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A FURTHER STUDIES ON THE SPECIES OF MONASCUS

作者:天曲生物 日期:2020-10-02 14:59:37 点击数:2

A FURTHER STUDIES ON THE SPECIES OF MONASCUS

 

LI Zhong-Qing   GUO Fang

 

(Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080)

 

 

Abstract: Taxonomy of Monascus was reviewed and the species were reinvestigated based on the type cultures and authentic strains preserved in ATCC, CBS, CGMCC, IFO, IMI and NRRL. Monascus albidulus, Monascus rutilus, Monascus fumeus and Monascus aurantiacus are described as new species. A key to twelve species of Monascus is provided..

Keywords: taxonomy, new species, key

 

 

           

 

李钟庆  郭芳

 

(中国科学院微生物研究所 北京 100080

 

摘要: 重温红曲菌属各家分类意见,对来自ATCCCBSCGMCCIFOIMINRRL的红曲菌属各个种模式菌株和可靠菌株再次进行了观察。描述了4个新种,它们是发白红曲菌,火红色红曲菌,烟灰色红曲菌和橙色红曲菌。提供了区别12种红曲菌的检索表。

 

关键词: 分类新种,检索表

 

 

Introduction 

The genus Monascus Tiegh. was established in 1884, and twenty three taxa were recorded until the 1970s. They are: M. ruber Tiegh., M. mucoroides Tiegh.(1884); M. heterosporus (Harz) J. Schröt. (1894), M. purpureus Went (1895), M. barkeri P.A. Dang.(1903), M. olei Pieb.(1910), M. paxii Lingelsh. (1916), M. albidus K. Sato, M. albidus var. glaber K. Sato, M. anka Nakaz. et K. Sato, M. anka var. rubellus K. Sato, M. araneosus K. Sato, M. fuliginosus K. Sato, M. major K. Sato, M. pilosus K. Sato, M. pubigerus K. Sato, M. rubiginosus K. Sato, M. rubropunctatus K. Sato, M. serorubescens K. Sato, M. vitreus K. Sato (1936), M. vini Savul. & Hulea (see Hawksworth & Pitt, 1983), M. bisporus (Fraser) Arx (1970), and M. kaoliang M.S. Tsai, T.H. Hseu & Y.S. Shen (1978). Hawksworth and Pitt (1983) excluded M. bisporus and M. mucoroides and merged 21 names into M. pilosus K.Sato ex Hawks. & Pitt, M. purpureus and M. ruber.

In the late 20th century, Li (1982) reported M. aurantiacus isolated from wheat ‘koji’ in Anhui, eastern China; Banard & Cannon (1987) reported  M. floridanus isolated from pine tissues in Florida, USA; Hocking and Pitt (1988) reported M. eremophilus isolated from mouldy prunes in Sydney; Cannon, Abdullah & Abbas (1995) reported M. pallens and M. sanguineus, isolated from sediment in Shatt-al-Arab River in Iraq; Udagawa & Baba (1998) reported M. lunisporus isolated from mouldy feeds in Tokyo.

The characteristics of colonies under given conditions are inheritable and stable and important  taxonomically, which have been used to identify Penicillium species by Raper & Thom (1949), and  Aspergillus species by Raper & Fennell (1965). These characteristics are also used by the authors in the present taxonomical studies of Monascus.

 

Materials and Methods

(A) Cultures

   The type cultures and authentic strains were obtained from ATCC,CBS, CGMCC, IFO,IMI, and NRRL.

The cultures isolated by K. Sato and deposited in former Dalian Scientific Institute were also obtained.

(B) Media

  (1) CYA: NaNO3 3.0g, K2HPO1.0g, KCl 0.5g, MgSO4·7H2O 0.05g, FeSO4·7H2O 0.01g, yeast extract 5.0g, sucrose 30g, agar 15g, distilled water to 1000ml.

