Microscopic Observations of Gills, Basidiospores and Mycelium in Agaricus brasiliensis

doi:10.3969/j.issn.1000-2561.2022.12.010

Abstract

Abstract:

Agaricus brasiliensis is a precious edible and medicinal mushroom, which is not only rich in nutrition but also has high medicinal value, and its industrial development has a broad prospect. However, the lack of germplasm resources of A. brasiliensis and insufficient knowledge of the genetic background of A. brasiliensis have hindered the development of its hybrid breeding technology, and the existing germplasm resources can hardly meet the practical needs of production. A. brasiliensis strains J1 was used as research materials, it is proposed to investigate the biological properties related to A. brasiliensis by micro-morphological observation of the reproduction-related structures of A. brasiliensis. The microscopic morphological characteristics of gills, basidiospores and mycelium in different growth and development stages were observed by scanning electron microscope and fluorescence microscope. The gills and trama were in the developmental stage and the hyphae lamellae were not obvious, and the gill sheets were not obvious during the morula state. The gill sheets were obviously observed, arranged parallel to each other, composed of trama and the fruiting layers on both sides, which were closely arranged, uniform in thickness, and a small amount of basidia were borne after the coral stage, but the trama was irregularly arranged. During the forming stage, the gills were clear and regular, and the inner veil was not ruptured, the gill space contains basidiospores and disorganized duct-like cells arranged in raised surface, and the duct-like cells in the gill were closely related. During the forming stage, the gill basidium was relatively full and wrinkled. There were 3 or 4 sporozoites on the gill basidia, and there were small connecting filaments between the basidiospores. The observation of basidiospore nuclei by DAPI staining showed that the most basidiospores were dinuclear spores, and a few were mononuclear and enucleated spores. The observation of mycelial nuclei by DAPI staining showed that the mycelium of A. brasiliensis mycelium was multinucleated and had septa, but there was no lock-like association. This study would clarify the microscopic morphological characteristics of gills, basidia, basidiospores and mycelium in different growth and development stages of A. brasiliensis, enrich the knowledge of biological studies and life history processes of A. brasiliensis, and lay a foundation for the hybrid breeding, basidiospore mating types, genetics transformation system of A. brasiliensis.

Key words: Agaricus brasiliensis, gills, basidiospore, nucleus

CLC Number:

S567.3

ZHOU Yu, HUANG Zaixing, BAO Xinglu, LI Jing, LIU Penghu. Microscopic Observations of Gills, Basidiospores and Mycelium in Agaricus brasiliensis[J]. Chinese Journal of Tropical Crops, 2022, 43(12): 2482-2488.

Figures/Tables 6

Fig. 1 Four developmental stages of A. brasiliensis A: Morula stage; B: Coral stage; C: Formation stage; D: Mature stage.

Fig. 2 Observation on morphology and structure of gill and basidia of A. brasiliensis by scanning electron microscopy A, B: Gills at the mulberry stage; C-F: Gills at the coral stage, a is the gill, b is the gill base.

Fig. 3 Observation on morphology and structure of gill gaps of A. brasiliensis by scanning electron microscopy A, B: Diagram of the gap between gills and gills; C, D: Vessel-like cells in the gills.

Fig. 4 Observation on morphology of basidia of A. brasiliensis by scanning electron microscopy A-D: Forming stage basidium; E-F: 3 or 4 sporozoites on basidium during the forming period, a and b are 4 sporozoites, c is 3 sporozoites.

Fig. 5 Fluorescence microscopic observation on basidiospore nucleus of A. brasiliensis A, B: Gill fragments (the small red circle represents binuclear spores); C, D: Fluorescence observation image (The red arrow points to the dinuclear spore, the white arrow points to the mononuclear spore, and the yellow arrow points to the seedless spore). Bars=100 μm.

Fig. 6 Fluorescence microscopic observation on hyphal nucleus of A. brasiliensis The red arrow points direct towards to the nucleus; the blue arrow points direct towards to the diaphragm. Bars=100 μm.

