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Effect of Different Fertilizers on Functional Diversity of Microbial Flora in Rhizospheric Soil Under Tobacco Monoculture
ACTA AGRONOMICA SINICA Volume 37, Issue 1, January 2011 Online English edition of the Chinese language journal Cite this article as: Acta Agron Sin, 2011, 37(1): 105–111.
RESEARCH PAPER
Effect of Different Fertilizers on Functional Diversity of Microbial Flora in Rhizospheric Soil Under Tobacco Monoculture YANG Yu-Hong1, CHEN Dong-Mei2, JIN Yan1, WANG Hai-Bin2, DUAN Yu-Qi1, GUO Xu-Kui2, HE Hai-Bin2, and LIN Wen-Xiong2,* 1
Yunnan Provincial Institute of Tobacco Research, Yuxi 653100, China
2
Institute of Agroecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Abstract: Continuous cropping obstacle is one the most important problems in tobacco (Nicotiana tabacum L.) production. To alleviate continuous cropping obstacle based on management of soil ecology, the effects of different fertilizers were tested using tobacco cultivar K326 growing in a field with 12-year consecutive cultivation. The rhizospheric soil was sampled to investigate the changes in functional diversity of microbial flora in different treatments. The results showed that the autotoxic allelopathic potential was maximal for the monoculture soil treated with traditional compound fertilizer, and minimal for the soil treated with farmyard manure. According to the result of BIOLOG analysis, traditional compound fertilizer was conducive to the growth of microbial flora feeding on amino acids and amine as carbon sources, the commercial organic fertilizer to the growth of microbial flora using carboxylic acids as a carbon source, and farmyard manure to the growth of microbial flora using carbohydrate, fatty acids, and phenolic acids as carbon sources. Principal component analysis indicated that the first 2 components were related to carbon sources, which accounted for 74.37% and 25.63% of the data variation. The carbon source of carbohydrate, fatty acids, and phenolic acids mainly contributed to the separation of the 2 principal components. The autotoxic allelopathic potential of tobacco rhizospheric soil was positively correlated with the average well color development (AWCD) value of microbial flora feeding on carbohydrate and phenolic acids as carbon sources, and negatively correlated with that of the microbial flora using the carbon source of fatty acids. In addition, for the growth of microbial flora in monoculture soil, farmyard manure was the best, followed by commercial organic fertilizer, and traditional compound fertilizer was the worst. Keywords: tobacco; consecutive cropping problem; microbial flora; functional diversity; BIOLOG
Continuous cropping results in serious reduction of crop biomass and quality, and increases the susceptibility of crop disease, which is known as continuous cropping obstacles. Tobacco (Nicotiana tabacum L.), a member of Solanaceae, is one of the important economic crops with high economic benefits and widely planted in the world [1]. However, continuous cropping obstacles always seriously restrict the sustainable development of tobacco production. The mechanism of continuous cropping obstacles is highly concerned because the problem becomes greater with the cropping years. Although no consistent conclusion is available regarding
the mechanism of continuous cropping obstacles, a general understanding points to several factors involved in the mechanism, such as imbalance of soil nutrients, autotoxic effect, and changing of soil microbial community [2–5]. These factors are all related to soil ecosystem. The physical and chemical processes in soil are very complex, and microorganisms play a crucial role in the processes, particularly in soil nutrient transformation, soil health, and sustainability of soil productivity. Pan et al. [6] found that continuous cropping tobacco affected the communities of bacteria, fungi, and actinomycosis in soil with the effect from large to small. Tobacco continuous cropping disturbed the balance of
Received: 15 June 2010; Accepted: 26 September 2010. * Corresponding author. E-mail: [email protected] Copyright © 2011, Crop Science Society of China and Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Published by Elsevier BV. All rights reserved. Chinese edition available online at http://www.chinacrops.org/zwxb/ DOI: 10.1016/S1875-2780(11)60003-5
YANG Yu-Hong et al. / Acta Agronomica Sinica, 2011, 37(1): 105–111
microbial communities and increased disease susceptibility of the crop [7]. Reasonable fertilization could optimize the structure of microbial communities in rhizospheric soil of tobacco in continuous cropping system, improve the microbial diversity, and alleviate continuous cropping obstacles [8–10]. Therefore, continuous cropping obstacles are considered to be closely related to soil fertility and microbial community. At present, numerous studies have been carried out to understand the characteristics of rhizosperic microbial florae in different crops or soil types, and the effects of different fertilizers on microbial flora in rhizospheric soil were reported in monoculture tobacco [11–13]. However, the quantitative changes of microorganisms in soils were emphasized in these studies, and the functional diversity of microbial flora was seldom mentioned. In the current study, we investigated the effects of different fertilizer types on functional diversity of microbial communities in a field with 12-year continuous cropping of tobacco. The result was expected to help understanding the mechanism of continuous cropping obstacles and provide feasible techniques for alleviating this problem in tobacco production.
