Microb Ecol (2014) 68:794–806 DOI 10.1007/s00248-014-0461-y
PLANT MICROBE INTERACTIONS
Bacterial Diversity in the Rhizosphere of Cucumbers Grown in Soils Covering a Wide Range of Cucumber Cropping Histories and Environmental Conditions Yongqiang Tian & Lihong Gao
Received: 29 April 2014 / Accepted: 4 July 2014 / Published online: 16 July 2014 # Springer Science+Business Media New York 2014
Abstract Rhizosphere microorganisms in soils are important for plant growth. However, the importance of rhizosphere microorganisms is still underestimated since many microorganisms associated with plant roots cannot be cultured and since the microbial diversity in the rhizosphere can be influenced by several factors, such as the cropping history, biogeography, and agricultural practice. Here, we characterized the rhizosphere bacterial diversity of cucumber plants grown in soils covering a wide range of cucumber cropping histories and environmental conditions by using pyrosequencing of bacterial 16S rRNA genes. We also tested the effects of compost addition and/or bacterial inoculation on the bacterial diversity in the rhizosphere. We identified an average of approximately 8,883 reads per sample, corresponding to around 4,993 molecular operational taxonomic units per sample. The Proteobacteria was the most abundant phylum in almost all soils. The abundances of the phyla Bacteroidetes, A c t i n o b a c t e r i a , F i r m i c u t e s, A c i d o b a c t e r i a , a n d Verrucomicrobia varied among the samples, and together with Proteobacteria, these phyla were the six most abundant phyla in almost all analyzed samples. Analyzing all the sample libraries together, the predominant genera found were F l a vo b ac t e r i um, O h t a e k w a n g i a , Opi tut us , Gp6 , Steroidobacter, and Acidovorax. Overall, compost and microbial amendments increased shoot biomass when compared to untreated soils. However, compost addition decreased the Electronic supplementary material The online version of this article (doi:10.1007/s00248-014-0461-y) contains supplementary material, which is available to authorized users. Y. Tian (*) : L. Gao (*) Beijing Key Laboratory of Growth and Developmental Regulation for Protected Vegetable Crops, Department of Vegetable Science, College of Agricultural and Biotechnology, China Agricultural University, No. 2 Yuanmingyuan Xilu, Beijing 100193, China e-mail:
[email protected] e-mail:
[email protected]
bacterial α-diversity in most soils (but for three soils compost increased diversity), and no statistical effect of microbial amendment on the bacterial α-diversity was found. Moreover, soil amendments did not significantly influence the bacterial β-diversity. Soil organic content appeared more important than compost and microbial amendments in shaping the structure of bacterial communities in the rhizosphere of cucumber.
Introduction Microbiota in the rhizosphere soils is generally different from that in bulk soil because growing roots provide niches for rhizosphere microorganisms [1, 2]. However, since many microorganisms associated with plant roots cannot be cultured [3] and the microbial diversity is enormous in the rhizosphere [4], it is still a challenge in both plant science and soil microbial ecology to relate microbial diversity to plant growth. The complex plant-associated microbial community is referred as the second genome of the plant since it is crucial for plant health [4]. Microorganisms in the rhizosphere can be endophytic, epiphytic, or closely associated and can develop in beneficial, neutral, or detrimental effects on plant growth [5]. Although most microorganisms in the rhizosphere may not directly interact with their host plant [6, 7], their effects on soil biogeochemical processes may have impacts on plant growth [8]. Plant-associated microorganisms can protect plants from pathogens [9], confer plant heat and/or salt tolerance [10], and improve plant growth by degrading plantproduced compounds that would otherwise be allelopathic or even autotoxic [1]. In addition, rhizosphere microorganisms often provide their host plants with critical nutrients because soil minerals generally present as unavailable forms to plants [1] and need to be mineralized by microorganisms into available forms [11]. The plant, in turn, influences microorganisms including those not touching roots through its root
Bacterial Diversity in the Rhizosphere of Cucumbers Grown in Soils
exudates in the rhizosphere soils [12, 13], leading to plant species-specific microbiome [4]. In addition, plant can also stimulate its microbiome through regulating the biotic and/or abiotic environments [14]. Microbial communities in the rhizosphere reflect plant cover type and cropping history [15–19]. For instance, Turner et al. [17] demonstrated that there were profound differences in the rhizosphere microbiome, particularly at the kingdom level between plants. A recent study even found that in the plant rhizosphere, a small but significant fraction of variation in microbial diversity could be attributed to host genetics [18]. Based on the results of these studies, rhizosphere microbial diversity can be partly influenced by the cropping history. For example, short rotations or monoculture provide soil microorganisms a near-continual supply of host materials, allowing some microorganisms continually infect or colonize the root of same crop [19]. It should be noted, however, that the rhizosphere microbial diversity can also be influenced by both the physical and chemical properties of the rhizosphere, some of which are determined by soil types [20, 21] and/or agricultural practices [21, 22]. In particular, soil bacterial biogeography has been found to be controlled primarily by edaphic variables [18, 23, 24]. For instance, Fierer and Jackson [23] demonstrated that the differences of soil