897

SEROLOGICAL RELATIONSHIPS OF SOIL ACTINOPHAGES

C. T. CORKE, J. 13. ROBINSON, AND R. J. DOUGLAS Department of il~icrobiology, University of Gzlelph, Guelph, O?ztario

Received July 2, 1964

Abstract Actinophages first appeared in soil perfusates 4 hours after perfusion began,

and, with certain species of Streplonzyces, titersas high a s loG plaque-forming units (PFU) per rnl of perfusate were reached in from 18 to 24 hours. Samples of soils collected in late summer and fall yielded larger nu~nbers of PFU than did samples collected in the spring. Antisera against two isolated S . scabies phages were used to characterize the "wild" phage populations appearing in soil perfusates. With S . scabies as the plating indicator most phages (92-9870) were found to be related serologically to phage L, and from 2.5% to 7.570 of the total PFU were related to phage S. Further experiments showed that phages related to L appear- ing in perfused soil were the do~ninant group acting on S . lavendz~lae and S . olivaceus.

The use of combined antisera for inactivation of perfusate permitted the isolation of new phages normally obscured by the dominant serological groups L and S.

Introduction An earlier publication fro111 this laboratory (7) dealt with numbers and host

ranges of actinophages developing in perfused soils. I t was found that each soil contained more viruses against streptomycete cultures isolated from the same soil than for cultures isolated from other soils. Further studies of the ecology of soil phages and their relationship to host systems using the perfusion technique have consistently shown large numbers of phages (greater than lo5 phage per milliliter of perfusate) against certain species of Streptomyces. Many of the phages isolated on different Streptomyces spp. were quite similar in their host range. This evidence of some homogeneity among "wild" actino- phages led to an examination of the possibility of characterizing these phage populations using a serological technique without the laborious procedures of isolating, purifying, and characterizing many individual phages.

This paper deals with the use of specific neutralizing antisera for two selected phages to examine differences in actinophage populations in soils.

Materials and Methods The evolution or appearance of phages from two soils was followed using the

perfusion technique described by Robinson and Corke (7). The soils selected were Guelph loam from the University of Guelph campus (PI-I 7.0-7.5) and Fox sandy loam from the Ontario Agricultural College Crops Research Station a t I-Iespeler, Ontario (pH 6.8-7.2). Fifty grams of air-dried soil (2 to 5 mm mesh) was perfused with 200 ml of distilled water a t room temperature (ca. 21 "C to 25 "C).

Plating Hosts Four species of Streptomyces were used, S . scabies, S . lavendz~lae, S . olivaceus,

and S . roseochromoge~zes. Cultures were maintained on slants of glucose-

Canadian Journal of Microbiology. Volume 10 (1964)

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898 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 10. 1964

asparagine agar. Inocula were prepared from 7- to 10-day-old cultures by rub- bing off spores into sterile 0.1% peptone-water. The spore preparations were filtered through sterile pads of fiberglass to remove cluinps of spores and large mycelial fragments.

Phage Plating The numbers of actinophage plaque-forining units (PFU) in soil were deter-

mined by plating perfusate a t intervals. Perfusates were rendered bacteria-free by filtering through a sterile Millipore filter (Grade HA) and dilutions of filtrate were then plated with 0.5 1111 of the spore suspensions using yeast-beef agar and the double-layer technique. All plates were incubated a t 25 "C for 24 hours, a t which time plaque counts were made.

Serum Inactivation Experiments Two actinophages isolated on S. scabies were used in the preparation of rab-

bit antisera. Phage L was isolated by Newbould and Garrard (6) and posses- sed a wide host range. Phage S, which has a inore restricted host range and a smaller plaque than phage L, was isolated by one of us (J. B. R.) from a soil a t St. Catharines, 0nt . l The two phages were unrelated serologically and some details of their properties are shown in Table I.

TABLE I Description of S . scabies phages

Phage Morphology Plaque type Susceptible Strepto7nyccs

L Ilead length, 72 m p Large, 3 mrn S . scabies, S . lave~tdzrlae, Tail length, 150 m p S . az~rezts, S . albz~s.

