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. 2013;8(2):e56190.
doi: 10.1371/journal.pone.0056190. Epub 2013 Feb 20.

Sclerotial formation of Polyporus umbellatus by low temperature treatment under artificial conditions

Yong-Mei Xing  1 , Li-Chun Zhang, Han-Qiao Liang, Jing Lv, Chao Song, Shun-Xing Guo, Chun-Lan Wang, Tae-Soo Lee, Min-Woong Lee
Affiliations

Sclerotial formation of Polyporus umbellatus by low temperature treatment under artificial conditions

Yong-Mei Xing et al. PLoS One. 2013.

Abstract

Background: Polyporus umbellatus sclerotia have been used as a diuretic agent in China for over two thousand years. A shortage of the natural P. umbellatus has prompted researchers to induce sclerotial formation in the laboratory.

Methodology/principal finding: P. umbellatus cultivation in a sawdust-based substrate was investigated to evaluate the effect of low temperature conditions on sclerotial formation. A phenol-sulfuric acid method was employed to determine the polysaccharide content of wild P. umbellatus sclerotia and mycelia and sclerotia grown in low-temperature treatments. In addition, reactive oxygen species (ROS) content, expressed as the fluorescence intensity of mycelia during sclerotial differentiation was determined. Analysis of ROS generation and sclerotial formation in mycelia after treatment with the antioxidants such as diphenyleneiodonium chloride (DPI), apocynin (Apo), or vitamin C were studied. Furthermore, macroscopic and microscopic characteristics of sclerotial differentiation were observed. Sclerotia were not induced by continuous cultivation at 25°C. The polysaccharide content of the artificial sclerotia is 78% of that of wild sclerotia. In the low-temperature treatment group, the fluorescent intensity of ROS was higher than that of the room temperature (25°C) group which did not induce sclerotial formation all through the cultivation. The antioxidants DPI and Apo reduced ROS levels and did not induce sclerotial formation. Although the concentration-dependent effects of vitamin C (5-15 mg mL(-1)) also reduced ROS generation and inhibited sclerotial formation, using a low concentration of vitamin C (1 mg mL(-1)) successfully induced sclerotial differentiation and increased ROS production.

Conclusions/significance: Exposure to low temperatures induced P. umbellatus sclerotial morphogenesis during cultivation. Low temperature treatment enhanced ROS in mycelia, which may be important in triggering sclerotial differentiation in P. umbellatus. Moreover, the application of antioxidants impaired ROS generation and inhibited sclerotial formation. Our findings may help to provide new insights into the biological mechanisms underlying sclerotial morphogenesis in P. umbellatus.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Different stages of sclerotia induced by low-temperature vs. mycelia in P. umbellatus cultivated at 25°C.
(A) Mycelia could not form sclerotia after cultivation at 25°C for 105 days in the control group. (B) SI stage of sclerotial differentiation at 60 days after cultivation in the low-temperature group. (C) and (D) SD stage of sclerotial formation at 90 days after cultivation in the low-temperature group. (E) and (F) SM stages of sclerotial formation with the total cultivating of 105 days in the low-temperature group. Representative images were from three independent experiments, with 30 replicates in each group.
Figure 2
Figure 2. Fluorescent image of ROS in P. umbellatus mycelial cells in the low temperature group.
ROS accumulation measured by CM-H2DCFDA staining of mycelia was observed at different time periods of cultivation. (A), (B), (C), (D), (E) and (F) respectively represented ROS content in mycelia after 45, 60, 75, 90, 90 and 105 days after cultivation in low-temperature group. These pictures were captured using a Zeiss fluorescent microscope at the 400×magnification. Scale bar, 5 µm. Representative images were from three independent experiments, with 50 replicates during each cultivation stage.
Figure 3
Figure 3. Fluorescent images of ROS in the mycelial cells of P. umbellatus at 25°C.
Mycelial ROS content was observed at different cultivation stages using CM-H2DCFDA staining. (A), (B), (C) and (D) respectively represented ROS levels in mycelia after 45, 75, 90 and 105 days of cultivation in the room temperature (control) group. These pictures were also taken under a Zeiss fluorescent microscope at the 400×magnification. Scale bar, 5 µm. Representative images were from three independent experiments, with 50 replicates during each cultivation stage.
Figure 4
Figure 4. ROS analysis of mycelial cells in the room temperature group vs. low temperature group.
The room temperature group was used as the control group. Independent T-test was used to analyze the data of the two groups at each cultivation phase. The data presented represent means ± SD from three independent experiments, with 50 replicates in each group.*P<0.01.
Figure 5
Figure 5. ROS images in low temperature group vs. different antioxidants groups after 90 days of cultivation.
(A) Fluorescent image of ROS in low temperature group (control). (B) VitaminC (1 mg mL−1 ) complemented group. (C) Group treated with vitaminC (5 mg mL−1 ). (D) Group complemented with vitaminC (10 mg mL−1 ). (E) VitaminC (15 mg mL−1 ) complemented group. (F) Fluorescent image of ROS in DPI (1 mM) or Apo (10 mM ) complemented group. The pictures were taken using a Zeiss fluorescent microscope. Images were representatives of three independent experiments, with 30 replicates in each group. Scale bar, 10 µm.
Figure 6
Figure 6. Microscopic characteristics of P. umbellatus mycelia during sclerotial formation.
P. umbellatus mycelia was subjected to morphological examination using Zeiss Axio Imager A1 microscope. (A) Mycelia with little branches at 30 days of cultivation. (B) Mycelia with multiple clamp connections 60 days after inoculation. (C) Thick and slim mycelia coexited at 90 days after inoculation. (D) Slim mycelia with multiple branches after 120 days of cultivation. (E) Colorless conidia and spore-producing structures were observed at 120 days of cultivation. (F) and (G) indicated the sclerine of the conidia (red arrows). Representative images were from three independent experiments. Scale bar, 10 µm.
Figure 7
Figure 7. Observation of P. umbellatus sclerotia by SEM 120 days after cultivation.
The pictures were observed using a JEOL JSM-6510 Scanning Electron Microscope. (A) Hyphae intertwined and fused were observed (red arrow). (B) Inflated mycelia like a ball at one end. (C) A cluster of multilateral crystalline structures. (D) Helical strikes on the surface of the mycelium with clamp connections (white arrow). (E) Conidia (arrows) and sclerine (* and **). (F) Local parts of mycelia swollen (arrows). (G) Sclerine residue on the surface of additional conidia. (H) Conidia formed with surrounding slim mycelia. Images were representatives of three independent experiments. Scale bar, (A), (B), (C), (E) and (G) 5 µm; (D) and (F) 2 µm; (H) 10 µm.
Figure 8
Figure 8. Microscopic structure of P. umbellatus artificial sclerotia at different stages of cultivation.
The pictures were examined using Zeiss Axio Imager A1 microscope. (A) The cultivation of artificial sclerotia for 105 day. (B) The cultivation of artificial sclerotia for 120 days. The outermost membrane in (A) and (B) was indicated with a red arrow. (C) The cultivation of artificial sclerotia for 150 days. 1 represented the loose hyphae near the outermost membrane, 2 represented the pigmented layer, and 3 represented the thick and interwoven hyphae. Representative images were from three independent experiments. Scale bar, (A) and (B) 10 µm; (C) 20 µm.
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Grants and funding

The research was financially supported by the National Natural Sciences Foundation of China (No.30830117, 31201666), the International Science and Technology Cooperation Projects of China (No. 2011DFA31260) and the National Research Foundation (NRF 2011–0016999). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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