References 23

[1]	CAMPELO M D S, NETO J F C, LIMA A B N, NETO F C D C, NÁGILA M P S R. Polysaccharides and extracts from Agaricus brasiliensis Murill-a comprehensive review[J]. International Journal of Biological Macromolecules, 2021, 183: 1697-1714.
[2]	KATSUYA T, HOICHI A, AKITOMO M, DAISUKE Y, KEN-ICHI I, YOSHIYUKI A, NAOHITO O. Outdoor-cultivated royal sun medicinal mushroom Agaricus brasiliensis KA21 (Agaricomycetes) reduces anticancer medicine side effects[J]. International Journal of Medicinal Mushrooms, 2021, 22(1): 31-43.
[3]	RUBEL R, SANTA H S D, SANTOS L, FERNANDES L, SOCCOL C. Immunomodulatory and antitumoral properties of Ganoderma lucidum and Agaricus brasiliensis mushrooms[J]. International Journal of Medicinal Mushrooms, 2018, 20(4): 393-403.
[4]	顾可飞, 邵毅, 李晓贝, 范婷婷, 刘志敏, 黄柳娟, 王红梅, 周昌艳. 松茸与姬松茸营养成分差异性研究[J]. 农产品质量与安全. 2017(1): 73-78.
	GU K F, SHAO Y, LI X B, FAN T T, LIU Z M, HUANG L J, WANG H M, ZHOU C Y. Analysis on differences in nutritional ingredients between Tricholoma matsutake and Agaricus blazei[J]. Quality and Safety of Agro-products, 2017(1): 73-78. (in Chinese)
[5]	LI Y, SHENG Y, LU X, GUO X, DU P. Isolation and purification of acidic polysaccharides from Agaricus blazei Murill and evaluation of their lipid-lowering mechanism[J]. International Journal of Biological Macromolecules, 2020, 157: 276-287
[6]	黄在兴, 王义祥, 赵光辉, 翁伯琦, 杨丽萍, 刘朋虎. 姬松茸育种研究进展[J]. 亚热带农业研究, 2020, 16(4): 273-278.
	HUANG Z X, WANG Y X, ZHAO G H, WENG B Q, YANG L P, LIU P H. Research progress on Agaricus brasiliensis breeding[J]. Subtropical Agriculture Research, 2020, 16 (4): 273-278. (in Chinese)
[7]	LIU P H, YUAN J, JIANG Z H, WANG Y X, WENG B Q, LI G X. A lower cadmium accumulating strain of Agaricus brasiliensis produced by ⁶⁰Co-γ-irradiation[J]. LWT-Food Science and Technology, 2019, 114: 108370.
[8]	鲍大鹏, 王南, 谭琦, 潘迎捷. 用荧光染色法对柱状田头菇(Agrocybe aegerita)子实体担子和担孢子的观察[J]. 南京农业大学学报, 2000(3): 57-60.
	BAO D P, WANG N, TAN Q, PAN Y J. Observation on basidia and basidiospores of Agrocybe aegerita by fluorescence staining[J]. Journal of Nanjing Agricultural University, 2000(3): 57-60. (in Chinese)
[9]	许昭仪, 李浩, 张平. 金针菇生活史各阶段核相研究[J]. 菌物学报, 2015, 34(3): 386-393.
	XU Z Y, LI H, ZHANG P. Behavior of nuclei in life cycle of Flammulina velutipes[J]. Mycosystema, 2015, 34(3): 386-393. (in Chinese)
[10]	王瑞娟, 万佳宁, 龚明, 茅文俊, 汪莹, 唐利华, 尚俊军, 杨慧, 鲍大鹏. 改良石蜡切片法观察金针菇子实体不同发育时期组织结构[J]. 食用菌学报, 2019, 26(2): 54-58.
	WANG R J, WAN J N, GONG M, MAO W J, WANG Y, TANG L H, SHANG J J, YANG H, BAO D P. Tissue structure observation of Flammulina filiformis fruiting bodies at different developmental stages by improved paraffin method[J]. Acta Edulis Fungi, 2019, 26(2): 54-58. (in Chinese)
[11]	董金福. 竹林地仿生栽培姬松茸技术研究[D]. 杭州: 浙江农林大学, 2011.
	DONG J F. The research of bionic cultivation technology of Agaricus Blazei in bamboo forest land[D]. Hangzhou: Zhejiang Agriculture and Forestry University, 2011. (in Chinese)
[12]	赵建霞, 冯伟林, 金群力, 沈颖越, 宋婷婷, 蔡为明. 双孢蘑菇担子与担孢子形态发育显微观测[J]. 园艺学报, 2019, 46(2): 356-364.
	ZHAO J X, FENG W L, JIN Q L, SHEN Y Y, SONG T T, CAI W M. Microscopic observation of the morphological development of basidia and basidiospores in Agaricus bisporus[J]. Acta Horticulturae Sinica, 2019, 46(2): 356-364. (in Chinese)
