A STUDY OF THE DISTRIBUTION AND THE EFFECTS OF BACTERIOPHAGE OF ROOT NODULE BACTERIA

IN THE SOIL'

Abstract 13acteriopliagc for clo\.cr noclulc bactcria can be fo~~ncl on roots and nod~rlcs of

all nat~lrall), gro\\frl clo\-cr plants a i ~ d also in tlic soil s~rrrounding tlic roots, bu t not in soil lvithout c l o ~ e r plallts. ;\lternati\.c hosts for the phage of clo\:er I~actcria arc pen I~actcrin, and \.ice 1-crsa. The I~actcria ancl the phage are lictcrogcncous i l l t11c sense that only a proportion of strains of clovcr I~acteria ancl of pea bactcria are susceptible to I\.sis Ily a $\:en race of phage, ililcl only a proportion of races of phage can I\.sc a gi\-en I)nctcrial strain. There cloes not sccni to be any associatiorl I~et\vccn tlic s~~sccptibilit), of bacterial strains to lysis by phage ancl any othcr features such as antigenic structure ancl ell'ectivcrlcss in nitrogen lisation. ?'here ma). 1)c an association \\it11 aviralcncc, i.c. irlability to irlfect the host plant. 7'he bcha\;ior of phage-1)ncterial iiiixtures clcpcncls or1 the s ~ ~ r r o ~ ~ n d i ~ i g n i e d i ~ ~ m . ?'lie longe\.ity of phage in soil or i l l a soil-lilce mcdiurn such as :I 1-crmiculitc mixtc~rc is rclati\.ely short, and tlic cfrect of phage can be localizccl so that pllage-s~~sccptible bacteria ni~tl the p11:lgc can exist close to cach other \ \ , i t l ~ o ~ ~ t any apparent intcractio~l. However, a s long as the pllagc is present, phage-resistant bacterial mutants are usciall\- present also. 7'he pliage- resistant mut;lnts may also be mutants i l l other respects s ~ ~ c h a s eSicctivcrless ill nitrogen fixation. 111 the prcsc~lcc of \vcal;e~lecl phage, bacterial mutants \\.ere

fo~lncl to occr~r that differ Iron1 tlie parent form ill c~fecti \~cncss but resemble i t ill susceptibility to the phage.

Introduction

De~~zo lo~ l and Dunez (1) founcl that bacteriophage of lucerne nodule bacteria is present in root noclules, I-oots, ancl stems of all old lucerne plants, ancl also i l l tlie soil su1-1-0~11lcling the roots, but not a t distances longer than about 30 cm. They also claimed that bacteriophage cIcstro~.s tlie bacteria in the root noclules ancl in the surrouncling soil, alld ~ I I L I S interferes \\.it11 ~zoclulation, with nitrogen fisatioll, ant1 11.itI1 normal development of the plants i l l nitrogerl-cleficie~~t soils or other mcclia. 'I'he soil collclition l;no\\~n as "lucerne sicl;ness of soil" (la fatigue tles IuzerlliPres) is, according to Demolon ancl Duiiez, caused by the clestruction of lucernc lloclule bacteria by bacterioplzage.

I t is not kno\vn whether the I-esults ol Demolon ancl Dunez apl~l>' to clover noclule bacteria ;lnd clover plants. That some of them do not seem to apply is she\\-n by the fact that GI-ijns (2) ancl Laird (7 ) f o ~ ~ n d that bacteriophage of clover bacteria hacl no eflect on noclulatioi~, nitrogen fixatio~l, and normal g1-01vt11 of clover plants. This paper describes a stucly of the distribution of l~actcriophage oi clover nocl~~lc bacteria in the soil ancl of tlze role it may plaj- there.

Methods

Tlze method of isolating phages from soil and from clover roots ancl nodules was similar to that described by Vanclecaveye and Iiatznelson (9). A clover plant was clug out together \\it11 a lump of soil. Tlze soil \\-as separated from

'l/I~lnuscript recei\-eel OcLol~cr 5, 1956. Contriblttion from the Soil hlicrobiology DeparLmcll~, Rothamstecl Espcrimerltal StaLio11,

I-Iarpenden, IHerLfordshire, E~lglancl.

