Pyrosequencing technology reveals the impact of different manure doses on the bacterial community in apple rhizosphere soil
Introduction
Apple trees are among the most important fruit trees in the world. China has the highest apple tree acreage (2.056 million hectares, 43.78% of the world's supply; FAOSTAT, 2010) and production (33.26 million tons, 47.86% of the world's supply; FAOSTAT, 2010). Northwest China and the Bohai Bay region are two of the leading apple-producing areas in China. Because many apple orchards are located in arid and semi-arid areas, the key problem for apple production is poor quality soil due to low soil organic matter (SOM) content. SOM can facilitate water entry, resist surface structure degradation and absorb minerals and nutrients (Fahey et al., 2011), all of which significantly impact fruit yield and quality (Weil and Magdoff, 2004). The average SOM content of apple orchard soil is 1.26% in northwest China and 1.14% in the Bohai Bay region, including Beijing (Li et al., 2007). This is much lower than the SOM content of apple orchard soil in the U.S. and Europe, which is approximately 2% (Zoppolo et al., 2012). Due to the low SOM content, manure is commonly used to improve the soil in many apple orchards in China. In recent years, due to the public's increasing concern about food safety, organic fruit production has been quickly increasing. In 2011, apples managed under certified organic farming systems accounted for approximately 6% of total U.S. farms (Slattery et al., 2011). Meanwhile, organic farming is in its infancy in China. Soil quality within apple orchards is the key factor hindering the conversion to organic management, as it does not allow for the use of chemical fertilizers and requires the use of more manure. In China, poultry manure is the most commonly used fertilizer in apple orchards because of its low cost and availability. However, the chosen quantity of manure is generally dictated by a farmer's experience rather than well-defined and quantitative criteria.
Different types and doses of fertilizers both raise the soil nutrient level and have a significant impact on soil microbes, including their biomass, activity and diversity. A previous study on paddy soil showed that the addition of organic manure strengthened the bacterial and fungal populations and their functional diversity but impoverished those of actinomycetes. Organic manure treatment also decreased the ratio of Gram-positive to Gram-negative bacteria, while chemical fertilizer showed less influence on soil microbes (Zhang et al., 2012). Apple orchard soil is a complex ecosystem that often harbors a diverse bacterial community that can be affected by tillage methods, microbicide application and soil fumigation (Peck et al., 2011, Yim et al., 2013). Microorganisms are key players in the cycle of nutrients in the soil as they represent soil quality and ecosystem sustainability (Verstraete and Mertens, 2004, Tipayno et al., 2012) and can affect soil nutrient recycling and physical properties (Young and Crawford, 2004). Thus, characterizing the ecological status of the microbial community within the soil is necessary to determine the long-term effects of changing soil quality.
Molecular biology-based studies on the ecology of soil microbes have used different testing methods. Yao et al. (2006) reported that soil fumigation and compost amendments in an apple replant site altered the soil microbial community composition but did not improve tree growth or yield. However, the microbial information from polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) analysis reflected only major strains (Fujimoto et al., 2003). Tipayno et al. (2012) characterized changes in soil bacterial communities in response to metal and metalloid contamination and initial phytoremediation using terminal restriction fragment length polymorphism (T-RFLP). Their results suggested that certain bacteria have potential applications in bioremediation, but it should be noted that T-RFLP analysis is based on simulated restriction enzyme digestion from a known molecular database rather than direct sequencing. Pyrosequencing is a new DNA sequencing method developed by Ronaghi et al. (1998) that differs from classical Sanger sequencing in that it relies on the detection of pyrophosphate release on nucleotide incorporation rather than chain termination with dideoxynucleotides. This method allows for the sequencing of a single strand of DNA by synthesizing the complementary strand one base pair at a time and detecting which base was actually added at each step until the DNA sequence of the single stranded template is determined. Because single stranded DNA is used as a template, pyrosequencing could be employed to sequence DNA mixtures isolated from environment samples. Compared with T-RFLP, pyrosequencing may provide more detailed information about the community because every read is sequenced and because the identification of individual species is more accurate (Lee et al., 2010). Given these advantages, pyrosequencing has been frequently used in two recent studies on soil microbial community structure (Lauber et al., 2009, Roesch et al., 2007). However, few pyrosequencing studies have been performed on soils in fruit orchards, and studies in apple orchard soil are uncommon.
In an attempt to further our understanding of the influences of manure application, we used pyrosequencing to investigate changes in the bacterial community within loam soil in apple orchards following different applications of manure contents. Understanding how manure influences the soil is crucial in regulating orchard soil to ensure high apple quality and yield. We used a microbial ecology view to determine the optimal amount of manure application for apple orchard loam soil. We also tested urease, saccharase and cellulase activity of soil because soil enzymatic activities have been related to the microbial community structure and have been considered for use as soil quality indicators.
Section snippets
Soil sampling sites and collection
In spring 2009, a one-year-old “Fuji” apple (’Red Delicious’ × ‘Ralls Genet’) nursery was planted with SH40 (Malus honanensis) as a dwarfing interstock and Malus hupehensis Var. Pingyiensis Jiang as a base stock. Each tree was planted into a transparent Plexiglas box (40 cm × 40 cm × 40 cm), and 10 replicates were planted per treatment. General use poultry manure (organic matter: 35.1%, total N: 1.82%, P2O5: 3.7%, K2O: 1.97%) was purchased from a local supplier, and different ratios of manure (0%, 5%,
Soil properties and enzyme activity
Three years after the manure amendment, the total nitrogen and organic matter contents of the apple rhizosphere soil correlated with manure ratio (Table 1). Available N, available P, available K, available Fe, and available B also showed an increasing trend with manure ratio, while little difference between treatments was found in available Ca, cation exchange capacity and pH. However, the soil enzyme activity followed a bell-shaped trend. Soil urease activity was highest with 10% manure
Discussion
Urease and saccharase were significantly affected by manure addition. Our results show that urease activity increased following manure addition when the ratio was below 15%, but in the 20% and 25% manure treatments, the urease activities decreased sharply despite the higher content of organic matter. Urease catalyzes the hydrolysis of urea to produce ammonia and carbamate and is thus recognized as important indicator of soil health. Previous reports showed that urease content exhibited a highly
Conclusions
Our study results indicate that manure application has a great impact on soil properties and bacterial communities and that an overdose of manure maintains different bacterial community structures and decreases enzymatic activity and plant growth. This study of the bacterial community investigated apple orchard soil quality and demonstrated that 10% manure refinement resulted in more growth mass and a significantly different microbial community pattern and was optimal for apple orchards in loam
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