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Effect of different long-term fertilization regimes on the viral community in an agricultural soil of Southern China
European Journal of Soil Biology 62 (2014) 121e126
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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Effect of different long-term fertilization regimes on the viral community in an agricultural soil of Southern China Lin Chen a, b, Weibing Xun a, b, Li Sun a, b, Nan Zhang a, b, Qirong Shen a, b, Ruifu Zhang a, b, * a b
Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing 210095, China National Engineering Research Center for Organic-based Fertilizers, Nanjing Agricultural University, Nanjing 210095, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2013 Received in revised form 26 March 2014 Accepted 26 March 2014 Available online 18 April 2014 Handling editor: Christoph Tebbe
Fertilization plays a pivotal role on soil biological process and affects the soil bacterial community, which act as hosts for viruses. The effect of fertilization on soil viral community has not been well explored. In this study, a Haplic Acrisol soil, which is the soil type for 13 provinces in Southern China, was analyzed after 22 years different fertilization regimes for their viral composition. The soil responded to organic fertilizations with an increased amount of soil organic matter (SOM) and pH (increased from 5.7 to 6.6), while with the decreased SOM and pH for chemical fertilization, especially for single nitrogen fertilization. The combined effects of SOM and pH caused by long-term different fertilization regimes on soil viral communities were investigated by direct calculation of virus-like particles (VLPs) through epifluorescence microscopy. The highest VLP abundance (13.1 107 per gram dry soil) was detected in soil applied with chemical and organic fertilizers. The viral and bacterial abundances of organic soil were 4 and 5 times higher than those of inorganic soil respectively. Transmission electron microscopy observation revealed a higher frequency of Myoviridae viruses in soil with organic amendments than without organic amendments, and vice versa for Podoviridae viruses. These results demonstrate that organic fertilizer could increase viral abundance and morphological diversity through suppressing soil acidification and improving soil organic matter. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Soil viral community Fertilization regimes Abundance Morphological diversity
1. Introduction Viruses are widely distributed in all ecosystems, and it is likely that their presence has important implications for maintaining the ecosystem functions. In soil, viruses, and among them especially bacteriophages influence the soil microbial community through various mechanisms, e.g. by directly influencing bacterial mortality through lysis, infection and energy acquisition from host cells [1e3], and the effect of the host genetic composition by phagemediated processes [4], such as lysogenic conversion and transduction after attaching and gene transfer (transduction) [5e8]. Soil viral adsorption and desorption can be influenced by many factors, including virus characteristics, land use, plant diversity, or soil physic-chemical features [9]. Several studies have focused on the viral abundance in different environments. Srinivasiah et al. [10] found that the viral abundance * Corresponding author. College of Resources & Environmental Science, Nanjing Agricultural University, Nanjing 210095, China. Tel.: þ86 25 84396477; fax: þ86 25 84396260. E-mail address: [email protected] (R. Zhang). http://dx.doi.org/10.1016/j.ejsobi.2014.03.006 1164-5563/Ó 2014 Elsevier Masson SAS. All rights reserved.
changed more than 2000 folds across marine system ranging from the deep sea to freshwater marshes. Another research revealed that viral abundance changed only 16 folds across different soils [11]. Williamson and co-workers [9] reported that a forest soil which contained more organic matter than an agricultural soil, also harbored more virus-like particles (VLPs). But comparison of viral abundances in the same agricultural soil with different management practices has, to your knowledge, not yet been well studied. In China, red soil, which belongs to the Haplic Acrisol (according to FAO-UNESICO WORLD MAP Revised Legend [12]) is one of the most important soil types which in fact covers 13 provinces in Southern China. The color of this soil is brick red or brown red, the fertility and productivity are usually low. The soil pH typically ranges from 5 to 6, and unbalanced fertilization especially excess nitrogen input may decrease the soil pH even further (acidification). In contrast, organic fertilization can stabilize the soil pH and increase crop yields through soil quality improvement [13]. Therefore, organic fertilization and chemical/organic combined fertilization are encouraged in this agricultural soil area. Soil pH is a key factor to drive the composition and activity of the soil bacterial community [14], which are the main hosts of soil
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viruses. Hence soil pH should also have strong effects on the soil viral community. For this study, a long-term (since 1990) fertilization research site located in Hunan Province of Southern China was selected. Different fertilization regimes for 22 years affected the soil in different ways: organic fertilizations not only increased the soil organic matter (SOM), but also stabilized or even increased the soil pH, while chemical fertilization, especially single nitrogen fertilization decreased SOM and soil pH. The combined effects of soil pH and SOM caused by long-term different fertilization regimes on soil viral communities were investigated by direct calculation of virus-like particles (VLPs) through epifluorescense microscopy (EFM). The morphological diversity of viruses was determined by direct observation using transmission electron microscopy (TEM). The results of this study demonstrate correlations between viral abundance and soil properties, especially SOM and pH. 2. Materials and methods 2.1. Field description and experimental design The field experiment is located in Qiyang, Hunan Province, in Southern China (111 520 3200 E, 26 450 1200 N), where the climate is subtropical monsoon climate with an average annual rainfall of 1250 mm and a mean annual temperature of 18 C. The potential evapotranspiration rates reach 1470 mm/year. This fertilization experiment was started in 1990 and included annual rotations of winter wheat (Triticum aestivum L.) and summer maize (Zea mays L.). Different fertilization treatments were implemented with two replicates in a random block design. Five fertilization regimes were chosen for this study: Control without fertilizers (CK), chemical fertilizers (nitrogen, phosphate, and potassium fertilizer, NPK), single nitrogen fertilizer (N), organic fertilizer (M), and chemical and organic fertilizer (NPKM). N, NPK, NPKM and M treatments received the same amount of nitrogen (456 kg/ha). Each plot was 20 m 40 m and the fertilization information is listed in Table 1. Soil properties were measured by standard methods [15]. 2.2. Sample collection and soil analysis Soil samples (depth of 0e20 cm) were collected in May, 2012. The temperature was between 23 and 28 C when sampling. Twelve randomly selected soil cores (approximately 5 cm in
Table 1 Detailed fertilization of different treatments (kg ha1). CKa N
Element b
N (urea) N (pig manure) Maize P (calcium superphosphate) K (potassium chloride) N (urea) N (pig manure) Wheat P (calcium superphosphate) K (potassium chloride)
NPK
NPKM
M
0 0 0
213(456) 213(456) 0 0 0 185(699)
64(137) 0 149(29200) 213(42000) 185(699) e
0
0
73(140)
0 0 0
91(195) 0 0
91(195) 27.3(58.5) 0 0 63.7(12500) 91(18000) 78.9(298) 78.9(298) e
0
0
31.4(60)
73(140)
31.4(60)
e
e
a Control without fertilizers (CK), single nitrogen fertilizer (N), chemical fertilizers (nitrogen, phosphate, and potassium fertilizer, NPK), chemical and organic fertilizer (NPKM), organic fertilizer (M). b Inside the brackets represent fertilizer applicated, and outsides indicate the N, P or K element content.Twenty-two-year annual fertilization of these soils were performed before soil sampling. Wheat (Triticum aestivum L.) was planted in winter while maize (Zea mays L.) in summer as rotation for every year on every soil field. 30% was fertilized in wheat season while 70% in maize season. In addition to control, the other 4 kinds of fertilizing soil receive the same rate of nitrogen.
