Comparison of rhizosphere and endophytic microbial communities of Chinese leek through high-throughput 16S rRNA gene Illumina sequencing

HUANG Yong-hong

College of Horticulture, Qingdao Agricultural University, Qingdao 266109, P.R.China

1. Introduction

Endophytes are microorganisms (bacteria, fungi and unicellular eukaryotes) that can live at least part of their life cycle inter- or intra-cellularly inside of plants, usually without inducing pathogenic symptoms (Murphyet al. 2014).Endophytes can cooperate with their host plant by producing secondary metabolites that can protect the plant, providing the ability to defend against predators and help their hosts to adapt to different stress conditions for survival (Abdalla and Matasyoh 2014). Diazotrophic endophytes of poplar and willow enhanced plant growth under N-limited conditions(Doty 2015). Two new strains of endophytic fungi isolated fromMoringa peregrinaproduced bioactive gibberellic acids(GAs) that increased the shoot length of GA-deficient mutantwaito-cand normal Dongjin-beyo rice seedlings (Khanet al.2014). Fourteen endophytic bacterial isolates from the rhizome ofCurcuma longaL. produced indole acetic acid(IAA) and effectively inhibited the growth ofEscherichia coliandKlebsiella pneumoniae, and fungal strains such asFusarium solaniandAlternaria alternata(Kumaret al.2016). Root endophytes isolated from wild populations ofHordeum murinumssp.murinumincreased grain yield in a nutrient-starved barley (Murphyet al. 2015a) and suppressed seed-borne infections in a barley cultivar (Murphyet al.2015b).Acremoniumsp. endophytes from root galls of tomato infected byMeloidogyne incognitareduced the incidence of root galls byca.60% (Yaoet al. 2015).

Chinese leek (Allium tuberosumRottler ex Sprengel) is a common vegetable in China. It significantly reduced the incidence of banana Fusarium wilt in the field (Huanget al.2012), and reduced the gall index of tomato plants caused byM. incognita(Huanget al.2016). Then researchers isolated several antifungal (Zuoet al.2015) and nematicidal components (Huang 2015). Further studies showed that Chinese leek altered the microbial composition of banana rhizosphere soil (Huanget al. 2012). These results led to an altered hypothesis that antifungal or antibacterial endophytes were associated with the Chinese leek.Bacterial endophytes were isolated that significantly inhibited the mycelial growth ofBotryosphaeria berengerianaandValsa mali, the pathogenic agents of apple ring rot and apple valsa canker, respectively (data not shown).

In this study, the rhizosphere and endophytic bacterial communities of Chinese leek were investigated using highthroughput Illumina sequencing of 16S rRNA genes. And the diversity of bacterial endophytes in the Chinese leek root and leaf was analyzed, then compared with that in Chinese leek rhizosphere soil, which was to identify the potential antifungal and nematicidal microbes.

2. Materials and methods

2.1. Sample collection

The Chinese leek used in the experiment was sampled from Xifu Town, Chengyang District, Qingdao City, China.The Chinese leek cultivar Dajingou was planted in field for 3 years. Then three composite samples were collected.Each composite sample included five clusters of whole plants with soil on root, which were collected randomly and at least 5 m apart from each other. All the collected samples were put into sterile plastic bags and brought to the laboratory.The redundant soil was removed through shaking the roots gently, then the soil sticked on the roots was collected with a brush and sieved with a 2-mm aperture sieve. The soil sticked on the roots was regarded as rhizosphere soil to analyze the rhizosphere bacterial community. Then the plants were washed with running water, then separated roots and leaves were cut into pieces of 2-cm long. The 2-cm long pieces were dipped into 75% ethyl alcohol for 2 min,then immersed into 3% sodium hypochlorite for 5 min, finally washed with sterile water for 5 times. The fifth washing water was coated onto potato dextrose agar (PDA) plate and incubated at 28°C for 7 d to determine the disinfection effect. Disinfection qualified leave and roots were used to analyze the endophytic bacterial community.

