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Chilean journal of agricultural research

On-line version ISSN 0718-5839

Chilean J. Agric. Res. vol.74 no.3 Chillán Sept. 2014

http://dx.doi.org/10.4067/S0718-58392014000300012 

RESEARCH

 

Effects of fertilization regimes on tea yields, soil fertility, and soil microbial diversity



Shan-Lian Qiu1, Li-Min Wang1, Dong-Feng Huang1, and Xin-Jian Lin1*

1Soil and Fertilizer Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003, China. *Corresponding author (xinjianlin@163.net).


Fertilization is an important agricultural practice for increasing crop yields and influencing soil properties. A field experiment was conducted in the period of 2006-2011 in southeastern China, to investigate the effects of fertilization regimes on tea (Camellia sinensis [L.] Kuntze) yields, soil chemical properties, and soil bacterial and fungal communities. The field experiment included six treatments: (1) unfertilized control (CON); (2) chemical fertilizers (NPK); (3) half-chemical fertilizers plus half-organic manure (1/2NPKOM); (4) organic manure fertilizers (OM); (5) half-chemical fertilizers plus half-organic manure plus legume stover returned (1/2NPKOM+L), and (6) chemical fertilizers plus legume stover returned (NPKL). Results showed that, compared to the control, NPK treatment showed no significant effect on soil organic matter (SOM), total N (TN), total P (TP), total K (TK), available N (AN), available K (AK) and tea yields, but showed the lowest bacterial Shannon index of 1.714 and the lowest value of 2.002 for fungal Shannon index. Organic manure treatment had the richest diversity of soil bacterial community with Shannon index of 2.542, and the highest levels of soil essential nutrients, including SOM (30.03%), TN (2.90 g kg-1), TP (1.35 g kg-1), AN (245.30 mg kg-1), AP (57.00 mg kg-1), and AK (271.80 mg kg-1), followed by 1/2NPKOM+L, which appeared the maximal tea yields of 6772 kg ha-1. Organic manure amendment was a key factor in determining soil properties and productivity. Base on soil quality and tea yields, both OM and 1/2NPKOM+L treatments were recommended as better choices of fertilization practices for tea soils in southeastern China. These findings provided a better understanding of the importance of fertilizations in promoting soil fertility, crop yields, and altering soil microbial diversity, leading to selection of scientific fertilization practices for sustainable development of agroecosystems.

Key words: Camellia sinensis, fertilization, soil chemical properties, soil microbial diversity, tea yields.


 

INTRODUCTION

Tea (Camellia sinensis [L.] Kuntze) is widely planted in China, covering 20 provinces from Hainan Province in the south to Shandong in the north, from Tibet in the west to Taiwan in the east. Fertilization, as an important agricultural practice, not only can increase crop yields and maintain soil fertility, but also result in shifts in the microbial communities, which play a critical role in maintaining the agroecosystem health through soil formation and biochemical processes, including residue decomposition and nutrient cycling. Many studies have focused on fertilization regimes on soil fertility (Yang et al., 2007; Liu et al., 2010), crop yields (Venkatesan et al., 2004; Zhu et al., 2007) and microbial communities (He et al., 2008; Shen et al., 2010), but, up to now, the results of these reports are still not unanimous, and even conflicted. Moreover, most studies were concerned about the soils for growing food crops, like rice (Wu et al., 2011), wheat (Shen et al., 2010), maize or their combination for rotation (Kumar and Yadav, 2001; Yang et al., 2007; Hu et al., 2011), while few about the soils for planting tea (Venkatesan et al., 2004). Hence, it is necessary to examine the effects of fertilization regimes on tea yields, soil fertility, and microbial communities. Particularly, soil microorganisms which are essential for maintaining the sustainability and productivity of terrestrial ecosystems (Naeem and Li, 1997; Costa et al., 2007), usually respond more rapidly to changing soil conditions than do either chemical or physical properties (Simek et al., 1999), and have been considered to be one of the most sensitive biological indicators for measuring soil quality (Kennedy, 1999). Therefore, studying the shifts of microbial communities under different fertilizations can help us to better understand the impact of fertilizers on soil quality. In this study, we took advantage of culture-independent methods including PCR and temperature gradient gel electrophoresis (TGGE) to detect the microbial diversity in a tea soil under different fertilization practices.

Knowledge about the shifts of soil fertility, crop yields, and microbial communities following different fertilizer management practices can improve our understanding of soil processes and thus help us to develop better management strategies which can maintain or improve soil fertility, increase tea yields and minimize damages to the agroecosystem. This study aimed to determine the effects of annual applications of inorganic and organic fertilizers on soil chemical properties, tea yields, and soil bacterial and fungal diversity in a tea soil in southeastern China.

