Ιmpact of Microbial Ιnoculants on Microbial Quantity， Enzyme Activity and Available Nutrient Content in Paddy Soil

Liu Xiao-jie， Duan Xue-jiao， Ma Na， Sun Tao， and Xu Jing-gang

1National Soybean Engineering Technology Research Center， Northeast Agricultural University， Harbin 150030， China

2College of Resources and Environment， Northeast Agricultural University， Harbin 150030， China

Ιmpact of Microbial Ιnoculants on Microbial Quantity， Enzyme Activity and Available Nutrient Content in Paddy Soil

Liu Xiao-jie1， Duan Xue-jiao2， Ma Na2， Sun Tao2， and Xu Jing-gang2

1National Soybean Engineering Technology Research Center， Northeast Agricultural University， Harbin 150030， China

2College of Resources and Environment， Northeast Agricultural University， Harbin 150030， China

The experiment was conducted to study the impact of application of microbial inoculants， compared with no microbial fertilizer， on enzyme activity， microbial biomass and available nutrient contents in paddy soil in Heilongjiang Province. The application of soil phosphorus activator was able to increase the quantity of bacteria and fungi in soil， but its effect on actinomycetes in soil was not significant. The application of microbial inoculants increased the urease and sucrase activities in soil over the growing season， but only at the maturing stage soil acid phosphatase activity was enhanced with the applying soil phosphorus activator. The application of soil phosphorus activator increased alkali-hydrolyzable nitrogen and available phosphorus contents in soil， but did not increase available potassium content in soil. The optimal microbial inoculant application rate as applied as soil phosphorus activator was 7.5 kg·hm-2.

microbial fertilizer， paddy soil， soil microorganism， soil enzyme activity

Ιntroduction

Since the reform and opening up， the agriculture is developing continuously fast and the use of chemical fertilizer is also increasing dramatically at the same time. At present， China is the country which is the most productive and using chemical fertilizers in the world. From the intensity of application of fertilizers，China is in the fourth place in the world， far higher than the world average （Guo et al.， 2010）. A large amount of chemical fertilizer application plus unreasonable structure of different fertilizers led to the emergence of large problems， such as pollution and high cost/low profit situation （Tand and Zheng， 2004）. The emergence of microbial fertilizer （or microbial inoculants） is one of the methods to solve the problems of chemical fertilizer over application， which is also one of the important indexes of soil quality and components of soil biochemical characteristics（Bhushan and Sharma， 2002； Zhang et al.， 2006；Jiao and Wei， 2009）. Reasonable application of microbial fertilizer combined with chemical fertilizers can not only improve the soil fertility， supply crop nutrients， promote crop growth and development，but also provide better environment for crop and microorganism living in soil.

The microbial fertilizer used in this experiment was called soil phosphorus activator， which provided the beneficial microbial strains， not only activating soil phosphorus， potassium and trace elements， improving fertilizer utilization efficiency， reducing the amount of fertilizer application rate， but also improving crop resistance， and enhancing the abilities of crops to resist disease. Many research results showed that the soil phosphorus activator could increase the yield ofrice and improve the rice taste （Wang et al.， 2003）. In addition， soil microorganisms play a major role in soil formation process， such as helping forming aggregated structure， driving nutrient cycling and energy transformation （Long et al.， 2003； 2004）.

The soil enzyme is abundant and widespread in soil and most of the soil enzymes were from microorganisms as well as a small part of plants（Perucci et al.， 2000； Burns， 1982）. Different fertilization methods have great influences on soil enzyme activities， which affect metabolism process in plantsoil ecosystem. Therefore， the relationship between the soil microorganism and soil enzyme activity need to be further studied. Also the soil available nutrients are related to microbial activity in soil and need to be clearly studied to provide scientific theoretical basis for the agricultural practice in the northeast cold region of China. In this paper， the soil phosphorus activator was used as a representative of microbial fertilizers and its effect on microbial quantity， enzyme activity and nutrient availability were studied.

