Roots from Distinct Plant Developmental Stages Are Capable of Rapidly Selecting Their Own Microbiome Without the Inﬂuence of Environmental and Soil Edaphic Factors

Soil Biology & Biochemistry 89 (2015) 206e209

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Soil Biology & Biochemistry

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Short communication Roots from distinct plant developmental stages are capable of rapidly selecting their own microbiome without the inﬂuence of environmental and soil edaphic factors

Jun Yuan a, b, 1, Jacqueline M. Chaparro b, 1, Daniel K. Manter c, Ruifu Zhang a, * ** Jorge M. Vivanco b, , Qirong Shen a, a Jiangsu Key Lab of Organic Solid Waste Utilization, Jiangsu Collaborative Innovation Center for Organic Solid Waste Utilization, Nanjing Agricultural University, Nanjing, 210095, China b Department of Horticulture and Landscape Architecture and Center for Rhizosphere Biology, Colorado State University, Fort Collins, CO, 80523, USA c USDA, Agricultural Research Services, Soil-Plant-Nutrient Research Unit, Fort Collins, CO, USA article info abstract

Article history: Soil microbes live in close association with plants and are crucial for plant health and fitness. Recent Received 19 March 2015 literature revealed that specific microbes were cultured at distinct developmental stages of Arabidopsis.It Received in revised form is not clear how fast the roots, depending on their developmental stage, can alter the root-associated 10 July 2015 microbiome. In this study, Arabidopsis, grown under sterile conditions at precisely distinct develop- Accepted 14 July 2015 mental stages were supplied with a soil microbial slurry. Within four days, roots selected specific mi- Available online 26 July 2015 croorganisms depending on plant development, and Proteobacteria among other bacterial groups were found to colonize the roots irrespective of developmental stage. Moreover, exposure to a microbiome Keywords: Arabidopsis thaliana resulted in modulation of phytohormone levels at different stages of Arabidopsis. © Developmental stages 2015 Elsevier Ltd. All rights reserved. Microbiome Phytohormone

