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Nitrogen utilisation and growth at reduced water activity by mucoralean fungi present in soil
S. Mr. J. 130t. 1999.65(5 & 6): 407-413
407
Nitrogen utilisation and growth at reduced water activity by mucoralean fungi present in soil B.O. Seabi1 , A. Botha*, B.C. Viljoen 1 and C. Roux 2 'Department of Microbiology and Biochemistry, University of the Orange Free State, P,Q Box 339, Bloemfontein , 9300 Republic of Sou th Africa *Department of Microbiology , University of Stellenbosch, Stellenbosch , 7600 Republic of South Africa 2Myco!ogy Unit, Biosystematics DIvi sion , ARC -PPRI , Private Bag X134 , Pretoria, 000 1 Repub lic of South Africa Reall!etf
r
JIIIII! 1991), I'e\'/sed 5 Ouuher 1999
Nine mucoralean funga l genera including 18 species were exam ined for their ability to utilise a series of nitrogen co ntaining compounds and to grow at an aw of 0.955 on solid media The nitrogen concentration in the media was 0 1
gil and the senes o f nitrogen containing compounds were ammonium chloride, asparagine , sod ium glutamate, sodium nitrite and potassi um nitrate . The gene ra we re Actmomucor Schostak. , 8 ackuseIJa Hesselt. & J .J. Ellis , Cunnmghamella Matr. . Gongronella Ri baldi, Mortierella Coem , Mucor Fresen .. Rhizomucor Luce! & Costantln , Rhizopus Ehrenb and Tham nostylum Arx & H. P. Upadhyay. Thirty~nine funga l strains obtained from cultu re collections (CBS, MUFS a nd PPRI) , as well as 12 sOil isolate s from the Karoo , were tested. All the species and stra ins te sted In this s tudy were able to utilise asparagine and glutamate . Strains bel ong in g to CunninghameJla, Mucor racemo sus Fresen ., Rhizopus microsporlls Tiegh. and Rhizopus st%nifer (Ehrenb .: Fr) VuHl were unable to utilise ammonium chloride. S trains of Cunninghamella, Mortierella, RhlZomacor, Rhizopus microsporus and Rhlzopus stoloniferwere una ble to g row o n nitra te as so le nitrogen s ource. Nitrite wa s found to be tOXIC to species be long ing to Cunnmghamella, Monlerefla , Rhizomucor, Rhizopus and Thamnoslylum . Members of Gongronella, Mortierella , Mucor racemosus, Rhizomucor and Th amnostyJum were unable to grow at an a w of 0.955. Key words: Nitrogen sources, phys iology, Mucorales , Upper Nama Karoo
"To whom correspondence shou ld be addressed. (E·ma il: [email protected] .ac.za).
Introduction t\ llIco rakall fungi are m ostl y saprophytes assoc iated with decay~ ing plant material. dun g or ot her o rgani c debr is in soil ( H essel ~ ti nc & Ellis 1973: DOlllsch Iff 01. 1980). T hese fungi are usuall y associated with moist environments , sllch as leafl itter in forests. and arc known to be rel atively into lerant to Iowan (Bro wn 1( 76). Grow th of Absidia...\[lIeor and Hlti::oplls occur above a\\ vtll ues of O.9::! to 0.93 (Ottaviani 1(93). However, muco ra lean fungi have "Iso bee n record ed in so il , deb ris and on plant roots fro m arid regions (Ste iman er al. 1995 ; Roux & Van Warlllelo
1997). Availabil ity of car bOil and nitrogen sources is known 10 playa major role in the composition and succession o f microbia l comm uniti es o n dl:caying organic matter (Daesche l ct al. 1987; HudSO il 1 9<)~) . ~ llIcora l e an fun gi are the first to coloni se decaying organic mate rial in soil, si nce these fungi are able to rapidly uti· lise the lim ited number of simple carb ohydrates that are usually availabl e. bdore other funga l gro ups take ove r the min erali sation of car bOil (Hesseltine & Ellis 1973). It is, there fo re, not surprising tha t srudies have indi ca ted that m ucora lean fungi are able to uti lise o rganic nit rogen as well as am lTIon iul11 salts (lnui el al. 1965: Aggelis el al. 198 7; Sajb idor el 0/. 1988; Tsuchiura & Sakura 1988 ). However, ce rtain authors found that some Ill llcoralean fun gi are able to utilize nitrate (Hanson & Dosta lek 1988; Certi k 1(93). T his characte ri sti c is not essential for a primary co lo nize r o f dead o rgani c malte r. s ince d uring the mi neralization of organic nitrogen, nitrifi cat ion o nly occurs after amm onific ation (Sparling 1(98). The spec ific positi on of particular species of l1lucora lcan fungi in the bi ogeochemical nitrogen cycle, howeve r. is unkn ow n since o nl y fragmentary information on the ut i· lisation of d iffe rent nitrogen conta ining compounds by these fungi, exists in literature. Wit h the Clbove as background , the aim of th is study was to
investigate the ecological nic he of 11111coralean fun gi in arid soil. \ . . itl1 spec ific reference to the posi ti on these: fungi occ upy il\ the biogeoc hemical cycle of nitroge n. Consequentl y. sckcted IIl U(O ~ ra lea n tax a occurri ng fre quently in soi l habi tats, includin g strai ns from culture collections. as well as isol ates obtain t:d from a so il samp le from arid Upper Nama Kamo (Low & Rcbdo 1(96). were used to eva lu ate in vitro growth to determi ne nitroge:n sources and a w tolerances .
Materials and Methods Strains used Thl! fungal strains and bolatcs uSl!d in this stu dy arl! list(:t\ in Tahk~ I and 3. The strai ns Wl!re: ohtainl!d from the Ccntraalhun:all \om Scili nlme icul ture:s (CBS). Nctherl ands. the: Mlicorakan I.:ult un: I.:ol~ kction of the Uni vcrs ity of the Orange Free Stah.' (!\.'IUFS). SOllth Africa. and from the Nation al (.:olkctill il of Fung i tpPR I). Prdon;l. SOllth Africa. Oth cr strain s \\e:r\! isola ted from it soil Sdlllrk origi nating from Upper Nama Karon (1,0\\ &. Rebel\) 1996).
Physiological properties Preparatiol1 o1"il1ocu/o A stcrih::. wet inoclJiating hll)r \\<1S llsed for I.:ach ["ungal strain 10 transfe r sporangiospore:; and/or h~ pllal Ibgllll.:nts from it IWO \\ l!1.:h. old cu lture on 2% (w/w) Di lco malt e.'tnll.:t agar (MEA) to 5 rnl stl!r~ ilc dist illed water. Fl)rty micro liters of the result ing suspells ion. C(ln~ tain ing circa 2 x 106 L'olony form lllg unit:-. pe:r Ill !. \\~b lI~l'd inocu late each delin l.!d medium. in Petri dishl!s.
l il
Nitrogen llIi1isaliol1 011 (/ solid de.fil1ed medillm A ser ies of solid ddine:d media \H\S prepared (Van d(:r Wall & Y ar ~ ro\,,. 1984). Each med ium. in PClri d ishes. consisted of 11.7 gi l Bal.:to yea.st c
S. At'r. 1. BOL 1999.65(5 & 6)
Table 1 The ability of strains representing different mucoralean genera to util ise the series of nitrogen containing compounds and to grow at 0.955 aw " Nitro;;c n S\l u rcC 1I1ilbalinn KN0 1
i\' r I. I CI
on
+++
++
or elt'gO/I.\" (Eidam) C. R. Iknj . &. Iks.;d t. !'. II IFS 121
1++
NaNO:
Strains Acl!lt()111IIU [el i /lO ll/ lit
I/" ("('gallS
(Eitlam) C R. lknj. &. l kssdL \1 11 FS
.-Ir I/IIIJIII I/mr (' /('g olls (EiJam) C it Iknj . &. Ik ssd L .\lI lFS
Hud-lISel/o 1IIIIIIJfOSpOro (Lf.:!lun .) Iknn) & R.!\. . Ik nj ]'vtt iFS OO}
If(/(" 11.\('/10
/(//111I/'OS/JO/'(/ (I . ~llJn.)
f((/( Au \{'II(I {UIIII Jrosl'(WlI (l.cnJn
Iknn) & I{ K. rknj. MI IFS OClX
+++
0
(!chimda({/ (l'lw:-..t.) Tha:-.:t rvll JI:S O()3
(Lclldn.) Pe, mnd & i);l l Vesl:o MI Ir s 2
0 +++
+ +++
l/orllt'n 'lIl1 (lII/oe/JfJidea W. G
()
l/o/'II('I'e/I" g/ohuli{em Rostt'up CBS 417.64
II
Y. Ling-Ynng CBS 430.76
I/ol'fl{'/'fI/lfllllljico/a
R.K. fk nj . C BS 292.63T
IflicorlCl gospamm
.\llIcorn/'! II/d/aide.\· Tiegh.
CBS 11 1J'(lR
+ ~+
•. +
(Tha xL) rhaxt. Ml lFS 00 I
(;ollgro/1!'lIa h lll/erI
t+
I-+.!-
(lha.\t.) rh:J\l. Ml lFS 002
& Dal Vesco MUFS
+~+
-j-+ +
( ' /il/I lI ngllll lll e{{a I!dlllJli/(/f(/
Pc~ronel
+
• ++
('/11 l11 illglwlJlc/{a ed/l/lIIl l1fll
(l.endn .)
+++
+++
(J
(' lIl1l11l1glllll/leJfa
.+
+++
J lknny &. R K . [!en.!. MI IFS OIl
(;01l,!!.1'01/1'1/(I hlli /ai
( rill
+++
+++
O.lJ):i;l"
,,
+ ++
nl)
ii i inl\\ III
/\S11
.~
.. .. .
., +
,
H
+H
+' .
•. ;'1-
l- + .
