The effect of application of micromycetes on plant growth , as well as soybean and barley yields *

The possibility of application of micromycetes (strains Penicillium bilaiae Pb14, Aureobasidium pullulans YA05 and Rhodotorula mucilaginosa YR07) to increase yields of soybean (Glycine max cv Almaty) and barley (Hordeum vulgare cv Arna) was estimated. It was shown that the most positive effect on germination energy and seed germination after seed treatment with liquid culture, supernatant and filtrate, is achieved at 1:5 dilution. In studying the influence of cell-associated and extracellular biologically active compounds of micromycetes (liquid culture and supernatant) on biometric parameters of seedlings, the maximum stimulating effect was observed in the variants with liquid culture. These strains of micromycetes were used as a bases for various compositions of preparations — application of each strain separately and application of micromycetes mixes. In microfield experiments, the increase of soybean yield ranged from 4.5 to 9.4 quintal/ha, barley — from 2.9 to 5.9 quintal/ha. A significant increase in various parameters of structure of the yield was shown in all experimental variants when compared to the control. It was found that an increase in soybean and barley yields and yield components was higher in the variant with a mix of micromycetes when compared to the separate application of each strain. The most efficient mixture was based on the mix of fungal strains (culture filtrate of P. bilaiae Pb14 diluted 1:5 + liquid cultures of A. pullulans YA05 and Rh. mucilaginosa YR07 in a 1:5 dilution).


