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Extraction of Microorganisms in the Cotton Rhizosphere and the Study of Their Practical Significance

1Ruzmetov Rasul, 2Abdullaev Ikram, 3Matyakubov Zafar, 4Sayyora Babajanova,

5Hulkar Babajanova

1PhD on Agricultural Sciences, Senior Research Fellow, Khorezm Mamun Academy, Uzbekistan.

E-mail: [email protected]

2Doctor of Biological Sciences, Professor, Chairman of the Khorezm Mamun Academy, Uzbekistan.

E-mail: [email protected]

3PhD on Biological Sciences, Senior Research Fellow at the Khorezm Mamun Academy, Uzbekistan.

E-mail: [email protected]

4Chair governor of the Department of Biotechnology, Urgench State University, Uzbekistan.

E-mail: [email protected]

5Biotechnology Internship Teacher, Urgench State University,Uzbekistan.

E-mail: [email protected]

Abstract: This article provides the research results on that in the conditions of Khorezm region’s dry climate and saline soils, pure cultures of bacteria involved in the mobilization of mineral phosphates were isolated, morphologically analyzed and found to belong to the genus Bacillus as gram-negative rods. Bacterial activity was monitored – solubility activity of water-soluble phosphate and antagonistic properties of soil- borne fungi such as Alternaria alternata Rhizoctonia solani, Fusarium solani Verticillium dahlia were studied. When cotton seeds were processed with the association of bacteria found to be active and encapsulated with composts, high yields of cotton were obtained in the bacterial-treated variants. The results obtained are important for the conditions of saline soils and can be used to prevent phosphorus

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deficiency in crops and to obtain high yields from cotton.

Keywords: rhizobacteria, mineral phosphates, phosphate mobilization, cotton, saline, antagonism, fungus.

Introduction

Similar to other organisms, plants coexist with many organisms in nature. These organisms can be fungi and bacteria that live in the soil. It is known that the life of a plant and its fertility depend on how microorganisms live in and around its body [2;8].

The plant controls the number of microorganisms that live around its roots. Due to the activity of these microorganisms, nutrients are easily absorbed by plants. At the same time, physiologically active substances that control the growth and development of various plants are synthesized by microorganisms and create conditions for normal plant nutrition [12]. In the rhizosphere, the number of microorganisms is 100 times greater than in soils where its root divisions do not reach. [15].

Complex of bacteria living in the rhizosphere varies depending on the plant species and averages 66 species. The most common types include Pseudomonas, Bacillus, Bacterium, Chromobacterium, mycobacterium, Mycococcus, Micrococus, Pseudobacterium, Sarcina, Promyxobacterium, Azotomanos, Lactobacterium [2; 4; 5;

6; 10; 11]. Cases of isolation of the genera Fusarium, Alternaria and Cladosporium were also recorded. On a number of individuals recorded a mixed infection, as we isolated B. tenella with Mucor sp.; B. tenella with Penicillium sp.; and B. tenella with Alternaria sp [1].The distributional geography of microorganisms depends on a complex of agroecological factors, such as humidity, temperature, substrate type, acidity, soil salinity, etc [7]. Associations of some specific microorganisms that dominate the soil develop [3]. Associative groups of certain bacteria convert organic and mineral phosphates, which are difficult to assimilate for plants, into easily digestible forms, and at the same time have the properties of increasing disease resistance [11]. Among the bacteria known to have such properties, almost 50% are

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rhizosphere bacterial strains [3; 9]. An in-depth study of these bacteria will enable the cultivation of ecologically clean products through the creation of biopreparations in agriculture and their application to crops. In the conditions of Khorezm region, where the ecological situation is complex, we studied the possibility of creating a biopreparation by extracting pure cultures of bacteria in the rhizosphere of cotton plants.

Methods

The research was conducted in cotton fields in the South Aral Sea region of Uzbekistan. During the study of the cotton root rhizosphere in selected fields, samples were obtained by means of a modified modification substantiated on the D.G.Zvyagintsev [14] method. In determining the number of microorganisms in the samples, we prepared a suspension from the soil, diluting it repeatedly and planting it in nutrient media. Microorganisms involved in the mobilization of mineral phosphates were studied in the Pikovsky nutrient medium.