diameter) were taken from each plot and mixed to form a composite sample. During the period of sending soil samples from Qiyang to the laboratory, fresh soil samples were stored with sterile bags, airtight and regular ventilation. The soil samples were stored at 4 C in the laboratory. Fresh, field-moist soils were stored at 4 C in the laboratory and processed within one week. All soil samples were homogenized and passed through a 4-mm sieve prior to use. 2.3. Extraction conditions Soil viruses were extracted from 10 subsamples of each soil following the methods reported by Wommack et al. [9,16]. In detail, samples (5.0 g) were weighed into 50-ml Teflon-coated polyethylene centrifuge tubes, and 15 ml of 1% potassium citrate solution (containing per liter, 10 g of potassium citrate, 1.44 g of Na2HPO4$7H2O, and 0.24 g of KH2PO4, pH 7, as described by Paul et al. [17]) was added into each tube. All tubes were vortexed for 3e 5 min (with each 10 s interrupted by 15 s of settling on ice) and allowed to stand for 15 min. After centrifugation at 5000 rpm (equal to 2991 g) for 15 min, the supernatants were passed through paper filter (Wo Hua) to remove small soil impurities. Subsequently, all tubes were centrifuged at 11,000 rpm (equal to 14,475 g) for 5 min to settle down some bacteria and soil particles. To eliminate bacteria and small soil impurities, supernatants were passed through 0.20-mm pore size mixed cellulose ester microporous membrane filter (Pall Corporation). To improve the extraction efficiency, soil particles, settled down by centrifugation at 5000 rpm, were resuspended in fresh eluant solution and the extraction process was repeated once. Finally, the combined filter liquor from double extractions was ultracentrifuged at 29,000 rpm (equal to 144000 g) for 60 min with a Beckman SW32Ti rotor, and the sedimentary bacteriophage was resuspended in SM buffer (100 mM NaCl, 10 mM MgSO4$6H2O, 50 mM Tris-Cl [pH 7.5] [16]) for further study. 2.4. Epifluorescence microscopy (EFM) VLPs were enumerated as described by Patel et al. [18]. After 100-fold dilution, the phage aliquots (10 ml) were suspended in 5 ml of 0.02 mm filter-sterilized MilliQ water. A 0.8-mm pore size, 25 mm diameter mixed cellulose ester membrane filter was wetted with filtered MilliQ water and placed onto the center of the filter holder. Next, a 0.02-mm pore size, 25 mm diameter Anodisc filter (Whatman International, Ltd., Dassel, Germany) was placed on top of the 0.8-mm pore size filter. The phage dilution was vacuum filtered through both filters. After drying by rubbing with a clean Kimwipe, the anodisc filter containing captured virus particles was stained by adding 100 ml of 1:400 SYBR Green I solution (1 ml SYBR Green I mixed with 400 ml 0.02 mm filter-sterilized Milli Q water). Filters were incubated for 18 min in the dark in a closed drawer. Then the filters were carefully picked up and dried with a clean Kimwipe in the dark, 30 ml of Fluorescence protectant (0.1% (vol/vol) p-phenylenediamine, 50% glycerin, 50% PBS (0.13 mol/L NaCl, 7.0 mmol/L Na2HPO4, 3.0 mmol/L NaH2PO4)) anti-fade mounting medium was used to fix the filters on the glass slide. The slides were analyzed by EFM with an upright biological microscope (Leica DM 5000 B). Twenty fields per sample were photographed at a magnification of 1000. 2.5. Transmission electron microscopy (TEM) VLPs morphological categorization was conducted as described by Williamson et al. [9,16,19]. Aliquots (5 ml) of re-suspended viruses in the SM buffer were dried on Formvar-coated 400 copper mesh Cu grids (3.05-mm diameter) using an incandescent lamp.
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The grids were then stained with 2% (wt/vol) phosphotungstic acid for 60e90 s, and dried under silica gel. The grids were examined at 100,000 magnification under a transmission electron microscope (Hitachi H-7650). Virus morphology was categorized as Siphoviridae, Myoviridae, Podoviridae, Tailless and Filamentous according to bacteriophage taxonomy guidelines [20]. Viruses were counted as phage with obvious tails (tailed phage), filamentous viruses and VLPs, which were similar to the head capsids of phage. Other phage morphologies were not recorded, because they accounted for small proportion of known virus morphological classification and were difficult to distinguish against the background debris in the samples. Twenty random fields in 3 replicate grids were counted, with about 200 VLPs being observed in each sample. The diversity of viruses was measured by the reciprocal Simpson’s index (1/D) ¼ 1/SPi2. 2.6. Extraction of DNA from soil samples Total soil DNA was extracted from 0.5 g (dry weight) soil using a PowerSoil DNA Isolation Kit (Mo Bio Laboratories Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. The extracts were evaluated on a 1% agarose gel. A NanoDrop ND-2000 spectrophotometer (NanoDrop, Wilmington, DE, USA) was used to determine the concentration and quality (A260/A280) of the extracted DNA. To minimize the DNA extraction bias, each soil was extracted 5 times using the same method, and all the extractions were pooled. DNA was 100-fold diluted for future work. 2.7. Quantification of soil bacteria Real-time quantitative polymerase chain reaction (RT-QPCR) was used for quantification of soil bacteria. The RT-QPCR was performed using a ABI 7500 Cycler (Applied Biosystems, Germany). The primer pair used was 347F: 50 -GGAGGCAGCAGTRRGGAAT-30 [21] and bact531R: 50 -CTNYGTMTTACCGCGGCTGC-30 [22]. The 20 ml reaction solution was prepared with SYBRÒ Premix EX TaqTM (TaKaRa) contained 10 ml of SYBRÒ Premix EX TaqTM (2), 0.4 ml of each primer (10 mM), 0.4 ml of ROX Reference DyeII (50), 2 ml of diluted DNA and 6.8 ml of ddH2O. Thermal conditions were set as follows: 30 s at 95 C for initial denaturation; 40 cycles of 5 s at 95 C, 34 s at 60 C. A standard curve was obtained using gradient diluted plasmid DNA containing a fragment of the 16S rRNA gene that was amplified from Bacillus subtilis 168 with an R2 value of 0.9999. The specificity of the amplification was verified by meltingcurve analysis and agarose gels electrophoresis. Copy numbers of were calculated from the Ct values correspond with the standard curve. 2.8. Statistical analysis Differences of VLPs among the treatments were calculated and statistically analyzed using analysis of variance (ANOVA) and Duncan’s multiple range tests (P < 0.05). 3. Results 3.1. Enumeration of VLPs from soils by EFM counts Based on double extractions, the highest VLP abundance was detected in the NPKM soil (1.306 108 VLPs/g dry soil), followed by the M soil (9.49 107 VLPs/g dry soil). The phage abundances in the other three treatments were all below 2.36 107 VLPs/g dry soil (Fig. 1), which was significant less than those in the NPKM and M soils (P < 0.05).
