Rice Bacterial Endophytes ; 16 S-Based Taxonomic 2 Profiling , Isolation and Simplified Endophytic 3 Community from Two Venezuelan Cultivars 4

Rice is currently the most important food crop in the world and we are only just 13 beginning to study the bacterial associated microbiome. It is of importance to perform screenings of 14 the core rice microbiota and also to develop new plant-microbe models and simplified 15 communities for increasing our understanding about the formation and function of its microbiome. 16 In order to begin to address this aspect, we have performed the isolation of bacterial strains from 17 the endorhizosphere of two rice cultivars from Venezuela. The validation of plant-growth 18 promoting bacterial activities in vitro has led us to select and characterize 15 isolates for in planta 19 studies such as germination test, endophytism ability and plant growth promotion. Consequently, 20 a set of 10 isolates was selected for the set-up of an endophytic consortium as a simplified model of 21 the natural rice bacterial endomicrobiota. Upon inoculation, the colonization and abundance of 22 each strain within the rice roots was tracked by a culture-independent technique in gnotobiotic 23 conditions in a 30 days period. Four strains belonging to Pseudomonas, Agrobacterium and Delftia 24 genera have shown a promising capacity for colonizing and coexistence in root tissues. On the 25 other hand, a bacterial community taxonomic profiling of the rhizosphere and the endorhizosphere 26 of both cultivars were obtained and are discussed. This study is part of a growing body of research 27 on core crops microbiome and simplified microbiomes, which strengthens the formation process of 28 the endophytic community leading to a better understanding of the rice microbiome. 29


Introduction
Rice is the staple food for more than a half of the world population and its production is dependent on chemical fertilizers and pesticides [1] which are in part responsive for global warming and groundwater pollution [2].To meet the world's demand for rice it is imperative to find environmentally sound ways that supplement the need for fertilizers [3].The use of microbial inoculants is attractive because they can complement and mitigate the use of the agrochemicals ensuring a healthier environment [2].
Microorganisms play an important role in agricultural systems where they live in close association with plants and can exert different kinds of positive effects on the crop's health and growth [4].The effects of this microbiota include (i) increased nutrient availability (biofertilization), (ii) the ability to compete with or inhibit/antagonize potential pathogens, or reduce their effects (antagonism), (iii) the ability to chemically stimulate the growth and/or tolerance of the host to abiotic stress (phytostimulation) and (iv) the ability to inactivate or degrade existing toxic substances 2 of 31 in the soil (detoxification) [5]- [7].Rhizosphere bacteria which live in the soil that is in intimate contact with the roots and are able to perform one or more of these functions are known as plant-growth promoting rhizobacteria or PGPR [8].Some rhizospheric bacteria are capable of penetrating the surface of the roots and colonize the internal tissues of the root, a niche also known as endorhizosphere [9].These bacterial endophytes overcome plant defenses and establish themselves as permanent inhabitants of internal tissues without causing harm to the host plant [10].
It is believed that bacteria colonizing the interior plant tissues could interact closely with the host having less competition for nutrients and living in a more protected environment [11].
Several studies have focused on the isolation and identification of rice bacterial endophytes from different locations and varieties [12].Moreover, a metagenomic analysis of the rice endophytic microbiome provided clues about its composition and functions for the plant host [13] and the dynamics changes during rice root-associated microbiomes have been described [14].More recently, an extensive isolation, identification and plant-growth promoting traits determination of rice bacterial endophytes has been performed [15], providing further information on bacterial diversity in the rice endosphere.Although also the composition of the endophytic microbiota of various plants is being studied [10], [16], [17], our knowledge of the endophytic bacterial ecology remains limited and the identification and characterization of novel beneficial endophytes is still needed.In addition, most studies involving PGPR and endophytic bacteria are mostly restricted to monostrain set-ups under laboratory conditions [18], and our understanding of the effect of entire microbial communities to plant growth remains at large unexplored.
The main objective of this study is to provide and to describe additional data regarding the bacterial endophytic diversity of rice, as well as to isolate and characterize promising strains with beneficial traits.In addition, we hypothesize that a simplified endophytic bacterial community can be designed and applied as bioinoculants, which constitutes a reductionist approach that can also facilitate the understanding of the plant-microbiota interaction.We have undertaken the 16S rDNA taxonomic bacterial profiling of the rhizosphere and endorhizosphere of two high-yield rice cultivars, Pionero 2010 FL and DANAC SD20A, extensively grown in Venezuela in 2014.Fifteen putative bacterial endophytes were then isolated from surface-sterilized roots and further studied for in vitro and in planta.We have performed inoculation of rice seedlings with a simplified community composed by 10 of the isolates and we have tracked them in the course of 30 days in greenhouse cultivation.The results obtained suggest that a group of them was able to significantly colonize together the rice endorhizospheres, indicating possible cooperation and ability to form a stable multispecies community.To our knowledge, this is the first study of its kind performed with Venezuelan rice.We believe this approach can be useful in the development of microbial solutions for a more sustainable agriculture.

