region: Aray, Zhenis, Astana, Aktobe, Kazakstan 10, Kazakhstan 20, Kazakstan 15 as well as from Turkey: Kutluk, Bezostaya, Sonmez, Katea. Aray - created by the method of individual selection from a hybrid population [(I- 269018 х Kyzylbas) х (I-276402 х Саратовская 29) х Казахстанская. Astana - created by hybridization in combination with transformation winter forms in spring (line Lutescens I-2959 x Tselinaya 90). Spring line Lutescens I-2959 is obtained from the Ilyichevka variety of winter wheat. Kazakhstan 15 - created by the method of individual selection from the F4 hybrid combination "Surf x Lutescens 49-71-62". Kazakhstan 10 - created by the method of individual selection from F3 Surfx Arrow. The variety is salt tolerant. As a material for selection work, 90 CIMMYT selection lines were used. In the basis of the nursery the varieties of the Siberian - Kazakstna lines are laid. The name and origin of the seeds are shown in the table, of which 32 are Kazakhstan varieties, 45 of them are Siberian varieties, 18 of them are varieties from other countries. (table 2)

25 Table 2 - Spring wheat from the Siberian-Kazakhstan lines studied in 2015-2017 in Turkey and Kazakhstan.

17ENT CNAME OC ORIGINATOR 16NURS 16ENT

1 2 3 4 5 6 1 SERI 16CBHL 2 2 STEPNAYA75 KAZ AKTOBE ARS 16CBHL 3 3 STEPNAYA1414 KAZ AKTOBE ARS 16CBHL 4 EAST-KAZAKHSTAN 4 GVK2055-1 KAZ ARI 16CBHL 5 5 LUTESTSENS2 KAZ KARABALYK ARS 16CBHL 9 KARABALYK ARS- 6 LINE-С-19SB KAZ CIMMYT 16CBHL 11 7 KARABALYKSKAYA 20 KAZ KARABALYK ARS 16CBHL 12 8 FANTAZIYA KAZ KARABALYK ARS 16CBHL 13 KARABALYK & KAZ RI 9 BOSTANDYK KAZ PLANT PROTACTİON 16CBHL 14 10 LUTESCENS 30 69/97 KAZ KARABALYK ARS 16CBHL 15 11 KARAGANDINSKAYA 30 KAZ KARAGANDA ARI 16CBHL 20 12 KARAGANDINSKAYA 31 KAZ KARAGANDA ARI 16CBHL 21 PAVLODARSKAYA 13 YUBILEYNAYA KAZ PAVLODAR ARI 16CBHL 24 KONDITERSKAYA 14 YAROVAYA KAZ PAVLODAR ARI 16CBHL 25 15 FITONС-50SB KAZ FITON-CIMMYT 16CBHL 27 16 FITON82 KAZ FITON 16CBHL 28 17 FITON-С-54SB KAZ FITON-CIMMYT 16CBHL 29 18 EKADA148 KAZ FITON-EKADA 16CBHL 30 19 EKADA 113 KAZ FITON 16CBHL 31 20 LYUBAVA KAZ FITON 16CBHL 33 21 FITON 41 KAZ FITON 16CBHL 34 22 FITON 204 KAZ FITON 16CBHL 35 23 VLADIMIR KAZ SHORTANDY ARI 16CBHL 36 24 TSELINA50 KAZ SHORTANDY ARI 16CBHL 37 25 TSELINNAYA NIVA KAZ SHORTANDY ARI 16CBHL 39 26 ASYLSAPA KAZ SHORTANDY ARI 16CBHL 40 27 AKMOLA 2 KAZ SHORTANDY ARI 16CBHL 41 28 AK ORDA KAZ SHORTANDY ARI 16CBHL 42 SHORTANDINSKAYA 29 2012 KAZ SHORTANDY ARI 16CBHL 43 30 TSELINNAYA 3S KAZ SHORTANDY ARI 16CBHL 44 31 ASTANA KAZ SHORTANDY ARI 16CBHL 45 32 ALTAISKAYA70 RUS ALTAY ARI 16CBHL 51 33 ALTAISKAYA110 RUS ALTAY ARI 16CBHL 52

26 Continuation of table – 2

1 2 3 4 5 6 34 TOBOLSKAYA RUS ALTAY ARI 16CBHL 53 35 ALTAYSKAYA ZHNITSA RUS ALTAY ARI 16CBHL 55 36 STEPNAYA VOLNA RUS ALTAY ARI 16CBHL 56 37 APASOVKA RUS ALTAY ARI 16CBHL 57 38 LUTENSCENS89-06 RUS OMGAU 16CBHL 58 39 DUET RUS OMGAU 16CBHL 59 40 PAVLOGRADKA RUS OMGAU 16CBHL 60 41 LUTESCENS29-12 RUS OMGAU 16CBHL 61 42 LUTESCENS106-11 RUS OMGAU 16CBHL 62 43 TULAIKOVSKAYA110 RUS SAMARA 16CBHL 65 44 LUTESCENS916 RUS SAMARA 16CBHL 66 45 GRECUM1003 RUS SAMARA 16CBHL 67 46 LUTESCENS1062 RUS SAMARA 16CBHL 68 47 LUTESCENS89-06 RUS OMGAU 16CBHL 69 48 ERITROSPERMUM85-08 RUS OMGAU 16CBHL 70 49 SEREBRISTAYA RUS SIB ARI 16CBHL 72 50 SERI 51 BOEVCHANKA RUS SIB ARI 16CBHL 73 52 OMSKAYA 37 RUS SIB ARI 16CBHL 75 53 LUTESTSENS7-04-4 RUS SIB ARI 16CBHL 76 54 LUTESTSENS197-04-7 RUS SIB ARI 16CBHL 79 55 LUTESTSENS220-03-45 RUS SIB ARI 16CBHL 81 56 TULAIKOVSKAYA 10 RUS SAMARA 16CBHL 85

57 TULAIKOVSKAYA ZOLOTISTAYA RUS SAMARA 16CBHL 86 58 TULAIKOVSK 100 RUS SAMARA 16CBHL 88 59 GREKUM 650 RUS SAMARA 16CBHL 89 60 LUTESCENS 920 RUS SAMARA 16CBHL 91 61 EKADA 121 RUS SAMARA 16CBHL 92 62 CIMMYT RUS SAMARA 16CBHL 97 63 P-23-17 RUS KURGAN 16CBHL 98 64 PAMYATI RUBA RUS CHELYABINSK 16CBHL 101 65 CHELYABA 75 RUS CHELYABINSK 16CBHL 102 66 ERITROSPERMUM 23707 RUS CHELYABINSK 16CBHL 104 US- 67 SY TYRA SYN US-SYN 16CBHL 112 US- 68 SY GOLIAD SYN US-SYN 16CBHL 113

69 US- SY SOREN SYN US-SYN 16CBHL 114

70 US- SY ROWYN SYN US-SYN 16CBHL 115

27 Continuation of table – 2

1 2 3 4 5 6 US- 72 SELECT SDSU US-SDSU 16CBHL 117

73 US- FORE FRONT SDSU US-SDSU 16CBHL 118

74 US- PREVAIL SDSU US-SDSU 16CBHL 119

75 US- ADVANCE SDSU US-SDSU 16CBHL 120

76 US- BRICK SDSU US-SDSU 16CBHL 121 77 CARBERRY CAN 16CBHL 122 78 MUCHMORE CAN 16CBHL 123 79 URALOSYBIRSKAYA RUS 16CBHL 130 80 TORNADO 22 KAZ FITON 16CBHL 132 81 LYUTESTSENS 1012 RUS ALTAY ARI 16CBHL 133 82 LYUTESTSENS 7-04-10 RUS KURGAN 16CBHL 134 83 LYUTESTSENS 208-08-4 RUS KURGAN 16CBHL 135 84 LYUTESTSENS 27-12 RUS OMGAU 16CBHL 136 85 ERITROSPERMUM 85-08 RUS OMGAU 16CBHL 137 86 LYUTESTSENS 6-04-4 RUS SIB ARI 16CBHL 138 87 LYUTESTSENS 186-04-61 RUS SIB ARI 16CBHL 139 88 CHEBARKULSKAYA 3 RUS CHELYABINSK 16CBHL 140 89 LINE D 25 RUS SARATOV 16CBHL 141 90 LINE 654 RUS SARATOV 16CBHL 142

28 3.2 Research Methods for root rot This study was carried out in 2015 - 2017 at two different locations: at the research institute Kazakh crop protection and Quarantaine and at the Transitional Zone Agriculture Research Institute (TZARI) in Eskisehir, Turkey, in collaboration with the International Wheat and Maize Improvement Centre (CIMMYT). Greenhouse assays conducted in Eskisehir, Turkey, study in Turkey utilized pathogenic F. culmorum Isolate 18 (isolated from a wheat crown near the city of Usak in the Agean region of Turkey) and Isolate 41 (isolated from a wheat crown in Konya, Central Anatolia Plateau of Turkey) [123]. Plant cultivars used in these experiments were chosen on the basis of their known levels of resistance or susceptibility (Table 3) [124, 125]. In the Kazakhstan, plants were grown in growth chambers for 52–70 days, with a day/night photoperiod of 12/12 h at a temperature of 25/15 C and a relative humidity of 60/80 (±5) %. In Turkey, plants were grown in the greenhouse for 49–56 days, with a day/night photoperiod of 16/8 h, a temperature of 25/15 C, and a relative humidity of 60/80 (±5) % for the duration of the experiments, similar to the methods of Mitter et al. [126]. Soil A standard potting mixture (SPM) 70:29:1 by volume of sand: field soil: natural organic matter (a mixture of sterilized animal manure and peat) was used in Turkey. The sand and the soil were sterilized at 110 C, and the organic matter at 70 C for four hours twice over the course of 2 days. In the USA, the potting mixture at a ratio of 70:29:1 was pasteurized at 70 C for 45 min. The organic source was a Sunshine potting mix #1 (Gro- Horticulture, Canada Ltd., Seba Beach, Canada).

Figure 1 - Soil preparation (CIMMYT,Turkey, 2017)

29 Seeds of each cultivar were surface disinfested with 95 % ethanol for 6 min, followed by rinsing in deionized water for 1 min, and then placed in 0.5 % NaOCl for 10 min, followed by six rinses (1 min each) in deionized water. Surface-disinfested seeds germinated on moist sterile blotting paper in plastic Petri dishes within 3–4 days at 23 C. Three-cm-long seedlings were used for further experimentation. In the USA, pre- germinated seeds were planted 1 cm deep into tubes (4 cm in diam. 9 20.5 cm in length) (Stuewe and Sons, Corvallis, OR), with 152 g of SPM. Plastic tubes (3 cm in diam. 9 12.5 cm in length) containing 100 g of SPM and sealed at the bottoms were used in Turkey [127]. Tubes were randomly arranged in plastic trays for all experiments. There was one seedling in each tube, and each tube was a replicate.

Figure 2 - Seed disinfestation and germination (CIMMYT,Turkey, 2017)

30 Isolates were grown on potato dextrose agar (PDA) for 10 days under fluorescent lights set at a 12-h photoperiod at 26 C for F. pseudograminearum and at 25 C for F. culmorum. For the millet seed inoculum in the USA, 200 g of white pearl millet seed (Pennisetum glaucum) were placed into half-liter Mason jars and autoclaved twice over a 2-day interval at a temperature of 121 C for 45 min at 103 kPa pressure. For oat seed (Avena sativa) inoculum in Turkey, grains were soaked in tap water overnight, and excess water was drained off. Polypropylene bags (20 cm 9 48 cm) with the aeration filters (Unicorn, Amsterdam, The Netherlands) were filled with soaked oat grains (250 g) and autoclaved at 121 C for 20 min and 103 kPa pressure three times over 3 days. After cooling, 4–5 cubes of 1 cm2 from 10-day-old PDA plates of isolates were added to the grain, mixed thoroughly, and incubated for 14–21 days at 22 ± 1 C temperature. After 3 days, flasks and bags were shaken daily to provide uniform colonization of grains.

Figure 3 - Inoculum preparation (CIMMYT,Turkey, 2017)

The three main methods of inoculation are summarized in radicles (4 days old) were soaked in a conidial suspension at a concentration of 1 9 106 macroconidia mL-1 of each respective isolate for one min and sown in plastic tubes containing potting mix. We tested a treatment using just distilled water and also adding 1 % methylcellulose, to make the solution more viscous so it would adhere to the seedlings. Stem base droplet: Pre-germinated seeds with emerged radicles were sown in plastic tubes containing standard potting mix. Each seedling was inoculated by placing a 10-lL droplet at a concentration of 106 macroconidia mL-1 suspension of a single Fusarium spp. isolate at the base of the stem, 0.5–1 cm away from the soil surface, 10– 14 days after sowing following the method of Mitter et al. [126]. We tested a treatment using just distilled water and also adding 1 % methylcellulose, to make the solution

31 more viscous so it would adhere to the seedlings. Inoculated tubes were covered with plastic and incubated at elevated relative humidity conditions by adding water to the bottom of the plastic trays in darkness for 48 h. After incubation, seedlings were removed from the tent or bins, randomized in trays, and left to grow until assessment time. Colonized grain: Approximately eight to ten millet seeds colonized by F. culmorum were placed 2.5 cm above the seed 6 days after planting (approximately 3 days after emergence) in the USA experiment. Two oat grains colonized by F. culmorum were added to the soil at the same time with the seed sown in each tube for the treatments in the Turkey experiment. The inoculum grains and wheat seed were covered with the soil mix and watered immediately to stimulate germination of the conidia on the millet and oats, respectively. Ten millet grains were soaked in 1 ml of water to estimate their relative conidial concentration using a hemocytometer, which averaged 5.0 9 105 macroconidia per mL for F. culmorum in Turkey, with an overall inoculum density of *2 9 105 macroconidia per mL. A non-inoculated sterilized control treatment for the conidial suspensions and colonized grain treatments was also included. Seedling-dipped non-inoculated control treatments were soaked in deionized water and sterile methyl cellulose (1 %) without conidia. For the stem base droplet method, control treatments consisted of placing a droplet of sterile distilled water or sterile methyl cellulose (1 %) at the base of the stem 1 cm from the soil line. Colonized grain control treatments consisted of inoculation with sterilized grain seed without conidia.

Figure 4 - Assessment ratings (CIMMYT,Turkey, 2017)

32 At 35–42 days (5–6 weeks) after inoculation (early tillering, Zadoks growth stage 14), plants were rated for crown rot severity. A rating system devised by Nicol et al. [127] was utilized, which was a 0–10 scale adapted from a 0–5 scale first proposed by Wildermuth and McNamara [128], based on an overall rating of the crown and the seedling base. The 0–10 symptom rating system was based on the following scale: 0 = no disease; 1–2 = minor symptoms on crown within the first internode region; 3–4 = obvious symptoms on crown within the first internode region; 5–6 = pronounced symptoms on crown with obvious darkened plant tissue due to infection penetrating to the third leaf; 8–9 = advanced darkened symptoms with severe stunting and near death due to disease infection and; 10 = dead plant with severe disease symptoms for FCR severity. Due to the infective nature of F. culmorum in Turkey on the roots [129], roots were also included in the assessment, whereas F. pseudograminearum in the USA was rated on the lower stem and the first internodes [120]. Growth chamber seedling screening Three runs of the growth chamber assay were conducted for the March/May; harvested and rated on March 20 (2017), May 20th (2017), Two growth chamber assays were conducted for the 17 CBHL; For the growth chamber assays, seeds of each cultivar were surface disinfested in 95% EtOH (ethanol) for 6 minutes, rinsed in de-ionized distilled water for 1 minute, then placed in 0.5% NaOCl for 10 minutes, followed by six rinses (1 minute each) in de-ionized distilled water. Following surface disinfestation, seeds were placed on moist filter paper and incubated for 3 days. Seedlings were placed in a 4 C for 4 to 5 days to obtain consistent germination. Seedlings were planted into a medium comprised of 50% sand and 50% peat (Greensmix Sphagnum Peat Moss, Wuapaca Northwoods LLC., Wuapaca, WI) (v/v) in 4 cm diameter x 20.5 cm long cone-tainers (Stuewe and Sons, Corvallis, OR) arranged in plastic trays and arranged in a randomized complete block design with 10 replications. The experiment was conducted in three 964 ft3 (Conviron, Winnipeg, CA.) growthrooms located at the Eskishehir, Turkey. Each growth chamber could only contain ~1,200 plants. Therefore, the 17 CBHL were split into two groups for testing with common check and parental genotypes included in each growth chamber. Genotypes within growth room sets were arranged in a randomized complete block design, with 10 replications. Seedlings were grown at 60/80 (±5)% day/night RH, a 12 hour photoperiod, and 25/15 oC day/night temperatures for the duration of the experiment, 35 days after inoculation. Plants were allowed to dry and symptoms were rated on dried tissue. Crown rot disease symptoms were rated using a 0 to 10 scale (0=no disease observed, 10 = stunted and dead) according to Nicol et al. (2004). The 0 to 10 symptom rating system was based on the following scale; 0=no disease; 1 to 2 = minor symptoms on crown within the first internode region; 3 to 4 = obvious symptoms on crown within the first internode region; 5 to 6 = pronounced symptoms on crown with obvious darkened plant tissue due to infection; 8 to 9 = advanced darkened symptoms with severe stunting and

33 near death due to disease infection; 10 = dead plant with severe disease symptoms) for crown rot severity. Seedling height from the base of the soil to the tip of the longest leaf was also recorded. Field screening A single field trial located near Mansfield WA was planted May 13th and 20th July 2017 for the 17CBHL respectively. Following seed inoculation as described above, approximately 30 seeds were planted in a 1.5 m linear row per to a depth of 5 cm with a deep furrow hand planting apparatus. Plots were spaced 25 cm apart. The experiments were arranged as randomized complete blocks with three replications for Sunco/Otis and two replications for 17 CBHL. After the growing season, when the wheat had senesced, approximately 10 plants were harvested per plot from which 5 stems were randomly selected and rated for crown rot severity on a scale from 0 to 10, similar to that of the growth chamber and outdoor terrace evaluations. Statistical analysis for crown rot disease screening A single rating scale was used (0 to 10; 10 = dead plant) for the growth chamber, outdoor terrace and field screens. Experimental units were individual plant stems that were scored for all growth room, terrace, and field data sets. The mean growth room, terrace, and field ratings from each respective screen and year based on the 0 to 10 scale were used for QTL mapping. Disease assessment and stratification survey methods This section provides descriptions of root disease survey procedures along with an overview on the suitability and application of each method. Field procedure Douglas-fir leading species survey variant (the default ILM survey). Stratify the selected area by tree cover, using aerial photos, biogeoclimatic and forest cover maps, and available history records. Determine the laminated root rot hazard rating and survey priority for each stratum, based on species composition. Establish a base line and place survey grid transects 40 to 70 m apart. Identify above-ground visible infection center boundaries using disease signs and symptoms. Use metric grid field sheets (or FS 1061 form) to record all intercept lengths. Optionally, one may map all infection centers and stratum boundaries. Summarize disease intercept and transect lengths per stratum. Infection center interception length is measured and recorded as the distance from the last healthy tree intercepted to the first healthy tree encountered beyond an infection centre. Infected trees must be adjacent to these healthy trees. Determine the disease incidence by summing the disease intercept lengths and transect lengths of each stratum (or all strata) using the following formula:

34

(1) Methods for recording the results of infection In contrast to the local, in common diseases it is relatively easy to establish the prevalence (percentage of diseased plants), but much more difficult - the intensity. Of course, when assessing the affection of wheat, it is sufficient to calculate the percentage of diseased ears, but in diseases such as root rot, plants can die completely, but they can turn yellow, burn, fall behind, etc. This is not the number of spots on the leaf or percentage of its coverage by stains; indicators of the intensity of the disease when taking into account root rot are much less objective. Therefore, the scales proposed for their evaluation by different researchers can be very different. For example, E. Gojman gives a very simple scale for assessing cereal involvement by Fusarium spp root rot in terms of plant appearance.

