Composition and Diversity Of Endophytic Bacterial Community in Seeds of Upland Rice Resources from Different Origin Habitats in China

doi:10.21203/rs.3.rs-1001992/v1

Upland rice has the characteristics of strong drought tolerance and wide adaptability. Cultivating upland rice with high yield and high quality can solve the contradiction between food shortage, water shortage, and population increase in countries all over the world, and is of great significance to the sustainable development of agriculture. In this study, high-throughput sequencing technology based on the Illumina Miseq platform was used to investigate the structure and diversity of endophytic bacterial communities using 12 upland rice variety seeds from different areas in Yunnan Province of China as materials. This study aims to reveal the "core microbiota" of the endophytic bacteria in upland rice seeds in the Yunnan Province of China by examining their diversity and community structures. The results showed that 39 endophytic OTUs were found to coexist in all samples. At the phylum level, the first dominant phyla in the 12 seed samples were Proteobacteria (66.92-99.98%). At the genus level, Pantoea (9.75-99.24%), Pseudomonas (0.11-37.24%), Curtobacterium (0.01-19.90%), Microbacterium (0.01-14.95%), Methylobacterium (0.40-5.86%), Agrobacterium (0.01-4.53%), Sphingomonas (0.04-1.56%), Aurantimonas (0.01-1.45%) and Rhodococcus (0.11-1.09%) served as the dominant genera that coexisted in all the upland rice seeds tested and represent the core microbiota in upland rice seeds. Through the correlation analysis with upland rice habitat environmental factors, the effects of climate and altitude on the structure and diversity of endophytic bacterial community in upland rice seeds were further revealed. The results showed that environmental factors such as temperature, precipitation and altitude have great influences on the structure of endophytic bacterial community in upland rice seeds. This study is of great significance to explore the relationship between upland rice and its endophytic bacteria and to tap the resources of drought-tolerant bacteria to improve the yield of local upland rice.

General Biochemistry

General Microbiology

Food Science & Technology

Yunnan Province of China

Upland rice seed

Endophytic bacteria

Diversity

Environmental factor

The food issue is a major issue related to the national economy and people's livelihood, and food security is an important part of national security. As we all know, the three major food crops in the world are wheat, rice and corn and wheat is the food crop with the largest sown area, the largest yield, and the most widely distributed in the world, while rice ranks second. According to statistics, rice is cultivated in 122 countries in the world, with a perennial cultivation area of 140-150 million hectares, and is widely distributed. However, droughts caused by persistent climate instability and unpredictable rainfall patterns have had a significant impact on rice cultivation, especially in sub-Saharan Africa and Southeast Asian countries (Khan et al. 2020). Rice is also the main food crop in China, and China is the country that produces the most rice in the world. However, the shortage of water resources in China has a great impact on rice cultivation and forces researchers to step up their research on upland rice to deal with the problem. Upland rice is a more drought-tolerant ecotype crop than rice and has the characteristics of drought tolerance, barren tolerance and wide adaptability. It is mainly planted in the Yellow-Huaihe River Basin of China and some places with insufficient water resources or uneven space and time.

Plant endophyte is a kind of important microbial resource that lives in various tissues and organs of healthy plants at a certain stage or all stages without causing infection symptoms. In the process of long-term interaction, plants and endophytes have formed a symbiotic unit and become an important part of plant evolution. The discovery of plant endophytes can be traced back to more than 100 years ago, but it has been ignored because of its characteristics, that is, it does not cause host plants to show symptoms similar to pathogen infection. Until the 1930s, many livestock were poisoned by eating herbage infected with endophytic fungi and it caused great losses to animal husbandry, and the study of plant endophytes attracted people's attention. Studies have shown that there are a large number of endophytic bacteria in plants. They mainly settle in the internal tissue of the host plant and form a series of mutually beneficial and symbiotic relationships with plants (Hassani et al. 2018). In addition to the biological functions of endophyte, such as promoting seedling germination, promoting plant growth, increasing yield, enhancing resistance and inducing the synthesis of secondary metabolites, their natural products have potential in pharmaceutical, agricultural, industrial and other fields (Ansary et al. 2018; Mohamad et al. 2018; Rangjaroen et al. 2019; White et al. 2019). And a large number of studies have shown that the establishment of plant endophyte diversity and community structure is closely related to plant variety, genotype, growth environment and geographical location (Edwards et al. 2015; Santoyo et al. 2016; Rybakova et al. 2017; Liu et al. 2017, 2019, 2020; Walitang et al. 2018 a, b; Zhang et al. 2018b; Raj et al. 2019).

