Effect of osmotic potential on streptomycete growth, antibiotic production and antagonism to fungi

EFFECT OF OSMOTIC POTENTIAL ON STREPTOMYCETE GROWTH, ANTIBIOTIC PRODUCTION AND ANTAGONISM TO FUNGI P. T. W. Department

of Forestry,

Australian

WONG*

and D. M.

National

(Accrptc’d

GRIFFIN

University.

14 March

Canberra.

A.C.T. 2600. Australia

1974

Summary--The growth ofeighteen isolates of Strrptorn~~s spp. on agars. the osmotic potentials of which were controlled by the addition of various solutes, showed that nine isolates were able to grow at ~ 100 bars and two of these grew at - 150 bars. This suggests that the water requirements of soil strcptomycctes are almost as diverse as those of soil fungi. In general, linear growth and sporulation declined with dccreasing osmotic potential. but one isolate grew optimally at potentials between -5 and ~ IO bars. Another isolate formed maximum amounts of a water-diffusible antibiotic per unit colony area at potentials between -20 and -35 bars. This appears to be the first report of osmotic potential inRuencing the production ofantihiotic by Srrrpro~~~_rcus. The increased antibiotic production at moderate potentials M;IS associated with inhibition of P~rhiur,l ulrimum. Rhi;ocfmirr .so/~~r~r and a Curculuritr sp. by the stt-cptomgcete at these potentials on osmotic agar plates. The possible ecological significance of this phcnomcnon in soil is discissed. ESI’ERIMENT4L

INTRODllCTlON

The effect of soil water on the activities of streptomycetes has not been widely studied. It has been observed that actinomycetes become more prevalent as soils dry out (Conn, 1932; Dommergues. 1962; Kouyeas, 1964). and that actinomycete spores and even mycelia are able to withstand considerable desiccation (Meiklejohn, 1957; Alexander. 1961; Kuster, 1968; Williams rr al., 1972). There is good evidence that streptomycetes survive in soil as spores (Subrahmanyan, 1929; Skinner. 1951 ; Pfennig. 1958; Lloyd. 1969: Williams ct al. 1972). but there is little information on the effect of water stress on their growth and activity in soil. Chen and Griffin (I 966) observed in passing that actinomycetes were only active in their experiments in soil at water potentials greater than - 55 bars. More recently. Williams c’r(II. (1972) showed that the growth of a Micromnosportr sp. and a number of Strrptornycrs spp. was severely limited at soil water potentials of about - 100 bars and absent at potentials of -700 bars. In many fungal studies. where solutes were added to an agar medium to control the water potential (Scott, 1956; Sommers c’tLII..1970: Adebayo and Harris, 1971: Dube. Dodman and FlenQe, 1971; Bruehl, Cunfer and Toiviainen. 1972; Cook. Papendick and Griffin, 1972). data obtained for growth rates correlated well with those obtained by employing other methods (Schneider, 1954; Kouyeas. 1964). This technique is used in the present investigation to study the effect of osmotic potential on streptomycete growth. antibiotic production and antagonism to fungi. * Present address: Biological and Chemical Research Institute. P.M.B. 10. Rydalmere. N.S.W. 21 16. 319

The streptomycetes were isolated from soil by the dilution plate technique, the agar medium containing anti-bacterial and anti-fungal antibiotics (Williams and Davies, 1965). Eighteen isolates. obtained from garden and cultivated soils and soils from semi-arid areas, were maintained at 25 C on oatmeal and potato dextrose agars.

Osmotk

poter7tiul cigar

The osmotic potential of the agar medium w’as co~itrolled by the addition to a basal medium (Sommcrs rt al., 1970) of calculated amounts of either (a) sucrose. (b) KC1 or (c) a salts mixture comprising NnCI, KCI and Na2S04 (Scott, 1956). The basal medium was composed of Na2HP0,. 0.75 g: KH,PO,. 0.75g. NaCI. O.lOg; MgS0,.7Hz0. Ol2g: NH,N03. 0.40g;glucose. l%Og; Difco yeast extract. O~lOg; Cornwall malt extract. I.00 g; agar, 15.0 g and distilled water, 1 litre. The total osmotic potential was the sum of the osmotic potential of the basal medium (-- I.2 bars) and that of the added solutes (Robinson and Stokes, 1955). All media were adjusted to pH 7.0 using 0.5 N NaOH. To conform with units of water potential currently favoured, osmotic potentials arc expressed in -bar units. Minus one bar potential equals - IO22cm of water potential or approximatclq pF 3.0. Detailed treatments of water potential may be found in the reviews of Griffin (1963. 1969, 1972) and Cook and Papendick (1970).

