Revealing the Tomato Endophyte Bacteria Communities Under Long-term Organic and Conventional Agricultural Practice System

Zeyu Zhang china agricultrual university Yabin Zhan College of Resources and Environmental Science, China Agricultural University, Beijing 100193, China Zengqiang Zhang College of Resources and Environmental Science, Northwest A&F University, Yanglin 712021, China Youzhou Liu Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210000, China Ting Xu College of Resources and Environmental Science, China Agricultural University, Beijing 100193, China Yuquan Wei College of Resources and Environmental Science, China Agricultural University, Beijing 100193, China Ji Li (  liji@cau.edu.cn ) College of Resources and Environmental Science, China Agricultural University, Beijing 100193, China

). Therefore, it is important to have a good understanding of the microbial communities. Bacterial can also live and thrive inside their host plants, which are called endophytes. Endophyte have been isolated from different parts of plants that are above and below ground including roots, stems, leaves, owers, fruits, tubers and seeds (Manzotti et al. 2020).
Bacterial endophyte normally completed their life cycle within host plants enhacing the plants to any tolerance to biotic and abiotic stresses (Kahremani et al. 2019). In addition, endophytic microorganisms can provide protection against fungal diseases, which can directly promote plant growth via phytohormone production, nutrient solubilization, and nitrogen xation and metabolism (Vector J et al.

2011).
Till now, little information is available about the bacterial community structure of tomato cultivated in a greenhouse environment, which has been widely used in tomato production. Exploring the structure of A long-term organic farming experiment were carried out in 2002 at the Quzhou Experimental Station in Hebei Province, China. All two farming system were under the same agricultural practice including 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 endophytic bacterial communities in plants grown under separate greenhouse. The structure of endophyte bacterial communities under different plant development stage (seeding stage, owering stage, fruiting stage and harvesting stage), organic and conventional agricultural practices and organ niche (root or stem) were explored in this study using 16s rRNA gene amplicion sequencing.
This study provides comprehensive insight into the bacterial communities associated with tomato cultivated in a greenhouse agro-ecosystem and provides useful information for the control of potential pathogens and promoting production in tomato cultivation.

Growth Conditions and Sample Collection
The eld experiment was conducted in Quzhou county, Hebei province (36°52′N, 115°01′E), and the longterm greenhouse experiment was set up in March 2002. The experiment consists of organic (ORG), lowinput (LOW), and conventional (CON) systems. Each system had three semi-round arch greenhouses (52 m in length and 7 m in width for each greenhouse). Crop varieties, irrigation, and tillage schemes are the same in all three systems in the same growing season.
Tomato samples (S. lycopersicum cultivar "Zhongza 302") were collected from the research greenhouse located in the organic and conventional greenhouse, and four plant 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. Thus 80 plant samples were sampled in total. Surface sterilization were performed in previously described (Gu et al. 2020), till no bacterial growth was detected after plating the roots on R 2 A agar at 30℃ for 7 days (

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 ).

Nucleotide Sequence Accession Numbers
The datasets supporting the conclusions of this article are available in the National Center for Biotechnology Information (NCBI) repository (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA316593/), under accession numbers SRR6214539. Data can be obtained from the BioSample database (https://www.ncbi.nlm.nih.gov/biosample).

Analysis of sequencing data
A total of 2,014,992 high-quality, non-plastid sequences were requested. The number of 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. Core microbiome analysis showed that total of 648 OTUs were consulted on 80 samples for the bacterial library, with 96 OTUs in common after ltering process (Fig.2).