Seeds are the reproductive organs of plants and the preservers and transmitters of plant genetic information. Seeds are also the adaptive strategy to ensure plant species reproduction under stress, and it is of great significance to plant reproduction and agricultural production (Steinbrecher and Leubner-Metzger 2017; Domergue et al. 2019). A large number of studies have proved that plant seeds are not only rich in endophytic resources, but also the medium to spread these microorganisms from maternal plants to offspring (Walitang et al. 2018a; Berg and Raaijmakers 2018; Li et al. 2019a; Liu et al. 2019). They are responsible for transferring beneficial bacteria from parent plants from generation to generation, thus achieve a direct or indirect impact on plant growth and development, health, quality, yield and functional components and other biological characteristics (Walitang et al. 2018a; Berg and Raaijmakers 2018; Li et al. 2019a; Liu et al. 2019). Endophytic bacteria are the most easily identified microorganisms from plant seeds by researchers. A large number of studies have found that the dominant endophytic bacteria in plant seeds are Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes phyla, and the dominant genera are Pantoea, Bacillus, Pseudomonas, Sphingomonas, Microbacterium and Acinetobacter (Truyens et al. 2015; Scott et al. 2018; Zhang et al. 2018b; Wang et al. 2020).

The conventional culture-dependent method, 16S rDNA clone library technique and denaturing gradient gel electrophoresis (DGGE) have been used to study endophytes in plant seeds (Hardoim et al. 2012; Jiang et al. 2013; Rybakova et al. 2017; Liu et al. 2017; Haimin et al. 2018). In addition to these technologies, high-throughput sequencing has gradually become a new method to evaluate and analyze the diversity and community structure of endophytes in plant seeds. In the previous research, the undergraduate research group also used high-throughput sequencing technology to explore the endophytic bacterial diversity and community structure of 14 upland rice seeds collected in different areas of China (Wang et al. 2020). However, compared with other plant tissue microorganisms, research on plant seed endophytes is still very limited, in particular, there are few studies on the effects of environmental factors related to plant growth on the diversity and community structure of endophytic bacteria in their seeds. In-depth study of endophytic bacteria in upland rice seed is not only conducive to the improvement of its subsequent yield, but also of great help to explore the mechanism of drought tolerance.

Based on the Illumina Miseq platform, the diversity and community structure of endophytic bacteria in 12 upland rice seeds from Yunnan Province of China were analyzed to explore the similarities and differences of endophytic bacteria in upland rice seeds growing in Yunnan Province of China. On this basis, combined with the temperature, humidity and altitude of each variety origin area, the effects of environmental factors of upland rice origin areas on the diversity and community structure of endophytic bacteria in upland rice seeds were studied. Besides, combined with our previous studies on the diversity and community structure of endophytic bacteria in upland rice seeds in different areas, we can further clarify the "core microbiota" of upland rice seeds.

The source of upland rice seeds

The 12 upland rice seed samples were provided by Hunan Hybrid Rice Research Center. The origin areas and related information of all samples are shown in Fig. 1 and Table 1. And all samples were uniformly planted on the Sanya Division Farm in Hainan Province and harvested on March 24, 2019. After that, the samples were transferred to sterile bags, sealed and stored at 4 ℃ until used (It can be preserved for a long time in a sealed environment).

Table 1

Summary of detailed information of upland rice seed samples
Sample ID	Variety names	Indica/Stem	Breeding places	Abbreviatios
19H011	Sanbangqishiluo	Stem	Dehong Prefecture, Yunnan Province	DH
19H012	Benbanggu	Stem	Gengma County, Yunnan Province	GM
19H013	Zimi	Stem	Jinping County, Yunnan Province	JP
19H014	Laozaogu	Indica	Jiangcheng County, Yunnan Province	JC
19H015	Xiaohonggu	Stem	Luchun County, Yunnan Province	LC
19H017	Mowanggunei	Stem	Mengla County, Yunnan Province	ML2
19H018	Jixuenuo	Stem	Gengma County, Yunnan Province	GM
19H019	Fanhaopi	Indica	Zhenkang County, Yunnan Province	ZK
19H022	Haobuka	Stem	Jinghong Country, Yunnan Province	JH
19H024	Haobayong 1	Stem	Menglian County, Yunnan Province	ML1
19H029	Lamujia	Stem	Ximeng County, Yunnan Province	XM
19H032	Haogelao	Stem	Menglian County, Yunnan Province	ML1