Fig. 1. Viral and bacterial abundance in 5 Qiyang agricultural soils. (a)VLPs abundance in 5 Qiyang agricultural soils based on duplicate extractions. (b)Bacterial abundance in 5 Qiyang agricultural soils based on quantitive PCR. Five fertilization regimes were chosen for this study, which include control without fertilizers (CK), chemical fertilizers (nitrogen, phosphate, and potassium fertilizer, NPK), single nitrogen fertilizer (N), organic fertilizer (M), and chemical and organic fertilizer (NPKM).
3.2. Effect of different fertilization regimes on soil characters and bacterial populations Application of chemical fertilizer (NPK), especially single nitrogen fertilizer (N), for 22 years contributed to the acidification of the soil in this research site, with the soil pH values decreasing to 4.19
Table 2 Chemical and biotic properties of the soils (0e20 cm depth) in different treatments. Treatmenta
Original CK soil
pH Available N(mg/kg dw) Total N(g/kg dw) Available P(mg/kg dw) Total P(g/kg dw) Available K(mg/kg dw) Total K(g/kg dw) SOMb (g/kg dw) Bacterial quantity (108 copies/g dw) VLPs (107/g dw) Viral diversityc
5.7 79.0 1.07 13.9 0.52 104 13.7 11.5 e e e
N
NPK
NPKM
M
5.10 4.26 4.19 6.08 6.58 87.8 146 129 155 151 0.866 0.880 1.000 1.58 1.60 8.0 7.1 69.2 113 112 0.475 0.481 1.29 1.68 1.40 72.5 60.0 225 378 328 12.97 12.96 14.22 14.84 14.26 12.8 12.5 16.2 26.7 25.2 6.81 2.09 4.86 27.2 40.5 2.25 2.68
1.34 2.95
2.36 3.43
13.1 3.15
9.49 3.65
a Chemical properties of soil in 1990 (Original soil); Control without fertilizers (CK), single nitrogen fertilizer (N), chemical fertilizers (nitrogen, phosphate, and potassium fertilizer, NPK), chemical and organic fertilizer (NPKM), organic fertilizer (M). b Soil organic matter content (% dry weight); c: Diversity was assessed by reciprocal Simpson’s index (1/D) ¼ 1/SPi2. c Diversity was assessed by reciprocal Simpson’s index (1/D) ¼ 1/SPi2.
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Fig. 2. Transmission electron microscopy images of virus-like particles directly isolated from agricultural soils. a, b, c tailless viruses; d, e Podoviridae; f, g, h, i Myoviridae; j filamentous virus; k, l, m, n Siphoviridae. Scale bars represent 100 nm.
L. Chen et al. / European Journal of Soil Biology 62 (2014) 121e126
and 4.26, respectively. Amendment of organic materials increased and stabilized the soil pH, as revealed by the pH value being 6.08 and 6.58 in NPKM and M treatments respectively (Table 2). Other nutrients contents (total N, available P, total P, available K and SOM) in NPKM and M were all higher than in the other treatments (Table 2). The differences of available N and total K between organic fertilizer soils and inorganic fertilizer soils were not significant. The highest abundance of bacteria was detected in the M soil (40.5 108 rRNA gene copies/g dry soil), followed by NPKM soil (27.2 108 copies/g dry soil). The bacterial quantities in the other 3 treatments ranged from 6.81 108 copies/g dry soil to 2.09 108 copies/g dry soil with N soil had the lowest bacterial population. 3.3. Viral diversity in soils with different fertilization regimes The viral morphologies in the 5 agricultural soils were observed under TEM (Fig. 2) and classified based on the typical morphotypes [20,23]. Two hundred randomly selected VLPs under TEM were examined for each soil. The tailless virus was dominant among other four viral morphotypes (Fig. 3). The frequency of Myoviridae viruses was much higher in soil with organic amendment (NPKM, M) than in other soils (CK, NPK, N). However, the frequency of Podoviridae viruses was significantly higher in soils without organic amendment (Fig. 3). Viral diversity calculated using the reciprocal Simpson’s index showed that M soil was the greatest with a value of 3.65, while that of CK soil was the lowest among all the treatments. The viral diversity of fertilizing soils was greater than in the control (Table 2). 4. Discussion The VLPs abundance in this study was, compared to other studies, relatively low [9,16]. Less bacterial host cells due to low nutrients and pH of this soil type in the research area may be one of the reasons [24]. There are many factors which potentially contribute to the differences of VLPs abundance in the 5 different fertilization soils. Twenty-two years different fertilization regimes did not only affect the overall soil organic matter (SOM), but also the pH values, the bacteria quantities and other soil characteristics. Soil pH and SOM may affect the viral community through influencing the interaction between viruses and soil particles, which mainly occurs between the virus protein capsids and the soil particles [25]. Soil particles are usually negatively charged.
