Isolation, identification, and functional verification of endophytic bacteria in Huanglian seeds
Seeds are important reproductive organs of plants and have significant importance in the preservation of germplasm resources and agricultural production. Endophytic bacteria are closely related to seeds and exist in different parts of the seed (seed coat, embryo, endosperm) as well as throughout the entire growth and development stage of the seed. The microbial taxa in seeds from different sources highly overlap, with endophytic bacteria found in seeds belonging to 4 phyla and 131 genera. Among them, Proteobacteria and Gamma Proteobacteria are the dominant groups in different seeds, while Bacillus, Pseudomonas, Bacillus like, Micrococcus, Staphylococcus, and Pseudomonas are the most common in different plant seeds. Danshen, rice, alfalfa and other seed endophytic bacteria are rich in species, carrying a large number of functional genes and promoting seed development through direct or indirect effects. Endophytic bacteria in seeds have various physiological functions related to seed development and germination. They promote seed development and plant growth by fixing nitrogen, dissolving phosphorus, producing plant hormones, ACC deaminase, producing iron carriers, and promoting nutrient absorption. They also produce antibiotics, hydrolytic enzymes (pectinase, amylase, cellulase, etc.), induce defense mechanisms, and promote the transformation of substances inside seeds, reducing the harm of biotic or abiotic stress to plants.
Coptis chinensis Franch. is a perennial herb of Coptis genus in Ranunculaceae, which uses dry rhizomes as medicine. It is one of the raw materials of many traditional Chinese patent medicines and simple preparations and has a large area of artificial cultivation. However, Huanglian seeds have morphological and physiological characteristics of post ripening, and require 9 months of variable temperature layering after falling off from the plant to mature and germinate, with a low germination rate; Not being able to withstand dehydration during storage poses difficulties for seed preservation and quality control, as well as for the introduction and cultivation of Huanglian, which in turn affects the yield and quality of medicinal materials. The preliminary metagenomic research of the research group found that Huanglian seeds have abundant endophytic bacteria, which may participate in pathways such as oxidative phosphorylation, tricarboxylic acid cycle, pentose phosphate, glycolysis gluconeogenesis, etc. These pathways are closely related to seed development and plant growth, and it is speculated that microorganisms may play a role in the post ripening and germination process of Huanglian seeds.
In order to further explore the promotion of endophytic bacteria in Huanglian seeds, this study obtained cultivable endophytic bacteria from Huanglian seeds through plate isolation. The endophytic bacteria in Huanglian seeds were identified by 16S rRNA, and their growth promoting characteristics such as phosphorus solubilization, nitrogen fixation, and IAA production were determined. The endophytic bacteria promotion function was verified by seed germination experiments. In order to screen for endophytic bacteria that are beneficial for seed ripening and germination, the aim is to provide a theoretical basis for revealing the promotion mechanism of plant endophytic bacteria and the mechanism of recalcitrant seed ripening and germination. At the same time, it also lays the foundation for the research and development of new and efficient microbial fertilizers.

 

 

Truyens et al. found that Bacillus and Pseudomonas are dominant groups in seeds of different plants, while Bacillus, Micrococcus, Staphylococcus, Pseudomonas, and Acinetobacter are also very common in endophytic bacteria in seeds. This study used Huanglian seeds as the object and isolated and identified 29 endophytic bacteria distributed in 13 genera, among which the dominant bacteria were Bacillus and Pseudomonas oligotrophic. Research on the growth promoting characteristics of Huanglian seeds shows that endophytic bacteria have the functions of producing IAA, solubilizing phosphorus, fixing nitrogen, and producing iron carriers. Some endophytic bacteria also have multiple growth promoting functions, such as Ba Toyonensis ZZ-4 can produce IAA and iron carriers, while Pr. megaterium ZZ-24 can produce IAA and has phosphorus solubilization function. The seed germination experiment further confirmed that the endophytic bacteria in Huanglian seeds with growth promoting functions are mostly distributed in the genus Bacillus. The research results of Gagne Bourgue et al. showed that Bacillus subtilis has the characteristics of producing IAA, cytokinins, and phosphorus solubilization. Lu et al. determined that Bacillus subtilis BN7 in Boju can hydrolyze phosphorus, potassium, and IAA. Therefore, the genus Bacillus may play an important role in the post ripening and germination process of Huanglian seeds, and has great development and utilization value in agricultural production.
Phosphorus exists in the soil in the form of insoluble inorganic phosphorus and organic phosphorus. Only a small portion of phosphorus is soluble in the soil and can be absorbed and utilized by plants. Endophytic bacteria in plants with the ability to dissolve phosphorus can convert phosphorus that is difficult for plants to absorb into easily absorbable forms, thereby promoting plant growth and development. Pseudomonas brassicacearum H9, isolated by Xie et al. from the rhizosphere soil of plants, is a bacterium with high phosphate solubilizing activity. Its bacterial suspension significantly promotes the germination of wheat and cucumber seeds. In addition, seed germination and seedling growth are regulated by IAA, and moderate exogenous IAA can promote seed germination and plant root development; Xu et al. isolated three endophytic bacteria capable of producing IAA from tomato and rice seeds, which colonize the roots, stems, and leaves of plants such as tomato, rice, rapeseed, and radish, and have varying degrees of growth promoting effects on seedlings. Khalaf et al. obtained 56 endophytic bacteria capable of secreting auxin from melon seeds, with Bacillus and Escherichia being dominant genera. The range of IAA secretion by endophytic bacteria that can produce auxin in the seeds of sesbania is 1.29-25.47mg/L. Ba. velezensis SC60 has the highest IAA production and significantly promotes embryonic root development and improves seed vitality. Six endophytic bacteria capable of secreting IAA were obtained in this experiment, among which ZZ-6, ZZ-21, ZZ-24, and ZZ-29 secreted more than 100mg/L of Pr. These bacteria can both dissolve phosphorus and produce high IAA Megaterium ZZ-24 has a significant promoting effect on the growth of Huanglian seeds. Further exploration of various growth promoting characteristics of endophytic bacteria in seeds and excavation of multifunctional endophytic bacteria will help to elucidate the mechanism of endophytic bacteria in seed development and germination.
There are various endophytic bacteria that promote growth in Huanglian seeds, and the dominant bacteria are mostly distributed in the genus Bacillus, which can dissolve phosphorus and produce high IAA in Pr Megaterium ZZ-24 has a good ability to promote the growth of Huanglian seeds, indicating its potential for research and development. Endophytic bacteria with multiple growth promoting abilities may play a role in the long-term post ripening process of Huanglian seeds, which may be an important factor for Huanglian seeds to complete post ripening and successfully germinate.