0
.+
• ++
0
-+
. ++
+ .•
Il
++ +
++ +
+>
,+
+ ,.
0
H
+'+
T+
...
++
'
~+
()
++
• -+
+-'- +
0
++ +
tt +
+++
Il
+H
+++
+++
II
++
. ++
H+
."
H
H +
. ++
H-
+++
+++
++ +
+H
+++
H+
H+
II
'"
H+
+ .....,
++ +
H+
++
,,+
H +
0
(Liml!. ) Schipp.:r M l lFS 00 I
II
+++
1-
~+
+++
II
Nhr::olll//cor /J/fsillus (Lind l. ) Schipper M l lFS 00)
II
+++
+.+
+H
II
T icgh. CBS 63 1.82
II
II
; ++
H +
+
T iegh. PI'RI 5560
()
II
++
+++
++
+
+++
+ ..... +
+ ++
++
III/cor/hII'll I 1\,(il1i.:r CBS 134.35 I hl(,()1' jJ/IIIII/ll'II.1'
Bononl CBS II Ul7
IllIcor 1'(/( l·/I/O.l'IIX
Fres CBS 115.08
NlII:::OII/I/( 01' I'lIsillllS
f?l1I:::OPliS 1/I/('I'OSporoU.l'
Rhcojlll.\ III/cms/wrolf.\·
Rl1I:::ofJlIs 01'1 ':::/1(' Went & Prins. Cier:rl. CBS 112.07
(Ehreno. : Fr.) Vuil!. COS 609.S2
II
()
+++
+++
++
/(/1/:::0/111 1' I/n/O /lUer (Ellrenb. : Fr.) Vui l!. CBS 31 t) .35
0
II
+ ++
+ ++
++
+++
+
.+
++
II
RhbJjlll.l' ." Ifl/olllfer
lhulII/w\'I\!/Il11jJirijorm e (Bainierl Arx & H.P LJp;luhyay t\·1UFS 025
"Nitrogen uti lisation. meas ured h) calculating th.: cololl) diameter ohlained nn the medi uOl with the parlicular nilrogen SOurl:e. as a percentage {If the l:OhUl~ d ialllt:tcr olll hc Illcd iulll \\ hieh b~st supporteu radia l gru\\1h orthe pmtic ular fungal stra in. S)lllhnls: 0 =: 0%: + "" 1-3.1 %: +.1. = .;4(,6%: +-... (,7- 100°1\): - = toxic . si ncc t h ~ CO ln ll ) densilY and d ia meter on thc part icular sol id medium w.:re less lhan Ilwl nbtaincd on the mcdiulll dc\ 'oid o ra nitrogt.:11 ~{l urc e: Abhre"i alil)1\s: Asn = Asparn gine: G!u = Sndiulll glutamate . #(iron-I ll at reduced '\". mca:-lIl'ed by ca lcul ating the colony diamcter on the medium wilh 0.955 a". as a percentagc nrth e w lony d iamch:: r ohtaill~d Oil a non water-stn.:ssetl control. Symon!s: () = 0%: + = 1- 33%: ++ = 34-f)o%: +++ = 67- 100% .
all had a pll n( circa 5.5 and \\'\.:re so lid ifi ed with 2%, (w/\\') \\ash~d pun fred agar. r hl' \\ilshcd aga r wa s pn:pan:d hy repctitiH:I~ \\ashing so lid ilicd agar block s ( I>ifco) in dt: m incral iscd waleI' (Van der Walt & Yarrow
di amctt.:r o btained after fivc days on the 111l.:d iullI containing the particu lar nitrogt:ll source. as a pl.:rccnlage of the wlony diamell..::l' ohta ined after the same period O il Ihe medium \\ hich best supported rad ia l growth o r the p'lrtic ula r fungal slr;un (Tah les I and 4).
I~X4)
t\ stlper1ici;iI wdl of H,() mm in diamete r. \\as made in th c center of the mediulll in cach Petri dish. using a hot g lass rou. Each Pcl!'i dish \\as 11111cu lated pipdting .1O ~t! or the illlll'u lulll into thl.: well
oy
amI then inwhatl.:d al 25°C ill1he dark. Thl.: d iallldc r or t h~ result ing colony \\'as measun.:d aHe r live days of inc ubat ion . A cont rol exp..::riment \\ as l'tIn ti uc\eJ in \\Ihid the abi lity to gnm Oil a solid med ium dc\uid 01' ;1 Ililrogen source was testl.:d All th l.: \!x pcr illlc:nt s \\el'l.: conducted III triplicate .
A1ea.'iuremenf (~rradial gr<)wlh ll'ilh different nitrogen source.\· Rad ial gnl\\ tho a s an in d icat ion of th e nlte of uti lisation o r the nllrog,en source. \\as measured. Thi s ",",IS done hy calculating the cnlon~
Growlh on (I medium with a redllced waleI' activity A solid me diulll was pre pared in Petri di shes. Th is lllediulll Ctl llsistcd of thc 100 hmin g (gil): IO.f). g.llH.:use: 5.0. peptollc: I .D. KII :! P O~: 0.5. MgS()~ . 7 11 :! () : nO.n. gl~ c aol: 15.0.
<1
pI I o r cirm
S .()
and
a"
orO.1)55. A sllper ti eial well in th e mcdiulll \"as pre rarcd and illllc lil atcd as descrihed ahove. T he illocul
or
S. Afr. J. Bot. 1999. 65(5 & 6)
4 09
Ih.: c'pcrlmo..'llts wcro.: conducted ill trip lica te. AfL'oSlfn...'1I11..'11l oj'radiClI grollllh on mediulI/ \1 '/111
lOll'
au
(iro\\ III \\ as me asured by calcul ating the colony diameter ohtalllcd artl.'f 11\ 1..: da~s on the m ed iulll \\ ilh 0 .955 i.l w' as it perce lit age of the c oh lll~ d ],11l1Clcr oht a incd \1 1' gl~ Cl'ft l] (
:lth:r thl. ? same peri od Oil thl..' l:olllrol dcyoid
l"ah1cs I and.t).
Determination of funga l taxa in soil 7i. (klllg of .'0" sample J h.; "a 1llpllll~ :-.1 Ie (lfll1l ~ ) is loc
,H url' h ] 3.J .e. Th e :-:i1 rn p1c was takl."l1 in mid-sumlll er. The su rface 11111.:r \\a~ li f'.. , scraped away to nxllll:C co nlal1linat lOll fromlhi s hahital ,\ S{II I "am ple Ill" ClrCII 2900 g , c o nsisti ng o r ni lH.: suhsamplcs. lakel1 at randolll (}\ er the are a of the site . each to a dcpth o!" 10 1.' 111 . I he , uh:- amplcs \\ere thoroughly mixed ill the labnratol')' 10 pro duce the :';lInpk. whi ch \\ uS !"urther processed . H<\-;
ag ar tRiolab): 0.02 gi l heno my l I Mdhy l 1-( bll l)' k ar h a lllll ~ I )-2-hcll/imida%Ole-carbamate. A.ldrich catalog no. JI-:. I SR-liJ was added heron: autoclaving. The sccond selec ti\ c med iulll \\'il S similar \(l the [irs!. except that it cOllta ined 10 gil SliCroSC as l:arhon sourcc ill~tcad stnrch . The relat ivel y nun -select iv e m..:d illlll (r,,1 YPps) (( 'arrcinl & Kuske 1(92 ). c on ta ined (gil ): Malt cxtrad (Dili.:o). 700: pc ptone ((h oi d). 1.00: )CaSl e xtrac t (D il'co). () 50: pe ni c il lin (i . 0.50: st re ptomyc in sulphate. 0. 50 :
or
or
jelen/ilicati()n
( 'hl'mical tllla~r.'iC:s o/s oi/ sample T he plioI' Ille snil sa mp le \\
or
Isolwio/l o/Iilllgi n~
lIsing the soil plate tcchnique of Ware up (19S(). two sdective Il h'dia (l"ollt ain ing henomy! and starch or sucrosl.! a s carbon source). as nell a:-. II rd ati vc non-sdeeti vt: medium. w e n~ USI.!(\ to iso late mlleo ra leiln fun g i from the soil sampll:. T he fi rst sel ective me dium COIl[;Iillcd (g/ I): st arch. 10.00: N H-ICI. 1.00: KH :!r0-l _ 1.00: MgS0-l ' 7 1 1 ~ ( ).
() 50:
~ ca s t
t:xlracl (D ileo). O.S (): allll c hloramph enicol. O.:W.
It a ls() cOlltil incd the tllllO\ving mineral salt s (mg/l ): FeSO.f. 7H 20.
111.OIl: InS)),. 711, 0. 10.UO: MnSO, . H,o. n.RO and CuSO,. 5H,O. n.()s Thc medium had it p H or 5.5 and was sol idi lied with 16 g/I
(~l/i{}/gi
Isol ates belonging to the h ighl.!r rungl were id e ntili ~ d up tn g cn us [lax ler l'1 al. ( 1994). Ellis (11)76 ) a mI Krcg. l'rk \'d using th c keys van Rij (1984). The lllul'ora1can fung i \\' c r~ ide nt iliell usi ng Ihc keys and des..:ripl iol1 s g ive n h~ ( ia ms ( I ( 77 ). I lcs:-d tinc & Fl lis ( I W1'I). !.lIllll & Ship toll ( 198 } ) a nd Schippl'r ( 1976. 1971-: and 19S·:t).
or
Results Utilisation of nitrogen sources by species and strains of the selected genera T he abi lity of species and slr::ains of the selected fungal genera to uti li se the series of ni trogen con tain ing co mpo un ds as sale n it rog~ n source is given in Table I. A ll the strai ns were ahle to uti lise the am ino acids, asparag ine and g lutamate . However. o nly 70% of the strains were ab le to utilise ammonium chloride. \-vhile 51% of the strains were ab le to grow on ni trale as sole nitrogcn source. The latter included strai ns of genera .·/c fin(JfllIIc()/", /3 ack1(sella. COllgronella, Mucor. fl!amnn.,IY/lIl1/, ::and Hhi=oj)/Is OJy=ae . The results obtained w ith nitrite, indicated that a t the concen trati ons used in thi s st udy, this co mpo ll nd was tox ic to species of ClInl1ing/wl1Iel/(I, Mo r/;erel/a, I?l!i=0I111U:UI', I?hi=opll.\· and 71WllllloslY/II/11.
