Tomato endophyte bacteria diversity under long-term organic agricultural manipulation systems

Purpose: Differences were observed in both microbial structure and physicochemical properties were observed in the organic and conventional agricultural systems. However, very little is known about the diversity of endophytic bacterial communities in plants grown in separate manipulation systems. The goal of this work was to get a broader overview of the diversity and dynamics of the tomato endophytic bacteria in the different agricultural manipulation systems. Methods: The structure of endophyte bacterial communities under different growth periods (seeding stage, �owering stage, fruiting stage and harvesting stage), agricultural manipulation practices (organic and conventional systems) and organ-type (root or stem) were explored by using 16Sr RNA gene pro�ling in this study. Results: A total of 2,014,992 part 16 SRRNA gene sequences were obtained. These sequences revealed large-scale functional taxonomy units (OTUs). That is, there are 648 different OTUS in libraries, and 96 OTUs are common. Tomato endophytes consisted mainly of four phylloxera, of which Proteobacteria was the most represented, followed by Formigiotes, bacteriophages, Actinobacteria, Gamma proteobacteria, the most abundant class of proteobacteria, bacteriobacteria, and so on. Proteobacteria are low. General enterobacteriaceae, Vecella, Bacillus, Mesorizobium and Chrysobacterium were shared by all growth periods. Rich endophytic bacterial diversity was observed at the seedling stage (DI), and endophytic bacterial diversity at the �owering stage and fruiting stage was low. Signi�cant difference in endophytic bacterial communities emerged from roots and different host biographical stages, and tomato exerts greater in�uence on endophyte bacteria compared to organ type (main) agricultural manipulation methods. Conclusions: Tomato endophyte microbiota have a distinct structure in different growth periods. Bacillus were enriched seeding stage and decreased in the fruiting stage while Mesorhizobium increased during in the fruiting stage. Tomato have distinct endosphere microbiota by comparing beta diversity of microbiota in different growth periods, compared with the manipulation resume and organ type. And a strong correlation was observed between the structure of the microbiota in the whole dataset and soil chemistry which indicated that the soil type and treatment affected the endosphere microbiota of tomato. Organ-type (niche) exert more in�uence on the tomato microbiota compared with manipulation treatments between organic-farming and conventional farming.


Introduction
Tomato (Solanum glycoprotein) are an important economic crop grown worldwide that are of commercial value and ecological importance.Evidence is increasing that soil microorganisms or those associated with crops play an important role in plant health (Zhao et al. 2016).Plant microorganisms, often representing plant identities and plant second genes, are essential for plant health (Rui et al. 2019).
Stabilization of microorganisms to increase plant nutrition and increase resistance to biological and aziotic stresses presents one of the few reservoirs that has not been used as an opportunity to address sustainability issues in agriculture (Cao et al. 2004).Therefore, it is important to have a good understanding of the endophytic bacterial communities in tomatoes.
Endophytic bacteria are a diverse group of bacteria that live inside plant tissues; These endophytes can live within cells, in the interstitium, or in the vascular system (Manzotti et al. 2020).Bene cial to the endophytic bacterial host plant, e.g., biological control of growth diseases or plant diseases (Kahremani et al. 2019), in addition, endophytic microorganisms can provide protection against fungal diseases (Vector J et al. 2011).Explaining the structure of endophytic bacteria can help clarify their function and potentially improve performance.Most plant species, including monocotyledonous dicotyledonous plants and endophytic microbiota, have been reported (Constantine et al. 2019).Good agricultural practices, e.g., agricultural microbial vaccines, for example, generally improve the yield of plants produced during organic farming and the growth of suitable plants (Hon et al. 2017).Endophytes microbiota can be affected by a variety of factors such as physical and chemical properties and good farming practices such as organic farming and microbial vaccination.E.g., and plant yields generally excel in organic farming suitable for plant growth (Dong et al. 2019).
A long-term greenhouse test was carried out in 2002 at the Kujo Experimental Station in Hebei Province, China.This experiment includes organic and conventional farming methods for evaluating the endophyte constitution of tomatoes.All three farming methods were under the same schemes like crop rotation, irrigation and plowing, but they differed in fertilization and plant protection management.Differences were found in both the microbial structure and the physicochemical properties (Han et al. 2017).However, little is known about the diversity of endophytic bacterial communities in plants grown under separate manipulation systems, and therefore, the structure (systems and organics) of endosperm bacterial communities under different maneuvers (sowing stage, owering stage, fruiting stage and harvest stage), and agricultural manipulation practices.Organ type (root or stem) was explored in this study using 16 SRNA gene amplicion sequencing, and the goal of this work was to obtain a comprehensive overview of the diversity and kinetics of tomato endophytic bacteria.Different agricultural manipulation systems.