320

P. T. W. W0h.c; and D. M. GROWTH

PATTERNS

OF

TWO

STREPTO11~CXTES

Streptomycetes isolates PI and P2 were used for a detailed study of their linear growth rates and density ofsporulation on the three osmotic agars. The streptomycetc inoculum was a concentrated spore suspension, obtained by transferring loopfuls of spores from a 3week-old culture grown at 25 C. to molten O.S”, water agar and vigorously shaking it. A 5.0 ~tl drop of the inoculum was dispensed onto the agar surface at the centre of the plate with an “Agla” Micrometer Syringe (Welcome Reagents Ltd.. England). A small circular inoculum area was obtained by using 0.5”,, water agar. which set on touching the agar surface. The inoculated plates were placed in plastic bags and scaled with rubber bands to prevent water loss. They were incubated at 25 + 1’C and examined after 2 and 3 weeks (linear growth phase) when the lincar growth was measured with callipers. and the density of sporulation noted. Four replicates were used in the cxperiments and two diameter measurements of the colonies were made at right angles to each other for each replicate. The growth rates of isolate PI on the different media were similar at the same osmotic potentials (Fig. I). When the data were tested for parallelism and equality of rcgrcssions. for the range of osmotic potentials bctacen ~ 3 and -40 bars. where the grap!ls appeared approximately linear. it was found that (I) a11 the three curves could not bc compared together as they were not a11 parallel. (2) when the sucrose and KCI curves wcrc compared. the growth rate of isolate P1 on sucrose media was significantly grcatcr (5 per cent level). In the isolate P2. the linear growth rates with the diffcrcnt solutes (Fig. 2) were found to bc not significantly difl‘erent at the 5 per cent level. The maximum growth rate in this isolate was not at the lowest potential as III PI but was between - 5 and - IO bars.

<;KIbtI\

The colonies of PI on basal medium had pink to buff aerial mycclium. often with white radial streaks and a white fringe, producing a powdery or matt surface over the colony. With decreasing potential, there was a rapid decline in the density of sporulation till about - 15 bars. beyond which there was a further gradual decline in sporulation. At potentials below -40 bars. sporulation was minimal or absent and colonies were raised and shiny in appearance. At -72 bars growth was so poor that the colonies which developed did not cover the inoculum drop even after 3 weeks incubation. There was no growth at - 78 bars. The sporulation pattern was similar on the three media but there was generally more abundant sporulation on the sucrose medium, especially at the higher potentials (4 to - I5 bars). The isolate P2 had greenish--cream aerial mycelium on basal medium. When sporulation was abundant. the surface of the colony was raised and uneven. With decreasing osmotic potential, the sporulation was less dense and a lighter colour. In general there was a more gradual decline in sporulation and spore colour with decreasing potential. compared to PI. There was also less marked differences in sporulation between the diffcrent media. At - 78 bars. discrete grey colonies were fol-med which did not cover the inoculum drop after 3 weeks’ incubation. Therefore, the osmotic potential limiting the growth of P2 would not be much less than ~ 7X bars. GROWTH

LIMITS OF SOME STREPTOMYCETES

As strcptomycete isolates P1 Bnd P2 ceased to grow on agar at osmotic potentials of about -80 bars, 16 other streptomycete isolates were tested for their ability to grow at lower osmotic potentials. Osmotic plates were prepared from the basal

Osmotic potential.

bars

Fig. I. Relationships bctwccn mean linear growth rate of strcptomgcctc isolate Pl and osmotic potential. controlled bq sucrose t-0). KU (m---B) and a salts mixture (V---V).

Osmotic

IO

potential

on

streptomycete

20

40

30

Osmotic

potential,

321

activities

50

a*.70

bars

Fig. 2. Relationships between mean linear growth rate of streptomycete isolate P2 and osmotic controlled by sucrose (e-0). KC1 (w---m) and a salts mixture (V---V).