Growth at 0.955 aw by species and strains of the selected genera Species and strains of the genera Adil1vl1Iw:or. /Juckltse//a.
Table 2
Characteri sti cs of the soil at sampling site
( 'h,l raC!c ristics ( 'helll ical prorcrlies
('lffl-
nil/Khamella and RIu':oplIs we re ab le to grow at 0.955 a w (Tahle I). The stra in of Mucor racC!l11o.ws. w as the o nl y strain of .\/ucor that was unab le to g row at 0,955 a \\. The struins cxtl llli ncd of ( /o llgnmel/a, Morlierel/(I, RhbJlJlttcor and Thal1l1lO.WY/llm were unable to grow at this aw value.
pi 1 n[" soil
7.25 ± 11.115
SnillllnlSlllrC content
6.66% ± O.22%)
Fungi present in the soil sample
()l'gal1ie carhnn
0.72%
Tilia l ni trogen
0.2%
Nll rllc
1. 1 ppm ± 0. 1 ppm
The fungi in the soi l sample, \vhieh were enumeraled lIsing the three isolation media, are li sted in T able 3. Strains of thirteen genera o f highe r fungi were fo und 10 occ ur in th e so il samp le . T he most frequentl y occurr ing of these were Trich oderma and ,. I.~pergil/us . Only four spec ies of mucoralean fun gi co uld he detected in the soil sample i.e. ACfil1ol11ncor elegans and llhi=oPIIS OIy::ae, wh ic h were the most numerous. and .\/o/'ficr/!I/u isuhellinCl and iHucor drCl"nel/oides
N itrate
18 .6 ppm :1:: 2.4 ppm
:\I11I11\1l1 llIl11
2.8 pplll ± 0.4 ppm
PhYSICll propcrties Ua~
30%
Silt
21%
Sa nd
48%
Utilisation of nitrogen sources by mucoralean isolates The ab il ity of mucoralcan isolates from the so il sa mple to uti li se the series of nitrogen con ta inin g co mpounds as sole nitrogen
s. Afr. 1.
·1111
Table 3
Bol. 1999.65(5 & 6)
Fungi isolated from the soil sample "'lcd.
[~(CFlJIg
MYPps (CFUlg soil)
Mell. A (CFUlg soil)
.-It f/1I01ll /lCOr elegc lIIs ( I:id arn) ( .It [knj . l le!'sc lt .
2... 0
760
760
1/(JrJ/el'el/a Isahel/ow OUlklll.
IXII 320
soil)
\ Iue-orale-.Il
1/IIU Jr Clrc:ille /loide,\'
Rlu:o/)/{.\ o r\"=al!
HW
SO
!2RO
II ()()
9~W
'1i('gh
W!.!111 & Prins. ( icerl.
[()TAL (;Clll'ra oruthcr funl!i
411
' (-rel/lIl11/ /l111
I'pergillus N(J/I".' "(/fl'lcJIlIIII
4XO :;60
( "/lIlio.ljl0rt lflll
611
("111"1'11/(// ' /(/
120
r//\W'/l/III
10
( il'O{l'Idllllll
1211
300
100
I ' l /(' (-i/I IIII,"C(,S
20
1'1.'III('JlliulJI
1211
20
60 I '(J~\ "\ ( ., "(( t/I/II/ N.liefle/rl/I/(I
I rJ( -hflderl/1(/
It HAL
411 71111
20
2180
200
42()
t T l}: Colony ii.mlling,uni ls: rv!Ypps: Relative non·sclcctive medium (Currciro & Koske 1992): Mea. A: Isolatinn medium A containing starch <1:' cilrhon source and 10 ppm hcnomyl; Mea. il: lsoialilln nH.:diulll 8 containing stl(;rn~(! as carboll source ,I11J 2IJ ppm henlllll YI.
source is given in Table 4. All the isolates were ab le to uti lise the amino acids . asparagine and gl utamate. as well as ammonium L'h loride . The iso lates of .\lOl'fh'rel/a i.w hellilJa were unable to utilise nitrate as sole nitrogen source and at the co ncent ration of l1it1'ih:.: used. this compound \\las toxic to the iso lates of .\/(}I'I/t!I'L'I/a i.~(1/)(:lIiI1(1 and Rhi; oplfs OIy;ae .
Growth at 0.955 aw by mucoralean isolates T he isolaks of , I ClillOllllfCOI' elc!R([J/s ..\lllco/' cin_-;lIelloides, and /(/11;01'".\ (l/Y;UC were able to grow at 0.955 a\\ (Tab le 4). Similar 10 the .\lor/lerdla strains from culture co llections (Tab le I ), th e isolate s of .llorrie/'e//a isabellina were unable to grow at 0.955
Discussion Nitrogen utilisation within the Mucorales :\ numher of workers testin g for the utilisation of defined nitro· gcll sources b) rnucoralcan fungi also included yeast extract in the culture media ( Hansen & Dosta lek 1988; Ccrt ik el al. 1993). Howevcr, ~ cast ex tract was found by Sajbidor el al. (1990) to be ablc to act as a nitrogen source on its own. This compli cates the intcrpretatilltl of results. Therefore, it was decided during the present ~tu d ~ to tcst for nitroge n uti li sation usi ng d efi ned med ia. III rtd d it ioll, in ord er 10 determine the n itrogcn req uirements of h) pltal cultun:s 0 11 solid s ub strates rather than those of sub· !l1l.! rged cultures in liquid subst rates , the media in th e present :-'ludy "en.: so lidi fi ed with washed agar . It is obviolls from the n:s ul ts obtai na bl e from literature, as wcll as the res ults depicted ill Tables i and 4, that within the Mucorales differences exist regarding the abi lity to utili se ox idised inorganic nitrogen .
inui el al. (1965) found that }?h;;opus species arc unable to uti· lise 21.0 gil sod ium nitrate (3.5 gil nitrogen) in a defin ed liquid mediu lll conta ined in stationary tes t tubcs . The res ults obtained in the present study are in agreement with these findings. However, it was found that the strain s and isolates representing Rhi;opus OIy:oe (Tables 1 and 4), were able to utilise nitrate when grown on the defi ned so lid media. Inte restingly, whe n the Rhi;oPIIS strains listed in Tabl e 1 were tested for g rowth and nitrate utilisation in defined liquid media with identical composi tions except for th e ab se nce of agar, none of the Rhi: opus strains cou ld grow on nitrate (resu lts no t s hown). T he di screpa ncy with the results of l!lui el al. ( 1965) can, therefore, be ascribed to the use of different culture conditions. The latter authors used s tationary liquid media containing 3.5 gil nitrogen, while so lid media con· ta ining on ly 0. 1 gil nitrogen we re used in the prese nt st udy. The result s of the present study indicate that R. (Jly:ae is less s pec ialised in its nitrogen source requirements than the other Rhi=rJl'lIS species tested in this study. The abi lity to g ro w on amm oniulll Illay be as a result of the presence o f a tran sport mechan ism for this compound (Jenn ings 1995). w hic h is abse nt in other melllbers of thi s genus. It may also be that spores of R. OIy;ae are more resistant toward s changes in pH up on germination in the ammon iulll containing medi um than the s pores of other s pec ies in thi s genu s (Je nni ngs 19(5). Generally, the All/CO/' species tested in thi s study were able to utilise in organic nitrogen compounds (Tab le I). However. .I/. o::ygospOI'IIS, a spec ies that can readily be distingu ished from the other ,Hllcur s pecies by the rormation or reddis h·brown thickwalled azygospores, did not ut ili se nitrate or nitrite, since tht.:sc compounds were toxic to the fungu s
S. A fr. J. Bot. 1999.65(5 & 6)
~ II
Table 4 The ability of mucoralean isolates from the arid soil sample to utilise the seri es of nitrogen containing compounds and to grow at 0.955 a. *Nilrogcll SIHln::c utilisation Strall1 s
Ac:linomllcor eiegum A 19
#(;n1\\lh
NaN()~
KNO,
NH., CI
Asn
(illl
f) ,,)55a,,
+++
+++
+++
++
+++
++
++
,·luiI/O Il/IIL'or e!egoll.\" B2X
+++
+++
+
+++
,,+
elegul/.\" [l (,)
+++
+++
++
+++
1++
++
..JCIIllOllllu.:or
"\/orfiel'cllo i.whellullI 15
()
++
++-
++ +
()
.lfomel'ellll /.\"((hel//I/(/ 35
()
+++
+++
+++
II
.to
()
++
+++
+,.