Growth Conditions and Sample Collection
Two sampling greenhouse was located in Quzhou (N 34 17" E 116 55", 476 m above sea level).Hebei province, China, of which manipulated by organic and conventional systems since 2002.Plants at four developmental stages, including seedling the stage, owering stage, fruiting stage, and harvesting stage were chosen during the growth period from March 2019 to August 2019.Five plants were randomly selected for each site at each of the various distinct growth stages, such that 80 plants were sampled in total.Roots were syndicated in Diagenode Bioruptor at low frequency for physical removal of microscopic soil aggregates and attached microbes (Vega et al. 2015).All eighty roots and stem gathered.Surface sterilization was made in previously described (Gu et al. 2020).No bacterial growth was detected after plating the roots on R 2 A agar at 30℃ for 7 days (Mcpherson et al. 2018).Moreover, no ampli cation of 16S rRNA genes was observed when the water used for the nal wash was used as a source of DNA.
Bar-Coded Pyrosequencing PCR optimization and Illmina-Hiseq2500 pyrotechnical were performed by the Amplicon Seq Service of the Biomak Biotechnology (China).Brie y, DNA aliquots (10 ng) were PCR-ampli ed at 94 for 30 s and then 30 cycles of 94 for 20 s, 45 for 20 s, and 65 for 60 s; with a nal extension at 72 for 5 min.V7-V9 hypervariable regions of the bacterial 16S rRNA gene were ampli ed with the primers 799 F and 1193 R containing the sequencing adapters and sample-speci c barcode (illmina-Hiseq2500 Sciences).The applicants were puri ed and quanti ed uorometrically with the Min Elute kit (Qiagen, Germany).

Sequence Analysis
The raw 16S rRNA gene sequencing reads were demultiplexed, quality-ltered by fastq version 0.20.0 and merged by FLASH version 1.2.7with the following criteria: (i) the 300 bp reads were truncated at any site receiving an average quality score of <20 over a 50 bp sliding window, and the truncated reads shorter than 50 bp were discarded, reads containing ambiguous characters were also discarded; (ii) only overlapping sequences longer than 10 bp were assembled according to their overlapped sequence.The maximum mismatch ratio of overlap region is 0.2.Reads that could not be assembled were discarded; (iii) Samples were distinguished according to the barcode and primers, and the sequence direction was adjusted, exact barcode matching, nucleotide mis match in primer matching (Chen et al. 2020).

Results
Bacterial Pro ling Analysis A total of 2,014,992 high-quality, non-plastid, and partial sequences were requested.The number of retained sequences per sample varied from 30,702 to 267,257 (Table.S1), and the rarefaction curves shown in Fig. 1, which suggested that these libraries detected a large majority of the endophytic bacterial diversity in the samples used in our study.648 OTUs were consulted on 80 samples for the bacterial library, with 96 OTUs in common after strict ltering process (Fig. 2).

Taxonomic Composition Analysis
The taxonomic summary of the diversity revealed that the tomato-associated endophyte bacteria mainly comprised four phyla (Fig. 3A), among which Proteobacteria was the most represented (40.63 %), followed by Firmicutes (21.76%),Actinobacteria (20.3%) and Bacteroidetes (8.7 %).A total of 253 different genera were identi ed across all samples, in addition, the relative abundance of bacterial classes of the top 10 most abundant genera in different growth periods was shown in Fig. 3B, that is, genera Weissella, Bacillus, Mesorhizobium and Chryseobacterium were shared by all growth periods.

Dynamics of endophytic bacteria in tomato during growth
The bacterial Shannon diversity index have no signi cant difference in different growth periods (Fig. 4A P-value cut off =0.05), while principal coordinate analysis (PCoA) of Bray-Curtis distance (Fig. 5B, permutations analysis of variance R 2 = 0.19, P < 0.001), and endosphere microbiota in four growth periods formed separated clusters in the rst two coordinate axes, and all of the pro les indicated that endosphere microbiota changed with the growth of tomato.
PCoA of bacterial microbiota using Bray-Curtis distance for organ sampling and long-term manipulation treatment factor in different growth periods (Fig. 5D-L).Tomato endosphere microbiota in different agricultural manipulation, and organ-type were also distinct.The seeding stage (TI), owering stage (T2), fruiting stage (T3) and harvesting stage (T4).Seeding stage (TI) datasets selected from the whole dataset were used to analyse the organ-type factor, and root (R) and stem (S) datasets from each growth period were used to analyse the agricultural manipulation treatment factor (FigS.5D-F).The beta diversity and PCoA analyse using Bray-Curtis distance matrices showed that bacterial microbiota of different organ types in four growth periods datasets were separated in the rst two coordinate axes (permutations analysis of variance R 2 = 0.354, P=0.001), however, treatment factors had no cluster in different root (R) or stem (S) datasets and were separated in the rst two coordinate axes (FigS.5D-F),and other root (R) or stem (S) datasets have the similar results (FigS.5G-N): a large variation was found in the endosphere microbiota of tomato with different organ types but a few agricultural manipulation treatments.