Table – 3 Scale of accounting for the intensity of damage to crops by root rot (according to E. Gojman)

Intensity of defeat, point Symptom of disease

0 The plant is healthy 1 Yellowish color of affected organs and parts 2 Coloration from yellowish to brownish, single brown streaks and spots 3 Strong ripening, partial shaking 4 Dying of tissues and organs

According to M.F. Grigoryev, it is necessary to excavate the plants under consideration, wash them from the soil and assess the involvement of the individual primary roots, the underground interstice, the secondary roots, the root neck and the base of the stem on a four-point scale, and then calculate the average index of damage Uneven, often focal, spread of the disease on the field is the next important accounting problem. In ecology it is common to divide all possible cases of mutual finding of objects into three groups: regular, uniform and contagious (focal) distribution (Figure 5).

Figure 5 - Types of distributions of objects in space: from left to right - regular, uniform, contagious*.

35 With a regular distribution, each object is at an equal distance from its neighbors. In nature, such a distribution of plants on the area does not happen, it is typical for artificial planting and planting (regular gardens, square-nesting crops). But even in regular orchards the distribution of diseased plants can not be regular. Uniform distribution provides an equal probability of presence or absence of the object at any point of the site. So, for example, wheat plants are distributed on the field. It is described by the Poisson equation:

1 - x = e-m (2)

where x is the number of infected plants (prevalence); m- average percentage of damage (intensity). With a contagious (focal) distribution, the distance between individual objects in the foci is less than the distance between objects outside the foci. It occurs most often and is described by the equation:

1 - x = (1 + m / k) -k, (3)

where k is the aggregation coefficient. Focal distribution of diseases in the area is very common and can be the cause of errors that occur when taking an average sample for analysis (if there are few foci, plants from them may not fall into the middle sample). For example, the main source of wheat late blight is the planting of infected tubers. If 1000 tubers were planted in the field, five of which were infected, and an infected plant grew from each infected tuber, then in the field among 1000 wheat bushes there will be five foci of the disease. Under favorable conditions, the spores that form on the infected plants will infect adjacent ones, and the foci will gradually expand, so that eventually the foci will merge and the spread of the disease will become uniform, but at the first counts, which are very important, diseased plants may fall out field of view of the observer. An equation that takes into account the average lesion of zero in the focal distribution of the disease was suggested by A.V. Filippov. For calculations, the following indicators are needed: - number of foci; - the average area of the hearth; - average lesion of the focus. Fungal genomic DNA extraction The genomic DNA was extracted from five to seven days old fungal cultures grown either in liquid broth or culture plates. The fungal mass from the culture plate was scraped out with the help of a fine spatula and fungal mass from the culture broth was obtained by filtering the culture broth through a 10 ml syringes containing glass wool that will allow the broth to pass through, while retaining the fungal mass. The fungal

36 mass obtained from the culture plate or broth was placed in a 2ml tube containing a ceramic pestle, 60–80 mg sterile glass beads (425–600 µM, Sigma) and lysis buffer (100 mM Tris HCl [pH8.0], 50mM EDTA, 3% SDS). Homogenization of fungal mass was done twice in a FastPrep®-24 tissue homogenizer (MP Biomedicals, USA) at 6 M/S for 60 sec. The resulting fungal tissue homogenate was centrifuge at 13,000 rpm for 10 min and supernatant was transferred to a fresh microcentrifuge tube. To the supernatant, 2 of RNase A (10mg/ml) was added and incubated at 37°C for 15 min. After the RNase A treatment, equal volume of phenol: chloroform: Isoamyl alcohol (25:24:1) was added and mixed well, followed by centrifugation at 13,000 rpm for 10 min (Note: this step can be repeated once more to completely get rid of proteins/cell debris). The upper aqueous layer was taken in a fresh micro centrifuge tube and then equal volume of 100% ethanol was added. Following precipitation at -20°C for 30 min, the whole content was centrifuged at 12,000 rpm for 10 min to pellet down the DNA. The DNA pellet was washed with 70% ethanol and centrifuged at 12,000 rpm for 5 min. The DNA pelletswere air dried and dissolved in 1× TE buffer. The tests were carried out at the experimental facility of Kazakh Research Institute of Plant Protection and Quarantine (“Kapal” farm situated in the village of “Akyn Sara” in the Eskeldy District of the Almaty Region). Spring wheat variety “Kazakhstan 10” was the research object in the current study. The mechanical composition of the soil was made up of dark brown soil, medium loamy soil, and humus (3.0 – 3.5%), pH 7.0. Forecrop was spring wheat plowed to the depth of 15-17 cm by “BD” disc header, pre-sowing cultivation, rolling after sowing. Sowing of seeds was produced on May 11 employing “SZ-3.6” tractor seeder. Seed application rate was 4.0 mln pcs/ha, while depth was 5-6 cm. Eight variants were tested in the experiment: 1. Control – seeds treated with water; 2. Vial TT disinfectant – 0.4 l/t; 3. Tank mix of Vial TT – 0.4 l/t + TS3 Strain – 0.25 l/t; 4. Tank mix of Vial TT – 0.4 l/t + GZ14 strain – 0.2 l/t; 5. Tank mix of Vial TT – 0.4 l/t + Extrasol– 0.1 l/ t; 6. Extrasol– 0.1 l/t; 7. Strain 2 – 0.15 l/t; 8. BL01 Strain – 0.05 l/t. The experiments were conducted under field conditions in four replications. The size of the plots was 0.25 ha. Biologics were applied using seed treatment technique before sowing. Seed pickling machine (PS-10) was employed for seeds treatment, water consumption rate was 10 l/t. Currently, environmentally focused technologies are increasingly used in crop production to achieve clean production, which is the most promising in the development of agriculture6. The lack of any substances in grain crops leads to disturbance in carbohydrate and nitrogen metabolism, protein synthesis, as well as reduces the resistance of plants to adverse environmental conditions7-8.

37 The aim of our study at this stage consisted in testing of biologics prepared on the basis of a rhizosphere bacteria strain of Bacillus suptilis. The experiments were laid out in the Almaty Region, in Talgar District, in the village of “Panfilov”, LLP “Bayserke Agro”, at the Experimental Base of Kazakh Research Institute of Plant Protection and Quarantine. Laboratory and field research methods were used when conducting experiments. “Baisheshek” barley variety was taken as a research object. Mechanical composition of the soil consisted of dark chestnut soil, medium loam soil, and humus (3.0 – 3.5%), pH was equal to 7.0. Sowing of barley seeds was produced on April 24 employing SZ-3.6 tractor seeder, seeding rate was 3.5 mln pcs/ha, at a seeding depth of 5-6 cm. Field experiments were carried out according to the following scheme: 1. Control – seeds treated with water; 2. Disinfectant Vial TT – 0.4 l/t; 3. Tank mix of Vial TT – 0.4 l/t + TS3 Strain –0.25 l/t; 4. Tank mix of Vial TT – 0.4 l/t + GZ14 Strain –0.2 l/t; 5. Tank mix of Vial TT – 0.4 l/t + Extrasol – 0.1 l/t; 6. Extrasol – 0.1 l/t; 7. Strain 2 – 0.15 l/t; 8. BL01 Strain – 0.05 l/t. Registration plot area was 0.25 ha. The experiments were carried out in four replications. Biologics were applied using seed treatment technique before sowing. Seed pickling machine (PS-10) was used for seeds treatment, water consumption rate was 10 l/t. Biological effectiveness of biologics and the infestation of plants with root rot are presented in Tables 21 and 22. The results of study have shown that in the pre-sowing treatment of barley seeds, biologics did not significantly affect the germination energy and laboratory seed germination, but at the same time slightly inhibited the infestation of seeds by mold fungi (15.8 – 86.8%). This resulted in increased seedling density by 7.3-13.6 pcs/m2. The biologics reduced the infestation of barley with root rot at the plant tillering phase by 31.5-66.3%. Besides, the biologics increase physiological growth of plants and contribute to accumulation of crops biomass in comparison with the control variant. In this experiment, treatment of seeds and crops by biologics positively affected the sowing qualities of the resulting seeds as compared to control. In Kazakhstan, wheat is the most important grain crop in terms of food significance and scale of production. Production of this crop in all continents amounts to 615 mln tones per year. About half of the global wheat grain production accounts for just five countries: Canada, USA, China, India, and Russia [130]. Winter wheat is a valuable crop in field rotation and a good forecrop for a number of crops (maize, sunflower, sugar beet, winter barley, stubble crops, etc.).

38 Yielding capacity refers to the average size of particular crop produced per unit of cultivated area, measured usually in hundredweight per hectare [131, 23 p.]. Yield characterizes the total amount of given crop production, while yielding capacity of the crop is its productivity under specific conditions of cultivation. In accordance with the specifics of these concepts, the yield is characterized by a number of indicators. These indicators include specific yield, yield before the beginning of timely harvesting, the actual yield, the so called crop in storage, and net yield. In the beginning, crop harvesting is estimated in the original recorded weight, and then in the actual weight of the grain after modifications, as well as based on the conversion with regard to standard humidity. Crops yield and yielding capacity are direct statistical characteristics of the level of crop production development and total agricultural production. Yield (bulk yield) is the total amount of production of any agricultural crops (group of crops) in real terms, obtained from the entire area of the crops. In relation to the importance of the crop yield index, we have investigated the plant biometrics and yield of winter wheat. Biometric analysis and yield estimate were conducted at the phase of full ripeness. To carry out proper recording, 50 plants were selected in four replications.

3.3 Research Methods for nematode Sampling Having observed symptoms that indicate possible or likely nematode infestation, the next stage is to collect samples from the affected plants and from the soil around the roots. These are then taken to the laboratory for analysis, to determine what nematodes are present and possibly their density. The following field characteristics have implications for the sampling method, and should therefore be considered at this stage: - Aggregated distribution of nematodes due to host root system and the seasonal behavior of the nematode - Crop type and history - Areas planted to different varieties - Soil moisture - Soil compaction - Soil type - Temperature and seasonal changes. Sampling tools Useful tools for sampling, some of which are shown in Fig. 20, include a spade, a hand trowel, a screwdriver, a soil auger (corer), knives (for cutting roots), scissors, polythene sample bags, tags. The soil auger or corer should have a blade 20–30 cm in length and 20–25 mm diameter, and can be either a complete cylinder or a half cylinder. Half cylinders help in removing soil from the corer. Marker pens for labeling the sample bag and a pencil and notebook are also necessary for recording information [132].

39

Figure 6 - Anassortment of tools for sampling nematodes*.

Number of samples Take enough samples to ensure they are representative of the situation in the field. The greater the number of sub-samples/cores combined for each field sample, the more accurate the assessment will be. A balance between available time and resources is, however, necessary. The sampling procedure and number of samples taken should allow for nematode variation or aggregation. From an area of 0.5 to 1 hectare, take a minimum of 10 core sub-samples, and even as many as 50. Combine these to make one composite sample to represent the field area sampled. Bulking of samples in this way helps to preserve them by maintaining the temperature and moisture of samples. Sampling pattern Nematodes are rarely distributed evenly in a field, and samples should therefore be collected from several areas within the field. Collect separate samples from both the poor growth areas and an area of relative good growth, where this is obvious, for comparison. Maintain a consistent sampling style and pattern during surveys and experiments to enable meaningful comparisons between fields, plots, treatments, etc. Sampling patterns can be random or systematic (Figure 21). Random sampling does not accommodate the patchy nature of nematode distribution, and is only representative if the sampling area is small. Systematic sampling is a more structured way to remove samples as it takes into consideration the nature of the field and nematode distribution.

Figure 7 - Sampling patterns for nematodes. (a) Random sampling; (b–d) systematic sampling*.

40

Time of sampling The optimum time for sampling varies between crops and is related to the growth stage of the crop and the objective of the sampling (predictive or diagnostic). Predictive sampling (not the focus of this guide) is often done early in the season, such as at or just before planting, or at the end of the previous cropping season to determine the number of nematodes (density). Many nematode species increase to high levels during the growing season and reduce during the off (dry) season; this is easier to see in annual crops than in perennial and tree crops. Samples should therefore ideally be collected in the middle of the season and/or at final harvest for diagnostic purposes. Perennials can be sampled during the active growing period such as during the rainy/growing season to identify the problem. Taking soil samples As a rule, avoid sampling very wet or very dry soil. However, where crops normally grow in, for example, swamp (e.g. paddy rice) or arid conditions (e.g. sisal), these should be sampled to represent these conditions. Divide fields larger than 1 hectare into 1 hectare (10,000 m2) plots and sample these plots separately. Take 10 to 50 sub-samples (cores) and combine them to make a composite sample that weighs 1–2 kg. Remove the soil sub-sample from the root zone using a trowel, auger, corer, spade or similar implement that is suitable for the crop being sampled [133].

Figure 8 - Taking and bagging samples*. Care of samples Collect samples in strong plastic bags, and label them clearly and systematically. Plastic labels marked with a water-resistant permanent marker or pencil can be placed in the sample bag (Figure 22), or alternatively write directly on the plastic bag with a permanent marker pen the sample number or reference. Paper labels are best attached to the outside of the bag with wire or twine. If using paper labels, use a pencil, not a pen (which will run or smudge when wet). But remember, paper labels deteriorate quickly during wet conditions. Record, where possible: - The crop and cultivar

41 - The sampling date - The farmer - The location (and GPS coordinates if possible) - A reference number (or plot) if within an experimental trial Sampling Nematodes are very sensitive and perishable, and it is very important that appropriate care is taken to keep them in good condition. Samples should noT be left in direct sunlight or in a closed vehicle in the sun. They should also not be left for long periods before processing. After collection, samples should be placed in a coolbox (insulated container), or packed in strong cardboard boxes and placed in a shaded area where conditions are cool. If unable to process immediately, samples can be stored in a refrigerator (approx. 10°C) for up to 2 weeks. Nematode survival decreases with time however, and nematodes from relatively hot environments can suffer chilling injury. Nematode extraction The next stage is to extract nematodes from the samples. This should be done as soon after collection as possible as samples deteriorate over time. There are four basic extraction techniques, which are covered in this guide: - Extraction tray method - Sieving method - Incubation method. Three further methods – elutriation, the Fenwick can and centrifugal flotation – r equire specialist equipment and are not described in this guide. Details on these methods can be found in publications listed in the References and Further Reading section. It is also possible in some cases to examine nematodes directly from plant tissue specimens [134]. Choosing an extraction method The choice of which method to use depends on the conditions and materials available, the sample type, and also the type of nematodes present. Some methods of extraction are more useful for specific types of nematode, while others are more general. This guide provides details of the most straightforward methods, including the extraction tray method, which is useful in the most basic conditions, provides a reasonable assessment of nematodes from soil, roots, seeds or plant tissue, and can be easily replicated [139, 23 p].

Table 4 – shows which extraction methods are suitable for which types of nematodes (sedentary or migratory) in soil or root/foliar samples

Incubation Soil sample Root/foliar sample Sedentary Migratory Sedentary Migratory nematodes nematodes nematodes nematodes 1 2 3 4 5 Extraction tray X X

42 Continuation of table – 4

1 2 3 4 5 continuation of the table - 4 Sieving X X Root/leaf X X maceration Incubation X X If the target nematode is known – such as in research experimental plots – it may be possible to precisely identify which material should be sampled and processed for the nematodes (soil, root, tuber, leaf, etc.). If you are not sure though, both roots and soil should be used for extraction with both sedentary and migratory nematodes considered.

Preparation of samples Dry soil should be properly mixed before sub-sampling for nematode extraction. Break up clumps and remove stones, roots and debris. Pass (dry) soil through a coarse sieve with holes of approx. 1–2 mm (Figure 9, step 1) into a suitable container and then mix thoroughly. Remove a sub-sample using a beaker or container of known volume; 100 ml soil is commonly used. From each bulked field sample two × 100 ml soil samples should be processed and the mean taken. Wet soil, such as from a rice paddy, needs to be removed from the field sample using small balls or clumps from various parts of the sample or from each root base, and measured using displacement of water to get samples of the same size. Fill the beaker to a set (marked) volume (e.g. 200 ml) and raise the volume to the required marked amount using soil clumps, e.g. raising 200 to 300 ml measures 100 ml of soil. Then use the sieving method to extract nematodes. Roots should be separated from soil and any soil adhering removed by gently tapping off, or rinsing gently under a tap or in a container of water. Dab the roots dry with paper towel, and then chop and extract according to the chosen extraction method [138]. Labeling It is important to use clear, correct and consistent labeling for samples (Figure 5). Label containers with either a waterproof marker or chinagraph pencil, or use labels that can be moved along with each stage of the extraction. Ensure all samples are correctly labeled at all times.

43

Figure 9 – Materials for and examples of labelling*.

Extraction tray method This method (or variations of it) is sometimes also called the modified Baermann technique, the pie-pan method, or the Whitehead tray method. Advantages: - Specialist equipment is not required - It is easy to adapt to basic circumstances using locally available materials - It xtracts a wide variety of mobile nematodes - It is a simple technique. Disadvantages: - Large and slow moving nematodes are not extracted very well - The extractions can sometimes be quite dirty (especially if the clay content of the soil is high) and therefore difficult to count - The proportion of nematodes extracted can vary with temperature, causing potential variation in results between samples extracted at different times - Maximum recovery takes 3–4 days. Equipment - A basket (or domestic sieve) made with coarse mesh - A dish/tray/plate, slightly larger than the basket - Tissue paper - Beakers or containers to wash the extraction into - Wash bottles - Waterproof pen - Knife/scissors - Weighing scales - Large bench space. Most items can be purchased ready-made or easily constructed from readily available materials. Funnels can be held in a rack or stand with rubber tubing attached to 44 the bottom and sealed with a clip or insect mesh attached to an ~10 cm section of ~15 cm diameter plastic piping can be used to construct the sieve. It is very important that the mesh and sieve base are raised slightly (~2 mm) from the bottom of the dish/plate using, for example, three or four small ‘feet’ glued to the base of the sieve. If this is not done, the nematodes cannot easily migrate into the water [137]. Sieving method Advantages:  Good extraction of all types of nematodes  Good for extraction of large and slow moving soil nematodes  Suitable for extracting nematodes from wet soil  Useful for cyst extraction from soil. Disadvantages:  Nematodesmay settle out with soil particles unlesssoil is well dispersed  Nematodescan be easily damaged.  Requires slightly more specialized equipment.  Equipment  For soil motile nematodes:  Beakers and buckets  Waterproof pen  Brass 2cm diameter sieves: 2 mm, 90µm (or53 µm) and 38 µm  Extraction tray equipment  For sedentary cyst (e.g. Heterodera) recovery:  As for soil motile nematodes, plus  Brass 20cm diameter sieves: 2mm, 250µm, 150µm Funnels  Filter paper (or paper towel/tissue) If it is not known whether sedentary nematodes are present, then all equipment should be used. Method For soil motile nematodes: Fill a bucket with about 6 liters of water. Mark a water line on the inside of the bucket with a waterproof pen for consistent water volume between samples. Place a sub-sample of sieved and mixed dry soil, or of wet soil measured by displacing water in a beaker (Fig.8) into the bucket (Figure 8). Mix the water thoroughly using your hand (Figure 8). Allow larger particles to settle for 30 seconds (Figure 8). Slowly pour off the upper ¾ of the water through the nested sieves: use a 2 mm sieve to catch debris for disposal, or just 90 µm and 38 µm ones to catch nematodes if there is no debris (Figure 8). This requires two people. Take great care to ensure that

45 water does not escape over the sides of the nested sieves (in between the stacked sieves) when pouring the bucket of water through, as nematodes will be lost in the escaping water. Pour slowly and tap the underside of the bottom sieve gently, if necessary, to help water flow through the the sieves. Refill the bucket to the marked line and repeat the process once or twice. Wash off the debris from the 90 and 38 µm sieves into a labeled beaker, ensuring that sieves are properly cleared by washing gently from behind [135]. Leave beakers for 2–3 hours for nematodes to settle to the bottom. If necessary gently pour off and discard excess water. For recovery of sedentary cysts: Air dry the soil sample before using for extraction. Fill a bucket with about 6 liters of water, and mark the water line on the inside of the bucket with a waterproof pen. Place the measured soil sub sample in the bucket. Mix the water thoroughly using your hand, then allow soil particles to settle for 60 seconds. Cysts should float. Slowly pour off the top ½ of water through the nested sieves: 2 mm to catch debris for disposal, and 250 µm and 150 µm to trap cysts. Wash off the debris from the 250 µm and 150 µm sieves into a labeled beaker. Refill the bucket to the marked line and repeat the process (steps 4–8) at least once, collecting all debris for each sample into the same beaker. Repeat this process as much as necessary until you are satisfied that no cysts remain in the bucket. Prepare and label a paper lining (filter paper, milk filter, paper towel etc.) for a funnel (i.e. in a cone shape) held in a stand or beaker. Pour the wash-off (sievings) in the beaker through the filter in the funnel. Allow water to drain through. Carefully remove filter papers from the funnel and place in a moistened tray to await direct observation under the microscope. Viewing can be done by gently opening the filter paper and spreading the contents across the filter paper, followed by viewing under stereomicroscope [136]. Screening method for Cyst nematode Two hundred and ninety spring wheat accessions including breeding lines, cultivars and landraces were tested (Table 1). The wheat accessions originated from Kazakhstan, Siberia. The material was provided by the International Winter Wheat Improvement Program. Nematode inoculum A pure growth room culture of H. filipjevi from Central Anatolian Plateau, Eskisehir (39.76665°N, 30.40552°E), was collected and cysts were extracted by Cobb’s decanting and sieving method (Cobb, 1918). Cysts were picked by hand and sterilised with 0.5% NaOCl for 10 min and rinsed several times with sterile distilled water. The surface-sterilised cysts were transferred into a funnel and stored at 4°C for hatching.