    (2) G25N: NaNO3 3.0g, K2HPO4 1.0g, KCl 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g,           yeast extract 5.0g, agar 15g, distilled water to 1000ml. After agar is dissolved, glyceral (analytical reagent grade) is added at the rate of 25% W/W, i.e. 250g glyceral is added to 750 ml medium.

    (3) MEA: malt extract powder 20g, peptone 1.0g, glucose 20g, agar 15g, distilled water to 1000 ml.

    (4)WA: filtrated fresh wort, albumen used as transparent agent, diluted to 15 Brix, and then added 1.5g/100ml agar.

    (5) MY50G: Malt extract, yeast extract and 50% glucose.

(C) Observation

Each of the strains was inoculated separately on the four kinds of media,and cultivated at 25oC for 7 days. The characters of colonies including form, size, color, outline, structure, texture, aerial hyphae, veins, exudate, substrate mycelium, and soluble pigment in media are observed macrographically. Morphology of hyphae, conidia, cleistothecia and ascospores are observed by microscope. The names of colors are based on A Guide to Colour Terminology edited by Committee of Editorial, Translating and Publishing Affairs, Chinese Academy of Sciences and published by Science Press in 1957.

 

Taxonomy

Monascus albidulus Z.Q. Li & F. Guo sp.nov.  (Figs. 1, and 5-6)

    Monascus albidus K. Sato, Bull. Agric. Chem. Soc. Japan 12:585, 1936 (nom. illegit.)

    Ab Monasco pallens differt colonia in agaro maltoso (WA) ad 25 post 7 dies 60 mm diam.,orbiculari, albida, velutina, reverse eburneo-flavida vel armeniaca; in MEA ad 25 post 7 dies 50 mm diam., albida, floccosa; in G25N ad 25 post 7 dies 25~30 mm diam., albida, coacta; in CYA ad 25 post 7dies 35 mm diam., albida, velutina vel floccosa.

    Holotypus HMAS 84447(in granis Oryzae sativae), cultura exsiccata ex cultura viva AS3.568 ex granis zymogenis Tritici aestivi in Dalian sinica isolata, in HMAS servatus.

Isotypus AS3.568, cultura lyophilisata in CGMCC servatus.

  Etymology: from latin, albidulus = whitish.     

    HMAS = Herbarium of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing.

    CGMCC = China General Microbiological Culture Collection, Institute of Microbiology, Chinese Academy of Sciences, Beijing.

 

On CYA, 25, 7 days, colonies 35 mm diam., white , aerial hyphae velvety to floccose.   

On G25N, 25, 7 days, colonies 25~30mm diam., white, aerial hyphae felted.

    On MEA, 25, 7 days, colonies 50 mm diam., white, aerial hyphae floccose, reverse ivory-yellowish.

    On WA, 25, 7 days, colonies 60 mm diam, white, plane, aerial hyphae velvety or felted, reverse ivory-yellowish or apricot colored..

Mycelium abundant, composed of irregularly branched, septate, smooth walled hyphae; hyphae uneven in width, 3~7.5 mainly 3~5µm wide, guttulate; substrate mycelium colorless or slightly colored. Conidia borne laterally on pedicel and terminally on hyphae, singly or two in short chain, globose or pyriform, hyaline, smooth, 7~9(~10)µm diam. Cleistothecia arising from the apex of the stalk like hyphae, (28~)38~48(~56)µm diam., peridium hyaline. Ascospores hyaline, smooth, ovulate or ellipsoid, 5.0~5.5×4.2~4.8µm.

Monascus albidus K. Sato (1936) based on living culture and Japanese description is illegitimate and therefore M. albidulus based on different type is erected here as a new species. The difference of M. albidulus from affined M. pallens Connon et al. is that the colonies of the former have aerial mycelia on CYA and MEA, while those of the latter have no aerial mycelium either on CYA or MEA.