MATERIALS AND METHODS

Site description, treatment, and soil sampling
A field trial was established in 2006 to determine the effects of different fertilizations on soil fertility, tea yields, and microbial communities. The study site is located at the Fu'an Experimental Station (26°41'-27°24' N, 119°23'-119°51' E) of Fujian Academy of Agricultural Sciences (FAAS), Fujian Province, southeastern China. This region has a subtropical marine monsoon climate with a mean annual precipitation of 1540 mm, temperature of 19.3 °C and sunshine of 1837 h. The tea cultivar was 'Huangguanyin'. Six fertilization treatments with triplicate for each treatment were designed as follows: (1) unfertilized control (CON), (2) chemical fertilizers (NPK), (3) half-organic manure plus half-chemical fertilizers (1/2NPKOM), (4) organic manure fertilizers (OM), (5) half-organic manure plus legume stover returned plus half-chemical fertilizers (1/2NPKOM+L), and (6) legume stover returned plus chemical fertilizers (NPKL) (Table 1). Treatments were allocated in the randomized complete block design. Band fertilization was carried out every winter. For NPK-containing treatments, N, P, K was applied in the form of urea, monoammonium phosphate, and potassium chloride, respectively. Organic manure fertilizers was the industrial farmyard manure made chiefly of chicken manure, with organic matter content of 368.90 g kg-1, total N (TN) 9.00 g kg-1, total P (TP) 22.90 g kg-1, total K (TK) 5.29 g kg-1. The legume plant (Cassia rotundifolia Pers.), using a seed rate of 7.5 kg ha1, was drilled into the top soil of spaces between tea tree rows. After the plant fades every winter, their mature seeds fell along the rows and naturally sprout in the next spring. Soil composite samples (0-20 cm) from each plot (size of 13.65 m2) were taken randomly in May 2011. The soil was sieved (< 2 mm) and stored at -20 °C for DNA extraction and 4 °C for other analyses.

Table 1. Fertilization treatments at the Fu'an experimental station.

Soil analysis
Soil pH was measured with a pH meter (EL20K, Mettler-Toledo, Columbus, Ohio, USA) using soil:water of 1:2.5 suspensions (Thomas, 1996). Soil organic matter (SOM) was determined by K2Cr2O7 oxidation-reduction titration method (Walkley and Black, 1934). Soil TN was measured using the Kjeldahl digestion-distillation method (Bremner, 1960). The TP was determined colorimetrically according to the molybdenum blue method, following persulfate digestion (Murphy and Riley, 1962). Soil TK was digested by HF-HOlO4 (Jackson, 1958) and determined by flame photometry. Available N (AN) and available P (AP) were determined by NaOH hydrolysable and molybdenum methods, respectively. Available K (AK) was extracted by ammonium acetate and determined by flame photometry (Carson, 1980). In this study, tea yields represented the cumulative tea production obtained between 2008 and 2011.

Soil DNA was extracted using a FastDNA SPIN Kit for soil (MP Biomedicals, Solon, Ohio, USA) according to the instructions of manufacturer. For soil bacteria, the 16S rDNA V3 fragments were amplified using the fD1/rD1 (Weisburg et al., 1991) and F341GC/R534 (Muyzer et al., 1993) primers with nested PCR. Amplification using primer pair fD1/rD1 was performed in 50-μL reaction mixtures, including 5 μL 10xPCR buffer with (NH4)2SO4, 3 μL 25 mM MgCl2, 4 μL 2.5 mM dNTPs, 0.5 μL 5 U μL-1 Taq DNA polymerase (MBI Fermentas, Burlington, Ontario, Canada), 1 μL 20 μM each primer, and 1 μL DNA template (10-50 ng). A touchdown PCR strategy was employed as follows: 5 min at 94 °C, 20 cycles of 1 min at 94 °C, 45 s at temperatures decreasing from 65 to 55 °C, 2 min at 72 °C, 10 cycles of 1 min at 94 °C, 45 s at 55 °C, 2 min at 72 °C, and final extension for 5 min at 72 °C. PCR amplification of a 16S rDNA V3 fragment using primer pair F341GC/R534 was carried out in a 25 μL containing 2.5 μL PCR buffer, 1.5 μL MgCl2, 2.8 μL dNTPs, 0.5 μL Taq DNA polymerase, 0.5 μL each primer, and 1 μL DNA template (Amplicons resulted from fD1/rD1 were diluted 1:100). Concentrations of PCR components and cycling conditions were the same as above. For fungi, fragments of 18S rDNA gene were amplified with primer pair FR1-GC/FF390 as described by Vainio and Hantula (2000). All PCR components (50 μL) were the same as fD1/rD1 reactions except for PCR buffer (10xbuffer with KCl) and primer pair. The PCR procedure was as follows: 3 min at 95 °C, then 35 cycles of 30 s at 95 °C, 45 s at 50 °C, 1 min at 72 °C, and final extension for 10 min at 72 °C (Beauregard et al., 2010). All reactions were carried out using a Whatman Biometra T1 96-well Thermocycler. Primer sequences are listed in Table 2.