INTRODUCTION
Barley is a valuable forage and food crop.The nutrient content of barley compares favorably with that of wheat, corn, oats, milo.Barley is the major cereal in many dry areas of the world.Barley yield depends on the growth conditions, agronomic practices, and on the properties of a cultivar.These conditions play a major role in improving the collection of production per unit area.The aboriginal cultivars play an important role (Vanova et al., 2006;Ullrich, 2011;Alireza, 2015;Tiwari, 2010).
Today, in almost all the regions of Kazakhstan, the trend of soil degradation is going on; processes leading to the loss of soil fertility are developed.Under these conditions the expansion of legumes cultivation can be a significant factor in increasing productivity of agrocenoses.The symbiotic relationships between plants and microorganisms play a crucial role in the life of plants, including legumes crops, and contribute to plant mineral nutrition, adaptation to abiotic stresses, protection against pathogens and pests (Morel et al., 2012;Kiers et al., 2003;Franche et al., 2009;Wells & Varel, 2011;Frey-Klett et al., 2011).Among legume crops, soybean occupies a unique position due to the extremely versatile use.The main feature of soybean, which distinguishes it from other legumes, is the chemical composition of the grain.The protein composition of this culture is the most complete of all vegetable proteins; it has well-balanced amino acid composition (Endres, 2001).Soy is widely used as food, forage and industrial plant due to its nutrient content.Soybean ranks first in cultivation areas and gross grain harvest among legumes and oilseeds crops in the world.
The most important task is ensuring environmental cleanliness and maintaining quality and food safety of grain and grain products.Crop yield formation depends on the sowing conditions in the optimal agronomic terms and at a high level, as well as the efficient use of fertilizers and plant protection products, in particular bio-fertilizers based on plant growth-promoting microorganisms (Singh et al., 2011;Pérez-Montano et al., 2014).
It is known that seed treatment with rhizosphere microorganisms is capable of increasing the biomass of the roots and nutrient uptake by the root system as well as stimulating seed germination due to the production of physiologically active substances such as vitamins, auxins, gibberellins, and inhibiting the development of pathogenic organisms (Morel et al., 2012).
The use of microorganisms with plant growth-promoting abilities isolated from natural habitats is very important.These microorganisms can be used to create highly effective biological preparations for agriculture.The application of bio-fertilizers is an effective alternative to chemical fertilizers.It helps to reduce chemical pollution (Singh, 2011;Pérez-Montano et al., 2014;Agamy et al., 2013).
There is evidence that the mixed cultures of rhizobacteria produce large amounts of phytohormones.In nature, microorganisms live in communities, so the biosynthesis of molecules that stimulate the growth and development of both plants and microorganisms themselves is, enhanced by their interaction.It is known that the precursors of phytohormones, such as tryptophan, are allocated in the rhizosphere with plant root exudates.
The strains were cultured for 5 days in Sabouraud dextrose broth with shaking (240 rpm) at 26°C.After incubation, the A. pullulans YA05 and Rh.mucilaginosa YR07 strains were centrifuged at 10 000 × g for 10 min.The culture of filamentous fungus P. bilaiae Pb14 was filtered through filter paper No. 4 (pore diameter 20-25 microns).
Determination of the optimal dilution.The liquid culture, culture supernatant and culture filtrate were used for soaking of previously sterilized seeds.Variants without dilution and 1:1, 1:3, 1:5, 1:10 dilutions (with water) were studied.Soybean (Glycine max cv Almaty) and barley (Hordeum vulgare cv Arna) seeds were used in the experiment.After daily soaking, seeds were placed in pots filled with a mixture of sand and sawdust.Seeds soaked in water were used as a control.Seeds were germinated for 14 days.At the end of the exposure, the germination energy and seed germination were determined.Germination energy was determined as a percent of number of seeds (grains) which germinated up to the time of peak germination in a 24 hour period.Seed germination was defined as a percent of number of seeds (grains) which germinated within 14 days under optimal conditions.Determination of plant growth-promoting effect of micromycetes.The optimal dilutions of liquid culture, culture supernatant and culture filtrate were used for seeds soaking.Seeds were germinated for 14 days.After exposure, biometric research was carried out.Length of stems and roots in the experiment and control was determined.
Microfield experiments.Microfield experiments were carried out in the Almaty region at the experimental plot of the "Turgen" agro-industrial company.Fields of the "Turgen" company are situated in the foothill steppe zone.The soil cover is represented by foothill dark chestnut soils.
Legume crop soybean (Glycine max cv Almaty) and grain crop barley (Hordeum vulgare cv Arna) were used in the experiment.Microfield experiments were conducted in small plots (1 × 1 m) in 4 replicates.
Control -without treatment.
A single-row arrangement of replicates was used in order to maximize the removal of similar plots.Soybean seeding depth was 3-5 cm, barley seeding depth -4-6 cm.The vegetative growth period of soybean (Glycine max cv Almaty) is 100-105 days.The vegetative growth period of barley (Hordeum vulgare cv Arna) is 75-85 days.
Analysis of the structure of soybean yield was carried out based on the following elements: plant height, number of pods and seeds per plant, seed weight per plant, weight of 1000 seeds.
The structure of the barley yield was evaluated based on the following components: plant height, ear length, number of grains per ear, grain weight per ear, weight of 1000 grains.
Plant height was determined by measuring the length of the main stem of each plant.
Ear length was determined by measuring ear length of the main stem from the base of the first spikelet excluding awns.
Two samples of 500 pieces from fraction of conditioned air-dry seeds were counted to determine the weight of 1000 seeds (grains).Each sample was weighed to the nearest 0.01 g and multiplied by two, and then the average weight of 1000 seeds to the nearest 0.1 g was calculated.The definition of the weight of 1000 seeds (grains) in terms of dry matter was carried out by the formula: A=B×(100-C)/100, where A -the absolute mass of seeds, g; B -weight of 1000 air-dry seeds, g; C -seed moisture, %.

Determination of the optimal dilution of liquid culture, culture supernatant and culture filtrate for seed treatment
Active strains of micromycetes P. bilaiae Pb14, A. pullulans YA05 and Rh.mucilaginosa YR07, previously selected in the studies for their biological properties, were used in experiments.These strains possess plant growth-promoting and antagonistic activity (Ignatova et al., 2015).
In the study of morphological characteristics of micromycetes, it was shown that the A. pullulans YA05 and Rh.mucilaginosa YR07 strains grow as single yeast cells, and the P. bilaiae Pb14 strain grows in the form of filaments.Accordingly, the following variants were used for seed treatment: liquid culture and culture supernatant of A. pullulans YA05 and Rh.mucilaginosa YR07 strains and culture filtrate of P. bilaiae Pb14.
The concentration of the biologically active compounds is important in the seed treatment with microorganisms.In studying optimal dilution of liquid culture and culture supernatant of the A. pullulans YA05 and Rh.mucilaginosa YR07 strains, it was shown that germination energy and seed germination achieved maximum at 1:5 dilution and were higher by 13-15% in a variant with soybean, and by 13-16% in a variant with barley when compared to the control.Similar results were recorded for the culture filtrate of P. bilaiae Pb14.The greatest positive effect was noted in the variant with 1:5 dilution (Table 1).