II
.I/orlierel/ol.wbelllllll
\Iucor ("Ircillt!l/o/(/es A4()
+++
+++
~+
++
+++
H-
,I fU("(1/" t.'/rCl/lIlllonles A ()()
+++
+++
+++
++
+++
+
Mucor clI"olleiloules B8
+++
+++
+++
++
++ +
+
54
++
+++
+++
+++
+
RJII::OPIIS OI:)'=lII! 79
+
+++
+++
+++
++
82
++
+++
+++
+++
+
Rhi::ojJus
Oly::lIl!
lUu::oJ)Il,\' O1 :r::(/C
*Nitrogcn utilisation. mcu:mn.:d by calcula ting the co lony diamC1cr obtained on the medium with the particular nitrogen source, as a pcn:cnlage of the clI]ony Jiumder on the mediulll which best su pported rad ial -l '" 1- 33%: ++ =; 34--60%,: +++ = 67- 100%: - ::... to.x ic. sinc~ t h ~ colony density aml d i;lI11ctcr on the part icular solid lllediulll wcrc lr.:ss than Ihat ohlil incd 1m Ihe medium dcvoid ofa n itrogen SllUrr.:e: Ahbreviat ions: I\sn = Asparagine: Gill = Sodium g lutamate . #(inm1h <1\ rcdl1ced ,t". measured b) ca1cu l;Hing. the colon) diillllell.:r on the mediulll with 0.955 a" . <1:-. a perccnt ag~ nJ" thc colony dia1llCII:r tlbt
growth orthc particular fu ngal strain. S)mhols: 0 = 0%:
cOIKclltr!nmell{/, ,\ fucor. Ilhi:upu.\" my:.ae and I1wmllo.\·(rlum. Interest in gly, it is known that ,\[/lcor and Ilhi:.opus Ill
IIsel/a lamp/'Ospora. ('wllling/ul/nella echilllt/a/a. Rhi:'oplls .\PP and mos t of the .\-/UCOI' strains. Comparing the results on the abi lity to gro w at 0.955 "1\' it can be co ncluded that tolerance toward s low wa ter activi ty may vary wi thin a species. Thi s is obviolls whcli com pari ng the datn on . J elcgl1l1s, M . ci"cil1Clloide.'i. R. micl"Ospol'lIS and R. OIy:.ae (Tables I and 4) . Howe ver. the difference in radial growth 0 11 the medium with low a w . between the culture collection strai ns of.1 elegam (Table I ) and the iso lates of this species (Table 4). llla) also reflect a Inck of selec ti on pressure duri ng the per iod of storage in the culture collection. It must be noted , however, that although the lllltCoralc.:an fungi menti oned above showed oSl11 otolcrance compared to the other lll 11coral can fungi , the rvlucorales arc known to be sign ificantl) less osmotolmant than higher fungi like Aspergillus
Fungi present in the soil sample The occ urrence of .lspert!illlIs and I'elllcillillill ill the so il salll plt: is not surpr ising since it is kn own th at membe rs of these ge nera frequ entl y occ ur in soi l from arid regions (G riffen 1963: Eid. cr el al. 1982 ; Allsopp ef 0/ 1987). Similarly. Cloc!o.\JJOI'illlll. Fusarium and PaecilolII.vces are kn own to occur cOl11monly in soil (G ilman 1959: \Vaid 1960: Eickcr 1970: Eicker 1974: Carreiro & Koske I CJCJ2). Isol ates represt: nting .·kremol1i lll1l. l3o/l)'(}Iric/l1Im. Curnt/arill and Geolrichllm. have also bl.!e n obtainl'd from southern African so il s (Eicker 1969: Eicker el (II. 1982: Strauss 1997).
The overall number of higher fun gal genera recorded in this study is sign ificantly less than the 135 genera reco rded by ROll.\ and Van Warmelo ( 1997) during an exte nsive survey of fu ngi assoc iated with plants and litter in a natural Karoa pas turl' at rvli ddelhurg in the Eastern Cape ProvilH:e, South Africa, How-
S. Mr. J. Bot. 1999.65(5 & 6) ever. it Illu st he born e in mi nd that in the presen t stu dy the fungal popu lations of olle soi l samp le w itho ut litter \\'crc investigated with met hntl s which were se lecti ve for Illll cora lea n fung i (Strauss 11)()7). In their survey of fungi assoc iated with plants and litter in the I\.aroo. Roux and Van WarmcJo ( 1997) fo und six mllcoralean gcncra. i.t!, ./criJlomllcor elegul1.L ClI/lllil1ghamel/l/ eciliulliola. _I for1ferel/u. I/lIcor, Rhi:opus siolon[ler and RJu":oll/lIcol". When strains of tiwsc taxa were tes ted for uti lisat io n of the se ri es of nitrogen containing compounds during the present study (Table I and 4). ollly rep resentatives of .·lctil1()IJ/llcor eleg(lIls and Mucor wert:
ganic oxi dised nitrogen compounds. In additio n, they were unable to grow at 0.955 a\\.. This restricts the potential nitrogen sources of these fung i to organic so urces co nta ining an nbll nda nce o f availabl e wnter. However, it wns found t hat isolates of A. elegans, AI circinelloides and R OJ:v:ae. obtained from the Karoo soil, were more osmotolorant and a ble to uti lise organic nitrogen sources as well as inorganic oxidised nitrogen produced du ri ng nitrifi cation. C urrentl y, the I11l1 coralean fungal po pulations present in soil from other arid regio ns ill South A frica are also being studied. The res ults of these studies will give us insight int o the d iversity of Illu coralean fungi associated with arid so il in South Africa.
References AGGELIS. G.. PINA. M .. RATOMA I IENINNA. R .. ARNAIIIJ . .\ .. GRA II.LL L GALZY , P.. MARTlN-p[~rVAT. P. 8.:.. PI:RRAl.!tJ . .1
P. 1987. Prouuction d'huiles riches en aciuc gammil [ino lcniqllc 11,11" uivcrscs souches de Ph ycomycetes. O/eagmelLr 42: 379-3 86. ALLSOP P. N .. OLIVI ER. D.L. & MI TCII ELL. D.T . 19R7. FlIng,l pop· ulations associated with rool systems tI l" proleaceolls seedl ings 011 ;I lowland fynbos site in South Africa ..\'. ,1/i·. 1. HOI. 53: 365-369. BARNARD. R.O .. BUYS. A.J .. CO nLEE. .I .G.K .. Dli I'REEZ. l'.C .. MEYER. J.H .. VAN DER MERWE. A..I .. VAN VlIliREN . .I AJ. & VOLSC HENK. J .E. 1990. Hand book ofsttlnuard soi l testing Illl.!l hod-; for .-.dv isory purposes. Soi l Sci. Soc. South A lrica. P.O . Bo.\ 30030.
Sllllll),sil.ic. Pretoria. BAXTER. A.I' .. RaNG. 1.11 .. RaUX. C.. SC HUlTE. I.. & VAN Il I R LINDE, E. 1994. Elelllt!lltary keys tn COlilmon rUllgi III Snuth ,\fi·icil. f'lnn! Pw lcction Rcst:arch Institute. Pretoria. South Ali·ic;1. BOTIIA . A.. PAU L. I.. ROLIX. C .. KOCK . .I. LL COETZ J:L. Il..l .. ST RAUSS . T. &. MA REE. C. 1999. Short cOl1ulllmicatillll. An isolatinn procedure /(lr arachidonic aciu producing ;l/orrierella ~p("ci("s. Aflfnnie
V(III
LeeulI'enhoek 75: 253- 256.
lJR OWN, A.D. 1976. Microbial
waleI' stress. Hocterio/ Re I'. -1-0: RO':;846. CARREIRO. M.M . & KOSKE. R.E. 1991. Rmllll Icmpl:ratll re isolations can hias against selection orin\\' h::mpl.!ratul"i.': micro l'ungi in tempcra t ~ forcs t so ils. ,\~v(."f)Jf)gi(l 84: 88(.-900. CERT[K. M., SAJBIDOR . .I . & STREDANSK!\, S .. 11)9':; . r::rr~Cl nr CII'hnn ond nitrogen Sllllrces ()J1 grO\vth. [ipi u production and liJ1ly acit.! composition of Mucur mllcedo . .llicmhios 74: 7-1 5. DAESC HEL. M.A .. ANDERSSON . ICE. & FL EM I NG. 11.1'. 1'187 Mkrobi<11 eco l og~ of I ~ nllcnting. pl ant milterials. FF.'\/.\· ,I/teronio/ Rev. 46: 357- 367. DOMSCH . K.I-J.. GAMS , W. 8.:.. ANDERSON. "1"-1 1. 19!W. ;1/(}!"ficl"/:/fu Coeillans 1863. 111: C01l1rcndiull1 oJ'snil fu ngi. eels K.IJ. Dl)lllsch. W. G
uon.
EICKE R. A. 1969. Micro fungi Irom surl:lCl! soi l 01" fo rest cn lll11l\1l1 ilic~ ill ZlIlul,mu. TrailS. H,. ,Ifvcol. So('. 53: 38 1·-391, rr CKER . /\ . 1970. EcoJ{lgi~al ohservations on suil rllngi . s. . I(r .I .\u. ()6: 327-33 4. EICKE R, A. 1974 . The mycoITora 01" an aJca[inc soil or Ihe 0PI.!H Savannah o r the Trans\'ClaL Tnf//s HI". .\~I ·("O/. Soc. 63: 2RI- 2}lR. EI CKER, A .. THER ON. Ci.K. & GRO B13 ELAA R. N. [982. ' 11 Mikrnh iolngie~e stuuic \'1111 'knal kollc' jll uic Gir ihcsvln ktc \'un Katl~nlalHl. S.\V,A.-Nam ibie. S. ,.//i· .J. BOI. I: 69- 74. ELLIS, M.B. 1976 .. More ucmatlaccous hyphom),cctcs , COllllllnm\'calth Mycological [nstitutc, Kcw. Sl1m~ y. Eng.land. GAMS. W. 1977. A key to the species Il l" MOl"liel"/: f/(/. 1'c:,..\·OOlIIll I) · 3R 1391.
Conclusio ns Alth ough more factors. playa ro le in g rowt h an d survival offu ng i in soi l, the resu lts showed the interp lay between awand nitrogen util isation. which prov id ed some po inte rs to the likely sllccessio ns of Ill uco ral ean fungi in soi l. Althoug h Morfierella species \Vere able to utilise amino ac ids, th ey were ullable to utilise in or-
(iI LMAN. J.e. 1959 . A manual 01" soli rllllgi. The Iowa Statc I're'is. Iowa. GRAY, T.R.G . & WILI .IAMS, S.T. 1979. Soi l microllnra and the uecomposition of ue.-.J organic maUer. In : Soil micro-organisms. eds T.R .G. Gray & S. T. Wi ll iams. pp. 75 -89. l.ong.IlHl1l. London . GR lr= r=EN. D.M. 1972. Eco log) nfsoil fu ngi. Clmpman anu IInl!. 11mdnn.