Taxonomic distributions of tomato endophytic bacteria
The taxonomic summary revealed that the tomato endophyte bacteria mainly comprised four phyla (Fig. 3A), among which Proteobacteria was the most represented (40.63 ± 2.68%), followed by Firmicutes (21.76 ± 1.66%), Actinobacteria (20.3± 3.62%) and Bacteroidetes (8.7± 4.45%). A total of 253 different genus were identi ed across all samples, in addition, the relative abundance of the top 10 most abundant genus was shown in Fig. 3B. The core members of genus within the tomato samples were Weissella, Bacillus, Mesorhizobium and Chryseobacterium.spp. A heatmap was constructed to illustrate the relative abundance dynamics of the 10 most abundant genus in the whole crop season (Figure3C,3D). The abundance of sphingobacterium.spp and Streptomyces.spp gradually increased in the tomato roots under the organic practice greenhouse, while the Bacillus.spp gradually decreased. And the similar results in the tomato stem endophyte showed that the abundance of Arthrobacter.spp, Rhizobium.spp gradually increased, while the Bacillus.spp gradually decreased at the late crop season. The abundance of Bacillus.spp and Acinetobacter.spp gradually decreased in tomato roots, and the abundance of sphingobacterium.spp and Streptomyces.spp gradually increased in the conventional agricultural greenhouse. And the abundance of Bacillus.spp gradually decreased in stem endophyte, and the abundance of Arthrobacter.spp and Rhizobium.spp gradually increased.
Endosphere communities are diverse among tomato organ niche The alpha diversity (Shannon index) of microbiota had no signi cant difference in different plant development stage (Fig. 4A). However, Shannon diversity index of microbiota from the endosphere-root sample were more diverse than those from the endosphere-stem sample either organic or conventional agricultural practice (P<0.001, Fig. 4B).
The beta diversity and PCoA analyses using Bray-Curtis distance matrices showed that bacterial microbiota of different organ niche in four plant growth periods datasets were separated in the rst two coordinate axes (Fig. 5A, permutations analysis of variance, Bray-Curtis distance, R 2 = 0.084, P =0.003). Tomato endosphere microbiota were distinguished from the seeding, owering, fruiting and harvesting stage. Principal coordinate analysis (PCoA) of Bray-Curtis distance showed that endosphere microbiota in four development stage separated clusters in the rst two coordinate axes (Fig. 5B, permutations analysis of variance, Bray-Curtis distance, R 2 = 0.173, P =0.001), and all of the pro les indicated that endosphere microbiota changed with the plant development time.
Tomato endosphere microbiota in different organ niche and agricultural practices treatments were also distinct. Speci cally, the seeding stage (TI, Fig. 5C-E), owering stage (T2, Fig. 5F-H), fruiting stage (T3, Fig.5I-K) and harvesting stage (T4, Fig. 5L-N) datasets selected were used to analysis the organ niche and agricultural practice treatment factor. Organic system root endophyte (ORGR), conventional system root endophyte (CONR), organic system stem endophyte (ORGS), conventional system stem endophyte (CONS)datasets from each plant development stage were used to analysis the agricultrual practice treatment factor. Seeding stage (TI) datasets selected from the whole dataset were used to analysis the organ niche factor, and root (R) and stem (S) datasets were used to analysis the agricultural practices system factor at seeding stage (Figs.5C-N). The beta diversity and PCoA analyse using Bray-Curtis distance showed that bacterial microbiota of different organ niche in four sampling time datasets were separated in the rst two coordinate axes (permutations analysis of variance R 2 = 0.283, P=0.001), and similar trend occurred in the owering stage, fruiting stage and harvesting stage. (Figs. 5F,5I, 5L).However, agricultural practices treatment factors had no cluster in different datasets and rarely separated in the rst two coordinate axes (Figs.5D,5E, 5G,5H, 5J,5K,5M,5N).