Sample collection and isolation of bacteria from rhizosphere and endorhizosphere
Three rice plants of cultivars Pionero 2010 FL (88 days after planting) and DANAC SD20A (90 days after planting) were collected in April 2014 from two fields in Acarigua (Portuguesa, Venezuela), packaged in sterile bags and cooled at 4 °C for 4 days until bacterial isolation.Five grams of roots with the adherent soil were gently vortexed for 5 minutes in 20 mL of sterile saline solution (0.85 % NaCl) and the rhizospheric soil suspensions were serially diluted and plated (100 µL) in triplicate on LB agar with cycloheximide (CHX) 50 mg/ml for determining the amount of rhizospheric colony-forming units (RCFU).The same 5 grams of rice roots were then surface sterilized in 70 % ethanol for 1 minute followed by 1.2 % hypochlorite for 15 minutes with agitation and finally washed 6 times with sterile distilled water.The extent of the sterilization was verified by plating the final wash concentrated to 100 µL on LB plates before proceeding maceration.Sterilized roots were then macerated using sterile mortar and pestle in 10 mL of 0.85 % NaCl sterile solution and different serial dilutions were plated in triplicate on LB/CHX plates for determining the 3 of 31 of putative endophytic colony-forming units (ECFU).The plates were incubated at 30 °C for 2 days.
Independent ECFU showing distinct colony morphology were picked and streaked again on LB plates to ensure purity of the culture.The remnants of macerated roots and rhizospheric soil suspensions were then used for DNA extraction.

Total bacterial diversity of rhizosphere and endorhizosphere
The rhizospheric and endorhizospheric DNA from the two rice cultivars were extracted using Soilmaster DNA Extraction Kit (Epicentre, USA) following the manufacturer's guidance.The quantity and quality of the DNA were assessed with Nanodrop (Thermo Fisher Scientific, USA) and electrophoresis in 0.7 %. agarose gel.The extracted DNA was used as template for the first amplification of the V4 variable region of the 16S rRNA by PCR using primers V4 515F, 802R, 806R tailed with two different GC rich sequences enabling barcoding with a second amplification.Each sample was amplified in triplicate in 20 µL volume reaction containing 8 µL HotMasterMix 5Prime (Quanta Bio, USA), 0,4 µL BSA 20X, 1 µL EvaGreen™ 20X (Biotium, USA), 0.5 µL 515F primer (10 µM modified with unitail 1), 0.25 µL 802R primer (10 µM modified with unitail 2), 0.25 µL 806R primer (10 µM modified with unitail 2), 0.5 µL MitoBlk_515F V4 mithocondrial blocking primer (100 µM,), 0.,5 µL ChloBlk_806R V4 chloroplast blocking primer (100 µM and 2 µL (10-50 ng) of DNA template.The PCR amplifications were performed with CFX 96™ PCR System (Bio-Rad, USA) with 34 cycles of 94 °C for 20 s, 52 °C for 20 s, 65 °C for 40 s and a final extension of 65 °C for 2 min.The primary amplification takes advantage of rice specific V4 blocking mitochondrial and chloroplast primers in order to increase amplification of prokaryotic sequences.The rationale for these blocking PCR reactions is described by [19].Deionized water was used in the negative controls.
The second PCR amplification (switch PCR) is required to attach the barcodes and was performed using a forward primer with the A adaptor (a sample-specific 10 bp barcode and the tail of the primary PCR primers) and a reverse primer with the P1 adaptor sequence and the reverse tail.We verified the size and the amount of the amplicons by agarose gel electrophoresis and then they were pooled in equimolar amounts.The library was purified by the E-Gel® SizeSelect™ (Invitrogen, USA) and verified the size and the amount with Agilent 2100 Bioanalyzer and a Qubit 1.0 fluorometer Q32857 (Thermo Fisher Scientific).
For sequencing the library was submitted to emulsion PCR on the Ion OneTouch™ 2 system using the Ion PGM™ Template Hi-Q OT2 View (Life Technologies, USA) according to the manufacturer's instructions.Ion sphere particles (ISP) were enriched using the E/S module.
Resultant live ISPs were loaded and sequenced on an Ion 316 chip (Life Technologies).This sequencing was done in the Life Science Department of the University of Trieste (Trieste, Italy).