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172 C;2NADI4N JOUR?i;2L OF MICROBIOLOGY. VOL. 3. 1957

the roots and sifted. The roots were cleaned of the remainder of the soil by washing in water. Twelve nodules were taken for isolation of bacteria and the remaining noclules together with the roots were ground in a mortar. Five milliliters of tap water were added to each gram of the sifted soil and to each gram of the ground roots and nodules. The mixtures were poured into flaslts and shaken vigorously by hand for 5 minutes. The), were then filtered through filter paper and the filtrates filtered again through Chamberland L3 porcelain filters. One milliliter of the final filtrate was added to each of a series of tubes each containing 10 ml. of a 24 hour culture of a bacterial test strain in the liquid medium. The tubes were incubated a t 28O C. for 3 days and the results read a t intervals of 24 hours. The absence of bacterial lysis after this time was taken as evidence of the absence of phage virulent for the particular bacterial test strain. Sixteen different bacterial test strains were usually used: four classical laboratory strains and 12 bacterial cultures freshly isolated from the ~lodules of the plants tested. When soils in which no clover plants had grown were tested, 16 bacterial test strains were also used: four classical laboratory strains and 12 bacterial cultures isolated from nodules of the nearest clover plants that grew in the neighborhood.

If any of the bacterial test strains were lysed by the filtrate of a soil extract, a quantitative assay of phage in the extract was made by the previously described plaque count method (3), using one of the susceptible strains.

The methods of isolatio~l of bacterial strains from root nodules and of testing bacterial strains for effectiveness of nitrogen fixation in root nodules, and the liquid and agar media, were the sallle as those used previously (4).

Results Occurrence and Distr ibz~t ion of Phage i n the Soi l

Phage was detected in all 10 extracts from clover roots and ~lodules and in all 10 extracts from samples of soil in which the clover plants had grown. No phage was detected in any of six extracts tested from soil in ~irhich no clover plants (or any other legumi~lous plants) had grown. Half the number of samples of each Itind were taken from fields and the other half fro111 pots kept in the greenhouse.

The phages that were present in the extracts tested did not lyse all the bacterial test strains. Out of the four classical laboratory strains, usually not more than three were lysecl, some more f req~~ent ly than others, and out of 12 bacterial cultures freshly isolated from root nodules for each particular case, usually not more than eight were l~rsed. Phage concentrations usually corresponclecl to 1-2 plaques per ml. of soil extract. The phage isolated from the esperinzental plot a t Rothamsted, where clover plants hacl continuo~~sly grown for 100 years, hacl an esceptio~lally wicle host range, lysing all the four classical bacterial strains and 10 out of 12 freshly isolated bacterial cultures. Phage co~lcentratio~l was also exceptionally high, corresponding to 20 plaques per 1111. of soil extract.

The 100 year clover field a t Rothan~stecl showed no signs of "clover sicltness", the plants being quite ~lormal in appearance and growth. Of all

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KLECZKO\VSKA: ROOT NODGLE BACTERIA 173

the other tested soils in which clover plants had grown, only those in four pots were "clover sick". The phages of these soils did not differ consistently from those of the other soils either in host range or concentration. Thus no evidence was obtained that "clover sickness" of soil is due to bacteriophage.

The implications of the above results may be interpreted after taking into account two facts: the loss of a proportion of phage during passag-e through a porcelai~l filter, and the heterogeileity of clover nodule bacteria and of their phages, which means that only a proportion of strains of the bacteria are lysed by a given "race" of phage and oilly a pl-oportion of "races" of phage for clover nodule bacteria can lyse a given bacterial strain. T o estimate the loss of phage during filtration, a phage stock culture was diluted in water and filtered through a Chamberland L3 filter. Phage was assayed in the fluid before and after filtration. The proportion of phage lost during filtration depended on phage concentration. T o obtain a filtrate with any phage concentration corresponding to bet~veen 1 and 10 plaques per ml. (as in filtered soil extracts), about 1.5 times more concentrated fluids had to be filtered. Thus about 30% of phage was lost by filtration. To minimize the uncertainty of results due to heterogeneit~. of phages and bacterial strains, 16 different bacterial strains were used for detecting the presence of phage in the tested samples. As 12 of the strains \\rere isolated from the same material or from material collected nenrb!., it seemed lilcely that a t least some of these strains ~vould be susceptible to the lytic effect of phage that inay be present in the sample tested. The fact that phage was detected in all the soils where clover plants had gro\vn rund in none of those without clover plants can be talcen as evidence that phage lor clover nodule bacteria ~ ~ s u a l l y occurs only in soils where clover plants gron-.