2.2. DNA extraction, PCR amplifications and lllumina library generation

Genomic DNA of the rhizosphere soil and the disinfection qualified leave and roots were extracted. The genomic DNA was stored at ?20°C until PCR amplification and metagenomic sequencing was carried out. The 16S rRNA gene consists of nine hypervariable regions flanked by regions of more conserved sequences. Because the V3–V4 region of the 16S rRNA gene provides ample information for taxonomic classification of microbial communities, this region was targeted for sequencing.

PCR amplification was conducted in a GeneAmp PCR System 9700 (Life Technologies, Carlsbad, CA, USA).PCR amplifications were performed using the KAPA HiFi HotStart ReadyMix PCR Kit (KAPA Biosystems,USA). Each reaction (25 μL) contained 12.5 μL 2× KAPA HiFi HotStart ReadyMix, 0.25 μmol L-1of each primer,and 10 ng of DNA template. Bacterial and archaeal 16S rRNA genes were amplified with the universal primer B341F (5′-CCTACGGGNGGCWGCAG-3′) and B805R(5′-GACTACHVGGGTATCTAATCC-3′) (Albertsenet al.2015; Nasrollahzadehet al. 2015) using the following conditions: initial denaturation at 95°C for 3 min, 25 cycles at 95°C for 30 s, 55°C for 30 s and 72°C for 30 s, and a final extension at 72°C for 5 min.

Indexes that allowed sample multiplexing during sequencing were incorporated between the Illumina Miseq adaptor and the reverse primer in the amplification reaction.

PCR amplicon libraries were prepared by combining the PCR products for each sample. After purification, the PCR products from the different samples were quantified using the Agilent 2100 Bioanalyzer System (Santa Clara, CA,USA) and then pooled at equal concentrations. Amplicon sequencing was performed on the Illumina Miseq platform at Beijing Ori-Gene Science and Technology (China).

2.3. Statistical and bioinformatics analysis

Heatmap figures were generated using custom R scripts.Canoco 4.5 was used to run principal component analysis(PCA). The community richness index, community diversity index, data preprocessing, operational taxonomic unit-based analysis and hypothesis tests were performed using Mothur Software (http://www.mothur.org/). The data were analyzed with a one-way ANOVA using SAS ver. 8.0 Software. The significance of the treatments was determined using Fisher’s least significant difference (LSD) test (P≤0.05).

3. Results

3.1. General analysis of the Illumina sequencing data

Using Mothur Software, the short, low-quality and ambiguous sequences were removed and a total of 79 261 high-quality sequences were obtained from the nine samples. The number of high-quality sequences per sample ranged from 4 238 to 15 977 (Table 1). Based on 97% species similarity,1 239 operational taxonomical units (OTUs) were found using USEARCH Software (http://www.drive5.com/usearch/)and 11 930 sequences (15.1% of the total sequences) were returned as unclassified.

3.2. Bacterial community composition

In the nine samples, 28 phyla were identified in total, with 2 unclassified. When the bacterial communities were compared at the phylum level, the bacterial community composition in the rhizosphere samples was different from the endophytic bacterial community composition in the leaf and root samples. All 28 phyla were presented in the rhizosphere soil samples, whereas only 17 of them were presented in the leaf and root samples.

The abundance of phyla in different samples were significantly different. In the rhizosphere soil, the five most abundant phyla were Proteobacteria, Acidobacteria,Bacteroidetes, Cyanobacteria, and Planctomycetes,which accounted for 71.97% of the total abundance phyla.And the percentage of them were 37.85, 10.99, 8.24,7.79 and 7.1%, respectively. In the endophytic bacterial communities, Cyanobacteria and Proteobacteria were the two most abundant phyla, which accounted for 98.17 and 96.48% in the leaf and root samples, respectively. And Cyanobacteria and Proteobacteria accunted for 75.44 and 21.04%, respectively in the leaf samples, 75.44 and 21.04%,respectively in the root samples.

Of the 26 classified phyla, only the abundance of Cyanobacteria in the leaf and root bacterial communities was significantly higher than that in the rhizosphere soil(F=125.32,P＜0.0001). The other 25 phyla in the leaf and root bacterial communities were significantly lower than that in the rhizosphere soil (Fig. 1).