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Adsorption of viruses to soil is affected by the viral isoelectric point and soil pH. When soil pH is greater than the viral isoelectric point, the viruses are negatively charged. Adsorption decreases with increasing of soil pH, which makes the virus dissociation and extraction process easier [26e28]. Also, humic substances, which are a component of the SOM, in soil are also negatively charged. Thus, SOM affected adsorption between viruses and soil particles. In addition, SOM also can restrain virus transport through enhancing hydrophobic interactions between the viruses and soil particle surfaces. But, overall, the main impact of SOM appears to be electrostatic rather than triggered by hydrophobic interactions [29]. A negative correlation was found in other studies between the content of SOM and the amount of viral adsorption [27,30]. When the amount of viruses adsorbed by soil particles decreased, as caused by organic fertilization, viruses could have more opportunities to reach new host cells [28,31]. Soil pH and SOM can both affect viral community through the microbial hosts. SOM content is closely related to soil fertility levels, and provides the main nutrient and energy sources for soil microorganism. Microbial abundance would increase with a higher SOM content in soil [32]. Soil pH is related to the soil base saturation, which is directly linked with soil nutrients and crop growth [13,33,34]. With pH values between 4 and 8, bacterial abundance and diversity are positively correlated to soil pH [14]. As the optimal growth pH ranges of bacteria are narrow, soil pH has a direct effect on bacterial community composition. Bacteria quantity, as a biotic factor, affects viral survival and production [4,31]. Under some circumstances, the absence of microorganism increased phage survival [9,35], since microorganisms would produce proteolytic enzymes to degrade viral protein capsids [36,37]. In opposite, another study reported the presence of microorganism increased phage survival [38]. In this study, a positive correlation between VLP abundance and bacterial abundance was found (Table 2), which is in accordance with several previous studies regarding a wide range of environments [39]. Our results indicate that the increasing of bacterial quantity might lead to higher viral quantity. Also Williamson et al. [40,41] revealed a clear correlation between bacterial and viral abundance. The content of SOM was much more in organic fertilizing soils, and the pH of soils with organic fertilization (NPKM and M soil) were higher than soils without organic fertilization (CK, N and NPK soil). Thus, viral abundance was much higher in soils with organic fertilization. According to the phage morphological distribution of 5500 known phage isolates, 96% of these viruses are tailed [20]. Filamentous viruses and tailless viruses are rarely reported morphotypes, which accounted for less than 1.5% and 2.0% of the total phages in environmental samples [42]. However, in this study, both filamentous and tailless VLPs could be easily detected under TEM in all 5 soil samples (Fig. 3), which suggests that VLPs distribution in this unique Haplic Acrisol soil was different from that of other environments. According to reciprocal Simpson’s index, viral diversity did not show consistent trend between inorganic fertilization and organic fertilization soils (as shown in Fig. 3). Viral diversities of fertilization soils were greater than those of control soil without any fertilization, which indicates viral diversity could be higher through organic fertilization. 5. Conclusions
Fig. 3. Frequency distributions of viral morphologies from 5 Qiyang agricultural soils. Viral categories include tailless (Tail-), Siphoviridae (Sipho), Myoviridae (Myo), Podoviridae (Podo) and filamentous (Fil). Histograms are based on measured value of 200 randomly selected individual viruses from each soil (3 replications). Five fertilization regimes were chosen for this study, which include control without fertilizers (CK), chemical fertilizers (nitrogen, phosphate, and potassium fertilizer, NPK), single nitrogen fertilizer (N), organic fertilizer (M), and chemical and organic fertilizer (NPKM).
Twenty-two years different fertilization regimes, including the use of chemical and organic fertilizers, changed many soil characteristics as well as the soil viral abundance and morphological diversity. The effects on the viral diversity appear to be affected by the virus type, as demonstrated in this study for the opposite effects of increased SOM on Myoviridae and Podoviridae viruses.
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Acknowledgments The authors thank the staff in Qiyang Red Soil Experimental Station for managing the field experiments and helping in collection of soil samples. This research was financially supported by the Chinese Ministry of Science and Technology (2013AA102802) and Chinese Ministry of Agriculture (201103004). R.Z and Q. S were also supported by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions and the 111 Project (B12009). References [1] M. Middelboe, N. Jorgensen, N. Kroer, Effects of viruses on nutrient turnover and growth efficiency of noninfected marine bacterioplankton, Appl. Environ. Microbiol. 62 (1996) 1991e1997. [2] K.E. Wommack, R.R. Colwell, Virioplankton: viruses in aquatic ecosystems, Microbiol. Mol. Biol. Rev. 64 (2000) 69e114. [3] F. Rohwer, D. Prangishvili, D. Lindell, Roles of viruses in the environment, Environ. Microbiol. 11 (2009) 2771e2774. [4] P. Gomez, A. Buckling, Bacteria-phage antagonistic coevolution in soil, Science 332 (2011) 106e109. [5] E.F. Boyd, B.M. Davis, B. Hochhut, Bacteriophage-bacteriophage interactions in the evolution of pathogenic bacteria, Trends Microbiol. 9 (2001) 137e144. [6] J. Holland, E. Domingo, Origin and evolution of viruses, Virus Genes. 16 (1998) 13e21. [7] M.K. Waldor, J.J. Mekalanos, Lysogenic conversion by a filamentous phage encoding cholera toxin, Science 272 (1996) 1910e1914. [8] E.V. Koonin, N. Yutin, Origin and evolution of eukaryotic large nucleocytoplasmic DNA viruses, Intervirology 53 (2010) 284e292. [9] K.E. Williamson, M. Radosevich, K.E. Wommack, Abundance and diversity of viruses in six Delaware soils, Appl. Environ. Microbiol. 71 (2005) 3119e3125. [10] S. Srinivasiah, J. Bhavsar, K. Thapar, M. Liles, T. Schoenfeld, K.E. Wommack, Phages across the biosphere: contrasts of viruses in soil and aquatic environments, Res. Microbiol. 159 (2008) 349e357. [11] K.E. Williamson, M. Radosevich, D.W. Smith, K.E. Wommack, Incidence of lysogeny within temperate and extreme soil environments, Environ. Microbiol. 9 (2007) 2563e2574. [12] X.J. Wang, Z.T. Gong, Assessment and analysis of soil quality changes after eleven years of reclamation in subtropical China, Geoderma 81 (1998) 339e355. [13] J.H. Guo, X.J. Liu, Y. Zhang, J.L. Shen, W.X. Han, W.F. Zhang, P. Christie, K.W. Goulding, P.M. Vitousek, F.S. Zhang, Significant acidification in major Chinese croplands, Science 327 (2010) 1008e1010. [14] J. Rousk, E. Baath, P.C. Brookes, C.L. Lauber, C. Lozupone, J.G. Caporaso, R. Knight, N. Fierer, Soil bacterial and fungal communities across a pH gradient in an arable soil, ISME J. 4 (2010) 1340e1351. [15] D.L. Sparks, Soil Science Society of America, American Society of Agronomy, Methods of Soil Analysis, in: Part 3, Chemical methods, Soil Science Society of America: American Society of Agronomy, Madison, Wis, 1996. [16] K.E. Williamson, K.E. Wommack, M. Radosevich, Sampling natural viral communities from soil for culture-independent analyses, Appl. Environ. Microbiol. 69 (2003) 6628e6633. [17] J.H. Paul, J.B. Rose, S.C. Jiang, C.A. Kellogg, L. Dickson, Distribution of viral abundance in the reef environment of Key Largo, Florida, Appl. Environ. Microbiol. 59 (1993) 718e724. [18] A. Patel, R.T. Noble, J.A. Steele, M.S. Schwalbach, I. Hewson, J.A. Fuhrman, Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I, Nat. Protoc. 2 (2007) 269e276. [19] K.E. Williamson, J.B. Schnitker, M. Radosevich, D.W. Smith, K.E. Wommack, Cultivation-based assessment of lysogeny among soil bacteria, Microb. Ecol. 56 (2008) 437e447.