Plants and soil beneficial microbes interact closely for disease (Chaparro et al., 2013, 2014). Similarly, the rhizosphere microbiome suppression, health, and nutritional aspects (Chaparro et al., 2012; identity of maize is altered by growth stage; Massilia, Fla- Lundberg et al., 2012). The rhizosphere microbial community is vobacterium, Arenimonas, and Ohtaekwangia were relatively abun- influenced by the physical and chemical properties of the soil dant at early stages, while Burkholderia, Ralstonia, Dyella, (Gottel et al., 2011; Schreiter et al., 2014), as well as by the genetics Chitinophaga, Sphingobium, Bradyrhizobium and Variovorax pop- of the host plant (Aira et al., 2010; Peiffer et al., 2013; Li et al., 2014). ulations were dominant at later stages (Li et al., 2014). Based on this Recently, it has been shown that distinct developmental stages of information, we could only say that at the time points when those Arabidopsis can culture specific microbial members of the rhizo- samples were taken the microbiome showed those characteristics; sphere microbiome. For instance, in the early stages (seedling and however, it is not clear how fast the microbial community is vegetative) Arabidopsis increases transcription of genes related to N responding to root growth and root exudate changes associated cycling in Nitrobacter, Rhodospirillum, Nitrosospira, Mesorhizobium, with developmental stage, and if concomitant changes in the soil and Azorhizobium, while in the later stages (bolting and flowering) matrix are also important for the interaction. The aim of this study the plant significantly induces transcription of genes belonging to was to determine the root's direct contribution in configuring the plant growth promoting rhizobacteria (PGPRs) such as Bacillus and microbiome irrespective of environmental and soil edaphic factors Burkholderia as well as Cyanothece spp. and Bradyrhizobium at different developmental stages. The experimental system consisted of growing Arabidopsis thaliana (Col-0) plants to different developmental stages (seedling, vegetative, and bolting) in sterile liquid medium (without the * Corresponding author. addition of sucrose). Once the desired plant developmental stage ** Corresponding author. E-mail addresses: [email protected] (J.M. Vivanco), [email protected]. was reached, the plants were transferred to new vessels and cn (Q. Shen). amended with a soil slurry (filtered and unfiltered) derived from a 1 Both authors contributed equally to this paper. http://dx.doi.org/10.1016/j.soilbio.2015.07.009 0038-0717/© 2015 Elsevier Ltd. All rights reserved. J. Yuan et al. / Soil Biology & Biochemistry 89 (2015) 206e209 207 native Arabidopsis soil which has previously been characterized by community in a short period of time. PCoA separated the com- Broeckling et al. (2008) and utilized in numerous experiments (Li munities associated with each developmental time point along et al., 2010; Badri et al., 2013; Chaparro et al., 2013, 2014). The PCoA 2 (Fig. 2A). Furthermore, we observed that the communities soil slurries were also added to no plant controls (media only) to changed in a successive manner as the plants developed. Seedling determine the potential role of the growing root on microbial and vegetative microbial communities were similar to each other, growth. Plants (and no plant controls) were exposed to the soil whereas the microbial community associated with bolting was slurry for a period of four days and roots/media were subsequently distinct (Fig. 2A). This phenomenon was also observed when Ara- collected and colony-forming units (CFU) of the active culturable bidopsis was grown in soil (Chaparro et al., 2014). The microbial microbial community were measured using a gradient dilution communities associated with the root were predominantly (James, 1990). The treatments amended with non-filtered soil comprised of Proteobacteria (Fig. 2B), which are consistently re- slurry showed bacterial growth as measured by CFU, while no CFUs ported to be associated with the rhizosphere microbial commu- were present in the no plant control (MS medium) or filtered soil nities of Arabidopsis (Bulgarelli et al., 2012; Chaparro et al., 2014) slurry treatments. Samples from plants at bolting stage (roots and and other plant species (Gottel et al., 2011; Peiffer et al., 2013; corresponding media) had significantly higher (p < 0.05, one-way Turner et al., 2013; Chaparro et al., 2014), and appear to use plant ANOVA, Tukey HSD post-hoc) bacterial growth as measured by cell-wall features to colonize the roots (Bulgarelli et al., 2012). CFUs per three plants or per 1 mL media (6.3 ± 1.8 Â 105 and Furthermore, the abundance of bacterial families within Proteo- 8.67 ± 2.01 Â 106) (data showed as means ± SD) compared to the bacteria belonging to Burkholderiaceae, Oxalobacteraceae, Rhizo- seedling (3.5 ± 0.51 Â 105 and 4.18 ± 1.75 Â 106) and the vegetative biaceae, Bacillaceae_1, Micrococcaceae, and Pseudomonadaceae were (3.7 ± 0.73 Â 105 and 5.89 ± 0.54 Â 106) stages, as well as the slurry significantly higher in the presence of a plant than in the initial soil which was added to liquid MS without plant (3.00 ± 1.08 Â 103). As slurry used as an inoculum (Supplemental Fig. S1). This indicates expected, these results are indicative of the importance of plants to that the plant selectively attracts these bacterial families to colonize promote microbial growth as the treatment with media alone its roots. The increased abundance of these bacterial families in the sustained significantly (p < 0.05, one-way ANOVA, Tukey HSD post- soil have also been associated with soil disease suppression hoc) lower microbial growth when compared to the treatments (Mendes et al., 2011). In contrast, Sphingomonadaceae, Gemmati- where a plant was present. monadaceae, Acidobacteria_Gp4_family_incertae_sedis, and unclas- We then examined the composition of the microbiome at each sified groups within Proteobacteria significantly decreased in developmental stage of Arabidopsis. Total RNA from the plant roots abundance in the presence of the plant (Supplemental Fig. S1). It that were exposed to native Arabidopsis soil slurry for a period of 4 should be noted that some of the root-associated microbes iden- days was isolated, subsequently converted to cDNA, amplified using tified in this study could be endophytes such as Actinocorallia sp. the dual indexed bacterial 16S rRNA V4 universal primers, and (Bulgarelli et al., 2012). However, due to our experimental set up subjected to Illumina MiSeq sequencing (Kozich et al., 2013) (see the root associated microbes and the endophytes were extracted Supplementary Material). Raw sequences were analyzed using together. Interestingly, Actinocorallia sp. was not found in our study. Mothur v1.32.0 (Schloss et al., 2009; Kozich et al., 2013). Sequences Root-associated microbial communities at each plant develop- were normalized to 22,100 sequences per sample in order to ensure mental stage were also distinct from one another. Specifically, at that differences in sequencing depth did not bias our analyses the phylum level, the microbial community associated with the (Lozupone et al., 2011). We also quantified the copy number of 16S seedling stage was significantly (p < 0.05, one-way ANOVA Tukey rRNA in each cDNA sample using qPCR of V1eV3 region of 16S rRNA HSD post-hoc) more abundant in Firmicutes when compared to the (see Supplementary Material) and the results showed the same vegetative and bolting stages (Fig. 2D), while the seedling stage was trend as with the CFU data; seedling stage showed the lowest significantly less abundant (p < 0.05, one-way ANOVA Tukey HSD (p < 0.0005, one-way ANOVA, Tukey HSD post-hoc) copy number of post-hoc) in Proteobacteria when compared to the vegetative and 16S rRNA (Fig. 1). Principal coordinates analysis (PCoA) of the mi- bolting stage (Fig. 2C). A closer look at the family level showed that crobial community revealed that the microbiome of the slurry Bacillacea_1 was responsible for the increase in Firmicutes at the alone was significantly different from those found in the roots at seedling stage (Supplemental Fig. S2F). Whereas, Bradyrhizobia- different developmental stages (AMOVA, Bonferonni adjustment, ceae, Comamonadaceae, and Rhizobiaceae were responsible for the p < 0.008) (Fig. 2A) indicating that the root selected members of the increase in abundance of Proteobacteria at late developmental stages (Supplemental Fig. S2A,C and E). Interestingly, Burkholder- iaceae and Erythrobacteraceae did not follow the trend observed by Proteobacteria (Supplemental Fig. S2B and D). Plant growth promoting rhizobacteria (PGPRs) are known to influence plant growth and defense by affecting hormonal and signaling processes. For instance, Luteibacter rhizovicinus MIMR1 (Guglielmetti et al., 2013) among other PGPRs (Hoffman et al., 2013) release indole-3-acetic acid (IAA) and its homologues into the rhizosphere promoting root and plant growth. Other PGPRs such as Bacillus pumilus SE34 and Pseudomonas fluorescens 89B61, increase plant defense by the induction of endogenous levels of salicylic acid (SA) or jasmonic acid (JA) (Yan et al., 2002). We tested plant phytohormone levels after plant exposure to soil slurries to determine whether bacteria colonizing the roots could provide any ef- fects on the plant and some trends were observed (Fig. S3). For example, plants showed significantly increased (p < 0.05, two-way ANOVA, Tukey post-hoc) levels of SA and JA when exposed to the Fig. 1. Real-time PCR quantification of 16S rRNA gene. The template used in qPCR non-filtered soil slurry (Fig. S3D). Furthermore, when the plants experiment was cDNA made from 200 ng RNA. The lower case letters indicate con- trasts that are significantly different (P < 0.05) among treatments. Each sample was aged, from vegetative to bolting, the levels of JA in the plant tissue measured in triplicate. Bars in this figure represent mean ± standard deviations. significantly decreased (p < 0.05, two-way ANOVA, Tukey post-hoc) 208 J. Yuan et al. / Soil Biology & Biochemistry 89 (2015) 206e209