According to the bacterial community abundance, the 26 classified phyla were clustered into 3 groups. All the phyla, belonged to Cyanobacteria, which was the most abundant bacterial phyla in the leaf and root communities,were classfied into one group. And all the phyla, belonged to Proteobacteria, which was the the most abundant bacterial phyla in the rhizosphere soil, were classfied into another group. The other 24 bacterial phyla were classfied into the third group (Fig. 2).

A total of 66 bacterial classes, 119 bacterial orders,and195 bacterial families were identified in total in the nine samples, the most five abundant class, order and family were accounted for 51.43, 29.24, and 29.06% in rhizosphere soil, 98.41, 97.72, and 97.63% in leave and 96.91, 95.15,and 95.05% in roots, respectively (Appendix A).

Two hundred and eighty-four bacterial genera were identified from the nine samples, 131 of which were unclassified. In the rhizosphere soil samples, the abundance of 23 bacterial genera was higher than 1%, accounting for 57.5% of the total. Among these bacterial genera, the four most abundant genera (7.74, 7.03, 6.07 and 4.39%) were unclassified. The five most abundant classified genera were Pseudomonas (3.32%),Flavobacterium(2.45%),Sphingomonas(1.89%),Gemmatimonas(1.81%) andDongia(1.70%). For the endophytic bacterial community in the leaf and root samples, two unclassified bacterial genera accounted for 97.1 and 93.95% of the total community,respectively. Their abundance was significantly higher in the endophytic communities than that in the rhizosphere soil community (Table 2).

Table 1 High-quality sequences that were used for further analysis after basic quality control for the three groups of Chinese leek samples

3.3. Bacterial α-diversity

Diversity parameters, such as the number of sequence analyxed, which were clustered into OTU, and good coverage were not significantly different between bacterial communities in the rhizosphere soil and in the leaf and root,whereas the other parameters were significantly different between them.

The number of OTUs in the rhizosphere soil community was 5.20 and 4.26 times higher than those in the leaf and root communities, respectively. The Chao1 estimator for the rhizosphere soil community was 2.54 and 2.39 times more than that in the leaf and root communities, respectively.Abundance-based coverage estimator (ACE) in the rhizosphere soil community was 1.79 and 1.62 times more than that in the leaf and root communities, respectively.These results showed that richness in the rhizosphere soil bacterial community was more than that in the endophytic bacterial communities.

Fig. 1 The relative abundance of different phyla in rhizosphere soil, leaf and root bacterial communities of Chinese leek. Bars represent the standard error of the three replicates and different letters above each phylum indicate significant differences at 0.05 probability according to the Fisher’s least significant difference (LSD) test.

The Shannon diversity index in the rhizosphere soil community was 6.42 and 4.33 times higher than that in the leaf and root communities, respectively, and the simpson diversity index in the rhizosphere soil community was 50.15 and 37.20 times lower than that in the leaf and root communities, respectively. These results showed that the diversity in the rhizosphere soil bacterial community was more than that in the endophytic bacterial communities.

The rank-abundance curve of the endophytic bacterial communities declined more sharply than that of the rhizosphere soil, which showed that the evenness in the rhizosphere soil bacterial community was higher than that in the endophytic bacterial communities (Appendix B).

Fig. 2 Hierarchical cluster tree of bacterial communities from the rhizosphere, leaf and root of Chinese leek at the phylum level.

Table 2 Significant differences of the bacterial genera among the three communities isolated from Chinese leek1)

3.4. Bacterial community structure

To analyze the relationships among the samples, OTUs were aligned with the SILVA database (https://www.arb-silva.de/)using PyNAST Software (https://sourceforge.net/projects/pynast/), and a phylogenetic tree was constructed using FastTree (Priceet al.2009). UniFrac Software (Lozupone and Knight 2005) was used to generate the distance matrix among bacterial communities. UniFrac analysis was carried out either unweighted, using only presence-absence information (Appendix C), or weighted, which took into account the relative proportions of each group (Appendix D).