Plant-growth promoting activities
Eighty-seven putative bacterial endophytes or EUFC were tested for indole-3-acetic acid (IAA) production in vitro.The IAA is a plant hormone secreted by plant-associated bacteria that increases the root elongation, root exudates and plant biomass (Etesami et al 2015).The bacterial cultures were grown in LB broth amended with tryptophan (100 µg/mL) at 30 °C for 4 days.The cells were sedimented by centrifugation and the supernatant (2 mL) was mixed with 4 mL of Salkowsky reagent (50 mL, 35 % perchloric acid, 1 mL 0.5 M FeCl3 solution) and incubated in darkness for 30 min.The appearance of a red-pink color indicated IAA production and OD530nm was recorded [20].
The concentration of IAA produced by cultures was measured with a calibration graph of commercial IAA obtained in the range of 10 -100 mg/mL and plotted in relation to the dry bacterial biomass.Fifteen bacterial isolates positive for the IAA production were chosen for further Pikovskaya agar [21].The phosphate solubilizing bacteria solubilize inorganic soil phosphorous, making it available to the plant and promoting the plant growth (Sharma et al 2013).The 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity was determined as described in [22], comparing the growth of bacteria on minimal medium (M9), M9 without N source and M9 with 30 µmol of ACC as sole N source.The ACC deamination lowers the hormone ethylene levels in the plant and promotes its growth (Glick 2015).N-acyl homoserine lactone quorum sensing signal assays were carried out as using Chromobacterium violaceum CV026 and C. violaceum CV017 as biosensors [23].Motility assay was performed as described by [24].The exopolysaccharide (EPS) production was assessed culturing the isolates on yeast extract mannitol medium as described in [25].The lipolytic activity was determined on 1/6 TSA medium amended with 1 % tributyrin [26] and proteolytic activity on 1/6 TSA medium amended with 2 % of powder milk [27].The quorum sensing signals, the motility, the EPA production and the enzymatic activities are important traits for endophytic colonization and lifestyle.The production of volatile hydrogen cyanide (HCN) was estimated qualitatively as previously described [28].HCN is an antifungal agent released by some beneficial bacteria.The antibacterial activity against rice pathogens (Dickeya zea, Pseudomonas fuscovaginae and Xanthomonas oryzae) was carried out plating the bacterial isolates on a bacterial lawn seeded with the pathogen.

Identification of selected isolates
Bacterial cells from 1 mL of overnight cultures in 2 mL of LB medium were sedimented by centrifugation and resuspended in sterile PSB 0.5 mL.The cells were boiled for 3 minutes, cooled in ice 3 minutes and centrifuged at maximum speed for 5 minutes.type strains/prokaryotic 16S ribosomal RNA database allowed the identification of the isolates.We considered > 97 % of identity for assigning species.The phylogenetic analysis was performed on the Phylogeny online platform.This software aligned the sequences with MUSCLE (v3.8.31), curated them with Gblocks (v0.91b), reconstructed the phylogenetic tree using the maximum likelihood method implemented in the PhyML program (v3.1/3.0 aLRT) and the tree rendering performed with TreeDyn (v198.3)[29].The isolates were deposited in the Venezuelan Center for Microorganisms Collection (Institute of Experimental Biology, Central University of Venezuela, Caracas) and the 16S rDNA sequences of the isolates were deposited in GenBank (NCBI).

Germination test, endophytism and plant-growth promotion assay
In order to track endorhizosphere bacterial colonization after inoculation in gnotobiotic conditions, the generation of rifampicin spontaneous resistant mutant was first achieved for the 15 selected isolates, as previously described [15], [30].Single colonies of endophytic isolates were grown on 5 mL of LB medium for 24 h at 30 °C and aliquots of 100 uL were then plated on LB agar containing rifampicin (Rif) 100 µg/mL and incubated 48 h at 30 °C.Single rifampicin resistant colonies were re-streaked on LB Rif, stored at -80 °C and used for in planta experiments.
The rice seeds of the Baldo cultivar have a germination rate > 97 % in untreated samples (data not shown) so the effect of the bacterial inoculation on seed germination was measured as the biomass of 4 days old seedlings.The seeds were surface sterilized for 30 minutes with 15 % hypochlorite solution and then rinsed six times with sterile water.Fifty sterilized seeds were germinated in a Petri dish containing 20 mL sterilized water plus 500 µL of an overnight culture of each strain in 1 mL of LB medium, separately.The plates with seeds were kept in the dark at 30 °C for 4 days, before determining the wet weight of 10 groups of 5 germinated seeds, randomly chosen and with the water excess uniformly absorbed with clean paper.A control plate with only water (20 mL) and LB (500 µL) was included.Individual seedlings were then transferred to a 50 mL tube containing 35 ml of semisolid (0.25 % agar) ½ Hoagland solution [31] and incubated at 28 °C, 75 % humidity, 16 h/8 h light-dark cycles.The seedlings were watered every two days using 1 /10 Hoagland solution.After 15 days, the inoculated plant roots were washed abundantly with tap water, dried with paper, separated from the aerial parts (cutting just below the cotyledon) and weighed.The root surface sterilization was performed as explained above and checked by plating the centrifuged sediment of the last wash (30 mL) on LB Rif 100 µg/mL.Then the roots were macerated with sterile pestle and mortar with 3 mL of phosphate buffered saline (PBS) sterile solution and 100 µL of the macerate was plated on LB/Rif plates, incubated at 30 °C for 48 h.The CFU of recovered bacteria were counted and the number of the putative bacterial endophytes was calculated as CFU per gram of root.The aerial parts of the plants were dried at 65 °C for 5 days for determining the plant growth promotion.A control group of plants without bacteria was included.
Five rice plants per treatment were harvested and processed.The mean of each treatment was compared to that in control with a two-tailed paired t-test (confidence interval 95%) using Graph Pad Prism version 5.0a.

