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BIOLOGICAL INDICATORS OF SOUTHERN CHERNOZEM DURING LONG-TERM APPLICATION OF MINERAL FERTILIZERS
Tsandur Nikolay Alexandrovich, doctor of agricultural Sciences, corresponding member of NAAS of Ukraine, Director of pilot farm Burykina Svetlana Ivanovna, candidate of agricultural Sciences head of Department of Agrochemistry and soil fertility Odessa state agricultural experimental station of NAAS E-mail: opitna_lab@ukr.net; burykina@ukr.net Naidenova Oksana Evgenyevna, candidate, of biological Sciences, senior researcher, National Scientific Center "Institute for Soil Science and Agrochemistry Research
named after A.N. Sokolovsky", Kharkov, Ukraine
BIOLOGICAL INDICATORS OF SOUTHERN CHERNOZEM DURING LONG-TERM APPLICATION OF MINERAL FERTILIZERS
Abstract: The article studies the change in the state of the microbial cenosis of southern chernozem during long-term use ofvarious doses of mineral fertilizers. Changes in enyzmatic activity and microbial population were noted: a decrease in the enzymatic activity of the soil at doses N120P30K30, N120P60K60, N180P30K30 and N180P60K60; a decrease in the number of microorganisms mobilizing mineral phosphates at dose of N180P60K60. No significant impact on the number of lumbricids was found.
Keywords: mineral fertilizers, microbial cenosis of the soil, enzymatic activity of soil, southern chernozem.
Introduction.
Mineral fertilizers still play a prominent role in not only increasing the production of agricultural products, but also in rising soil fertility. Long-term use of mineral fertilizers leads to significant changes in agrophysical, agrochemical and biological parameters of soil. The changes in the first two parameters were studied by many researchers, but the structural and functional changes in the microbial cenosis of the southern chernozem, its biological activity when the various doses of mineral fertilizers are systematically used in the bogharic conditions of Black Sea region steppes of Ukraine is insufficiently studied.
A number of authors indicate an increase in the biogen-esity of the soil when using mineral and organo-mineral fertilization programs. Therefore, studies conducted in the third rotation of a six-field crop rotation on gray forest soil, indicate activation of mineralization processes of soil nitrogen on intensive and high-intensive mineral backgrounds [1, 557]. Similar results were obtained on chernozem soils of the Left-Bank Forest-Steppe of Ukraine: usage of mineral fertilizers in doses of N „ „„P K resulted in an increase in the number
30-90 30-60 30-60
of microorganisms assimilating organic and mineral nitrogen, compared to nil treatment variant by 7-9% (podzolized chernozems) and 3.9% (ordinary chernozem) [2, 17]. It is noted that the resistance of the microbial cenosis to the effects of
fertilizers depends on the type of soil: the microbial system of the leached chernozem has greater stability in comparison with sod-podzolic [3, 19].
Observation of development of soil toxicosis and inhibition of microflora in cases of prolonged systematic use of high doses of mineral fertilizers have been made [4, 65-82; 5, 133]. The use of high doses of mineral fertilizers causes a disruption in the structure of microbial communities, especially when use long-continually [6, 111-113]. In most cases, the use of mineral fertilizers in moderate doses positively affects the microorganisms and biological activity of the soil, and increased doses often inhibit the activity of many microbiological processes (nitrogen fixation, nitrification), while the mineralization processes, humus destruction, and the scale of denitrification increase. The inhibition of the development of soil microflora under winter wheat was noted with a dose of fertilizers N90P90K90 [7, 130-150]. It was also found that the use of high doses of mineral fertilizers causes an increase in the number of fungi, and the proportion of phytopathogenic and toxin-forming species [8, 175-176]. In addition, some data on the 65-year-old application of balanced doses of mineral fertilizers without negative changes in microbiological soil indicators exists [9, 116].
The systematic usage of large quantities of mineral fertilizers, an unbalanced ratio of nitrogen, phosphorus and po-
tassium can cause disbalance in the biogeochemical cycle of these elements in soils. Therefore, for each specific soil and climatic conditions and crops, it is necessary to determine the optimum doses of mineral fertilizers, which will not cause negative biological processes in soils and affect the microflora of soils negatively with increase in yields.
One of the indicators of soils' biological activity is their enzymatic activity. Enzymes such as dehydrogenase, invertase and polyphenol oxidase are important in the formation of soil fertility. Their activity is widely used as a biodiagnostic indicator when assessing the influence of various factors on soils.
Soil invertebrates, earthworms (lumbricides) in particular, can also serve as indicators of soil properties change under the influence of mineral fertilizers.
The purpose of our studies was to determine the changes in microbiological and biochemical indicators of southern chernozem and the number of lumbricidae depending on the doses of mineral fertilizers with long-term use.