Plant growth-promoting activity of micromycetes strains
Determination of plant growth-promoting effect of micromycetes was carried out by seed soaking method.The influence of the filtrate of P. bilaiae Pb14 in 1:5 dilution, cell-associated and extracellular biologically active Effect of micromycetes on plant growth and yield compounds of the A. pullulans YA05 and Rh.mucilaginosa YR07 strains (liquid culture and supernatant in 1:5 dilution) was studied.
It has been observed that the stimulating effect of micromycetes could be detected at the earliest stages of plant development, starting from seed germination.
In variants with A. pullulans YA05 and Rh.mucilaginosa YR07, it was noted that stem length of soybean increased 1.2-1.5 fold, and root length 1.2-2.6 fold when compared to the control seedlings.A 1.3-1.5 fold increase in stem length and 1.1-1.3fold increase in root length was observed in experiments with barley (Table 2).The best results were obtained by seed treatment with liquid cultures.The maximum stimulating effect was achieved in a variant with a mix of "A.pullulans YA05 + Rh. mucilaginosa YR07" (Fig. 1).
Statistically significant increase in biometric parameters of two-week seedlings (the increase of shoot and root length) was observed after seed treatment with fil-trate of P. bilaiae Pb14.The data presented in Table 2 show that length of stems and roots of barley increased 1.5-fold when compared to the control.A 1.5-fold increase in stem length and 2.6-fold increase in root length were obtained in variants with soybean seedlings.

Evaluation of the properties of biological preparations in microfield experiments
Liquid cultures of micromycetes strains: A. pullulans YA05 + Rh. mucilaginosa YR07, and fungal filtrate of P. bilaiae Pb14 diluted 1:5 were used to create test samples of bio-preparations.
The results of microfield studies indicated that soybean yield ranged from 26.4±0.6 quintal/ha to 35.8±1.1 quintal/ha depending on the variant of the experiment.The increase of yield in the experimental variants ranged from 4.5 to 9.4 quintal/ha.The highest soybean yield was obtained in the variant No. 3 -35.8±1.1 quintal/ ha (Fig. 2).
Table 1.Influence of various options of dilution on germination energy and seed germination.Liquid cultures and culture supernatants of the A. pullulans YA05 and Rh.mucilaginosa YR07 strains and culture filtrate of P. bilaiae Pb14 were diluted 1:1, 1:3, 1:5, 1:10 to determine the optimal dilution for seed treatment.The germination energy and germination of soybean and barley seeds were determined.As can be seen from the data presented in Table 3, there were significant differences between plant heights in the experimental variants and the untreated one.The maximum plant height was recorded in variants No. 1 and No. 3, where it reached 83.4±1.5 and 85.2±1.8 cm respectively.

Variant of experiment
The largest number of pods and seeds per plant were formed under the influence of biological preparation based on the mix of micromycetes and amounted to 73.6±1.6 and 179.5±5.6,respectively.Application of the tested compositions of bio-preparations promoted the formation of higher seed weight per plant and provided an increase in weight by 18-24% compared with an untreated variant.The seed weight values ranged from 26.5±0.6 g to 32.9±0.7 g.The maximum positive effect was recorded in variant No. 3.There was a significant increase in the weight of 1000 seeds in all experimental variants compared to the control.The highest weight of 1000 seeds was observed during treatment with bio-preparation No. 3, where the value of this parameter reached 230.7±5.7 (Table 3).
In conducting the microfield research, the yield of barley amounted to 19.4±0.5 quintal/ha in the control variant.The maximum yield was registered for the variant No. 3, where the increase in yield was 5.9 quintal/ha compared to the variant without treatment (Fig. 3).
Maximum height of barley plants was characterized in variants No. 1 and No. 3 -89.5±2.5 and 90.4±1.5 cm respectively.The ear length ranged from 6.1±0.1 cm in the variant without treatment to 7.7±0.3cm in variant No. 3. The increase in the ear length by 10-26% depending on the composition of bio-preparations was observed in all experimental variants (Table 4).
Investigated plants significantly differed in the number of grains per ear, this parameter ranged from 15.4±0.5 to 19.3±0.7 pc.Data analysis allows us to conclude that application of preparations increases grain weight by 20-24% per ear.The grain weight amounted to 0.9±0.04g per ear in the variant without treatment, while it was significantly higher in the experimental variants with maximum values in variant No. 3 -1.1±0.02g.Weight of 1000 grain amounted to 37.9±1.3g in the untreated variant, which was lower by 16-21% compared to the experimental variants.In variant No. 3, the weight of 1000 grains reached the maximum of 45.5±0.5 g (Table 4).