46

Figure 10 – Extraction of soil nematodes using the sieving method*.

For recovery of sedentary cysts: Air-dry the soil sample before using for extraction.

47 Fill a bucket with about 6 liters of water, and mark the water line on the inside of the bucket with a waterproof pen. Place the measured soil sub-sample in the bucket. Mix the water thoroughly using your hand, then allow soil particles to settle for 60 seconds. Cysts should float. Slowly pour off the top ½ of water through the nested sieves: 2 mm to catch debris for disposal, and 250 µm and 150 µm to trap cysts. Wash off the debris from the 250 µm and 150 µm sieves into a labeled beaker. Refill the bucket to the marked line and repeat the process (steps 4–8) at least once, collecting all debris for each sample into the same beaker. Repeat this process as much as necessary until you are satisfied that no cysts remain in the bucket. Prepare and label a paper lining (filter paper, milk filter, paper towel etc.) for a funnel (i.e. in a cone shape) held in a stand or beaker. Pour the wash-off (sievings) in the beaker through the filter in the funnel. Allow water to drain through. Carefully remove filter papers from the funnel and place in a moistened tray to await direct observation under the microscope. Viewing can be done by gently opening the filter paper and spreading the contents across the filter paper, followed by viewing under stereomicroscope. Screening method for Cyst nematode Two hundred and ninety spring wheat accessions including breeding lines, cultivars and landraces were tested (Table 1). The wheat accessions originated from Kazakhstan, Siberia. The material was provided by the International Winter Wheat Improvement Program. Nematode inoculum A pure growth room culture of H. filipjevi from Central Anatolian Plateau, Eskisehir (39.76665°N, 30.40552°E), was collected and cysts were extracted by Cobb’s decanting and sieving method [140]. Cysts were picked by hand and sterilised with 0.5% NaOCl for 10 min and rinsed several times with sterile distilled water. The surface- sterilised cysts were transferred into a funnel and stored at 4°C for hatching. Freshly hatched juveniles after 2 days (48 h) were used as inoculum. We performed a polymerase chain reaction-restriction fragment length polymorphism analysis to confirm the species of identification [141]. Screening assay of wheat accessions Six spikes of each of the 94 wheat accessions were picked by hand and one representative spike was selected from each accessions. A susceptible wheat cv. Bezostaya 1 was used as control. Seven seeds from each spike were germinated in moistened tissue in Petri dishes for 3 days at 22°C. After germination, five seedlings of a similar phenotype were selected. A sterilised potting mixture of sand, field soil and organic matter (70:29:1, v/v/v) was filled in RLC4-pine tubes (25 × 160 mm, Ray Leach Cone-tainer™; Stuewe & Sons). One germinated seed was planted per tube in a 200 tube

48 rack (RL200; Ray Leach Cone-tainer™) and plants were organised in a randomised block design. Each plant was inoculated with 250 freshly hatched J2 of H. filipjevi in 1 ml water into three holes around the shoot base 7 days after transplanting. Plants were grown in a growth room at 26°C and 65% RH. Twenty-five days after planting, plants were fertilised with water-soluble Nitrophoska® Solub/Hakaphos® (20:19:19 NPK including micro elements such as P2O5, K2O, B, Cu, Fe, Mn, Mo and Zn; COMPO) at 1 g l–1. Plants were harvested at 63 days post infection (dpi) to collect the cyst from the soil and the roots. The soil from each tube was collected in a 2 l beaker filled with water and the soil mixture was stirred, then left for about 30 s to allow the heavy sand and soil debris to settle down. Roots were washed very gently on the upper sieve to free any females and cysts left attached to the root system. The soil mixture was poured through 850 and 250 μm sieves. This process was repeated three times to ensure all females and cysts were collected. Females and cysts from both roots and soil were captured on a 250 μm sieve and counted under a dissecting microscope. The roots were further checked for females and cysts that had not been dislodged during the washing process. The host status of the tested wheat accessions was determined and categorised into five groups based on mean number of females and cysts present per plant [142]. The following ranking was used on a per plant basis: resistant (R) = <5 females and cysts; moderately resistant (MR) = 5-10 females and cysts; moderately susceptible (MS) = 11-15 females and cysts; susceptible (S) = 1619 females and cysts; and highly susceptible (HS) = >20 females and cysts. The widely grown winter wheat cv. Bezostaya 1 in Turkey was used as the susceptible control. Resistance assay The experimental method described above for the screening assay of wheat accessions was used for the resistance assay. For plant growth measurements, non- inoculated (NI) and inoculated (I) plants were analysed. All treatments were repeated 3 times and performed in a completely randomised design. To monitor nematode infection and nematode development, roots were stained with acid fuchsin at 2, 5, 10 and 15 dpi [143]. To determine nematode reproduction, plants were harvested at 63 dpi, female and cysts were extracted from roots and soil, and the numbers of eggs and J2 determined after gently crushing the cysts. To measure cyst size, cysts were transferred to 2% water agar and photographed with a DM2000 dissection microscope (Leica Microsystems). The largest optical section of the cysts area was calculated using LAS software (Leica Microsystems). To assess growth parameters, the plants were washed gently, remaining soil particles were removed, and the root surface was dried with soft paper towel. Immediately after drying, the fresh plant weight and root weight was recorded. Root volume was measured volumetrically [144].

49 4 RESULTS OF THE RESEARCH

4.1 Microbial Identification of fusarium solani The goal of this research was to identify fungal samples by using the validated MicroSeq® system from Applied Biosystems. This automated system is based on PCR amplification and DNA sequencing of fungal 16S or D2-LSU rRNA genes. After extraction and purification of genomic DNA from the sample, the 16S ribosomal RNA- (bacterial samples) or the D2-LSU gene (fungal samples) is amplified by PCR. The PCR is next followed by a sequencing step. Product is detected and analysed using capillary electrophoresis and results are analysed against the validated MicroSEQ library. Because micro organisms have their own specific variations in their ribosomal genes, the system can accurately identify thousands of microbial species, including bacteria and fungi. This service meets the requirements of regulatory agencies in Europe and the US. Samples In the table below the samples received by BaseClear are summarized, including date of entrance and date of analysis. Further more the type of sample as indicated by the client on the order form is noted. Based on this designation the samples were analyzed using either the 16SrRNA- (Bacteria) or the D2-LSU procedure (Fungi).

Figure11 – Pure strain of Fusarium solani (KazRIPPQ, 2016)

In the table below the results of the analysis are detailed. Per sample only the specimen with the highest matching score is giving or the specimens are given that have 50 a score <0.1% from the highest matching score. In the enclosure, a more detailed analysis is giving including the top 5 matching specimens.

Table 5 – Specimen Analysis (Base clear, Netherlands, 2016)

Code Name % Match Organism Type Comments Name F2847 fusarium- 100,00 Fusarium S - solani- 100,00 anthophilum F1-0168 100,00 Fusarium 100,00 napiforme Fusarium nygamai Fusarium proliferatum var. proliferatum CBS=134.95

Table 6 – Specimen analysis table (Base clear, Netherlands, 2016)

Speci Sam Basecal Filter Assembly Specimen Top Match % Cons Librar men ples ling Score Matc ensus y h Lengt Entry h Lengt h fusari 2 43 Fusarium 100. 278 278 um− anthophilum 00 solani −F1− 0168 NCF 2 0

PCF 2 44 Saccharomyc 100. 281 281

es cerevisiae 00 ATCC=9763

Table 7 – Phylogenetic tree table (Base clear, Netherlands, 2016)

Specimen % Match Sequence Entry Library fusarium−solani−F1−0168 100.00 Fusarium anthophilum AB_FungalLib_2013 100.00 Fusarium napiforme AB_FungalLib_2013 100.00 Fusarium nygamai AB_FungalLib_2013 100.00 Fusarium proliferatum AB_FungalLib_2013 var. proliferatum CBS=134.95

51 Table 8 – Phylogenetic tree (Base clear, Netherlands, 2016)

Specimen % Match Sequence Entry Library 99.30 Fusarium xylarioides AB_FungalLib_2013 NCF No Libraries Searched Against PCF 100.00 Saccharomyces cerevisiae AB_FungalLib_2013 ATCC=9763 99.98 Saccharomyces cerevisiae AB_FungalLib_2013 ATCC=18824 96.75 Saccharomyces bayanus AB_FungalLib_2013 96.75 Saccharomyces pastorianus AB_FungalLib_2013 94.86 Zygosaccharomyces AB_FungalLib_2013 microellipsoides

Specimen : fusarium−solani−F1−0168 N.Join: 1.0%

Fusarium nygamai fusarium−solani−F1−0168 Fusarium xylarioides Fusarium napiforme Fusarium proliferatum var. proliferatum CBS=134.95 Fusarium anthophilum

Specimen : PCF N.Join: 2.5%

PCF Saccharomyces cerevisiae ATCC=9763 Saccharomyces cerevisiae ATCC=18824 Zygosaccharomyces microellipsoides Saccharomyces bayanus Saccharomyces pastorianus (5)

Figure12 – Phylogenetic tree (Base clear, Netherlands, 2016)

52 4.1.1 Assessment of screening methods to identify resistance to root rot (Fusarium colmorum) in wheat The crown rot (foot rot, root rot) which caused by Fusarium culmorum, F. pseudograminearum (formerly F. graminearum group 1), F. graminearum (formerly F. graminearum group 2) are important disease of cereals around the globe and occur wherever cereal productions systems exist especially under drought conditions [129]. Crown rot is associated with reduced yields in wheat production and can be caused by numerous factors. Fusarium culmorum is a ubiquitous soil borne pathogen that infects cereals, especially in wheat. Fusarium has a substantial impact upon the yield of infected plants as it increases wheat susceptibility to crown rot, a disease in wheat characterized by brown discoloration of the stem base and crown. Severe Fusarium infection can render the wheat crown dysfunctional. Crown rot also stunts plant growth and seed production. Fusarium culmorum is an important disease due to the worldwide prevalence of losses due to crown rot. Most often found root rot disease in the main areas of spring wheat cultivation - in the Volga region, the Altai Territory, Kazakhstan, Western and Eastern Siberia. In the United States Pacific Northwest a yield loss of 9% in 1994 was attributed to Fusarium associated with crown rot (Hogg). In Turkey crown rot has caused yield losses exceeding 50%. From 1998 to 2008 yield losses due to crown rot have increased by 9%, 3% and 1.2% in the northern, southern, and western wheat growing regions of Australia, respectively. All these countries rely heavily upon their wheat industries, and these losses have a detrimental impact upon the farmers and economy[130]. Plants were grown in growth room for 8 weeks. After 8 weeks plants harvested and scored for disease symptoms. Each plant was given a rating of 1-5 (depending browning percentage on stem) based on a standardized scale for disease reaction. From the bellow on table -9 dates of 98 lines to the disease root rot, pathogen Fusarium culmorum.

Table 9 – Screening of genotypes of wheat to the disease root rot, pathogen Fusarium colmorium (crouth room, CIMMYT, Turkey, 2017)

C-name No R1 R2 R3 Mean Grou- No R1 R2 R3 Mean Grou - Selec- ping ping tioon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Seri 1 3 4 4 3,7 4 1 3 3 3 3,0 3 Stepnaya75 2 3 3 4 3,3 3 2 3 3 3 3,0 3 Stepnaya1414 3 3 3 4 3,3 3 3 3 3 3 3,0 3 Gvk2055-1 4 4 3 3 3,3 3 4 4 3 3 3,3 3 Lutestsens2 5 1 2 1 1,3 1 5 1 1 1 1,0 1

53 Continuation of table – 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Line-с-19sb 6 3 3 3 3,0 3 6 3 4 4 3,7 4 Karabalykskaya 20 7 3 3 4 3,3 3 7 3 3 4 3,3 3 Fantaziya 8 4 4 4 4,0 4 8 3 3 3 3,0 3 Bostandyk 9 4 4 3 3,7 4 9 4 4 4 4,0 4 Lutescens30 69/97 10 3 3 4 3,3 3 10 4 4 5 4,3 5 Karagandinskaya 11 4 4 4 4,0 4 11 3 3 3 3,0 3

Karagandinskaya 12 4 4 4 4,0 4 12 3 4 4 3,7 4 31 Pavlodarskaya 13 2 2 3 2,3 2 13 2 3 3 2,7 3 yubileynaya Konditerskaya 14 3 3 4 3,3 3 14 3 3 3 3,0 3 yarovaya Fitonс-50sb 15 3 3 3 3,0 3 15 3 3 4 3,3 3 Fiton82 16 3 3 3 3,0 3 16 4 3 3 3,3 3 Fiton-с-54sb 17 3 3 3 3,0 3 17 2 2 3 2,3 2 Ekada148 18 1 1 1 1,0 1 18 1 2 1 1,3 1 x Ekada 113 19 3 3 4 3,3 3 19 4 4 4 4,0 4 Lyubava 20 4 4 4 4,0 4 20 5 4 4 4,3 5 Fiton 41 21 3 3 3 3,0 3 21 3 3 3 3,0 3 Fiton 204 22 2 2 3 2,3 2 22 4 3 3 3,3 3 Vladimir 23 3 4 3 3,3 3 23 3 4 3 3,3 3 Tselina50 24 4 4 4 4,0 4 24 4 4 4 4,0 4 Tselinnaya niva 25 3 4 3 3,3 3 25 4 4 4 4,0 4 Asylsapa 26 3 3 3 3,0 3 26 3 3 2 2,7 3 Akmola 2 27 3 4 3 3,3 3 27 4 4 4 4,0 4 Ak orda 28 3 3 4 3,3 3 28 3 3 3 3,0 3 Shortandinskaya 29 4 3 4 3,7 4 29 4 4 4 4,0 4 2012 Tselinnaya 3s 30 4 3 3 3,3 3 30 4 3 3 3,3 3

54 Continuation of table – 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Astana 31 4 4 4 4,0 4 31 4 4 4 4,0 4 Altaiskaya70 32 3 3 3 3,0 3 32 4 3 4 3,7 4 Altaiskaya110 33 3 2 3 2,7 3 33 2 2 2 2,0 2 Tobolskaya 34 3 4 4 3,7 4 34 4 4 4 4,0 4 Altayskaya 35 4 3 3 3,3 3 35 3 4 3 3,3 3 Stepnaya volna 36 3 3 3 3,0 3 36 3 3 4 3,3 3 Apasovka 37 4 4 4 4,0 4 37 5 4 4 4,3 5 Lutenscens89-06 38 4 4 4 4,0 4 38 3 4 4 3,7 4 Duet 39 4 4 3 3,7 4 39 4 4 4 4,0 4 Pavlogradka 40 3 3 3 3,0 3 40 3 4 4 3,7 4 Lutescens29-12 41 4 4 4 4,0 4 41 3 4 4 3,7 4 Lutescens106-11 42 2 3 3 2,7 3 42 3 3 3 3,0 3 Tulaikovskaya110 43 3 4 3 3,3 3 43 3 3 3 3,0 3 Lutescens916 44 2 2 3 2,3 2 44 2 3 2 2,3 2 x Grecum1003 45 1 2 1 1,3 1 45 2 1 1 1,3 1 x Lutescens1062 46 4 4 4 4,0 4 46 3 3 3 3,0 3 Lutescens89-06 47 2 2 3 2,3 2 47 3 2 2 2,3 2 x Eritrospermum85-08 48 3 3 4 3,3 3 48 3 3 4 3,3 3 Serebristaya 49 4 3 3 3,3 3 49 4 3 3 3,3 3 Seri 50 3 3 4 3,3 3 50 4 4 3 3,7 4 Boevchanka 51 3 3 4 3,3 3 51 4 3 4 3,7 4 Omskaya 37 52 5 3 4 4,0 4 52 4 4 4 4,0 4 Lutestsens7-04-4 53 3 3 3 3,0 3 53 3 3 3 3,0 3 Lutestsens197-04-7 54 4 4 3 3,7 4 54 3 4 4 3,7 4 Lutestsens220 55 4 4 5 4,3 5 55 4 4 3 3,7 4 Tulaikovskaya 10 56 3 3 3 3,0 3 56 4 3 3 3,3 3 Tulaikovskaya 57 4 3 4 3,7 4 57 3 3 3 3,0 3 zolotistaya

55 Continuation of table – 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Tulaikovsk 100 58 1 1 1 1,0 1 58 1 2 1 1,3 1 x Grekum 650 59 3 3 4 3,3 3 59 4 4 4 4,0 4 Lutescens 920 60 4 4 4 4,0 4 60 4 3 4 3,7 4

Ekada 121 61 4 4 4 4,0 4 61 5 4 4 4,3 5 Cimmyt 62 1 1 1 1,0 1 62 1 2 2 1,7 1 x P-23-17 63 4 4 3 3,7 4 63 3 3 3 3,0 3 Pamyati ruba 64 4 3 4 3,7 4 64 3 4 4 3,7 4 Chelyaba 75 65 4 3 3 3,3 3 65 3 3 3 3,0 3 Eritrospermum 66 4 4 4 4,0 4 66 3 3 4 3,3 3 23707 Sy tyra 67 1 1 2 1,3 1 67 1 1 1 1,0 1 x Sy goliad 68 3 4 3 3,3 3 68 3 3 3 3,0 3 Sy soren 69 4 3 3 3,3 3 69 3 3 4 3,3 3 Sy rowyn 70 1 1 1 1,0 1 70 2 1 1 1,3 1 x Sy ingmar 71 1 1 1 1,0 1 71 1 2 1 1,3 1 x Select 72 1 2 1 1,3 1 72 1 1 1 1,0 1 x Fore front 73 3 3 3 3,0 3 73 3 4 3 3,3 3 Prevail 74 3 4 3 3,3 3 74 2 3 3 2,7 3 Advance 75 3 3 4 3,3 3 75 4 3 3 3,3 3 Brick 76 3 3 3 3,0 3 76 4 3 3 3,3 3 Carberry 77 3 4 3 3,3 3 77 3 3 3 3,0 3 Muchmore 78 2 1 1 1,3 1 78 1 2 2 1,7 1 x Uralosybirskaya 79 2 2 1 1,7 1 79 1 1 1 1,0 1 x Tornado 22 80 3 2 2 2,3 2 80 2 3 3 2,7 3 Lyutestsens 1012 81 3 3 4 3,3 3 81 3 3 3 3,0 3 Lyutestsens 208-08-4 83 3 4 3 3,3 3 83 3 3 3 3,0 3 Lyutestsens 27-12 84 2 2 1 1,7 1 84 1 1 1 1,0 1 x Eritrospermum85-08 85 4 4 4 4,0 4 85 3 3 3 3,0 3

56 Continuation of table – 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Lyutestsens6-04- 86 1 2 1 1,3 1 86 2 2 1 1,7 1 x

Lyutestsens 186- 87 4 4 4 4,0 4 87 3 3 3 3,0 3 04-61 Chebarkulskaya 3 88 4 4 4 4,0 4 88 4 5 4 4,3 5 Line d 25 89 4 4 4 4,0 4 89 3 4 3 3,3 3 Line 654 90 4 3 3 3,3 3 90 5 4 4 4,3 5 C 249 (mr) 91 2 1 2 1,7 1 2 2 2 2,0 2 C altay (mr) 92 2 3 2 2,3 2 2 2 3 2,3 2 C seri (s) 93 4 4 4 4,0 4 4 3 4 3,7 4 C sunco (mr) 94 3 2 2 2,3 2 2 2 2 2,0 2 C kutluk (s) 95 4 4 4 4,0 4 4 5 4 4,3 5 C suzen (s) 96 4 5 4 4,3 5 4 4 4 4,0 4 C kiziltan 97 4 5 4 4,3 5 4 4 4 4,0 4

The trials were conducted between March and May. The experiments are arranged as lattice design and each entry is replicated 6 times. Plants are scored for typical symptoms of root rot using a 0-5 scale.