Sample surface sterilization and treatment

Three replicates of each sample were selected in this study. Firstly, the husks of each upland rice seed sample were removed by a small sheller. Then, under aseptic conditions the following operations were performed in the order listed: husked seeds were washed three times with prepared sterile water; 5 g of seeds were placed in a clean and sterile 50-mL tube containing 25 mL of phosphate buffer (per liter: 7.15 g of NaH₂PO₄·2H₂O, 22.04 g of Na₂HPO₄·12H₂O, 200 µL of Silwet L-77) (Zhang et al. 2018b; Liu et al. 2019), and the seeds were sonicated twice by an Ultrasonic Processor Scientz-IID sonicator (NingBo Scientz Biotechnology Co., Ltd., China) at low power (237.5 W; 950 W × 25%) in an ice bath for 5 min (alternating thirty 2-s bursts and thirty 2-s rests) (Zhang et al. 2018b; Liu et al. 2019). To validate that the surface was sterilized, sterile tweezers were used to press surface-sterilized seeds into LB medium (LUQIAO), and the samples were incubated at 30°C for 72 h.

DNA extraction

Five grams of surface-sterilized upland rice seeds from each sample was frozen with liquid nitrogen and was quickly ground into a fine powder with a pre-cooled sterile mortar, and then the DNA was extracted using the FastDNA® SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA) following the manufacturer’s instructions of the Kit.

Amplicon library preparation and sequencing

All PCR amplifications were performed using TransStartFastPfu DNA Polymerase (TransGen, Beijing, China). For rice seeds, 799F (5’-AACAGGATTAGATACCCTG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) were used for the first-round amplification. Then the 750 bp fragment amplified from endophytic bacteria was used as the template for the second-round amplification for the V6-V8 region (968F: 5’-AACGCGAAGAACCTTAC-3’ and 1378R: 5’-CGGTGTGTACAAGGCCCGGGAACG-3’) (4-7bp barcode was added to the 5' primer of 968F and 1378R). All of the thermocycling steps were as follows: 5 min at 95 ℃; 20 cycles of 45 sec at 95 ℃, 30 sec at 57 ℃, and 30 sec at 72 ℃. The amplified products were purified and mixed in equivalent amounts. After using the TruePrep® DNA Library Prep Kit V2 for Illumina (Vazyme, China) to perform the library, all samples were sequenced by 300-bp-paired-end sequencing with a MiSeq platform using the MiSeq® Reagent Kit v3 (600 cycles) (Illumina).

Sequence data processing

The assembly of paired FASTQ files was performed by Mothur (version 1.39.0) (Schloss et al. 2011). Briefly, paired sequence reads were assembled after removing raw reads with ambiguous bases or low quality, such as read length < 50 bp, average Qscore < 25, or reads not matching the primer (pdiffs = 0) and barcode (bdiffs = 0). The high-quality DNA sequences were aligned to the SILVA reference database (V119) (Quast et al. 2013), and using chimera. uchime module to remove chimera sequences. Then the reads were classified and grouped into OTUs (operational taxonomic units) under the threshold of 97% identity.

Data statistics

Community richness, evenness and diversity analysis (Shannon, Simpson, ACE, Chao and Good’s coverage) were performed using Mothur. Both PCoA (Principal Co-ordinates Analysis) and NMDS (Non-metric Multidimensional Scaling) were analyzed based on the tayc matrix by mothur. The t-test (with 95% confidence intervals) was used to determine whether the means of evaluation indices were statistical difference, and p-value < 0.05 was considered as a significant standard. Taxonomy was assigned using the online software RDP classifier (Wang et al. 2007) at default parameter (80% threshold) based on the Ribosomal Database Project (Cole et al. 2009). Genera and family abundance differences between samples were analyzed by Metastats (White et al. 2009). Spearman correlation coefficient between two variables was calculated using the R command "cor.test". RDA analysis based on genus level was performed by "vegan" package in R. Average annual precipitation, average annual temperature and altitude were selected to be variable parameters.

Sequence accession numbers

The raw high-throughput sequencing data were submitted to the NCBI database with Accession number SRR13319808-SRR13319843 and BioProject number PRJNA688367.