DISCUSSION
The plant growth-promoting microorganisms promote plant growth and development, increase yield, enhance phosphorus, nitrogen, potassium and other elements' up-
It is known that intensive metabolism and biochemical transformations start at the earliest stages of seed germination.Therefore the impact of nutrients on the plant, its growth processes, is directly related to the penetration of these biogenic substances into the seeds during germination.One of the important conditions for normal plant development is the optimal concentration of physiologically active substances in the environment (Miransari & Smith, 2014;Kucera et al., 2005).It has been observed that in all experimental variants, plants have more developed leaf blades compared to the control.The most positive effect was obtained in the variant with 1:5 dilution of liquid culture, culture supernatant and fungal filtrate.Probably, in such ratio the optimal concentration of physiologically active substances is achieved.
In studying the influence of cell-associated and extracellular biologically active compounds of micromycetes, the plant growth-promoting activity of liquid culture was higher than the activity of supernatant.Furthermore, it would exclude a centrifugation step which could reduce the cost of the preparation.Localization of these biologically active compounds with plant growth-promoting activity indicates the physiology of interaction between microorganisms and plants, and helps to choose the direction of the biotechnological production of strain-producers and technology of plant treatment.
Microfield experiments on soybean (Glycine max cv Almaty) and barley (Hordeum vulgare cv Arna) were carried out to evaluate the properties of test samples of biopreparations.The yield and yield components of these crops were investigated.
The yield is the main integrating criterion of cultivation of any crop.The yield depends on soil fertility, specific weather conditions, the technology used and properties of a cultivar.High yield of investigated crops was obtained after application of tested bio-preparations.Yield of soybean and barley increased by 17-35% and 15-30%, respectively, when compared to the untreated variant.
Weight of 1000 seeds (grains) is an important quality parameter, which reflects the amount of nutrients contained in the seeds and its size.Large seeds contain more nutrients that provide faster plant development in the first phases of growth, higher yields, and more powerful and productive plants.Compositions of bio-preparations had a positive influence on the value of 1000-seeds (grains) weight.There was a significant increase in the weight of 1000 seeds by 7-14% in experiments with soybean and by 15-20% in variants with barley.
Analysis of the data revealed a positive effect of test samples of preparations on the growth characteristics of soybean, which is reflected in increasing of plant height by 21-29%, increase in the number of pods and seeds per plant by 19-26% and 21-29%, respectively, in the experimental variants compared to the control.These results are in agreement with the studies of other authors (Morel et al., 2012;Cassán et al., 2009;Silva et al., 2013).
In the study of the structure of barley yield in the experimental variants, the increases in the plant height by 13-16%, ear length by 10-26%, number of grains per ear by 16-25%, grain weight per ear by 14-22%, were achieved.Similar studies on the influence of microorganisms on the barley yield and yield components were conducted by other researchers (Nabti et al., 2014;Mehrvarz et al., 2006).
The study of yield components of forage crops such as soybean (Glycine max cv Almaty) and barley (Hordeum vulgare cv Arna) determining its size and quality, has allowed to more fully estimate the efficiency and potential of the various compositions of bio-preparations.It is noted that preparations had a positive influence on all main yield components.The most effective bio-preparation was based on the mix of fungal strains (culture filtrate of P. bilaiae Pb14 + liquid cultures of A. pullulans YA05 and Rh.mucilaginosa YR07, in dilution 1:5).It was found that application of mix of micromycetes was the most effective compared to their separate use.The results obtained are in agreement with literature data (Cassán et al., 2009;Kawai & Yamamoto, 2006).
The increased yield and improving of various yield parameters due to use of a mix of micromycetes (composition of bio-preparation No. 3) compared to their separate use (samples No. 1 and No. 2) allows to make an assumption about the possibility of synergistic relationship between the components of micromycetes composition.

Table 2 . Influence of seed treatment with micromycetes strains on seedling growth.
Liquid cultures and culture supernatants of the A. pullulans YA05 and Rh.mucilaginosa YR07 strains (each strain separately and their mixture) and fungal filtrate of P. bilaiae Pb14 were used for seed treatment to determine their plant growth-promoting effect.Stem and root length of soybean and barley seedlings was measured in experiments and control (treatment with water).