.AhTIBIOTIC

medium using KC1 only to control the osmotic potential. A range of potentials from -60 to - 150 bars. in steps of -10 bars, was obtained, and inoculated as before. There were three replicates per potential for each streptomycete isolate. at 25 ? 1YI and examined

The plates

were

incubated

hibition zones. l-2 cm wide. wcrc formed when the streptomycete and one of the fungi wcrc grown for a week on the Same agar plate at 25 C. The fungal inhibition was due to a diffusible antibiotic and not to nutrient competition, since an agar plug taken from

Table

PI P2 P3 P4 P5 P6 P7 P8 P9 PI0 PI I P12 Bl B2 83 B4 Sl s2 * No growth

at - 60 bars.

PKODLICTIOh

The streptomycete isolate PI was antagonistic to the following fungi: Pqthium ulrirnurn Trow. Rhix~onirr solani Kuhn, Ph~rophthorn cim~urnorni Rands. S&roti~rn rolfisii Sacc. and a Cm&LLr1’((sp. similar to (‘IIIYYIrtritr irltc,r..st’rllirfclrtr Berk. and Ravn. (Gilman. 1957). In-

weekly for growth. The osmotic potentials at which growth of the streptomycetes was last detected i.e. growth ceased between this potential and - IO bars below it, arc shown in Table I. Of the 18 isolates examined weekly for a month, nine could grow at - 100 bars and two of these grew at - I50 bars.

Streptomycete isolate

potential,

I Osmotic potential at which growth last detected

Origin

of soil

Pine Forest, Lidsdale, N.S.W. Garden, Mosman. N.S.W. Garden. Mosman. N.S.W. Garden. Mosman. N.S.W. Garden, Mosman, N.S.W. Garden. Mosman, N.S.W. Garden. Turramurra. N.S.W. Garden. Turramurra. N.S.W. Jarrah Forrest. W.A. Aerial contaminant. Sydney. N.S.W. Aerial contaminant, Sydney. N.S.W. Aerial contaminant, Sydney, N.S.W. Semi-arid scrub, Balranald. N.S.W. Semi-arld scrub. Balranald. N.S.W. Semi-arid scrub, Balranald. N.S.W. Semi-arid scrub, Balranald. N.S.W. Semi-arid scrub. Whylla. S.A. Semi-arid scrub. Port Augusta. S.A.

( ~ bars) 70 x0 70 70 90

I IO 60’ 70 60 90

IO0 I so IN I 20 140 I 50 I IO I40

P. T. W. WONC and D. M. GtuFFih

32

the inhibition zone further inhibited the fungus when placed on t&h agar (Hsu and Lockwood, 1969). The drcct of osmotic potential on the production of antibiotic by this streptomycete was studied. A ranse ofosmotic potentials from - 1.2 bars to -i5 bars m steps of ~ 5 bars was obtained by the addition of KCI to liquid basal medium. Duplicate 100 ml

conical flasks containing

30 ml of sterile liquid media

at the various potentials loopful ofstrcptomycetc

were

turn grown scricd

each

inoculated

with

a

spores from a i-week-old culat 25 C. Uninoculated osmotic media

as controls.

The tlasks were shaken

on a mechanical shaker in a room at 25 + I ‘c’ for 2 weeks. The contents of the duplicate flasks were pooled and liltcred through a 0.3 btrn millipore filter and the antibiotic activity of the filtrates was bioassayed with P. I,/ri/rflr,rl. T\\o ml of the filtrate was pipetted into a 9.0 cm sterilc plastic Petri dish and 13.0 ml of ?I,, molten basal ;IZ;LI- medium were then added. Thr contents were n;uxl :md the plates were kept for 23 h at 25 C to allow the antibiotic to diffuse uniformly through the aga-. Control plates were similarly prepared for the control Ilasks. A 6.0 mm agar plug of P. ~lti~tx~m from the margin of a colony was then placed at the centrc of each plate. The plates were incubated at 25 + I ‘C. m-tc~ 24 and 3s h. the diameter or the tkllgal colonies WI-C mcasurcd. controlled-tcmperaturc

The linear gf.oM#th rates in the two series are shown inhibition of P. ~r/tir~u~r~occurred at osmotic potentials between -20 bars and -35 bars. in Fig. 3. Maxm~um

70-_--_,

.