Diversity analysis of endophytic bacteria in upland rice seeds

According to the information of barcode and front-end primers, the quality control sequences were divided into 36 groups of sequence files, and a total of 2,089,709 high-quality sequences were obtained, with an average of 58,047 sequences per sample (Supplementary Table S1). Because of the large sample size, we use the added value of the repeated samples as the final calculation. The original diversity data are shown in Supplementary Table S2. According to the difference of distance between the sequences, 16S rRNA genes obtained were clustered into OTUs for species classification under the similarity level of 97%. A total of 5,704 OTUs were generated from all samples, with the number of OTUs in each sample ranging from 322 to 1,527 (Table 2). The statistical results showed that 39 endophytic OTUs (0.68%) coexisted in the 12 sample seeds. And each sample contained its unique OTUs. The proportions of unique OTUs were 41.23%, 28.28%, 47.20%, 34.26%, 33.82%, 42.31%, 32.47%, 31.78%, 27.93%, 37.43%, 33.29% and 29.07% in sample 19H011, 19H012, 19H013, 19H014, 19H015, 19H017, 19H018, 19H019, 19H022, 19H024, 19H029 and 19H032, respectively. These results indicated that the endophytic bacteria in the seeds of different upland rice varieties were different.

Table 2

Statistical results of endophytic bacterial alpha-diversity in each sample
Samples	OTUs	ACE	Chao	Shannon	Simpson
19H011	1237	2152.41±283.50^a	1450.72±138.08^ab	2.49±0.11^abc	0.25±0.03^de
19H012	548	993.58±109.81^c	681.90±76.09^d	1.34±0.09^e	0.44±0.04^bc
19H013	322	1493.91±194.63^bc	565.28±59.46^d	0.35±0.03^f	0.85±0.01^a
19H014	540	1128.40±144.40^bc	824.16±157.67^cd	1.29±0.00^e	0.40±0.00^bc
19H015	1236	2178.22±93.51^a	1481.45±145.92^ab	2.11±0.56^cd	0.35±0.15^cd
19H017	1527	2503.33±524.37^a	1824.46±385.30^a	2.86±0.49^a	0.18±0.09^e
19H018	1281	2187.95±121.99^a	1547.97±116.65^a	2.65±0.07^ab	0.18±0.02^e
19H019	774	1274.84±101.17^bc	830.52±119.33^cd	1.50±0.07^e	0.49±0.02^b
19H022	752	1371.88±160.68^bc	895.02±38.10^cd	1.54±0.08^e	0.44±0.04^bc
19H024	1488	2636.47±208.10^a	1758.92±99.96^a	2.27±0.14^bc	0.34±0.04^cd
19H029	706	1301.53±22.02^bc	882.11±40.84^cd	1.65±0.00^de	0.38±0.00^bcd
19H032	860	1544.71±195.49^b	1101.41±119.54^bc	1.67±0.07^de	0.35±0.01^cd
Note: The difference of bacterial alpha-diversity based on a 16S rDNA sequence assignment dataset with a 97% sequence similarity threshold in upland rice seeds. Values were tested in analysis of variance (ANOVA) with three replicates in each treatment. Data within the same diversity indexes followed by the same lowercase letters are not significantly different at p = 0.05 level.

The diversity (α diversity) indexes of samples include ACE, Chao, Shannon and Simpson values, and the ACE and Chao values are used for sample abundance assessment, and Shannon and Simpson values are used for sample diversity assessment. In general, there are some differences in diversity and abundance of each sample, and the sample 19H011, 19H015, 19H017, 19H018 and 19H024 have significant differences in ACE, Chao, Shannon and Simpson values from other samples (Table 2). The values of ACE, Chao and Shannon of sample 19H017 were significantly higher than those of other samples, and the values of sample 19H013 were the lowest. These results implied that the diversity and richness of endophytic bacteria in sample 19H017 were the highest among all samples, and that of sample 19H013 was the lowest. The above results are consistent with the results of Rarefaction curve and Rank abundance curve of all samples in the case of OUT = 0.03 (Supplementary Fig. S1 and Fig. S2).