S . antibioticus, S . roseockro7nogenes

S Head length, 80 m p Small, 1 mrn S . scabies, S . albzts Tail length, 320 lnp

In experiments involving the characterization of phage populations appear- ing in soil perfusate, antisera for phages L and S were added to perfusates a t higher concentrations than ilormally employed in routine inactivation of homologous phages. This was done to ensure that inost phages related to either phage would be effectively inactivated. The protocol for such experiments was as follows.

Reaction tube 1 coilsisted of equal volumes of a 1 : 1000 dilution of normal rabbit serum in yeast beef (YB) broth, and undiluted soil perfusate. Plating this mixture with host cells gave a total couilt of phages active on the specific plating host. Preliminary experiinents indicated that the presence of normal rabbit serum in diluent always resulted in a slightly higher couilt than when the broth alone was used.

Reaction tube 2 was coinposed of equal voluines of perfusate and anti-L serum (diluted 1:1000 with YB broth). When plated, the contents of this tube gave a count of phages not inactivated by this antiseruin and therefore serologically unrelated to phage L. The nuinber of phages related to phage L could then be calculated by difference from the control count.

'In a previous publicatior~ (7) phages L and S were referred to as NPlf and P2F2 respectively.

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CORICE ET AL.: SOIL ACTINOPHAGES 899

Reaction tube 3 was made up of equal voluines of perfusate and anti-S serum (1:500 dilution with YB broth). This tube gave a count of phages not related to S, and provided by difference froin the numbers in tube 1 the count of phages related to the S phage.

Reaction tube 4 consisted of perfusate with both antisera a t the above- mentioned dilutions, which, when plated with host cells, yielded those phages present in the perfusate which were unrelated to either phage.

The reaction tubes were incubated for 1 hour a t 30 OC, whereupon dilutions were made and residual PFU determined by the double-layer technique.

Results The phages detected in these experiments originated from the lysis of sus-

ceptible cells which developed naturally in the soil during perfusion since no inoculum was added to the soils. In inost cases the first PFU were detected 4 hours after initiation of perfusion of air-dried Fox loam (Fig. 1). Counts increased rapidly, reaching a ~naximuin in another 10 to 20 hours depending on the Streptomyces sp. used as plating host. Titers of lo5 to 10"er ml of perfusate were often reached. Using S . roseockronzogenes, the first phage was detected after 8 hours' perfusion and a lower maximum count was reached amounting to about lo4 PFU per ml of perfusate. The pattern of phage evolution against these hosts was about the same in Guelph loam. High titers were not observed on all Streptomyces spp; for example, with S. fradiae as host, perfusates rarely yielded plaques.

In these experiments marked differences in phage content of samples of a single soil type were observed, which may possibly be related to the time of

/ .>'-s. lavendulae --\

i ' S. roseochrornogenes I/ i 1.

TIME OF PERFUSION IN HOURS

FIG. 1. Time o f appearance o f phages active on S. scabies, S. lave?zdz~lae, and S. roseo- chro?noge?zcs in perfusates o f Fox loam.

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CANADIAN JOURNAL O F MICROBIOLOGY. VOL. 10, 1964

2 4 6 8 10 12 14 16 18 2 0 22 24 TIME OF PERFUSION I N HOURS

FIG. 2. Effect of time of sampling soils on phage content of perfusate. Upper group of curves represent P F U active on S. scabies from samples collected a t various times in the fall. The lower group represents P F U from spring sample collections of Guelph loam.

Total Phoge Count

2 __---- A/ Phage Related Ta to L

/' $ -.-.-.-. Reloted to S o ,o/*' ____-___--------

J X

/' x---*-- /'

/' Not Reloted to L o r S

i i 0

I 2 4 6 8 10 12 14 16 18 20

T I M E OF PERFUSION I N HOURS

FIG. 3. The appearance in a soil perfusate of serological groups of S. scabies phage during 20 hours perfusion (see text for experimental details).