Simplified community colonization assay
Ten bacterial strains were cultured for 48 h at room temperature in 10 mL of LB medium and diluted to OD600nm of 2.0.The cells were then sedimented by centrifugation, washed with sterile 10 mL PBS and resuspended in 3 ml PBS. 2 mL of each bacterial/PBS suspension were mixed and finally, 30 mL of PBS were added bringing the final volume to 50 mL. 2 mL of this mixed suspension were used for DNA extraction and the remaining 48 mL were added to 800 mL of semisolid ½ Hoagland solution.A control without bacteria (only with LB broth) was included.
One-week-old Baldo rice individual seedlings (sterilized and germinated as described above) were transferred to 40 mL (in Falcon tubes) of this community-containing semisolid Hoagland solution incubated and watered as described above.Three plants from the control and the treatment were recovered at 10, 20 and 30 days after planting, for a total of 18 plants harvested.The roots and aerial parts were separated and weighed.The roots were then sterilized and macerated with liquid nitrogen.The resulting root powder was used for DNA extraction and a 16S rRNA gene library was constructed and sequenced exactly as described in Material and Methods 2.2, for carrying on the amplicon-based taxonomic profiling.The general stepwise procedure is shown in Figure 1.

Biodiversity of Venezuelan rice rhizosphere and endorhizosphere communities by culture-independent methods
In order to obtain a picture of the taxonomic diversity of the two Venezuelan rice cultivars, the population of the total rhizospheric and endorhizospheric bacterial community was assessed.It was analyzed in 6 plants that were harvested from two fields, 3 plants belonging to Pionero 2010 FL  1.After the removal of plant-derived, anonymous and singletons OTUs, the high-quality reads were clustered in a total of 341 different OTUs with a taxonomic assignment evaluated with > 97% sequence identity as the cutoff.As expected, the snapshot of the total bacterial community showed a greater abundance and diversity of bacterial species in the rhizosphere than in the endorhizosphere, as suggested by the richness and diversity estimators shown in Table 2.The rhizosphere of DANAC SD20A cultivar was colonized by a larger bacterial community than that of Pionero 2010 FL.  341 OTUs in total were binned to a taxonomical category and their distribution within the samples is summarized in Figure 3 and the complete list is in Supplementary Table

Isolation of culturable bacteria from rhizosphere and endorhizosphere
The adherent soil of 5 grams of roots (i.e. the rhizospheric soil) was serially diluted and plated in triplicate on LB/CHX plates.The estimated average number of culturable bacteria recovered was 5.5 x 10 7 CFU per gram of rhizospheric soil.On the other hand, the 5 grams of roots yielded from 1420 to 361120, with an average of 121076 CFU per gram of sterilized-macerated roots.In order to perform the plant-growth promoting tests, 87 putative endophytic bacterial isolates were chosen based on color and colony morphology differences.

Germination test, endophytism assay, and plant-growth promotion
The 15 isolates were in planta assayed for germination, endophytic colonization, and plant growth promotion.For these experiments, we created spontaneous rifampicin resistant mutant derivatives in order to select them after their recovery from colonized plant tissues.Only 2 strains significantly increased the germination rate of the seeds; Agrobacterium sp.E2315-germinated seeds were 7.6 % higher on average than control seeds and Serratia glossinae E2309 with a 7.3 % germination increase (Figure 5A).Of the 15 isolates tested, only 1 could be recovered after inoculation from the endorhizosphere, this was Pseudomonas fluorescens E1308.The CFU of this strain ranged from 170 to 44000 CFU per gram of surface-sterilized roots.This isolate was also the best promoter of plant growth since the plants displayed an increase of 110 % of the aerial parts dry weight when compared to the control plants (p < 0.05) (Figure 5B).Also, other 8 strains showed a statistically significant positive effect on plant growth promotion, namely P. mendocina E1108 (103 %), Rhizobium sp.E2315 (103 %), Serratia fonticola E2105 (79 %), P. jessenii E2333 (67 %), Delftia tsuruhatensis E2330 (65 %), Bacillus amyloliquefaciens E1101 (59 %), P. pseudoalcaligenes E1205 (37 %) and Pseudomonas sp.E1201 (37 %).