1 1.1
Materials and methods Experimental design
The field experiment was conducted in Yuxi Experiment Base of Yunnan Tobacco Research Institute (Yuxi, Yunnan Province, China) from 2008 to 2009. The tobacco accession K326 was planted in the field where tobacco has been grown continuously for 12 years. The nutrient basis of soil (pH 7.6) before planting was as follows: organic matter 2.2%, total nitrogen 0.95 g kg1, total phosphorus 1.70 g kg1, total potassium 10.0 g kg1, available nitrogen 45.9 mg kg1, available phosphorus, 69.1 mg kg1, and available potassium 160.5 mg kg1. The cation exchange capacity was 4.44 cmol kg1. Three treatments were designed with different fertilizer types, i.e., traditional compound fertilizer (T1, control), traditional compound fertilizer plus farmyard manure (T2), and traditional compound fertilizer plus commercial organic fertilizer (T3). The farmyard manure was the pig manure deposited for 3 months (nitrogen content of 0.4%). All treatments had the same ratio of nutrient components as that of the traditional compound fertilizer, which was 12 12 24 for N P2O5 K2O, and the amount of nitrogen applied in each treatment was about 75 kg ha1. Each treatment had 3 replicates, and the whole experiment contained 9 plots. The plot area was 28 m2. Tobacco seedlings were transplanted on April 25, and the density was 56 seedlings per plot. A half of traditional compound fertilizer was used as the basal fertilizer on April 26, which was placed 10 cm distant from the hill and covered
with 10–15 cm soil; another 10 g per plant was given with irrigation water on May 5; and the remaining fertilizer was applied on May 25. The farmyard manure in T2 treatment was broadcasted before planting at the rate of 1 kg per plant. The rest of fertilizer was applied in the same method as mentioned above (T1 and T3). After harvest, the rhizospheric soils of tobacco were collected using quartering method and preserved at 4 qC. 1.2 Toxic effect of tobacco rhizosphere soil from continuous cropping system The toxic effects of tobacco rhizospheric soil on lettuce (Lactuca sativa L.) and tobacco were estimated using the soil-agar sandwich method [14]. Fifteen grams of the soil sample were mixed with 30 mL agarose (0.8%) when the agarose temperature was about 45qC. After solidification of the agarose, extra 2 mL of agarose (0.5%) was added onto the surface. Then, 10 pre-germinated seeds of lettuce or tobacco were placed onto the agarose surface for germination and growth. The cultural medium without rhizospheric soil was used as the control. Each treatment had 5 replicates. The lettuce seeds were incubated for 3 d under 25qC and 12 h photoperiod, and the tobacco seeds were under 30qC and 12 h photoperiod for 10 d. The root lengths of lettuce and tobacco were measured. The relative inhibition rate (IR) was calculated using the formula IR = [(value of control value of treatment) / value of control] × 100%. 1.3 Functional diversity of microbial community in tobacco rhizospheric soil The BIOLOG ECO microplate was used to determine the microbial function diversity of soil [14]. Five grams of freshly-collected soil was put into an autoclaved flask with 100 mL NaCl (0.85%). The flask was shaken at 120 rpm for 3 min, and then cooled on ice for 2 min. Subsequently, 5 mL of supernatant was transferred into another flask with 45 mL sterile distilled water. After 3 times of dilution, the 1:1000 extract was used for enzyme-linked immunosorbent assay (ELISA). An aliquot of 150 ȝL extract solution was added into each well of a BIOLOG ECO plate that was prewarmed to 25 qC. The plate was incubated at 28 qC, and the absorbance at 590 nm was recorded everyday until the seventh day using the ELISA reaction plate reader (Multiscan MK3, Thermo Fisher Scientific, MA, USA). Each well of the BIOLOG ECO plate (Biolog, Hayward, CA, USA) was loaded with a single-carbon source. Thirty-one single-carbon sources in 6 classes were used in the analysis, such as carbohydrate, fatty acid, amino acid, amine, carboxylic acid, and phenolic acid. ELISA reaction of microorganisms was presented as the average well color development (AWCD) for each microplate, which was calculated using the formula AWCD = [(C R)] / N, where C is the absorbance of the 31 carbon source wells, R
YANG Yu-Hong et al. / Acta Agronomica Sinica, 2011, 37(1): 105–111
is the absorbance of the corresponding control wells, and N refers to the number of carbon source. 1.4
Data analysis
Statistical analysis, principal component analysis (PCA), and correlation analysis were conducted using SSPS 11.5 software.
2
Results
2.1 Toxic effect of tobacco rhizospheric soil from continuous cropping system Compared to the control, the root lengths of lettuce and tobacco seedlings were significantly inhibited in the rhizospheric soil of continuous cropping tobacco, and the inhibitory effect (IR) varied with the fertilizer supply (Fig. 1). For lettuce, the IRs were 45.15%, 18.03%, and 21.70% in treatments T1, T2, and T3, respectively; for tobacco, the IRs were 57.52%, 33.18%, and 37.81%, respectively. The result showed that the autotoxic effect of tobacco in the continuous cropping system was regulated by different fertilizers, and farmyard manure had the best effect, whereas traditional compound fertilizer had the worst effect. In addition, the autotoxic effect of tobacco rhizospheric soil on lettuce seedlings was greater than that on tobacco seedlings. 2.2 Functional diversity of microbial community in rhizospheric soil under tobacco monoculture The AWCD values of microflora increased with cultural time regardless their different feeding carbon sources. The microflora feeding on different carbon resources had signifi-
Inhibition rate (%)
80 60
Tobacco
Lettuce a
a b
40 c
20 0
T1
T2
b
b
T3
T1
T2
T3
Treatment
Fig. 1 Autotoxic effect of soil mediated by continuous cropping tobacco on root lengths of tobacco and lettuce under different fertilization treatments T1: Traditional compound fertilizer; T2: Traditional compound fertilizer plus farmyard manure; T3: Traditional compound fertilizer plus commercial organic fertilizer. Error bar shows the derivation of 5 replicates. Different letters above the bars indicate significant difference (P < 0.05) among treatments.
