Effects of Biological Bacterial Fertilizer on Carbon Metabolism Characteristics of Rhizosphere Soil Bacteria in Rice

ZHAO Jie, ZHU Xiao-lei*, YE Ming, HE Xing-wu, HUANG Wu-jian

1. Suyu Agricultural Technology Extension Center, Suqian 223800, PRC;

2. Wolvbao Bioscience Co. Inc, Suqian 223800, PRC

Abstract The effects of biological bacterial fertilizer and chemical fertilizer on carbon metabolism characteristics of rhizosphere soil bacteria in rice were studied through a plot experiment. The results showed that the number and Mcintosh index of bacteria in rice rhizosphere soil increased significantly with the application of biological bacterial fertilizer. It was found that the AWCD (average well color development) value of the bacteria remarkably increased and the decomposition of carboxylic acids, amines and heterozygotes were accelerated when adding biological bacterial fertilizer at a proper weight percent. All in all, proper addition of biological bacterial fertilizer could increase the number and carbon metabolism of bacteria. The most appropriate application rate was 70% chemical fertilizer nitrogen+30% biological bacterial fertilizer nitrogen for rice production in Northern Jiangsu Province.

Key words Biological bacterial fertilizer; Rhizosphere soil; Bacteria; Carbon metabolism characteristics

1. Introduction

The ecological environment of soil is critical to the growth of crops. Good soil environment can result in better quality and yield of crops[1-2]. However, the overuse of pesticides and fertilizers in recent years has deteriorated the soil ecological environment, causing a range of problems, such as soil hardening, unbalanced nutrients, poor soil fertility, and dysbacteriosis,etc[3-5]. The application of biological bacterial fertilizer can improve the physicochemical characters of soil, increase soil fertility, and activate soil microorganism, which is receiving more and more attention and recognition from scholars in the field[6-7]. Of the numerous types of biological bacterial fertilizers, very few were reported to be suitable for Northern Jiangsu Province, and few researches were conducted on the effects of biological bacterial fertilizer on carbon metabolism characteristics of rhizosphere soil bacteria in rice.

Biolog microplate technology is one of the mainstream methods of studying metabolism characteristics of microbial community. This method is convenient, fast and sensitive, which can directly obtain the metabolism characteristics of soil microbial community[8-10]. In this study, a plot experiment using Biolog was conducted to explore the effects of biological bacterial fertilizer on carbon metabolism characteristics of rhizosphere soil bacteria in rice. The biological bacterial fertilizers in the experiment were provided by Wolvbao Bioscience Co. Inc. The objective of the study is to elaborate the effects of biological bacterial fertilizer on the micro-ecological environment of soil and define a proper application amount of biological bacterial fertilizer, thus providing a theoretical basis and technical support for the reasonable application of biological bacterial fertilizer in rice production.

2. Materials and Methods

2.1. Experiment field

The plot experiment was conducted from June to September 2019 in Suyu District, Suqian, Jiangsu Province. Soil properties: sandy loam; pH 7.2; organic content 25.60 g/kg; available nitrogen 123.19 mg/kg; available phosphorus 16.32 mg/kg; available pota- ssium 127.47 mg/kg.

2.2. Experiment materials

Rice variety: Jingliangyouhuanglizhan; nitrogen fertilizer: urea (N 46%); phosphorus fertilizer: calcium superphosphate (P2O512%); potassic fertilizer: potassium sulfate (K2O 60%); the biological bacterial fertilizers were provided by Wolvbao Bioscience Co. Inc. (N 12%), effective live bacteria content 109CFU/g.

2.3. Experiment design

The experiment involved 4 treatments: T0, T15, T30 and T45. Each treatment used the same total nitrogen dosage of 267 kg/hm2. T0: 100% chemical fertilizer nitrogen+0 biological bacterial fertilizer nitrogen; T15: 85% chemical fertilizer nitrogen+15% biological bacterial fertilizer nitrogen; T30: 70% chemical fertilizer nitrogen+30% biological bacterial fertilizer nitrogen; T45: 55% chemical fertilizer nitrogen+45% biological bacterial fertilizer nitrogen. Each treatment was repeated three times, hence totally 12 plots were set. Each plot had an area of 20 m2, and the ridges were built according to the plot dimension of 4 m×5 m. The ridges between the plots were covered with plastic films before the application of base fertilizer to avoid leakage. Single irrigation and drainage method was adopted until harvest, and a protection row was set for each plot.

2.4. Sample collection and measurement

After the rice is ripe and harvested, five holes of rice from each plot were selected, the soil was shaken to get the rhizosphere soil of rice, and the number of the rhizosphere soil bacteria was measured. The bacteria were then cultured in beef extract peptone medium. The quantitative analysis was conducted using dilution method of plate counting[11]. The carbon metabolism characteristics of soil bacteria was determined by the Biolog method of GARLAND J Let al.[12].

2.5. Data analysis

Microsoft Excel 2007 was used for data processing. SPSS 17.0 software was used for variance analysis, correlation analysis and principal component analysis. Based on previous research results[13-14], three diversity indexes, namely Richness index S, Mcintosh index U and Simpson index D were mainly analyzed.