S. Ati·. 1. Bol. 1999.65(5 & 6) IIAt\SS()N . I.. & I)OSTALEK. M. 19!18. Effect ot'culture conditions nn Ill)cd ial gnmlh ami production of linoknic ac.:id hy the fun g.us . \/(J"'I~'rdl(lI "(///J(lIIl/ i(l/llI . ..11'1'1. ,llIcmhw! Bunec:hl/ol. 2R: 24()- 2.t6. I r ES"'EJ:lI ~ E. C.\\'. & ELLI S. J ..L 1973. Mucora les . In : The fungi. an nth illll,:CU tn.:atise. ells. G.G. AinS\Vlllth. F.K. SparrO\\ & A.S . Sussman. ell I [ . pro I X7-217 . Academic Pn:ss. Inc .. Orl ando. r1( , I)S( )N . 11 .. 1. 1992. Fungal Binlogy. Carnhridgc IJnivt:rsi t) I'n.:ss, ('amhri dge IN t I!. " . T:\t\.U):\. Y. &: II.ZtJKA. I I. 1965. Taxonom ical stwh .!s on genus !?llo/lms. J Gell. App/ Mi('l'oblOl. II (suppkml!nt): 1- 20, .!I'.NNli\(iS. I). 11. 1995. Til.: physiolog) of funga l llutrition. Camhridge lini \ c.:rslt~ Jln.:ss. CUl11bndgc .
"-.RU iI· R- V ·\N RI.I. N.J. W. 1984 . The )easts • a taxonomic study. Else· \ ier SCH.:m:t! Publishers 13. V.. Atn!">tcrdam, The Netherlands. I.o\\'. A. B. & RE HE LO. A.G. 1996. Vegetation or South Africa, l.eSOlhll and Swaziland. Departmt:nt or Environmt::nta! An'airs am! ["nuriSlll. Pn:toria. South AJs·ica. I liNN. .J /\ . 8.:. SH IPTON. W.A. 19~3. l{c-cvaluatiol\ orluxonomic crikri>! in ( ·lIJ lIIil/ghamclla. '/I'am' HI' A(vc:ol.So(:. 81: 303-3 12. OII AV IANI. F. 1993 . Moulds in fond spoilage. In: Enqt.:itJpacd ia of [(lOt! SI..:I\:11I.:1.:. lilod technology and nutrition. cds. R. Macrae. R.K . [{llhinsllll & M.J . Sadler. Vol. 7. pp. 4338--4344 . Acadcmic Press . 1.{Jnuon. 1{(H IX. C. & VAN W/\RMELO. K.T. 1997. A surv..::)' urthe lllycobiota ora n.lltl ral K'lroO pasture. Bot/llllia27(2): 167- 183. SAlB IJ)OR . .l.. CERTI K. M . & DORBRONOVA. S. 1988. Intlucncc o f dilrt::rcnl e lrbon sources on gn)\Hh, lipid content and lally acil..! composition III jelUr str'.Iln!"> hdongill g to Mucllnllcs . BiotechI/O!. I. Cll. 10: 3--17 - 350 . . . . A.lBID()R .. 1.. [)OBRONOVA. S. & CERTIK. M. 199(). Arachiunnic aciu pn)duct itlll by Mnrticrdlu sr. S-1 7: Inlluence of C.N ratio. 1110techl/ol I. t'lt 12.455---4%. SCIl IPP I:: R. M.A.'\. 1976. On Muc:or drdnefloide.I, Jlltc:or rac:e11l0SIIS anu n:lah;u ~pcciL!s. SltId. I\~V{;(JI. 12: 1-40.
413 SCHIPPER. M.A.!\. 1978. On certain spccies of Mucor with a kc) to all llcccpLed spccil!s. SluJ A-fvcol. 17: I- 52. SC I1IPPER. M.A.A. 1984 . " revision lIl" the genus RlJI:'0l'"s I. 'Ihe Rh l"f%lltjcr-group ano Nil ory=ae ..)·tur/. ,\~n.:ol. 25: 1-19. SPARLING. G.P 1998. Soil mierohial biomass. activity ano lllltricnt cycling as indicators of soil health. In: I ~ill!tlgi ca l inrJicatl)f~ or soil health , cds. C.E. Pankhurst. n.M . Doubc & V.V.S.R. (iupla . pp 97119. CAD Internat ional. Oxon. SPOTTS. R.A . & eERV ANTES , L A. 19~6 . PopUlati ons tl L l/llall" I'1rI/orm;.\· in soi l of rear orchards in the Ih llJd River val k)" of Orcgon flam disease 70: 935- 937 .
STEIMAN. R.. GUIRAlJD. 1' .. Si\(;l'. 1... SEJGL F-M IJRilNIlL F. & LAFOND, J-L 1995. My-conura of soil arounu the Dead Sea. I Ascom ycetes (includi ng ,hpergillus anI..! PeniCilliuJII). B;Jsidiolll) cctcs. Zygomycetes. System App/ ,\ficrohlOl. 18: 3 10-317. STRAUSS. T. 1997. The i!">o!alion of gamnl
407
Nitrogen utilisation and growth at reduced water activity by mucoralean fungi present in soil B.O. Seabi1 , A. Botha*, B.C. Viljoen 1 and C. Roux 2 'Department of Microbiology and Biochemistry, University of the Orange Free State, P,Q Box 339, Bloemfontein , 9300 Republic of Sou th Africa *Department of Microbiology , University of Stellenbosch, Stellenbosch , 7600 Republic of South Africa 2Myco!ogy Unit, Biosystematics DIvi sion , ARC -PPRI , Private Bag X134 , Pretoria, 000 1 Repub lic of South Africa Reall!etf
r
JIIIII! 1991), I'e\'/sed 5 Ouuher 1999
Nine mucoralean funga l genera including 18 species were exam ined for their ability to utilise a series of nitrogen co ntaining compounds and to grow at an aw of 0.955 on solid media The nitrogen concentration in the media was 0 1
gil and the senes o f nitrogen containing compounds were ammonium chloride, asparagine , sod ium glutamate, sodium nitrite and potassi um nitrate . The gene ra we re Actmomucor Schostak. , 8 ackuseIJa Hesselt. & J .J. Ellis , Cunnmghamella Matr. . Gongronella Ri baldi, Mortierella Coem , Mucor Fresen .. Rhizomucor Luce! & Costantln , Rhizopus Ehrenb and Tham nostylum Arx & H. P. Upadhyay. Thirty~nine funga l strains obtained from cultu re collections (CBS, MUFS a nd PPRI) , as well as 12 sOil isolate s from the Karoo , were tested. All the species and stra ins te sted In this s tudy were able to utilise asparagine and glutamate . Strains bel ong in g to CunninghameJla, Mucor racemo sus Fresen ., Rhizopus microsporlls Tiegh. and Rhizopus st%nifer (Ehrenb .: Fr) VuHl were unable to utilise ammonium chloride. S trains of Cunninghamella, Mortierella, RhlZomacor, Rhizopus microsporus and Rhlzopus stoloniferwere una ble to g row o n nitra te as so le nitrogen s ource. Nitrite wa s found to be tOXIC to species be long ing to Cunnmghamella, Monlerefla , Rhizomucor, Rhizopus and Thamnoslylum . Members of Gongronella, Mortierella , Mucor racemosus, Rhizomucor and Th amnostyJum were unable to grow at an a w of 0.955. Key words: Nitrogen sources, phys iology, Mucorales , Upper Nama Karoo
"To whom correspondence shou ld be addressed. (E·ma il: [email protected] .ac.za).
Introduction t\ llIco rakall fungi are m ostl y saprophytes assoc iated with decay~ ing plant material. dun g or ot her o rgani c debr is in soil ( H essel ~ ti nc & Ellis 1973: DOlllsch Iff 01. 1980). T hese fungi are usuall y associated with moist environments , sllch as leafl itter in forests. and arc known to be rel atively into lerant to Iowan (Bro wn 1( 76). Grow th of Absidia...\[lIeor and Hlti::oplls occur above a\\ vtll ues of O.9::! to 0.93 (Ottaviani 1(93). However, muco ra lean fungi have "Iso bee n record ed in so il , deb ris and on plant roots fro m arid regions (Ste iman er al. 1995 ; Roux & Van Warlllelo
1997). Availabil ity of car bOil and nitrogen sources is known 10 playa major role in the composition and succession o f microbia l comm uniti es o n dl:caying organic matter (Daesche l ct al. 1987; HudSO il 1 9<)~) . ~ llIcora l e an fun gi are the first to coloni se decaying organic mate rial in soil, si nce these fungi are able to rapidly uti· lise the lim ited number of simple carb ohydrates that are usually availabl e. bdore other funga l gro ups take ove r the min erali sation of car bOil (Hesseltine & Ellis 1973). It is, there fo re, not surprising tha t srudies have indi ca ted that m ucora lean fungi are able to uti lise o rganic nit rogen as well as am lTIon iul11 salts (lnui el al. 1965: Aggelis el al. 198 7; Sajb idor el 0/. 1988; Tsuchiura & Sakura 1988 ). However, ce rtain authors found that some Ill llcoralean fun gi are able to utilize nitrate (Hanson & Dosta lek 1988; Certi k 1(93). T his characte ri sti c is not essential for a primary co lo nize r o f dead o rgani c malte r. s ince d uring the mi neralization of organic nitrogen, nitrifi cat ion o nly occurs after amm onific ation (Sparling 1(98). The spec ific positi on of particular species of l1lucora lcan fungi in the bi ogeochemical nitrogen cycle, howeve r. is unkn ow n since o nl y fragmentary information on the ut i· lisation of d iffe rent nitrogen conta ining compounds by these fungi, exists in literature. Wit h the Clbove as background , the aim of th is study was to
investigate the ecological nic he of 11111coralean fun gi in arid soil. \ . . itl1 spec ific reference to the posi ti on these: fungi occ upy il\ the biogeoc hemical cycle of nitroge n. Consequentl y. sckcted IIl U(O ~ ra lea n tax a occurri ng fre quently in soi l habi tats, includin g strai ns from culture collections. as well as isol ates obtain t:d from a so il samp le from arid Upper Nama Kamo (Low & Rcbdo 1(96). were used to eva lu ate in vitro growth to determi ne nitroge:n sources and a w tolerances .