cantly different AWCD values in the 3 treatments. When carbohydrate, fatty acid, or phenolic acid was used as the carbon resource, treatment T2 exhibited the largest effects on AWCD values with the simulative equation gradients of 0.320, 0.289, and 0.222, respectively; comparatively, the gradients of AWCD simulative equations were 0.318, 0.272, and 0.196 for treatment T3 and of 0.293, 0.264, and 0.176 for treatment T1, respectively. When amino acid was used as the carbon source, the AWCD values of microflora were affected by fertilization treatments in the order of T1 (0.365) > T3 (0.353) > T2 (0.344). For amide and carboxylic acid used as the carbon sources, the effects on AWCD value by fertilizers showed the orders of T1 (0.370) > T2 (0.360) > T3 (0.344) and T3 (0.373) > T1 (0.332) > T2 (0.314), respectively (Fig. 2). Therefore, the traditional compound fertilizer was conducive to the growth of the microflora feeding on amino acids and amine as carbon sources, and the commercial organic fertilizer and farmyard manure were favorable for the microflora using carboxylic acids and carbohydrate, fatty acids, and phenolic acids as the carbon sources. 2.3 PCA of microflora in tobacco rhizospheric soil under continuous cropping system Based on AWCD values of microflora cultural for 7 d, the functional diversity of microflora was analyzed using PCA method. The utilization property of carbon source was separated into 2 principal components, which explained 74.4% and 25.6% of the total variation. The soil samples from T1, T2, and T3 treatments were clustered on different positions in the PCA graph (Fig. 3). According to the comparative analysis between PCA and AWCD value of single-carbon source, the component 1 was significantly correlated with 16 single-carbon sources, of which 12 had positive correlations and 4 had negative correlation. The positive carbon sources were mainly involved in carbohydrate and phenolic acid, and the negative carbon sources were mainly in carbohydrate and fatty acid classes. The component 2 contained each one carbon source with significantly positive (fatty acid) and negative (phenolic acid) correlations (Table 1). Clearly, the major carbon sources were carbohydrate, fatty acid, and phenolic acid, which resulted in the differentiation of the principal components. 2.4 Correlation between microflora using different carbon sources and autotoxic effect of tobacco rhizospheric soil The correlation coefficient of autotoxic potential between tobacco and lettuce was 1.00 (P < 0.01). On both tobacco and lettuce in different fertilization treatments, the autotoxic effects of rhizospperic soils from tobacco continuous cropping field were positively correlated with the AWCD values of microflora using carbohydrate and phenolic acid, but negatively correlated with the AWCD values of microflora using fatty acid. In addi-
YANG Yu-Hong et al. / Acta Agronomica Sinica, 2011, 37(1): 105–111
3
2
2
1
1
0
0 1
3
AWCD value
3
A
2
3
4
5
6
7
1
8 3
C
2
2
1
1
0
2
3
4
5
6
7
8
2
3
4
5
6
7
8
2
3
4
5
6
7
8
D
0 1
3
AWCD value
B
2
3
4
5
6
7
8
1 3
E
2
2
1
1
0
F
0 1
2
3
4
5
6
7
8
1
Days of microorganism culture
T1
Days of microorganism culture
T2
T3
Fig. 2 Changes in averages of well color development (AWCD) values of tobacco rhizospheric microflora with different carbon sources A: Carbohydrate; T1: y = 0.293x + 0.1544 (R2 = 0.8603), T2: y = 0.320x + 0.1493 (R2 = 0.8553), T3: y = 0.318x 0.0545 (R2 = 0.9611); B: Fatty acid; T1: y = 0.264x + 0.1017 (R2 = 0.9196), T2: y = 0.289x 0.0052 (R2 = 0.9609), T3: y = 0.272x + 0.2061 (R2 = 0.8172); C: Amino acid; T1: y = 0.365x 0.0948 (R2 = 0.9381), T2: y = 0.344x + 0.0907 (R2 = 0.8838), T3: y = 0.353x 0.0267 (R2 = 0.9422); D: Amide; T1: y = 0.370x 0.0060 (R2 = 0.9340); T2: y = 0.360x + 0.0114 (R2 = 0.9055), T3: y = 0.344x + 0.0794 (R2 = 0.8809); E: Carboxylic acid; T1: y = 0.332x 0.0087 (R2 = 0.8788), T2: y = 0.314x + 0.0732 (R2 = 0.9129), T3: y = 0.373x 0.0221 (R2 = 0.9256); F: Phenolic acide; T1: y = 0.176x + 0.2972 (R2 = 0.7935), T2: y = 0.222x + 0.2284 (R2 = 0.7420), T3: y = 0.196x + 0.2209 (R2 = 0.7964). T1: Traditional compound fertilizer; T2: Traditional compound fertilizer plus farmyard manure; T3: Traditional compound fertilizer plus with commercial organic fertilizer.
YANG Yu-Hong et al. / Acta Agronomica Sinica, 2011, 37(1): 105–111
Table 1 Carbon sources mainly correlated with components of principal component analysis Carbon source
Class of carbon source
Correlation coefficient
Principal component 1
Fig. 3 Loadings for principal component analysis of tobacco rhizospheric microflora under continuous cropping system T1: Traditional compound fertilizer; T2: Traditional compound fertilizer plus farmyard manure; T3: Traditional compound fertilizer plus with commercial organic fertilizer.
tion, the AWCD values of microflora using phenolic acid was negatively correlated with that using fatty acid (Table 2). This result indicated that the autotoxic effect of tobacco rhizosphere soil was closely correlated with the microflora using carbohydrate and phenolic acid as carbon sources.