[13]	胡晓棣, 李熠, 任蜀豫, 姚一建. 冬虫夏草、蛹虫草菌丝隔膜和细胞核荧光染色[J]. 菌物学报, 2016, 35(9): 1099-1105.
	HU X D, LI Y, REN S Y, YAO Y J. Fluorescent staining of septa and nuclei in Ophiocordyceps sinensis and Cordyceps militaris[J]. Mycosystema, 2016, 35(9): 1099-1105. (in Chinese)
[14]	HERRRIRA K M, ALVES E, COSTA M D, DIAS E S. Electron microscopy studies of basidios porogenesis in Agaricus brasiliensis[J]. Mycologia, 2012, 104(6): 1272-1280
[15]	谭伟, 周洁, 曹雪莲, 苗人云, 贾定洪, 黄忠乾, 甘炳成, 彭卫红, 郑林用. 姬菇258和金凤2-1杂交构建姬菇新菌株[J]. 西南农业学报, 2015, 28(6): 2655-2660.
	TAN W, ZHOU J, CAO X L, MIAO R Y, JIA D H, HUANG Z Q, GAN B C, PENG W H, ZHENG L Y. Hybrid strains breeding by Pleurotus cornucopiae No.258 and Pleurotus sp Jinfeng2-1[J]. Southwest China Journal of Agricultural Sciences, 2015, 28(6): 2655-2660. (in Chinese)
[16]	于浩, 葛志豪, 徐丽丽, 郭立忠. 双单杂交技术选育长根菇高品质抗病新菌株[J]. 南方农业学报, 2019, 50(12): 2621-2628.
	YU H, GE Z H, XU L L, GUO L Z. Breeding of high oudenone production and Trichoderma resistant Oudemansiella raphanipes strain by dikaryon monokaryon matin[J]. Journal of Southern Agriculture, 2019, 50(12): 2621-2628. (in Chinese)
[17]	任纪帆, 朱静娴, 王庆佶, 高馨梅, 姜淑霞. 大球盖菇“山农球盖3号”品种的选育[J]. 菌物学报, 2020, 39(6): 977-982.
	REN J F, ZHU J X, WANG Q J, GAO X M, JIANG S X. Breeding of Stropharia rugosoannulata “Shannongqiugai No. 3”[J]. Mycosystema, 2020, 39(6): 977-982. (in Chinese)
[18]	陈美元, 廖剑华, 郭仲杰, 李洪荣, 卢政辉, 蔡丹凤, 王泽生. 双孢蘑菇耐热相关基因的表达载体构建及转化研究[J]. 菌物学报, 2009, 28(6): 797-801.
	CHEN M Y, LIAO J H, GUO Z J, LI H R, LU Z H, CAI D F, WANG Z S. The expression vector construction and transformation of thermotolerance-related gene of Agaricus bisporus[J]. Mycosystema, 2009, 28(6): 797-801. (in Chinese)
[19]	乔燕楠, 林星雨, 高雅, 闫静, 张岚岚, 李南羿. 根癌农杆菌介导的双孢蘑菇转基因体系的建立[J]. 食用菌学报, 2021, 28(1): 48-54.
	QIAO Y N, LIN X Y, GAO Y, YAN J, ZHANG L L, LI N Y. Establishment of a high-efficiency agrobacterum mediated transformation system in Agaricus bisporus[J]. Acta Edulis Fungi, 2021, 28(1): 48-54. (in Chinese)
[20]	喻义赣. 香菇农杆菌介导转化条件优化及BCA基因转化研究[D]. 福州: 福建农林大学, 2009.
	YU Y G. Research of the agrobacterium-mediated transformation condition optimization and transformation of BCA gene in Lentinus edodes[D]. Fuzhou: Fujian Agriculture and Forestry University, 2009. (in Chinese)
[21]	杨利梅. 砖红绒盖牛肝菌子实体、菌丝和原基的显微及超微结构观察[D]. 昆明: 云南大学, 2017.
	YANG L M. Microstructure and ultrastructure observation of fruit bodies, mycelia and primordia of Xerocomus spadiceus[D]. Kunming: Yunnan University, 2017. (in Chinese)
[22]	郭勇, 叶小金, 甘炳成, 彭卫红. 不同温度和光照培养条件下3种食用菌菌丝的菌落及菌丝形态的研究[J]. 西南农业学报, 2011, 24(6): 2301-2306.
	GUO Y, YE X J, GAN B C, PENG W H. Study on three kinds of edible fungi hyphae colony and hyphae form in different temperature and light training conditions[J]. Southwest China Journal of Agricultural Sciences, 2011, 24(6): 2301-2306. (in Chinese)
[23]	徐思佳, 万佳宁, 李焱, 李燕, 尚俊军, 鲍大鹏. 刺芹侧耳交配型基因敲入单核体后锁状联合和核相的表征观察[J]. 菌物学报, 2020, 39(6): 1130-1138.
	XU S J, WAN J N, LI Y, LI Y, SHANG J J, BAO D P. Characterization of clamps and nuclear phases in the monokaryons with transformed mating genes in Pleurotus eryngii[J]. Mycosystema, 2020, 39(6): 1130-1138. (in Chinese)