[20] H.W. Ackermann, 5500 Phages examined in the electron microscope, Arch. Virol. 152 (2007) 227e243. [21] B.S. Kim, B.K. Kim, J.H. Lee, M. Kim, Y.W. Lim, J. Chun, Rapid phylogenetic dissection of prokaryotic community structure in tidal flat using pyrosequencing, J. Microbiol. 46 (2008) 357e363. [22] C.W. Nossa, W.E. Oberdorf, L.Y. Yang, J.A. Aas, B.J. Paster, T.Z. DeSantis, E.L. Brodie, D. Malamud, M.A. Poles, Z.H. Pei, Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome, World J. Gastroenterol. 16 (2010) 4135e4144. [23] H.W. Ackermann, Phage classification and characterization, Methods Mol. Biol. 501 (2009) 127e140. [24] R. Danovaro, E. Manini, A. Dell’Anno, Higher abundance of bacteria than of viruses in deep Mediterranean sediments, Appl. Environ. Microbiol. 68 (2002) 1468e1472. [25] S.E. Dowd, S.D. Pillai, S.Y. Wang, M.Y. Corapcioglu, Delineating the specific influence of virus isoelectric point and size on virus adsorption and transport through sandy soils, Appl. Environ. Microbiol. 64 (1998) 405e410. [26] J. Lukasik, T.M. Scott, D. Andryshak, S.R. Farrah, Influence of salts on virus adsorption to microporous filters, Appl. Environ. Microbiol. 66 (2000) 2914e 2920. [27] Y.J. Chu, Y. Jin, T. Baumann, M.V. Yates, Effect of soil properties on saturated and unsaturated virus transport through columns, J. Environ. Qual. 32 (2003) 2017e2025. [28] B.Z. Zhao, H. Zhang, J.B. Zhang, Y. Jin, Virus adsorption and inactivation in soil as influenced by autochthonous microorganisms and water content, Soil. Biol. Biochem. 40 (2008) 649e659. [29] J. Zhuang, Y. Jin, Virus retention and transport as influenced by different forms of soil organic matter, J. Environ. Qual. 32 (2003) 816e823. [30] R.L. LaBelle, C.P. Gerba, Influence of pH, salinity, and organic matter on the adsorption of enteric viruses to estuarine sediment, Appl. Environ. Microbiol. 38 (1979) 93e101. [31] K.A. Lawson, Y.M. Barnet, C.A. McGilchrist, Environmental factors influencing numbers of Rhizobium leguminosarum biovar trifolii and its bacteriophages in two field soils, Appl. Environ. Microbiol. 53 (1987) 1125e1131. [32] J. Six, H. Bossuyt, S. Degryze, K. Denef, A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics, Soil. Tillage Res. 79 (2004) 7e31. [33] S.T. Berthrong, E.G. Jobbagy, R.B. Jackson, A global meta-analysis of soil exchangeable cations, pH, carbon, and nitrogen with afforestation, Ecol. Appl. 19 (2009) 2228e2241. [34] T. Larssen, G.R. Carmichael, Acid rain and acidification in China: the importance of base cation deposition, Environ. Pollut. 110 (2000) 89e102. [35] A.M. Nasser, R. Glozman, Y. Nitzan, Contribution of microbial activity to virus reduction in saturated soil, Water Res. 36 (2002) 2589e2595. [36] C.J. Hurst, Influence of aerobic microorganisms upon virus survival in soil, Can. J. Microbiol. 34 (1988) 696e699. [37] M.V. Yates, L.D. Stetzenbach, C.P. Gerba, N.A. Sinclair, The effect of indigenous bacteria on virus survival in ground-water, J. Environ. Sci. Health A 25 (1990) 81e100. [38] M. Pantasticocaldas, K.E. Duncan, C.A. Istock, J.A. Bell, Population-Dynamics of bacteriophage and Bacillus-Subtilis in soil, Ecology 73 (1992) 1888e1902. [39] S. Jiang, G. Steward, R. Jellison, W. Chu, S. Choi, Abundance, distribution, and diversity of viruses in alkaline, hypersaline Mono Lake, California, Microb. Ecol. 47 (2004) 9e17. [40] D.M. Winget, K.E. Williamson, R.R. Helton, K.E. Wommack, Tangential flow diafiltration: an improved technique for estimation of virioplankton production, Aquat. Microb. Ecol. 41 (2005) 221e232. [41] S.R. Bench, T.E. Hanson, K.E. Williamson, D. Ghosh, M. Radosovich, K. Wang, K.E. Wommack, Metagenomic characterization of Chesapeake bay virioplankton, Appl. Environ. Microbiol. 73 (2007) 7629e7641. [42] H.W. Ackermann, Bacteriophage taxonomy, Microbiol. Aust. (2011) 90e94.
Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Effect of different long-term fertilization regimes on the viral community in an agricultural soil of Southern China Lin Chen a, b, Weibing Xun a, b, Li Sun a, b, Nan Zhang a, b, Qirong Shen a, b, Ruifu Zhang a, b, * a b
Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing 210095, China National Engineering Research Center for Organic-based Fertilizers, Nanjing Agricultural University, Nanjing 210095, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2013 Received in revised form 26 March 2014 Accepted 26 March 2014 Available online 18 April 2014 Handling editor: Christoph Tebbe
Fertilization plays a pivotal role on soil biological process and affects the soil bacterial community, which act as hosts for viruses. The effect of fertilization on soil viral community has not been well explored. In this study, a Haplic Acrisol soil, which is the soil type for 13 provinces in Southern China, was analyzed after 22 years different fertilization regimes for their viral composition. The soil responded to organic fertilizations with an increased amount of soil organic matter (SOM) and pH (increased from 5.7 to 6.6), while with the decreased SOM and pH for chemical fertilization, especially for single nitrogen fertilization. The combined effects of SOM and pH caused by long-term different fertilization regimes on soil viral communities were investigated by direct calculation of virus-like particles (VLPs) through epifluorescence microscopy. The highest VLP abundance (13.1 107 per gram dry soil) was detected in soil applied with chemical and organic fertilizers. The viral and bacterial abundances of organic soil were 4 and 5 times higher than those of inorganic soil respectively. Transmission electron microscopy observation revealed a higher frequency of Myoviridae viruses in soil with organic amendments than without organic amendments, and vice versa for Podoviridae viruses. These results demonstrate that organic fertilizer could increase viral abundance and morphological diversity through suppressing soil acidification and improving soil organic matter. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Soil viral community Fertilization regimes Abundance Morphological diversity
1. Introduction Viruses are widely distributed in all ecosystems, and it is likely that their presence has important implications for maintaining the ecosystem functions. In soil, viruses, and among them especially bacteriophages influence the soil microbial community through various mechanisms, e.g. by directly influencing bacterial mortality through lysis, infection and energy acquisition from host cells [1e3], and the effect of the host genetic composition by phagemediated processes [4], such as lysogenic conversion and transduction after attaching and gene transfer (transduction) [5e8]. Soil viral adsorption and desorption can be influenced by many factors, including virus characteristics, land use, plant diversity, or soil physic-chemical features [9]. Several studies have focused on the viral abundance in different environments. Srinivasiah et al. [10] found that the viral abundance * Corresponding author. College of Resources & Environmental Science, Nanjing Agricultural University, Nanjing 210095, China. Tel.: þ86 25 84396477; fax: þ86 25 84396260. E-mail address: [email protected] (R. Zhang). http://dx.doi.org/10.1016/j.ejsobi.2014.03.006 1164-5563/Ó 2014 Elsevier Masson SAS. All rights reserved.