Materials and Methods

Experimental design

The field experiment was located in Acheng District，Harbin City， Heilongjiang Province， where the soil was a typical black soil with organic matter content 45.4 g · kg-1， available phosphorus content 35.2 mg · kg-1，available potassium content 142 mg · kg-1. The alkali solution nitrogen content in soil was 20.9 mg · kg-1and pH was 5.45. The variety of rice used in the experiment was Daohuaxiang-Ⅱ. The microbial inoculants used were the soil phosphorus activator produced by Heilongjiang New-Green Biological Co.， Ltd. The soil samples were collected at the tillering stage， jointing stage， heading stage and maturing stage respectively at five points in each experimental plot at each sampling time. In order to avoid the influence of air on the surface soil microorganism，0-5 cm soil were removed before each sampling and 5-15 cm soil were sampled and roots and rocks in the samples were picked and separated from soil by hands. The microbiological properties of soil were determined from the fresh soil as soon as possible after each sampling or placed in the refrigerator under 4℃. The physical and chemical properties of another part of soil samples were determined in the laboratory.

The random block design was used in this experiment. Traditional fertilization was used as the background fertilization treatment. The application amount of microbial fertilizers was set up into three treatments: without microbial fertilizer （CK）；7.5 kg · hm-2phosphorus activator （P1）； 15 kg · hm-2phosphorus activator （P2） with three replications. Each experimental cell was 10 m long and 6.7 m wide， totally 66.7 m2. The cell was separated with plastic sheet with 10 cm underground and 35 cm aboveground. The experiment cell was irrigated and drained independently.

Experimental methods

The basic physical and chemical properties of soil included pH （1 : 2.5 soil to water ratio）， soil organic matter content （SOM）， total nitrogen， total phosphorus，alkali hydrolizable nitrogen， available phosphorus，available potassium in soil were determined using conventional method （Jiao and Wei， 2009）.

Microbial quantity was determined by dilution plate counting method （Long et al.， 2003）. Bacteria，fungi and actinomycetes were cultured with beef extract peptone culture medium， Bengal red culture medium and Gao culture medium （starch medium），respectively.

The urease activity in soil was determined by phenol sodium hypochlorite colorimetric method. It was expressed by the quantity （mg） of NH3-N in 1 g soil at 37℃ after 24 h. The acid phosphatase activity in soil was determined by sodium phosphate two sodium colorimetric methods. The sucrase activity was determined by 3， 5-two nitro acid colorimetric method. The sucrase activity was expressed by the quantity （mg） of glucose formed in 1 g soil at 37℃after 24 h （Long et al.， 2004）. The acid phosphatase activity was expressed by the quantity （mg） of phenol in 1 g soil at 37℃ after 24 h.

Data analyses

The experimental data was statistically analyzed by EXCELL and SPSS software. Differences among treatments were set at 95% confidence levels.

Results

lmpact of microbial fertilizer on bacterium quantity in soil

Overall， the number of bacteria in soil increased from the tillering stage to the jointing stage then decreased，with the maximum at the jointing stage， but the lowest at the maturing stage over the whole growing season（Table 1）. The amount of bacteria in soil increased gradually with the increase of the amount of the soil phosphorus activator used in the experiment. The number of soil bacteria under P2 was significantly higher than that of CK by 68.7%， 53.4%， 44.95% and 126.36% at the tillering， jointing， heading and maturing stages， respectively.

lmpact of microbial fertilizer on fungus quantity in soil

The number of the soil fungi increased slowly over the growing season and reached the maximum at the jointing stage under CK and P1， but it reached maximum at the heading stage under P2 （Table 2）. However， the number of fungi increased significantly with the increase of the amount of the soil phosphorus activator used in the experiment at each stage over the growing season. The number of fungi under P1 was significantly higher than that of CK by 58.65%，56.82%， 31.65% and 48.70% at the tillering， joining，heading and maturing stages， respectively.