Simplified community inoculation, colonization, and plant growth promotion
It was of interest to perform in planta studies with a bacterial consortium in order to determine possible bacterial inter-species community effects on host colonization.We decided to use a bacterial equivalent to OD600nm of 2.0 of each culture was used for the mixed bacterial inoculum.This inoculum was included in the semisolid Hoagland solution where plants were grown.After 30 days, there was a significant increase of 15 % (p < 0.05) in the wet weight of the inoculated plants compared to control non-inoculated, both in the roots and in aerial parts (Figure 6).A cultivation-independent tracking, using 16S rDNA amplicon sequencing, was carried out in order to obtain insight into the colonization ability of the 10-strain simplified community over time.
The numbers of reads obtained, bacterial-and plant-derived, are shown in Table 1 section B.
Regarding the total bacterial endophytic abundance, it was noted that the uninoculated plants were systematically lower in bacterial populations at each time point compared to that in inoculated plants (Supplementary figure 2) The composition of the cell mix (the pooled bacterial cultures that were then used as inoculum) varied from 36 reads (P.chengduensis E1108) to 13145 reads (S. glossinae E2309) in a total of 45246 reads, as shown in Figure 7A.In order to track the abundance of each strain of the bacterial consortium within the plants, their 16S sequences were used against the total 16S rDNA library sequenced.This was also performed for the control plants in order to determine if any seed-borne bacterial endophyte was taxonomically close enough to the strains used in the consortium, which could lead to false positives.The abundance of the simplified bacterial community was tracked in control and inoculated plants and it is represented as relative abundances in Figure 7B.The abundance and identity of the reads suggested that taxonomically related strains to P. pseudoalcaligenes E1205, P. gessardii E1308, S. glossinae E2309 and Agrobacterium sp.E2321 were present in the control plants in low abundance.In the inoculated plants, at least 8 out of 10 bacterial strains were detected within the plant roots.Only 4 strains were however detected after 30 days of cultivation, namely: P. pseudoalcaligenes E1205, Agrobacterium sp.E2321, D. lacustris E2330 and P.
jessenii E2333.This dataset suggested that these strains were capable to colonize together the rice roots.

Discussion
It is of great importance to study the microbiota diversity and functionality on the main agricultural crops [34], as well as to develop models for the study of plant-microbe interaction through simplified microbiota [35].In this study, (i) we have performed a survey on the total bacterial endophytic community in Oryza sativa cv.Pionero FL 2010 and O. sativa cv.DANAC SD20A, (ii) we have carried out the isolation and partial characterization of 15 putative bacterial endophytes, and (iii) we have narrowed a 4-strains simplified microbiota as a starting point for a working model for bacteria-bacteria and bacteria-plant interactions in rice, towards a future efficient bioinoculant formulation possibly based on a mixed inoculum.

Amplicon-based taxonomic profiling.
Profiling the bacterial communities allowed us to determine that the rhizospheres of the sampled plants were more diverse than the endorhizospheres, an observation widely documented [14], [36], [37].The use of blocking primers was successful since > 99.9 % of the endorhizospheric reads belonged to bacteria.Proteobacteria were by far the most predominant group in both compartments of both rice varieties, and this is in agreement with several previous studies [14], [15], [38], [39].However, members of Deltaproteobacteria and Epsilonproteobacteria class were not detected in the endorhizospheres analyzed here; this is in contrast to what has been reported in a previous report of rice microbiome in Italy [15] and Philippines [38].We further compared the OTUs abundance differentially distributed between the rhizosphere and the endorhizosphere of each rice cultivar.We identified members of Cellvibrio genus as being highly predominant inhabitants in both endorhizospheres.The members of this genus are known as obligates aerobic cellulolytic bacteria and other complex carbohydrates degraders [40] which are believed to be key activities necessary for the colonization of the plant endosphere.Cellvibrio spp.have been reported as members of the rice endosphere [15], however with a lower abundance (between 0.01 and < 1 %) than in our study.Some Cellvibrio species are nitrogen-fixing bacteria, especially the Cellvibrio diazotrophicus [41].Other species enriched in both endospheres were P. pseudoalcaligenes, Agrobacterium sp. and Opitutus sp.
Endophytic P. pseudoalcaligenes and Agrobacterium sp. have been previously reported in rice [42], [43] and they have also been frequently isolated from different plant types and tissues [44]- [47].Opitutus sp. has been reported as an inhabitant of anoxic rice paddy soils [48] and as a rice endophyte [15], moreover, members of Verrucomicrobiae in the rice endosphere have also been reported by [38].
Interesting Opitutus sp. is obligate anaerobic with a fermentative metabolism that utilizes rice plant-derived carbons [36].The presence of anaerobic microbes within the plant, an environment which is O2-rich, seems paradoxical and was also reported by [14].
In the Pionero FL 2010 cultivar, Pedobacter, Variovorax and Devosia genus were enriched in the endorhizosphere with respect to the rhizosphere.Pedobacter sp. has been previously isolated from rice paddy soil [49].Variovorax sp. is a versatile PGP bacterium able to colonize the plant endosphere [50] including rice [51].Devosia sp. is a soil bacterium from the Rhizobiales family, nodule-forming and nitrogen fixing [52].Bacteria belonging to these three genera have been detected in the rice endosphere of rice grown in Italy [15].
Two bacterial species counted for half of the total bacterial population in the endosphere of Pionero FL 2010.First, Microvirgula aerodenitrificans, the most abundant one, is an aerobic denitrifier [53] and has been reported previously as a rice endorhizosphere inhabitant [15].Secondly Caulobacter sp., which has also been reported to be associated rice in two other parts of the world [54][55] [15] and to have PGP properties [44].In the endorhizosphere of the DANAC SD20A cultivar, strains belonging to the Azospirillum, Acinetobacter and Citrobacter genera were dominant.
Azospirillum and Acinetobacter are diazotrophic plant-growth promoting bacteria that can modulate the phytohormone balance [56], [57].To our knowledge, there is just one report of the isolation of Citrobacter as rice endophyte [58], although the rice metagenomic study most likely revealed loci which belong to Citrobacter sp.[38].Apart from Cellvibrio, P. pseudoalcaligenes, and Opitupus sp., the endosphere of the DANAC SDS20A cultivar was highly enriched by Rhodoferax sp., a nitrate reducer bacterium [59].
It is important to mention that this analysis was subjected to the intrinsic bias of the amplification and sequencing techniques, as well as the data processing [34], thus some taxa could not be appropriately represented in our study.On the other hand, the number of plants sampled (three for each cultivar) would not reflect the real bacterial endophytic microbiota of each cultivar.
Nevertheless, the taxonomic range of putative endophytic microbiota of rice has been extended with this work, making an important contribution to the rice microbiome research, improving the progress towards the elucidation of the rice core microbiota.