Figure 13 - Assessment scale for root rot disease (crouth room, CIMMYT, Turkey, 2017)

57 The results of stability evaluation from the 17CBHL, 98 varieties lines are presented. Six of them are standard and medium standard varieties of spring wheat to root rot disease. Donors of resistance with 17CBHL gene lines were found on the following scale: stable, Resistant, 1: 1-9%, moderately resistant, 2: 10-29%, moderately susceptible, 3: 30-69%, susceptible, 4:70-89%, and highly susceptible, 5: 90-99%, and also the results of their use in breeding. Under laboratory conditions, the most aggressive strains of Fusarium culmorum were used for inoculation, which was collected from the wheat fields of the Central Anatolian Plateau, Kirsehir, Turkey. However, tolerant reaction is being estimated by evaluating the yield under both infested versus non infested plots. The desired lines are those having both resistant and tolerance reactions to soil borne disease. By this research work shown, the efficiency of using genes under laboratory conditions for disease root rot in spring wheat breeding along the Kazakhstan and Siberian lines [131, 15 p]. By results of experiments were selected next gens of wheat genotypes: Ekada148, Lutescens916, Grecum1003, Lutescens89-06, Tulaikovsk 100, Cimmyt, Sy tyra, Sy rowyn, Sy Ingmar, Select, Muchmore, Uralosybirskaya, Lyutestsens 27-12, Lyutestsens 6-04-4.

Table 10– Screening of genotypes of wheat to the disease root rot, pathogen Fusarium colmorium (field condition, Kaskelen experimental station 2017)

№ Gene Number Number Length Length Number Root rot 1000 of plants of of the of ear of ear (selection) seeds steams growth (cm) weight (cm) 1 2 3 4 5 6 7 8 9 19 1 6 54 9 14 1 2 4 38 8 13 0 3 5 48 8 14 0 103 4 2 50 10 14 1 5 2 50,5 10,5 18 0 6 5 48 8 13 0 x 2 45 1 13 37 6 5 0 2 12 54 9 15 0 36 x 3 46 1 14 75 10 18 0 172 x

58 Continuation of table – 10

1 2 3 4 5 6 7 8 9 4 48 1 6 59 10,5 16 1 2 4 53 9 15 0 3 5 67 9 17 1 4 4 66 9 13 0 135 5 9 47 9 15 0 6 11 48 8 13 0 x 5 59 1 5 71,5 9 14 0 2 3 58 10 15 0 60 x 6 63 1 3 54 8 14 0 2 5 55 10 16 0 70 3 2 46 7 15 0 x 7 68 1 2 64 11 18 1 2 1 39 6 10 0 84 3 6 53 7 13 0 4 4 54 9 15 0

1 2 3 4 5 6 7 8 9 8 71 1 1 56 11 18 0 2 1 41 8 16 1 3 2 50 8 14 0 72 4 5 49 8 14 0 x 9 72 1 6 59 11 13 0 2 2 43 9 12 0 120 3 4 38 8 11 0 x 10 73 1 2 61 10 20 0 2 6 87 14 21 1 86 3 6 32,5 6 11 0 x 11 79 1 6 32 6 13 0 2 2 30 7 11 0 3 5 32 5 6 1 121 4 5 44 8 13 1 5 3 59 10 15 0 x

59 Continuation of table – 10

1 2 3 4 6 7 8 9 10 12 80 1 2 60 8 14 1 2 3 42 6 10 1 136 3 5 55 8 11 0 4 7 46 7 12 0 5 7 44 4 8 0 6 4 36 6 9 1 7 3 40 6 10 0 x 13 85 1 5 45 8 12 0 2 4 40 6 11 1 69 3 4 42 7 12 0 x 14 87 1 1 45 9 15 0 2 1 37 7 8 0 49 3 4 40 7 7 1 x

At field condition seeding was done mid of May. For field experiment was used 107 varieties of wheat. The mechanical composition of the soil was made up of dark brown soil, medium loamy soil, and humus (3.0 – 3.5%), pH 7.0. Forecrop was spring wheat plowed to the depth of 15-17 cm by “BD” disc header, pre sowing cultivation, rolling after sowing. Seed application rate was 4.0 mln pcs/ha, while depth was 5-6 cm. By results of experiments were selected next gens of wheat genotypes: Ekada148, Lutescens916, Grecum1003, Lutescens89-06, Tulaikovsk 100, Cimmyt, Sy tyra, Sy rowyn, Sy Ingmar, Select, Muchmore, Uralosybirskaya, Lyutestsens 27-12, Lyutestsens 6-04-4 [145].

4.1.2 Assessment of screening methods to identify resistance to Heterodera filipjevis in wheat Resistant wheat cultivars can be very effective in controlling cyst nematodes. Research to identify resistance sources and to characterise molecular markers for resistant phenotypes is ongoing in wheat and its wild relatives. However, there are very few studies focusing on the mechanism of resistance in wheat-nematode interactions. Wheat landraces and domesticated genotypes possess genetic variation including resistance to biotic and abiotic stresses (Kimber & Feldman, 1987). The screening of 94 winter wheat accessions resulted in identifying 15.54% as resistant, 7.77% as moderately resistant, 25.56% as moderately susceptible, 25.56% as susceptible and 25.56% as highly susceptible to H. filipjevis.

60 Table 11– Screening of genotypes of wheat to the disease of root, nematode Heterodera filipjevis (crouth room, CIMMYT, Turkey, 2017)

Gene name R1 R2 R3 Mean Grouping Selection Seri 13 15 13 13,7 4 Stepnaya75 25 22 18 21,7 5 Stepnaya1414 16 10 15 13,7 4 Gvk2055-1 28 25 26 26,3 5 Lutestsens2 16 12 14 14,0 4 Line-с-19sb 10 12 11 11,0 3 Karabalykskaya 20 10 14 10 11,3 3 Fantaziya 11 12 13 12,0 3 Bostandyk 9 10 13 10,7 3 Lutescens 30 69/97 3 3 2 2,7 1 x Karagandinskaya 30 21 24 26 23,7 5 Karagandinskaya 31 13 23 15 17,0 5 Pavlodarskaya yubileynaya 22 21 16 19,7 5 Konditerskaya yarovaya 18 25 24 22,3 5 Fitonс-50sb 8 13 10 10,3 3 Fiton82 10 14 9 11,0 3 Fiton-с-54sb 16 16 13 15,0 4 Ekada148 4 8 5 5,0 2 x Ekada 113 13 11 14 12,7 4 Lyubava 11 12 18 13,7 4 Fiton 41 5 5 6 5,3 2 x Fiton 204 4 5 6 5,0 2 x Vladimir 16 12 14 14,0 4 Tselina50 13 13 10 12,0 3 Asylsapa 4 3 3 3,3 1 x Akmola 2 5 5 3 4,3 1 x Ak orda 15 20 14 16,3 4 Shortandinskaya 2012 14 12 10 12,0 3 Tselinnaya 3s 3 3 3 3,0 1 x Astana 6 5 4 5,0 2 x Altaiskaya70 4 2 3 3,0 1 x Altaiskaya110 16 15 20 17,0 5 Tobolskaya 25 17 20 20,7 5 Altayskaya zhnitsa 14 10 20 14,7 4 Stepnaya volna 14 10 19 14,3 4 Apasovka 18 17 20 18,3 5 Lutenscens89-06 21 22 13 18,7 5 Duet 19 10 15 14,7 4 Pavlogradka 12 9 14 11,7 3 Lutescens29-12 4 5 4 4,3 1 x Lutescens106-11 7 5 3 5,0 2 x Tulaikovskaya110 10 9 5 8,0 3

61 Continuation of table – 11

1 2 3 4 5 6 7 Lutescens916 8 3 5 5,3 2 x Grecum1003 4 5 4 4,3 1 x Lutescens1062 26 19 20 21,7 5 Lutescens89-06 3 5 5 4,3 1 x Eritrospermum85-08 4 4 3 3,7 1 x Serebristaya 14 13 19 15,3 4 Seri 10 11 13 11,3 3 Boevchanka 12 14 13 13,0 4 Omskaya 37 20 22 25 22,3 5 Lutestsens7-04-4 4 7 9 6,7 3 Lutestsens197-04-7 12 12 10 11,3 3 Lutestsens220-03-45 4 3 3 3,3 1 x Tulaikovskaya 10 14 10 10 11,3 3 Tulaikovskaya zolotistaya 12 12 10 11,3 3 Tulaikovsk 100 15 13 10 12,7 4 Grekum 650 4 4 3 3,7 1 x Lutescens 920 4 5 6 5,0 2 x Ekada 121 14 15 21 16,7 4 Cimmyt 25 27 15 22,3 5 P-23-17 16 19 15 16,7 4 Pamyati ruba 6 8 5 6,3 3 Chelyaba 75 10 11 12 11,0 3 Eritrospermum 23707 21 23 13 19,0 5 Sy tyra 26 23 22 23,7 5 Sy goliad 15 10 14 13,0 4 Sy soren 14 21 13 16,0 4 Sy rowyn 12 15 13 13,3 4 Sy ingmar 30 25 24 26,3 5 Select 2 3 5 3,3 1 x Fore front 10 13 11 11,3 3 Prevail 10 12 13 11,7 3 Advance 24 23 26 24,3 5 Brick 24 20 22 22,0 5 Carberry 11 13 12 12,0 3 Muchmore 25 17 24 22,0 5 Uralosybirskaya 11 15 12 12,7 4 Tornado 22 18 20 13 17,0 5 Lyutestsens 1012 13 14 11 12,7 4 Lyutestsens 7-04-10 21 16 15 17,3 5 Lyutestsens 208-08-4 4 4 5 4,3 1 x Lyutestsens 27-12 11 15 12 12,7 4 Eritrospermum 85-08 20 14 23 19,0 5 Lyutestsens 6-04-4 15 12 11 12,7 4

62 Continuation of table – 11

1 2 3 4 6 7 8 Lyutestsens 186-04-61 11 12 14 12,3 3 Chebarkulskaya 3 8 13 12 11,0 3 Line d 25 25 27 17 23,0 5 Line 654 12 15 10 12,3 3 Bezostaya (s) 14 11 13 12,7 4 Katea (R) 4 3 3 3,3 1 Kutluk (HS) 17 15 19 17,0 5 Sonmez (MR) 7 6 5 6,0 2

The trials were conducted between March and May. The experiments are arranged as lattice design and each entry is replicated 3 times. Plants are scored by caunting cysts. By the results determined: resistant – 14 genotypes of wheat, as moderately resistant – 7, as moderately susceptible - 23, as susceptible - 23 and as highly susceptible - 23 to H. filipjevis.

Figure 14 – Assessment scale for Heterodera filipjevis to 94 lines of wheat genotypes (crouth room, CIMMYT, Turkey, 2017)

The screening of 94 winter wheat accessions resulted in identifying 15.54% as resistant, 7.77% as moderately resistant, 25.56% as moderately susceptible, 25.56% as susceptible and 25.56% as highly susceptible to H. filipjevis. By the experiment selected 21 resistant genotypes of wheat to H. filipjevis. They are: lutescens 30 69/97, ekada148, fiton 41, fiton 204, tselinnaya niva, asylsapa, akmola 2, tselinnaya 3s, astana, altaiskaya70, lutescens29-12, lutescens106-11, lutescens916,

63 grecum1003, lutescens89-06, eritrospermum85-08, lutestsens220-03-45, grekum 650, lutescens 920, select, lyutestsens 208-08-4 From the results of screening experiment, confirmed that wheat accessions 21 possess resistance and can subsequently be crossed with high yielding cultivars improving their genetic resistance to CCNs.

4.2 Survey and determenation of cyst nematodes (Heterodera spp) in some cereal growing regions of Kazakhstan Kazakhstan is an important producer and exporter of high quality wheat. Average annual production is about 13 million tons, but output is highly dependent on weather and in recent years has fluctuated between 10 and 17 million tons. Between 2 and 8 million tons is exported annually, mainly to destinations in Europe (including Russia and Ukraine), northern Africa, and Central Asia. Accordingly, the greatest attention in Kazakhstan (as well as throughout the country) was paid to the study of nematode fauna of the most important crops (cereals, vegetables, vegetables, fruits, etc). The main purpose of such studies was to maximize the species diversity of nematodes associated with specific agricultural crops, mainly on a qualitative basis. The detection of the presence of harmful phytoparasitic species in this case was secondary and disordered. For example, as a result of long-term studies of the nematode fauna of cereal crops, 62 species of these worms belonging to 14 families and 4 orders were identified, phytoparasitic species among them were only 7 species from the family of hopllyaimids, and cystogenic and root nematodes were not detected. In the Kustanai region, 116 species of nematodes related to 49 genera, 25 families, and 5 orders were detected on wheat, but only 24 phytohelminths without information on their harmfulness and prevalence. Thereofere, at the time, the most harmful pathogenis are cereal cyst and root lesion nematode, which have been documented to cause economic yield loss on rainfed wheat production systems in several part of the world. Nematodes are tiny but complex unsegmented roundworms that are anatomically differentiated for feeding, digestion, locomotion, and reproduction. These small animals occur worldwide in all environments. Most species are beneficial to agriculture. They make important contributions to organic matter decomposition and the food chain. One type of plant-parasitic nematode forms cysts that contain eggs from which juvenile nematodes hatch to damage and reduce yields of many agriculturally important crops. The cyst nematode genus Heterodera contains as many as 70 species, including a complex of 12 species known as the Heterodera avenae group. Species in this group invade and reproduce only in living roots of cereals and many species of grasses. They do not reproduce on any broadleaf plant. Three species in the H. avenae group (H. avenae, H. filipjevi, and H. latipons) cause important economic losses in small grain crops worldwide and are known as the cereal cyst nematodes.

64 Table 12 – Distribution of cyst nematodes in some fields of Shortandy region, 2017.

№ CNAME OC ORIGINATOR Place of CCN swoing 1 SERI Astana 1 2 LUTESTSENS2 KAZ KARABALYK ARS Astana 1 3 FITON-С-54SB KAZ FITON-CIMMYT Astana 1 4 EKADA148 KAZ FITON-EKADA Astana 1 5 SHORTANDINSKAYA 2012 KAZ SHORTANDY ARI Astana 1 6 TSELINNAYA 3S KAZ SHORTANDY ARI Astana 1 7 ASTANA KAZ SHORTANDY ARI Astana 1 8 LUTESCENS29-12 RUS OMGAU Astana 1 9 LUTESCENS106-11 RUS OMGAU Astana 1 10 LUTESCENS89-06 RUS OMGAU Astana 2 11 SEREBRISTAYA RUS SIB ARI Astana 1 12 LUTESTSENS7-04-4 RUS SIB ARI Astana 1 13 TULAIKOVSKAYA RUS SAMARA Astana 1 ZOLOTISTAYA 14 TULAIKOVSK 100 RUS SAMARA Astana 1 15 P-23-17 RUS KURGAN Astana 1 16 PAMYATI RUBA RUS CHELYABINSK Astana 1 17 SY TYRA US-SYN US-SYN Astana 1 18 ADVANCE US-SDSU US-SDSU Astana 1 19 BRICK US-SDSU US-SDSU Astana 1 20 MUCHMORE CAN Astana 1 21 URALOSYBIRSKAYA RUS Astana 1 22 LYUTESTSENS 27-12 RUS OMGAU Astana 1 23 ERITROSPERMUM 85-08 RUS OMGAU Astana 1 24 LYUTESTSENS 6-04-4 RUS SIB ARI Astana 1 25 LINE D 25 RUS SARATOV Astana 1 26 LINE 654 RUS SARATOV Astana 1

65

Figure 14 – Distribution of cyst nematodes in some fields of Shortandy region, 2017.

In 2017 by doing survey some main wheat growing areas (North Kazakhstan – province Shortandy) was taken 90 soil samples. As a result, was found 27 cyst nematodes from 90 siol samples at province of Shortandy. Performing microscopic identification of intercepted nematodes, the following species of plant parasitic nematodes were identified – Heterodera spp. Damage from cereal cyst nematodes is greatest when susceptible crops are produced annually. Cereal cysts nematodes are also capable of reproducing on a wide range of economically important grasses that include bentgrass, bluegrass, fescue, ryegrass, brome, orchard grass, canary grass, timothy, and sorghum. These crops should not precede wheat, barley, or oat in crop rotations on fields where cereal cyst nematodes are known to be present [146]. Cereal cyst nematodes (CCN) are a global economic problem for cereal production. Heterodera filipjevi is one of the most commonly identified and widespread CCN species found in many wheat production regions of the world. Cereal cyst nematodes are more readily detected on the roots of seed lings than on adult plants. The roots of infested plants develop frequent branches (picture at left) and swellings (cysts). The adult females, which are the size of a pinhead, are colored off white when young, turning into dark brown cysts as they age (picture in middle). Yield loss has been documented on cereals in Europe, north western India, southern Australia, Pakistan, Saudi Arabia, and the Pacific Northwest of the USA, but documented distribution has occurred in many more countries throughout the world.