 

Monascus rutilus Z.Q. Li & F. Guo sp.nov.  (Figs. 2, and 7-8)

Monascus anka Nakaz. & K. Sato, Bull. Agric. Chem. Soc. Japan 12:585, 1936 (nom. illegit.)

Ab Monasco purpureo Went differt colonia in MEA ad 25 post 7 dies 8 mm diam., ferrugineo-fusca, superficie non flocculosa, margine crenata; ad 25 post 25 dies 20 mm diam., scabrosa, fusca, margine irregulariter dentata, superficie non flocculosa; in G25N non crescente; cleistotheciis parvis, 30~50μm diam.

Holotypus HMAS 84448, cultura exsiccata ex granis Oryzae sativae rubro-mucedinosis (‘ang-kak’) e Fujian provincia sinica isolata, in HMAS servatus.

Isotypus AS 3.2636, cultura lyophilisata in CGMCC servatus.

  Etymology: from latin, rutilus = glowing red

 

On CYA, 25, 7 days, colonies 25mm diam., Pink Color (33’).

On G25N, 25, 7 days no growth.

On MEA, 25, 7 days, colonies 8 mm diam., Iron Brown (b47’); 25, 25 days,colonies 20 mm diam., Ochre Color (d67’).

On WA, 25, 7 days, colonies 22mm diam,, Brick Red (c37’); 25, 25days, colonies 56 mm diam., scabrous, Lycii Red (75’), edge with crenate, centre gaped.

Mycelium composed of irregularly branched, septate, smooth-walled, oleous, hyaline or redish, 3~5 µ m wide hyphae, substrate mycelium redish. Conidia singly or in short chains, borne on the apex of hyphae,  ovariform or pyriform, 7.8~10×7.8 µm. Cleistothecia globose, 30~50 µm diam., redish. Ascospores ellipsoid, hyaline or redish, 6~7×5~5.5 µm.

Monascus anka K. Sato (1936) based on living culture and Japanese description is illegitimate and therefore M. rutilus based on different type is erected here as a new species. M. rutilus differs from M. purpureus Went in that colonies of the former are ‘lycii red’, with crenate edge, gaped at the centre, not growing on G25N, while those of the latter are purplish, with smooth outline and floccose aerial mycelium, and growable on G25N.

 

Monascus fumeus Z.Q. Li & F. Guo sp.nov.  (Figs. 3, and 9-11)

Monascus fuliginosus K. Sato, Bull. Agric. Chem. Soc. Japan 12:585, 1936 (nom. illegit.)

Ab Monasco ruber Tiegh. differt colonia in agaro maltoso (WA) ad 25 post 7 dies 65 mm diam., orbiculari, plana, radiatim venosa, velutina, olivaceo-grisea; ad 25 post 25 dies 85 mm diam., plana, molybdea vel fuliginosa; ascosporis ellipsoideis, brunneis.

Holotypus HMAS 84449, cultura exsiccata ex vino Motai zymogeno in Guizhou provincia sinica isolata, in HMAS servatus.

Isotypus AS 3.2093, cultura lyophilisata in CGMCC servatus.      

Etymology: from latin, fumeus = sooty-coloured.

 

On CYA, 25, 7 days, colonies 25 mm diam., white, thin and sparse.          

    On G25N, 25, 7 days, colonies 6~7 mm diam., white and sparse.

    On MEA, 25, 7 days, colonies 45 mm diam., Tea Brown (d76’), plane, with sparse aerial hyphae; 25 days colonies 75 mm diam., Copper Brown (d 77’).

    On WA, 25, 7 days, colonies 65 mm diam., plane, with mass radial veins, Olive Grey (c 74’), aerial hyphae white-grey; 25 days,colonies 85 mm diam., velvety, Lead Grey ( 43’), with mass radial veins.