Table 2. Primers used in this study.
GC clampa represents CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G.
GC clampb represents CCC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GCC G.

PCR products were loaded on 0.45-mm thick 8% denaturing gels (8% polyacrylamide gel [Acr/Bis = 37.5:1], lxTAE [40 mM Tris-Cl, 1 mM EDTA], 2% glycerol, and 8 M urea). Temperature gradient gel electrophoresis (TGGE) was performed using a TGGE system (Whatman Biometra, Goettingen, Germany). All gels were run at 130 V for 3 h with 1xTAE buffer. Temperature gradient was optimized at 56-69 °C for bacteria and 49-61 °C for fungi. After electrophoresis, gels were silver-stained using the procedures devised by Bassam and Caetano-Anolles (1993). Gel images were captured using the Cannon camera and analyzed by the Bio-rad Quantity One software. The diversity indices (Shannon index) were calculated according to TGGE patterns. Each resolved band was considered as a specific phylotype. The pixel intensity of each band detected by Quantity One software represented the abundance of a specific phylotype for diversity estimations. The Shannon-Weaver indices (H') (Shannon and Weaver, 1963) were calculated using the following Equation [1]:

H = -∑pi ln pi [1]

where pi = ni /∑n, ni is the abundance of the ith phylotype per lane, and Sn is the total abundance of all phylotypes per lane.


Finally, cluster analysis according to the TGGE patterns was employed to produce the dendrograms by the unweighted pair-group method with arithmetic average (UPGMA) method using Quantity One software.

Statistical analysis
One-way ANOVA was employed to analyze differences of all soil indices. Post hoc tests for each variable were made using LSD comparisons. Significant differences for all statistical tests were evaluated at the level of P< 0.05. The Pearson correlation analysis was utilized to determine the correlations between all the measured parameters. All data analyses were conducted with the SPSS software (SPSS for Windows, Version 18.0, Chicago, Illinois, USA).

RESULTS AND DISCUSSION

Effect of different fertilizations on soil chemical properties and tea yields
Soil pH and nutrient contents are summarized in Table 3. Soil pH ranging from 5.40 to 5.68 was not significantly different across all treatments, indicating that there was no noticeable effect of fertilizers on soil pH after 5 yr of continuous annual fertilizer applications. Yang et al. (2007) and Gu et al. (2009) also reported that inorganic fertilizer and organic amendment had no effect on soil pH after 22 yr of continuous annual fertilizer applications. However, Shen et al. (2010) and Hu et al. (2011) showed that inorganic and organic fertilization significantly decreased soil pH after long-term fertilizer applications. These inconsistent results could be due to different soil types, its properties, climate, duration of the experiment, etc.

Different fertilizer applications caused significant changes in soil chemical properties (Table 3). SOM contents varied from 9.00% to 30.03%, and the highest value occurred in OM treatment and the lowest in NPK treatment. Furthermore, SOM contents in the treatments (OM, 1/2NPKOM, and 1/2NPKOM+L) with organic manure were significantly (P< 0.05) higher than that in the unfertilized control (CON) and NPK treatment. Similarly, the contents of TN, TP, AN, AP, and AK were the highest in OM treatment, the second highest in 1/2NPKOM+L treatment but the lowest in CON treatment. Obviously, the OM-containing treatments (OM, 1/2NPKOM, and 1/2NPKOM+L) showed much greater increase in soil chemical fertility, which could be attributed to the regular and greater addition of organic manure for 5 yr. However, application of inorganic fertilizer alone (NPK) showed no significant effect on soil chemical properties (except for AP) compared to CON treatment, this was consistent with the findings at Xinhua and Ningxiang in Hunan, China (Hao et al., 2008).