\ ,,

,Control <

‘. ‘1

\

\

suggesting that maximum occurred at these potentials. STREPTOMYCETE

antibiotic

production

AI\;TAC;ONISM TO FUNGI

The antagonism by streptomyccte isolate Pl to P. ul~irnurtz. R. sohi and the Curz~ularic~sp, was also studied on osmotic agar plates. Agar plates. osmotically adjusted with sucrose, KC1 and the salts mixture were prepared as before to give a range of osmotic potentials from - I .2 bars to - 54.7 bars. The streptomycetc was inoculated at a point 2.0 cm from the periphery of the agar plate. The plates were scaled in plastic bags and incubated at 25 + I C. After 5 days, four replicates at each potential were inoculated with 6mm discs of rungal inoculum. placed 4.0 cm from the centre of the streptomycetc colonies. The plates were rewrapped in plastic bags and incubated at 25 f 1 ‘C. At daily intervals. the diameter of the streptotnycete colony (Xl) and the inhibition zone (.Y2) were measured with callipcrs. Morphological changes in the fungal colonies were also noted. If the area of fungal inhibition around the streptomycete colony (42) is assumed to be circular, then it may be calculated from XI and X2 (Fig. 4). Since the areas of the streptomyccte colonies (A I) at the different potentials differed greatly. the fungal inhibition was round to be most satisfactorily calculated as inhibition per unit area of the streptomycete. i.e. AZ:,4 1. Initially, an attempt was made to measure the areas around the streptomycete colonies in which the fungus was inhibited. Such measurements could be made when the potential was higher than -20 bars. When the potential was less than this value, no accurate measurements could be made because of the slow growth of the fungus. A comparison of the measured and calculated areas of inhibition showed that the diffcrcnce between the values was less than about 10 per

\

Key F 5 x, x2 Osmotic

potential

of sterile

filtrates,

= = = =

Fungol inoculum disc Streptomycete colony Diameter of streptomycete Inhibition zone

colony

bars m=

Fungal

colony

Fig. 3. Diagram showing fungal inhibition by streptomycctc isolntr PI on osmatic agar plate.

Osmotic

potential

on streptomycete

Osmotic

potentiol.

323

activities

bars

Fir. 5. Relationshitx between inhibition of Pvthiurn u/tinlu/)l Der unit colonv area of streptomvcete isolate n ) ,tnd a salts mixture (V V). P land osmotic potential, controlled by s&se (e -0). K’CI (m :

cent. It was. therefore, decided that the calculated area (A2) gave an accurate and easily obtained measure of the true inhibition area, and was used in the analysis. The measurements used for calculating A2/Al were made at least 72 h after inoculating with the fungi, as this period was necessary for inhibition zones to be clearly observed at the lower potentials. The relationships between A2/AI and the osmotic potential controlled by the three solutes are shown in Fig. 5. The curves showing the inhibition of P. ultinlurfl on KC1 and the salts mixture media were similar while that on sucrose medium was markedly reduced at the lower potentials. In general. fungal inhibition increased with decreasing potential, with a maximum at about - 35 bars. Inhibition at lower potentials was not measured because of the limited growth of P. ultimurn at these potentials. When R. ,so/r~r~i and the Curouhria sp. were used in similar experiments using only KC1 to control the

potential, they were found to show the same inhibition pattern (Fig. 6). With these fungi, where considerable growth occurred below -30 bars. a peak inhibition was observed between - 30 and -40 bars. Inhibition of R. solarzi was reduced at -42 bars and no measurements could be made at lower potentials where growth was minimal. In the Cu~c&rirr sp. where growth was still vigorous below - 50 bars. inhibition was found to further decline.

OBSERVATIONS

OF FL hiCAL

I’VHIBITIOR

Microscopically. fungal inhibition was mainly expressed in the form of lysis of the hyphal tips. These were vacuolated or devoid of cytoplasm. and cxtrudcd cytoplasm was sometimes observed beside them. Some hyphal tips were swollen to form apical knobs and others were variously distorted. Some branch hyphae turned away from the advancing front. Observations over an extended period indicated that the fungi were not completely inhibited by the antibiotic but were retarded in growth. All the three fungi progressed slowly towards the streptomycetc colony. especially at potentials above - IO bars and below 41 bars.