66

Figure 15 – CCNs comprise a number of closely related species and are found in most regions where cereals are produced (map maked by Abdelfattah Amer Dababat, CIMMYT, Turkey, 2016)

67

Yield losses can also become very high in 2-year rotations (cereals with summer fallow or a crop such as potato) and 3-year rotations (e.g., winter wheat, a spring cereal, and a nonhost broadleaf crop or fallow). Crop rotations that include several years of broadleaf crops, corn, fallow, or resistant wheat, barley, or oat varieties can greatly reduce the nematode density. In general, growing a susceptible host only once during a 3- to 4-year period can dramatically reduce the density of H. avenae in soil.

Table 13 – Distribution of cyst nematodes in some fields of Uralsk region, 2017

№ CNAME OC ORIGINATOR Place of CCN swoing 9 LUTESCENS106-11 RUS OMGAU Uralsk 1 17 SY TYRA US-SYN US-SYN Uralsk 2 18 ADVANCE US-SDSU US-SDSU Uralsk 2 21 URALOSYBIRSKAYA RUS Uralsk 1 70 SY ROWYN US-SYN US-SYN Uralsk 1 89 LINE D 25 RUS SARATOV Uralsk 1 90 LINE 654 RUS SARATOV Uralsk 2

Figure 16 – Distribution of cyst nematodes in some fields of Uralsk, 2017.

From West Kazakhstan, Uralsk research station was taken 90 soil sample by 350 gr, collected 9 cysts. Performing microscopic identification of intercepted nematodes, the following species of plant parasitic nematodes were identified – Heterodera spp. The results were given on the table 13, Figure 16.

68 Table 14 – Distribution of cyst nematodes in some fields of Almaty region, 2017

№ CNAME OC ORIGINATOR Place of CCN swoing 1 2 3 4 5 6 718 SERI Almaty 2 2 STEPNAYA75 KAZ AKTOBE ARS Almaty 1 3 STEPNAYA1414 KAZ AKTOBE ARS Almaty 4 4 GVK2055-1 KAZ EAST-KAZAKHSTAN ARI Almaty 3 5 LUTESTSENS2 KAZ KARABALYK ARS Almaty 3 KARABALYK ARS- Almaty 1 6 LINE-С-19SB KAZ CIMMYT 7 KARABALYKSKAYA 20 KAZ KARABALYK ARS Almaty 5 8 FANTAZIYA KAZ KARABALYK ARS Almaty 4 KARABALYK & KAZ RI Almaty 4 9 BOSTANDYK KAZ PLANT PROTACTİON 10 LUTESCENS 30 69/97 KAZ KARABALYK ARS Almaty 6 11 KARAGANDINSKAYA 30 KAZ KARAGANDA ARI Almaty 10 12 KARAGANDINSKAYA 31 KAZ KARAGANDA ARI Almaty 5 PAVLODARSKAYA Almaty 5 13 YUBILEYNAYA KAZ PAVLODAR ARI KONDITERSKAYA Almaty 3 14 YAROVAYA KAZ PAVLODAR ARI 15 FITONС-50SB KAZ FITON-CIMMYT Almaty 5 16 FITON82 KAZ FITON Almaty 2 17 FITON-С-54SB KAZ FITON-CIMMYT Almaty 0 18 EKADA148 KAZ FITON-EKADA Almaty 4 19 EKADA 113 KAZ FITON Almaty 1 20 LYUBAVA KAZ FITON Almaty 5 21 FITON 41 KAZ FITON Almaty 1 22 FITON 204 KAZ FITON Almaty 2 23 VLADIMIR KAZ SHORTANDY ARI Almaty 3 24 TSELINA50 KAZ SHORTANDY ARI Almaty 5 25 TSELINNAYA NIVA KAZ SHORTANDY ARI Almaty 5 26 ASYLSAPA KAZ SHORTANDY ARI Almaty 1 27 AKMOLA 2 KAZ SHORTANDY ARI Almaty 4 28 AK ORDA KAZ SHORTANDY ARI Almaty 2 29 SHORTANDINSKAYA 2012 KAZ SHORTANDY ARI Almaty 5 30 TSELINNAYA 3S KAZ SHORTANDY ARI Almaty 3 31 ASTANA KAZ SHORTANDY ARI Almaty 4 32 ALTAISKAYA70 RUS ALTAY ARI Almaty 3 33 ALTAISKAYA110 RUS ALTAY ARI Almaty 2 34 TOBOLSKAYA RUS ALTAY ARI Almaty 5 35 ALTAYSKAYA ZHNITSA RUS ALTAY ARI Almaty 1 36 STEPNAYA VOLNA RUS ALTAY ARI Almaty 1

69 Continuation of table – 14

1 2 3 4 5 6 37 APASOVKA RUS ALTAY ARI Almaty 1 38 LUTENSCENS89-06 RUS OMGAU Almaty 1 39 DUET RUS OMGAU Almaty 1 40 PAVLOGRADKA RUS OMGAU Almaty 1 41 LUTESCENS29-12 RUS OMGAU Almaty 2 42 LUTESCENS106-11 RUS OMGAU Almaty 3 43 TULAIKOVSKAYA110 RUS SAMARA Almaty 1 44 LUTESCENS916 RUS SAMARA Almaty 4 45 GRECUM1003 RUS SAMARA Almaty 1 46 LUTESCENS1062 RUS SAMARA Almaty 1 59 GREKUM 650 RUS SAMARA Almaty 1 60 LUTESCENS 920 RUS SAMARA Almaty 1 61 EKADA 121 RUS SAMARA Almaty 2 62 CIMMYT RUS SAMARA Almaty 2 63 P-23-17 RUS KURGAN Almaty 2 64 PAMYATI RUBA RUS CHELYABINSK Almaty 1 74 PREVAIL US-SDSU US-SDSU Almaty 4 88 CHEBARKULSKAYA 3 RUS CHELYABINSK Almaty 1 89 LINE D 25 RUS SARATOV Almaty 2 90 LINE 654 RUS SARATOV Almaty 1

Figure 17 – Distribution of cyst nematodes in some fields of Almaty, 2017.

In 2017 by doing survey some main wheat growing areas (South Kazakhstan – Almaty region) collected 150 cyst nematodes from 90 soil samples. Performing

70 microscopic identification of intercepted nematodes, the following species of plant parasitic nematodes were identified – Heterodera spp [147]. Cereal cysts nematodes are also capable of reproducing on a wide range of economically important grasses that include bentgrass, bluegrass, fescue, ryegrass, brome, orchard grass, canary grass, timothy, and sorghum. These crops should not precede wheat, barley, or oat in crop rotations on fields where cereal cyst nematodes are known to be present.

4.3 Identification and spreading of plant parasite nematodes in wheat growing areas of west and south – east part of Kazakhstan Nematodes are a diverse group of worm-like animals. They are found in virtually every environment, both as parasites and as free-living organisms. They are generally minute, but some species can reach several meters in length. This guide focuses specifically on plant parasitic nematodes, which are very small or microscopic, can cause significant damage to crops, and are extremely widespread. Plant parasitic nematodes are mostly thread-like worms ranging from 0.25 mm to >1.0 mm long, with some up to 4.0 mm. Although most taper toward the head and tail, they come in a variety of shapes and sizes. Females of some species lose their worm-like shape as they mature, becoming enlarged and pear-, lemon- or kidney-shaped or spherical as adults. Plant parasitic nematodes are ever-present and are incidental with plant growth and crop production. They are significant constraints to sustainable agriculture and can be difficult to control. Determining the importance of individual nematode species, nematode communities and nematodes in combination with other problems is not a simple task at the best of times, but is more difficult in tropical than in temperate climates. Species previously not known to cause crop damage are continually being discovered, particularly as agriculture changes to suit changing needs and new crops are introduced. By doing survey in west and south – East part of Kazakhstan was taken 180 soil samples. From West Kazakhstan, Uralsk research station was taken 90 soil sample by 150 gr, also from South – East Kazakhstan, Kaskelen research station was 90 soil samples. The results were given on the table – 13

Table 15 – spreading of plant parasite nematodes in Uralsk and Kaskelen research station, 2017 year

N Place free Aphe Aphe Dory Tych Filen Pratylen Paraty Dity sam- livin len len lai elenchu chus chus lenchus len- ple g chus choides med s chus 1 2 3 4 5 6 7 8 9 10 11 1 Almaty 40 10 10 4 Almaty 20 20 10 10

71 Continuation of table – 15

1 2 3 4 5 6 7 8 9 10 11 5 Almaty 40 70 8 Almaty 60 30 20 30 20 9 Almaty 30 10 30 10 10 Almaty 20 10 20 10 10 11 Almaty 110 10 60 10 13 Almaty 60 20 60 20 19 Almaty 40 20 10 10 10 20 Almaty 10 10 10 10 10 21 Almaty 70 30 20 22 Almaty 10 10 10 24 Almaty 30 25 Almaty 50 10 20 20 10 29 Almaty 80 31 Almaty 20 20 10 34 Almaty 50 20 10 36 Almaty 130 20 37 Almaty 20 10 10 38 Almaty 40 10 10 10 39 Almaty 180 100 70 10 47 Almaty 90 10 30 48 Almaty 30 20 20 10 50 50 Almaty 50 10 52 Almaty 40 10 20 10 20 74 Almaty 20 20 10 30 77 Almaty 50 30 10 30 10 30 10 78 Almaty 79 Almaty 30 10 10 10 10 81 Almaty 20 10 30 20 10 20 91 Uralsk 30 10 10 95 Uralsk 50 10 10 10 96 Uralsk 40 10 100 Uralsk 50 101 Uralsk 40 30 102 Uralsk 70 106 Uralsk 80 20 10 107 Uralsk 50 10 10 20 108 Uralsk 30 10

72 Continuation of table – 15

1 2 3 4 5 6 7 8 9 10 11 109 Uralsk 20 10 10 110 Uralsk 30 10 10 10 111 Uralsk 60 10 113 Uralsk 80 30 30 10 114 Uralsk 90 10 118 Uralsk 20 20 20 119 Uralsk 20 20 121 Uralsk 10 10 125 Uralsk 40 10 12 Uralsk 30 30 134 Uralsk 90 30 20 30 137 Uralsk 50 40 10 10 10 143 Uralsk 110 50 10 144 Uralsk 30 20 10 145 Uralsk 10 146 Uralsk 100 40 10 30 147 Uralsk 10 10 167 Uralsk 20 10 40 172 Uralsk 20 10 174 Uralsk 10 10 10 10 176 Uralsk 10 10 177 Uralsk 80 10

Figure 18 – spreading of PPN at Kaskelen research station, 2017 year

73

Figure 19 – spreading of PPN at Uralsk research station, 2017 year)

Figure 20 – Pratylenchus spp (Bolu, Turkey, 2017year)

74

Figure 21 – Pratylenchus spp (Bolu, Turkey, 2017year)

Figure 22 – Pratylenchus spp (Bolu, Turkey, 2017year)

75

Figure 23 – Aphelenchoides spp (Bolu, Turkey, 2017year

Figure 24 – Pratylenchus spp (Bolu, Turkey, 2017year) 76 Aphelenchus (Aphelenchus) Bastian, 1865. (Cobb, 1927). From the family Aphelenchidae: Body tapering anteriorly. Cuticle transversely striated. Lateral field with numerous incisures. Deirids present at about level of the excretory pore. Head slightly offset. Spear shaft with slight thickenings at the base. Procorpus cylindrical, constricted slightly where it joins the ovoid median oesophageal bulb which contains prominent, median, crescentic valve plates. Oesophageal glands usually with, sometimes without a lobe overlapping the intestine dorso laterally and joining the alimentary canal where the nerve ring surrounds it just posterior to the median bulb. Excretory pore about opposite nerve ring. Intestine joined to median bulb by a short isthmus about 11/2 body widths long. Vulva posterior, ovary outstretched, prodelphic; a post vulval sac present, rather obscure but usually reaching about half-way from vulva to anus.Vagina with thickened walls. Rectum about one to two body-widths long. Tail between on and four anal body-widths long, cylindrical to a rounded end. Phasmids subterminal. Male with bursa usually supported by one pre anal and about three post anal, subterminal pairs of ribs. Spicules paired, slender, ventrally slightly arcuate, proximally slightly cephalated. Gubernaculum about a third as long as the spicules. Bionomics: Mycophagous, soil inhabiting. Aphelenchoides is a genus of plant pathogenic foliar nematodes. In 1961 Sanwal listed 33 species and provided a key. The most important species of these are Aphelenchoides ritzemabosi, the chrysanthemum foliar nematode; Aphelenchoides fragariae, the spring crimp or spring dwarf nematode of strawberry, which also attacks many ornamentals; and Aphelenchoides besseyi, causing summer crimp or dwarf of strawberry and white tip of rice. Several species of this genus feed ectoparasitically and endoparasitically on aboveground plant parts. Genus Tylenchus (Bastian, 1865). Small nemas rarely over 1.0 mm long. Tails of both sexes similar, elongate conoid to filiform. Spear knobbed. Median bulb with valvular apparatus. Cardia present. Vulva well posterior to middle of body. Anterioovary outstretched. Posterior uterine branch rudimentary. Spermatheca a definite pouch in uterine branch. Bursa adanal. Phasmids not visible. Genus Filenchus (Andrassy, 1954). Head sclerotization delicate, stylet usually <15um; cone less than half total stylet length. Transverse striae usually extend onto head up to small labial plate which is squarish with rounded corners; four cephalic sensilla present or absent. Amphidial apertures usually elongate slits begining near oral disc or at edge of labial plate, extending laterally through three or four head annuli; rarely small elliptical aperture confined to labial plate. Body annuli fine to coarse. Lateral field two lines setting off a single plain band or three or four lines. Tail elongate conoid, curved or straight, to effilate/filiform even hair-like in outline. Pratylenchus is a genus of nematodes known commonly as lesion nematodes.They are parasitic on plants and are responsible for root lesion disease on many taxa of host plants in temperate regions around the world. Lesion nematodes are migratory endoparasites that feed and reproduce in the root and move around, unlike the cyst or

77 root knot nematodes, which may stay in one place. They usually only feed on the cortex of the root.Species are distinguished primarily by the morphology of the stylets. Ditylenchus dipsaci is an plant pathogenic nematode that primarily infects onion and garlic.It is commonly known as the stem nematode, the stem and bulb eel worm, or onion bloat (in the United Kingdom). Symptoms of infection include stunted growth, discoloration of bulbs, and swollen stems. D. dipsaci is a migratory endoparasite that has a five stage life cycle and the ability to enter into a dormancy stage. D. dipsaci enters through stomata or plant wounds and creates galls or malformations in plant growth. This allows for the entrance of secondary pathogens such as fungi and bacteria. Management of disease is maintained through seed sanitation, heat treatment, crop rotation, and fumigation of fields. D. dipsaci is economically detrimental because infected crops are unmarketable [3, 50 p]. As mentioned before was taken 180 soil samples from both regions, by doing microscopically identification from 64 soil samples we found free living nematodes and plant parasite nematodes. Performing microscopic identification of intercepted nematodes, the following species of plant parasitic nematodes were identified – Aphelenchus spp – 260 pieces, Aphelenchoides spp – 290 pieces, Tylenchus spp – 50 pieces , Filenchus spp - 30 pieces, Pratylenchus spp - 30 pieces, Parapratylenchus spp – 10 pieces, Ditylenchus spp – 100 pieces at the province of Ural; also at the province of Almaty were identified - Aphelenchus spp – 303 pieces, Aphelenchoides spp – 570 pieces, Tylenchus spp – 110 pieces, Filenchus spp – 30 pieces, Pratylenchus spp – 170 pieces, Parapratylenchus spp – 90 piesec, Ditylenchus spp – 90 pieces [148].

4.4 Survey and identification of cyst nematodes from destination Astana to Kostanay In 2017 on August was taken 34 soil samples from every 15km from the distance Astana – Kostanay, collected 418 cyst nematodes. By doing survey determained infected places of CCN, GPS (coordinates): N 51.79737 E69.07957 , N 51.92537 E67.36339 , N 52.08652 E65.82018 , N 52.13801 E65.57913 .

Table 16 – Spreading of cereal cyst nematodes by distance Astana – Kostanay, 2017year

Country Region Province GPS (coordinates) sample weig CCN (gr) 1 2 3 4 5 6 7 Kazakhstan Astana Vozdvijenka N 51.25695 E71.13594 soil -1 302 1 Kazakhstan Astana Dolmatova N 51.31070 E70.95541 soil -2 519 0 Kazakhstan Astana Novoishimka N 51.33357 E70.74667 soil -3 307 3 Kazakhstan Astana Akmechit N 51.34506 E70.61421 soil -4 448 1

78 Continuation of table – 16

1 2 3 4 5 6 7 Kazakhstan Astana Dolmatova N 51.31316 E70.33214 soil -5 297 0 Kazakhstan Astana Dolmatova N 51.31316 E70.33214 soil -6 297 0 Kazakhstan Astana Pervomaik N 51.33424 E70.09822 soil -7 421 1

Kazakhstan Astana Novocherk N 51.40010 E69.93914 soil -8 300 0 e Kazakhstan Astana Astrahanka N 51.50440 E69.81050 soil -9 260 0 Kazakhstan Astana Zhaltyr N 51.63478 E69.85533 soil -10 323 0 Kazakhstan Astana Dolmatova N 51.67275 E69.67598 soil -11 291 5

Kazakhstan Astana Dolmatova N 51.76732 E69.49433 soil -12 325 0 Kazakhstan Astana Dolmatova N 51.80022 E69.33849 soil -13 356 8 Kazakhstan Astana Dolmatova N 51.79737 E69.07957 soil -14 425 31 Kazakhstan Astana Dolmatova N 51.80130 E68.87138 soil -15 302 0 Kazakhstan Astana Dolmatova N 51.81328 E68.63663 soil -16 300 7 Kazakhstan Astana Atbasar N 51.80207 E68.42405 soil -17 556 0 Kazakhstan Astana Atbasar N 51.74756 E68.29964 soil -18 341 0 Kazakhstan Astana Atbasar N 51.78610 E70.26752 soil -19 460 5 Kazakhstan Astana Perekatnoe N 51.78362 E68.09285 soil -20 571 2 Kazakhstan Astana Kairakty N 51.83607 E67.90561 soil -21 550 0 Kazakhstan Astana Dolmatova N 51.82668 E67.70901 soil -22 457 0 Kazakhstan Astana Dolmatova N 51.88147 E67.49546 soil -23 450 Kazakhstan Astana Dolmatova N 51.92537 E67.36339 soil -24 365 48 Kazakhstan Astana Dolmatova N 51.90625 E67.13081 soil -25 320 0 Kazakhstan Astana Dolmatova N 51.90327 E66.95406 soil -26 318 6 Kazakhstan Astana Dolmatova N 51.91494 E66.64499 soil -27 300 0 Kazakhstan Kostanay Esil N 51.94514 E66.48064 soil -28 491 0 Kazakhstan Kostanay Buzuluk N 51.87778 E66.21957 soil -29 415 6 Kazakhstan Kostanay Janyspai N 52.01166 E66.21959 soil -30 379 0 Kazakhstan Kostanay Janyspai N 52.03396 E66.02906 soil -31 440 2 Kazakhstan Kostanay Chelgashy N 52.08652 E65.82018 soil -32 561 262

79 Continuation of table – 16

1 2 3 4 5 6 7 Kazakhstan Kostan Dolmatova N 52.13801 E65.57913 soil -33 473 25

Kazakhstan Kostan Dolmatova N 52.18036 E65.36450 soil-34 355 5

80

Figure 25 – map by coordinates direction from Astana to Kostanay, 2017.