Bacterial endophytes community compositions and structures of upland rice seeds

The endophytic bacterial community composition of 12 upland rice seed samples from different areas in Yunnan Province of China is shown in Fig. 2 at the phylum level. The endophytic bacterial community structure of 12 samples had low diversity at the phylum level, mainly including Proteobacteria and Actinobacteria. The relative abundance of Proteobacteria was relatively high, and the relative abundance of different samples was between 66.92% and 99.98%. And followed by Actinomycetes with abundance ranging from 0.01–32.21%. At the genus level, 148 genera were covered by endophytic bacteria in all upland rice seed samples, and the main bacteria with high relative abundance were Pantoea, Pseudomonas, Curtobacterium, Microbacterium, Methylobacterium, Agrobacterium, Sphingomonas, Aurantimonas and Rhodococcus, and the proportion ranges from 9.75-99.24%, 0.11-37.24%, 0.01-19.90%, 0.01-14.95%, 0.40-5.86%, 0.01-4.53%, 0.04-1.56%, 0.01-1.45% and 0.11-1.09%, respectively (Fig. 3). Table 3 listed in detail the dominant genera and proportion of each upland rice seed sample. According to the results, we can know that there were significant differences in the abundance distribution of endophytic bacteria in different upland rice seed samples. The classification of samples at the level of 97% sequence similarity (genus, top10) in Fig. 4 also showed that the abundance distribution of endophytic bacteria in each seed sample was different at the genus level.

Table 3

Statistical results of dominant genera in each upland rice seed sample
Taxonomy	19H011	19H012	19H013	19H014	19H015	19H017
Pantoea	50.70%	78.83%	99.24%	90.28%	63.48%	9.75%
Pseudomonas	4.11%	13.56%	0.11%	1.64%	3.11%	37.24%
Curtobacterium	3.91%	0.22%	0.01%	2.64%	1.18%	19.16%
Microbacterium	14.95%	0.37%	0.01%	2.12%	14.49%	6.05%
Methylobacterium	2.51%	0.19%	0.01%	0.24%	1.93%	5.86%
Agrobacterium	4.53%	0.23%	0.11%	0.10%	2.03%	0.52%
Sphingomonas	0.77%	0.24%	0.04%	0.05%	1.56%	0.96%
Aurantimonas	0.69%	0.22%	0.01%	0.07%	1.06%	0.75%
Rhodococcus	0.07%	0.04%	0.01%	0.01%	0.10%	1.09%
Acidovorax	0.74%	0.02%	0.01%	0.01%	0.23%	0.03%
Taxonomy	19H018	19H019	19H022	19H024	19H029	19H032
Pantoea	30.49%	74.82%	73.06%	68.47%	67.13%	83.78%
Pseudomonas	33.47%	2.84%	14.23%	2.40%	5.21%	5.14%
Curtobacterium	5.05%	13.95%	2.11%	1.50%	19.90%	0.22%
Microbacterium	7.05%	1.21%	0.75%	7.26%	0.36%	0.50%
Methylobacterium	2.17%	0.24%	0.25%	3.34%	0.32%	0.44%
Agrobacterium	1.47%	0.07%	0.22%	2.29%	0.01%	0.61%
Sphingomonas	0.84%	0.17%	0.25%	1.48%	0.11%	0.50%
Aurantimonas	1.17%	0.08%	0.18%	1.45%	0.11%	0.42%
Rhodococcus	0.29%	0.06%	0.06%	0.04%	0.08%	0.01%
Acidovorax	0.03%	0.01%	0.01%	0.12%	0.01%	0

To explore the differences in the community structure of endophytic bacteria in the 12 upland rice seed samples, PCoA and NMDS methods were used to draw the two-dimensional distribution diagram of seed samples (Fig. 5 and Fig. 6). The distance of each sample in the two-dimensional diagram can reflect the degree of community structure similarity, and the closer the distance between sample points is, the more similar the community structure is. The results of PCoA and NMDS showed that the distance between sample 19H019 and 19H029 was close, and the distance between sample 19H012, 19H015, 19H022 and 19H024 was close. The relult implied that the endophytic bacterial community structure of sample 19H019 and 19H029 was similar, and the endophytic bacterial community structure of sample 19H012, 19H015, 19H022 and 19H024 was similar.

Analysis of environmental factors affecting the community structure and diversity of endophytic bacteria in upland rice seeds

In order to further explore the impact of environmental factors of origin areas on the endophytic bacterial community structure and diversity of the sample seeds, we inquired about the temperature, precipitation and altitude of the corresponding areas in Yunnan Province of China. The specific data are shown in Table 4 and Fig. 1B. Then we showed the relationship between environmental factors and sample distribution and the main dominant bacteria by RDA (Redundancy Analysis), taking the average annual precipitation, average annual temperature and altitude as variables (Fig. 7). The effect of environmental factors on endophytic bacteria in seeds in the RDA diagram is mainly characterized by the length of environmental factors, while the influence degree of environmental factors on each strain is reflected by the cosine value of the angle. Temperature, precipitation and altitude have great effects on endophytic bacteria in upland rice seeds, and precipitation and altitude are the main influencing factors (Fig. 7). There was a significant positive correlation between the main dominant bacteria Pantoea and precipitation, temperature and altitude, and the correlation between Pantoea and altitude was the strongest. However, there was a negative correlation between other dominant bacteria and environmental factors. The proportion of other bacteria is low and no further analysis is made.