Mycelium composed of irregularly branched, septate, thin- and smooth-walled, granuligerous, light brown-colored or hyaline, 2~7μm wide hyphae. Conidia borne terminally on pedicels, 2~8 in chain, light brown or hyaline, globose or obpyriform, 5~10×5~8μm. Cleistothecia globose, arising terminally from the apex of short hyphae, 46~70μm diam, brown. Ascospore ellipsoidal, 5~5.5×4.5μm, brown.

Monascus fuliginosus K. Sato (1936) based on living culture and Japanese description is not valid nomenclaturally and here reduced as a synonym. M. fumeus based on different type is therefore erected here as a new species. M. fumeus differs from M. ruber Tiegh. in having grey-colored colonies and brown ascospores. The latter has brown or brown-orange colonies and hyaline ascospores.

                                                                            

Monascus aurantiacus Z.Q. Li ex Z.Q. Li & F. Guo sp.nov.   (Fig. 4)  

    Monascus aurantiacus Z.Q. Li, Acta Microbiol. Sinica 22(2):120,1982. (based on living culture)

    Ab Monasco purpureus Went differt colonia in agaro maltoso (WA) ad 25 post 7 dies 33 mm diam., dilute chromia, scabrosa, margine fimbriata, lacunosa in centro; 25 post 25 dies 75 mm diam., dilute aurantiaca, scabrosa, lacunosa in centro; in agaro G25N non crescente; ascosporis ovalibus vel ellipsoideis, 6~7.5×4~5.5μm, leniter galbis.

Holotypus HMAS 84450, cultura exsiccata ex granis Tritici aestivi zymogenis in Anhui provincia sinica isolata, in HMAS servatus.

Isotypus AS 3.4384, cultura lyophilisata in CGMCC servatus.

    Etymology: from latin aurantiacus = orange-colored.

 

    On CYA, 25, 7 days, colonies 35 mm diam., white, sparse.

    On G25N, 25, 7 days, no growth.

    On MEA, 25, 7 days, colonies 18~20 mm diam., Pale Oriole Yellow (d26’), with little floccose aerial hyphae; 25, 25 days, colonies 55 mm diam., margin denticulate, Pale Orange (65’).

    On WA, 25, 7 days, colonies 33 mm diam., Light Chrome (b27’), margin fimbriate; 25, 25 days, colonies 75 mm diam., Pale Orange (67’), scabrous, with sparse aerial hyphae, gaped in the centre.

Mycelium composed of hyaline or yellowish, irregularly branched, septate, guttulate and vacuolate, thin-walled, 3~6μm wide hyphae, substrate mycelium 5~8(~12)μm wide, yellowish. Conidia rare, singly, obpyriform, 10~12×8~10μm. Cleistothecia arising singly from the apex of stalk-like hyphae, yellowish or redish, globose, 30~60(~75)μm diam. Ascospores light greenish-yellow, ovale or ellipsoidal, 6~7.5×4~5.5μm. The morphological drawing and microphotograph, and colony color see Li (1982).

M. aurantiacus is distinguished from M. purpureus Went by its colony color, structure and growth rate on MEA. In M. aurantiacus the colonies are chrome or yellowish-orange, without floccose aerial hyphae, and not growing on G25N; the ascospores are light green-yellow. In M. purpureus the colonies are redish-orange or purple, usually with floccose aerial hyphae, and growing on G25N; the ascospores are hyaline. M.aurantiacus is also distinguished from M. ruber Tiegh. and M. pilosus K.Sato ex Hawksworth & Pitt by its slower growth rate on MEA and no growth on G25N. M. ruber and M. pilosus are able to grow on G25N and the colonies are redish or pinkish.

 

 

Key TO Species Of The Genus Monascus 

Based on our own research results and investigation made by Barnard & Cannon (1987), Cannon, Abdullah & Abbas (1995), Hawksworth & Pitt (1983), Hocking & Pitt (1988), and Udagawa & Baba (1998).