DIS<‘LSSIDN

I 0

I

IO Osmotic

I

I

I

I

20

30

40

50

potentials.

I 60

bars

Fig. 6. Relationships between inhibition of Pyrhium ultimum (*-0). Rhkoctonia solani (W-U) and a Curdaria sp. (V---V) by streptomycete isolate PI and osmotic potential, controlled by KCI.

The use of solutes to control the osmotic potential ofa medium is a convenient method to stud) the water requirements for microbial growth. Its advantages and disadvantages have been discussed (Sommers. (‘I trl.. 1970; Griffin. 1972). In these experiments. the response of two Streptotnyces isolates to decreasing osmotic potential was not affected by the nature of the solute. with the exception that sucrose increased the growth rate and sporulation of isolate PI. This is likely to be a nutritional effect, as has been observed in fungi (Sommers et ul., 1970). Fungal inhibition was also reduced

324

P. T. W. WOI\(; and D. M.

on sucrose plates in the antagonism experiment (Fig. 5). There arc a number of possible reasons for this. Firstly, sucrose may have a direct &cct in depressing antibiotic production. Secondly, the obscrvcd increase in the vigour of P. ulfirmu probably rcducrd the cffectivcness of the antibiotic since inhibitors arc known to be more effective when fungal growth is reduced (Brown. 1922; Tomkins. 1Y29). Finally. and most importantlc. the increased growth rate of the streptomycete on sucrose plates increased the colony arca. ‘41. and therefore depressed the inhibition index 12 ‘.4I. Thus. with a view to eliminating the nutritional cffcct of sucrose. KCI was used in much of the other work. It was also more conveincnt to use than the salts mixture. In a survey of the water requirements for the growth of eighteen isolates of St~c~prorf1~~5 spp. from divcrsc environments. half of them were able to grow at - 100 bars and two of these grew at - 150 bars. This indicatcs that the water requirements of soil streptom>.cetes arc almost as diverse as those of soil fungi (Grlltin. 1963. 1969. 1972). All the six isolates from semi-arid areas grew at potentials below - 100 bars (Table I ), while the growth of most isolates from garden soils was limited at higher potentials. This suggests a correlation between the ability of soil strcptomycctcs to grow at low potentials and the aridity of their origins. but ;i more extensive survey will bc required to confirm this. Chen and Griffin (1966). however. fiiiled to demonstrate such 3 correlation for fungi. Our results arc similar to those of Klevenskaya ( 1960).who recorded sonic soul actinomjcetcs grow ing at osmotic pressures of I 17 atmospheres (about ~ I IS bars). All these values. howebcr. may be over-cstimatcs of their growth limits in soil since Adcbayo and Harris (1971) have shown that osmotic potential \\;Ls 1css scvcrc than matric potential on the growth of P/IJ~~o/)/Ifl~or.o cirmr~w~ti Rands and .-l/rc~~u~.itr /VIIUI.\Nccs in soil. They found that the metric potentials at which fungal gro\\ th ccascd were numerically one-half to txo-thirds the osmotic potentials that limited fungal growth. They suggested that the anomaly was prohab1) due to reduced solute transport at low matric potentials. Similar cfrects ha\;c been rcportcd on Q~hioho/r~.s gnrmiffi,5 (Sacc.) Sncc. and F~r.sc~~~irfm ro.sw~i~ (Lk.) Snyd. and Hans. f. sp. c,c,rctr/i.\ ((‘kc.) Sn!d. and Hans. “Culmorum” (Cook. Papcndick and Grittin. 1972). Ifstrcptom~cctesarcsimil~~rl~ afTccLcd. then m:lnq \+ill ccasc to grow in soil at potentials hclou - IO0 barA. C’hcn and Gritiin (1966) had ohscrtcd no actinomycctc activitv in soils nt matric potentials of --55 bars. while W-illiams (‘r ol. (1972) have shown that strcptomycete groM th was greatly reduced at ~ I5 bars and minimal at about - 100 bars (pF 5.1). Even so. it is cbident that many streptomycctcs will interact \vith a large number of soil fungi. which arc active in this range of soil potentials. In general. the linear growth of strcptomycetes dcclincd with dccrcasing osmotic potential. Howcvcr. the optimum growth rate of isolate P2 wa5 not at the highest potential ( - 1.2 bars). but at potentials hct\\ccn - 5