Minerals and techniques. Microbiological and biochemical studies were conducted in the sixth rotation of the seven-field crop rotation of a long-term stationary field experiment of the science and technology in the field of ag-rochemistry and soil fertility department of the Odessa State Agricultural Experimental Station in 2015-2017. Experiment was conducted in 1971 on low-humus loamy southern chernozem over loess. Experiment was conducted in 4 fields, entry into the crop rotation was carried out on one field, the repeatability of the options in the experiment is triple, the placement is systematic. The area of the experimental plot is 240 sq.m., area of the accounting plot.
17 fertilizer systems are studied; mineral fertilizers used are: ammonium nitrate, granulated single superphosphate, potassium salt and ammonium nitrate phosphate fertilizer. Dosages are listed in the tables to show the results of the study.
Soil samples were taken in the 0-25 cm layer on winter wheat crops in the stage of earing after two precursors: bare fallow and winter rapeseed and determined the following biological indicators: the number of microorganisms of the main ecological-functional and taxonomic groups by microbiological seeding of soil suspension of the appropriate dilution on dense nutrients medium [10-13]: organotrophic bacterium - on meat-and-peptone agar (MPA), microorganisms that assimilate nitrogen mineral compounds and actinomycetes - on starch-and-ammonia agar (SAA) oligotrophic microorganisms - on starvation agar (SA), fungi - on Richter's medium, microorganisms that mobilize mineral phosphates - on Muromtsev's medium, organo-phosphates - on Menkina's medium. Estimates, in particular mineralization [14], oligotrophy [15] and microbial transformation of soil organic matter (MTSOM) [16, 13-16],
characterized by the intensity of mineralization processes and the trophic levels of the soil, were determined by the ratio of individual groups of microorganisms, the total biological index (TBI) - a method of relative values by G. Azzi [17, 242-243]. Biochemical activity of soil enzymes, such as invertase (by photocolorimetric method, described by D. G. Zvyagintsev et al. [10, 157-158]), dehydrogenases (by A. Sh Galstyan [18,39-40, 19, 1-7] and polyphenol oxidase (by L. A. Karyagina and N. A. Mikhailovskaya [20, 40-41, 21, 1-6]).
The number of earthworms was determined by tecnique of M. S. Gilyarov [22] in the soil layer of 10 cm on the plots of 50 x 50 cm.
The authencity of the data obtained during the research was evaluated by analysis of variance using the standard package of programs "Statistica 7.0".
Solution and consideration. The abundance of microorganisms of the studied groups in the soil against the background of all fertilizer systems when growing winter wheat after winter rapeseed was higher than in the nil treatment variant (Table 1). The maximal abundance of microflora (twice the nil control index) was observed when N180P60K60 was applied. Nitrogenous fertilizers treatment in a dose of on the average increased the number of microorganisms by 38% compared to nil treatment variant, with a dose of N180, the abundance of microorganisms in the soil differed little from the nil control variant. When N60P30K30 was applied, the abundance of microflora was on average higher than in nil treatment variant by 58%. The increase in the dose of nitrogen (N180P30K30) resulted in a lessening of effect. P60K60 treatment gave an increase in the abundance of microflora by 46% relative to nil treatment, the application of these doses of phosphorus and potassium fertilizers with the minimum norm of nitrogen (N60P60K60) had a less positive effect, with the exception of N180P60K60, where, as already noted, there was the highest number of microorganisms in mainly due to the bacteria that assimilate mineral nitrogen. When nitrogen fertilizers were treated in a dose of
N180 (N1s0 N180P30K30, N180P60K60) the abundance of micromy-cetes was twice that of nil treatment variant. The oligotrophy
index on fertilized variants was predominantly lower than in the nil treatment variant, which indicates an improvement in the nutritional regime. The mineralization coefficient also had lower values than those in the nil treatment variant, except for the N180P60K60 variant. The coefficient of microbial transformation of soil organic matter with the introduction of mineral fertilizers was significantly higher than in the nil treatment. Generally, the number of physiologically active microorganisms mobilizing mineral phosphates was lower than the values at the nil treatment, with increasing doses the degree decreased (Table 2).