Isolation of putative endophytic bacteria, determination of its PGP traits, and plant colonization.
Beneficial endophytic bacteria play important roles that positively affect directly or indirectly plant growth and development [60].In this study, we selected 15 putative bacterial endophytes isolated from Venezuelan rice because they were IAA producers.IAA is the main auxin in plants, controlling the roots architecture, thereby improving nutrient acquisition [61]- [63].Our estimations of the produced IAA are related to milligrams of dry bacterial biomass, instead of milliliters of culture, since we think it could be more useful for future comparisons.
Two Bacillus strains (Firmicutes phylum), B amyloliquefaciens E1101 and B. altitudinis E2315, were identified among our isolates.Although these two strains did not affect the germination rate of the surface-sterilized rice seeds, they positively influenced the plant growth however our inoculation experiments did not reveal them as endophytes.Bacillus spp.are widely used commercially as biofertilizer and biocontrol agents in agriculture due to their spore-forming ability and stability in their formulations.In our work, B. amyloliquefaciens has shown the most potent antibacterial activity, antagonizing or inhibiting the growth of 14 bacterial species (data not shown).
B. amyloliquefaciens is known to produce surfactins and an array of secondary metabolites and is considered a model for unraveling plant-microbe interactions and biocontrol [71].It is interesting to note that in our taxonomic profiling, Bacilli abundance was extremely low in the four compartments analyzed, with a maximum abundance of 0.016 % of the total reads.It cannot be excluded that the isolation procedure favored the growth of non-abundant Bacillus spp. or alternatively that the PCR for 16S-based taxonomic profiling was not so efficient for this bacterial group.
The other 13 isolates belong to Proteobacteria, the most abundant phylum in the taxonomic analysis.The alfaproteobacteria Agrobacterium sp.E2321 had the most positive impact on the germination rate, but this did not translate into a plant growth promotion.This strain displayed a number of PGP traits in vitro, however, was not able to perform beneficial effects in planta; this contradiction was discussed by [64] when they found similar discordance when analyzed the effect of rhizobacteria on the growth of barley under salt stress.These results would suggest that the current in vitro PGP screening methods may need to be re-evaluated.The isolate Serratia glossinae E2309 was the only bacterial inoculum that increased the germination rate and also plants growth.
Others Serratia spp.have been previously reported as PGP strains [65]- [67] and could, therefore, be a good candidate to further study.However, the other S. glossinae isolated (E2105), did not promote the plant growth.Interestingly our two S. glossinae isolates displayed a different profile of in vitro activities thus despite being to the same species, probably there are differences between the two isolates which affect the PGP performance.In our taxonomic profiling, Serratia spp.were not detected in the endorhizospheres of DANAC SD20A cultivar but were detected in low abundance in the rhizosphere of Pionero 2010 FL.This discrepancy could be explained by cultivation and or PCR amplification bias.
Other isolates such as Delftia sp.E2330 and another Pseudomonas spp.did not affect the germination rate but promoted the plant growth.Delftia sp.has been isolated from the rhizosphere of rice and is considered as a PGP bacterium [68].In our taxonomic survey, Delftia spp.were present in low abundance in both compartments of DANAC SD20A cultivar.Our isolate Delftia sp.E2330 showed the strongest quorum quenching activity in vitro.Since Delftia sp.VM4 was reported to possess AHL-acylase activity [69], we speculate that our isolate could also possess this enzyme activity as quorum sensing interference.Pseudomonas spp., are very abundant members of the rice endorhizospheres [38], [55], [70], [71], however, only P. aeruginosa E1103 displayed some PGP traits in the conditions that we have tested.Maybe P. aeruginosa could be included in the category of Pseudomonas_OTHER or Pseudomonas sp. in our total community determination.The Aeromonas spp.
isolates did not show PGP activity or improved germination; Aeromonas isolates have however been reported to have PGP activity in and rice [72], [73].Cultivation media and/or the genotype of the host could be influencing this.
Of the 15 isolates re-inoculated, only P. fluorescens E1308 could be re-isolated from the endosphere of the 5 plants harvested.The other strains could be in low abundance not enough for cultivation.The recovery of rifampicin spontaneous mutants is an approach used elsewhere with this aim and has shown to be a valid way and stable approach for the detection [15], [30].
We should mention some limitations of our methods and analysis.For instance, endophytic strains were isolated from two rice cultivars genotypically different from the one used in the in planta experiments hence it is possible that plant genotype influences endosphere colonization/microbiota, as stated by [35], [39], [74]- [76].On the other hand, the number of sampled plants per treatment (5 plants) could be insufficient for the objectives.