Table 1 Dynamics of bacterium in soil

Table 2 Dynamics of fungi in soil

lmpact of microbial fertilizer on actinomycetes quantity in soil

The effect of the soil phosphorus activator on the number of soil actinomycetes was analyzed and the results is shown in Table 3. The number of soil actinomycetes was the highest at the heading stage and the lowest at the tillering stage over the growing season. The number of soil actinomycetes under P2 compared with CK was 8.1%， 4.39% and 8.09% higher， respectively at the tillering， heading and maturing stages. The number of soil actinomycetes was not signi-ficantly different under P1 and P2 at the heading stages. Overall， the soil phosphorus activator had no significant effect on the number of actinomycetes in soil.

lmpact of microbial fertilizer on urease activity in soil

The dynamics of urease activity in soil over the growing season are shown in Fig. 1. The urease activity decreased first from the tillering stage to the heading stage and then increased to the maturing stage. The urease activity was higher under the treatment with the soil phosphorus activator applied than that of CK and there was significant difference between P1 and P2 over the growing season except at the jointing stage.

lmpact of microbial fertilizer on sucrase activity in soil

The sucrase activity first increased and then decreased from the tillering stage to the jointing stage and from the heading stage to the maturing stage. The sucrase activity at the jointing stage was significantly higher than that at other stages. Compared with CK， the sucrase activity increased by 82.99% and 88.99% when the soil phosphorus activator was applied at the jointing stage， 59.7%-1.55% at the heading stage， 8.78%-19.61% at the heading stage and 71.96%-92.26% at the maturing stage under P1 and P2， respectively. The sucrase activity greatly increased by the soil phosphorus activator over the growing season except at the heading stage （Fig. 2）.

The changes of sucrase activity were different at different stages under the same treatment. Sucrase activity in soil increased to the highest at the jointing stage， and then decreased at the heading and the maturing stages for CK， while under P1 and P2 treatments， sucrase activity reached the maximum at the jointing stage and then decreased dramatically over the rest of the growing season. The sucrase activity under P2 treatment was greater than that under P1 at each stage over the growing season.

lmpact of microbial fertilizer on acid phosphatase activity in soil

The dynamics of soil acid phosphatase activity at different growing stages are shown in Fig. 3. The changes of acid phosphatase activity were not significant at the tillering， jointing and heading stages，but significant at the maturing stage. Compared with CK， the soil acid phosphatase activity increased 20.98%-66.61% by using the soil phosphorus activator under P1 and P2， respectively at the maturing stage.

Table 3 Dynamics of actinomycetes in soil

The acid phosphatase activity under the same treatment at different stages was not the same. The soil acid phosphatase activity between CK and P1 plus P2 treatments was significant at each stage over the growing season， but the difference between P1 and P2 was significant at the tillering and the maturing stages， but not significant at the jointing and the heading stages.

lmpact of microbial fertilizer on alkalihydrolyzable nitrogen content in soil

Application of the soil phosphorus activator could improve soil alkali-hydrolyzable nitrogen content. And the effect of the soil phosphorus activator on thecontent of soil alkali-hydrolyzable nitrogen at different growth stages is shown in Fig. 4. The content of soil alkali-hydrolyzable nitrogen under P1 treatment was the highest， by 12.35% compared to CK， and increased by 11.2% compared with P2 treatment. The difference between P2 and P1 treatments was not significant. At the jointing stage， the content of soil alkalihydrolyzable nitrogen under P1 treatment increased by 19.94% compared with CK.

At the heading stage， the content of soil alkalihydrolyzable nitrogen under P2 treatment was the highest and increased by 19.75% compared with CK and increased by 6.16% compared with P1 treatment. At the maturing stage， the content of soil alkalihydrolyzable nitrogen under P2 treatment was the highest， by 19.34% compared with CK.

Fig. 1 Dynamics of urease activity in soil

Fig. 2 Dynamics of sucrase activity in soil

Fig. 3 Dynamics of acid phosphatase activity in soil

lmpact of microbial fertilizer on available phosphorus content in soil

The dynamics of soil available phosphorus content at different stages are shown in Fig. 5. At the tillering stage， the content of available phosphorus under P1 treatment was the highest， by 157.21% compared with that of CK. The content of available phosphorusunder P2 treatment increased by 123.48% compared with that of CK. The difference was not significant between P2 and P1. At the jointing stage， the content of available phosphorus under P1 and P2 treatments was higher than that of CK， but the difference was not significant between P2 and P1. At the heading stage， the contents of available phosphorus under P2 and P1 were 102.53% and 93.59% higher than those of CK. The difference was not significant between P2 and P1. At the maturing stage， the content of available phosphorus under P1 treatment was the highest and increased by 163.88% compared with that of CK. The application of the soil phosphorus activator could obviously improve the content of available phosphorus in soil， but when the amount of P1 treatment reached the highest level， continuing to increase the amount of the soil phosphorus activator in soil could reduce the content of soil available phosphorus content in soil.