Į-D-lactose
Carbohydrate
1.00**
D-cellobiose
Carbohydrate
1.00**
Glycogen
Carbohydrate
1.00**
Į-cyclodextrin
Carbohydrate
1.00**
D,L-Į-glycetol phosphate
Carbohydrate
1.00**
Glucose-1-phosphate
Carbohydrate
1.00**
D-galacturonic acid
Carboxylic acid derivatives
1.00**
N-acetyl-D-glucosamine
Amine
1.00**
D-mannitol
Carbohydrate
1.00**
D-xylose
Carbohydrate
1.00**
ȕ-methyl-D-glucose
Carbohydrate
1.00**
4-hydroxy benzoic acid
Phenolic acid
Itaconic acid
Carboxylic acid derivatives
0.98* 1.00**
1-Erythritol
Carbohydrate
1.00**
D-galacturonic acid
Carboxylic acid derivatives
1.00**
D-glucosaminic acid
Amino acid
1.00**
Principal component 2 Ȗ-hydroxybutyric acid
Carboxylic acid derivatives
4-Hydroxy benzoic acid
Phenolic acid
0.98* 1.00**
* Significant at P < 0.05. ** Significant at P < 0.01.
Table 2 Correlation analysis between autotoxic allelopathic potential of tobacco rhizospheric soil and AWCD values of microflora in continuous cropping system Carbon source
Autotoxic potential Tobacco
Lettuce
Carbohydrate
0.99**
0.98*
Phenolic acid
0.98*
0.99*
AWCD value of microflora cultured in single-carbon source Carbohydrate
Phenolic acid
Fatty acid
Carboxylic acid
Amide
Amino acid
1.00** 0.94
1.00**
Fatty acid
0.94
0.96*
0.89
0.99**
1.00**
Carboxylic acid
0.57
0.51
0.66
0.37
0.25
Amide
0.24
0.18
0.36
0.03
0.11
0.94
1.00**
Amino acid
0.64
0.68
0.55
0.79
0.87
0.27
0.59
1.00** 1.00**
Data are the averages of treatments T1, T2, and T3. * Significant at P < 0.05. ** Significant at P < 0.01.
3
Discussion
Continuous monoculture results in serious decline of biomass and quality of tobacco and enhances occurrence of disease in production [1]. It is found that fertilization management can alleviate this continuous cropping obstacle to some extent, such as proper quantities of fertilizers and application regime [8–10]. This effect was confirmed in this study, in which farmyard manure and commercial organic fertilizer showed positive effects on reducing autotoxic potential of rhizospheric soil under tobacco continuous cropping system, particularly, the farmyard manure. Fertilization is able to change the microflora populations in
tobacco rhizopheric soil, and thus affect autotoxicity of the soil [8–10]. Microorganism is a component of soil ecology and plays an important role in nutrient transformation and decomposition. The structure of soil microbial community refers to the quantity and proportion of main microflora in soil. Changes in the community structure directly affect the functions of rhizospheric microorganisms; as a result, plant growth is regulated due to variation of soil substance and energy conversion. Therefore, the effect of fertilizer on microbial function diversity is a research focus for eliminating autotoxicity in soil of continuous cropping tobacco. We found that the soil microflora was increased significantly in number after fertilization, but its function diversity varied with
YANG Yu-Hong et al. / Acta Agronomica Sinica, 2011, 37(1): 105–111
fertilizer types. Based on AWCD values of the single-carbon species, the functional diversity of microflora can be explained with 2 principal components, and 3 carbon sources (carbohydrate, fatty acid, and phenolic acid) contributed mainly to this separation. According to correlation analysis, the AWCD values of microflora using carbohydrates and phenolic acid as carbon sources were correlated with the autotoxic effect of tobacco in the continuous cropping soil. This finding is helpful to understand the crucial factors in the complex soil microecosystem of continuous cropping tobacco that may influence the microbial functional diversity. We concluded that the microorganisms using carbohydrates and phenolic acids as carbon sources play a key role in the autotoxic effect of tobacco in continuous cropping soil. Plant continuous cropping may cause variations of chemical substances in rhizospheric soils. For example, the continuous cropping of poplar (Populus deltoides Bart. ex. Marsh.) increased the phenolic acid accumulation and reduced the ammonifier in soils [15]; in rhizosphere of continuous cropping soybean (Glycine max L. Merr.), phenolic acids were observed in accumulation [16]. In this study, we found that the rhizospheric microflora using carbohydrate and phenolic acid were closely correlated with the autotoxic effect of tobacco. Therefore, we speculated that the accumulations of phenolic acid and carbohydrate in rhizospheric soil might be a reason for the continuous cropping obstacle in tobacco. However, the phenolic acid-dependent microorganism can be stimulated when farmyard manure is applied, resulting in the consumption of phenolic acid in soil and alleviation of autotoxic effect ultimately. Comparatively, traditional compound fertilizer was favorable for microflora feeding on amino acid, fatty acid, carboxylic acid, and amine as carbon sources, but inhibited the growth of microflora using phenolic acid and carbohydrate (Table 2). The result of this study showed that farmyard manure was the best type of fertilizer in the continuous cropping system of tobacco, whereas, the traditional compound fertilizer was the worst. In the complex rhizospheric microecosytem of tobacco, a variety of microorganisms are involved. We need to know what species could use carbohydrate and phenolic acid as carbon sources and their interactions. Besides, the mechanism of signal conduction may explain the stimulation and inhabitation of favorable microfloar in rhizosphereic soil of tobacco. These issues should be disclosed in future studies.
4
Conclusions
Fertilizer management could optimize the structure of microbial community in rhizospheric soil of tobacco and mitigate the continuous cropping obstacle. Farmyard manure was the most suitable fertilizer to alleviate such obstacle, followed by commercial organic fertilizer, and traditional compound fertilizer was the worst. Farmyard manure was
favorable to the growth of the microflora feeding on carbohydrate and phenolic acid as carbon sources, which might be the key factors for mitigating the continuous cropping obstacle of tobacco.
