Temperature relations in Monascus

Trans. Br. mycol. Soc. 57 (3), 465-472 (197 1 ) Printed in Great Britain

TEMPERATURE RELATIONS IN MONASCUS By KESHARI L. MANANDHAR* AND A. E. APINIS

Department ofBotany, The University, Nottingham The temperature requirements in growth and development (production of cleistothecia and conidia) and other cultural characteristics on agar media are reported for 37 species and strains of Monascus. These include several isolates obtained from silage, stored grain, soil and grassland vegetation in Great Britain. The isolates differed in their minimal, optimal and maximal temperatures in various growth aspects, and in pigment production. Most of the isolates tested were microthermophilic, growing in a temperature range of 18-45°C with optima at 30---38°, but a few which may be considered as transitional mesophiles are unable to grow at 40°.

Van Tieghem (1884) found that Monascus tuber grows well at 35°e. Harz ( I 890) reported the optimum temperature for the growth of Physomyces (Monascus) heterosporus as 30-31°. Barker (1903) agreed that the optimum temperature for growth was 30° but that the fungus grew very slowly below 20°. Piedallu (1909/10), in his comparative study of various Monascus isolates, used optimum temperature for growth as a basis for classification. M. barkeri had an optimum of about 25° but scarcely grew at 30°. M. purpureus and M. olei showed growth optima between 30 and 40°. The two latter species were distinguished by their maximum growth temperatures. Whereas M. purpureus continued to grow at temperatures Young (1930) up to 47°, but not at 48°, M. olei grew at 46° but not at 4 found that growth of these fungi, most rapid at 30, 35 and 40°, is limited by the higher temperature, while the cultures below 30° are slightly slower in development and those below room temperature are considerably delayed. Nakazawa & Sato (1930) found that the optimum temperature for growth of their isolates was 33-35°. with a maximum over 37° and a minimum below 30°. Growth was always very slow above 40°. Apinis (1963a) reported M. purpureus from alluvial grassland soil as a psychrotolerant thermophile growing within a temperature range of 17-48°. Thirumalachar, Whitehead & Mathur (1964) reported that Backusia (Monascus) terricola grew rapidly at 37°. Subsequently Cole & Kendrick (1968) found that M. ruber and B. terricola both had optimum temperatures for growth of 30° but produced more conidia and cleistothecia at st. It is evident that most Monascus species and strains prefer higher temperatures for growth than most mesophilic fungi and in this respect they are related to thermophilous fungi. This investigation was carried out to obtain further information on temperature ranges for growth and development of Monascus spp.

t.

* Present address: Department of Agricultural Education and Research, Khumal Tar, Lalitpur, Nepal.

