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| Establishment and Optimization of the Artificial System for the Formation of Jiaobai |
| YIN Yu-Mei1,*, ZHANG Ya-Fen1,*, CAO Qian-Chao2, HU Peng1, XIA Wen-Qiang1, CUI Hai-Feng1, YU Xiao-Ping1, YE Zi-Hong1,** |
1 Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine/College of Life Sciences, China Jiliang University, Hangzhou 310018, China;
2 Microbiological Inspection Center, Sichuan Institute for Food and Drug Control, Chengdu 611731, China |
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Abstract Jiaobai, the swollen stem of Zizania latifolia, formed by infection with the biotrophic basidiomycete fungus Ustilago esculenta, has become the second largest aquatic vegetables in China. Traditional methods of Jiaobai in planting and breeding not only cost a lot of labor and financial resources but also cause variety degeneration, producing inedible grey Jiaobai and male Jiaobai in the fields, which seriously affect the economic benefits. As a typical dimorphic fungus, closely related to Ustilago maydis, compatible haploid strains of scale pathogenicity were first selected and identified from different strains and combinations of U. esculenta status. Next, leaves, leaf sheaths and tubular roots were inoculated, separately to determine the best inoculation site-leaf sheaths. Then, the inoculation method (stem base injection method, tissue infiltration method with stem base and tissue infiltration method) inoculation serious suspension culture concentration pathogen cell (OD600 value of 2.0, 1.5, 1.0, 0.5) and the cultivation environment of seedlings after inoculation were screened. Results showed that the appropriate inoculation method was stem base injection method, the appropriate cell concentration was OD600 value of 2.0, and the better cultivation environment was kept in greenhouse or transplanted to field in mid-March or mid-September. An optimized artificial inoculation system was established, which could induce the swollen of stems. Firstly, tubular roots of Z. latifolia were germinated in greenhouse under a 12/12 h light/dark cycle at 25/22 ℃ for 10~20 d; Secondly, compatible strains UET1 and UET2 of U. esculenta with an OD600 value of 2.0 were syringe-inoculated into the base of seedling stems, kept in greenhouse or transplanted to field in mid-March or mid-September. Under this artificial inoculation system, plant survival rate was about 80% with swollen stem accounts for more than 80%, tissue slice of leaf sheath and stem apex, collected at early infection stages, analyzed by laser scanning confocal microscopy after staining with wheat germ agglutinin-Alexa Fluor 488 and propidium iodide-Alexa Fluor 561, showed that a large amount of invasion hyphae were formed and expanded to stem apex and full of black masses of teliospores. In order to stop the telispores formation, MT strains from white Jiaobai (swollen stem without telispores) and T strains from grey Jiaobai (swollen stem with full of telispores) were collected and analysed by transcriptome. Differentially expressed genes were screened and selected for functional verification. Fortunately, a key gene Itd1 (interfered factors in teliospores development)(GenBank No. MK164419) related to teliospores formation in U. esculenta was found, encoding 852 amino acids and acted only late stages during infection. Itd1 deletion mutants from the compatible U. esculenta strains UET1ΔItd1 (CGMCC No.16723) and UET2ΔItd1 (CGMCC No.16724) showed a defect in telisorores formation for inoculation. Combining artificial inoculation method and successful genetic modification, an artificial system for U. esculenta-induced swollen stem formation without black teliospores was established. The above study provided a reliable theoretical and technical basis to establish a new efficient and stable breeding way for white Jiaobai. At the same time, it laid a foundation for further research on the dimorphism transition, filamentous growth and teliospores formation of U. esculenta.
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Received: 07 December 2018
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Corresponding Authors:
**, zhye@cjlu.edu.cn
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| About author:: * Authors who contribute equally |
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