Acknowledgment The study was supported by the projects of Yunnan Provincial Tobacco Monopoly Bureau, Kunming, China.
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RESEARCH PAPER
Effect of Different Fertilizers on Functional Diversity of Microbial Flora in Rhizospheric Soil Under Tobacco Monoculture YANG Yu-Hong1, CHEN Dong-Mei2, JIN Yan1, WANG Hai-Bin2, DUAN Yu-Qi1, GUO Xu-Kui2, HE Hai-Bin2, and LIN Wen-Xiong2,* 1
Yunnan Provincial Institute of Tobacco Research, Yuxi 653100, China
2
Institute of Agroecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Abstract: Continuous cropping obstacle is one the most important problems in tobacco (Nicotiana tabacum L.) production. To alleviate continuous cropping obstacle based on management of soil ecology, the effects of different fertilizers were tested using tobacco cultivar K326 growing in a field with 12-year consecutive cultivation. The rhizospheric soil was sampled to investigate the changes in functional diversity of microbial flora in different treatments. The results showed that the autotoxic allelopathic potential was maximal for the monoculture soil treated with traditional compound fertilizer, and minimal for the soil treated with farmyard manure. According to the result of BIOLOG analysis, traditional compound fertilizer was conducive to the growth of microbial flora feeding on amino acids and amine as carbon sources, the commercial organic fertilizer to the growth of microbial flora using carboxylic acids as a carbon source, and farmyard manure to the growth of microbial flora using carbohydrate, fatty acids, and phenolic acids as carbon sources. Principal component analysis indicated that the first 2 components were related to carbon sources, which accounted for 74.37% and 25.63% of the data variation. The carbon source of carbohydrate, fatty acids, and phenolic acids mainly contributed to the separation of the 2 principal components. The autotoxic allelopathic potential of tobacco rhizospheric soil was positively correlated with the average well color development (AWCD) value of microbial flora feeding on carbohydrate and phenolic acids as carbon sources, and negatively correlated with that of the microbial flora using the carbon source of fatty acids. In addition, for the growth of microbial flora in monoculture soil, farmyard manure was the best, followed by commercial organic fertilizer, and traditional compound fertilizer was the worst. Keywords: tobacco; consecutive cropping problem; microbial flora; functional diversity; BIOLOG
Continuous cropping results in serious reduction of crop biomass and quality, and increases the susceptibility of crop disease, which is known as continuous cropping obstacles. Tobacco (Nicotiana tabacum L.), a member of Solanaceae, is one of the important economic crops with high economic benefits and widely planted in the world [1]. However, continuous cropping obstacles always seriously restrict the sustainable development of tobacco production. The mechanism of continuous cropping obstacles is highly concerned because the problem becomes greater with the cropping years. Although no consistent conclusion is available regarding
the mechanism of continuous cropping obstacles, a general understanding points to several factors involved in the mechanism, such as imbalance of soil nutrients, autotoxic effect, and changing of soil microbial community [2–5]. These factors are all related to soil ecosystem. The physical and chemical processes in soil are very complex, and microorganisms play a crucial role in the processes, particularly in soil nutrient transformation, soil health, and sustainability of soil productivity. Pan et al. [6] found that continuous cropping tobacco affected the communities of bacteria, fungi, and actinomycosis in soil with the effect from large to small. Tobacco continuous cropping disturbed the balance of
Received: 15 June 2010; Accepted: 26 September 2010. * Corresponding author. E-mail: [email protected] Copyright © 2011, Crop Science Society of China and Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Published by Elsevier BV. All rights reserved. Chinese edition available online at http://www.chinacrops.org/zwxb/ DOI: 10.1016/S1875-2780(11)60003-5
YANG Yu-Hong et al. / Acta Agronomica Sinica, 2011, 37(1): 105–111
microbial communities and increased disease susceptibility of the crop [7]. Reasonable fertilization could optimize the structure of microbial communities in rhizospheric soil of tobacco in continuous cropping system, improve the microbial diversity, and alleviate continuous cropping obstacles [8–10]. Therefore, continuous cropping obstacles are considered to be closely related to soil fertility and microbial community. At present, numerous studies have been carried out to understand the characteristics of rhizosperic microbial florae in different crops or soil types, and the effects of different fertilizers on microbial flora in rhizospheric soil were reported in monoculture tobacco [11–13]. However, the quantitative changes of microorganisms in soils were emphasized in these studies, and the functional diversity of microbial flora was seldom mentioned. In the current study, we investigated the effects of different fertilizer types on functional diversity of microbial communities in a field with 12-year continuous cropping of tobacco. The result was expected to help understanding the mechanism of continuous cropping obstacles and provide feasible techniques for alleviating this problem in tobacco production.