Transactions British Mycological Society MATERIALS AND METHODS

Monascus species and strains were obtained from the culture collections of the Department of Botany, The University of Nottingham (BDUN), Commonwealth Mycological Institute (IMI), Centraal Bureau voor Schimmelcultures (CBS), The National Agricultural Advisory Service Laboratories in Newcastle upon Tyne and Shardlow (NAAS) and the University of Waterloo, Canada. The names for the cultures are quoted as they were given on the labels when received. Details of isolates are as follows: isolate no. I. M . purpureus Went, BDUN 85, soil, G.B., 1950; 2 . Monascus sp., BDUN 329, silage, NAAS, G.B., 1967; 3. Monascus sp., BDUN 331, barley grain, GB, 1967; 4. Monascus sp., BDUN 330, barley grain, G.B., 1967; 5. Backusia terncola Thirum., Whitehead & Mathur, soil, India; 6. M. ruber Van Tieghem, IMI 57972, 1954; 7. M. purpureus, Triticum sp., G.B., IMI 62570, 1956; 8. M. purpureus, Theobroma cacao, Ghana,IMI 77365,1958; 9. M.purpureus, G.B., IMI 89281, lactose, 1932; 10. M. purpureus, G.B., IMI 89282, lactose, 1945; I I. B. terricola, soil, India, IMI 81596; 12. B. terricola, soil, India, IMI 10973°; 13. M.purpureus, soil, India, IMI 123954; 14. Monascus sp. BDUN 333, barley grain, G.B., 1966; 15. Monascus sp. BDUN 334, silage, G.B., 1968; 16. M. purpureus, BDUN 335, barley grain, G.B., 1968; 17. B. terricola, 176, soil, India via Waterloo (Canada); 18. M. ruber, 177 soil, India, via Waterloo (Canada); 19. Monascus sp. NAAS, silage, G.B., 1968; 20. Monascus sp., NAAS, silage, G.B., 1968; 21. Monascus sp., IMI 134169, 1969; 22. M. albidus, Sato, CBS 280, 1934; 23. M. albidus var. glaber Sato, CBS 281, 1934; 24. M. albus, CBS 282, 1934; 25. M. anka Nakazawa & Sato, CBS 283, 1934; 26. M. pilosus Sato, CBS 286, 1934; 27. M. araneous Sato, CBS 284, 1934; 28. M. rubiginosus Sato, CBS 288, J934; 29. M. rubropunctatus Sato, CBS 289, 1934; 30. M. serorubescens Sato, CBS 290, 1934; 31. M. oitreus Sato, CBS 291, 1934; 32. M. major Sato, CBS 283, 1934; 33. M. pubigerus Sato, CBS 287, 1934; 34. Monascus sp., NAAS, silage, G.B., 1969; 35. Monascus sp., NAAS, G.B., 1969; 36. M. purpureus, cereals, BDUN, G.B., 1966; and 37. M. purpureus, Zea mays, IMI 138019, Nigeria, J969. The temperature range of growth of Monascus species was ascertained by growing the fungi on potato dextrose agar (PDA) and malt extract agar (MA). For inoculation, 90 mm perspex Petri dishes, each containing 20 ml of agar, were used. Inoculum disks 6 (5'5) mm were cut from actively growing r o-day-old cultures and placed in the middle of Petri dishes used in the test series. Triplicate Petri dishes of each isolate on the two abovementioned media were used for each temperature. Inoculated dishes were incubated at 12°, 15°, 18°, 25°, 30°, 37°, 40° and 45°, and the rate of growth noted according to the diameter of the colony. Conidium and cleistothecium production at various temperatures was noted with the microscope while pigment production was recorded using Ostwald's colour chart (Steller & Ostwald, 1939).

Monascus. Keshari L. Manandhar and A. E. Apinis

467

RESULTS

The mean linear growth rates of the strains are summarized in Table I. None of the species and strains was able to grow at 12°. Strains I, 6, 12, 14, IS, 23 and 30 grew very slowly at ISo during 18 days but most did not show any visible growth during the first 7 days, and subsequently grew slowly. Most strains and species grew well at 2So, 30°, 3t and 40° with optimum temperatures either at 30° or and with maxima at 4So.

sr

Table

I.

Linear growth rate (mmjday) qfMonascus strains nos. 1-37 at various temperatures on malt extract agar Average growth rate (mmJday)

,

Strain no.

18° (days 13-26)

2So (days 6--16)

30° (days 4-13)

37° (days 4-13)

I 2 3 4 S 6 7 8 9 10

o'7S 1'16 I'OS 1'19 1'20 1'28 0'97 0,60 0,62 o,8S o'9S 1'23 0'94 1,88 1,68 0,67 O'S7 o'6S o'6S o'So o'S6 0'7 1 0,63 0'47 o'6S 1"04 1"01 0'7 8 1'00 0'93 1'27 0,66 1'36 1'76 1'47 1,67 1'34

3'27 4'12 4'26 4,60 4,80 4'20 3,80 4'So 3'73 3'97 4'21 4'46 3'79 4'44 4'17 3'42 4'SI 5'°S 4'SS 4'7 8 4'44 2'94 3'59 3'22 3'12 4'7 0 4,60 2'27 4,83 4'5 6 4'28 3,66 4'7 2 4'S7 3,87 3'9 8 3'S4

