High-throughput sequence analysis reveals variation in the relative abundance of components of the bacterial and fungal microbiota in the rhizosphere of Ginkgo biloba

Background The narrow region of soil, in contact with and directly influenced by plant roots, is called the rhizosphere. Microbes living in the rhizosphere are considered to be important factors for the normal growth and development of plants. In this research, the structural and functional diversities of microbiota between the Ginkgo biloba root rhizosphere and the corresponding bulk soil were investigated. Methods Three independent replicate sites were selected, and triplicate soil samples were collected from the rhizosphere and the bulk soil at each sampling site. The communities of bacteria and fungi were investigated using high-throughput sequencing of the 16S rRNA gene and the internal transcribed spacer (ITS) of the rRNA gene, respectively. Results A number of bacterial genera showed significantly different abundance in the rhizosphere compared to the bulk soil, including Bradyrhizobium, Rhizobium, Sphingomonas, Streptomyces and Nitrospira. Functional enrichment analysis of bacterial microbiota revealed consistently increased abundance of ATP-binding cassette (ABC) transporters and decreased abundance of two-component systems in the rhizosphere community, compared to the bulk soil community. In contrast, the situation was more complex and inconsistent for fungi, indicating the independency of the rhizosphere fungal community on the local microenvironment.

239 relationship network of these genera indicated a complex functional collaboration within the 240 microbiota. To analyze the functional diversities of bacteria, the KEGG functional enrichment 241 analysis of bacterial microbiota was compared between the rhizosphere and the bulk soil. The 242 frequency of ATP-binding cassette (ABC) transporters was enriched significantly in the 243 rhizosphere while the frequency of the two-component system decreased significantly in the 244 rhizosphere ( Figure S10). This indicated a functional divergence of bacterial microbiota in 245 response to the rhizosphere of ginkgo roots. 246 247 Discussion 248 With the development of the ginkgo-based pharmaceutical industry and of ginkgo horticulture, it 249 is increasingly important to fully understand the different aspects of ginkgo biology, including 250 the relationship with its root microbiota. Most of the previous research on ginkgo had focused on 251 the biosynthesis pathways of various bioactive compounds present in ginkgo leaves, which are 252 raw materials for the pharmaceutical industry. On the other hand, little research has been carried 253 out on ginkgo roots. To our knowledge, this research represents the first report on the 254 relationship between ginkgo roots and the soil microbiota.

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In this study, the 16S rDNA in bacteria and the ITS rDNA sequences in fungi were amplified 256 and sequenced. We did not find the contaminant sequences from chloroplast, mitochondrial or 257 nuclear DNA, which have frequently occurred in related research (Beckers et al. 2016). This 258 finding showed that our PCR approach was optimized and suitable for this research. Considering 259 the complex nature of the soil environment, which may cause changes in microbiota 260 composition, we chose three different and independent sites for sample collection. Only those 261 changes which occurred in all three sites were discussed.

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One of the important findings in this study was the accumulation in the rhizosphere of species 263 of Rhizobium and its fellow rhizobial genus Bradyrhizobium. Rhizobium and Bradyrhizobium are 264 nitrogen-fixing bacteria which induce the development of nodules in the roots of legume plant 265 hosts (Long 1996). In addition, flavonoids are widely accepted to be regulators of symbiotic 266 interactions, acting as specific signals between plant hosts and Rhizobium (Mierziak et al. 2014). 267 Considering that flavonoids are one of the most important groups of secondary plant metabolites 268 in ginkgo (Kleijnen & Knipschild 1992), it is possible that ginkgo root cells secrete flavonoids, 269 which act as specific signals to attract the accumulation of Rhizobium and Bradyrhizobium in the 270 rhizosphere. However, despite the accumulation of Rhizobium and Bradyrhizobium in the 271 rhizosphere, we did not find any nodules on ginkgo roots. This might be due to the lack of Nod 272 factor receptors or to defects of the subsequent kinase cascade in ginkgo, which is crucial to , it would be interesting to identify the missing 275 steps in ginkgo which are associated with its inability to form nodules. 276 We also observed an accumulation of Sphingomonas in the ginkgo rhizosphere. Bacteria 277 within the genus Sphingomonas share the common capacity to degrade a broad range of aromatic 278 compounds (Fredrickson et al. 1995). Thus, the accumulation of Sphingomonas in the 279 rhizosphere indirectly suggested the secretion of different aromatic secondary metabolites from 280 ginkgo roots, which attracted the accumulation of aromatics-consuming Sphingomonas species.

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Streptomyces is the largest genus of the Actinobacteria, with more than 500 species having 282 been described (Euzeby 2008). Streptomyces not only produces a volatile metabolite, geosmin, 283 which result in the distinct "earthy" odor of soil, but also produces antibiotics, which they use to 284 compete with other bacteria for resources. A number of them have been developed as 285 antifungals, antibiotics and chemotherapeutic drugs to improve human health (Raja & 286 Prabakarana 2011). The benefits to ginkgo of Streptomyces accumulation in the rhizosphere are 287 currently unknown, but this phenomenon could increase the complexity of the composition of the 288 microbiota in the rhizosphere.

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In contrast to those bacterial genera which accumulated in the ginkgo rhizosphere, there were 290 also some genera which decreased in the rhizosphere compared to the bulk soil. The genus 291 Nitrospira consists of a group of species which are widely distributed in many natural 292 environments (Bartosch et al. 2002), and they are considered to play important roles in the 293 nitrogen cycle in both water and soil (Hayatsu et al. 2008). Despite the potential advantage of an 294 exogenous nitrate supply to ginkgo, Nitrospira did not accumulate in the rhizosphere but rather 295 was present at lower frequencies in the rhizosphere than in the bulk soil. This decline may due to 296 the inhibitory effects of antibiotics produced by other rhizobacteria (Streptomyces, for instance), 297 which accumulated in the rhizosphere. Alternatively, the decline may be caused by the complex 298 secondary metabolites secreted by ginkgo roots. It has been reported that flavonoids, secreted by 299 root cells, had both positive and negative effects on nodule formation by nitrogen-fixing bacteria  (Hoch 2000). For the bacteria in the rhizosphere, the frequency of 312 two-component systems decreased compared to that in the bulk soil. We propose that bacteria 313 must cope with different environmental stimuli in the soil. For the bacteria in the rhizosphere, the 314 microenvironment is greatly affected by the plants. These bacteria are partially "protected" by 315 the rhizosphere, and there is no need for these bacteria to employ numerous two-component 316 systems to cope with the different challenges from the ever-changing environment as would be 317 the case for the bacteria in the bulk soil.
Compared with the clear changes in bacterial frequency between the rhizosphere and bulk soil, 319 the responses of fungal frequency between the two soil types varied between the different 320 collection sites. Many fungal genera accumulated in the rhizosphere of one collection site, yet 321 decreased at other collection sites ( Figure S8). Considering the different and complex subcellular 322 structures of fungi, it is possible that the substances secreted by root cells and bacteria have 323 relatively little effect on the distribution of the fungal microbiota.