Analysis of the composition and differences of endophytic bacterial communities in different dormant states of Qianghuo seeds
Notopterygium incisum Ting ex H. T. Chang is a plant species in the Umbelliferae family. Its dried rhizomes are used as medicine and are commonly used in traditional Chinese and Tibetan medicine, commonly known as black medicine. It has a high market demand. At present, Qianghuo mainly comes from wild resources and is mainly distributed in shrub grassland areas at an altitude of 3000-4500m in Sichuan, Gansu, Qinghai and other places. In recent years, excessive excavation has caused great damage to the ecological environment, and wild Qiang live resources are approaching depletion. Therefore, artificial domestication and cultivation are urgently needed.
When the seeds of Qianghuo mature and land, the embryo development is incomplete, and there is often no embryo or a primitive embryo phenomenon. It has a dual dormancy characteristic of morphology and physiology, and belongs to deep dormancy seeds. The germination rate of Qianghuo seeds in natural environments is extremely low (0.52%). The current effective method for relieving the dormancy of Qianghuo seeds is GA+variable temperature sand storage method. Firstly, warm temperature sand storage method is used to relieve their morphological dormancy, and then low-temperature sand storage treatment is required to relieve their physiological dormancy before the seeds can have germination ability. The seed treatment time can last for more than 8 months. Using this method to treat Qianghuo seeds, the highest germination rate is 21.2%. In addition, the growth cycle of Qianghuo medicinal herbs is over 4 years, and due to the long investment cycle and slow effectiveness, the development of artificial cultivation is very slow. Currently, only sporadic cultivation is available, which cannot meet the sustainable development needs of the Qianghuo market. The technology of manually releasing the dormancy of Qianghuo seeds has not made breakthrough progress in the past decade, which is one of the main problems restricting the large-scale cultivation of Qianghuo.
Endophytic bacteria in plants refer to fungi or bacteria that live within various tissues and organs of healthy plants at certain or all stages of their life cycle. They form a complex and special mutualistic relationship with the host plant in long-term coexistence, and promote cell division, root development, and seedling growth by increasing the metabolism of auxin and mitogens in the plant. The dormancy of seeds is controlled by the balance between gibberellin growth promoters and abscisic acid growth inhibitors. Research has found that endophytic bacteria can regulate seed dormancy by regulating or inducing the synthesis of endogenous hormones in plants, providing a theoretical basis for exploring endophytic bacteria resources with growth promoting functions in seeds. Pu Fengya et al. found that endophytic bacteria in Job’s tears can increase the germination rate of Job’s tears, rice, and wheat seeds, and promote seedling growth by inducing root growth. Lei Ruifeng discovered that endophytic bacteria in silver sand locust seeds promote seed germination by regulating seed metabolism. Xiang Yiqing and others found that endophytic bacteria in Huanglian seeds can significantly improve the germination rate of Huanglian seeds. The endophytic bacteria of Sichuan pepper (Capsicum annuum), rice (Oryza sativa L.), and Dendrobium catenatum directly promote the growth and development of host plants by secreting endogenous hormones such as auxin, abscisic acid, cytokinin, gibberellic acid, etc. The endophytic bacteria isolated from the seeds of Achnatherum inebrians, Dendrobium nobile, and Lolium rigidum, as well as the endophytic bacteria isolated from the roots of Polygonatum multiflorum L., regulate seed dormancy by secreting endogenous hormones such as cytokinin, significantly improving their germination and survival rates. However, can the endophytic bacteria in Qianghuo seeds promote the release of dormancy by promoting the growth of its embryo? Based on this, this experiment takes Qianghuo seeds as the research object and uses Illumina MiSeq culture free high-throughput sequencing technology to study the composition and differences of endophytic bacterial communities in different dormancy states of Qianghuo seeds. This provides a theoretical basis for further research on the use of microorganisms to relieve Qianghuo seed dormancy and also provides important theoretical references for the cultivation of plants with similar dormancy characteristics.