(;ICIF~I\

and - IO bars (Fig. 2). This is not surprising since it is commonplace among fungi (Cook. 1Y71) and has hccn recorded for actinomycetes (Klevenskaqa. 1960). With declining lincar growth. thcrc was a concomitant dcclinc in sporulation but many isolates continued to sporulatc at osmotic potentials of -60 bars. It is not known whcthcr streptomycetes will in fact sporulatc at thcsc potentials in natural soil. In liquid osmotic media. isolate PI produced most antibiotic per unit volume of media at potentials bctwccn -20 bars and -35 bars (Fig. 3). The inhibition of the three fungi by this isolate on osmotic agal also retlcctcd this increase in antibiotic production per unit arca of P1 at thcsc potentials (Fig. 6). Thib appears to bc the first report of osmotic potential intluencing the production of antibiotics in :I Srr’c/‘/orlr!,c,c,\sp. It is noteworthy that maximum amounts of antibiotic wcrc produced at potentials where the growth rate was Icss than half the maximum growth rate. This is bt no scam unique in antibiotic-producing microorgamsms sinco an antibiotic-producing isolate of C~J/I~U/O,S~XI,-i[f/~ rpwtrirwm Nisikado and Ikata. produced most antibiotic rctative to fungal growth at osmotic potcntials between - 32 and - 55 bars when its growth rate I+;IS less than one-third the maximum growth rate (Bruchl (21trl.. I973 They also showed that Ihr production of antibiotic at these potentials contributed to the grcatcr survival of the fungus in wheat straw buried in soil.

The importance of hater-soluble antibiotics in the of soil microorganisms is still being debated but they can bc significant in rich substrates (Skinner. 1956: Wright. IY56) and in rnicrocnvironmelits in which they accumulate (Brian. 1957). The apparent shift in the metabolism of isolate PI from one of active linear gro\\ th and sporulation at high potentials to one of increased antibiotic production at potentials below - IO bars suggests a possible ccolo$cal role. As with C’. +o,ui/lcllr/rr (Bruehl. Cunfcr and roiviaincn. I972), the production of most antibiotic at modcratc potcntials may permit the strcptom)cctc to maintain possession of coloniscd substrata at those potentials. Morco\cr. ;IS strcptoniycctcs siirvilc in soil principallj’as sports. the prcsencc of antibiotics in the suhstratc ma) bc more important Lvhcn fuvoul-able conditions return. for thcj ma! then provide the gcrminatin~ sports with 2 hricf immunit! from microbial antagonl5m.

ccolo~y

Osmotic

potential

on streptomycete

centrations of oxygen and carbon dioxide. AII~I. Bat. (Land.) 36, 257-283. BRUEHL G. W.. CUNFER B. ANI) TOIWAINEN M. (1972) Influence of water potential on growth. antibiotic production and survival of Crphalosporium yrcrminuutn. Curl. J. Pht Sci. 52, 417-423. CHEN A. W.-C. and GRIFFIN D. M. (1966) Soil physical fattars and the ecology of fungi-V: Further studies in relatively dry soils. Tru,ls. Br. rll~,co/. Sot,. 49, 4 19-426. CONN J. H. (1932) A microscopic study of changes in the microflora of soil. Tech. &r/l. ,“\;.): St. .+rrc. c’sp. Sr!l. 204. Coor< R. J. and PAPFUIXCK R. I. (1970) Effect of soil water on microbial growth, antagonism and nutrient availability in relation to soil-borne fungal diseases of plants. In Roar Disru.ses LJ~I Soil-borrw Pafhogc~m (T. A. Toussoun. R. V. Bcga and P. E. Nelson. Eds) pp. XI-XX. University of California Press. Los Angeles. COOK R. J.. PAP~NIX(.I< R. 1. and GRIFFIN D. M. (1972) Growth of two root-rot fungi as affected by osmotic and matric water potentials. Soil Sci. Sot. .~I,I. Plot. 36, 7X 82. Coorc R. J. (1973) Influence of low plant and soil water potentials on diseases caused by soil-borne fungi. Phyroptrh/o~~~ 63, 45 I -45X. DOMM~K(;~NI:SY. Contribution it l’etude dc la dynamiquc microbiCnne des sols en Tone semi-aride et en s&he. .4,1/t. A
.4usr.