Table 4

Environmental data statistics of origin areas of upland rice
Breeding places	Average annual precipitation/mm	Average annual temperature/℃	Altitude/m
DH	1500	19.2	800-2000
GM	1300	18.8	450-3323
JP	2400	22	1470-5958
JC	2300	18.7	317-2207
LC	2400	16.6	320-2637
ML2	1700	21.8	480-2023
ZK	1600	18.7	510-2468
JH	1400	20.3	485-2196.8
ML1	1400	19.6	497-2603
XM	2800	15.2	590-2458.9

At present, the research on upland rice is mainly focused on its drought tolerance and genetic improvement (Xia et al. 2019; Abdirad et al. 2020; Luo et al. 2020b; Uddin and Fukuta 2020). Studies on the root microbial diversity of upland rice and its effects on the growth and drought tolerance of upland rice have also been reported (Pang et al. 2020). Our research group also explored the diversity and community structure of endophytic bacteria in upland rice seeds in different regions of China and preliminarily revealed the core microbiota of endophytic bacteria in upland rice seeds (Wang et al. 2020). In this study, the data obtained by sequencing and analysis can reflect the composition of endophytic bacteria in all upland seed samples, and the results showed that Proteobacteria and Actinomycetes represented the main dominant phyla of endogenous bacteria in upland rice seeds in this study. A large number of research reports also pointed out that Proteobacteria was the main dominant bacteria in plant seeds (Truyens et al. 2015; Scott et al. 2018; Zhang et al. 2018b; Wang et al. 2020).

Overall, this study found that the main endophytic bacterial groups and community structure of the 12 upland rice seed samples in Yunnan Province of China were similar. The overlapping and non-overlapping areas of the Venn diagram are usually used to represent the shared OTUs and unique OTUs existing in each group of samples or each sample (Vandenkoornhuyse et al. 2015), but due to the large number of samples, the Venn diagram could not be drawn. Through the statistics of Venn diagram data (Supplementary Table S3), it is found that the shared OTUs of all samples was 0.68%, and the abundance accounted for > 2.55% of the total OTUs in each seed sample. The primary shared genera were Pantoea, Pseudomonas, Curtobacterium, Microbacterium, Methylobacterium, Agrobacterium, Sphingomonas, Aurantimonas and Rhodococcus. Pantoea (9.75-99.24%) was the first dominant genus shared by all 12 seed samples, while other shared genera were represented by Pseudomonas (0.11-37.24%), Curtobacterium (0.01-19.90%), Microbacterium (0.01-14.95%), Methylobacterium(0.40-5.86%), Agrobacterium (0.01-4.53%), Sphingomonas (0.04-1.56%), Aurantimonas (0.01-1.45%) and Rhodococcus (0.11-1.09%). Although most species of Pantoea are usually well-known plant pathogens, in our previous studies on the endophytic bacteria of upland rice seeds collected in different regions, we also found that Pantoea was the first dominant genus in all samples (Wang et al. 2020). Besides, a large number of studies have also reported that Pantoea is the endophytic dominant genus in rice, barley, maize, rape, hemp and Salvia miltiorrhiza seeds (Granér et al. 2003; Liu et al. 2017, 2019; Shahzad et al. 2017; Walitang et al. 2017; Chen et al. 2018; Rahman et al. 2018; Scott et al. 2018; Zhang et al. 2018b; Raj et al. 2019). As a dominant endophyte, potentially pathogenic bacteria Pantoea exists in healthy upland rice and other plant seeds, but does not show disease symptoms, indicating that it should have other effects on plants. It has been proved that some species of Pantoea do play an important role in promoting plant growth. Luziatelli et al. (2020) have found that Pantoea agglomerans C1 isolated from lettuce not only has biological functions such as producing indole-3-acetic acid (IAA), dissolving phosphate and inhibiting plant pathogens, but also has high biotechnology potential as a plant growth-promoting bacteria in heavy metal contaminated soil. Another study has also found that endophytic bacteria Pantoeaagglomerans can well alleviate abiotic stresses such as drought and salt in durum wheat (Cherif-Silini et al. 2019). Besides, a large number of reports show that Pantoea is of great help to plant growth, yield increase and disease resistance (Megías et al. 2016; Lumactud and Fulthorpe 2018; Zhang et al. 2018a).