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CORKE ET AL.: SOIL ACTINOPEIAGES 901

sampling. Figure 2 shows the phage counts against S. scabies in perfusates of Guelph loam. The upper three curves represent the phage appearing in per- fusate from plot samples collected from late summer to early fall, while the lower group depicts the phage counts from soil collected from the same plot area from April to June. Because of the higher counts of phage in the fall samples, these soils were used for seruin inactivation experiinents.

In preliminary experiinents involviilg serum inactivation of "wild phage" in perfusates, S. scabies was used as the plating host. I t was in~inediately ap- parent that the doininant group of phages present was related serologically to phage L, with only slight inactivation of phages in perfusate by anti-S serum. The use of the coinbined antisera in these experiinents indicated that the total number of P F U inactivated was about equal to the total inactivated by the two sera acting individually. T o confirm that plaq~ies appearing on the plates after treatment of perfusate with both sera represented "new" phages, a series of purified phage clones was propagated from plaques appearing on plates of inactivated perfusate. Routine seruin inactivatioil experiments as described by Alexander and McCoy (1) were carried out with anti-S and anti-L sera. Fourteen phages isolated in this way were found to be serologically un- related to either of our two phages. Four isolated froin plates inactivated with only a n t i 3 serum were related to phage L. This indicated that this technique was capable of fractionating the "wild phage population" into broad serological groups.

The results of one of the experiinents on inactivation of phages in perfusate of Guelph loan1 using S. scabies as a plating indicator are shown in Fig. 3. Most phages were found to be related to our phage L (92-98yo), while those related to S constituted from 2.5 to 7.5% of the total population. The use of coinbilled antisera resulted in a slightly greater inactivation than L alone, and this fraction of the population (new phages) constituted less than 3% of the total count.

The results of a series of experiments as described above led us to determine whether, by using hosts other than S. scabies, shifts in these percentages of related phages could be detected. Phage-containing perfusate from Fox loam was inactivated and plated with S. scabies, S. lave7zdulae, and S. olivacezts. Most of the PFU active on each of these three species was related to L. Anti- L-serum inactivated 99.4% of PFU active on S. scabies, 99.4 to 99.8% of phage active on S. lavendztlae, and from 60 to 80yo of PFU active on S. oliu- aceus. A sinall percentage of the total PFU active on S. scabies and S. lavefz- dulae was related to phage S (2 to 6%) but none of the PFU detected by S. olivaceus appeared to be related to phage S.

Discussion The importance of bacterial and actinomycete phages as components of the

soil population, and their possible effects on the growth of host cells in a natural environment has been little studied. Anderson (2) suggested the pos- sibility that bacteriophages in a natural environment could modify popula- tions of susceptible cells by such effects as production of phage-resistant mutants and lysogenization and by transduction. Similarly, Kleczkowska (5) observed interesting changes in Rhizobiz~m caused by soil phages. She observed

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902 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 10, 1964

that many of the phage-resistant mutants lost their ability to participate in the symbiotic nitrogen fixation process, while a few lost their ability to initiate nodule formation. concept of the importance of these phage-Rhizobinm relationships was that lysis of R h i z o b i z ~ m was not responsible for "soil fatigue" but that the phage could cause this condition by increasing the proportion of ineffective bacteria.

The relationships of actinophages to their hosts in soil must be very com- plex. Adany phages isolated from soil show wide host ranges (3, 9) while others, such as reported by Ichavina and Rautenshtein (4) for S. olivacez~s, may be quite specific in their host range. The appearance of actinophages from soils in such numbers as we have reported must obviously depend on a large num- ber of cells in that soil in a physiological state suitable for multiplication of the viruses. Some of our experiments, for example, have resulted in titers against S. scabies as high as 1.06 X loG PFU per milliliter of perfusate, which is equiv- alent to 4.3 X loG PFU Der grain of soil. If the data of Alexander and i\iIcCov * - (1) reported for S. griseus are considered as typical for other Streptomyces phage systems, an average burst size of 200 particles per lysed cell (germ tube or equivalent) can be postulated. Using this as a basis, therefore, a t least 21,000 host cells per gram of soil must be destroyed to yield these titers. This is prob- ably a minilnuin figure since some phages may adsorb readily to soil. I t may be pertinent here to note that the S phage was observed to be much more rapidly adsorbed to soil than was phage L (7) . This may help to explain the relative paucity of group S phages in soil perfusates.