S'73 6'7 1 6'37 6'13 7'02 7'08 6'21 6'42 S,8S 5'96 6,82 6'79 4'27 4'70 3'09 5,68 6'02 6'44 6'02 6'I1 S·67 4'37 4'3 0 4,63 4'4 6 6'97 6'9 0 2'35 S'28 S'9 1 6'IS 3'S9 6'9 8 7'09 7'01 7'7 1 6'47

S'27 S'94 6'14 6'9 0 7'48 5'07 6'S3 6'79 5'96 6'11 6,88 7'17 S'S2 2,84 0'99 S'86 6'10 7"35 6'81 6'17 5,63 5,82 6'00 3'48 6,so 8'3 6 7,62 2'7 8 4,63 6'13 6'28 4,82 9'13 6'2S 6'04 7'19 6'33

II

12 13 14 IS 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 3S 36 37

4So (days 9-16) 1,66 1'24 1'4S 1'21 1,62 2'18 1'77 l'S9 I'S3 1'03 1,84 I'S7 1'17 0 0 1'70 0'79 I'OS 1'4 2 0'93 1'4 2 1,88 1'2S 0 I'7S 0'9 0 0'94 1'77 0 1,67 0 o'9S 1'30 0 0 0 0

The growth of most strains was not significantly different at 30° and

3t but the growth rate in general at 2So and 40° was lower. At 4So and 18° growth was very slow. Strains 14 and 15 showed rapid growth at low temperatures but did not grow at temperatures above 40°. Strain 4 grew well at 18° but very slowly

468

Transactions British Mycological Society

at 45°, while strains 16, 25 and 32 grew very little at 18° but somewhat better at 45°, and strain 6 grew equally well at both temperatures. Similarly, strains I, 5, II and 12 grew reasonably well at both lower and higher temperatures (T a ble I). Other cultural characteristics of individual isolates at various temperatures are displayed in Table 2 which shows the formation of conidia and cleistothecia. In general, colonies at or near the optimum temperature show very similar characteristics. In most cases a culture is more densely overgrown with a gregarious aerial mycelium and the colour is more intense at 37 0 than at 30°. Cultures at extreme temperatures are quite different from those at optimal growth temperature. Strains 2, 4, 5, 12, 17, 18 and 19 at 45° produce light to dark grey, wrinkled colonies thickly covered by short but compact aerial mycelia. Sporulation of different strains is variable (Table 2). Strains I, 14, 15, 16,29 and 32 did not produce conidia on ordinary media, whereas 22,23, 26, 29, 32 and 33 produced cleistothecia. Production of conidia and cleistothecia does not differ at 250, 300 or 37 0 (Table 2). Cultures grown at 45 0 for 15 days did not produce cleistothecia but strains 3, 4, 7, 12, 24, 25, 28 and 30 produced a few conidia. In contrast to this, conidia were richly produced by strains 17, 18, 19, 20 and 2 I at this temperature. At 4 00 strains 5, 6, 12, 13, 24, 27, 28, 30 and 31 produced cleistothecia. Most of th e strains produced numerous conidia at 400 except for 6, 22, 23, 24, 27, 28 and 32. At 180 most strains produced cleistothecia and conidia. Strains 4, 2 I and 24 failed to produce cleistothecia but formed conidia only at 180 during 20 da ys. The growth on MA at various temperatures is slower than that on PDA but, in general, the growth pattern of individual strains at various temperatures is similar to that on PDA. Temperature considera bly affects pigment production. Most of the isolates which produced a brown colour at an optimum temperature only produced yellow to yellowish orange colour at 180. At a temperature abo ve 400, pigment production is reduced considerably. At optimal growth temperatures of 25 0 , 30° and 37 0 on PDA (T able 3) strains 2, 3, 4, 5, 24, 25 and 33 produced colonies in reverse yellow, whereas 22, 23 and 26 produced colourless colonies in revers e. Others including 8-12, 13, 16-20, 29, 34 and 35 were brown in reverse. Only strains 16 and 25 produced pink aerial colonies. Colonies at 18° are generally less dense and the colour of the colony reverse differs from that of cultures grown at 30°. Strains 5,8,9, 10, I I, 12, 17,20 and 35 which produced a brown aerial colony at optimum temperature produced a yellow to yellowish orange reverse when grown at 180 • Some produced a colourless reverse (strain 6) or light pink reverse (strains 7, 16, 25 and 36) if grown at 18°. DISCUSSION

In general, the temperatures required for growth of Monascus species and strains tested are within the range of 18-450 C. Certain variations have been observed in growth patterns at optimal temperatures, and the strains

Table 20 day~ at

Strain no.