lmpact of microbial fertilizer on available potassium content in soil

The dynamic change of soil available potassium in soil is shown in Fig. 6. After application of the soil phosphorus activator， the content of available potassium in soil was stable， implying that the soil phosphorus activator might not have significant effects on improving the available potassium content in soil.

Fig. 4 Dynamics of alkali-hydrolyzable nitrogen content in soil

Fig. 5 Dynamics of available phosphorus content in soil

Fig. 6 Dynamics of soil available potassium content in soil

Discussion

Microbial fertilizer （or inoculants） could change microbial varieties and population in soil. With the increase of the amount of the soil phosphorus activator used in soil， the number of bacteria and fungi in soil increased， and the number of actinomycetes changed little. The increase in the number of soil bacteria and fungi might be due to the increase in available phosphorus in soil caused by the soil phosphorus activator， since previous studies indicated that an appropriate increase of phosphorus element could increase the number of soil bacteria and fungi （Shi et al.， 2014； Liu et al.， 2012； De Vries et al.， 2006）. The soil phosphorus activator treatment had no effects on the number of the soil actinomycetes， indicating that actinomycetes in soil were not sensitive to phosphorus content in soil.

Soil urease and sucrase activities increased with the increase of the soil phosphorus activator used in the experiment， but the soil acid phosphatase activity was lower at the tillering， jointing and heading stages. The activity of acid phosphatase increased rapidly. Li et al. （2006） also got the similar results in the study. Applying the soil phosphorus activator had few effects on the activity of soil acid phosphorus at the tillering，jointing and heading stages. But at the maturing stage，soil acid phosphatase activity would be enhanced with increasing the amount of fertilizers， which might due to the maturing stage， rice growth needed more phosphorus. The increase of soil enzyme activity might be related to the increase of microbial fertilizers. The increase of soil enzyme activity could increase the rate of biochemical reactions in soil （Sun et al.， 2003）.

The soil phosphorus activator could improve the content of alkali-hydrolyzable nitrogen and available phosphorus in soil. But excessive application could not have the same results. The increase in the number of microorganisms in soil could improve the soil microbial activity， so as to accelerate the conversion of nutrient elements from insoluble to water soluble in soil， and improve the plant use efficiency for the nutrients. Although the microorganism itself did not contain nutrients， it could increase nutrient availability in soil and promote plant use efficiency for nutrients， so it had some functions of fertilizers. In conclusion， applying the soil phosphorus activator could increase soil available nitrogen and available phosphorus contents， but not the content of soil available potassium. So selection of microbial fertilizers according to use purposes is also an important task for farmers.

Conclusions

The soil phosphorus activator increased the number of microorganisms in soil. The effect of the soil phosphorus activator increased the number of bacteria and fungi more significantly than that of actinomycetes.

Application of the soil phosphorus activator enhanced the urease and invertase activities in soil；however， its effect on the activity of soil acid phosphates was not significant.

Application of the soil phosphorus activator improved soil alkali-hydrolyzable nitrogen content and available phosphorus content， implying that different microbial inoculants did not have exactly the same effects on soil microorganism quantity， enzyme activity， and biomass and soil available nutrient. Selection of microbial fertilizers or inoculants according to the agricultural situation is important in farming practice.

The suitable application rate of the soil phosphorus activator at 7.5 kg · hm-2was recommended in the farming practice according to the experimental results. Excessive application of microbial fertilizers should be prohibited， since it not only reduced output/input ratio，but also produced some toxic effects on crops.

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S144； S155 Document code： A Article lD： 1006-8104（2015）-04-0007-08

Received 16 July 2015

Liu Xiao-jie （1963-）， female， researcher， engaged in the research of crop breeding and cultivation. E-mail: xjlmarry@163.com