Table 3a. Effects of different fertilization treatments on soil chemical properties and tea yields.
CON: unfertilized control; NPK: chemical fertilizers; 1/2NPKOM: half-chemical fertilizers plus half-organic manure; OM: organic manure fertilizers; 1/2NPKOM+L: half-chemical fertilizers plus half-organic manure plus legume stover returned; NPKL: chemical fertilizers plus legume stover returned; SOM: soil organic matter; TN: total N; TP: total P; TK: total K.

Table 3b. Effects of different fertilization treatments on soil chemical properties and tea yields.
Values are given as means ± SD (n = 3); Values within a column followed by different lowercase letters are significantly different (n = 3, LSD, P < 0.05).
CON: unfertilized control; NPK: chemical fertilizers; 1/2NPKOM: half-chemical fertilizers plus half-organic manure; OM: organic manure fertilizers; 1/2NPKOM+L: half-chemical fertilizers plus half-organic manure plus legume stover returned; NPKL: chemical fertilizers plus legume stover returned; SOM: soil organic matter; TN: total N; TP: total P; TK: total K; AN: available N; AP: available P; AK: available K.

The cumulative yield data of tea obtained from 2008 to 2011 were analyzed statistically and also shown in Table 3, ranging from 2309 to 6772 kg ha-1. In comparison with CON treatment, the four organic-containing treatments (i.e., 1/2NPKOM, OM, 1/2NPKOM+L, and NPKL) significantly (P < 0.05) increased tea production. The maximum tea yields were observed in the 1/2NPKOM+L treatment and nearly twice higher than that in CON treatment. Among all the treatments, the tea productions were ranked as follows: 1/2NPKOM+L > OM > 1/2NPKOM > NPKL > NPK > CON.

Effect of different fertilizations on soil microbial communities
The TGGE profiles of the bacterial and fungal communities of the samples are shown in Figures 1a and 1b, respectively. For both bacteria and fungi, there were obviously fewer bands detected in NPK than in other treatments, suggesting that application of chemical fertilizer alone declined bacterial and fungal richness and diversity. In addition, the bacterial Shannon index in NPK treatment had the lowest value of 1.714, which was in accordance with Kamaa et al. (2011), who showed that the bacterial diversity was negatively affected by inorganic fertilizer alone. In this experiment, the OM treatment had the maximum bacterial diversity, followed by 1/2NPKOM+L and 1/2NPKOM, this confirmed many earlier findings that organic amendments promoted the bacterial diversity (Peacock et al., 2001; Maeder et al., 2002; Sun et al., 2004; Gu et al., 2009), probably due to input of foreign microorganisms and improvement of soil properties under organic manure application. For fungi, all fertilization treatments appeared a trend towards distinct fungal community structures (Figure 1b), and significantly (P < 0.05) lower diversity (Table 4) when compared to CON treatment. This is in conflict with Kamaa et al. (2011), who showed that the fungal diversity was the lowest in fallow and control treatments and significantly lower than the other treatments. The reasons for this inconsistent result could be due to different physical texture and (or) chemical nutrient elements and (or) crop cover types in different soil types, which were suggested to be the dominant factors, controlling the general microbial community composition (Larkin et al., 2006).


Figure 1. Temperature gradient gel electrophoresis (TGGE) profiles of (a) 16S rRNA and (b) 18S rRNA genes of soil samples under different fertilization practices.
CON: unfertilized control; NPK: chemical fertilizers; 1/2NPKOM: half-chemical fertilizers plus half-organic manure; OM: organic manure fertilizers; 1/2NPKOM+L: half-chemical fertilizers plus half-organic manure plus legume stover returned; NPKL: chemical fertilizers plus legume stover returned.

Table 4. Effects of different fertilization treatments on soil bacterial and fungal diversity.
Values are given as means ± SD (n = 3).
Values within a column followed by different lower-case letters are significantly different (n = 3, LSD, P < 0.05).
CON: Unfertilized control; NPK: chemical fertilizers; 1/2NPKOM: half-chemical fertilizers plus half-organic manure; OM: organic manure fertilizers; 1/2NPKOM+L: half-chemical fertilizers plus half-organic manure plus legume stover returned; NPKL: chemical fertilizers plus legume stover returned.