 

1. On MEA and CYA at 25 in 14 days colonies not growing; on MY50G at 25 in 14 days colonies 6-9mm………………………….…….....Monascus eremophilus

1. On MEA and CYA at 25 in 7 days colonies growing…………  …………….2

2. On MEA colonies white…………… ……………..………………   ………….3

2. On MEA colonies of other color…....…..……      ………………………...4

3. On MEA at 25 in 7 days colonies 30 mm diam., circular, with rugose aerial hyphae, felted, white, reverse slightly yellow; on CYA at 25 in 7 days colonies 10 mm diam., mycelium thin and sparse………………. …………    ………………………....Monascus albidulus

3. On MEA and CYA at 25 in 7days colonies 9~10 mm diam., circular, plane, aerial mycelium almost absent, hyaline on obverse and reverse…   ...Monascus pallens

4. On MEA at 25 in 7 days the diam. of colonies less than 20mm………………..5

4. On MEA at 25 in 7 days the diam. of colonies more than 20mm…………  …7

5. On MEA at 25 in 7 days colonies 16.5~18.5 mm diam., outline circular, aerial hyphae floccose to lanose, pale brown at margin, central portion dull green; on WA at 25 in 7 days colonies 18 mm diam., Pale White Green (c31’), aerial hyphae velvety…………………………………………………………Monascus floridans

5. On MEA at 25 in 7 days the diam. of colonies less than 10mm…………………………………………………………………… ……….6

6. On MEA at 25 in 7 days colonies 8 mm diam., Iron Brown (b47’); on WA at 25 in 7days colonies 22mm diam., Brick Red (c37’), margin crenate, scabrous, gaped in centre, Lycii Red(75’) when age…………………………………………………………….…..Monascus rutilus

6. On MEA at 25 in 7 days colonies 8~10 mm diam., circular, plane, Light Ochre Color (a65’), aerial hyphae velvety to floccose; on WA at 25 in 7 days colonies 22 mm diam., Ochre Color (d67’), aerial hyphae velvety……………………………………………….…...….Monascus sanguineus

7. On MEA at 25 in 7 days colonies 18~20 mm diam., margin fimbriate, Light Chrome (b27’); on WA at 25 in 7 days colonies 33 mm diam., Light Orange (65’), slightly scabrosity………………………………….Monascus aurantiacus

7. On MEA and WA colonies of other color………………………………………..8

          8. On MEA at 25 in 7 days colonies 28 mm diam., light orange, aerial hyphae floccose; on WA at 25 in 7 days colonies 50 mm diam., Rose Pink (a34’)…....………………………………………………...…Monascus pilosus

          8. On MEA and WA colonies of other color……….…….……………………….9

          9. Colonies in tones of orange to brown………....….……………………………10

          9. Colonies in tones of olive or grey…………….………………………………..11

         10. On MEA at 25 in 7 days colonies 32mm diam., Yellowish roseate (d26’), aerial hyphae velvety; on WA at 25 in 7 days colonies 48 mm diam., Pale Cocoa Brown (b57’)..……………………………………………………Monascus ruber

         10. On MEA at 25 in 7 days colonies 22~28mm diam., Deer Brown (b45’), aerial hyphae velvety; on WA 25 in 7 days colonies 26~34 mm diam., Iron Brown (b47’), aerial hyphae velvety to floccose, Light Red Sandal Purple (75’) when age……………………….….………………..…...….Monascus purpureus

         11. On MEA at 25 in 7 days colonies 18~23 mm diam., olive brown, radially sulcate, ascospores lunate or ellipsoid…………………………………………..…………Monascus lunisporas

         11. On MEA at 25 in 7 days colonies 45 mm diam., Tea Brown (76’); on WA at 25 in 7 days colonies 65 mm diam., Olive Grey (c74’), Lead Grey (43’) when age…...……..…………………………………………….Monascus fumeus

 

 

Acknowledgement  

We would like to express our gratitude to Professor Yong-Nian Yu and Professor Jian-Yun Zhuang for their valuable suggestions to this paper.