Assays of phage lor clover nodule bacteria in the soil give only minimum values. -\ssuming that every particle of phage that can lyse the bacterial strain used for plating forlns a plaque, the assays \ \ r i l l still give lour values because of the loss of phage during filtration ant1 because ol heterogeneity of phag-es and bacterial strains. The loss during filtration can be allo\ved for by multiplying the resulting figures by a factor (in this case by 1.5), but no such factor can be found to a1101v for heterogeneity. If a soil sample contains a i n i s t ~ ~ r e of different "races" of phages for clover nodule bacteria, the phage particles ol only those "races" that can lyse the bacterial strain usccl for plating will form plaques. The proportions of numbers of particles of such phages to the total numbers of particles of all phages for clover nodule bacteria in different soil samples are ~lnlcno\\rn. The figures that are obtained from the assays can, therefore, apply only to concentrations of phage that can lyse the bacterial strain used for plating.

Phage Susceptibility Relationships Anzong Nodule Bacteria o j Different Host Inocz~lation Groups

The problem of distribution of phages for clover nodule bacteria in the soil is connected with the problem of alternative hosts. Table I shows that pea nodule bacteria are alternative hosts. Two different phages lysed widely

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TABLE I

Inoculation groups of bacteria tested

Clover Pea Lucerne Lupine Soybean

Total number oi strains tested 30 16 5 5 4

Number of strains Iysed by phage: I 3 6 1 2 0 0 0

I I 3 2 0 0 0

XOTE: Phage I \\-as isolatecl with a strain of clol-er bacteria, ant1 phage I1 wit-h a strain of pea bacteria. r\ll the strains that \\-ere Iysed bl- phage I1 \\.ere also Iysecl by phage I .

different proportions of tested bacterial strains, but each lysed about an equal propor t io~~ of strains of each of the t\vo inoculation groups, although one of the phages (I) was originally isolatetl as a phage for clover nodule bacteria and the other (11) for pea nodule bacteria. As each phage was virulent against about equal proportions of strains of both i~~oculation groups of bacteria, there does not seem to be any reason to clistinguish between phages for clover bacteria and for pea bacteria. The phages clicl not lyse any of the testecl strains of nodule bacteria of three other inoculation groups: lucerne, lupine, and so).bean. IHo\\.evcr, as the nu~nbers of these strains \\rere small, xeneralizations ~ ~ o u l t l not be safe.

The similarity in phage susceptibilit~. between strains of clover and pea inoculation KI-oups of bacteria is paralleled by serological relationships. I<leczlio~~~ski and 'I'hornton (6) found that clover and pea bacteria are not serologically disting~~ishable. Both groups are serologically heterogcneo~~s, containing Kroups of strains sel-ologically relatecl among themselves but unrelatecl to strains of other g r o ~ p s . 'I-he groups of serologically relatecl strains contain strains of both clover and pea bacteria. Ho\vever, no relation- ship between antigenic structure and susceptibility to I\,sis b ~ r a phage nras foi111cI. 3'Iarshall ant1 Vincent (8) on the other hand, n.orl;ing with other strains of clover bacteria ant1 other "I-aces" of phage, tlitl fincl such a relation- ship. I t is possible that different structural elements that conclition susceptibility of bacterial cells to clifferent phages ma\. or may not be associated ~vith antigenic structures. Further evidence of the close I-elationship between clovel- ancl pea bacteria is the fact that some of the strains can cross-infect (10, 5).