1 1.1
Materials and methods Experimental design
The field experiment was conducted in Yuxi Experiment Base of Yunnan Tobacco Research Institute (Yuxi, Yunnan Province, China) from 2008 to 2009. The tobacco accession K326 was planted in the field where tobacco has been grown continuously for 12 years. The nutrient basis of soil (pH 7.6) before planting was as follows: organic matter 2.2%, total nitrogen 0.95 g kg1, total phosphorus 1.70 g kg1, total potassium 10.0 g kg1, available nitrogen 45.9 mg kg1, available phosphorus, 69.1 mg kg1, and available potassium 160.5 mg kg1. The cation exchange capacity was 4.44 cmol kg1. Three treatments were designed with different fertilizer types, i.e., traditional compound fertilizer (T1, control), traditional compound fertilizer plus farmyard manure (T2), and traditional compound fertilizer plus commercial organic fertilizer (T3). The farmyard manure was the pig manure deposited for 3 months (nitrogen content of 0.4%). All treatments had the same ratio of nutrient components as that of the traditional compound fertilizer, which was 12 12 24 for N P2O5 K2O, and the amount of nitrogen applied in each treatment was about 75 kg ha1. Each treatment had 3 replicates, and the whole experiment contained 9 plots. The plot area was 28 m2. Tobacco seedlings were transplanted on April 25, and the density was 56 seedlings per plot. A half of traditional compound fertilizer was used as the basal fertilizer on April 26, which was placed 10 cm distant from the hill and covered
with 10–15 cm soil; another 10 g per plant was given with irrigation water on May 5; and the remaining fertilizer was applied on May 25. The farmyard manure in T2 treatment was broadcasted before planting at the rate of 1 kg per plant. The rest of fertilizer was applied in the same method as mentioned above (T1 and T3). After harvest, the rhizospheric soils of tobacco were collected using quartering method and preserved at 4 qC. 1.2 Toxic effect of tobacco rhizosphere soil from continuous cropping system The toxic effects of tobacco rhizospheric soil on lettuce (Lactuca sativa L.) and tobacco were estimated using the soil-agar sandwich method [14]. Fifteen grams of the soil sample were mixed with 30 mL agarose (0.8%) when the agarose temperature was about 45qC. After solidification of the agarose, extra 2 mL of agarose (0.5%) was added onto the surface. Then, 10 pre-germinated seeds of lettuce or tobacco were placed onto the agarose surface for germination and growth. The cultural medium without rhizospheric soil was used as the control. Each treatment had 5 replicates. The lettuce seeds were incubated for 3 d under 25qC and 12 h photoperiod, and the tobacco seeds were under 30qC and 12 h photoperiod for 10 d. The root lengths of lettuce and tobacco were measured. The relative inhibition rate (IR) was calculated using the formula IR = [(value of control value of treatment) / value of control] × 100%. 1.3 Functional diversity of microbial community in tobacco rhizospheric soil The BIOLOG ECO microplate was used to determine the microbial function diversity of soil [14]. Five grams of freshly-collected soil was put into an autoclaved flask with 100 mL NaCl (0.85%). The flask was shaken at 120 rpm for 3 min, and then cooled on ice for 2 min. Subsequently, 5 mL of supernatant was transferred into another flask with 45 mL sterile distilled water. After 3 times of dilution, the 1:1000 extract was used for enzyme-linked immunosorbent assay (ELISA). An aliquot of 150 ȝL extract solution was added into each well of a BIOLOG ECO plate that was prewarmed to 25 qC. The plate was incubated at 28 qC, and the absorbance at 590 nm was recorded everyday until the seventh day using the ELISA reaction plate reader (Multiscan MK3, Thermo Fisher Scientific, MA, USA). Each well of the BIOLOG ECO plate (Biolog, Hayward, CA, USA) was loaded with a single-carbon source. Thirty-one single-carbon sources in 6 classes were used in the analysis, such as carbohydrate, fatty acid, amino acid, amine, carboxylic acid, and phenolic acid. ELISA reaction of microorganisms was presented as the average well color development (AWCD) for each microplate, which was calculated using the formula AWCD = [(C R)] / N, where C is the absorbance of the 31 carbon source wells, R
YANG Yu-Hong et al. / Acta Agronomica Sinica, 2011, 37(1): 105–111
is the absorbance of the corresponding control wells, and N refers to the number of carbon source. 1.4
Data analysis
Statistical analysis, principal component analysis (PCA), and correlation analysis were conducted using SSPS 11.5 software.
2
Results
2.1 Toxic effect of tobacco rhizospheric soil from continuous cropping system Compared to the control, the root lengths of lettuce and tobacco seedlings were significantly inhibited in the rhizospheric soil of continuous cropping tobacco, and the inhibitory effect (IR) varied with the fertilizer supply (Fig. 1). For lettuce, the IRs were 45.15%, 18.03%, and 21.70% in treatments T1, T2, and T3, respectively; for tobacco, the IRs were 57.52%, 33.18%, and 37.81%, respectively. The result showed that the autotoxic effect of tobacco in the continuous cropping system was regulated by different fertilizers, and farmyard manure had the best effect, whereas traditional compound fertilizer had the worst effect. In addition, the autotoxic effect of tobacco rhizospheric soil on lettuce seedlings was greater than that on tobacco seedlings. 2.2 Functional diversity of microbial community in rhizospheric soil under tobacco monoculture The AWCD values of microflora increased with cultural time regardless their different feeding carbon sources. The microflora feeding on different carbon resources had signifi-
Inhibition rate (%)
80 60
Tobacco
Lettuce a
a b
40 c
20 0
T1
T2
b
b
T3
T1
T2
T3
Treatment
Fig. 1 Autotoxic effect of soil mediated by continuous cropping tobacco on root lengths of tobacco and lettuce under different fertilization treatments T1: Traditional compound fertilizer; T2: Traditional compound fertilizer plus farmyard manure; T3: Traditional compound fertilizer plus commercial organic fertilizer. Error bar shows the derivation of 5 replicates. Different letters above the bars indicate significant difference (P < 0.05) among treatments.