81

Figure 26 – Spreading of CCN by direction Astana - Kostanay, 2017.

Figure 27 – Heterodera spp under microscope, KazNAU, Alamty, 2017.

4.5 Effectiveness of Biologics Application Against Root Rot of Grain Crops In today’s environment it is the challenge of finding new promising areas in agriculture for the use of technology safe for human health, animals, and the biosphere in general. In this regard, there is a gradual transition from intensive industrial agricultural production to alternative production (biological or environmental, in particular), which provides for the rational use of energy resources and reduction of environmental pollution, production of high quality agricultural products, the preservation and increase of soil fertility, and non-waste use of agricultural products.

82 An integral part of ecological agriculture is the application of biologics, which improve plant nutrition, as well as biological means of plant protection. BioHumate is one of such biologics, produced through phytogenic waste bioconversion. Since cereals are major source of food, enzymes, trace elements, mineral salts and other biologically active substances and are the components of indispensable products of human rational nutrition, the requirements concerning their quality is quite high2-3. To ensure high quantitative and qualitative productivity indicators it is necessary to observe technology of cropper cultivation. Application of biologics at cultivation of grain crops initiates plants growth and development, improves nitrogen and phosphorus nutrition, increases their resistance against pathogens and as a result helps to increase productivity and product quality, gives the opportunity not only to save significant amounts of energy, but also creates a favorable environment for agriculture in general, since it helps to increase fertility of soils using much smaller amounts of mineral fertilizers and, consequently, reducing the level of environmental pollution4-5. In this regard, we have conducted laboratory and field experiments. Objects and research methods An analysis of the antifungal activity of Bacillus subtilis showed that the maximum inhibitory activity against the phytopathogenic fungus Fusarium graminearum is noted in the living bacterial culture. The culture fluid after separation of bacterial cells had activity, 2.5 times lower than the living culture, and antifungal the activity of the total fraction of metabolites extracted from the culture liquid was 2 times less active than the live culture (Figure – 15,16,17,18,19,20). It can be assumed that additional factors contribute to the total antagonistic activity of Bacillus subtilis, for example, exoenzymes.

Figure 28 – control Figure 29 – seeds treated with Vial

83

Figure 30 – seeds treated with Figure 31 – seeds treated with Ekstrasol Ekstrasol + Vial

Figure 32 – seeds treated with Figure 33 – seeds treated with BL01 Vial + GZ14

Figure 34 – seeds treated with Strain 2

Note * All of the figures [20,21,22,23,24, 25,26] was taken from the laboratory “Bisolbi” company, Saint Petersburg, 2015.

84 The tests were carried out at the experimental facility of Kazakh Research Institute of Plant Protection and Quarantine (“Kapal” farm situated in the village of “Akyn Sara” in the Eskeldy District of the Almaty Region). Spring wheat variety “Kazakhstan 10” was the research object in the current study. The mechanical composition of the soil was made up of dark brown soil, medium loamy soil, and humus (3.0 – 3.5%), pH 7.0. Forecrop was spring wheat plowed to the depth of 15-17 cm by “BD” disc header, pre-sowing cultivation, rolling after sowing. Sowing of seeds was produced on May 11 employing “SZ-3.6” tractor seeder. Seed application rate was 4.0 mln pcs/ha, while depth was 5-6 cm. Eight variants were tested in the experiment: 1. Control – seeds treated with water; 2. Vial TT disinfectant – 0.4 l/t; 3. Tank mix of Vial TT – 0.4 l/t + TS3 Strain –0.25 l/t; 4. Tank mix of Vial TT – 0.4 l/t + GZ14 strain –0.2 l/t; 5. Tank mix of Vial TT – 0.4 l/t + Extrasol– 0.1 l/ t; 6. Extrasol– 0.1 l/t; 7. Strain 2 – 0.15 l/t; 8. BL01 Strain – 0.05 l/t. The experiments were conducted under field conditions in four replications. The size of the plots was 0.25 ha. Biologics were applied using seed treatment technique before sowing. Seed pickling machine (PS-10) was employed for seeds treatment, water consumption rate was 10 l/t. The results of biological effectiveness are presented in Tables 1 and 2. Currently, environmentally focused technologies are increasingly used in crop production to achieve clean production, which is the most promising in the development of agriculture6. The lack of any substances in grain crops leads to disturbance in carbohydrate and nitrogen metabolism, protein synthesis, as well as reduces the resistance of plants to adverse environmental conditions7-8. The aim of our study at this stage consisted in testing of biologics prepared on the basis of a rhizosphere bacteria strain of Bacillus suptilis. The experiments were laid out in the Almaty Region, in Talgar District, in the village of “Panfilov”, LLP “Bayserke Agro”, at the Experimental Base of Kazakh Research Institute of Plant Protection and Quarantine. Laboratory and field research methods were used when conducting experiments. “Baisheshek” barley variety was taken as a research object. Mechanical composition of the soil consisted of dark chestnut soil, medium loam soil, and humus (3.0 – 3.5%), pH was equal to 7.0. Sowing of barley seeds was produced on April 24 employing SZ-3.6 tractor seeder, seeding rate was 3.5 mln pcs/ha, at a seeding depth of 5-6 cm. Field experiments were carried out according to the following scheme: 1. Control – seeds treated with water; 2. Disinfectant Vial TT – 0.4 l/t; 3. Tank mix of Vial TT – 0.4 l/t + TS3 Strain – 0.25 l/t; 4. Tank mix of Vial TT – 0.4 l/t + GZ14 Strain –0.2 l/t; 5. Tank mix of Vial TT – 0.4 l/t + Extrasol – 0.1 l/t; 6. Extrasol – 0.1 l/t;

85 7. Strain 2 – 0.15 l/t; 8. BL01 Strain – 0.05 l/t. Registration plot area was 0.25 ha. The experiments were carried out in four replications. Biologics were applied using seed treatment technique before sowing. Seed pickling machine (PS-10) was used for seeds treatment, water consumption rate was 10 l/t. Biological effectiveness of biologics and the infestation of plants with root rot are presented in Tables 17 and 18. The results of study have shown that in the pre-sowing treatment of barley seeds, biologics did not significantly affect the germination energy and laboratory seed germination, but at the same time slightly inhibited the infestation of seeds by mold fungi (15.8 – 86.8%). This resulted in increased seedling density by 7.3-13.6 pcs/m2. The biologics reduced the infestation of barley with root rot at the plant tillering phase by 31.5-66.3% (Table 3). Besides, the biologics increase physiological growth of plants and contribute to accumulation of crops biomass in comparison with the control variant (Table 18). In this experiment, treatment of seeds and crops by biologics positively affected the sowing qualities of the resulting seeds as compared to control. In Kazakhstan, wheat is the most important grain crop in terms of food significance and scale of production. Production of this crop in all continents amounts to 615 mln tones per year. About half of the global wheat grain production accounts for just five countries: Canada, USA, China, India, and Russia. Winter wheat is a valuable crop in field rotation and a good forecrop for a number of crops (maize, sunflower, sugar beet, winter barley, stubble crops, etc.). Yielding capacity refers to the average size of particular crop produced per unit of cultivated area, measured usually in hundredweight per hectare. Yield characterizes the total amount of given crop production, while yielding capacity of the crop is its productivity under specific conditions of cultivation. In accordance with the specifics of these concepts, the yield is characterized by a number of indicators. These indicators include specific yield, yield before the beginning of timely harvesting, the actual yield, the so-called crop in storage, and net yield. In the beginning, crop harvesting is estimated in the original recorded weight, and then in the actual weight of the grain after modifications, as well as based on the conversion with regard to standard humidity. Crops yield and yielding capacity are direct statistical characteristics of the level of crop production development and total agricultural production. Yield (bulk yield) is the total amount of production of any agricultural crops (group of crops) in real terms, obtained from the entire area of the crops12. In relation to the importance of the crop yield index, we have investigated the plant biometrics and yield of winter wheat. Biometric analysis and yield estimate were conducted at the phase of full ripeness. To carry out proper recording, 50 plants were selected in four replications. The results of the biometric analysis and the yield index are shown in Tables 17. The results of field and laboratory studies, presented in Table 1, have shown that pre-sowing treatment of wheat seeds with biologics slightly increases laboratory germination of seeds, enhances field germination rate (7.8-15.4 pcs/m2), inhibits the development of mold fungi by 26.990.4% and root rot – by 29.4-66.7%. Besides, the

86 biologics increase physiological growth of plants and accumulation of crops biomass in comparison with the control variant.

Table 17 - Effect of biologics on spring wheat seeds germination and the infestation of plants with root rot (LPP «Baiserke Agro», 2015)

Product Applicatio Seed quality indicators, % Plant Plant Biological n rat, kg/t, The Laborat Infecti density, infection efficacy 2 l/t germinati ory on pcs./м percentage against root on germina rate with root rot disease, capacity tion rot in the (%) mid- tillering stage, (%) Vial TT 0.4 90.5 93.8 0.25 270.5 2.8 62.7 Vial TT + Strain 0.4 + 0.25 92.0 94.9 0.5 276.8 2.7 64.0 TS3 Vial TT + Strain 0.4 + 0.2 91.9 96.1 0.75 277.5 2.5 66.7 GZ14 Vial TT + 0.4 + 0.1 91.8 95.5 0.75 275.9 2.6 65.3 Extrasol Extrasol 0.1 90.9 94.0 1.8 271.6 4.7 37.3 Strain 2 0.15 91.2 93.9 1.7 269.9 5.0 33.3 Strain BL01 0.5 90.8 95.1 1.9 271.0 5.3 29.4

Control without - 89.7 92.1 2.6 262.1 7.5 - treatment

Table 18 - Effect of products on plant growth and biomass accumulation of spring wheat at mid-tillering stage (LPP «Baiserke Agro», 2015)

Product Application rat, Plant height, Plant biomass (50 kg/t, cm plants), g l/t Vial TT 0.4 18.9 63.5 Vial TT + Strain TS3 0.4 + 0.25 21.7 66.6 Vial TT + Strain GZ14 0.4 + 0.2 21.5 67.1 Vial TT + Extrasol 0.4 + 0.1 22.1 65.9 Extrasol 0.1 19.4 64.1 Strain 2 0.15 18.7 62.9 Strain BL01 0.5 19.2 63.8 Control without treatment - 16.2 61.0

87 Nowadays the environmentally friendly technologies are often used in plant breeding in order to obtain clean product, and thus, it is the most promising direction in the development of agriculture [6]. A lack of any substance in cereal crops leads to disorders of the carbohydrate and nitrogen metabolism, as well as protein biosynthesis, also decreases the plant resistance to adverse environmental conditions [7-8]. At this stage, the aim of this study was to test biological products based on the bacterial Bacillus suptilis strain. Experiments were laid out in the LLP “Baiserke-Agro”, which is another experimental farm of the Kazakh Research Institute for Plant Protection and Quarantine, located in the “Panfilov” village, Talgar region, Almaty province. During the experiment were used the laboratory and field research methods. The object of this study was the barley variety “Baysheshek”. Soil texture was dark brown or chestnut brown, medium loam, humus content 3.0-3.5%, pH 7.0. Sowing seeds was done on the 24th of April by a seed sowing machine CZ-3.6, sowing rate 3.5 million pcs./ha, at the depth of 5-6 cm. Field experiments were performed by using the following treatments: 1. Control (water treated seeds); 2. Seed disinfectant Vial TT (0.4 l/t); 3. A tank-mix of Vial TT (0.4 l/t) and Strain TS3 (0.25 l/t); 4. A tank-mix of Vial TT (0.4 l/t) and Strain GZ14 (0.2 l/t); 5. A tank-mix of Vial TT (0.4 l/t) and Extrasol (0.1 l/t); 6. Extrasol (0.1 l/t); 7. Strain 2 (0.15 l/t); 8. Strain BL01 (0.05 l/t). The experiment was laid out in split-plots designed with four repetitions and each plot size of 0.25 ha. Products were applied by seed treatment method prior to the sowing process. Seed treatment machinery (PS-10) was used for treatment process with water flow rate of 10 l/t. Biological efficacy results and infection rate with a root rot disease are presented in the Table 3 and Table 4.

Table 19 – Effect of products on the seed germination of barley and the infection rate with plant disease ((LPP «Baiserke Agro», 2015)

Product Applica Seed quality indicators, % Plant Plant Biological tion rat, density, infection efficacy against The Laborat Infecti 2 kg/t, germin ory on pcs./м percentage root rot disease, l/t ation germina rate with root rot (%) capacit tion diseas in the y mid-tillering stage, (%)

1 2 3 4 5 6 7 8 Vial TT 0.4 92.5 99.3 0.5 165.8 3.2 64.1

88 Continuation of table – 19

Vial TT + Strain 0.4 + 94.1 98.7 0.25 167.7 3.4 61.8 TS3 0.25 Vial TT + Strain 0.4 + 93.7 98.7 0.25 169.2 3.1 65.2 GZ14 0.2 Vial TT + 0.4 + 95.2 98.7 0.5 170.1 3.0 66.3 Extrasol 0.1 Extrasol 0.1 92.8 98.5 1.4 164.9 5.4 39.3 Strain 2 0.15 93.1 98.3 1.5 163.8 5.6 37.1 Strain BL01 0.5 92.5 99.5 1.6 165.1 6.1 31.5 Control without - 91.9 98.7 1.9 156.5 8.9 - treatment

The findings of this study shows that the applied products for the pre-sowing seed treatment of barley crops did not have significant effect on the seed germination capacity and laboratory germination. However, it did have some effect on the inhibition of the fungal growth on the seeds (15.8 – 86.8%), and thus, the plant density increased up to 7.3-13.6 pcs/м2. The products decreased the barley infection with root rot disease at the mid-tillering stage for 31.5-66.3% (Table 16). Furthermore, the products helped to increase the physiological plant growth and biomass accumulation compared to control (Table 17).

Table 20 – Effect of products on plant growth and biomass accumulation of barley crops at mid-tillering stage (LPP «Baiserke Agro», 2015)

Product Application rat, Plant height, Plant biomass (50 kg/t, l/t cm plants), g

Vial TT 0.4 21.8 70.9 Vial TT + Strain TS3 0.4 + 0.25 23.1 72.2 Vial TT + Strain GZ14 0.4 + 0.2 22.9 71.2 Vial TT + Extrasol 0.4 + 0.1 24.3 73.1 Extrasol 0.1 21.9 70.6 Strain 2 0.15 21.7 68.8 Strain BL01 0.5 20.9 67.9 Control without treatment - 19.1 66.1

The result of this experiment showed that the biological seed treatment had positive effect on the seed yields and quality compared to control treatment. Winter wheat is an important crop in crop rotation and regarded as a good predecessor for some crops, e.g. corn, sunflower, sugar beet, winter barley. Crop productivity is the mean number of the amount of a crop that was harvested per unit of cultivated area and is normally measured in centners per hectare. Crop yield is a total volume of produced crop, whereas crop productivity refers to the 89 amount of yield under certain conditions of crop cultivation. In accordance with above definition of the crop yield, there are several indicators that details crop yield, such as crop species, harvested yield prior a planned harvesting, the actual harvest is the so-called granary, and clean harvest. Initial harvesting includes the measurement of the first collected weight, and then the actual weight after the grain processing, as well as during standard humidity. Crop yield and crop productivity of agricultural crops are the main statistical characteristics of the level of development for crop production and agriculture in general. Crop yield (gross yield) is the total volume of products of any agricultural crop (or group of crops) naturally harvested from total sowing area of crops. At this stage, the study was done on biometric indicators and yield counting of winter wheat crop under salinity soil conditions to test biological products in Almaty province. Biometric analysis and yield counting were performed at the stage of crop’s full ripeness. Fifty plants were selected in four repetitions for registration. The results of biometric analysis and yield indicators are shown in Table 21 and Table 22.

Table 21 – Biometric analysis of winter wheat crop treated with biological products (LPP «Baiserke Agro», 2015)

Product Plant Ear Quantity Plant density, Root rot Plant Seed height, cm length, of pcs disease, biomass biomass cm productive grade (50 (1000 shoots plants), seeds), g g

Strain GZ14 85.2 7.046 1.12 12.8 0.2 162 48 Strain BL01 86.564 7.88 1.54 14.48 0.14 239 46 Extrasol 83.06 8.716 3.36 15.86 0.18 369 48 Strain TS3 87.424 8.704 1.74 15.94 0.14 291 48 MEGA 82.298 7.582 1.18 14.48 0.02 176 46 Control 71.99 6.8 1.08 11.24 0.14 108 42 without treatment

Table 22 – Yield indicators of winter wheat crop treated with biological products under salinity conditions (LPP «Baiserke Agro», 2015)

Products Area (per ha) Crop yields (centners/ha) 1 2 3 Strain GZ14 1 34.00 Strain BL01 1 31.00 Extrasol 1 28.50 Strain TS3 1 25.00 MEGA 1 20.00 Control without 1 31.50 treatment

90 The findings of field and laboratory study showed that the applied products for the pre-sowing seed treatment of wheat crops had slight effect on the seed laboratory germination, increased field seed germination (7.8-15.4 pcs /м2), and inhibited the fungal growth on the seeds (26.9 – 90.4%), as well as decreased the infection with root rot disease for 29.4-66.7% (Table 1). Furthermore, the products helped to increase the physiological plant growth and biomass accumulation compared to control (Table 2). According to the above-mentioned data, the Extrasol and the strain TS3 showed optimal results compared to control treatments. Treatments with the MEGA and the strain GZ14 were less affected by root rot disease in accordance with indicators. Treatment with the strain GZ14 illustrated the highest results based on the findings of this study. Thus, based on the results of our experiments, the following products recommended applying for crop production: Extrasol and the strain GZ14. To sum up, the seed disinfectant Vial TT showed 178.1% efficacy compared to control, whereas the tank-mix of Vial TT and strain TS3 showed 161.8% efficacy compared to control. The tank-mix of Vial TT and strain GZ14 showed 187.0% efficacy, while tank-mix of Vial TT and Extrasol showed 166.7% efficacy, only Extrasol showed 64.8% efficacy, only Strain 2 showed 58.9% efficacy and only Strain BL0 showed 45.9% efficacy. Treatment with Vial TT and strain GZ14 showed the lowest infection rate with root rot disease in accordance with the results, whereas the highest result showed the treatment with the Strain BL01 [149]. Research on seed germination and infection rate of agricultural crops with various biological products has an important role in Kazakhstan and worldwide. This is because world population require healthy food and high yields of agricultural crops.

91 5 ECONOMIC EFFICIENCY

For the purpose of achieving the economical efficiency of winter wheat production: the yield of winter wheat obtained, c / ha; Price per 1 t of winter wheat, KZT; price of winter wheat, tenge; expenses on production of winter wheat, KZT; net income, tenge; Profitability level,% Disclosed (paragraph 27).