Conidia

18°

Cleistotheci~

2.

Production of conidia and cleistothecia at different temperatures

10 day~ ,--- - - - -

Conidia

at 25°

r

IS days at 45°

IS days at 40°

8 daysA at 37°

,

Cleistothecia

A

Cleistothe-;;~

f

"-

Cleistotheci~

++++ ++++ ++++ ++++

++++ ++++ + +

++++ ++++ ++ +++

+ ++++ +

+++ ++ +

++++ +++ ++++ ++++ +++ ++++

++++ +++ ++++ ++ +++

++++ +++ ++++ ++++ ++++ +++ ++++

+++ +++ ++ + ++

+

f~

+

::I.

, Conidia

Conidia

I

2

3 4

~

7 8 9

10 II 12

13

+

+

+ +

+++ + ++ ++ + ++ +

14

IS 16 17 18 19 20 21

22

23 24 25

+++ ++ + +

++++ ++ ++ +

+++

++ ++ +++ ++++ +++ ++ +++

++

+++ + + + + ++ ++

+++ +++ ++++ ++

+ + +

++++ ++++

+

+

+

26 27

28 29 30 31 32 33 34 35 36 37

++

+++

+ + + +

+ +

+

++

+

+

+++ +++ +++

+

+++ +++ ++++ ++++ +++ ++

++++

++ + +

++++ + +++

++

++ + +++ +++ +++ ++++ ++++ ++++ ++++ ++++ ++++

++ + ++

++ +

+ +

+ ++++ ++

++

+++ ++

+

+ ++

+++

++++ +++ ++++ ++

Key: -. absent; +. very few;

\

Conidia

++++

++++ ++++ ++++ ++++ ++ +++ +++ ++ ++ ++

+

++ ++

+

++++ +++ +++ ++++ ++++ ++++ ++++ ++++ ++++

+

++++ ++++ ++++ ++++

+ +

+++

++

+++ ++++

Cleistothecia

~

~

.t'-..

+ ++++ +

++++ ++++ ++++ +++ ++++ +++

t

++

++ ++

~

+++ ++++ ++

++

++++ + ++++ ++++ ++++ + +++ +++ +++ ++ ++++ ++++ +++ ++++ ++++ + +. few; + + +, moderate; + + + +. abundant.

+++

t

.~ ~

~ ~.

$

Table 3. Colour of colony reverse on PDA at different temperatures (Colour standard-Ostwald in Steller & Ostwald 1939) 30· in , 8 days

25· in 10 days

,

A

Strain no, I 2 3 4 5 6

A 9 10 II 12 13 14 IS

16 17 18 19 20 21 22 23 24

Colony diam (em) 4'2 5'3 5'4 5'5 5'6

S'§

4'

5'7 5'0

4 '7 5'5 5'6 4'5

7'1 6 '1 3 '7 6'0 6 '4 5'8

6'0 6'0 2'9 3'8 3'2

Colour XIV II VI VIII VI VIII VIII VI VI VI VIII VI IV II II VI VIII IV VI II VI

In general 4pa 3ig 4ga 4ig 3ga 6ia 6lc 4pi Spi Spi

4Pg 41e Spl ISig Isig sng 4Pg sni

6pi

snl

s ga

a

YG YP YO YP DP DP B B B B YB DB DG OG B B B B GB OP

,

Colony diam (cm) 5"3

5'8

6'9 6'9 7'6 7'7 6'5

7"26'4 6'5

7'6 7'6 5'9

6'1 4'9

5'7

6'6 7'2 6'3 6'6 6'2 4'2

W W W

5'1

DP LY

5'7

Colour XII II VI VIII VI XIV VI VIII VIII VI VIII VI VIII II II VI II II VI II VIII IV VIII IV VIII X VI XII VIII VI IV IV IV VI VI

In general

s!'a

31g 7l1a 31a 7ga 7pa 7ga 3n e 4Pg 41e 3n e 7ga 4pg IS!g ISlg sng

snl snl Snr sn sIc

R GB DP YO DP C DP YB B B YB DP DB DG DG B GB GB B GB PO

W W

~

-.....l

,

0

.