Seedling inoculation with a simplified bacterial community.
Microorganisms do not act as individuals but rather act as a dynamically changing microbial community, where cells interact and communicate with one another.This communication influences bacterial behavior significantly affecting the phenotypes of the microbial community [77].It is therefore of importance to developing new model systems for incorporating communities of microorganisms in plant microbiota research [35].The use of traceable simplified ecosystems reduces the complexity of naturally complex microbiota and its investigation increase our knowledge regarding factors that shape and influence microbial communities.We, therefore, performed rice inoculations with a 10 strain simplified community in order to assess its potential for host colonization and possible differences compared to single strain inoculations.We did not use strains which possessed strong in vitro antibacterial activity.Assessing colonization via 16S rDNA gene community profiling showed that 8 strains were detected in the endorhizosphere.Within this group, P. pseudoalcaligenes E1205, Agrobacterium sp.E2321, Delftia sp.E2330 and P. jessenii E2333 remained in the endorhizospheres after 30 days of plant growth.The isolate P. fluorescens E1308, the only one recovered from surface-sterilized inoculated rice plants in the single-strain in planta tests, was surprisingly not detected when co-inoculated with the 9 other strains.The bacterial community can be influencing the endophytic colonization of this strain or the host plant favored the colonization of other strains.The design of simplified microbial communities has been recently considered as a priority for harnessing the plant microbiome in sustainable agriculture [35] and this approach has been addressed in Arabidopsis [78] and in maize [79].In this work, we initiated PGP and colonization studies of a simplified community of 10 bacterial strains and initial results encourage further studies of synergistic, signaling and cooperative behavior of a multispecies consortium as well as the role of the plant genotype.
The reaction was performed in25 µL volume containing 10 µL HotMasterMix 5Prime, 1.25 µL EvaGreen™ 20X, 1.5 µl barcoded primer (10 µM), 1 µl of the first PCR product with the following conditions: 8 cycles of 94 °C for 10 s, 60°C for 10 s, 65 °C for 40 s and a final extension of 72 °C for 3 min.The list of oligonucleotides used and its sequences characteristics are shown in supplementary table 1.

Table 1 .
Sequences characteristics.The number (#) and its corresponding percentage (%) of plant-derived and bacterial-derived 16S reads sequenced, as well as the average length in bp, are listed.A) Results for the 16S-based taxonomic profiling of the two rice cultivars.B) Results for the simplified community assay.Microbiome analysis by phylum distribution and frequency (expressed as the percentage on the total number of OTUs) is summarized in Figure2.Representatives of Proteobacteria, the most abundant phylum, were 71 % to 87 % of the total OTUs.Also, the proteobacterial classes were considered: Gammaproteobacteria was most abundant, followed by Betaproteobacteria and Alfaproteobacteria, while representatives of Deltaproteobacteria and Epsilonproteobacteria were not detected in the endorhizospheres.Other abundant phyla were Bacteroidetes, which were nearly equally distributed among the samples.Verrucomicrobia were enriched in the endorhizosphere of Pionero 2010 FL whereas Actinobacteria, Cyanobacteria, Fibrobacteres and Spirochaetes were equally distributed among the samples.Acidobacteria, Chloroflexi, Nitrospirae and Planctomycetes phyla were only detected in the rhizospheres.

Figure 2 .Table 2 .
Figure 2. Frequency distribution of the bacterial phyla in the rhizosphere (R) and endorhizosphere (E) of the sampled rice roots.Bar graphs of the taxonomic annotation of bacterial reads among the distribution of the most abundant phyla.The classes of Proteobacteria phylum are also shown in shades of blue.