As an important reproductive organ of plants, seeds can serve as a medium for the transmission of beneficial microorganisms from mother plants to the next generation through vertical propagation, thereby promoting seed germination and seedling growth. Endophytic bacteria in the seeds of grasses such as rice and corn play an important role in seed germination, seedling growth and development, and host plant growth. The seeds of Dendrobium nobile Lindl. exhibit post ripening and lack endosperm tissue, requiring symbiosis with microorganisms for germination. The dominant endophytic bacteria are mainly Proteobacteria and Cyanobacteria. This study found that Qianghuo seeds are in three different dormant states, and the types of endophytic bacterial communities are similar at the phylum level. The endophytic bacteria are mainly composed of Proteobacteria, Actinobacteria, and Firmicutes, which is consistent with the dominant endophytic bacterial phyla of other plant seeds. However, compared with Dendrobium officinale seeds, its dominant endophytic bacterial phyla has differences. The types of plant secretions and the ability of microorganisms to colonize, survive, and spread to seeds vary, resulting in differences in endophytic bacteria in different plant seeds. Because endophytic bacteria in seeds come from different plant organs or meristematic tissues, they transfer and colonize the seed embryo and endosperm through microtubule connections or gametes, and spread vertically. The seeds of medicinal plants Qianghuo and Dendrobium officinale both have difficulty germinating due to their dormancy characteristics. However, studies have found that the dominant endophytic bacterial phyla of the two have differences, which may be due to their different sources of endophytic bacteria. Qianghuo seeds are dormant due to incomplete embryo development, and their endophytic bacteria mainly come from the endosperm, while Dendrobium officinale seeds are dormant due to the lack of endosperm. Their endophytic bacteria mainly come from the embryo, so there are differences in the phylum types between the two. It is speculated that Qianghuo seeds also need to coexist with microorganisms to germinate. A small amount of endophytic bacteria belonging to the phylum Bacteroidetes (2.01%) appeared in the seeds of Qianghuo in the GSC state, while unknown endophytic fungi belonging to the phylum Bacteroidetes appeared in 24.33% and 7.74% of the seeds in the GSB and GSC states, respectively. These endophytic bacteria may play an important role in promoting seed embryo growth and releasing seed dormancy, and will be the focus of future research. Endophytic fungi have high abundance and diversity in the seeds of many plants. The endophytic fungi in Qianghuo seeds are mainly composed of Ascomycota and Basidiomycota, which is consistent with the dominant endophytic fungi in Phaseolus vulgaris L. seeds and Dendrobium nobile seeds.

At the genus level, the composition of endophytic bacterial communities changes from GSA to GSB status, and the types of endophytic bacterial communities are similar from GSB to GSC. Through Alpha diversity analysis, it was found that the diversity of endophytic bacteria in GSA was significantly lower than that in GSB and GSC, while there was no significant difference in the diversity of endophytic bacteria between GSB and GSC. The main reason may be that in the GSA state, the Proteobacteria phylum had an absolute advantage, accounting for 97.08%, thereby inhibiting the growth of other bacteria. In contrast, the seeds in the GSB and GSC states were in a relatively hypoxic environment for a long time, resulting in relatively less competition among microorganisms and no significant inhibitory effect between microorganisms, leading to a higher diversity index than in the GSA state. There was no significant difference in the diversity of endophytic fungal communities during the entire dormancy release process of seeds. Through Beta diversity PCoA analysis, it was found that the community structure of endophytic bacteria underwent significant changes from GSA to GSB and then to GSC state, while the community structure of endophytic fungi did not change significantly from GSA to GSB until the physiological dormancy of seeds was released. Qianghuo seeds mature and land, and only have germination ability after long-term warm and low-temperature stratification treatment. During this period, the structure of the endophytic bacterial community in the seeds changes, indicating that this is one of the main influencing factors for Qianghuo seeds to have germination ability, and its mechanism of action needs further research.