S u s c e p t ~ b ~ l ~ t y to Lysis by Pl~ages a?zd Other Characf(~ristics o j Bacterial Strains Table I1 was compilecl to determine whether there is any association

between susceptibility to 1) sis by a phage ancl other characteristics of bacterial strains, such as antigenic structure, ab i l i t~ to infect the host plant (virulence), ancl effectiveness in nitrogen fiuation i l l root nodules. The antiserum used for the agglutinatio~~ tests \\as prepared against a bacterial strain that was highly effective in nitrogen fixation ancl susceptible to lysis by the phage ~ ~ s e d

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I;LECZKO\VSI;:l: ROOT SOTIUT,IS B.lCTI<RI.l

I>rs.rnlnv~r~os 01. v ~ ~ r o u s clran,\c-rrsnls.rlcs ;\)ro\-c; 17 STRAIYS or; CLOVliR SODTLE H:\C'I'I:KI:i

Sitrogen lisation ;\gglutillatioil ----- -----------

Lysis by phage 1)). alltiserum Ellecti\,e lilcKccti\.c A-\vir~~letit

+ + 3 1 0

+ - 3 2 0 - + 1 0 1 - - 2 1 3

Nom: ?'hc ligures slio\\. nlimbers of strains that [all into groups oi dilicrcnt combinations of ieat~rrcs.

in the tests. 'Tliei-c \\.ere susceptible mid resistant strains as \\re11 as effective ancl ineffective ones among those that were ant1 those tliat \\?ere not agglutinated by- tlie antiserum, and also among tliose that \\rere nncl those that urcrc not Iysed b ~ , the phage. I t can be concluclecl, therefore, tliat neither susce~~tibility to lysis by phage, nor effectiveness in nitrogen fixation, is associatccl with any particular antigenic structure, ai~cl tliat there is no association bct\\,een susceptibility to phage ancl effectiveness or ineffectiveness in nitrogen fisation. Virulence, i.e. the nl~ility of bacterial strains to infect the host plant (irrespective of effectiveness or ineffectiveness in nitrogen fixation), \\;as sho\\;n by strains tliat were and were not l~~secl by tlie phage, ancl by strains that were and were not agglutinatecl by the antiserum. Viriilence is not, therefore, associatecl with any particular antigenic structure, or \\lit11 susceptibility or resistance to phage. .\virulent bacterial strains, on the other hancl, occurred among tliose that were resistant to lysis by phage. -1s there \\:ere four avir i~le~it strains, the probability of this occurring by cliance was 1/16. I t is possible, tliereforc, that avirulence might be associatecl with resistance to Iysis by phage, altliougli the reverse is obvious l~~ not true. This could be further testecl because there were a t Rotliamstecl a number of virulent and avirulent successive derivatives of one bacterial strain. Out of six avirulent derivatives only one was l~lsecl by the phage, ancl out of six virulent derivatives only one \vas not I!.secl. Thus tlie loss of virulence may be associatecl with tlie loss of susceptibility to lysis by phage.

i7fect o f Environnzental Cond.itions and tlze Behavior of Phage-Iost 11bixlu.res As phages ancl susceptible bacteria are founcl in tlie same samples of soil

and in clover plants growing in it, conditions in tlie soil seem to be such tliat phages ancl bacteria can exist sicle by side ~vithout affecting each other. TO see how far external conclitions can affect phage-host interactions, comparisons were made between the behavior of phage-host mixtures in nutrient liqi~itl or agar meclium, in sterilizetl soil, and i l l a vermiculite ~nisture. The soil \\;as "garden loa~n" with pH 6.2. The vermiculite mixture consisted of two parts of vermiculite, one part of sand, and one part of crushed flint. Both were

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'I'ABLE 111 + U 0\

EFFECTS OF EXTERNAL CONDITIOSS ON GRO\VTII 017 I'IIAGE-BACTERIAL MIXTURES A N D Oh' APPEAR4NCE OF PHAGE-RESISTANT MUTANTS

Bacterial strain "A121 11" Bacterial strain "Coryn" (cffccti\re in nitrogen fixation) (ineffective in nitrogen fixation)

--- --

Percentagest of bacteria Percentagest of bacteria Conc.* of: tha t were phage: Co~lc." of: tha t were phage:

Time of - -- "; i n c ~ ~ b a tion Medium I'hage Bact. S ~ ~ s c c p t . Resist. Phage Bact. Suscept. Resist. ?