Figure 3 .
Figure 3. Microbiota composition of the two rice cultivars.A total of 341 OTUs were identified by 16S rRNA sequencing profiling , using a 97 % of similarity against the database.326426 high-quality reads were obtained, 256701 from Pionero 2010 FL (A) and 69795 from DANAC SD20A (B) cultivar.The values in the Venn diagrams indicate the number of OTUs found exclusively in the rhizosphere (R), in the endosphere (E) or those found in both compartments, and the number in parenthesis indicates the relative abundance of those OTUs.The 20 most abundant species detected in each compartment and their abundance are shown (%).The length of the color bars represents the value in the cell.

3. 5
in vitro assays of plant beneficial traits It was of interest to determine whether the 15 IAA-producing putative rice bacterial endophytes possessed other important plant beneficial traits such as nitrogen fixation, phosphate solubilization, ACC deaminase activity, HCN production and antibacterial activities.Other relevant traits for endophytic lifestyle like quorum sensing acyl-homoserine lactone (AHL) production, quorum quenching activity, exopolysaccharide (EPS) production, motility and secretion of enzymes were also assayed.The results of these assays are summarized in Figure 4B.

Figure 4 .
Figure 4. Putative endophytic bacteria isolated from surface-sterilized rice roots.A) The bacterial isolates were putatively identified by 16S sequencing and the rDNA sequences (average length 1518 bp) were used for constructing the cladogram.B) Plant-growth promoting activities and antibacterial activities detected in in vitro tests (IAA, indole acetic acid production; N2, nitrogen fixation; P, phosphorous solubilization; ACCD, ACC deaminase activity; AHL, acyl homoserine lactone production; QQ, quorum quencher activity.;HCN,hydrogen cyanide production; EPS, exopolysaccharide production; Swim and swarming and motility; Lipolytic and proteolytic activity; antibacterial activity against Dickeya zea, Pseudomonas fuscovaginae and Xanthomonas oryzae.The assays were performed in biological triplicates.

Figure 5 .
Figure 5. Plant growth promotion by single-strain inoculation.A) Germination rate.The wet weight of 4 days old germinated seeds was determine.Each dot represents the average weight of 5 germinated seeds in the dispersion graph.The average and standard deviation are shown as red lines.B) Plant growing rate.The dry weight of the aerial parts (stems and leaves) was determined.The averages are shown relative to the control (arbitrarily 100) with its standard deviation.The values were obtained from 5 different inoculated plants cultivated during 15 days.The red asterisks indicate statistical significance (p<0.05).

Figure 6 .
Figure 6.Effect of the bacterial consortium in plant growth.One-week old rice seedlings were inoculated with a mixture of 10 bacterial strains and grown in controlled conditions for 30 days.Each ten days, 3 plants were harvested, cut in the two parts shown, and weighted.A control without bacterial inoculation was included.The asterisk indicates statistic significance (p<0.05).

PreprintsFigure 7 .
Figure 7. Composition of the 10-strains simplified community and its abundance during 30 days growth of rice seedlings.A) The cell mix represents the 10 species mixed and used as inoculum.The relative abundance of each strain is shown in brackets.The total number of reads was n = 45246.B) The relative abundance of each consortium strain was tracked at 10, 20 and 30 days after the inoculation of the rice seedlings.The results for non-inoculated and inoculated plants are shown in the colored bars.The total number of reads was n = 111291.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 November 2017 doi:10.20944/preprints201711.0135.v1 Peer
-reviewed version available at Microorganisms 2018, 6, 14; doi:10.3390/microorganisms6010014 Figure 1.Methods workflow.Stepwise approach for determining the taxonomic profile of the bacterial endophytic microbiota of two rice cultivars and the setup of a simplified community based on in vitro and in planta performance of the isolates.2.7 Analyses of sequencing data.Reads were initially mapped against O. sativa mithocondrial (NC_011033) and plastidial genomes (NC_001320).Unmapped reads were further processed.We used CloVR 1.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 November 2017 doi:10.20944/preprints201711.0135.v1
Peer-reviewed version available at Microorganisms 2018, 6, 14; doi:10.3390/microorganisms6010014cultivarand the other 3 to DANAC SD20A cultivar.The total DNA from rhizosphere and endorhizosphere was extracted for performing 16S rDNA amplicon library sequencing.We obtained 326496 high-quality bacterial reads of 248 bp length in average.The reads count per sample, as well as those obtained from plant organelles, are shown in Table1, section A. The relation of the number of reads per OTU detected is shown in the rarefaction curve in Supplementary Figure

Table 3 . Molecular identification of the putative bacterial endophytes isolated from the two rice cultivars.
The 16S rRNA gene were sequenced and compared to the rRNA type prokaryotic strains database.The accession number to the NCBI (A), the accession number to the Venezuelan Center for