cantly different AWCD values in the 3 treatments. When carbohydrate, fatty acid, or phenolic acid was used as the carbon resource, treatment T2 exhibited the largest effects on AWCD values with the simulative equation gradients of 0.320, 0.289, and 0.222, respectively; comparatively, the gradients of AWCD simulative equations were 0.318, 0.272, and 0.196 for treatment T3 and of 0.293, 0.264, and 0.176 for treatment T1, respectively. When amino acid was used as the carbon source, the AWCD values of microflora were affected by fertilization treatments in the order of T1 (0.365) > T3 (0.353) > T2 (0.344). For amide and carboxylic acid used as the carbon sources, the effects on AWCD value by fertilizers showed the orders of T1 (0.370) > T2 (0.360) > T3 (0.344) and T3 (0.373) > T1 (0.332) > T2 (0.314), respectively (Fig. 2). Therefore, the traditional compound fertilizer was conducive to the growth of the microflora feeding on amino acids and amine as carbon sources, and the commercial organic fertilizer and farmyard manure were favorable for the microflora using carboxylic acids and carbohydrate, fatty acids, and phenolic acids as the carbon sources. 2.3 PCA of microflora in tobacco rhizospheric soil under continuous cropping system Based on AWCD values of microflora cultural for 7 d, the functional diversity of microflora was analyzed using PCA method. The utilization property of carbon source was separated into 2 principal components, which explained 74.4% and 25.6% of the total variation. The soil samples from T1, T2, and T3 treatments were clustered on different positions in the PCA graph (Fig. 3). According to the comparative analysis between PCA and AWCD value of single-carbon source, the component 1 was significantly correlated with 16 single-carbon sources, of which 12 had positive correlations and 4 had negative correlation. The positive carbon sources were mainly involved in carbohydrate and phenolic acid, and the negative carbon sources were mainly in carbohydrate and fatty acid classes. The component 2 contained each one carbon source with significantly positive (fatty acid) and negative (phenolic acid) correlations (Table 1). Clearly, the major carbon sources were carbohydrate, fatty acid, and phenolic acid, which resulted in the differentiation of the principal components. 2.4 Correlation between microflora using different carbon sources and autotoxic effect of tobacco rhizospheric soil The correlation coefficient of autotoxic potential between tobacco and lettuce was 1.00 (P < 0.01). On both tobacco and lettuce in different fertilization treatments, the autotoxic effects of rhizospperic soils from tobacco continuous cropping field were positively correlated with the AWCD values of microflora using carbohydrate and phenolic acid, but negatively correlated with the AWCD values of microflora using fatty acid. In addi-
YANG Yu-Hong et al. / Acta Agronomica Sinica, 2011, 37(1): 105–111
3
2
2
1
1
0
0 1
3
AWCD value
3
A
2
3
4
5
6
7
1
8 3
C
2
2
1
1
0
2
3
4
5
6
7
8
2
3
4
5
6
7
8
2
3
4
5
6
7
8
D
0 1
3
AWCD value
B
2
3
4
5
6
7
8
1 3
E
2
2
1
1
0
F
0 1
2
3
4
5
6
7
8
1
Days of microorganism culture
T1
Days of microorganism culture
T2
T3
Fig. 2 Changes in averages of well color development (AWCD) values of tobacco rhizospheric microflora with different carbon sources A: Carbohydrate; T1: y = 0.293x + 0.1544 (R2 = 0.8603), T2: y = 0.320x + 0.1493 (R2 = 0.8553), T3: y = 0.318x 0.0545 (R2 = 0.9611); B: Fatty acid; T1: y = 0.264x + 0.1017 (R2 = 0.9196), T2: y = 0.289x 0.0052 (R2 = 0.9609), T3: y = 0.272x + 0.2061 (R2 = 0.8172); C: Amino acid; T1: y = 0.365x 0.0948 (R2 = 0.9381), T2: y = 0.344x + 0.0907 (R2 = 0.8838), T3: y = 0.353x 0.0267 (R2 = 0.9422); D: Amide; T1: y = 0.370x 0.0060 (R2 = 0.9340); T2: y = 0.360x + 0.0114 (R2 = 0.9055), T3: y = 0.344x + 0.0794 (R2 = 0.8809); E: Carboxylic acid; T1: y = 0.332x 0.0087 (R2 = 0.8788), T2: y = 0.314x + 0.0732 (R2 = 0.9129), T3: y = 0.373x 0.0221 (R2 = 0.9256); F: Phenolic acide; T1: y = 0.176x + 0.2972 (R2 = 0.7935), T2: y = 0.222x + 0.2284 (R2 = 0.7420), T3: y = 0.196x + 0.2209 (R2 = 0.7964). T1: Traditional compound fertilizer; T2: Traditional compound fertilizer plus farmyard manure; T3: Traditional compound fertilizer plus with commercial organic fertilizer.
YANG Yu-Hong et al. / Acta Agronomica Sinica, 2011, 37(1): 105–111
Table 1 Carbon sources mainly correlated with components of principal component analysis Carbon source
Class of carbon source
Correlation coefficient
Principal component 1
Fig. 3 Loadings for principal component analysis of tobacco rhizospheric microflora under continuous cropping system T1: Traditional compound fertilizer; T2: Traditional compound fertilizer plus farmyard manure; T3: Traditional compound fertilizer plus with commercial organic fertilizer.
tion, the AWCD values of microflora using phenolic acid was negatively correlated with that using fatty acid (Table 2). This result indicated that the autotoxic effect of tobacco rhizosphere soil was closely correlated with the microflora using carbohydrate and phenolic acid as carbon sources.