~ - +- n

1 month Soil 0 7 X 10L 100 0 Vermiculite nlixtllre 3 X 10L 2 X 105 64 36 Liquid nletlium 2X108 9 x 1 0 " 0 100

3 months Soil 0 3 - - 0 0 - - C

200 100 0 2 X 10' 500 45 55 w

Ver~lliculite mixture 10 Z Liquid ~llediunl 5X10G 3 x 1 0 ' 0 100 5 X l o 7 3 X l o 7 0 100 r +

*The concentrations are gi\en i l l terms of nu~nbers of phage particles (plaque count) and of viable bacterial cells (colony count) per ml. of $ the liquid medium or per g. of each ol the other media. The i?tilial c o ~ ~ c r ~ ~ l r o l i o ~ ~ s \\-ere about 5 X l o 4 for the bacterial strains and about 5 X l o 5 for ,r - the phages. -

tBased on the r e s ~ ~ l t s of testing 30-40 cultllres isolated from single colonies. Thc same c ~ l l t ~ ~ r e s were also tested on plants for erfecti\leness in nitrogcn fixation. The results of these tests are given in Ta l~ le I\[. o

m TABLE IV

r 0 0 EFFECT OF EXTERNAL COSDITIONS ON APPE.\R.ZXCE OF MUTASTS I S EFFECTIVENESS IN XITROGEN FIXATION IN PHAGE-BACTERIAL MIXTURES , <

Bacterial strain "A1211 I", Bacterial strain "Coryl~", 2 originally effective originally ineffective r

-- -- W

Erfectiveiless in nitrogen fixation: Effectiveness in nitrogen fixation: - --- w - VI -4

Treatment of phage-bacterial l l l i s t~~res Effect. Intermed. Ineffect. Eflect. Intermed. Ineffect. -

3 months' illcubation in the liquitl lnedium 0 0 40(r) 0 0 40 (r) 3 months' illcubation ill the vermiculite 111ist~1re 35(s) 0 l ( s ) 9 (s) 2 (s) 29(rs) Surface iiloculation ol acrar 111ates 17 0 3 3 0 6 47 -

NOTE: The Iigurcs are the i ~ ~ ~ n t b e r s of isolated bacterial culturcs that fell under the three hcadings. 'I'hc sillle cultures were tested for susccptibility to the phages: (r) all cultules \\.ere phagc resistant; (s) all c u l t ~ ~ r e s \yere phage susceptible; (rs) 22 cultures were phage resistant and 7 phage susceptible.

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I<LECZI;OIYSK.\: ROOT XODULE BACTERI.~ 177

sterilized by autoclaving twice for 1 hour a t 15 lb. pressure. Two bacterial strains were used, one of which ("A12111") was effective and the other ("Coryn") ineffective in nitrogen fixation, and two corresponding virulent phages.

Twenty-five milliliters of a water suspension of bacteria grown on agar slopes (ca. 6XlO"acteria per ml.) and 2.5 1111. of a phage stocl; (ca. 6X108 plaques per ml.) were added to 250 ml. of a sterile soil extract. The mixture was kept for 3 hour a t room temperature to allow for combination between phage and bacteria, and then 1 1111. of the mixture was added to 10 ml. of the l i q ~ ~ i d medium or to 10 g. of soil or vermiculite mixture, whose water content was then increased by acldi~lg another 1 inl. of the soil estract. All the media were incubatecl a t rooin tcinperature in tubes plugged with cotton wool. Determinations were made after 1 month and after 3 months of incubation; the results are given in Tables I11 and IV. The liquid m e d i ~ ~ m was tested directly, whereas water was added to the other media (soil and vermiculite mixture) in the amount of 1 ml. for each gram, and the m in clarified water extracts thus obtained were used directly. Table 111 shows that in the soil the phage disappeared completely during the 1st month. Bacteria that escapcd the effect of the phage initially present probably multiplied for some time within the 1st month of incubation and were all susceptible to lysis by the phage. However, after the initial period of multiplication, the bacteria died out in the sterilized soil and only very fe~v were detected after 3 months of incubation.