Table 23 – The Effectiveness of Seeds Before Sowing Seeds of Winter Wheat (Talgar District, Almaty, Bayserke Agro LLP, 2015)

Ecological features Control without Extrasol, ц/га Strain GZ14, treatment, ц/га (эталон) ц/га The yield of winter wheat 31.5 28.5 34.0 obtained, c / ha Price per 1 cent of wheat, tenge 6000.0 6000.0 6000.0 Price of winter wheat received, 189000.0 171000.0 204000.0 tenge Costs for winter wheat production, 104000.0 105500.0 109900.0 tenge Pure bottom, tenge 85000.0 65500.0 94100.0 Profitability level,% 81.7 62.1 85.6

The yield of wheat obtained - Control without treatment, c / hectare - 31,5 c / ha; Extrasol, c / ha (standard) - 28.5 c / hectare and Strain GZ14 - 34.0 c / hectare. Today the price of 1 kg of wheat is 6000 tenge. The price of the product was increased by 1 t of wheat grades: Control without treatment - 189000.0 tenge; Extrasol (standard) - 171000.0 tenge and Strain GZ14 model - 204000.0 tenge. Production costs for production of wheat in the production: Control without treatment - 104000,0 tenge; Extrasol - 105500.0 Tenge and Strain GZ14 - 109900.0 Tenge. Net income is the expense recognized as a difference in the cost of production of wheat in the price of the product obtained. The monetary aggregate of the cash flow: ТТ - pure class; SEL - total cost of the product; MS - cost. TT = HDP – MS (6) The following information is available on the 23rd cite: Control without treatment - 85000.0 tenge; Extrasol (standard) - 65500.0 tenge and Strain GZ14 model - 94100.0 tenge. Profitability level is a business indicator of the business. Apart from the costs involved in the production of baked products, it also provides pure income. The profitability of the market is expressed as a percentage per annum on its annual annual yield of the coin with its annual net profit. Determines the profitability of a particular product by dividing its earnings by its own cost.

92 The main factors of profitability improvement are labor productivity improvement, defining the use of the main products, diminishing the cost of the product, defining the product.

DD = TP * 100 / LP (7)

Where: DD - Profitability level,%; TP - net profit, tenge; LP - total loss, tenge. Profitability level: Control without treatment - 81.7%; Extrasol (standard) - 62.1% and Strain GZ14 - 85.6%. Reachability, in particular the Strain GZ14 model It's time to come. These samples are economically feasible and can also be used against the top of the specimen, and against the rash.

93 CONCLUSION

During the study, MicroSeq® from Applied Biosystems was used to identify the pathogen sample, as a result the Fusarium solani pathogen has been identified. By results of experiments were selected next gens of wheat genotypes: EKADA148, LUTESCENS916, GRECUM1003, LUTESCENS89-06, TULAIKOVSK 100, CIMMYT, SY TYRA, SY ROWYN, SY INGMAR, SELECT, MUCHMORE, URALOSYBIRSKAYA, LYUTESTSENS 27-12, LYUTESTSENS 6-04-4. resistant donors with gene lines of 17CBHL, which can later be used in breeding, have been identified. In the laboratory, the most aggressive strain of Fusarium culmorum was used for inoculation, which was harvested from the wheat fields of the Central Anatolian Plateau (Kirshekhir, Turkey). Successful identification of resistant genotypes of wheat will increase yield and reduce the need for chemical plant protection products. The target lines are those that have both resistant and tolerant reactions to root rot. Shown the efficiency of using genes in laboratory conditions for root rot, which can be used in selection of spring wheat of the Siberian- Kazakhstan lines. By the screening selected 21 resistant genotypes of wheat to Heterodera. Filipjevis. They are: LUTESCENS 30 69/97, EKADA148, FITON 41, FITON 204, TSELINNAYA NIVA, ASYLSAPA, AKMOLA 2, TSELINNAYA 3S, ASTANA, ALTAISKAYA70, LUTESCENS29-12, LUTESCENS106-11, LUTESCENS916, GRECUM1003, LUTESCENS89-06, ERITROSPERMUM85-08, LUTESTSENS220- 03- 45, GREKUM 650, LUTESCENS 920, SELECT, LYUTESTSENS 208-08-4 Conducting surveys in the major cereal crop growing areas of Northern Kazakhstan – Shortandy, at A.I. Baraev research centre was taken 90 soil samples, by the result was the first time found 24 cereal cyst nematodes, in western Kazakhstan, Uralsk experimental station was taken 90 soil samples, found 9 cereal cyst nematodes in the South - Eastern Kazakhstan, Kaskelen research development stations were taken 90 soil samples of the soil samples was found 150 cereal cyst nematodes. Heterodera spp - for microscopic identifications intercepted nematodes, the following types of parasitic nematodes have been identified. Three species in the H. avenae group (H. avenae, H. filipjevis, and H. latipons) cause important economic losses in small grain crops worldwide and are known as the cereal cyst nematodes. In a route survey by the direction of Astana-Kostanay, 34 soil samples were taken every 15 km on spring wheat crops, of which 17 (50%) were isolated from the genus Heterodera spp. Were collected under a microscope 418 pcs nematodes. Plant parasitic nematodes are ever present and are incidental with plant growth and crop production. They are significant constraints to sustainable agriculture and can be difficult to control. Determining the importance of individual nematode species, nematode communities and nematodes in combination with other problems is not a simple task at the best of times, but is more difficult in tropical than in temperate climates. From wheat growing areas of west and south – east part of Kazakhstan was taken 180 soil samples from both regions, by doing microscopically identification from 64 soil samples we found free living nematodes and plant parasite nematodes. Performing microscopic identification of intercepted nematodes, the following species of plant

94 parasitic nematodes were identified – Aphelenchus spp – 260 pieces, Aphelenchoides spp – 290 pieces, Tylenchus spp – 50 pieces , Filenchus spp - 30 pieces, Pratylenchus spp - 30 pieces, Parapratylenchus spp – 10 pieces, Ditylenchus spp – 100 pieces at the province of Ural; also at the province of Almaty were identified - Aphelenchus spp – 303 pieces, Aphelenchoides spp – 570 pieces, Tylenchus spp – 110 pieces, Filenchus spp – 30 pieces, Pratylenchus spp – 170 pieces, Parapratylenchus spp – 90 piesec, Ditylenchus spp – 90 pieces. Processing of spring wheat seeds before sowing with various treatments showed the following results of biological efficiency before harvesting: chemical etchant Vial-TT, vs (0.4 l / t) - 62.7%, tank mixture: chemical etchant Vial-TT, vs.c. (0,4 l / t) + bacterial preparation strain TS3 (0,25 l / t) - 64,0%, tank mixture: chemical etchant Vial-TT, vs (0,4 l / t) + bacterial preparation GZ14 (0,1 l / t) - 64,0%, tank mixture: chemical etchant Vial-TT, vs (0.4 l / t) + strain Extrasol (0.1 l / t) - 65.3%, strain Extrasol (0.1 l / t) - 37.3%, strain 2 (0.15 l / t) - 33.3% and strain BL01 (0.5 l / ton) - 29.4%.

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106 ANNEXES A

INITIAL DATA

Table А 1 - Factors

Factors Repetitions (groups) 1 2 3 4 1 2955 3210 3280 3155 2 2740 2880 2840 2940 3 3510 3335 3430 3325

Table А 2 - Source Data Parameters

Factors Number Amounts Average Disper. Average Relative Conf. main (groups) Repet. Error Error value 1 4 12600 3150 19516 69.8 2.22 3150 +- 193.48 2 4 11400 2850 7066,67 42 1.47 2850 +- 116.43 3 4 13600 3400 7616 43.6 1.28 3400 +- 120.87

Total Average M= 3133.333 The total variance S= 64477.09 Error of mean m= 73.30137 Accuracy of experience g%= 2.339405

RESULTS OF THE DISPERSIONAL ANALYSIS

Table А 3 - Analysis of variance

Sources of Sum of Degree of Dispersion Criterion Variation quadratic freed Fcalc Ftabule deviations variation By repl. 606664 2 303332 33.27 5.57 Factor 20520 3 6840 1.33 4.03 residual 82064 9 9118.22 - - 709248 11 - - -

Significance level Z= 0.05

107 Table А 4 - Analysis of the force of influence of factor grading

Indicators According to According to Plokhinsky to Snedekor Correlation of Rate % 86 89 the criteria are reliable. (F-Fisher criteria)| 26.61 33.27 number of degree a)2 a)2 freedom b)9 b)9

NOTE: The table value of the Fisher's criteria F = 5.57 Level of significance Z = 0.05

Correlation ratio in percent shows the variation studied trait due to the influence of the gradation of factor A.

COMPARISON OF GROUP MEDIUM DISPERSONAL COMPLEX

Table А 5 - According to the method of J. Tuke

Comparing Maximal Calculate Table groups difference tq Qst X( 3 )-X( 2 ) 550 9.979999

NOTE: For the number of groups (n = 3) and degrees freedom (K = 9), as well as for the 5% level value, define the table value Qst.

Table А 6 - According to the method of H. Sheffe

Comparing Maximal Calculate Table groups difference tq Qst X( 3 )-X( 2 ) 550 9.000001 E-02 3.34

NOTE: Fixed-Corrected Tabular the meaning of the Fisher Criterion

108 CALCULATION OF THE LOWEREST SUBSTANTIAL DIFFERENCE (NDS)

Experience Error Sxcp = 47.74469 Error of difference of means Sd = 67.52118 Table value of the Student's test t (0.05) = 2.26 The absolute value of the HSR (0.05) = 152.6 The relative value of HCP (0.05) = 4.87%

109 ANNEXES B

Table B1 – Screening of genotypes of wheat to the disease root rot, pathogen Fusarium colmorium (field condition, full table, Kaskelen, 2017)

№ Gene Number Number Length Length Number Root rot 1000 of plants of of the of ear of ear (selection) seeds steams growth (cm) weight (cm) 1 2 3 4 5 6 7 8 9 1 1 1 3 58 10 13 1 2 4 64 10 16 2 3 5 62 6 8 1 4 3 55,5 9 13 0 5 2 61 7 8 1 343 6 4 61,1 10,3 15 1 7 4 58,5 9,5 15 1 8 2 44,5 8,9 12 1 9 4 59,5 9,5 15 0 10 1 54 8,3 11 1 1 2 3 4 5 6 7 8 9 2 2 1 7 39 7 8 1 2 3 36 5 6 0 45 3 3 49 4,5 5 0 4 2 39 8 8 0 3 3 1 4 67 10 16 2 2 4 53 8 13 2 3 4 40 9 12 2 4 3 60 10 16 2 153 5 3 43 9 15 2 6 4 53 10 16 3 7 4 42 9 15 3 4 4 1 5 58 8 15 1 2 3 43 7 13 2 58 3 3 65 7 14 1 5 5 6 6 1 8 52 9 14 0 2 9 47 11 19 0 3 9 44 8 15 0 4 6 37 6 13 1 129 5 4 41 7 15 1 6 4 43 8 14 2 7 5 39 8 14 0 8 2 30 4 11 0

110 Continuation of table – B 1

7 7 1 9 46 4 5 1 2 2 50 10 15 2 3 7 54 7 9 1 4 2 43 9,5 15 0 5 5 57 9 12 2 79 6 5 45 9 14 2 7 4 29 7 11 1 8 3 30 6 10 2 9 2 40 7 10 0 8 8 1 3 69 9 15 2 2 1 55 13 16 1 3 2 40 7 11 2 84 4 3 46 6 11 0 5 4 45 7,2 11 3 9 9 10 10 1 4 47 5 9 1 2 4 40 8 12 1 3 6 36 5 5 0 4 2 36 6 6 1 58 5 5 39 7 12 2 6 4 30 6 9 0 7 4 42 7 9 1 1 2 3 4 5 6 7 8 9 11 11 1 3 49 9 13 1 21 12 12 1 4 42,3 7,3 10 1 2 2 57 7 12 1 3 2 46 5,5 9 0 88 4 3 35 3,7 9 1 5 3 49,5 10,3 16 0 6 8 41,4 7 11 2 13 13 1 6 36 8,2 14 0 2 6 48 8 8 1 3 5 58,5 9 17 1 4 4 64 10 19 2 100 5 2 34 6,7 11 1 6 12 48,2 5 7 1 14 14 15 15 1 2 42 8,2 14 1 2 3 47 9 16 2 3 3 42 9 12 2 4 5 55 9,5 13 0 108 5 3 37 8 13 1 6 3 42 8 13 0 7 3 33 6,5 10 0 8 1 28 4 6 0 9 4 29 7,5 8 0 10 2 38 9 15 1

111 Continuation of table – B 1

1 2 3 4 5 6 7 8 9 16 16 17 17 1 5 32 8 12 1 6 18 18 1 4 36 5 10 0 2 6 26 5 9 2 59 3 11 62 11 17 1 19 19 1 6 54 9 14 1 2 4 38 8 13 0 3 5 48 8 14 0 103 4 2 50 10 14 1 5 2 50,5 10,5 18 0 6 5 48 8 13 0 x 20 20 1 8 44 11 16 3 12 21 21 1 2 33 7 16 1 5 22 22 1 5 40 9,5 12 1 2 1 48 8 11 1 3 2 37 8 14 0 93 4 3 50 11 19 3 5 2 34 5 8 2 6 2 44 7 13 0 7 3 52 11 16 2 8 1 53 4 5 1 1 2 3 4 5 6 7 8 9

23 23 1 5 45 7 12 0 2 8 54 7,5 13 2 82 3 7 49 9 16 1 4 7 50 8 15 0 24 24 1 10 66 8 16 2 2 5 55 9 17 2 3 3 57 9 16 1 4 2 42 6 10 2 159 5 4 51 8 14 2 6 2 41 5 10 1 7 7 40 7 15 2 8 5 52 6 12 3 9 9 53 8 15 3 25 25 1 5 43 9,5 17 2 2 2 42 8 13 1 3 2 45 6,5 11 0 136 4 2 42 6 13 2 5 3 34 7 16 2 26 26 1 5 69 9,5 16 1 5 2 9 58 18 16 0

112 Continuation of table – B 1

1 2 3 4 5 6 7 8 9 27 27 1 5 51 9 15 0 2 3 60 9,4 17 1 3 4 62,5 10 16 3 109 4 3 58,5 8 14 1 5 2 50 8 14 2 6 4 54 10,5 14 0 28 28 1 9 54 9 15 0 2 7 57 9 15 3 3 7 44 8 14 3 67 4 3 52 9 13 0 29 29 1 4 35 5 15 1 2 8 68 8 16 1 3 16 64 10 15 2 164 4 4 58 8 13 1 5 7 62 8 15 1 6 17 5 7 16 1 7 8 57 8 14 1 8 16 60 6 13 1 30 30 1 4 59 9 15 1 2 6 67 9,5 15 1 3 4 69 9 15 1 194 4 3 45 7 11 1 5 8 81 9 16 2 6 5 62 10 15 0 31 31 1 4 50 6,5 15 2 2 4 29,5 3,5 10 2 3 9 53,5 5,5 8 2 108 4 3 38,5 5 9 0 5 8 51,5 8,5 13 0 6 17 76,5 9 15 2 32 32 1 6 68 7,5 12 1 2 6 72 7 13 2 3 7 51 7 13 1 80 4 4 51 9 12 1 5 2 53 9 15 3 6 6 78,5 8,5 19 3 33 33 1 4 60 11 19 2 2 3 43,5 7,6 13 1 3 12 68 12 20 2 255 4 7 55 11,5 20 2 5 3 45 8,5 15 0 6 2 46 8,5 15 2 7 2 35 7 8 1 8 3 40 6,5 13 1 9 3 66,5 12 18 2 113 Continuation of table – B 1

1 2 3 4 5 6 7 8 9 34 34 1 9 43 6,5 12 1 2 11 53 8 13 2 3 9 52 7 13 2 133 4 10 42 6 11 3 5 8 62 9 16 1 6 3 39 6 7 1 35 35 1 5 60 11 17 1 2 8 57 10 13 1 95 3 4 62 11 17 2 4 4 57 10 16 1 36 36 1 5 35 4,5 9 2 2 3 59 12 19 1 61 3 4 51 10 11 1 4 4 38 5,5 12 0 37 37 1 2 51 9,5 17 2 2 7 43 5,5 10 1 3 7 45 8 13 2 14 4 2 49 10 12 0 5 3 39 5 8 2 38 38 1 5 51,5 10,5 16 1 2 6 55 11 18 2 3 4 36,5 6 12 3 89 4 3 87,5 12,1 19 1 5 10 33 6 9 1 39 39 1 7 74 10 16 1 2 7 52 10 17 0 3 6 57 9 17 2 190 4 5 68 8 16 2 5 4 59 9 12 1 6 6 69 9 16 1 7 11 58 9 19 3 40 40 41 41 1 5 69 9 16 2 2 6 57 7 15 1 68 3 3 47 7 12 2 42 42 1 6 56 8,5 17 2 2 4 55,5 8 12 1 3 3 38 7,5 16 1 137 4 5 43 7,5 14 1 5 10 42 6 12 0 6 10 60 9,5 18 1 43 43

158

114 Continuation of table – B 1

1 2 3 4 5 6 7 8 9 44 44 1 1 35 6 8 1 2 1 32 7 11 2 3 4 55 9 18 2 145 4 3 40 8 13 2 5 10 76 10 15 2 6 9 58 9 15 1 45 45 1 13 37 6 5 0 2 12 54 9 15 0 36 x 46 46 1 14 75 10 18 0 172 x 47 47 1 5 64 9 17 1 2 8 51 9 14 1 3 4 62 10 16 1 172 4 4 65 5 19 2 5 14 42 9,5 15 2 48 48 1 6 59 10,5 16 1 2 4 53 9 15 0 3 5 67 9 17 1 4 4 66 9 13 0 135 5 9 47 9 15 0 6 11 48 8 13 0 x 1 2 3 4 5 6 7 8 9 49 49 1 9 52 7 14 0 2 5 45 8 15 3 3 6 56 9,5 13 1 100 4 5 49 6 15 1 5 6 60 9 15 1 50 50 51- 51-52 1 3 52 11 14 0 52 2 5 35 7,5 6 0 34 3 2 45 12 11 1 53 53 1 2 37 7 15 1 2 1 33 7 11 0 3 3 41 6 12 1 4 3 36 6 12 1 5 3 37 6 10 0 6 1 37 7 7 0 54 54 1 3 43 7 13 1 2 2 40 8 14 1 68 3 3 49 9 16 1 4 3 52 7 13 1 5 2 60 9,5 16 2

115 Continuation of table – B 1

1 2 3 4 5 6 7 8 9 55 55 1 2 56 9 16 1 2 3 46 9 16 2 2 3 2 44 8 16 2 56 56 1 6 58 8 12 1 2 3 59 4 7 0 95 3 1 54 8 15 0 4 3 39 8 15 2 5 4 68 8 16 2 6 2 39 4,5 13 1 57 57 1 2 20 7 9 0 2 58 58 1 2 48 9 18 0 2 8 54 9 16 0 72 3 5 38 5 14 1 59 59 1 5 71,5 9 14 0 2 3 58 10 15 0 60 x 60 60 1 4 43 3 4 1 2 3 35 4 7 0 3 3 53 8,5 14 0 105 4 4 55 8 12 0 61 61 1 4 61 9 15 0 2 2 41 7 11 1 56 62 62 1 5 31 5,5 10 1 2 8 27 7 12 2 16 3 3 36 7 12 2 63 63 1 3 54 8 14 0 2 5 55 10 16 0 70 3 2 46 7 15 0 x 64 64 65 65 1 5 80 9 12 1 2 2 66 9 14 1 3 3 49 8 13 0 4 4 49 8,5 13 1 5 5 75 10 15 1 6 3 65 8 13 1 7 4 55 9,5 14 1 8 3 66 9 14 0 9 6 57 8 14 0 67 67 1 2 55 9 14 1 2 2 64 10 16 1 3 1 51 12 16 3 103 4 2 51 6,5 12 0 5 2 63 3 13 1

