Diploid mycelia of Ustilago esculenta fails to maintain sustainable proliferation in host plant.

Smut fungi display a uniform life cycle including two phases: a saprophytic phase in vitro and a parasitic phase in host plants. Several apathogenic smut fungi are found, lacking suitable hosts in their habitat. Interestingly, MT-type Ustilago esculenta was found to maintain a parasitic life, lacking the saprophytic phase. Its long period of asexual proliferation in plant tissue results in severe defects in certain functions. In this study, the growth dynamics of U. esculenta in plant tissues were carefully observed. The mycelia of T- and MT-type U. esculenta exhibit rapid growth after karyogamy and aggregate between cells. While T-type U. esculenta successfully forms teliospores after aggregation, the aggregated mycelia of MT-type U. esculenta gradually disappeared after a short period of massive proliferation. It may be resulted by the lack of nutrition such as glucose and sucrose. After overwintering, infected Zizania latifolia plants no longer contained diploid mycelia resulting from karyogamy. This indicated that diploid mycelia failed to survive in plant tissues. It seems that diploid mycelium only serves to generate teliospores. Notably, MT-type U. esculenta keeps the normal function of karyogamy, though it is not necessary for its asexual life in plant tissue. Further investigations are required to uncover the underlying mechanism, which would improve our understanding of the life cycle of smut fungi and help the breeding of Z. latifolia.

First Report of Leaf Sheath Disease, Caused by Microdochium seminicola, on Zizania latifolia in China.

Zizania latifolia, which belongs to the tribe Oryzea, has been widely grown over thousands of years as an aquatic vegetable in China. In the autumn of 2019, the disease harmful to the leaf sheaths of Z. latifolia was found in Jinhua City and Lishui City, Zhejiang Province. The disease caused brown oval-shaped spots (3~5 cm long, 0.5~1 cm wide) on leaf sheaths (Figure 1A). The infected plants produced non-commercial Jiaobai. The surface of the Jiaobai was pale yellow instead of white, and the length of the Jiaobai was 25% shorter than the uninfected plants. To 2022, the disease has been found in most planting areas of Z. latifolia in Zhejiang, Jiangsu, Fujian, Jiangxi and Anhui provinces in China. About 20%~50% incidence of the disease in these main production areas has seriously threatened the yield and quality of Jiaobai. To identify the causal agent, diseased leaf sheaths were collected. Symptomatic leaf sheath tissue was sterilized with 75% alcohol for 30 sec, and then washed three times with sterile distilled water for 1 min. A total of 37 strains were isolated on potato dextrose agar (PDA). We selected 10 strains for further morphological identification. An abundant mass of aerial mycelia was formed in 8 days (Figure 1B). The mycelium is transparent, septate and smooth. Conidia is fusiform, straight or curved, 0~3 septa, hyaline, tapering and rounding at the apex (Figure 1C and 1D). Strain XYQ3 was obtained through multi-generation culture. To further identify the pathogen of this fungus, genomic DNA was extracted and internal transcribed spacer (ITS), large subunit rDNA (LSU), ß-tubulin (BTUB) and DNA-directed RNA polymerase II subunit (RPB2) of strain XYQ3 were amplified. These sequences were obtained and submitted to GenBank (accession numbers MT605000, OP585914, MZ619085, MZ619084, respectively). After alignment analysis of sequencing data, we found that the ITS sequence indicated 100% identity with M. seminicola (GenBank acc no. KP859007). The LSU sequence showed 100% identity with M. seminicola (GenBank acc no. KP858943). The BTUB sequence showed 99.47% identity with M. seminicola (GenBank acc no. KP859070). The RPB2 sequence has higher identity with M. seminicola, reaching 99.76% with 4 sequences (GenBank acc nos. KP859140, KP859116, KP859134 and KP859130). Strain XYQ3 and a total of 16 other Microdochium isolates were selected for phylogenetic analysis using the ITS, LSU, BTUB and RPB2 sequences (Figure 2). Phylogenetic tree analysis revealed that strain XYQ3 was clustered with the M. seminicola clade, including M. seminicola strains MFG 60131, MFG 60132, CBS 122706 and KAS 1527. Therefore, this fungus was identified as M. seminicola based on morphological observation, multiple sequences alignment analysis and phylogenetic analysis. Strain XYQ3 was used in pathogenicity tests. According to Koch's postulates, 10 fresh leaf sheaths and leaves of host plants were inoculated by spraying conidial suspension at the concentration of 1×106 conidia/mL. Sterile water was sprayed as negative control. Inoculated plants were cultured under 25℃ light for 10 h, darkness for 14 h, covered with plastic film to maintain high humidity above 65%, and observed daily for disease development. After 6 d, symptoms similar to those observed in the field (Figure 1E and 1F). The pathogen of this fungus was re-isolated from the symptomatic inoculated leaf sheath and re-identified as strain XYQ3. Many Microdochium species are important pathogens of Poaceae (Liang et al., 2019). M. seminicola is classified as a new species in the Xylariales family by the report of Hernández-Restrepo (Hernandez-Restrepo et al., 2016) and was only found in wheat, oats and barley so far (Gagkaeva et al., 2020; Gavrilova et al., 2020). To the best of our knowledge, this fungus is the first report in the disease of Z. latifolia. As this disease is prevalent in main planting areas of Z. latifolia and has seriously threatened the production of Jiaobai, it is very important to isolate and identify the pathogen for the subsequent drug screening and control.

Cloning and disruption of the UeArginase in Ustilago esculenta: evidence for a role of arginine in its dimorphic transition.

BACKGROUND: Ustilago esculenta, a typical dimorphic fungus could infect Zizania latifolia and induce host stem swollen to form an edible vegetable called Jiaobai in China. The strains differentiation especially in the mating ability and pathogenicity is closely related to different phenotypes of Jiaobai formed in the fields. Dimorphic switching, a tightly regulated processes, is essential for the pathogenetic development of dimorphic fungi. In responses to environment cues, dimorphic switching can be activated through two conserved cell signaling pathways-PKA and MAPK pathways. Previous study indicated that exogenous arginine could induce hyphal formation in several dimorphic fungi through hydrolysis by arginase, but inhibit the dimorphic transition of U. esculenta. We conducted this study to reveal the function of arginine on dimorphic transition of U. esculenta. RESULTS: In this study, we found that arginine, but not its anabolites, could slow down the dimorphic transition of U. esculenta proportionally to the concentration of arginine. Besides, UeArginase, predicated coding arginase in U. esculenta was cloned and characterized. UeArginase mutants could actually increase the content of endogenous arginine, and slow down the dimorphic transition on either nutritious rich or poor medium. Either adding exogenous arginine or UeArginase deletion lead to down regulated expressions of UePkaC, UePrf1, mfa1.2, mfa2.1, pra1 and pra2, along with an increased content of arginine during mating process. CONCLUSION: Results of this study indicated a direct role of arginine itself on the inhibition of dimorphic transition of U. esculenta, independent of its hydrolysis by UeArginase.