The cluster heatmap using the unweighted (Fig. 3-A) or weighted distance (Fig. 3-B) showed that the rhizosphere soil samples grouped together, and the leaf and root samples grouped together. PCA using the unweighted (Fig. 4-A)or weighted distance (Fig. 4-B) also revealed that thefirst principal component separated the soil samples from the leaf and root samples. The cluster heatmap and the PCA confirmed the differences between the rhizosphere soil bacterial community and the endophytic bacterial communities.

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Fig. 3 Heatmap of similarities between the rhizosphere soil bacterial communities and the endophytic bacterial communities of Chinese leek based on the unweighted (A) and weighted(B) UniFrac distance. The color gradient from blue to red indicated increasing similarity.S, rhizosphere soil; L, leaf; R, root.

4. Discussion

High throughput sequencing of 16S rRNA gene fragments has been successfully applied for in-depth analysis of bacterial communities associated with the animal gut (Guoet al. 2016;Jiaet al. 2016), water (Jiaoet al. 2016) and soil (Huaet al. 2015; Wanget al. 2016). In this paper, we adopted this technique to analyze the rhizosphere soil and endophytic (leaf and root) bacterial communities of Chinese leek, to characterize the unseen majority of these bacterial communities and to achieve a high throughput and deep insight into these bacterial communities, providing a basis for the development and utilization of Chinese leek endophytic or rhizospheric bacterial communities.

In the rhizosphere soil, among 26 classified phyla, Proteobacteria (37.85%) was the most dominant phylum across all 3 rhizosphere soil samples, followed by Acidobacteria (10.99%) and Bacteroidetes (8.24%). The results were in agreement with those of previous studies. Sunet al.(2014) found in the apple rhizosphere soil of a replant site and a new planting site, Proteobacteria,Acidobacteria and Bacteroidetes were the three dominant phyla, accounting for almost 50% of the reads (Sunet al.2014). In a banana mono-culture orchard, Proteobacteria,Acidobacteria and Bacteroidetes were the three most abundant phyla at relative abundances of approximately 35,15 and 10%, respectively (Shenet al. 2014). Among the banana rhizosphere microbial community manipulated by 2 years of consecutive biofertilizer application, Proteobacteria accounted for 70.1% of the total bacterial 16S rRNA gene sequences, followed by Bacteroidetes and Acidobacteria(Shenet al. 2015). Proteobacteria (accounting for 35.7–97.4% of species) was also the most abundant phylum in the tomato rhizosphere (Liet al. 2014). The results of these previous studies and this study revealed Proteobacteria was the dominant bacterial phylum in the rhizosphere soil. In the endophytic bacterial community of Chinese leek, Cyanobacteria and Proteobacteria were the two most abundant phyla, accounting for 98.17 and 96.48% of the total community in the leaf and root, respectively. This showed that the composition and abundance of endophytic bacterial communities were different from those of the rhizosphere soil.

In many previous studies, culture-based approaches were used to study microbial communities (Compantet al.2011; Koukolet al. 2012; Venkatachalamet al. 2015), but these methods suffered from organismal bias and were far too labor-intensive to adequately assess highly diverse microbial communities. In addition, in many cases, the vast majority of microbes are uncultivable using traditional methods. Direct DNA clone sequencing avoids culturing bias and has the potential to detect rare taxa, and has made it feasible to capture the often hidden microbial diversity (Masukoet al. 2013). Compared with the traditional culture-based methods, high-throughput sequencing can detect many minor members of the microbial community,many of which are not presented in the existing database(unclassified sequences). In this study, many unclassified sequences/phyla/genera were identified, including: 11 930(15.1%) of 79 261 high-quality sequences were unclassified;2 (7.14%) of 28 identified phyla were unclassified; and 135(52.46%) of the detected 284 genera were unclassified.In the rhizosphere soil, the top 4 genera, accounting for 25.22% of the total abundance, were all unclassified. In the endophytic communities, the top 2 genera were unclassified,which accounted for 97.1 and 93.95% of the total community in the leaf and in the root, respectively. In previous studies,substantial proportions of unclassified sequences were also identified (Shenet al. 2014, 2015; Wuet al. 2016). In a banana orchard soil, 12 845 (9.3% of the total sequences)of 137 646 sequences were unclassified (Shenet al. 2014).Among all bacterial and fungal sequences in banana rhizosphere soil, 4 179 bacterial sequences (4%) and 89 332 fungal sequences (58%) were returned as unclassified(Shenet al. 2015). At the genus level, unclassified bacteria in four fecal microbiota samples of dholes accounted for 39.36, 52.29, 37.34, and 25.66% of the total reads (Wuet al. 2016). The identification of unclassified members of the bacterial community in this study and the previous studies shows the efficiency of high-throughput sequencing in detecting bacteria, especially minor, rare and uncultured species, in a range of samples. In addition, our results showed that there were many unknown members of the rhizosphere soil and endophytic bacterial communities of Chinese leek, indicating the great potential of this resource for development of beneficial bacterial communities.

