Anaerobic Growth of Fungi

Notes and Brief Articles

299

Rhizopus spp. occur commonly in soil. They grow rapidly by comparison with other soil fungi and they are among the primary colonizers of organic matter incorporated into soil. These results, indicating that low concentrations of ' Gramoxone' W might reduce the ability of spores of R. stolonifer to colonize a substrate containing residues of the herbicide, suggest that

A

~ 10p m

B

I-:-:--i 10pm

Fig. 1, R. stolonifer. A, Spores germinating in liquid medium containing 10 p.p.m. of paraquat as ' Gra m oxone ' Wj B, a spore germinating in control medium (both 20 h after inocula tion).

patterns of saprophytic colonization in the field may be affected by herbicides. The concentrations of ' Gramoxone' W used in these experiments are well within the range likely to occur in sprayed vegetation in the field. VALERIE WILKINSON and R. L. LUCAS, Department of Agricultural Science, University of Oxford ANAEROBIC GROWTH OF FUNGI

J. W. Foster (1949) said: ' O ne of the major metabolic differences between fungi and bacteria is that there are no anaerobic moulds either obligate or facultative.' Anaerobic growth has, however, been achieved in recent years with species of Mucor and Fusarium (Bartnicki-Garcia & Nickerson, 1962; Gunner & Alexander, 1964) and other fungi (Stotzky & Goos, 1965). Trans. Br. mycol. Soc. 53 (2), (1969). Printed in Great Britain

Table

I.

Anaerobic growth

offungi

No . of cu ltures Growth

No growth

II

12

0 I

I

'~"

Class

ttl :<

Phycomycetes

Mu cor ales

Ascomycetes

Endomycetales Eu rot iales Erysipha les Hypocreal es

0

I

6

0

Spha eria les

0

5

H elotia les Ust ilaginales Agar icales Nidulariales Sp ha eropsidales

0

I

0 0

I

Melanconi ales Moniliales Cryptococca ceae Moniliaceae

~

~

~ ~

~

:e <0 C7>

e

~ ::sO

...s·

~

C') ~

~

~ ~.

s·

Species

~

:;l

Basidiomycetes

Fun gi I mperfecti

Dem atiaceae

Tuber cul ariaceae Total

I

3

0

I

I

4

0 0

2

I

0

43

2

21

12

5

38

102

No growth

8

M ucor hiemalis, M. pusillus. 111. ramannianus, Choanephora cucurbitarum, Absidia spinosa, A . orchidis, Mortierella alpina, Syncephalastrum raamosum, Thamnidium elegans Cephaloascusfragrans Arthroderma multifidum Anixiopsis peruoiana

$:I

Gr owth

Mucor abundans, M . circinelloides, M . erectus, M . f ragilis, M. griseocyanus, M. variallS, Zygorhynchus moelleri, Z. ouilleminii

-

Lilliputia rufula Gibberellafujikuroi, G. intricans, G. saubinetti, Nectria ditissima

-

Macrophomina phaseoli

I

4

W

Breitanomyces bruxillensis Cephalosporium acremanium, Cephalosporium sp., Geotrichum candidum

Monotospora sp., Papularia sphaerospetma

Cylindrocarpon radicicola, Fusarium conglutinans, F. moniliforme, F. oxysporum

-

Chaetomium globosum, C. cochliodes, Petriella asymmetrica, Subbaromyces splendens, Amphisphaeria incrustans Lambertella comi-maris Ustilago violacea Poria vaporia, Lenzites sepioria, Aleurodiscus afr icanus lll osporium oliva/rum Phomalingam, P. pigmentivora, Cy lopleajug/andis, Ascochyta tarda Colletomchum limicola, Pesta/otia rhododendron Calcitalna acraspora Acrocylindrium oryzae, Aspergillus candidus, Acfutnaricus, A sfumigatus, A. niger, A. terreus, Beauuaria bassiana, Botrytisf abae, Cephalosporium sp., Gliocladium uirens, Penicillium aurantiooirens, P. breoicompadum, P. charlesii, P. gladioli, P. griseofuloum, P. melinii, P. oryzae, P. paxillae, P. piscarum, P. rubrum, P. spinulosum, P. steckii, Scopulariopsis breuicaulis, Tilachidum nigrescms, Trichoderma viride, Verticillium dahliae Alternaria solani, Botryotrichum pululiferum, Cladosporium cellare, Corynesporium casseicola, Curoularia andropogonis, Glimastix conooluta, Haplographium fuligineum, Helminthosporium satioum, H. dematioideum, Humicolafuscoatra, Mammaria echinobottyoides, Memnoniella echinata, Stachybotrys atra, S. alternans, Stemphylium radicinum Epicoccus purpurescens, Fusarium solani, Illosporium oliva/rum, Myrothecium t errucaria

~

l! ~

~. b::l

;:1. ....

~

~ g S'

()'Cj

§.

~

~. Q

Notes and Brief Articles

301

Since variation of medium constituents affects the secondary metabolism of fungi it seemed possible that changing the oxygen tension might also produce such effects. Various fungi were cultured anaerobically with the object of obtaining new metabolites or modifying the production ofknown ones. Production of secondary metabolites was inhibited by anaerobic conditions so that this aim was not achieved, but the results show a range of fungi which grew anaerobically, thus confirming the conclusions of Stotzky & Goos. The fungi were incubated for 7 days at 25 DC on a medium containing peptone (10 g), yeast extract (3 g), and glucose (20 g) in I I adjusted to pH 5'5 before autoclaving and dispensed in I ml portions in small test tubes or 100 ml portions in 500 ml conical flasks. The tubes were placed in a Macintosh-Fildes jar which was evacuated and flushed out with N 2 several times. Where no anaerobic growth was observed in tubes, subsequent aerobic growth confirmed the viability of the inoculum. The flasks were fitted to a rotary shaker, with a slow stream of N, (0'061/min/flask) containing 2-4 % CO 2 passing through continuously. Control aerobic cultures were also incubated. 'White spot' nitrogen was used in all cases, and anaerobic conditions were confirmed with methylene blue and thioglycollic acid tubes (Baird and Tatlock Ltd.), Dry weights were obtained by washing the mycelium from each flask and drying in a vacuum oven at 80 0 • Strains of fungi used were derived from the Akers culture collection. Table

2.