J. hid.

Sci. 24, 57 65.

GILMAN J. C. (1957) A Ma~lutrl c!f Sorl F~~Iz<~I. 2nd Edn. The Iowa State University Press, Iowa. GRIFFIN D. M. (1963) Soil moisture and the ecology of soil fungi. Bar. Rw. Cunlhridcgc, Phil. Sot. 38, 141~ 166. GKIFFIN D. M. (1969) Soil water in the ecology of fungi. .1)2!1 Rev. Phytopmh. 7, 289~ 3 IO. GKWIN D. M. (1972) E&X/!. of Soil Furyi. Chapman & Hall. London. HS[I S. C. and LO~KWOOI, J. L. (1969) Mechanisms of inhibition of fung in agar by streptomycetes. J. gc,i. :\ffcrobio/. 57, 149~_158. KI.I VLZIWAYA I. L. (1960) Development of soil actinomycetcs in media with various osmotic pressures. ,Miwobioh/w 29. 2 I5 2 19.

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activities

325

V. (1964) An approach to the study of moisture relations of soil fungi. Pltrur Soil 20, 351-763. KL STIK E. (1968) Taxonomy of soil actinomycetes and related organisms. In Tlzc, Ec&)c/y (!I Soil Btrc!crltr (T. K. ti. Ciraj and D. Parkinson, Eds) pp. 322 336. Li\#crpool University Press. LLO\~I) A. B. (1969) Behaviour of streptomycetes in soil. J. qr’~. .2fic,ohio/. 56, I65 170. MI.IIcctes in a’Kenvn soil. J. Soil. Ser. 8, 24&247. PFI NUI~ N. (195;) Beobachtu_een dcs Wachstums~erh;~ltcns von Streptomyceten auf Rossi-Cholodneq-Atlfwtlchsplatten im Bodcn. .-1rch. .2fih,ohic~/. 31, 206 2 16. Rouirso~ R. A. :ind STOKI s R. H. (1955) ~~/c~~r&~~~~, %/I,rim.\. Academic Press. New York. S(.HVLII)~K R. (195-l) Untcrsuchungcn iiber icuchtlgkeltsanspruche pnrasitiacher Pilie. Ph~~tr~prrhd. Z. 21, 6 I 78. S<.o~.r W. J. (1956) Water relations of food spoilage microorganisms. -Idrtrn. loot/ Rrzs. 7, X3. 177. SKINNIX F. A. (1951) A method for distinguishing between viable spores and mycelial fragments of actinomycctes in soil. J. qcn. .2li~~robio/. 5, I59 166. SKINN~K F. A. (1956) Inhibition of growth of fungi by Srrcpto,n~‘cr’.s spp. in relation to nutrient condltlons. .I germ. .~fkrobio/. 14. 381-392. SoMhftKs L. E.. HARKIS R. F.. DAI 1ou F. N. and GARDWR W. R. (1970) Water relations of three root-infecting P/I),fopthrr species. Plr!,roputllo/ot!, 60. 937 934. sl I%KAH>IANyANV. (19291StlldieS on soi] ;fCtfnOfTI)cctcS -11: Their mode of occurrence in soil. .1. /JIM/.I,I,s~. S?,. 12, 57. TOMPKINSR. G. (1929) Studies of the growth of moulds I. Pwc. R. Sot,.. Lord. B. 105. 375 401. WII.LIA~~SS. T. and DAVII s F. L. (1966) Llsc ofantibIotIcs forselective isolation and enumcratlon of actinomycctcs in soil. J. +,c,i. hfic~robiol. 38, 25 I 161. WlLLfAhfS s. T.. SHi\MI I MIILLAfJ M.. WA ISO\ I.. T. and MA’I.I.II.LI)C. I. (1972) Studies on the ecology of nctinomycetes in soil- VI: The Influence of moisture tension on growth and survival. Soil. Bid. Bwc~/wr~. 4. 21 S 275. WKI(;HT J. M. (1956) The productlon of antibiotics m soil IV: Production of antibiotics m coats of seeds sown in 5011. ..l,r!l. ~rppl Hit)/. 44, ihI_ 566.