Table 1.- Influence of different doses of mineral fertilizers on the abundance of microorganisms of basic ecological and functional groups in southern chernozem soil, stationary field experiment
Experiment variant Moisture content % Nitrogen-assimilating microorganisms, million/g Actino-mycetes, million/g Fungi, thousand/ g Oligo-trophs, million/g Eutrophs, million/g Indicators
organic mineral oligotrophy mineralization MTSOM TBI,%
total bacterium
Field No. 1, crop-winter wheat, Knopa variety, development phase - earing (precursor-winter rapeseed)
Nil treatment 13.82 7.27 24.41 19.06 5.35 34.65 45.56 31.71 1.44 3.36 9.44 100
N6o 12.75 24.25 31.12 20.70 10.42 25.19 45.71 55.40 0.83 1.28 43.15 138
N1So 11.22 9.56 23.54 18.53 5.01 62.64 46.00 33.16 1.39 2.46 13.44 103
N60P30K30 13.34 16.29 39.92 28.65 11.27 21.90 63.55 56.23 1.13 2.45 22.94 158
"^1S0P30"^"30 11.61 14.97 30.89 21.64 9.25 69.19 41.79 45.93 0.91 2.06 22.22 119
P60K60 13.06 14.36 41.70 33.13 8.57 32.42 52.20 56.09 0.93 2.90 19.31 146
N60P60K60 13.17 13.92 36.30 31.41 4.89 25.66 43.75 50.25 0.87 2.61 19.26 127
N P K ISO 60 60 11.52 14.27 59.90 48.61 11.29 69.87 68.84 74.24 0.93 4.20 17.67 193
HCPo,o5 - 1.22 3.10 - 1.04 4.26 4.48 - - - - -
Field No. 2, crop - winter wheat, Knopa variety, phase of development - earing (precursor - bare fallow)
Nil treatment 12.00 28.46 51.67 41.63 10.04 41.81 33.40 80.17 0.42 1.82 44.14 100
N60 11.05 23.68 25.38 17.16 7.77 27.03 12.21 49.09 0.25 1.07 45.77 49
N1So 14.68 22.16 28.44 23.49 4.95 30.59 34.16 50.63 0.67 1.28 39.43 83
N60P30K30 11.94 23.00 27.60 21.52 6.08 23.87 15.85 50.62 0.31 1.20 42.17 55
■^1S0P30"^"30 11.28 21.16 37.07 27.30 9.77 21.87 14.80 58.25 0.25 1.75 33.24 59
P60K60 12.59 31.19 35.78 26.89 8.89 26.27 21.55 67.00 0.32 1.15 58.38 75
N60P60K60 10.66 20.27 20.43 16.69 3.74 22.51 19.86 40.72 0.49 1.01 40.38 55
N P K ISO 60 60 12.19 21.58 30.09 23.63 6.42 17.95 34.72 51.69 0.67 1.39 37.06 84
HCPo,o5 - 1.10 3.24 - 1.06 6.00 4.03 - - - - -
Notes:
fO 0) CO
1 - Hereinafter, the number of microorganisms is expressed in millions (fungi - in thousands) of colony forming units (CFU) per 1 g of dry soil;
2 - MTSOM - microbial transformation of soil organic matter coefficient;
3 - TBI-total biological index
Table 2.- Influence of different doses of mineral fertilizers on the abundance of phosphate-mobilizing microorganisms in southern chernozem
Experiment variant Phosphate-mobilizing microorganisms, million/g
mineral organic
total with Ca3(PO4)2 dissolution zones total with phytin dissolution zones
Field No. 1
nil treatment 24.08 11.37 16.47 8.95
N60 18.07 12.74 25.79 10.54
N180 23.31 7.81 21.99 10.49
N60P30K30 26.72 8.57 26.72 12.59
N180P30K30 28.93 11.52 15.17 2.35
P60K60 24.09 6.25 29.81 15.21
N60P60K60 15.99 6.30 30.83 15.99
N P K 180 60 60 16.31 2.59 18.89 6.94
HCP005 2.06 1.40 2.60 1.36
Field No. 2
nil treatment 21.17 11.05 18.74 5.72
N60 15.69 7.18 14.80 7.33
N,80 14.26 5.18 23.00 13.65
N60P30K30 13.22 8.05 6.65 3.45
N180P30K30 15.10 7.40 17.69 8.88
P60K60 16.72 7.80 18.91 8.66
N60P60K60 17.66 7.49 22.87 10.91
N P K 180 60 60 12.32 5.90 23.45 11.80
HCP005 3.15 1.28 2.82 1.49
The abundance of microorganisms, mobilizing organic phosphates, significantly increased. Exceptions are physiologically active forms on variants N180P30K30 and N180P60K60, where their abundance was much lower.