changed more than 2000 folds across marine system ranging from the deep sea to freshwater marshes. Another research revealed that viral abundance changed only 16 folds across different soils [11]. Williamson and co-workers [9] reported that a forest soil which contained more organic matter than an agricultural soil, also harbored more virus-like particles (VLPs). But comparison of viral abundances in the same agricultural soil with different management practices has, to your knowledge, not yet been well studied. In China, red soil, which belongs to the Haplic Acrisol (according to FAO-UNESICO WORLD MAP Revised Legend [12]) is one of the most important soil types which in fact covers 13 provinces in Southern China. The color of this soil is brick red or brown red, the fertility and productivity are usually low. The soil pH typically ranges from 5 to 6, and unbalanced fertilization especially excess nitrogen input may decrease the soil pH even further (acidification). In contrast, organic fertilization can stabilize the soil pH and increase crop yields through soil quality improvement [13]. Therefore, organic fertilization and chemical/organic combined fertilization are encouraged in this agricultural soil area. Soil pH is a key factor to drive the composition and activity of the soil bacterial community [14], which are the main hosts of soil
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viruses. Hence soil pH should also have strong effects on the soil viral community. For this study, a long-term (since 1990) fertilization research site located in Hunan Province of Southern China was selected. Different fertilization regimes for 22 years affected the soil in different ways: organic fertilizations not only increased the soil organic matter (SOM), but also stabilized or even increased the soil pH, while chemical fertilization, especially single nitrogen fertilization decreased SOM and soil pH. The combined effects of soil pH and SOM caused by long-term different fertilization regimes on soil viral communities were investigated by direct calculation of virus-like particles (VLPs) through epifluorescense microscopy (EFM). The morphological diversity of viruses was determined by direct observation using transmission electron microscopy (TEM). The results of this study demonstrate correlations between viral abundance and soil properties, especially SOM and pH. 2. Materials and methods 2.1. Field description and experimental design The field experiment is located in Qiyang, Hunan Province, in Southern China (111 520 3200 E, 26 450 1200 N), where the climate is subtropical monsoon climate with an average annual rainfall of 1250 mm and a mean annual temperature of 18 C. The potential evapotranspiration rates reach 1470 mm/year. This fertilization experiment was started in 1990 and included annual rotations of winter wheat (Triticum aestivum L.) and summer maize (Zea mays L.). Different fertilization treatments were implemented with two replicates in a random block design. Five fertilization regimes were chosen for this study: Control without fertilizers (CK), chemical fertilizers (nitrogen, phosphate, and potassium fertilizer, NPK), single nitrogen fertilizer (N), organic fertilizer (M), and chemical and organic fertilizer (NPKM). N, NPK, NPKM and M treatments received the same amount of nitrogen (456 kg/ha). Each plot was 20 m 40 m and the fertilization information is listed in Table 1. Soil properties were measured by standard methods [15]. 2.2. Sample collection and soil analysis Soil samples (depth of 0e20 cm) were collected in May, 2012. The temperature was between 23 and 28 C when sampling. Twelve randomly selected soil cores (approximately 5 cm in
Table 1 Detailed fertilization of different treatments (kg ha1). CKa N
Element b
N (urea) N (pig manure) Maize P (calcium superphosphate) K (potassium chloride) N (urea) N (pig manure) Wheat P (calcium superphosphate) K (potassium chloride)
NPK
NPKM
M
0 0 0
213(456) 213(456) 0 0 0 185(699)
64(137) 0 149(29200) 213(42000) 185(699) e
0
0
73(140)
0 0 0
91(195) 0 0
91(195) 27.3(58.5) 0 0 63.7(12500) 91(18000) 78.9(298) 78.9(298) e
0
0
31.4(60)
73(140)
31.4(60)
e
e
a Control without fertilizers (CK), single nitrogen fertilizer (N), chemical fertilizers (nitrogen, phosphate, and potassium fertilizer, NPK), chemical and organic fertilizer (NPKM), organic fertilizer (M). b Inside the brackets represent fertilizer applicated, and outsides indicate the N, P or K element content.Twenty-two-year annual fertilization of these soils were performed before soil sampling. Wheat (Triticum aestivum L.) was planted in winter while maize (Zea mays L.) in summer as rotation for every year on every soil field. 30% was fertilized in wheat season while 70% in maize season. In addition to control, the other 4 kinds of fertilizing soil receive the same rate of nitrogen.
diameter) were taken from each plot and mixed to form a composite sample. During the period of sending soil samples from Qiyang to the laboratory, fresh soil samples were stored with sterile bags, airtight and regular ventilation. The soil samples were stored at 4 C in the laboratory. Fresh, field-moist soils were stored at 4 C in the laboratory and processed within one week. All soil samples were homogenized and passed through a 4-mm sieve prior to use. 2.3. Extraction conditions Soil viruses were extracted from 10 subsamples of each soil following the methods reported by Wommack et al. [9,16]. In detail, samples (5.0 g) were weighed into 50-ml Teflon-coated polyethylene centrifuge tubes, and 15 ml of 1% potassium citrate solution (containing per liter, 10 g of potassium citrate, 1.44 g of Na2HPO4$7H2O, and 0.24 g of KH2PO4, pH 7, as described by Paul et al. [17]) was added into each tube. All tubes were vortexed for 3e 5 min (with each 10 s interrupted by 15 s of settling on ice) and allowed to stand for 15 min. After centrifugation at 5000 rpm (equal to 2991 g) for 15 min, the supernatants were passed through paper filter (Wo Hua) to remove small soil impurities. Subsequently, all tubes were centrifuged at 11,000 rpm (equal to 14,475 g) for 5 min to settle down some bacteria and soil particles. To eliminate bacteria and small soil impurities, supernatants were passed through 0.20-mm pore size mixed cellulose ester microporous membrane filter (Pall Corporation). To improve the extraction efficiency, soil particles, settled down by centrifugation at 5000 rpm, were resuspended in fresh eluant solution and the extraction process was repeated once. Finally, the combined filter liquor from double extractions was ultracentrifuged at 29,000 rpm (equal to 144000 g) for 60 min with a Beckman SW32Ti rotor, and the sedimentary bacteriophage was resuspended in SM buffer (100 mM NaCl, 10 mM MgSO4$6H2O, 50 mM Tris-Cl [pH 7.5] [16]) for further study. 2.4. Epifluorescence microscopy (EFM) VLPs were enumerated as described by Patel et al. [18]. After 100-fold dilution, the phage aliquots (10 ml) were suspended in 5 ml of 0.02 mm filter-sterilized MilliQ water. A 0.8-mm pore size, 25 mm diameter mixed cellulose ester membrane filter was wetted with filtered MilliQ water and placed onto the center of the filter holder. Next, a 0.02-mm pore size, 25 mm diameter Anodisc filter (Whatman International, Ltd., Dassel, Germany) was placed on top of the 0.8-mm pore size filter. The phage dilution was vacuum filtered through both filters. After drying by rubbing with a clean Kimwipe, the anodisc filter containing captured virus particles was stained by adding 100 ml of 1:400 SYBR Green I solution (1 ml SYBR Green I mixed with 400 ml 0.02 mm filter-sterilized Milli Q water). Filters were incubated for 18 min in the dark in a closed drawer. Then the filters were carefully picked up and dried with a clean Kimwipe in the dark, 30 ml of Fluorescence protectant (0.1% (vol/vol) p-phenylenediamine, 50% glycerin, 50% PBS (0.13 mol/L NaCl, 7.0 mmol/L Na2HPO4, 3.0 mmol/L NaH2PO4)) anti-fade mounting medium was used to fix the filters on the glass slide. The slides were analyzed by EFM with an upright biological microscope (Leica DM 5000 B). Twenty fields per sample were photographed at a magnification of 1000. 2.5. Transmission electron microscopy (TEM) VLPs morphological categorization was conducted as described by Williamson et al. [9,16,19]. Aliquots (5 ml) of re-suspended viruses in the SM buffer were dried on Formvar-coated 400 copper mesh Cu grids (3.05-mm diameter) using an incandescent lamp.