In the vermiculite mixture the two phage-bacterial systems behaved somewhat c1ifferentl~-, although the general trend was similar. In one system the c o ~ ~ c e n t r a t i o ~ ~ of phage after 1 month was about l/lOth of the original, and alillost no phage was detected after 3 months. The concentration of viable bacteria increased during the 1st month and about 40% of the bacteria were phage-resistant mutants. After 3 n~onths most bacteria had died out, but those that sui-vivecl were a11 phagc susceptible. In the other system phage concentration after 1 non nth \xT,\s 10 times the original, and after 3 months i ~ ~ s t unclcr l/lOth ol the original. The concentration of viable bacteria increascd during the 1st month, about 10% being phage-resistant nuta ants; after 3 inonths inost of the bxter ia had died out, about half of those that

- 7 survived being phage resistcult. I IILIS, in the vermiculite mix t~~res bacteria susceptible to lj.sis by a phage can exist side by side with the phage. When thc phage is a t a sizable concentration, phage-resistant bacterial mutants are usually present. If phage dies out, the phage-resistmt mutants seem to revert to susceptibility or die out soorier than phage-susceptible bacteria.

The liquid medium contained an abundant population of bacteria that appeared as seconclary growth alter the original bacterial population was lysed. All the bacteria were phage-re~ist~unt mutants and their n~imbers increased between 1 and 3 inonths of incubation. 'I'he concentration of active phage decreased during this time, but did not fall to the low level to which it usually falls in similar conditions in filtered cult~ii-es lrec ol bacteria (i.e. to

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1 i 8 C.11-.\DI.-\V J O U I I S \I, O F MICROBIOLOGY. VOL. 3. 1957

about 10". This may be due to the fact t:hat the population of phage- resistant bacteria occasionally producecl phage-susceptible cells, or because accumulating bacterial metabolic proclucts protectecl phage from inactivation.

Scarcity of nuti-ients for bacteria i l l the soil aucl in the vermiculite mixture could have been responsible for the results obtainecl. An experiment nras set up with the soil and verrniculite mixture exactl~l as clescl-ibecl above, except that a l i q ~ ~ i d nutrient rnediu~n was substituted for the soil estract. In the presence of the nutrients the bacteria all became resistant as they did i l l liquicl media, suggesting that in the earlier experiment the persistence of susceptible bacteria liras clue to lacli of nutl-ients. But the concentration of phage fell between 1 and 3 months of incubation to the level to which it i~sually falls in filtered liq~iicl cultures free oi bacteria. This coulcl be esplainecl b>- assuming either that bacterial metabolic proclucts that may protect phage from inactivation in the liquid meclium mere :~clsorbecl b ~ , soil particles, or that phage was adsorbed.

I n adclitio~l to the tests shown in Table 111, the bactcl-ial cultui-es isolated after 3 months of incubation wit11 phages in cliffel-eilt media were also testecl on plants for effectiveness i l l uitrogeu fixation. The results are shown i l l

Table 117, ~v l~ i ch also inclucles results of an experimc~lt in \vhich phage-bacterial mixtures mere inoculatecl on the surface of agar plates, as described previously (4). There is eviclence that phage-resistant mutants arise indepenclently of any action of the phage, which merel~. reveals their presence by destroj-ing susceptible bacteria. There is also evidence that wheil n bacterium is in a state of cliseq~~ilibri~im I-esulting in a nuta at ion i l l one feature, a mutation in another ieature is also lil;cl>, to occur, although the illutatioils ma). be inclepenclent of each other (4). There may also be a reversal of one m u t a t i o ~ ~ \vithout a reversal of another. Thus phage-resistant mutants may becoine susceptible again, but an accompan)~iilg m ~ ~ t a t i o n , for example, in efr'ective~less in nitrogen fisation, may not be reversecl. Thus thc bacterial mutations i l l

the phage-bacterial mixtures, sholv~i in Table IV, call not be consiclerecl to be caused by phage, but may merely- be selectecl bj- it. I t shoulcl be borne in mind, ho~vever, that the rates of mutation of the t\vo bacterial strains (iiA12111" and "COS~II") must be extremely lolv, so that in the abse~lce of a selective factor such as phage, the). appear perfectly stable.