3. Results and Analysis

3.1. Effects of biological bacterial fertilizer on the number of rhizosphere soil bacteria in rice

Bacterial community was the largest group of soil microorganisms, accounting for 70%～90% of the total soil microorganisms[15]. It played a critical role in the process of nutrient conversion and energy cycles in soil. Fig. 1 showed that different treatments presented different effects on the number of rhizosphere soil bacteria in rice. The number of the bacteria increased with the application of biological bacterial fertilizer. The order of the increasing bacteria under different treatments was in the order: T45>T30>T15>T0, and the differences between the treatments were significant. The results indicated a huge facilitation of biological bacterial fertilizer on the number of rhizosphere soil bacteria in rice, and T45 had the best treatment effect.

Fig. 1 Effects of different treatments on the number of soil bacteria

3.2. Kinetic characteristics of rhizosphere soil bacteria in utilizing carbon sources

AWCD (average well color development) was an important parameter to assess the average ability of microorganisms in utilizing carbon source substrates. The higher the AWCD, the stronger the carbon source utilization ability, and the better the activity of the rhizosphere soil bacteria will be[16]. According to Fig. 2, as the incubation time increased, the carbon source utilization ability of bacterial communities first increased and then became stable. The AWCD changed slightly in the first 24 h of the incubation, indicating a low utilization of carbon sources; whereas it increased tremendously in 24～72 h, which meant that the soil microorganisms metabolized actively and carbon sources were utilized heavily; after 72 h, the AWCD tended to be stable. The overall performances of the treatments in AWCD were in the order: T30>T15>T45>T0; T15, T30 and T45 preceded T0 significantly, indicating that the application of biological bacterial fertilizer can improve the carbon metabolic activity of bacteria and promote their ability in carbon sources utilization.

Fig. 2 AWCD dynamics over incubation time

3.3. Rhizosphere soil bacteria’s utilization of different carbon sources

According to Table 1, after 72 h of culturing, the AWCD performances of different types of carbon catabolites of the 4 treatments were as follows: T30 had the highest utilization of the three carbon sources of carbohydrate, amino acids and polymers, followed by T0, T15 and T45 which showed no significant difference; T0 had the lowest utilization of carboxylic acids and heterozygotes, followed by T15, T30 and T45 which had a significant increase in utilization after application of biological bacterial fertilizer; T30 had the highest utilization of amine carbon sources, followed by T15, T45 and at last T0. Therefore, the application of biological bacterial fertilizer can significantly improve bacteria’s utilization of the three carbon sources of carboxylic acids, amines and heterozygotes, and accelerate their decomposition. T30 had the best treatment effect among the 4 treatments.

3.4. Principle component analysis of carbon source utilization of rhizosphere soil bacteria

After 72 h culturing, the AWCDs were standardized and the principle components were analyzed. Totally three principle components were extracted, the cumulative contribution rate of which was 89.135%.Specifically, the variance contribution rates of the first, second and third principle components were 72.855%, 9.445% and 6.835%, respectively (Table 2).

Table 1 Utilization of different carbon sources

Table 2 Contribution rate of principal component

The loading value of carbon sources reflected the correlation between utilization of carbon sources and the principle components. The higher the loading value, the larger impact it had on the principlecomponents[17]. As shown in Table 3, 30 carbon sources presented large contribution to the first principle component (loading value>0.7). Of them, the carboxylic acids accounted for 30%, the carbohydrates accounted for 23.3%, the amino acids accounted for 20%, the polymers accounted for 13.3%, and the amines and heterozygotes were all 6.7%;L-Arginine that belonged to amino acid carbon source had a large contribution to the second principle component. Therefore, the carbon sources utilized by rhizosphere soil bacteria in rice in this study were mainly carboxylic acids, carbohydrates and amino acids.

Table 3 Loading values of principal components of 31 sole carbon sources

3.5. Diversity indices of carbon sources utilization

According to Table 4, there was no significant difference between the Richness index S and Simpson index D; meanwhile, as the application of biological bacterial fertilizer increased, the Mcintosh index U kept increasing. T15, T30 and T45 had an increase of 8.5%, 18.1% and 19.8% respectively compared with T0, and the difference between T30 and T45 was not significant. To sum up, the application of biological bacterial fertilizer can significantly increase the Mcintosh index U of the rhizosphere soil bacteria in rice, and the most appropriate application rate was 70% chemical fertilizer nitrogen+30% biological bacterial fertilizer nitrogen.

Table 4 Diversity indices

4. Conclusion and Discussion

The number and Mcintosh index U of bacteria in rice rhizosphere soil increased significantly with the increase of biological bacterial fertilizer. The main reason was that the live bacteria count of this biological bacterial fertilizer was as much as 109CFU/g, which might contain a large amount of useful bacteria to the growth of rice.

The experiment showed that the performances of the rhizosphere soil bacteria in using carbon sources under different treatments were in the order: T30>T15>T45>T0. The main reason was that the application of fertilizer and biological bacterial fertilizer could increase the content of soil nutrient and provide a good environment for the growth of bacteria; the application of biological bacterial fertilizer could also accelerate the decomposition of the three carbon sources of carboxylic acids, amines and heterozygotes, bring a large number of live bacteria, improve the micro-ecological environment for the bacteria, and enhance their ability in utilizing certain carbon sources.

The principle component analysis indicated that the major carbon sources utilized by rhizosphere soil bacteria in rice in this study were carboxylic acids, carbohydrates and amino acids. Therefore, farmyard manures containing high content of carboxylic acids, carbohydrates and amino acids, such as rotten grass or leaves fertilizer, seed cake fertilizer and manure fertilizer, or new biological fertilizers were recommended as the fertilizer for rice production.