In addition to Pantoea, the main genus of shared endophytic bacteria, some dominant genera, including Pseudomonas, Curtobacterium, Microbacterium, Methylobacterium, Agrobacterium, Sphingomonas, Aurantimonas and Rhodococcus, are also shared by all 12 upland rice seeds. This result is basically consistent with our previous studies on endophytic bacteria in upland rice seeds and once again strongly indicates that these bacteria are the core endophytic bacterial communities in upland rice seeds. Pseudomonas is usually the dominant genus in the seeds of barley (Rahman et al. 2018), rice (Walitang et al. 2017; Liu et al. 2019), maize (Liu et al. 2012; Liu et al. 2020), rape (Granér et al. 2003), sugar beet (Kanivets and Pishchur 2001), peanuts (Sobolev et al. 2013), browntop millet (Verma and White 2018), Marama beans (Chimwamurombe et al. 2016), hemp (Scott et al. 2018), tobacco (Li et al. 2019b) and Salvia miltiorrhiza (Chen et al. 2018). Besides, Curtobacterium, Microbacterium, Methylobacterium, Agrobacterium, Sphingomonas and Aurantimonas are often found in the seeds of these plants as endophytic dominant genus. It is also reported that many species of these bacteria have important contributions in promoting plant growth and improving plant nitrogen fixation, resistance to harmful bacteria, cold resistance, drought resistance and tolerance to heavy metals. For example, Kumawat et al. (2019) found that the synergism of Pseudomonas aeruginosa and Bradyrhizobium sp. can improve plant growth, nutrient acquisition and soil health in soybean. Another study found that endophytic bacteria Pseudomonas stutzeri A15 isolated from rice had strong nitrogen fixation ability and was significantly better than that of chemical nitrogen fertilizer after inoculation of rice (Pham et al. 2017). The researchers also found that endophytic bacteria Curtobacterium, Microbacterium and Methylobacterium carried in plant tissues generally can promote host plants to produce auxin, dissolve phosphate and inhibit Fusarium and other fungal pathogens (Sánchez-López et al. 2018; Verma and White 2018; Passari et al. 2019). Through the study, Feng et al. (2021) found that rice would actively recruit more degradable endophytes for symbiotic interaction under pesticide chlorpyrifos exposure, to ensure the normal growth of rice plants under pollution stress. Of course, for upland rice, the ability of endophytic dominant bacteria in their seeds to withstand the abiotic stress of drought is undoubtedly the most important. According to the existing research, it can be found that Pseudomonas, Microbacterium, Methylobacterium and Sphingomonas isolated from plants play a great role in improving the drought tolerance and drought resistance of plants (Wang et al. 2014; Egamberdieva et al. 2015; García-Fontana et al. 2020; Luo et al. 2020a; Zhang et al. 2020). Therefore, as dominant genera, these bacteria participate in the community structure of endophytic bacteria in upland rice seeds and may be of great significance to the drought resistance of upland rice. As for the drought-resistant bacteria, we speculate that some of them may have been actively recruited from the environment by upland rice under drought stress. It is worth mentioning that different from other genera of endophytic dominant bacteria, Rhodococcus is rarely reported as a dominant genus in plant endophytes. The ability of Rhodococcus to degrade a variety of toxic chemicals and produce bioactive substances is the most frequently reported, and more and more attention has been paid to it. Reports on the Rhodococcus also show that some of its species not only have the function of producing cytokinin but also can colonize the root system and increase the plant biomass in polychlorinated biphenyl (PCBs) contaminated soil (Jameson 2019; Vergani et al. 2019).