Materials and Methods Strains used Thl! fungal strains and bolatcs uSl!d in this stu dy arl! list(:t\ in Tahk~ I and 3. The strai ns Wl!re: ohtainl!d from the Ccntraalhun:all \om Scili nlme icul ture:s (CBS). Nctherl ands. the: Mlicorakan I.:ult un: I.:ol~ kction of the Uni vcrs ity of the Orange Free Stah.' (!\.'IUFS). SOllth Africa. and from the Nation al (.:olkctill il of Fung i tpPR I). Prdon;l. SOllth Africa. Oth cr strain s \\e:r\! isola ted from it soil Sdlllrk origi nating from Upper Nama Karon (1,0\\ &. Rebel\) 1996).
Physiological properties Preparatiol1 o1"il1ocu/o A stcrih::. wet inoclJiating hll)r \\<1S llsed for I.:ach ["ungal strain 10 transfe r sporangiospore:; and/or h~ pllal Ibgllll.:nts from it IWO \\ l!1.:h. old cu lture on 2% (w/w) Di lco malt e.'tnll.:t agar (MEA) to 5 rnl stl!r~ ilc dist illed water. Fl)rty micro liters of the result ing suspells ion. C(ln~ tain ing circa 2 x 106 L'olony form lllg unit:-. pe:r Ill !. \\~b lI~l'd inocu late each delin l.!d medium. in Petri dishl!s.
l il
Nitrogen llIi1isaliol1 011 (/ solid de.fil1ed medillm A ser ies of solid ddine:d media \H\S prepared (Van d(:r Wall & Y ar ~ ro\,,. 1984). Each med ium. in PClri d ishes. consisted of 11.7 gi l Bal.:to yea.st c
S. At'r. 1. BOL 1999.65(5 & 6)
Table 1 The ability of strains representing different mucoralean genera to util ise the series of nitrogen containing compounds and to grow at 0.955 aw " Nitro;;c n S\l u rcC 1I1ilbalinn KN0 1
i\' r I. I CI
on
+++
++
or elt'gO/I.\" (Eidam) C. R. Iknj . &. Iks.;d t. !'. II IFS 121
1++
NaNO:
Strains Acl!lt()111IIU [el i /lO ll/ lit
I/" ("('gallS
(Eitlam) C R. lknj. &. l kssdL \1 11 FS
.-Ir I/IIIJIII I/mr (' /('g olls (EiJam) C it Iknj . &. Ik ssd L .\lI lFS
Hud-lISel/o 1IIIIIIJfOSpOro (Lf.:!lun .) Iknn) & R.!\. . Ik nj ]'vtt iFS OO}
If(/(" 11.\('/10
/(//111I/'OS/JO/'(/ (I . ~llJn.)
f((/( Au \{'II(I {UIIII Jrosl'(WlI (l.cnJn
Iknn) & I{ K. rknj. MI IFS OClX
+++
0
(!chimda({/ (l'lw:-..t.) Tha:-.:t rvll JI:S O()3
(Lclldn.) Pe, mnd & i);l l Vesl:o MI Ir s 2
0 +++
+ +++
l/orllt'n 'lIl1 (lII/oe/JfJidea W. G
()
l/o/'II('I'e/I" g/ohuli{em Rostt'up CBS 417.64
II
Y. Ling-Ynng CBS 430.76
I/ol'fl{'/'fI/lfllllljico/a
R.K. fk nj . C BS 292.63T
IflicorlCl gospamm
.\llIcorn/'! II/d/aide.\· Tiegh.
CBS 11 1J'(lR
+ ~+
•. +
(Tha xL) rhaxt. Ml lFS 00 I
(;ollgro/1!'lIa h lll/erI
t+
I-+.!-
(lha.\t.) rh:J\l. Ml lFS 002
& Dal Vesco MUFS
+~+
-j-+ +
( ' /il/I lI ngllll lll e{{a I!dlllJli/(/f(/
Pc~ronel
+
• ++
('/11 l11 illglwlJlc/{a ed/l/lIIl l1fll
(l.endn .)
+++
+++
(J
(' lIl1l11l1glllll/leJfa
.+
+++
J lknny &. R K . [!en.!. MI IFS OIl
(;01l,!!.1'01/1'1/(I hlli /ai
( rill
+++
+++
O.lJ):i;l"
,,
+ ++
nl)
ii i inl\\ III
/\S11
.~
.. .. .
., +
,
H
+H
+' .
•. ;'1-
l- + .
0
.+
• ++
0
-+
. ++
+ .•
Il
++ +
++ +
+>
,+
+ ,.
0
H
+'+
T+
...
++
'
~+
()
++
• -+
+-'- +
0
++ +
tt +
+++
Il
+H
+++
+++
II
++
. ++
H+
."
H
H +
. ++
H-
+++
+++
++ +
+H
+++
H+
H+
II
'"
H+
+ .....,
++ +
H+
++
,,+
H +
0
(Liml!. ) Schipp.:r M l lFS 00 I
II
+++
1-
~+
+++
II
Nhr::olll//cor /J/fsillus (Lind l. ) Schipper M l lFS 00)
II
+++
+.+
+H
II
T icgh. CBS 63 1.82
II
II
; ++
H +
+
T iegh. PI'RI 5560
()
II
++
+++
++
+
+++
+ ..... +
+ ++
++
III/cor/hII'll I 1\,(il1i.:r CBS 134.35 I hl(,()1' jJ/IIIII/ll'II.1'
Bononl CBS II Ul7
IllIcor 1'(/( l·/I/O.l'IIX
Fres CBS 115.08
NlII:::OII/I/( 01' I'lIsillllS
f?l1I:::OPliS 1/I/('I'OSporoU.l'
Rhcojlll.\ III/cms/wrolf.\·
Rl1I:::ofJlIs 01'1 ':::/1(' Went & Prins. Cier:rl. CBS 112.07
(Ehreno. : Fr.) Vuil!. COS 609.S2
II
()
+++
+++
++
/(/1/:::0/111 1' I/n/O /lUer (Ellrenb. : Fr.) Vui l!. CBS 31 t) .35
0
II
+ ++
+ ++
++
+++
+
.+
++
II
RhbJjlll.l' ." Ifl/olllfer
lhulII/w\'I\!/Il11jJirijorm e (Bainierl Arx & H.P LJp;luhyay t\·1UFS 025
"Nitrogen uti lisation. meas ured h) calculating th.: cololl) diameter ohlained nn the medi uOl with the parlicular nilrogen SOurl:e. as a percentage {If the l:OhUl~ d ialllt:tcr olll hc Illcd iulll \\ hieh b~st supporteu radia l gru\\1h orthe pmtic ular fungal stra in. S)lllhnls: 0 =: 0%: + "" 1-3.1 %: +.1. = .;4(,6%: +-... (,7- 100°1\): - = toxic . si ncc t h ~ CO ln ll ) densilY and d ia meter on thc part icular sol id medium w.:re less lhan Ilwl nbtaincd on the mcdiulll dc\ 'oid o ra nitrogt.:11 ~{l urc e: Abhre"i alil)1\s: Asn = Asparn gine: G!u = Sndiulll glutamate . #(iron-I ll at reduced '\". mca:-lIl'ed by ca lcul ating the colony diamcter on the medium wilh 0.955 a". as a percentagc nrth e w lony d iamch:: r ohtaill~d Oil a non water-stn.:ssetl control. Symon!s: () = 0%: + = 1- 33%: ++ = 34-f)o%: +++ = 67- 100% .
all had a pll n( circa 5.5 and \\'\.:re so lid ifi ed with 2%, (w/\\') \\ash~d pun fred agar. r hl' \\ilshcd aga r wa s pn:pan:d hy repctitiH:I~ \\ashing so lid ilicd agar block s ( I>ifco) in dt: m incral iscd waleI' (Van der Walt & Yarrow
di amctt.:r o btained after fivc days on the 111l.:d iullI containing the particu lar nitrogt:ll source. as a pl.:rccnlage of the wlony diamell..::l' ohta ined after the same period O il Ihe medium \\ hich best supported rad ia l growth o r the p'lrtic ula r fungal slr;un (Tah les I and 4).
I~X4)
t\ stlper1ici;iI wdl of H,() mm in diamete r. \\as made in th c center of the mediulll in cach Petri dish. using a hot g lass rou. Each Pcl!'i dish \\as 11111cu lated pipdting .1O ~t! or the illlll'u lulll into thl.: well
oy
amI then inwhatl.:d al 25°C ill1he dark. Thl.: d iallldc r or t h~ result ing colony \\'as measun.:d aHe r live days of inc ubat ion . A cont rol exp..::riment \\ as l'tIn ti uc\eJ in \\Ihid the abi lity to gnm Oil a solid med ium dc\uid 01' ;1 Ililrogen source was testl.:d All th l.: \!x pcr illlc:nt s \\el'l.: conducted III triplicate .