Fig. 2. (A) Principal coordinates analysis (PCoA) of the pairwise community dissimilarity (BrayeCurtis index) of the microbial community (control [slurry], seedling, vegetative, and bolting) analyzed by MiSeq sequencing. (B) Relative abundance (%) of the major bacterial phyla present in the root-associated microbial community. * indicate statistically significant differences between the root associated microbial communities (seedling, vegetative, and bolting) and the control (slurry). Underline indicates statistically significant differences between the root associated microbial communities (p < 0.05 one-way ANOVA Tukey HSD post-hoc). (C) Proteobacteria and (D) Firmicutes. Average relative abundance (RA ± SEM) of the bacterial phyla found in plant root bacterial communities. Three replicates per treatment were used. Distinct letters above bars indicate statistically significant differences between the root associated microbial communities (seedling, vegetative, and bolting) (p < 0.05 one-way ANOVA Tukey HSD post-hoc). in the microbial treatment (Fig. 3). These results indicate that the orchestrated by a combination of physical (root structure charac- microbial community specifically and plant developmental stage teristics) and chemical (root exudation) changes that occur alters plant hormone levels. We also performed ANOVA tests using throughout plant development. all of the data across all hormones, stages of development, and microbial community exposure. These tests found the soil slurry 1. Brief methodology (microbial exposure) to be a significant factor after controlling for all others (p < 0.001, two-way ANOVA Tukey HSD post-hoc). Arabidopsis seeds were surface sterilized then plated and grown Prior evidence suggests that the plant attracts bacteria that aid on full strength MS (supplemented with 3% sucrose) agar (1.5%) in nutrient uptake at later stages (Chaparro et al., 2014), and in our square plates until they reached 7, 14, or 21 days (seeding, vege- study, we found evidence that supported this observation. Bacteria tative, or bolting stages, respectively). Arabidopsis plants at capable of nitrogen acquisition such as Rhizobiaceae significantly different stages were transferred to 6-well plates (3 plants/well; 18 increased with plant development (Supplemental Fig. S2E). The plants per replicate for a total of 54 plants per treatment) con- same trend was also observed with the abundance of Proteobac- taining fresh 2.7 mL of liquid MS media supplemented with 0.3 mL teria (Fig. 2C) which harbor many N-fixing microbes (Saravanan of filtered soil slurry, unfiltered soil slurry or MS media (control). et al., 2008). Furthermore, PICRUSt analysis based on 16S rRNA For root microbiome analysis, roots of 3 plants grown in a well were sequencing data also showed that the abundance of N fixing mi- harvested after four days of inoculation for CFU assay and RNA crobes (i.e., nifH abundance) increased significantly (p < 0.05, one- isolation. 200 ng of total RNA from each treatment was reverse way ANOVA Tukey HSD post-hoc) with each successive plant transcribed into cDNA. The cDNA was used for 16S rRNA quantifi- developmental stage (Supplemental Fig. S4). cation via q-PCR and MiSeq sequencing analysis. Sequences were These results highlight the direct ability of the root at distinct analyzed using Mothur v.1.33.3 (Schloss et al., 2009; Kozich et al., developmental stages to rapidly re-organize the surrounding mi- 2013) and the raw sequence data have been deposited into the crobial community. Although, we did not study the specific root NCBI Sequence Read Archive (SRA) as study ID SRP059551. For the attributes responsible for the microbiome change, it is likely phytohormone analysis, plants were harvested after seven days of J. Yuan et al. / Soil Biology & Biochemistry 89 (2015) 206e209 209

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