Effect ofanaerobiosis ongrowth in stationary test tubes and shake flasks Growth after 7 days incubation

,

Shake flasks A.C.C. culture no, 3 29 27 24 27 28 546 962 238 1 3 26 1634 1822 9 17 I I 60 174 32 27 13 806 1137 818 2123

Tubes Visual Fungus Mucor erectus Mi fragilis M. abundans Zygorhyncus moelleri Z. vuilleminii Absidia spinose Syncephalastrum racemosum Lilliputia rufula Arthroderma multifidum Gibberella fujikuroi Gibberella fujikuroi Chaetomium globosum Aspergillus niger Geotriehum eandidum Penicillium melinii Alternaria solani Fusarium oxysporum Fusarium oxysporum

Visual

~

N

A

I

4 3 3 3 3 4 3 3 3 3 3 3 4 3 3 3 3 4

I I I

2 0 0 2 0 I

2 0 0

2 0 0

2 I

~

(

,---A----,

N

2 2 I

2

,

0 2 0

2 I

0 0 I

0 0

2 2

\

Dry wt. (rngjrnl) ,------"-----,

A

N

A

3 4 3 4 3 2 3 3 2 3 3 2 4 3 4 4 2 3

0'44 2'4 2 1'29 0'69 1'16 0'18

5" 8'8 3'9 7'0 7.8 8·8

0'37

8'1

0'67 0'65

7'7 5"6

0'39

8'S

1'79 2'13

9'4 12'7

N = anaerobic, A = aerobic, A.C.C. = Akers culture collection. o = no growth, I = slight growth, 2 = fair growth, 3 = good growth, 4 = very good growth. Trans. Br. mycol. Soc. 53 (2), (1969). Printed in Great Britain

302

Transactions British Mycological Society

Of 140 cultures tested 38 grew anaerobically (Table I). Such growth was found particularly in members of the Mucorales, Hypocreales and Tuberculariales and sporadically in other groups. In all cases of growth, results were confirmed in tubes or shake flasks, as were a few cases where growth failed (Table 2). Dry weights up to 2'4 mg/ml were obtained. This is a wider range of fungi showing anaerobic growth than has been previously reported. REF ERENCES

BARTNICKI-GARCIA, S. & NICKERSON, W. J. (1962). Nutrition, growth and morphogenesis of MIKoT rouxii. ]. Bact. 84, 841-858. FOSTER, J. W. (1949). Chemical activities offungi, p. 162. New York: Academic Press. GUNNER, H . B. & ALEXANDER, M. (1964). Anaerobic growth of Fusarium osysporum. ]. Bact. 87, 130g-1316. STOTZKY, G. & Goos, R. D. (1965). Effect of high CO 2 and 02 tensions on the soil microbiota. Can. ] . M icrobiol. 11,853-868. P. J. CURTIS, Imperial Chemical Industries Ltd., Pharmaceuticals Division, Alderley Park, Macclesfield

OCCURRENCE OF PSILOCYBIN IN THE SPOROPHORES OF PSILOCYBE SEMILANCEATA

The nature of the active principles of the sporophores of Mexican hallucinogenic Agaricales (Psilocybe mexicana Heim and related species) has been determined by Hofmann, Heim, Brack & Kobel (1958) and by Hofmann, Frey, Ott, Petrzilka & Troxler (1958). The principal active substance is psilocybin (4-phosphoryl-N,N-dimethyl-tryptamine) although psilocin (4-hydroxyl-N,N-dimethyltryptamine) occurs in trace amounts. Conocybe siligineoides Heim has also been shown to contain psilocybin (Benedict, Brady, Smith & Tyler, 1962). The occurrence of these substances in British Agaricales has not previously been established, although there have been several instances of suspected poisoning by sporophores of Panaeolina and Psilocybe spp. Air-dried samples of the sporophores of Panaeolinafoenisecii (Pers. ex Fr.) Maire (1·65 g) and Psilocybe semilanceata (Fr. ex Secr.) Kummer (2'35 g), collected by Miss M. Holden at Rothamsted Experimental Station, Harpenden, Herts., have been investigated for the presence of psilocybin and psilocin. The finely milled tissue was extracted with methanol (2 x 20 ml) and the combined methanol extracts reduced to small volume in vacuo. An aliquot of each extract was chromatographed with authentic psilocybin and psilocin on silica gel G in a chloroform/methanol/ammonia (80: 20: 0'2) solvent system, followed by spraying with 3 % p-dimethylaminobenzaldehyde in concentrated Hfll to detect indole derivatives by .their blue-grey colour. This system separates psilocybin from psilocin although the former does not move from the origin. The Panaeolinafoenisecii extract contained no detectable psilocybin or psilocin, and psilocin was also undetectable in the Psilocybe semilanceata extract. However, the P. semilanceata extract contained a substance which was Trans, Br. "!~col, Soc. 53 (2), (196g). Printed in Great Britain