In many experiinents with both soils the highest numbers of phages were obtained when S. scabies was used as the plating host, followed in diminishing order by S. lavendulae, S. ipomoeae, S. olivacez~s, and S. roseochromogenes. Whether these differences arose because fewer phages can infect these hosts, or because of species differences in efficiency of plating is still unknown. The seasonal differences observed with phages for S. scabies suggest that the num- bers of hosts in the soil vary a t different times of the year. Walrsman (8) pointed out that actinomycetes show the highest counts in the fall. The data bn phage numbers, however, will depend not only on numbers of cells a t the time of soil collection but also on their survival during air-drying, and also on their ability to resume growth when the soil is rewetted.

Most of the phages active on the Streptomyces spp. that we have used belong to the L serological group. Perhaps this could be expected since this phage appears to have a very wide host range. However, i t is possible that ineinbers of the L group, while serologically related, may differ in other characteristics such as morphology and host range. In four different experiments with Guelph loam using S. scabies the percentage of PFU related to L was froin 90 to 94% while with Fox loam the analogous percentages were greater than 99%. Using Fox loam, 99% of the PFU appearing on S. lavendulae were related to L while only 60 to 80% of the PFU on S . olivaceus were related to this phage. Froin 2 to 6% of the total phages active on S. scabies and S. lavendz~lae were found to be related to phage S , which has a narrow host range. Over a period of 24 hours there does not appear to be any appreciable change in the per- centage of the serological types detected by this procedure. By extending the host systems, more inforination of this type might be obtained and should

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CORICE ET AL.: SOIL ACTINOPHAGES 903

contribute to the knowledge of the importance and distribution of phage in soil.

The use of the combined antisera has provided us with a technique for the isolation of phages nornlally obscured by the phage systems already discovered. In our experience, nornlal procedures of isolation of phages for S. scabies and S. lavendzrlae would allnost invariably lead to isolation of serological relatives of L since ill the two soils exarnined so far this serological type icdorninant.

References ALEXANDER, R. R. and McCoy, E. Characterization of Streptomyces grisezrs bacteriophage.

J. Bacteriol. 72, 378-386 (1956). ANDERSON, E. S. The relations of bacteriophages to bacterial ecology. I n Microbial ecology,

7th Svrnp. Soc. Gen. Microbiol. 1957. GILMOUR, C. hi. and BUTHALA, D. The isolation and study of actinophage from soil.

Bacteriol. Proc. (Soc. Am. Bacteriologists), 50, 17 (1950). I~HAVINA, E. S. and RAUTEXSHTEIX, IA. I. Act . olivacezts actinophages and lysogenesis

arnong cu l t~~res of this species. Microbiology USSR Engl. 'Fransl. 27, 433-441 (1958). ~(LECZI~OWSI<A, J. A study of phage-resistant mutants of R l ~ i z o b i z l ~ ~ a trifolii . J. Gen. Micro-

biol. 4, 298-310 (1950). NEWUOULD, F. H. S. and GARRARD, E. H. Studies on actinophage for Strepton~yces scabies

CThaxt.) Walcsman and Mer~rici. Can. 1. Botanv. 32. 386-391 (1954). , , . R O U I N ~ O N , J. 'B. and CORI~E, C. T. ~re l imin i ry studies on the distributioi; of actinophages

in soil. Can. J. Microbiol. 5, 479-484 (1959). WAI~SMAN, S. A. Principles of soil microbiology. Williams Sr Wilkins Co., Baltimore. 1927. WELSCH, M. Actinophages dans les ~nilieux naturels. Cornpt. Rend. Soc. Biol. 150, 1496-

1499 (1956).

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