Fig. 4 Principal component analysis (PCA) of the rhizosphere soil bacterial communities and the endophytic bacterial communities of Chinese leek based on the unweighted (A) and weighted (B) UniFrac distance.

In the α-diversity analysis, parameters evaluating microbial richness such as Chao1, ACE, Shannon,Npshannon,Simpson and the rank-abundance curve revealed that microbial diversity, richness, and evenness were higher in the rhizosphere soil bacterial community than in the endophytic bacterial communities. This might be due to the rhizosphere acts as a bridge connecting plant roots and their surrounding soil environment and it is an important place where complex chemical, physical, and biological interactions occur. Interactions associated with the rhizosphere include root-root, root-insect, and root-microbe interactions (Baiset al. 2006). These complex interactions need more bacteria with different functions to finish, which led to increased diversity, richness and abundance of beneficial soil microbes. Compare with the complicated rhizosphere areas, relatively stable single environment in plants resulted in relatively single microbial population. That is to say, the rhizosphere bacterial community diversity and the relative simplification of the endophytic bacterial community are the results of their environment and functional adaptation.

From the PCA, the three samples from the rhizosphere soil were clustered into one group, and the six samples from the endophytic bacterial communities (leaf and root) were clustered into together, which showed that the rhizosphere soil bacterial community was different from the endophytic bacterial communities. Although differences existed between the two types of bacterial communities,the abundances of the major components of the bacterial communities were similar, that was, Proteobacteria and Cyanobacteria were the two most abundant bacterial phyla in rhizosphere soil and in the endophytic bacterial communities, which was in agreement with the following studies. Endophytic community composition isolated from four different teosintes and ten different maize varieties varied in relation to plant host phylogeny, however, a core microbiota of endophytes was conserved inZeaseeds across boundaries of evolution, ethnography and ecology(David and Raizada 2011).

Chinese leek has antifungal (Huanget al. 2012) and nematicidal activities (Huanget al. 2016), which were got by our research program. In this paper, we also analyzed the rhizosphere soil bacterial community and endophytic bacterial communities. The finding of this study would contribute to the effort to isolate and identify the nematicidal and antifungal bacterial communities in Chinese leek.

5. Conclusion

Illumina sequencing of 16S rRNA genes of rhizosphere soil, leaf and root bacterial communities revealed that the rhizosphere bacterial community was significantly different from the endophytic bacterial communities. Microbial diversity, richness and evenness in the rhizosphere soil bacterial community were higher than those in the endophytic bacterial communities. The bacterial community was composed of five dominant phyla (Proteobacteria,Acidobacteria, Bacteroidetes, Cyanobacteria and Planctomycetes) in the rhizosphere and two dominant phyla (Cyanobacteria and Proteobacteria) in the root and leaf. The endophytic bacterial communities from the leaf and the root were slightly, but not significantly different from each other. The findings of this study will contribute to the goal of isolation and identification of the nematicidal and antifungal bacterial communities in Chinese leek.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31471864 and 31272151) and the Qingdao Agricultural University High-level Personnel Startup Fund, China (6631115024). We thank Beijing Ori-Gene Science and Technology (China) for providing the Illumina Miseq platform.

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