Determination of the number of microorganisms that mobilize mineral phosphates in the soil was carried out in fields with different mechanical composition and different levels of salinity. The amount of microorganisms belonging to different physiological groups was studied in soils with the most common average sandy mechanical composition in the conditions of Khorezm region. In order to determine the number of microorganisms belonging to different physiological groups, MPA (Meat peptone agar-agar), Chapek, Vinogradsky, Giltay, Ashby environments were used. The MPA nutrient medium was delivered readily.

After enumerating colonies of microorganisms grown from suspensions planted in Pikovsky nutrient media, the areas of calcium phosphate dissolved around their colonies were studied. Calcium phosphate-dissolved microorganisms were extracted in more areas around the colonies. Their morphological properties were studied using an immersion 1800x lens of a microscope. The studied microorganisms were stored at 5–

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700С in the refrigerator in a nutrient medium of the potato-agar. Among bacteria, the most acid-forming microorganisms were studied and their cultures were numbered. 2 cultures were found to be active and propagated in liquid Chapek medium and their effect on seed germination was studied in vegetative containers under laboratory conditions. Dissolution of calcium phosphates in cultures of Pikovsky in 50 ml volumetric flasks of cultures was studied.

The antagonistic properties of phosphorylating bacteria were studied on the example of fungi that cause fusarium wilt and rhizoctonia. We used the “a pit” method.

For this, we placed a suspension of FMB in a pit on the agar where phytopathogenic fungi grow. As a result, it was observed that zones of fungal growth were formed around the pit [14].

Field experiments were carried out on the basis of generally accepted methods in Uzbekistan, in 4 repetitions and 6 variants, in field parts of the size 50 m2, according to the following scheme. The seeds in the control variant were sown in water for 13 hours. The experiments were conducted in saline fields.

1. The seeds in the control variant were sown in water for 13 hours.

2. In the second variant, without fertilizer, the seeds were soaked in phosphorylating bacteria for 13 hours and encapsulated in biocompost.

3. In the third variant, with the application of 50% fertilizer (N100P70K50), seeds were soaked in water for 13 hours.

4. In the fourth variant, with the application of 50% fertilizer (N100P70 K50), seeds were soaked in phosphorylating bacteria for 13 hours and encapsulated in biocompost.

5. In the fifth variant, with the application of 100% fertilizer (N200P140 K100), seeds were soaked in water for 13 hours.

6. In the sixth variant, with the application of 100% fertilizer (N200P140 K100), seeds were soaked in phosphorylating bacteria for 13 hours and encapsulated in

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biocompost.

Phenological observations of the cotton were carried out in accordance with the methods adopted by the General Uzbek Cotton Research Institute.

The agrochemical composition of the field was studied in the laboratory conditions before the experiment. It was studied using generally accepted methods.

Table 1

Agrochemical composition of the field that the experiment conducted Dept

h

Humu s

Total % Easily assimilated ones, mg. kg Nitroge

n

Phosphor us

Potassiu m

Amm onium

Nitrate Phosph orus

Potass ium Agrochemical composition of saline washed field

0 -30 0,91 0,047 0,190 1,40 17,8 6,3 37,2 360 30-

60

0,80 0,045 0,149 1,53 15,3 5,0 10,4 288

Analysis of experimental data

The amount of humus in the soil was determined by the method of V.I.Tyurin, the total amount of nitrogen was determined by means of Keldal method and the amount of phosphorus was found out using Meshyakov method [13]. The results obtained are presented in Table 1.

In Khorezm region, mainly saline soils are prevalent, the soil salinity is washed away in autumn and spring, and the dry climate and very hot summer months lead to secondary salinization of soils. Given the prevalence of salinity in the region, samples were taken from the cotton rhizosphere growing in saline soils, and 21 acid-forming bacteria were isolated. According to the results of monitoring, two bacteria were extracted because of their strong antagonistic properties against active acid-forming and phytpathogenic fungi (Fig. 1) and their fish peptone agar-grown colonies were

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photographed. These bacteria were studied by gram staining and found to be susceptible to gram-negative bacteria. When the spores were stained, they were found to have spore-forming properties. Relationships to carbohydrates and amino acids were also analyzed and it was found that these bacteria belong to Bacillus sp, Baccilus subtillis and species.