In the liquicl meclium all the bacteria that were testecl appe;lrecl uniiorm, all being phage resistant ant1 all i~leffective in ilitrogeli fixation, although one of the original strains was effective. The other turo meclia, OIL the other halld, appmrcd to contain bacteria that clifferecl in effectiveness in nitrogen fixation zund in phage susccptibi1it)r (see also Table 111).

The uniform it)^ of the bacterial populatioils in the liquicl meclium as cornparecl with agar call be explained by the ease of diffusion. The nen~ly formecl mutant cells are a t first very slolv growing, although eventually they grow as fast as their parent forms (4). 111 a liquid medium all bacteria except those that are phage-resistant mutants are lysecl. Xoii~, one of the slolv- clevelopiilg mutants may multiply faster than the others, ant1 so its progeny

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KLECZKO\\ SK.1 ROOT SODI'LIC B.\CTl<RI.\ 179

may eventually suppress m~~ltiplication of the other mutant hacteria ancl dominate the \\.hole of the ~necliu~n. 'Thus all {-he culture may be uniform, ancl all the bacteria will be like or ilnlilie the parent strain in effectiveness oE nitrogen fixation, clepencling on \\,hether or not the successiul mutant to phage resistance happenecl to be also a mutant in effectiveness in nitrogen fixation. On agar plates, on the other hand, the progenies of all the mutant bacteria arc localized as colonies ancl so clo not interfere niith each other's m~~ltiplication. I n the vermiculite misture, whose \\rater content \ \us just sufficie~lt to malie it ~nois t , progenies of different bacteria presumab1~- \\rere sl~fficieiitly localizecl so as not to interfere with each other as much 21s the)- clo in a liquid, ancl so bacteria \\;it11 clifiere~it features ct~ii easily be fountl. 'I'lie sallle reasoning applies to the soil. The presence of phage-susceptible hacteria i n the same vermiculite mistures 01- in the same soil cultures ~vhich also contain the phage call also be explainccl by assuming localizatio~i of the phage ancl atlsorl)tio~l on soil or vel-mic~~lite particles.

Mutants in effectiveness in nitrogen fixation which were fount1 in the vermiculite mistu~-es \\-ere all phage susceptible. This can perhaps be esplainecl by as sum in^ that they \\rere clescenclai~ts of bacteria that luutatecl to pliage resistance ancl also in effectiveness in nitrogen fixatioti, but subse- quently revertecl to phage susceptibility \\;bile I-etaining the other ncc~uirecl feature.

Discussion

'I'he clistribution sti~clies clescri1~ecl above she\\; that the presence of phage in soil is closely relatccl to that of the plaiit that harbors the host bacteria, but that its presence cloes not result in the elimination of its bactel-ial host.

The espei-iments \\.it11 the ~rermiculite mistuse sho\\:ecl that phage ancl bacteria aclcletl to such a ~ n i s t ~ ~ r e coulcl cocsist proviclecl t11:1t no nutrients are aclcled. 'The hactei-ia protlucecl some resistant r n ~ ~ t a n t s but susceptible cells \\,ere not eliminatecl. l'he ph;~ge clestro\-ecl all but the resistant mutants in the soil \\;hen ~lutrients \\;ere aclclecl. 'I'he pel-sistence of susceptible forms \\ilie~-e no ~ i ~ ~ t r i e n t was sup~~liecl \\rould seen1 therefore to be clue to stai-vation, I-ccluci~ig ~nultiplication of the bacteria ancl hence of the 11li:~gc.