Discussion
During the long history of organic farming experiment from Quzhou county in 2002, three agricultural manipulations were carried out.Here, we explore the endophyte constitution under two farming methods.
We reported that the reproductive period is associated with the enrichment of endophytes.In general, the difference from rhizosphere to rhizosphere to phylloxera to endosphere is minimal (Lee et al. 2018), so low concentration and diversity in root and stem endophyte bacterial specimens are understood in this study, and no signi cance was found in diversity over different time periods (Fig. 2A).The bacteria in the stems come from the environmental soil, air, air or water, which are enriched by microorganisms (Doju et al. 2018).This may explain the greater richness in the roots compared to the stems (Zhuang et al. 2020).Over time, endophytic bacteria from the roots can migrate or be transported to the upper parts of plants (Lamo et al. 2018).In this study, the relatively closed environment provided by the greenhouse may have been another reason for the low diversity of stem bacteria.Furthermore, the bacterial richness and diversity of endophytes vary in different tissues, resulting in greater bacterial diversity in the roots.Similar results were reported for other plant hosts such as Arabidopsis, rice and agave species (Paul Schulz et al. 2018).
Additional studies showed that organ-type varieties recruit distinct endophytic microbiota (FigS.3D-L), suggesting that the niche has a considerable impact on endophyte microbiota establishment, while agricultural manipulation exert limited in uence on the constitution endophytes bacteria.As the plant organ-type (niche) explained 29.0% of the differential abundance in endophytic microbiota, and it will be essential to provide a clue for screening the bene cial microbiota involved in promoting growth and resisting the diseases (Valenzuela et al. 2018).
We may assume that the dynamics of endophytic bacteria communities are due to differing microenvironments in vivo and in vitro under different plant developmental stages, and the similar results showed that structure of endosphere microbiome was affected by the assembly process and plants growing periods (Ye et al. 2018).Most endophytic bacteria are recruited from the soil and rhizosphere bacteria in plant growth processes, maintaining the help with the suppression of pathogen, nitrogen xation, and this nding was consistent with the previous reports in other plant species, including Arabidopsis, soybean, rice and some of its relatives (Constantin et al. 2019).
Coordination between the host plant and the microbiota is critical to plant growth in natural environments.We found that roots of tomato recruited a higher proportion of Bacillus and Mesorhizoium (Fig. 2), indicating that the Firmicutes probably involved in the nurtrients uptake and plant metabolism, which could explain the majority of bene cial biocontrol-microbiota belong to this phylum in the root environment (Silvina et al. 2018).
Endophytic bacterial diversity studies indicate that taxonomic groups such as Bacteroidetes, Actinobacteria, Acidobacteria, Proteobacteria and Firmicutes are possibly involved in growth by enhancing nutrient acquisition and tolerance to biotic and abiotic stresses (Silvina et al. 2020).In the vegetative tissues (roots, stems and leaves), the bacterial genera Acinetobacter, Enterobacter, Pseudomonas and Pantoea were abundantly present in these samples, albeit in different amounts (Bulgarelli et al. 2012).
In the present study, bacterial communities varied among planting developmental stages.Numerous reports showed that rice genotype in shaping the root microbiota using a limited set of two to four rice varieties (Wei et al. 2020).Salt-accumulating halophyte S. European would depend on microbiota, which allow optimal growth in saline conditions (Sun et al. 2008).Plants not only actively in uence the microbiome structure in diverse niches by metabolites (Zhao et al. 2015), but also increase the abundance of bene cial communities in the endosphere at speci c growth periods (Romero et al. 2014).
In summary, the present study provides a holistic view of the composition, diversity and in uential factors shaping the endophytic bacterial communities associated with greenhouse grown tomato plants.This is still a case study and the knowledge gained in other long-term experiments should be veri ed to gain a comprehensive understanding of the role of soil microorganisms in plant health, as it is mainly designed with each speci c environmental signi cance (Zhao et al. 2016).Some bene cial bacterial strains have been isolated in our laboratories and their proper functions in tomato growth and health will be studied in the future.These efforts will provide an important data base for the further utilization of bene cial bacteria in tomato production.

Conclusion
Tomato endophyte microbiota have a distinct structure in different growth periods.Bacillus were enriched seeding stage and decreased in the fruiting stage while Mesorhizobium increased during in the fruiting stage.Tomato have distinct endosphere microbiota by comparing beta diversity of microbiota in different growth periods, compared with the manipulation resume and organ type.And a strong correlation was observed between the structure of the microbiota in the whole dataset and soil chemistry which indicated that the soil type and treatment affected the endosphere microbiota of tomato.Organ-type (niche)exert more in uence on the tomato microbiota compared with manipulation treatments between organicfarming and conventional farming.