The appearance of Bacillus sp colonies The appearance of Baccilus subtillis colonies

Gram staining of Bacillus sp Gram staining of Baccilus subtillis Figure 1. Some morphological features of rhizosphere bacteria

These bacteria were propagated in the laboratory and their antagonism against the pathogens Rhizoctonia sola, Fusarium sola, Verticillium dahliae, Alternaria alternata was studied. In this case, the diameter of the lysis zone around the phytopathogenic fungus culture was determined by the percentage of the total area of

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the lysis zone when the middle part of the petri dish was filled with a phosphor- mobilizing bacterial suspension. (Table 2) In this case, when the antagonism of Bacillus subtillis was studied against the phytopathogenic fungus Rhizoctonia solani, it formed a 65 mm lysis zone and accounted for 70%.

Table 2

Inhibition of the development of phytopathogenic fungi by phosphorylating bacteria

Cultures of rhizobacter

ia

Inhibition of the development of phytopathogenic fungi Rhizoctonia

solani

Fusarium solani

Verticillium dahliae

Alternaria alternatа d,

mm

% d, mm

% d, mm % d,

mm

%

Bacillus subtillis

65 70 60 66 40 44 50 55

Bacillus sp 60 70 60 66 25 30 15 16

In the study of the antagonism of Fusarium solani against the fungus, the lysis zone was 60 mm and accounted for 66%. The antagonism of Verticillium dahlia was slightly less than that of the fungus 40 mm: 44%. Alternaria alternatа was 50 mm relative to the alternate fungus and accounted for 55%. When Bacillus sp was studied, similar results were obtained for the fungi Rhizoctonia solani, Fusarium solani.

Verticillium dahlia was observed to be less atogenic than Bacillus subtillis compared to Alternaria alternata fungi.

Bacterial calcium phosphate solubility activity was studied. (Table 3) In this case, less soluble phosphates in water were dissolved in the early stages of Bacillus subtillis - 14 days after inoculation of bacteria than in Bacillus sp. After 21–28 days,

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the amount of water-soluble phosphates in the Bacillus subtillis culture medium was moderate compared to the Bacillus sp culture medium. It was observed in the experimental results that the amount of soluble phosphates, which accelerated the formation of acid, which can be explained by the change in pH, also increased. No solubility of phosphates was observed in the control variants in which bacteria were not cultured. When bacteria isolated from the conditions of Khorezm region were cultured in Pikovsky nutrient medium for the dissolution of calcium phosphate, it was observed that a lysis zone was formed around 100% of the colonies of these bacteria, indicating the dissolution of calcium phosphate.

Table 3

Dissolution of Ca3(PO4)2 in bacterial suspension (mg/50 ml in 50 ml suspension)

Varian ts

After 7 days After 14 days After 21days After 28 days The

number of bacteria

, mln

рН Р2О

5

The number

of bacteria

, mln

рН Р2О

5

The number

of bacteria

, mln

рН Р2О

5

The number

of bacteria

, mln

рН Р2О

5

Contro l

0 6,6 0,46 0 6,7 0,5 0 6,6 6,9 0 6,6 6,9

Bacillu s subtilli

s

7,6 5,3 22,0 38,0 5.1 52,0 35,0 4,8 46,0 32,0 4,8 27,0

Bacillu s sp

7,6 5,2 25,0 34,0 5,0 54,5 38,0 4,7 42,0 28,5 4,8 25,0

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The seeds were mixed with the studied bacteria in a 106-titre suspension of the bacterial association in a sheltered place from the sun until they were completely covered with compost made from straw (Fig. 2). The purpose of covering the top of the bacterized seeds with compost is to keep them in a dry climate without losing a certain amount of moisture, along with protecting them from sunlight. The climate of Khorezm region is dry and moisture plays an important role in the development of biological processes in the soil.