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The grids were then stained with 2% (wt/vol) phosphotungstic acid for 60e90 s, and dried under silica gel. The grids were examined at 100,000 magnification under a transmission electron microscope (Hitachi H-7650). Virus morphology was categorized as Siphoviridae, Myoviridae, Podoviridae, Tailless and Filamentous according to bacteriophage taxonomy guidelines [20]. Viruses were counted as phage with obvious tails (tailed phage), filamentous viruses and VLPs, which were similar to the head capsids of phage. Other phage morphologies were not recorded, because they accounted for small proportion of known virus morphological classification and were difficult to distinguish against the background debris in the samples. Twenty random fields in 3 replicate grids were counted, with about 200 VLPs being observed in each sample. The diversity of viruses was measured by the reciprocal Simpson’s index (1/D) ¼ 1/SPi2. 2.6. Extraction of DNA from soil samples Total soil DNA was extracted from 0.5 g (dry weight) soil using a PowerSoil DNA Isolation Kit (Mo Bio Laboratories Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. The extracts were evaluated on a 1% agarose gel. A NanoDrop ND-2000 spectrophotometer (NanoDrop, Wilmington, DE, USA) was used to determine the concentration and quality (A260/A280) of the extracted DNA. To minimize the DNA extraction bias, each soil was extracted 5 times using the same method, and all the extractions were pooled. DNA was 100-fold diluted for future work. 2.7. Quantification of soil bacteria Real-time quantitative polymerase chain reaction (RT-QPCR) was used for quantification of soil bacteria. The RT-QPCR was performed using a ABI 7500 Cycler (Applied Biosystems, Germany). The primer pair used was 347F: 50 -GGAGGCAGCAGTRRGGAAT-30 [21] and bact531R: 50 -CTNYGTMTTACCGCGGCTGC-30 [22]. The 20 ml reaction solution was prepared with SYBRÒ Premix EX TaqTM (TaKaRa) contained 10 ml of SYBRÒ Premix EX TaqTM (2), 0.4 ml of each primer (10 mM), 0.4 ml of ROX Reference DyeII (50), 2 ml of diluted DNA and 6.8 ml of ddH2O. Thermal conditions were set as follows: 30 s at 95 C for initial denaturation; 40 cycles of 5 s at 95 C, 34 s at 60 C. A standard curve was obtained using gradient diluted plasmid DNA containing a fragment of the 16S rRNA gene that was amplified from Bacillus subtilis 168 with an R2 value of 0.9999. The specificity of the amplification was verified by meltingcurve analysis and agarose gels electrophoresis. Copy numbers of were calculated from the Ct values correspond with the standard curve. 2.8. Statistical analysis Differences of VLPs among the treatments were calculated and statistically analyzed using analysis of variance (ANOVA) and Duncan’s multiple range tests (P < 0.05). 3. Results 3.1. Enumeration of VLPs from soils by EFM counts Based on double extractions, the highest VLP abundance was detected in the NPKM soil (1.306 108 VLPs/g dry soil), followed by the M soil (9.49 107 VLPs/g dry soil). The phage abundances in the other three treatments were all below 2.36 107 VLPs/g dry soil (Fig. 1), which was significant less than those in the NPKM and M soils (P < 0.05).
Fig. 1. Viral and bacterial abundance in 5 Qiyang agricultural soils. (a)VLPs abundance in 5 Qiyang agricultural soils based on duplicate extractions. (b)Bacterial abundance in 5 Qiyang agricultural soils based on quantitive PCR. Five fertilization regimes were chosen for this study, which include control without fertilizers (CK), chemical fertilizers (nitrogen, phosphate, and potassium fertilizer, NPK), single nitrogen fertilizer (N), organic fertilizer (M), and chemical and organic fertilizer (NPKM).
3.2. Effect of different fertilization regimes on soil characters and bacterial populations Application of chemical fertilizer (NPK), especially single nitrogen fertilizer (N), for 22 years contributed to the acidification of the soil in this research site, with the soil pH values decreasing to 4.19
Table 2 Chemical and biotic properties of the soils (0e20 cm depth) in different treatments. Treatmenta
Original CK soil
pH Available N(mg/kg dw) Total N(g/kg dw) Available P(mg/kg dw) Total P(g/kg dw) Available K(mg/kg dw) Total K(g/kg dw) SOMb (g/kg dw) Bacterial quantity (108 copies/g dw) VLPs (107/g dw) Viral diversityc
5.7 79.0 1.07 13.9 0.52 104 13.7 11.5 e e e
N
NPK
NPKM
M
5.10 4.26 4.19 6.08 6.58 87.8 146 129 155 151 0.866 0.880 1.000 1.58 1.60 8.0 7.1 69.2 113 112 0.475 0.481 1.29 1.68 1.40 72.5 60.0 225 378 328 12.97 12.96 14.22 14.84 14.26 12.8 12.5 16.2 26.7 25.2 6.81 2.09 4.86 27.2 40.5 2.25 2.68
1.34 2.95
2.36 3.43
13.1 3.15
9.49 3.65
a Chemical properties of soil in 1990 (Original soil); Control without fertilizers (CK), single nitrogen fertilizer (N), chemical fertilizers (nitrogen, phosphate, and potassium fertilizer, NPK), chemical and organic fertilizer (NPKM), organic fertilizer (M). b Soil organic matter content (% dry weight); c: Diversity was assessed by reciprocal Simpson’s index (1/D) ¼ 1/SPi2. c Diversity was assessed by reciprocal Simpson’s index (1/D) ¼ 1/SPi2.