I'revious \\;orlc by Iileczlco\\~slca (4) llas sho\vn that some of the resistant nuta ants of Rlzizobil~nz appeal-ing in the presencc of phage differ also from the parent form in the effectiveness of the nocl~~les t11;~t the)- procluce. Similar changes in effectiveness ma!- occur anio~ig-st phage-susceptible bacteria in the absence of phage, but such mutants arc mi~ch more abundant a ~ n o ~ i g s t phage- resistant strains.

'The bchavior of the "C'or)rn" s t~ - a i~ i s aclclecl with phage to the vermiculite mistui-e was thus at variance wit11 past esperie~ice in that the stl-ain proclucecl a consiclerable l~roport io~i of effective or partly effective niutants (:~11 of which were phage susceptible) but no effective mutants that lvere phage resistant.

With a n effective parent strain, a large proportion oE the mutants clevelopecl i ~ i the presence of pl~ag-c have bee11 founcl to be ineffective, \\,hereas \\;it11 all ineffective parent strain, relatively few effective resistant mutants appear

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180 Cf\NADIAN JOURXAL O F hIICROBIOLOGY. VOL. 3. 195i

after treatment with phage. The presence of phage in soil carrying clover should thus result in the accum~il,~tion of ineffective, resistant mutants and hence in a correlation of resistance and ineffectivity in the wild pop~~lation. But if susceptible as well as resistant inutants to effectiveness tend to appear in the presence of reduced phage activity no such correlation would be expected and in fact none has been found. The appearance of mutants influencing nodule behavior in the presence of phage may bear on the occurrence of ineffective strains in field soils, but apart froin this, the present work supports the conclusion of Grijns (2) and Laird (7) that phage is not likely to have an immediately h a r m f ~ ~ l effect on a clover crop.

Acknowledgment

The author xvishes to express her appreciatioil to Dr. H. G. Thornton, For. Sec. R.S., for his interest in this work.

References 1. DEMOLON, A. and Duxaz, A. Recherches sur la r61e dl1 bactCriophage dans la fatigue

des luzernihrs. Ann. agron. New Ser. 5, 89-1 11 (1935). 2. GRIJNS, A. Clover plants in sterile cultivation do not produce a bacteriophage. Zcntr.

Baliteriol. Abt. 11, 71, 248-251 (1927). 3. KLEC~I~OWSI<A, J. The production of plaques by Rhizobiunz bacteriophage in poured

plates and its value as a counting methocl. J . Bacteriol. 50; 71-79 (1945). 4. I<LBCZKOWSKA, J. rZ study of phage resistant mutants of Rhzzobiz~m trifolii. J. Gen.

Microbial. 4, 298-310 (1950). 5. I<LECLKOWSI~A, J., NUTMAY, 1'. S., aud BOND, G. Kote on the ability of certain strains

of Rhizob iz~?)~ from peas and clo\er to infect each others host plants. J. Bacteriol. 48, 673-675 (1944).

6. I<LECZKO%~SI~I, A. and THORNTOY, H. G. serological study of root nodule bacteria from pea and clo\rer inoculation groups J. Racterioi. 48, 661-672 (1944).

7. LAIRD, D. G. Bacteriophage and root nodule bacteria. Arch. Milcrobiol. 3, 159-193 (1912\ ,- --,.

8. ~\'~ARSIIAI~I., I<. C. and VINCBST, J. hI. Relationship betnreen the somatic antigens of Rhizobizrih trifolii and susceptibility to bacteriophage. ,\ustralia~l J . Sci. 17, 68-69 (10<4\

9. VAXDECAVEYB, S. C. and I<ATZNELSOX, H. Bacteriophage as related to the root r~otlule bacteria of alfalfa. I. Bacteriol. 31. 46.5-177 (1936). < ~,

10. \YILSON, J . K . ~ e g t ~ ~ n i & u s pla~its and'tl,eir associated organislns. Cornell U~li'i.. Agr. Espt. Sta. R'Iem. 221 (1939).

Can

. J. M

icro

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. Dow

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