The role of endophytic bacteria in plant seeds has not been elucidated, but a large number of studies have shown that endophytic bacteria have regulatory effects on host plants and seeds. The endophytic bacteria in ryegrass can produce cytokinins and interact with plant hormones produced by the plant, thereby regulating seed dormancy. Endophytic bacteria in seeds can also promote plant growth by producing plant hormones or facilitating the absorption of plant nutrients, especially nitrogen and phosphorus. This study further analyzed the characteristic microorganisms of GSA, GSB, and GSC at the genus level. There were a total of 15 endophytic bacteria and no characteristic microorganisms in endophytic fungi. These microorganisms were relatively abundant in their respective dormancy stages compared to other dormancy stages, and may play an important role in the release of morphological and physiological dormancy of Qiangfu seeds, making them a key microbial population for future research. Among them, the dominant bacterial genera in GSA status are Erwinia and Pseudomonas. Pseudomonas is involved in the spoilage process of many fruits and vegetables, with about 50% to 90% of spoilage bacteria being Pseudomonas. The pectin acid lyase and pectin methylgalacturonate enzyme secreted by Erwinia have a synergistic effect on softening plant tissues, which can degrade the pectin on the plant cell wall and cause softening and spoilage. The results of this study indicate that fresh harvested Qiangfu seeds must be promptly stored in sand to prevent spoilage and affect seed germination. Huang Zhiyuan et al. found that the relative abundance of Erwinia genus in Phyllostachys nuda bamboo shoots decreased with increasing storage temperature during storage. This study found that the abundance of fresh harvested Qiangfu seeds significantly decreased after warm temperature treatment (15 ℃), which is consistent with previous research results. This may be related to the fact that the endosperm of the seeds was subjected to warm temperature and sand storage treatment, and the dominant bacterial genera that appeared decomposed the endosperm produced a large amount of energy for embryo development, indirectly inhibiting their growth. When the Qianghuo seeds mature and land, the embryo has not yet developed and needs to be stored in warm sand for up to 8 months to release its morphological dormancy, which is related to the presence of difficult to biodegrade germination inhibitors in the endosperm. The red swimming bacteria genus has denitrification ability and is a functional bacterium that can degrade organic matter and some nitrogen-containing compounds that are difficult to biodegrade. This study found that in the GSB state, the dominant endophytic bacteria are the red motile bacteria genus and Catellatospora genus, which may play a role in degrading germination inhibitors and promoting embryo growth, and are the key microbial groups for future research. The dominant bacterial genera in GSC status are the genera of Deinococcus, Steridobacterium, Rhizobium, Mucococcus, Mycobacterium, Hydrobacteria, Micromonospora, Actinobacteria, Bacillus, Flavobacterium, and Myceticola. Research has found that the genus Dvoras is a nitrogen fixing bacterium; Slow growing rhizobia have the ability to dissolve inorganic phosphates, which can increase the pigment content of plant photosynthesis; The Mycetocola strain was initially discovered on the Mi Dui Glacier and is a cold resistant microorganism. After being released from dormancy by warm sand storage, Qianghuo seeds do not have the ability to germinate. They must be treated with low-temperature sand storage before they can germinate. It is speculated that after the release of dormancy, the endosperm will be exhausted and unable to meet the energy required for further growth of the embryo and embryo root. The nitrogen products produced by the degradation of nitrogen-containing germination inhibitors by red motile microorganisms accumulated during the dormancy release stage cannot be directly absorbed by the embryo. They must be converted into nitrogen that can be absorbed by the plant by nitrogen fixing microorganisms to promote embryo growth and embryo root breakthrough through the seed coat. It is speculated that nitrogen fixing microorganisms need to cooperate with Myceticola microorganisms to grow normally, while Myceticola microorganisms can only grow normally in low-temperature environments. Therefore, Qianghuo seeds must undergo a period of time after the release of dormancy. Only after low-temperature treatment can the embryonic root break through the seed coat, Seeds have the ability to germinate. It is speculated that the characteristic microorganisms of slow growing rhizobia in GSC state embryos can increase the content of photosynthetic pigments in embryos, preparing for photosynthesis in embryos.
Due to the fact that Qianghuo seeds mature and land as prototype embryos or without embryos, the growth of seed embryos during artificial cultivation is very slow, resulting in long germination time and difficult germination process of Qianghuo seeds. It takes up to 11 months to germinate in a natural state. It is urgent to find microorganisms from the perspective of microorganisms that promote the growth of seed embryos to release their morphological and physiological dormancy, providing strong support for the development of artificial cultivation of Qianghuo. This experiment conducted an in-depth study on the community structure composition and differences of endophytic bacteria and fungi in different dormancy states of Qiangfu seeds through cultivation free high-throughput sequencing methods. The characteristic microorganisms of each dormancy state were analyzed. This paper provides a new idea for using microbial methods to relieve the dormancy of Qiangfu seeds, fills the gap in the research field of microbial promotion of seed dormancy release, and lays the foundation for further utilizing these potential endophytic bacterial resources with dormancy release promoting effects to improve the germination rate of Qiangfu seeds and promote their standardized development in artificial cultivation. It is expected to provide theoretical guidance for the use of microbial methods such as engineering strains or microbial preparations to quickly relieve seed dormancy.