Į-D-lactose
Carbohydrate
1.00**
D-cellobiose
Carbohydrate
1.00**
Glycogen
Carbohydrate
1.00**
Į-cyclodextrin
Carbohydrate
1.00**
D,L-Į-glycetol phosphate
Carbohydrate
1.00**
Glucose-1-phosphate
Carbohydrate
1.00**
D-galacturonic acid
Carboxylic acid derivatives
1.00**
N-acetyl-D-glucosamine
Amine
1.00**
D-mannitol
Carbohydrate
1.00**
D-xylose
Carbohydrate
1.00**
ȕ-methyl-D-glucose
Carbohydrate
1.00**
4-hydroxy benzoic acid
Phenolic acid
Itaconic acid
Carboxylic acid derivatives
0.98* 1.00**
1-Erythritol
Carbohydrate
1.00**
D-galacturonic acid
Carboxylic acid derivatives
1.00**
D-glucosaminic acid
Amino acid
1.00**
Principal component 2 Ȗ-hydroxybutyric acid
Carboxylic acid derivatives
4-Hydroxy benzoic acid
Phenolic acid
0.98* 1.00**
* Significant at P < 0.05. ** Significant at P < 0.01.
Table 2 Correlation analysis between autotoxic allelopathic potential of tobacco rhizospheric soil and AWCD values of microflora in continuous cropping system Carbon source
Autotoxic potential Tobacco
Lettuce
Carbohydrate
0.99**
0.98*
Phenolic acid
0.98*
0.99*
AWCD value of microflora cultured in single-carbon source Carbohydrate
Phenolic acid
Fatty acid
Carboxylic acid
Amide
Amino acid
1.00** 0.94
1.00**
Fatty acid
0.94
0.96*
0.89
0.99**
1.00**
Carboxylic acid
0.57
0.51
0.66
0.37
0.25
Amide
0.24
0.18
0.36
0.03
0.11
0.94
1.00**
Amino acid
0.64
0.68
0.55
0.79
0.87
0.27
0.59
1.00** 1.00**
Data are the averages of treatments T1, T2, and T3. * Significant at P < 0.05. ** Significant at P < 0.01.
3
Discussion
Continuous monoculture results in serious decline of biomass and quality of tobacco and enhances occurrence of disease in production [1]. It is found that fertilization management can alleviate this continuous cropping obstacle to some extent, such as proper quantities of fertilizers and application regime [8–10]. This effect was confirmed in this study, in which farmyard manure and commercial organic fertilizer showed positive effects on reducing autotoxic potential of rhizospheric soil under tobacco continuous cropping system, particularly, the farmyard manure. Fertilization is able to change the microflora populations in
tobacco rhizopheric soil, and thus affect autotoxicity of the soil [8–10]. Microorganism is a component of soil ecology and plays an important role in nutrient transformation and decomposition. The structure of soil microbial community refers to the quantity and proportion of main microflora in soil. Changes in the community structure directly affect the functions of rhizospheric microorganisms; as a result, plant growth is regulated due to variation of soil substance and energy conversion. Therefore, the effect of fertilizer on microbial function diversity is a research focus for eliminating autotoxicity in soil of continuous cropping tobacco. We found that the soil microflora was increased significantly in number after fertilization, but its function diversity varied with
YANG Yu-Hong et al. / Acta Agronomica Sinica, 2011, 37(1): 105–111
fertilizer types. Based on AWCD values of the single-carbon species, the functional diversity of microflora can be explained with 2 principal components, and 3 carbon sources (carbohydrate, fatty acid, and phenolic acid) contributed mainly to this separation. According to correlation analysis, the AWCD values of microflora using carbohydrates and phenolic acid as carbon sources were correlated with the autotoxic effect of tobacco in the continuous cropping soil. This finding is helpful to understand the crucial factors in the complex soil microecosystem of continuous cropping tobacco that may influence the microbial functional diversity. We concluded that the microorganisms using carbohydrates and phenolic acids as carbon sources play a key role in the autotoxic effect of tobacco in continuous cropping soil. Plant continuous cropping may cause variations of chemical substances in rhizospheric soils. For example, the continuous cropping of poplar (Populus deltoides Bart. ex. Marsh.) increased the phenolic acid accumulation and reduced the ammonifier in soils [15]; in rhizosphere of continuous cropping soybean (Glycine max L. Merr.), phenolic acids were observed in accumulation [16]. In this study, we found that the rhizospheric microflora using carbohydrate and phenolic acid were closely correlated with the autotoxic effect of tobacco. Therefore, we speculated that the accumulations of phenolic acid and carbohydrate in rhizospheric soil might be a reason for the continuous cropping obstacle in tobacco. However, the phenolic acid-dependent microorganism can be stimulated when farmyard manure is applied, resulting in the consumption of phenolic acid in soil and alleviation of autotoxic effect ultimately. Comparatively, traditional compound fertilizer was favorable for microflora feeding on amino acid, fatty acid, carboxylic acid, and amine as carbon sources, but inhibited the growth of microflora using phenolic acid and carbohydrate (Table 2). The result of this study showed that farmyard manure was the best type of fertilizer in the continuous cropping system of tobacco, whereas, the traditional compound fertilizer was the worst. In the complex rhizospheric microecosytem of tobacco, a variety of microorganisms are involved. We need to know what species could use carbohydrate and phenolic acid as carbon sources and their interactions. Besides, the mechanism of signal conduction may explain the stimulation and inhabitation of favorable microfloar in rhizosphereic soil of tobacco. These issues should be disclosed in future studies.
4
Conclusions
Fertilizer management could optimize the structure of microbial community in rhizospheric soil of tobacco and mitigate the continuous cropping obstacle. Farmyard manure was the most suitable fertilizer to alleviate such obstacle, followed by commercial organic fertilizer, and traditional compound fertilizer was the worst. Farmyard manure was
favorable to the growth of the microflora feeding on carbohydrate and phenolic acid as carbon sources, which might be the key factors for mitigating the continuous cropping obstacle of tobacco.
Acknowledgment The study was supported by the projects of Yunnan Provincial Tobacco Monopoly Bureau, Kunming, China.
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