Through this study, we also found that although there were significant differences in endophytic bacterial diversity, abundance and community structure in some of the 12 upland rice samples from origin areas of Yunnan Province of China, the difference of some of the samples was very small. The results of PCoA and NMDS showed that all samples could be separated in PC1-PC2 or NMDS1-NMDS2 coordinate system (Fig. 5 and Fig. 6), and there were differences among different upland rice seeds by comparing the diversity index of different upland rice varieties (Table 2). This further shows that the differences in varieties and genotypes of upland rice do have a certain effect on the diversity and community structure of endophytic bacteria in upland rice seeds. A large number of studies on the community structure and diversity of endophytic bacteria in plant seeds have also found that although there are coexisting endophytic dominant bacterial groups among different varieties of plant seeds, at the same time, they have significant differences in endophytic bacterial community structure and diversity (Xu et al. 2014; Rybakova et al. 2017; Liu et al. 2017, 2019, 2020; López et al. 2018; Walitang et al. 2018 ab; Raj et al. 2019). Although some studies have found that the endophytic bacterial community structure and diversity among different varieties of seeds are very similar, there are still some differences in the abundance of bacterial groups (Zhang et al. 2018b; Wang et al. 2020). However, there are no significant differences in endophytic bacterial diversity and community structure in some upland rice seeds, and they are particularly close in PCoA and NMDS results (Fig. 5 and Fig. 6). Therefore, the diversity and community structure of endophytic bacteria in upland rice seeds should be affected not only by variety differences, but also by other factors, such as environmental factors of origin areas, structure and chemical composition of samples (Torres-Cortés et al. 2018; Girsowicz et al. 2019; Kuźniar et al. 2020).

In order to further explore the influencing factors of endophytic bacterial community structure and diversity in upland rice seed samples, we investigated and compared the environmental factors such as temperature, precipitation and altitude of origin areas in Yunnan Province of China. There were differences in these environmental factors among the origin areas, and through RDA analysis, it was further found that temperature, precipitation and altitude had great effects on endophytic bacteria. Among them, the altitude at the origin areas had the greatest influence on the main dominant bacteria Pantoea in upland rice seeds. Thus it can be seen that the community structure and diversity of endophytic bacteria in upland rice seeds are affected not only by upland rice varieties and genotypes, but also by environmental factors such as temperature, precipitation and altitude. We can also conclude that the community structure and composition of endophytic bacteria in upland rice seeds are caused by upland rice varieties, genotypes and environment, rather than by a single factor.

The actual living state of plants in nature is the state of microorganisms and plants, and plant breeding is the cultivation of symbiotes between plants and microorganisms (Wang et al. 2015). Upland rice has the characteristics of drought resistance and drought tolerance, and the symbiotic microorganisms should also have corresponding drought tolerance characteristics to adapt to the local environment. Using high-throughput sequencing technology to explore the community structure and diversity of endophytic bacteria in upland rice seeds from origin areas of Yunnan Province of China is of great significance for the subsequent excavation of drought-tolerant bacteria resources and the improvement of local upland rice yield. At the same time, the mechanism of drought tolerance at the microbial level of upland rice can be reflected by comparing the microbial differences between upland rice and rice, and this study lays a foundation for this.

Exploring the endophytic microbial community structure and diversity of upland rice seeds is the basis for understanding the synergistic effect of endophytic bacteria in upland rice and the new functions and new substances produced by the synergy. Pantoea, Pseudomonas, Curtobacterium, Microbacterium, Methylobacterium, Agrobacterium, Sphingomonas, Aurantimonasand Rhodococcusserved as major core endophytic bacteria in twelve upland rice seed samples in this study. Overall, there were some differences in endophytic bacterial community structure and diversity among all seed samples, but the differences in some samples were not significant. The differences of endophytic bacterial community structure and diversity in upland rice seeds are not only related to the varieties of the samples themselves, but also affected by local temperature, precipitation, altitude and other environmental factors.

OTU: Operational Taxonomic Units; DGGE: Denaturing Gradient Gel Electrophoresis; PCoA: Principal Coordinates Analysis; NMDS: Non-metric Multidimensional Scaling; RDA: Redundancy Analysis

Acknowledgments

We would like to thank the research team of Prof. Huajun Zheng of Shanghai-MOST Key Laboratory of Health and Disease Genomics of Chinese National Human Genome Center at Shanghai for its platform and technical support in high-throughput sequencing and bioinformatics analysis.

Authors’ Contributions

Wang ZS and Zhu YQ designed and participated in all experimental procedures, performed data analysis, and drafted the manuscript. Sun BQ participated in the samples collection and preparation. Li N and Liu H participated in the plant samples cultivation. Zheng HJ, Wang WP and Liu Y supervised the study and critically revised the manuscript. All authors read and approved the final manuscript.

Funding

The research was supported by the Fundamental Research Funds for the Central Universities (No. FRF-TP-18-012A1; FRF-BR-19-003B) and the Open Research Fund of State Key Laboratory of Hybrid Rice (Hunan Hybrid Rice Research Center) (No. 2017KF04).

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no competing interests.

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Supplementarydata.docx

Composition and Diversity Of Endophytic Bacterial Community in Seeds of Upland Rice Resources from Different Origin Habitats in China

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