Discussion
During the long-term organic farming experiment from Quzhou county in 2002, three agricultural practice were carried out. Here, we explore the endophyte constitution under two agricultural practice systems. In general, the diverse from rhizosphere to rhizosphere to phylloxera to endosphere is minimal (Lee et al. 2018), so low abundance and diversity in root and stem endophyte bacterial are reasonable in this study.
No signi cance was found in alpha diversity over different plant development time (Fig. 3A). It is widely accepted that assembly of endosphere microbiome in plant is driven by many aspects, including climate environment, soil source, host developmental stage, cultivation practice, and root architecture (Ye et al. 2018), and our research showed that organ niche varieties as well as different plant development stage recruited distinct endophyte microbiota (Figs.5B,5C,5F,5I,5L), suggesting that the niche had an signi cant impact on endophyte microbiota establishment, while agricultural practice exerted limited in uence on the constitution endophytes bacteria under greenhouse condition. 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 Coordination between the host plant and the microbiota is critical for plant growth in natural environments. We found that roots recruited a higher proportion of Bacillus and Mesorhizoium (Fig. 3), 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).
We have demonstrated the prevalent role of organ niche in shaping the assembly and composition of the endosphere microbiome, and PERMANOVA for beta diversity based on the combined data from the organ In summary, this study provided a comprehensive view of the organ niche and agricultural practice factors shaping the endophyte bacterial communities associated with tomato under greenhouse. Some bene cial endophyte bacterial strains have been isolated in this study and their functions in promoting growth and health will be studied in the future. These efforts will provide an important clue for the application of bene cial endophyte bacteria into tomato agricultural production. The interaction between the plant and related microbiota research could pave the way for technologies to modulate the plant microbiota that increase crop productivity and resist plant pathogen infection.

Conclusion
Tomato endophyte microbiota have a distinct structure in different plant development stage. Bacillus.spp were enriched seeding stage and decreased in the fruiting stage while Mesorhizobium increased during in the fruiting stage. Organ niche had a signi cant impact on the endosphere microbial communities of tomato.

Declarations
Availability of data and materials All data analyzed during this study are included in this published article and its supplementary information les.

Funding
This work was nancially supported by the National Natural Science Foundation of China (201503010311304).
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable. Rarefaction curves for bacterial OTUs, clustering at 97 % rRNA sequence similarity. TIORGR organic system root endophyte in the seeding stage, T2ORGR: organic system root endophyte in the owering stage, T3 ORGR: organic system root endophyte in the fruiting stage, T4ORGR: organic system root endophyte in the harvesting stage. TICONR: conventional system root endophyte in the seeding stage, T2CONR: organic system root endophyte in the owering stage, T3 CONR: organic system root endophyte in the fruiting stage, T4CONR: organic system root endophyte in the harvesting stage. TIORGS: organic system stem endophyte in the seeding stage, T2ORGS: organic system stem endophyte in the owering stage, T3 ORGS: organic system stem endophyte in the fruiting stage, T4ORGS: organic system stem endophyte in the harvesting stage. TICONS: conventional system stem endophyte in the seeding stage, T2CONS: conventional system stem endophyte in the owering stage, T3 CONS: conventional system stem endophyte in the fruiting stage, T4CONS: organic system stem endophyte in the harvesting stage.

Figure 2
Venn diagram showing the shared OTUs among the 80 samples. TIORGR organic system root endophyte in the seeding stage, T2ORGR: organic system root endophyte in the owering stage, T3 ORGR: organic system root endophyte in the fruiting stage, T4ORGR: organic system root endophyte in the harvesting stage. TICONR: conventional system root endophyte in the seeding stage, T2CONR: organic system root endophyte in the owering stage, T3 CONR: organic system root endophyte in the fruiting stage, T4CONR: organic system root endophyte in the harvesting stage. TIORGS: organic system stem endophyte in the seeding stage, T2ORGS: organic system stem endophyte in the owering stage, T3 ORGS: organic system stem endophyte in the fruiting stage, T4ORGS: organic system stem endophyte in the harvesting stage. TICONS: conventional system stem endophyte in the seeding stage, T2CONS: conventional system stem endophyte in the owering stage, T3 CONS: conventional system stem endophyte in the fruiting stage, T4CONS: organic system stem endophyte in the harvesting stage.