A1ea.'iuremenf (~rradial gr<)wlh ll'ilh different nitrogen source.\· Rad ial gnl\\ tho a s an in d icat ion of th e nlte of uti lisation o r the nllrog,en source. \\as measured. Thi s ",",IS done hy calculating the cnlon~
Growlh on (I medium with a redllced waleI' activity A solid me diulll was pre pared in Petri di shes. Th is lllediulll Ctl llsistcd of thc 100 hmin g (gil): IO.f). g.llH.:use: 5.0. peptollc: I .D. KII :! P O~: 0.5. MgS()~ . 7 11 :! () : nO.n. gl~ c aol: 15.0.
<1
pI I o r cirm
S .()
and
a"
orO.1)55. A sllper ti eial well in th e mcdiulll \"as pre rarcd and illllc lil atcd as descrihed ahove. T he illocul
or
S. Afr. J. Bot. 1999. 65(5 & 6)
4 09
Ih.: c'pcrlmo..'llts wcro.: conducted ill trip lica te. AfL'oSlfn...'1I11..'11l oj'radiClI grollllh on mediulI/ \1 '/111
lOll'
au
(iro\\ III \\ as me asured by calcul ating the colony diameter ohtalllcd artl.'f 11\ 1..: da~s on the m ed iulll \\ ilh 0 .955 i.l w' as it perce lit age of the c oh lll~ d ],11l1Clcr oht a incd \1 1' gl~ Cl'ft l] (
:lth:r thl. ? same peri od Oil thl..' l:olllrol dcyoid
l"ah1cs I and.t).
Determination of funga l taxa in soil 7i. (klllg of .'0" sample J h.; "a 1llpllll~ :-.1 Ie (lfll1l ~ ) is loc
,H url' h ] 3.J .e. Th e :-:i1 rn p1c was takl."l1 in mid-sumlll er. The su rface 11111.:r \\a~ li f'.. , scraped away to nxllll:C co nlal1linat lOll fromlhi s hahital ,\ S{II I "am ple Ill" ClrCII 2900 g , c o nsisti ng o r ni lH.: suhsamplcs. lakel1 at randolll (}\ er the are a of the site . each to a dcpth o!" 10 1.' 111 . I he , uh:- amplcs \\ere thoroughly mixed ill the labnratol')' 10 pro duce the :';lInpk. whi ch \\ uS !"urther processed . H<\-;
ag ar tRiolab): 0.02 gi l heno my l I Mdhy l 1-( bll l)' k ar h a lllll ~ I )-2-hcll/imida%Ole-carbamate. A.ldrich catalog no. JI-:. I SR-liJ was added heron: autoclaving. The sccond selec ti\ c med iulll \\'il S similar \(l the [irs!. except that it cOllta ined 10 gil SliCroSC as l:arhon sourcc ill~tcad stnrch . The relat ivel y nun -select iv e m..:d illlll (r,,1 YPps) (( 'arrcinl & Kuske 1(92 ). c on ta ined (gil ): Malt cxtrad (Dili.:o). 700: pc ptone ((h oi d). 1.00: )CaSl e xtrac t (D il'co). () 50: pe ni c il lin (i . 0.50: st re ptomyc in sulphate. 0. 50 :
or
or
jelen/ilicati()n
( 'hl'mical tllla~r.'iC:s o/s oi/ sample T he plioI' Ille snil sa mp le \\
or
Isolwio/l o/Iilllgi n~
lIsing the soil plate tcchnique of Ware up (19S(). two sdective Il h'dia (l"ollt ain ing henomy! and starch or sucrosl.! a s carbon source). as nell a:-. II rd ati vc non-sdeeti vt: medium. w e n~ USI.!(\ to iso late mlleo ra leiln fun g i from the soil sampll:. T he fi rst sel ective me dium COIl[;Iillcd (g/ I): st arch. 10.00: N H-ICI. 1.00: KH :!r0-l _ 1.00: MgS0-l ' 7 1 1 ~ ( ).
() 50:
~ ca s t
t:xlracl (D ileo). O.S (): allll c hloramph enicol. O.:W.
It a ls() cOlltil incd the tllllO\ving mineral salt s (mg/l ): FeSO.f. 7H 20.
111.OIl: InS)),. 711, 0. 10.UO: MnSO, . H,o. n.RO and CuSO,. 5H,O. n.()s Thc medium had it p H or 5.5 and was sol idi lied with 16 g/I
(~l/i{}/gi
Isol ates belonging to the h ighl.!r rungl were id e ntili ~ d up tn g cn us [lax ler l'1 al. ( 1994). Ellis (11)76 ) a mI Krcg. l'rk \'d using th c keys van Rij (1984). The lllul'ora1can fung i \\' c r~ ide nt iliell usi ng Ihc keys and des..:ripl iol1 s g ive n h~ ( ia ms ( I ( 77 ). I lcs:-d tinc & Fl lis ( I W1'I). !.lIllll & Ship toll ( 198 } ) a nd Schippl'r ( 1976. 1971-: and 19S·:t).
or
Results Utilisation of nitrogen sources by species and strains of the selected genera T he abi lity of species and slr::ains of the selected fungal genera to uti li se the series of ni trogen con tain ing co mpo un ds as sale n it rog~ n source is given in Table I. A ll the strai ns were ahle to uti lise the am ino acids, asparag ine and g lutamate . However. o nly 70% of the strains were ab le to utilise ammonium chloride. \-vhile 51% of the strains were ab le to grow on ni trale as sole nitrogcn source. The latter included strai ns of genera .·/c fin(JfllIIc()/", /3 ack1(sella. COllgronella, Mucor. fl!amnn.,IY/lIl1/, ::and Hhi=oj)/Is OJy=ae . The results obtained w ith nitrite, indicated that a t the concen trati ons used in thi s st udy, this co mpo ll nd was tox ic to species of ClInl1ing/wl1Iel/(I, Mo r/;erel/a, I?l!i=0I111U:UI', I?hi=opll.\· and 71WllllloslY/II/11.
Growth at 0.955 aw by species and strains of the selected genera Species and strains of the genera Adil1vl1Iw:or. /Juckltse//a.
Table 2
Characteri sti cs of the soil at sampling site
( 'h,l raC!c ristics ( 'helll ical prorcrlies
('lffl-
nil/Khamella and RIu':oplIs we re ab le to grow at 0.955 a w (Tahle I). The stra in of Mucor racC!l11o.ws. w as the o nl y strain of .\/ucor that was unab le to g row at 0,955 a \\. The struins cxtl llli ncd of ( /o llgnmel/a, Morlierel/(I, RhbJlJlttcor and Thal1l1lO.WY/llm were unable to grow at this aw value.
pi 1 n[" soil
7.25 ± 11.115
SnillllnlSlllrC content
6.66% ± O.22%)
Fungi present in the soil sample
()l'gal1ie carhnn
0.72%
Tilia l ni trogen
0.2%
Nll rllc
1. 1 ppm ± 0. 1 ppm
The fungi in the soi l sample, \vhieh were enumeraled lIsing the three isolation media, are li sted in T able 3. Strains of thirteen genera o f highe r fungi were fo und 10 occ ur in th e so il samp le . T he most frequentl y occurr ing of these were Trich oderma and ,. I.~pergil/us . Only four spec ies of mucoralean fun gi co uld he detected in the soil sample i.e. ACfil1ol11ncor elegans and llhi=oPIIS OIy::ae, wh ic h were the most numerous. and .\/o/'ficr/!I/u isuhellinCl and iHucor drCl"nel/oides
N itrate
18 .6 ppm :1:: 2.4 ppm
:\I11I11\1l1 llIl11
2.8 pplll ± 0.4 ppm
PhYSICll propcrties Ua~
30%
Silt
21%
Sa nd
48%
Utilisation of nitrogen sources by mucoralean isolates The ab il ity of mucoralcan isolates from the so il sa mple to uti li se the series of nitrogen con ta inin g co mpounds as sole nitrogen
s. Afr. 1.
·1111
Table 3
Bol. 1999.65(5 & 6)
Fungi isolated from the soil sample "'lcd.
[~(CFlJIg
MYPps (CFUlg soil)
Mell. A (CFUlg soil)
.-It f/1I01ll /lCOr elegc lIIs ( I:id arn) ( .It [knj . l le!'sc lt .
2... 0
760
760
1/(JrJ/el'el/a Isahel/ow OUlklll.
IXII 320
soil)
\ Iue-orale-.Il
1/IIU Jr Clrc:ille /loide,\'
Rlu:o/)/{.\ o r\"=al!
HW
SO
!2RO
II ()()
9~W
'1i('gh
W!.!111 & Prins. ( icerl.
[()TAL (;Clll'ra oruthcr funl!i
411
' (-rel/lIl11/ /l111
I'pergillus N(J/I".' "(/fl'lcJIlIIII
4XO :;60
( "/lIlio.ljl0rt lflll
611
("111"1'11/(// ' /(/
120
r//\W'/l/III
10
( il'O{l'Idllllll
1211
300
100
I ' l /(' (-i/I IIII,"C(,S
20
1'1.'III('JlliulJI
1211
20
60 I '(J~\ "\ ( ., "(( t/I/II/ N.liefle/rl/I/(I
I rJ( -hflderl/1(/
It HAL
411 71111
20
2180
200
42()
t T l}: Colony ii.mlling,uni ls: rv!Ypps: Relative non·sclcctive medium (Currciro & Koske 1992): Mea. A: Isolatinn medium A containing starch <1:' cilrhon source and 10 ppm hcnomyl; Mea. il: lsoialilln nH.:diulll 8 containing stl(;rn~(! as carboll source ,I11J 2IJ ppm henlllll YI.
source is given in Table 4. All the isolates were ab le to uti lise the amino acids . asparagine and gl utamate. as well as ammonium L'h loride . The iso lates of .\lOl'fh'rel/a i.w hellilJa were unable to utilise nitrate as sole nitrogen source and at the co ncent ration of l1it1'ih:.: used. this compound \\las toxic to the iso lates of .\/(}I'I/t!I'L'I/a i.~(1/)(:lIiI1(1 and Rhi; oplfs OIy;ae .
