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.