37 · in 8 days

, Colony diam (cm) 5'1 7'0 7'2 7'5

8'0 8'2 7'0

7'5

7'0 7'0 7'8 8'3 6'5 3'5

2'2

5'8

6'2 7'5

6,8 6,8 7'0 5'0 5'4

Colour XII II VI

vru

VI XIV VI VIII VIII VI VIII VI VIII II II VI IV VI IV II VI

In general

sna 3ig 7ga 3ia 7ga 7pa 7ga 3ne 4Pg 41e 3n e 7ga 4Pg ISig ISig Spi sni

Spi sni

ronl sga

OR GB OP YO DP C OP YB B LB YB OP B DG OG B DB B DB DG PO

W W

LY IV 3'0 3ea LY 3~a ara LY VIII aia 6'5 LY 26 lea LY 6'7 7'8 W a PO 7'0 VIII 27 7'8 4- i a a 4 ia R 61a R 28 2'0 X 1'7 6la 2'3 R LG 6'2 YB 3ic VI 1e 6'2 4'6 29 YB 3 C 6na C XII 6'5 6na 6 '3 C 30 5'9 6,8 6,8 LP 6ia DP VIII 6 '1 sia PO 31 2,8 OP 6ga DP VI 6ga 3'2 32 4'8 OP g,o LP 6'6 YP IV 4ea 4cg LP 5'8 33 DB DB 3pl II 2nl 7'5 7'6 4'2 DG 34DB DB II anl 7'5 3Pl 3'6 35 7'4 OG DP 6ga OP VI 7'4 7'2 4ga YO 36 3'7 PB PB 6'9 VI 4-1e PB 7'8 37 5'4 41e General colour key: B, Brown; C, Ca rmine; DB, Dark Brown; OG, Dark Grey; OP, Deep Pink; G, Grey; G B, Greyish Brown ; LB , Light Brown; LG, Light Grey; LP LilJ,ht Pink; LY, Light Yellow; 0, Orange ; OP, Orange Pink; OR, Orange Red ; OY, Orange Yellow; P, Pink; PO, Pinkish Orange; PR, Pinkish Red; R, Red; W, Colourless; Y, Yellow, YO, YellOWIsh Orange, 25

3'5 5'6 5'8

VIII IV VIIl XII II XII IV VIIl IV IV IV IV VI

61c lea 4 ia s na lee 6na 4 ea 61c 4ea 3pl 3pl 6ga 4ng

3'2

s~

~ .... ~.

~ b;:l ;:I.

....

~

~ C"I

(;)

<:l O'Q

[ ~ ~.