116 Continuation of table – B 1

1 2 3 4 5 6 7 8 9 68 68 1 2 64 11 18 1 2 1 39 6 10 0 84 3 6 53 7 13 0 4 4 54 9 15 0 x 69 69 1 3 51 7 11 0 2 3 50 7 12 3 13 70 70 1 3 37 4,9 9 3 33 71 71 1 1 56 11 18 0 2 1 41 8 16 1 3 2 50 8 14 0 72 4 5 49 8 14 0 72 72 1 6 59 11 13 0 2 2 43 9 12 0 120 3 4 38 8 11 0 x 73 73 1 2 61 10 20 0 2 6 87 14 21 1 86 3 6 32,5 6 11 0 74 74 1 3 63 7 11 1 2 5 65 8,5 14 1 3 8 66 8 13 1 4 2 50 7 13 1 286 5 4 60 8 13 0 6 5 73 10 17 0 7 4 70 10 16 1 8 5 71 9,5 15 2 9 6 71 7,5 13 0 10 6 73 10 17 1 11 3 62,5 10 12 1 75 75 1 3 43 7 13 1 2 8 53 9,5 16 0 3 5 51 8 14 0 170 4 5 48 7 12 0 5 4 46 7 12 1 6 5 53 3,5 5 0 7 3 44 7 14 0 76 76 1 3 41 6,5 12 1 24 77 77 1 2 38 7,5 13 1 2 2 33 6 12 1 3 2 35 6 11 1 142 4 6 43 8,5 13 0 5 3 36 6 10 0 6 3 36 7 13 0 7 6 45 7 12 1 117 Continuation of table – B 1

1 2 3 4 5 6 7 8 9 78 78 No data 79 79 1 6 32 6 13 0 2 2 30 7 11 0 3 5 32 5 6 1 121 4 5 44 8 13 1 5 3 59 10 15 0 x 80 80 1 2 60 8 14 1 2 3 42 6 10 1 136 3 5 55 8 11 0 4 7 46 7 12 0 5 7 44 4 8 0 6 4 36 6 9 1 7 3 40 6 10 0 x 1 2 3 4 5 6 7 8 9 81 81 1 3 45 6 10 1 2 2 47 7 12 1 98 3 3 35 5,5 8 1 4 4 34 5 10 1 5 4 51 6 11 1 82 82 1 2 37 6 11 2 2 9 46 7 13 3 3 8 57 8 16 1 128 4 3 39 6 15 1 5 2 30 7,3 15 1 83 83 1 8 38 7,5 12 0 2 3 47 9 15 0 71 84 84 1 3 46 6 10 1 17 85 85 1 5 45 8 12 0 2 4 40 6 11 1 69 3 4 42 7 12 0 x 86 86 1 1 51 6 9 1 2 3 45 9 11 1 59 3 1 26 4 7 0 87 87 1 1 45 9 15 0 2 1 37 7 8 0 49 3 4 40 7 7 1 88 88 1 2 52 7 11 2 22 2 3 43 8 11 0 89 89 1 4 64 14 17 1 2 5 62 11 19 2 59 3 5 39 8 15 1

118 Continuation of table – B 1

1 2 3 4 5 6 7 8 9 90 90 1 3 42 6 11 0 2 2 47 5,5 8 1 3 1 30 4 7 2 91 91 1 3 47 6 10 0 2 1 49 7 11 0 3 2 43 6,5 12 1 92- 92-93 1 5 51 11 16 1 93 2 1 48 9 14 1 3 1 53 10 17 2 4 3 47 5,5 11 1 127 5 3 53 10 15 1 6 3 59 11,5 17 0 94 94 1 4 30 9 21 1 67 2 3 60 8 16 0 95 95 1 6 49 9 15 2 2 4 48 8 11 1 30 3 2 40 6 11 0 96 96 1 2 38,5 7,5 13 2 2 1 53,5 3,5 8 2 3 2 60 8,2 14 3 106 4 1 55 11 18 0 97 97 1 2 44,5 7,5 14 2 2 2 42 7 14 1 3 5 52,5 8 15 2 112 4 1 42 7 16 1 5 2 42 9,5 16 2 6 2 42 7 14 2 7 2 39 7 14 2 98 98 1 2 67 7 11 0 31 2 1 46 9 16 3 99 99 1 1 51 7 12 2 2 2 44 7 11 1 3 3 36 4,5 9 0 4 2 68 9,5 14 1 5 1 60 9 14 0 6 4 33 5 8 1 7 2 40 5 7 3

119 Continuation of table – B 1

1 2 3 4 5 6 7 8 9 100 100 1 2 54 10,5 16 2 2 3 28 4,5 7 1 3 3 74 12 19 1 4 5 58 10 15 2 5 3 55 6 9 0 6 4 75 16 9 1 7 2 70 9 15 1 8 3 50 7,5 11 1 225 9 4 63 9 17 1 10 2 75 11 16 1 11 2 64 7,5 15 1 12 4 55 9 16 1 13 3 51 9 16 0 14 1 52 10 15 0 101 101 1 2 66 10,5 17 1 2 3 67,5 10 19 2 3 1 53 11 17 1 172 4 1 48 7,5 14 2 5 2 63 10 16 1 6 5 65 12 20 3 7 2 47 10 17 0 8 3 30 7 13 0 102 102 1 4 54 10,5 12 2 2 5 67 11 16 0 70 3 3 63 13 18 1 103 103 1 3 60 9 12 1 2 5 55 9 16 1 70 3 5 50 8 14 0 4 3 51 7 14 0 5 3 49 8 15 0 104 104 1 2 62 9,5 17 1 2 3 62,5 11 18 1 3 2 45 10 19 2 194 4 3 71 11 18 2 5 3 63 11,5 18 0 6 3 64 10,5 17 0 7 3 63 10,5 16 1 8 4 72 9 13 0 9 4 40 8,5 13 0 105 105 106 106 1 4 46 7 12 2 18 107 107 1 2 37 7 14 1 17

120 ANNEXES C

Table C 1 – Distribution of cyst nematodes in some fields of Shortandy region, 2017.

17 CNAME OC ORIGINATOR 16NURS 16ENT Place of CCN ENT swoing 1 2 3 4 5 6 7 8 1 SERI 16CBHL 2 Astana 1 2 STEPNAYA75 KAZ AKTOBE ARS 16CBHL 3 Astana 0 3 STEPNAYA1414 KAZ AKTOBE ARS 16CBHL 4 Astana 0 4 GVK2055-1 KAZ EAST- 16CBHL 5 Astana 0 KAZAKHSTAN 5 LUTESTSENS2 KAZ KARABALYK 16CBHL 9 Astana 1 ARS 6 LINE-С-19SB KAZ KARABALYK 16CBHL 11 Astana 0 ARS-CIMMYT 7 KARABALYKSKAYA KAZ KARABALYK 16CBHL 12 Astana 0 20 ARS 8 FANTAZIYA KAZ KARABALYK 16CBHL 13 Astana 0 ARS 9 BOSTANDYK KAZ KARABALYK 16CBHL 14 Astana 0 & KAZ RI PLANT PROTACTİON 10 LUTESCENS 30 69/97 KAZ KARABALYK 16CBHL 15 Astana 0 ARS 11 KARAGANDINSKAYA KAZ KARAGANDA 16CBHL 20 Astana 0 30 ARI 12 KARAGANDINSKAYA KAZ KARAGANDA 16CBHL 21 Astana 0 31 ARI 13 PAVLODARSKAYA KAZ PAVLODAR 16CBHL 24 Astana 0 YUBILEYNAYA ARI 14 KONDITERSKAYA KAZ PAVLODAR 16CBHL 25 Astana 0 YAROVAYA ARI

15 FITONС-50SB KAZ FITON- 16CBHL 27 Astana 0 CIMMYT 16 FITON82 KAZ FITON 16CBHL 28 Astana 0 17 FITON-С-54SB KAZ FITON- 16CBHL 29 Astana 1 CIMMYT 18 EKADA148 KAZ FITON-EKADA 16CBHL 30 Astana 1 19 EKADA 113 KAZ FITON 16CBHL 31 Astana 0 20 LYUBAVA KAZ FITON 16CBHL 33 Astana 0 21 FITON 41 KAZ FITON 16CBHL 34 Astana 0

121 Continuation of table – C 1

1 2 3 4 5 6 7 8 22 FITON 204 KAZ FITON 16CBHL 35 Astana 0 23 VLADIMIR KAZ SHORTANDY 16CBHL 36 Astana 0 ARI 24 TSELINA50 KAZ SHORTANDY 16CBHL 37 Astana 0 ARI 25 TSELINNAYA NIVA KAZ SHORTANDY 16CBHL 39 Astana 0 ARI 26 ASYLSAPA KAZ SHORTANDY 16CBHL 40 Astana 0 ARI 27 AKMOLA 2 KAZ SHORTANDY 16CBHL 41 Astana 0 ARI 28 AK ORDA KAZ SHORTANDY 16CBHL 42 Astana 0 ARI 29 SHORTANDINSKAYA KAZ SHORTANDY 16CBHL 43 Astana 1 2012 ARI 30 TSELINNAYA 3S KAZ SHORTANDY 16CBHL 44 Astana 1 ARI 31 ASTANA KAZ SHORTANDY 16CBHL 45 Astana 1 ARI 32 ALTAISKAYA70 RUS ALTAY ARI 16CBHL 51 Astana 0 33 ALTAISKAYA110 RUS ALTAY ARI 16CBHL 52 Astana 0 34 TOBOLSKAYA RUS ALTAY ARI 16CBHL 53 Astana 0 35 ALTAYSKAYA RUS ALTAY ARI 16CBHL 55 Astana 0 ZHNITSA 36 STEPNAYA VOLNA RUS ALTAY ARI 16CBHL 56 Astana 0 37 APASOVKA RUS ALTAY ARI 16CBHL 57 Astana 0 38 LUTENSCENS89-06 RUS OMGAU 16CBHL 58 Astana 0 39 DUET RUS OMGAU 16CBHL 59 Astana 0 40 PAVLOGRADKA RUS OMGAU 16CBHL 60 Astana 0 41 LUTESCENS29-12 RUS OMGAU 16CBHL 61 Astana 1 42 LUTESCENS106-11 RUS OMGAU 16CBHL 62 Astana 1 43 TULAIKOVSKAYA110 RUS SAMARA 16CBHL 65 Astana 0 44 LUTESCENS916 RUS SAMARA 16CBHL 66 Astana 0 45 GRECUM1003 RUS SAMARA 16CBHL 67 Astana 0 46 LUTESCENS1062 RUS SAMARA 16CBHL 68 Astana 0 47 LUTESCENS89-06 RUS OMGAU 16CBHL 69 Astana 2 48 ERITROSPERMUM85- RUS OMGAU 16CBHL 70 Astana 0 08 49 SEREBRISTAYA RUS SIB ARI 16CBHL 72 Astana 1 50 SERI Astana 0 51 BOEVCHANKA RUS SIB ARI 16CBHL 73 Astana 0 52 OMSKAYA 37 RUS SIB ARI 16CBHL 75 Astana 0 53 LUTESTSENS7-04-4 RUS SIB ARI 16CBHL 76 Astana 1

122 Continuation of table – C 1

1 2 3 4 5 6 7 8 54 LUTESTSENS197-04-7 RUS SIB ARI 16CBHL 79 Astana 0 55 LUTESTSENS220-03-45 RUS SIB ARI 16CBHL 81 Astana 0 56 TULAIKOVSKAYA 10 RUS SAMARA 16CBHL 85 Astana 0 57 TULAIKOVSKAYA RUS SAMARA 16CBHL 86 Astana 1 ZOLOTISTAYA

58 TULAIKOVSK 100 RUS SAMARA 16CBHL 88 Astana 1 59 GREKUM 650 RUS SAMARA 16CBHL 89 Astana 0 60 LUTESCENS 920 RUS SAMARA 16CBHL 91 Astana 0 61 EKADA 121 RUS SAMARA 16CBHL 92 Astana 0 62 CIMMYT RUS SAMARA 16CBHL 97 Astana 0 63 P-23-17 RUS KURGAN 16CBHL 98 Astana 1 64 PAMYATI RUBA RUS CHELYABINSK 16CBHL 101 Astana 1 65 CHELYABA 75 RUS CHELYABINSK 16CBHL 102 Astana 0 66 ERITROSPERMUM RUS CHELYABINSK 16CBHL 104 Astana 0 23707 67 SY TYRA US- US-SYN 16CBHL 112 Astana 1 SYN 68 SY GOLIAD US- US-SYN 16CBHL 113 Astana 0 SYN 69 SY SOREN US- US-SYN 16CBHL 114 Astana 0 SYN 70 SY ROWYN US- US-SYN 16CBHL 115 Astana 0 SYN 71 SY INGMAR US- US-SYN 16CBHL 116 Astana 0 SYN 72 SELECT US- US-SDSU 16CBHL 117 Astana 0 SDSU 73 FORE FRONT US- US-SDSU 16CBHL 118 Astana 0 SDSU 74 PREVAIL US- US-SDSU 16CBHL 119 Astana 0 SDSU 75 ADVANCE US- US-SDSU 16CBHL 120 Astana 1 SDSU 76 BRICK US- US-SDSU 16CBHL 121 Astana 1 SDSU 77 CARBERRY CAN 16CBHL 122 Astana 0 78 MUCHMORE CAN 16CBHL 123 Astana 1 79 URALOSYBIRSKAYA RUS 16CBHL 130 Astana 1 80 TORNADO 22 KAZ FITON 16CBHL 132 Astana 0 81 LYUTESTSENS 1012 RUS ALTAY ARI 16CBHL 133 Astana 0 82 LYUTESTSENS 7-04-10 RUS KURGAN 16CBHL 134 Astana 0 123 Continuation of table – C 1

1 2 3 4 5 6 7 8 83 LYUTESTSENS 208-08- RUS KURGAN 16CBHL 135 Astana 0 4 84 LYUTESTSENS 27-12 RUS OMGAU 16CBHL 136 Astana 1 85 ERITROSPERMUM 85- RUS OMGAU 16CBHL 137 Astana 1 08 86 LYUTESTSENS 6-04-4 RUS SIB ARI 16CBHL 138 Astana 1 87 LYUTESTSENS 186-04- RUS SIB ARI 16CBHL 139 Astana 0 61 88 CHEBARKULSKAYA RUS CHELYABINSK 16CBHL 140 Astana 0 89 LINE D 25 RUS SARATOV 16CBHL 141 Astana 1 90 LINE 654 RUS SARATOV 16CBHL 142 Astana 1

124 Table – C 2 Determenation of cyst nematodes (Heterodera spp) in some cereal growing regions of Kazakhstan (Almaty, Uralsk, 2017 year)

№ Place (country, region) CCN Place (country, region) CCN 1 2 3 4 5 1 West Kazakhstan- Uralsk 0 South – east 2 Kazakhstan, Almaty 2 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 3 West Kazakhstan- Uralsk 0 South – east 4 Kazakhstan, Almaty 4 West Kazakhstan- Uralsk 0 South – east 3 Kazakhstan, Almaty 5 West Kazakhstan- Uralsk 0 South – east 3 Kazakhstan, Almaty 6 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 7 West Kazakhstan- Uralsk 0 South – east 5 Kazakhstan, Almaty 8 West Kazakhstan- Uralsk 0 South – east 4 Kazakhstan, Almaty 9 West Kazakhstan- Uralsk 1 South – east 4 Kazakhstan, Almaty 10 West Kazakhstan- Uralsk 0 South – east 6 Kazakhstan, Almaty 11 West Kazakhstan- Uralsk 0 South – east 10 Kazakhstan, Almaty 12 West Kazakhstan- 0 South – east 5 Kazakhstan, Almaty 13 West Kazakhstan- Uralsk 0 South – east 5 Kazakhstan, Almaty 14 West Kazakhstan- Uralsk 0 South – east 3 Kazakhstan, Almaty 15 West Kazakhstan- Uralsk 0 South – east 5 Kazakhstan, Almaty 16 West Kazakhstan- Uralsk 0 South – east 2 Kazakhstan, Almaty 17 West Kazakhstan- Uralsk 2 South – east 0 Kazakhstan, Almaty 18 West Kazakhstan- Uralsk 2 South – east 4 Kazakhstan, Almaty

19 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 20 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty

125 Continuation of table – C 2

1 2 3 4 5 21 West Kazakhstan- Uralsk 0 South – east 5 Kazakhstan, Almaty 22 West Kazakhstan- Uralsk 1 South – east 1 Kazakhstan, Almaty 23 West Kazakhstan- Uralsk 0 South – east 2 Kazakhstan, Almaty 24 West Kazakhstan- Uralsk 0 South – east 3 Kazakhstan, Almaty 25 West Kazakhstan- Uralsk 0 South – east 5 Kazakhstan, Almaty 26 West Kazakhstan- Url 0 South – east 5 Kazakhstan, Almaty 27 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 28 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 29 West Kazakhstan- Uralsk 0 South – east 4 Kazakhstan, Almaty 30 West Kazakhstan- Uralsk 0 South – east 2 Kazakhstan, Almaty 31 West Kazakhstan- Uralsk 0 South – east 5 Kazakhstan, Almaty 32 West Kazakhstan- Uralsk 0 South – east 3 Kazakhstan, Almaty 33 West Kazakhstan- Uralsk 0 South – east 4 Kazakhstan, Almaty 34 West Kazakhstan- Uralsk 0 South – east 3 Kazakhstan, Almaty 35 West Kazakhstan- Uralsk 0 South – east 2 Kazakhstan, Almaty 36 West Kazakhstan- Uralsk 0 South – east 5 Kazakhstan, Almaty 37 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 38 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 39 West Kazakhstan- Url 0 South – east 0 Kazakhstan, Almaty 40 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 41 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty

126 Continuation of table – C 2

1 2 3 4 5 42 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 43 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 44 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 45 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 46 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 47 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 48 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 49 West Kazakhstan- Uralsk 0 South – east 2 Kazakhstan, Almaty 50 West Kazakhstan- Uralsk 0 South – east 3 Kazakhstan, Almaty 51 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 52 West Kazakhstan- Url 0 South – east 0 Kazakhstan, Almaty 53 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 54 West Kazakhstan- Uralsk 0 South – east 4 Kazakhstan, Almaty 55 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 56 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 57 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 58 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 59 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 60 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 61 West Kazakhstan- Uralsk 0 South – east 2 Kazakhstan, Almaty 62 West Kazakhstan- Uralsk 0 South – east 2 Kazakhstan, Almaty 63 West Kazakhstan- Uralsk 0 South – east 2 Kazakhstan, Almaty

127 Continuation of table – C 2

1 2 3 4 5 64 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 65 West Kazakhstan- Url 0 South – east 0 Kazakhstan, Almaty 66 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 67 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 68 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 69 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 70 West Kazakhstan- Uralsk 1 South – east 0 Kazakhstan, Almaty 71 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 72 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 73 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 74 West Kazakhstan- Uralsk 0 South – east 4 Kazakhstan, Almaty 75 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 76 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 77 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 78 West Kazakhstan- Url 0 South – east 0 Kazakhstan, Almaty 79 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 80 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 81 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 82 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 83 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 84 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 85 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty

128 Continuation of table – C 2

1 2 3 4 5 86 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 87 West Kazakhstan- Uralsk 0 South – east 0 Kazakhstan, Almaty 88 West Kazakhstan- Uralsk 0 South – east 1 Kazakhstan, Almaty 89 West Kazakhstan- Uralsk 1 South – east 2 Kazakhstan, Almaty 90 West Kazakhstan- Uralsk 2 South – east 1 Kazakhstan, Almaty

129

Figure C 1 - collecting cyst nematodes under microscope ( Ankara, Turkey ,2017)

Figure C 2 - Biometrical analaysis ( KAZNAU, Kazakhstan, 2017)

130

Figure C 3 - Harvest time (Kaskelen, Kazakhstan,2017)

Figure C 4 - Harvest time (Kaskelen, Kazakhstan,2017)

131 ANNEXES D

132 133

134

135

136

137

138

139

140

141

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142

143

144