In the soil of all variants of fertilizer systems of field No. 2, where winter wheat was grown after bare fallow, the microflora was lower in number than in the nil treatment, and the degree of decrease in nil treatment became lower with increase of fertilizers doses. The highest abundance of microflora among the fertilized variants, according to the TBI
(as well as in field No. 1), is noted on the variant N, P,
x 180 60 60
The oligotrophy index on the fertilized variants of field No. 2 was also lower than in the nil treatment variant, with the exception of the variants where the nitrogen fertilizer dose was at the level of 180 kg/active material. The same tendency is observed in the values of the mineralization index. The MTSOM coeffiaent at doses ^ N6oPзoKзo, N60P60K60 did not differ significantly from the values at the nil treatment, at
doses N180 N180P30K3<y N180P60K60 had lower values. The total
number of bacteria mobilizing organic phosphates, as well as
the number of their active forms in the soil of fertilized field variants, where the precursor was bare fallow, was lower than in the nil treatment variant. The number of the latter was twice lower in the variants with a dose of mineral nitrogen of 180 kg/active material (N180, N180P60K60). The number of bacteria dissolving organic phosphates in fertilizer applications was higher than in nil treatment soil.
One of the indicators of soil quality is the level of its enzymatic activity. The dehydrogenase activity in the soil of the fertilized variants of field No. 1 did not differ significantly from the nil treatment varient, the activity of invertase and polyphenol oxidase was reduced (Table 3). The maximum decrease in polyphenol oxidase activity was noted in variants
N N P K
N180, N180P60K60.
In the soil of the fertilized variants of field No. 2, the activity of all three enzymes studied decreased. The lowest activity
of dehydr°genase had the variants N180P30K30, N180P60K60, N60P60K60. A noticeable decrease in invertase activity was observed wl0en doses N180P30K30, N180P60K60, P60K60 were applied, polyphenol - on variants, where N, N180P60K60 were applied.
BIOLOGICAL INDICATORS OF SOUTHERN CHERNOZEM DURING LONG-TERM APPLICATION OF MINERAL FERTILIZERS
Table 3.- Influence of different doses of mineral fertilizers on the enzymatic activity of southern chernozem
Experiment variant Dehydrogenase, mg TFP / 100 g for 24 hours, Invertase, mg glucose / 1g for 24 hours Polyphenol oxidase, mg 1.4-n-benzoquinone / 10 g for hour
Field No. 1
nil treatment 147.00 2.34 1440
N60 136.50 1.34 936
N180 135.00 0.74 504
N60P30K,0 166.50 1.59 1296
N.80P,0K30 144.00 1.84 456
P60K60 150.00 0.69 1008
N60P60K60 162.00 1.79 1320
N P K 180 60^60 141.00 0.89 432
HCP005 20.14 1.40 66.80
Field No. 2
nil treatment 187.00 2.59 3456
N60 177.30 2.55 2160
N180 154.00 2.46 1752
N60P30K,0 171.80 1.64 2184
N180P30K30 151.20 1.41 1344
P60K60 180.10 1.50 2088
N60P60K60 156.70 2.96 1800
N P K 180 60^60 152.60 1.86 1368
HCP005 22.60 1.26 70.24
Note: TPF - triphenylformazan
In the soil of the treated variants of field No. 2, the activity of all three enzymes studied decreased. The lowest activity of
dehydrogenase had the variants N180, N180P30K30» N180P60K60,
N120P30K3<y N120P60K60' N60P60K60. A n°ticeable deCrease in in-
vertase activity was noted when doses N180P30K30, N180P60K60, P60K60, were applied; decrease in polyphenol oxidase ac-
tivety - in variants where N120, N180, N120P30K30, N120P60K60,
N180P60K60 were applied.
The applied fertilizer doses were influenced by the number of earthworms in the following way: a significant increase is noted with a minimum rate of full and phosphorus-potassium fertilizer and a decrease in N180 both in pure form and
against the background of P30-60K30-60 (table 4).
Table 4.- The number of earthworms in southern chernozem when different doses of mineral fertilizers are applied (in the 0-10 cm layer, the average for the two fields)
Variant nil treament N60 N180 N60P30K30 N180P30K30 P60K60 N60P60K60 N P K 180 60 60 НСР
number/m2 30 32 26 35 24 37 34 27 4
Conclusion. The effect of long-term use of different doses of mineral fertilizers on the microflora of southern chernozem manifested itself differently in two experimental fields. The state of the microbial cenosis of the soil of the fertilized variants under the same culture differed depending on the predecessor. Common trends for both microbial cenosis changes under the influence of systematic application of mineral fertilizers were a decrease in enzymatic activity at doses of N180P30K30 and N180P60K60, a decrease in the number ofmicroorganisms mobilizing mineral phosphates was visible when N180P60K60 was applied.
The number of earthworms increased significantly at the minimum norm of full and phosphorus-potassium fertilizer and decreased - at N180 both in pure form and against the background of P30-60K30-60.
For the further research of regularities and direction of changes in the state of microbial coenoses and the number of individual representatives of the mesofauna of southern chernozem under the influence of long-term fertilization, many years of data are needed.
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