Q

Monascus. Keshari L. Manandhar and A. E. Apinis

471

tested can be placed into five groups based on their optimal temperature requirements: (I) isolates which grew best at 25°, e.g. 14, 15 and 28; (2) isolates with an optimum of 25-30°, such as 29; (3) isolates with best spreading growth at 30°, such as 19, 20, 24, 34 and 35; (4) isolates possessing optimal spreading growth at 30-37°, such as I, 2, 3, II, 12, 16,17, 30, 31, 36 and 37; and (5) best spreading growth at 37° such as 4-10, 13, 18, 21-23, 25-27, 32 and 33. Most of the Monascus isolates studied did not grow at 48° on PDA or MA, but a number of them grew reasonably well at 45°. Some of the strains showed maxima below 45°. According to their maximum temperatures the various isolates studied can be regarded either as transitional mesophiles (Apinis, I963a, b) such as 14 and 15 which did not grow at 40° but grew well at 37°, or as microthermophiles (Apinis, I963b) which grew at 45° but not at 48°, comprising all the other isolates studied. Almost all the strains grew at 18°, although some very little, but no growth was observed at 12° in any strain. At 15° some strains (I, 2,4,6 12, 14, 15, 17, 19, 22-3 1 and 33-37) grew while others (3, 5, 7, 8, 9, I I, 13, 16, 18, 20, 21 and 32) did not. Strains growing at 15° can be further distinguished according to the degree of growth at 15° after 20 days. Poor growth was observed in strains I, 2, 4, 10, 12, 17, 19, 22, 23, 25 and 30-36, but reasonably good growth took place in isolates 6, 14, 15,24,26,27,28,29 and 37. There is a marked distinction between the strains which exhibited good growth at low temperatures and poor growth at high temperatures, and the strains which showed good growth at high temperatures and poor growth at low temperatures. Certain cultures on PDA and MA kept at 48° for 15 days without showing any growth were placed at 30° and commenced growth after 3 days (isolates 1-5, 7-13, 16-21, 24, 27 and 34-37). Other cultures (6, 22, 23, 25,26 and 28-33), however, did not grow any more even after incubation for 10 days at 30°. It seems probable that for certain strains the temperature of 48° is too high. Our thanks are due to Dr J. A. von Arx (Centraalbureau voor Schimmelcultures), Professor C. G. C. Chesters (Department of Botany, University of Nottingham) , Miss Evelyn Forsyth (NAAS, Newcastle-upon-Tyne), Dr J. Harrison (NAAS, Shardlow), Mr A. Johnston and Dr Agnes H. S. Onions (Commonwealth Mycological Institute, Kew) and Dr W. B. Kendrick (University of Waterloo, Canada), and to the laboratory staff of the Department of Botany, University of Nottingham.

REFERENCES

APINIS, A. E. (1963 a). Occurrence of thermophilous microfungi in certain alluvial soils near Nottingham. Nova Hedwigia 5,57-78. APINIS, A. E. (1963 b). Thermophilous fungi of coastal grasslands. Soil Organisms (ed. by ]. Doeksen and]. van der Drift), pp. 427-438. North Holland Publ, Co., Amsterdam. BARKER, B. T. P. (1903). The morphology and development of the ascocarp in Monascus. Annals of Botany, London 7.7, 168-236.

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Transactions British Mycological Society

COLE, G. T. & KENDRICK, W. B. (1968). Conidium ontogeny in hyphomycetes. The imperfect state of Monascus ruber and its meristem arthrospores. Canadian Journal rif Botany 46, 987-992. HARz, C. O. (1890). Physomyces heterosporus. Botanisches Zentralblatt 4 1 , 378-379, 405-41 I. NAKAZAWA, R. & SAT6, K. (1930). On the Monascus of Taiwan, the red seed of rice. Nihon Nogei Kagakkai-Shi (Journal rif the Agricultural Chemical Society rif Japan) 6, 352-35 8. PIEDALLU, A. (1909). Sur une nouvelle moisissure du tannage a l'huile, Monascus purpureus. Compte rendu hebdomadaire des seances deI'Acadlmie des Sciences, Paris 147, 510-513. PIEDALLu, A. (1910). Sur une nouvelle moisissure du tannage a l'huile, Monascus olei. Compte rendu hebdomadaire des seances de l'Academie des Sciences, Paris 151, 397-399' STELLER, G. & OSTWALD, G. (1939). Die Kleine Farbmesstafel nach Wilhelm Ostwald. Gottingen: K. G. Muster-Schmidt. THIRUMALACHAR, M.J., WHITEHEAD, M. D. & MATHUR, P. N. (1964). A new genus of Eurotiaceae from soil. Mycologia 56, 8og--815. VAN TmGHEM, P. (1884)' Monascus, genre nouveau de l'ordre des Ascomycetes. Bulletin Societe botanique deFrance 31, 226-23 I. YOUNG, E. M. (1930). Physiological studies in relation to the taxonomy of Monascus species. Transactions rifthe Wisconsin Academy rifSciences, Arts and Letters 25, 227-244.

(Acceptedfor publication 3 May 1971)