Cluster analyses (Figure 2a) based on bacterial TGGE profiles showed the three OM-containing (OM, 1/2NPKOM, 1/2NPKOM+L) had a relatively similar banding pattern and clustered together in Cluster 2. Within Cluster 1, the triplicate within NPK treatment further grouped into a smaller cluster, indicating different bacterial community structures in the NPK soil. Two clusters of bacterial dendrogram clearly distinguished between soils treated with organic manure input and soils without organic manure input, suggesting that applications of organic manure had important effects on soil bacterial communities. For fungal TGGE profiles, cluster analyses showed that the CON treatment was separated at the first branch (Figure 2b) and clearly distinguished itself from the treatments with fertilizer, indicating that the highest difference of fungal community existed between CON and other fertilization treatments, and thus, inorganic, organic inputs and their combination significantly altered soil fungal community structures. The treatments of NPKL, 1/2NPKOM, and 1/2NPKOM+L were more closely combined in one sub-group (Figure 2b), which indicated that combined use of organic input with inorganic fertilizers lead to more similar fungal communities. Unexpectedly, we observed that within NPKL treatment, Sample 17 could not be closely clustered into a group containing Samples 16 and 18 (Figures 2a and 2b), suggesting that the experimental replication in the same treatment was not good in this field trial, possibly due to spatial variation of hilly area in tea fields.

Figure 2. Cluster analysis of temperature gradient gel electrophoresis (TGGE) fingerprints in Figure 1 (a) 16S rRNA and (b) 18S rRNA genes.
CON: unfertilized control; NPK: chemical fertilizers; 1/2NPKOM: half-chemical fertilizers plus half-organic manure; OM: organic manure fertilizers; 1/2NPKOM+L: half-chemical fertilizers plus half-organic manure plus legume stover returned; NPKL: chemical fertilizers plus legume stover returned.

Relationships among soil properties, tea yields, and microbial diversity indices
Soil pH is one of the most influential factors affecting the microbial community in soil (Rousk et al., 2009; 2010). In this study, soil pH was positively correlated with the Shannon indices of bacteria (r = 0.541, P < 0.05, Table 5). Soil organic matter (SOM), as an important component of the soil, plays a key role in determining soil physical, chemical, and biological properties that strongly affect crop production (Powlson et al., 2012; Peng et al., 2014). Therefore, maintaining and increasing SOM content in agricultural soils is of paramount importance. Usually, balance application of inorganic fertilizers and organic amendments increased organic input to the soil, then enhanced SOM content (Kanchikerimath and Singh, 2001; Hai et al., 2010), improved soil fertility and crop production (Zhu et al., 2007; Liu et al., 2010). In our study, SOM contents were positively correlated with contents of TN (r = 0.987, P < 0.01), TP (r = 0.964, P < 0.01), AN (r = 0.914, P < 0.01), AP (r = 0.867, P < 0.01), and AK (r = 0.904, P < 0.01), and with tea yields (r = 0.589, P < 0.05) as well as the Shannon indices of bacteria (r = 0.484, P < 0.05, Table 5). Tea yields were also significantly and positively correlated with contents of TN (r = 0.512, P < 0.05), TP (r = 0.499, P < 0.05), AN (r = 0.701, P < 0.01), AP (r = 0.679, P < 0.01), and AK (r = 0.707, P < 0.01, Table 5) besides with SOM. So we know that, SOM contents increased with increasing levels of organic amendment, which in turn caused increases in TN, TP, AN, AP, AK contents in soils and soil bacterial diversity (Table 3). Subsequently, the improved soil in fertility provided more available nutrients for tea growth, and directly led to increase in tea yields. Therefore, organic amendment was a key factor in determining soil properties and productivity.

Table 5. Pearson correlation among microbial diversity indices, soil properties and tea yields.
*P = 0.05; **P = 0.01.
SOM: Soil organic matter; TN: total N; TP: total P; TK: total K; AN: available N; AP: available P; AK: available K; H'b: Bacterial Shannon-Weaver index; H'f: fungal Shannon-Weaver index.

CONCLUSIONS

The results of the field experiment revealed that both OM and 1/2NPKOM+L treatments showed much greater increase in tea yields, more effective improvement in soil chemical fertility, and higher diversity of bacterial community, compared to the other fertilizer treatments. Therefore, both OM and 1/2NPKOM+L treatments could be considered and recommended as better fertilization practices in tea soil in southeastern China.

ACKNOWLEDGEMENTS

This work was financially supported by the Special Fund (2010R1024-4) of fundamental scientific research at nonprofit research institutions in Fujian, the Natural Science Foundation (2011J05057) of Fujian Province, China and the Doctoral Research Fund (2010BS-7) of Fujian Academy of Agricultural Sciences, China.

 

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Received: 26 October 2013.
Accepted: 9 June 2014.