Growth at 0.955 aw by mucoralean isolates T he isolaks of , I ClillOllllfCOI' elc!R([J/s ..\lllco/' cin_-;lIelloides, and /(/11;01'".\ (l/Y;UC were able to grow at 0.955 a\\ (Tab le 4). Similar 10 the .\lor/lerdla strains from culture co llections (Tab le I ), th e isolate s of .llorrie/'e//a isabellina were unable to grow at 0.955
Discussion Nitrogen utilisation within the Mucorales :\ numher of workers testin g for the utilisation of defined nitro· gcll sources b) rnucoralcan fungi also included yeast extract in the culture media ( Hansen & Dosta lek 1988; Ccrt ik el al. 1993). Howevcr, ~ cast ex tract was found by Sajbidor el al. (1990) to be ablc to act as a nitrogen source on its own. This compli cates the intcrpretatilltl of results. Therefore, it was decided during the present ~tu d ~ to tcst for nitroge n uti li sation usi ng d efi ned med ia. III rtd d it ioll, in ord er 10 determine the n itrogcn req uirements of h) pltal cultun:s 0 11 solid s ub strates rather than those of sub· !l1l.! rged cultures in liquid subst rates , the media in th e present :-'ludy "en.: so lidi fi ed with washed agar . It is obviolls from the n:s ul ts obtai na bl e from literature, as wcll as the res ults depicted ill Tables i and 4, that within the Mucorales differences exist regarding the abi lity to utili se ox idised inorganic nitrogen .
inui el al. (1965) found that }?h;;opus species arc unable to uti· lise 21.0 gil sod ium nitrate (3.5 gil nitrogen) in a defin ed liquid mediu lll conta ined in stationary tes t tubcs . The res ults obtained in the present study are in agreement with these findings. However, it was found that the strain s and isolates representing Rhi;opus OIy:oe (Tables 1 and 4), were able to utilise nitrate when grown on the defi ned so lid media. Inte restingly, whe n the Rhi;oPIIS strains listed in Tabl e 1 were tested for g rowth and nitrate utilisation in defined liquid media with identical composi tions except for th e ab se nce of agar, none of the Rhi: opus strains cou ld grow on nitrate (resu lts no t s hown). T he di screpa ncy with the results of l!lui el al. ( 1965) can, therefore, be ascribed to the use of different culture conditions. The latter authors used s tationary liquid media containing 3.5 gil nitrogen, while so lid media con· ta ining on ly 0. 1 gil nitrogen we re used in the prese nt st udy. The result s of the present study indicate that R. (Jly:ae is less s pec ialised in its nitrogen source requirements than the other Rhi=rJl'lIS species tested in this study. The abi lity to g ro w on amm oniulll Illay be as a result of the presence o f a tran sport mechan ism for this compound (Jenn ings 1995). w hic h is abse nt in other melllbers of thi s genus. It may also be that spores of R. OIy;ae are more resistant toward s changes in pH up on germination in the ammon iulll containing medi um than the s pores of other s pec ies in thi s genu s (Je nni ngs 19(5). Generally, the All/CO/' species tested in thi s study were able to utilise in organic nitrogen compounds (Tab le I). However. .I/. o::ygospOI'IIS, a spec ies that can readily be distingu ished from the other ,Hllcur s pecies by the rormation or reddis h·brown thickwalled azygospores, did not ut ili se nitrate or nitrite, since tht.:sc compounds were toxic to the fungu s
S. A fr. J. Bot. 1999.65(5 & 6)
~ II
Table 4 The ability of mucoralean isolates from the arid soil sample to utilise the seri es of nitrogen containing compounds and to grow at 0.955 a. *Nilrogcll SIHln::c utilisation Strall1 s
Ac:linomllcor eiegum A 19
#(;n1\\lh
NaN()~
KNO,
NH., CI
Asn
(illl
f) ,,)55a,,
+++
+++
+++
++
+++
++
++
,·luiI/O Il/IIL'or e!egoll.\" B2X
+++
+++
+
+++
,,+
elegul/.\" [l (,)
+++
+++
++
+++
1++
++
..JCIIllOllllu.:or
"\/orfiel'cllo i.whellullI 15
()
++
++-
++ +
()
.lfomel'ellll /.\"((hel//I/(/ 35
()
+++
+++
+++
II
.to
()
++
+++
+,.
II
.I/orlierel/ol.wbelllllll
\Iucor ("Ircillt!l/o/(/es A4()
+++
+++
~+
++
+++
H-
,I fU("(1/" t.'/rCl/lIlllonles A ()()
+++
+++
+++
++
+++
+
Mucor clI"olleiloules B8
+++
+++
+++
++
++ +
+
54
++
+++
+++
+++
+
RJII::OPIIS OI:)'=lII! 79
+
+++
+++
+++
++
82
++
+++
+++
+++
+
Rhi::ojJus
Oly::lIl!
lUu::oJ)Il,\' O1 :r::(/C
*Nitrogcn utilisation. mcu:mn.:d by calcula ting the co lony diamC1cr obtained on the medium with the particular nitrogen source, as a pcn:cnlage of the clI]ony Jiumder on the mediulll which best su pported rad ial -l '" 1- 33%: ++ =; 34--60%,: +++ = 67- 100%: - ::... to.x ic. sinc~ t h ~ colony density aml d i;lI11ctcr on the part icular solid lllediulll wcrc lr.:ss than Ihat ohlil incd 1m Ihe medium dcvoid ofa n itrogen SllUrr.:e: Ahbreviat ions: I\sn = Asparagine: Gill = Sodium g lutamate . #(inm1h <1\ rcdl1ced ,t". measured b) ca1cu l;Hing. the colon) diillllell.:r on the mediulll with 0.955 a" . <1:-. a perccnt ag~ nJ" thc colony dia1llCII:r tlbt
growth orthc particular fu ngal strain. S)mhols: 0 = 0%:
cOIKclltr
IIsel/a lamp/'Ospora. ('wllling/ul/nella echilllt/a/a. Rhi:'oplls .\PP and mos t of the .\-/UCOI' strains. Comparing the results on the abi lity to gro w at 0.955 "1\' it can be co ncluded that tolerance toward s low wa ter activi ty may vary wi thin a species. Thi s is obviolls whcli com pari ng the datn on . J elcgl1l1s, M . ci"cil1Clloide.'i. R. micl"Ospol'lIS and R. OIy:.ae (Tables I and 4) . Howe ver. the difference in radial growth 0 11 the medium with low a w . between the culture collection strai ns of.1 elegam (Table I ) and the iso lates of this species (Table 4). llla) also reflect a Inck of selec ti on pressure duri ng the per iod of storage in the culture collection. It must be noted , however, that although the lllltCoralc.:an fungi menti oned above showed oSl11 otolcrance compared to the other lll 11coral can fungi , the rvlucorales arc known to be sign ificantl) less osmotolmant than higher fungi like Aspergillus
Fungi present in the soil sample The occ urrence of .lspert!illlIs and I'elllcillillill ill the so il salll plt: is not surpr ising since it is kn own th at membe rs of these ge nera frequ entl y occ ur in soi l from arid regions (G riffen 1963: Eid. cr el al. 1982 ; Allsopp ef 0/ 1987). Similarly. Cloc!o.\JJOI'illlll. Fusarium and PaecilolII.vces are kn own to occur cOl11monly in soil (G ilman 1959: \Vaid 1960: Eickcr 1970: Eicker 1974: Carreiro & Koske I CJCJ2). Isol ates represt: nting .·kremol1i lll1l. l3o/l)'(}Iric/l1Im. Curnt/arill and Geolrichllm. have also bl.!e n obtainl'd from southern African so il s (Eicker 1969: Eicker el (II. 1982: Strauss 1997).
The overall number of higher fun gal genera recorded in this study is sign ificantly less than the 135 genera reco rded by ROll.\ and Van Warmelo ( 1997) during an exte nsive survey of fu ngi assoc iated with plants and litter in a natural Karoa pas turl' at rvli ddelhurg in the Eastern Cape ProvilH:e, South Africa, How-
S. Mr. J. Bot. 1999.65(5 & 6) ever. it Illu st he born e in mi nd that in the presen t stu dy the fungal popu lations of olle soi l samp le w itho ut litter \\'crc investigated with met hntl s which were se lecti ve for Illll cora lea n fung i (Strauss 11)()7). In their survey of fungi assoc iated with plants and litter in the I\.aroo. Roux and Van WarmcJo ( 1997) fo und six mllcoralean gcncra. i.t!, ./criJlomllcor elegul1.L ClI/lllil1ghamel/l/ eciliulliola. _I for1ferel/u. I/lIcor, Rhi:opus siolon[ler and RJu":oll/lIcol". When strains of tiwsc taxa were tes ted for uti lisat io n of the se ri es of nitrogen containing compounds during the present study (Table I and 4). ollly rep resentatives of .·lctil1()IJ/llcor eleg(lIls and Mucor wert:
ganic oxi dised nitrogen compounds. In additio n, they were unable to grow at 0.955 a\\.. This restricts the potential nitrogen sources of these fung i to organic so urces co nta ining an nbll nda nce o f availabl e wnter. However, it wns found t hat isolates of A. elegans, AI circinelloides and R OJ:v:ae. obtained from the Karoo soil, were more osmotolorant and a ble to uti lise organic nitrogen sources as well as inorganic oxidised nitrogen produced du ri ng nitrifi cation. C urrentl y, the I11l1 coralean fungal po pulations present in soil from other arid regio ns ill South A frica are also being studied. The res ults of these studies will give us insight int o the d iversity of Illu coralean fungi associated with arid so il in South Africa.
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