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Fig. 2. Transmission electron microscopy images of virus-like particles directly isolated from agricultural soils. a, b, c tailless viruses; d, e Podoviridae; f, g, h, i Myoviridae; j filamentous virus; k, l, m, n Siphoviridae. Scale bars represent 100 nm.
L. Chen et al. / European Journal of Soil Biology 62 (2014) 121e126
and 4.26, respectively. Amendment of organic materials increased and stabilized the soil pH, as revealed by the pH value being 6.08 and 6.58 in NPKM and M treatments respectively (Table 2). Other nutrients contents (total N, available P, total P, available K and SOM) in NPKM and M were all higher than in the other treatments (Table 2). The differences of available N and total K between organic fertilizer soils and inorganic fertilizer soils were not significant. The highest abundance of bacteria was detected in the M soil (40.5 108 rRNA gene copies/g dry soil), followed by NPKM soil (27.2 108 copies/g dry soil). The bacterial quantities in the other 3 treatments ranged from 6.81 108 copies/g dry soil to 2.09 108 copies/g dry soil with N soil had the lowest bacterial population. 3.3. Viral diversity in soils with different fertilization regimes The viral morphologies in the 5 agricultural soils were observed under TEM (Fig. 2) and classified based on the typical morphotypes [20,23]. Two hundred randomly selected VLPs under TEM were examined for each soil. The tailless virus was dominant among other four viral morphotypes (Fig. 3). The frequency of Myoviridae viruses was much higher in soil with organic amendment (NPKM, M) than in other soils (CK, NPK, N). However, the frequency of Podoviridae viruses was significantly higher in soils without organic amendment (Fig. 3). Viral diversity calculated using the reciprocal Simpson’s index showed that M soil was the greatest with a value of 3.65, while that of CK soil was the lowest among all the treatments. The viral diversity of fertilizing soils was greater than in the control (Table 2). 4. Discussion The VLPs abundance in this study was, compared to other studies, relatively low [9,16]. Less bacterial host cells due to low nutrients and pH of this soil type in the research area may be one of the reasons [24]. There are many factors which potentially contribute to the differences of VLPs abundance in the 5 different fertilization soils. Twenty-two years different fertilization regimes did not only affect the overall soil organic matter (SOM), but also the pH values, the bacteria quantities and other soil characteristics. Soil pH and SOM may affect the viral community through influencing the interaction between viruses and soil particles, which mainly occurs between the virus protein capsids and the soil particles [25]. Soil particles are usually negatively charged.
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Adsorption of viruses to soil is affected by the viral isoelectric point and soil pH. When soil pH is greater than the viral isoelectric point, the viruses are negatively charged. Adsorption decreases with increasing of soil pH, which makes the virus dissociation and extraction process easier [26e28]. Also, humic substances, which are a component of the SOM, in soil are also negatively charged. Thus, SOM affected adsorption between viruses and soil particles. In addition, SOM also can restrain virus transport through enhancing hydrophobic interactions between the viruses and soil particle surfaces. But, overall, the main impact of SOM appears to be electrostatic rather than triggered by hydrophobic interactions [29]. A negative correlation was found in other studies between the content of SOM and the amount of viral adsorption [27,30]. When the amount of viruses adsorbed by soil particles decreased, as caused by organic fertilization, viruses could have more opportunities to reach new host cells [28,31]. Soil pH and SOM can both affect viral community through the microbial hosts. SOM content is closely related to soil fertility levels, and provides the main nutrient and energy sources for soil microorganism. Microbial abundance would increase with a higher SOM content in soil [32]. Soil pH is related to the soil base saturation, which is directly linked with soil nutrients and crop growth [13,33,34]. With pH values between 4 and 8, bacterial abundance and diversity are positively correlated to soil pH [14]. As the optimal growth pH ranges of bacteria are narrow, soil pH has a direct effect on bacterial community composition. Bacteria quantity, as a biotic factor, affects viral survival and production [4,31]. Under some circumstances, the absence of microorganism increased phage survival [9,35], since microorganisms would produce proteolytic enzymes to degrade viral protein capsids [36,37]. In opposite, another study reported the presence of microorganism increased phage survival [38]. In this study, a positive correlation between VLP abundance and bacterial abundance was found (Table 2), which is in accordance with several previous studies regarding a wide range of environments [39]. Our results indicate that the increasing of bacterial quantity might lead to higher viral quantity. Also Williamson et al. [40,41] revealed a clear correlation between bacterial and viral abundance. The content of SOM was much more in organic fertilizing soils, and the pH of soils with organic fertilization (NPKM and M soil) were higher than soils without organic fertilization (CK, N and NPK soil). Thus, viral abundance was much higher in soils with organic fertilization. According to the phage morphological distribution of 5500 known phage isolates, 96% of these viruses are tailed [20]. Filamentous viruses and tailless viruses are rarely reported morphotypes, which accounted for less than 1.5% and 2.0% of the total phages in environmental samples [42]. However, in this study, both filamentous and tailless VLPs could be easily detected under TEM in all 5 soil samples (Fig. 3), which suggests that VLPs distribution in this unique Haplic Acrisol soil was different from that of other environments. According to reciprocal Simpson’s index, viral diversity did not show consistent trend between inorganic fertilization and organic fertilization soils (as shown in Fig. 3). Viral diversities of fertilization soils were greater than those of control soil without any fertilization, which indicates viral diversity could be higher through organic fertilization. 5. Conclusions
Fig. 3. Frequency distributions of viral morphologies from 5 Qiyang agricultural soils. Viral categories include tailless (Tail-), Siphoviridae (Sipho), Myoviridae (Myo), Podoviridae (Podo) and filamentous (Fil). Histograms are based on measured value of 200 randomly selected individual viruses from each soil (3 replications). Five fertilization regimes were chosen for this study, which include control without fertilizers (CK), chemical fertilizers (nitrogen, phosphate, and potassium fertilizer, NPK), single nitrogen fertilizer (N), organic fertilizer (M), and chemical and organic fertilizer (NPKM).
Twenty-two years different fertilization regimes, including the use of chemical and organic fertilizers, changed many soil characteristics as well as the soil viral abundance and morphological diversity. The effects on the viral diversity appear to be affected by the virus type, as demonstrated in this study for the opposite effects of increased SOM on Myoviridae and Podoviridae viruses.
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