Figure 2. Seeds that have been bacterized and covered with compost

It was taken into account that humid microorganisms and seeds provide moisture in the conditions of the desert zone, where the climate is dry, and protect from bactericidal sunlight during involuntary sowing. Seeds were sown in special drills. Rhizosphere microorganisms were analyzed for variants during each growth period of the plant. (Table 4) Experimental results showed that the number of bacteria that grow if the meat peptone agar is high during the seedling and cocoon formation phase of the cotton. MPA-Meat peptone agar was higher in the number of bacteria growing than in the control variant or in the variants used in phosphorbacteria in the 3- 4 leaf extraction phase. This process was observed to be higher in the control variant as opposed to in the variants in which fungi were used. The number of actinomycetes and cellulose decomposers in the rhizosphere of cotton decreased to the bactericidal variants before the seedling phase. During the phases of flowering and the formation of cotton balls, a massive decrease in their number was observed. An increase in the

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number of bacteria belonging to another group studied was observed.

Table 4

Distribution of agronomically important microorganisms in the root system of cotton by its growth phases in the unwashed saline field

Variants Physiological groups of microorganisms MP

A

Fungi Actinomyc etes

Nitrifier Cellulos e

breakers

Ashby

Sprouting 3- 4 leaves Control 135

0

60 81 53 60 100

FMB 149

0

55 110 55 30 480

50% fertilizer 195 3

12.5 92 55 60 300

50%+ FMB 142

0

51 96 76 77 450

100% fertilizer 149 0

86 82 102 30 510

100% fertilizer + FMB

131 9

45 96 180 32 510

Seedling phase

Control 140

0

87 65 100 55 150

FMB 280

0

66 96 200 28 520

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50% fertilizer 212 0

58 90 150 55 380

50%+ FMB 195

0

47 86 200 53 720

100% fertilizer 195 0

95 71 200 30 600

100% fertilizer + FMB

286 0

60 85 220 28 680

Flowering phase

Control 130

0

89 110 50 10 92

FMB 245

0

57 270 72 32 680

50% fertilizer 149 0

58 270 87 25 420

50%+ FMB 142

0

42 360 92 53 740

100% fertilizer 174 0

71 320 110 30 740

100% fertilizer + FMB

256 0

51 830 102 55 940

Phase of cotton ball formation

Control 240

0

79 120 28 100 86

FMB 426

0

82 380 180 200 620

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50% fertilizer 330 0

90 260 55 100 360

50%+ FMB 560

0

118 360 76 180 640

100% fertilizer 360 0

77 330 96 200 420

100% fertilizer + FMB

470 0

89 380 180 300 620

During the flowering phase, the number of cellulose-degrading bacteria increased only in the bacterized variants. There was also a small increase in the number of bacteria growing in the Ashby nutrient medium.

The effect of FMB on cotton yield was studied (Fig. 5) in which an increase in yield was observed with the use of phosphorylating bacteria. In all the variants studied, the application of FMB yielded high in its unused 50% fertilizer and 100%

fertilizer applied variants.

0 5 10 15 20 25 30 35

Yield, c/ha

Control FMB 50% fertilizer 50% fertilizer + FMB 100% fertilizer 100% fertilizer + FMB

Figure 5. Influence of FMB on cotton yield

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Mineral fertilizers were not used in the control and FMB variant. At the same time, only in the variant using FMB, the yield increased by 2.7 centner. When (N100P70K50) -50% fertilizer was used, the yield increased by 2.2 centner compared to the usage of FMB with 50% fertilizer. When (N200 P 140 K100) -100% fertilizer was used, the yield was 2.4 centner higher than when compared to the variant using FMB applied with 100% fertilizer.

Conclusion

Bacteria that mobilize active mineral phosphates in the cotton rhizosphere of saline soils of Khorezm region are gram-negative bacteria that belong to the genus Bacillus, they belong to the genus Baccilus sp. Baccilus subtillis.

They form a lysis zone of 66-70% of Rhizoctonia sola, Fusarium sola phytopathogenic fungi. They form a lysis zone of 44–55% and 16–30%, respectively, of Verticillium dahlia, Alternaria alternata phytopathogenic fungi.

They were observed to increase the water solubility of phosphates as a result of the acidic conditions of Rn in aqueous solution.

It was observed that the number of rhizosphere microorganisms in the growth phases of cotton increased with the number of microorganisms of agrobiological importance in the variants using phosphobilizing bacteria. An increase in cotton yield was observed in the variants using FMB.

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