bacterial communities among different ecosystem types could be largely explained by soil pH. Understanding interactions between microbiota and their host plants growing in soils with different environmental conditions under different agricultural practices and identifying the plant and soil factors controlling these interactions could be helpful in both plant science and soil microbial ecology to relate microbial diversity to plant growth. Cucumber (Cucumis sativus L.) is one of the most economically significant vegetable crops in the world, as well as a model system for sex determination studies and plant vascular biology [25]. Cucumber root exudates, such as amino acids, organic acids, sugars, phenolics, polysaccharides, and proteins [1, 26], can interact with soil microorganisms [27, 28]. For example, cucumber is a well-known allelopathic plant producing secondary metabolites that not only inhibit the growth of conspecific plants (i.e., autotoxicity) [26] but also may promote the incidence of microbial pathogens (e.g., Fusarium wilt pathogen) [27]. In addition, microbial diversity in cucumber rhizosphere can be substantially influenced by soil conditions and agricultural practices [29, 30]. Despite the agricultural and biological importance of cucumber, knowledge of cucumber-associated rhizosphere microbiome is currently very limited. To data, the rhizosphere microbiome of mature cucumber plants growing in field soils remain poorly characterized, and many of the roles and interactions of the soil conditions and agricultural practices remain to be elucidated. Here, we investigated 12 different soils covering a wide range of cucumber cropping histories and environmental
795
conditions from Northern China. In China, monoculture is a common practice in cucumber production. This agricultural practice generally can result in soil degradation and eventually lead to cucumber yield decline. Many agricultural practices have been suggested and evaluated to improve soil quality and to overcome this yield decline. One effective practice is to amend soil with organic composts. Organic composts improve the overall soil environment (including microbial environment) so that it is better able to enhance crop growth [19]. This benefit has been sufficiently proven. However, since most organic composts must be formulated to improve their physicochemical properties, there is some limitation in the capacity of composts to improve soil microbial environment. Thus, the addition of biological control agents (e.g., Bacillus subtilis used in this study) to composts is a viable strategy to enhance plant growth when compost is used to amend soils [31]. However, few studies look at the interaction between microbial treatments and soil quality in terms of microbial communities. We therefore treated all soils with/without compost addition and/or bacterial inoculation and then tested their effects on the cucumber rhizosphere bacterial microbiota, which was characterized by pyrosequencing 16S rRNA gene amplicons. The aims of this study were (1) to describe the characteristics of bacterial communities in cucumber rhizosphere soils and understand the forcing environmental and artificial factors that shape bacterial diversity and (2) to detect the main taxa that can be considered as “indicator species” for cucumber plants. The results of this study show that overall plants growing in soils treated with compost or microbial amendments had higher shoot biomass than plants growing in untreated soils; however, soil organic content appeared more important than compost and microbial amendments in shaping the structure of bacterial communities in the rhizosphere of cucumber.
Materials and Methods Site Description and Experimental Design Soils from twelve diverse fields located in Northern China were selected to cover a wide range of cucumber cropping histories (i.e., from 1 to 22 years) and soil types (e.g., pH 5.87–7.52, organic matter 9.1–29.3 g kg−1). A detailed description of each soil and compost was given in Table S1. In February 2012, all soils were treated with zero or more of the following amendments to create treatments that included (1) the untreated soil (soil treatment (S), control), (2) 6 % compost (soil plus compost treatment (SC)), (3) 6 % compost plus 109 spores of B. subtilis per liter soil (soil plus compost plus inoculation treatment (SCI)), and (4) 109 spores of B. subtilis per liter soil (soil plus inoculation treatment (SI)). The compost was prepared from plant straw and chicken manure (for
796
more information, see Table S1). B. subtilis (isolate LY-A) was obtained from China Center of Industrial Culture Collection, the member of World Federation for Culture Collections. This strain was isolated from a cucumber soil based on its ability to adhere to cucumber root and was selected according to its ability to inhibit growth of Fusarium oxysporum mycelia in vitro, as determined by dual-confrontation tests. Preparation of this strain was described by Chung et al. [31]. Cucumber (C. sativus cv. “Zhongnong No. 16,” Beijing, China) seeds were surface sterilized as described in Ofek et al. [32] and were germinated and grown in PVC pots (25-cm diameter, 30-cm height, one plant per pot) filled with 5 kg of soil for 80 days in a greenhouse with a 10:14-h light/dark cycle, and the mean temperature was kept at 26 °C during the day and 15 °C during the night, with 80 % relative humidity. The experiment was a completely randomized block design with four replicates. Cucumber plants were harvested 80 days after treatments, when rhizosphere soil samples were collected for analyses. Plants were carefully removed from the soil using a small sterilized drain spade, and the root systems were vigorously shaken to dislodge loosely adhering soil, which was discarded. The root-adherent soil particles (