..


 

Reference

[1]Barnard EL, Cannon PF, 1987. A new species of Monascus from pine tissues in Florida. Mycologia 79:479~484

[2]Bridge PD, Hawksworth DL, 1985. Biochemical tests as an aid to the identification of Monascus species. Letters in Applied Microbiology 1:25~29

[3]Cannon PF, Abdullah SK, Abbas BA, 1995. Two new species of Monascus from Iraq, with a key to known species of the genus. Mycological Research 99:659~662

[4]Dangeard PA, 1907. L’origine du perithece chez les ascomycetes. Botaniste 10:5

[5]Hawksworth DL, Pitt JI, 1983. A new taxonomy for Monascus species based on cultural and microscopical characters. Aust Bot 31:51~61

[6]Hocking AD, Pitt JI, 1988. Two new species of xerophilic fungi and a further record of Eurotium halophilicumMycologia 80:82~88

[7]Iizuka H, Lin Ch F, 1980. On the genus Monascus of Asia and its specific characteristics. Adv Biotech 2:555~561

[8]Li ZQ, 1982. A new species of the genus Monascus, Monascus aurantiacusActa Microbiol Sinica .22:331-342

[9]Li ZQ, Guo F, 2003. The Morphology and Taxonomy of Monascus. Beijing: China Light Industry Publisher. 1~65 (in Chinese)

[10]Lingelsheim A, 1916. Eine neue pigmentbildender MonascusHedwigia 57:253~254

[11]Nakazawa R, Sato K, 1930. Monascus from Taiwan red rice. J Agric Chem Soc Japan 6:352

[12]Nishikawa J, Iizuka H, 1993. Numerical taxonomy of Monascus species based on the electrophoretic analysis of intracellular enzymes. J Basic Microbiol 33: 331~342

[13]Piedallu A, 1910. Sur une nouvelle moisissure du tannage a l’huile le Monascus olei. C R Acad Sci 151:397~399

[14]Raper KB, Thom C, 1949. Manual of Penicillia. Baltimore:Willians & Wilkins Co. 1~875

[15]Raper KB, Fennell DI, 1965. The Genus Aspergillus. Baltimore:The Willians and Wilkins Co. 1~686

[16]Sato K, 1930. Monascus from Chinese North-East and Korean wheat leaven (report No.1). J Agric Chem Soc Japan 6:957~965

[17]Sato K, 1932. Monascus from fermented bean curd (report No.2). J Ferment Tech 10:544

[18]Sato K, 1933. Monascus from Chinese wheat leaven and fremented products (report No.3). J Agric Chem Soc Japan 11:494~503

[18]Sato K, 1934. Monascus from Chinese fermented products (report No.4). J Fermen Tech 12:439~445

[19]Sato K, 1936. Classsification of oriental Monascus (Appendix: Monascus nomenclature). J Agric Chem Soc Japan 12:583-586

[20]Schröter J, 1894. Hemiascineae. in Engler A, Prantl K (eds.) Die Natürlichen Pflanzenfamilien, Erster Teil, Abt. 1. Leipzig: Wilhelm Engelmann. 143~149

[21]Tsai MS, Hseu TH, Shen YS, 1978. Purification and characterization of acid protease from Monascus kaoliangInt J Pept Protein Res 12:293-302  

[22]Udagawa S, Baba H, 1998. Monascus lunisporus, a new species isolated from mouldy feeds. Cryptogamie Mycology 19:269-276

[23]van Arx JA, 1970. The Genera of Fungi Sporulating in Pure Culture. J.Carmer Lehre. 1~84

[24]van Tieghem PEL, 1884. Monascus genre nouveau de l’ordre des ascomycetes. Bull Soc Bot Fr 31:226~231

[25]Went FAFC, 1895. Monascus purpureus, le Champignon de l’ang-quac. Une nouvelle Thelebolee. Ann Sci Nat Bot Ser 8:1~18


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