Polyvirulent rhizobiophage from a soybean rhizosphere soil

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Soil Biol. Biochem. Vol. 30, No. 14, pp. 2171±2175, 1998 # 1998 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain S0038-0717(98)00048-0 0038-0717/98 $19.00 + 0.00

SHORT COMMUNICATION POLYVIRULENT RHIZOBIOPHAGE FROM A SOYBEAN RHIZOSPHERE SOIL F. S. ALI,1 T. E. LOYNACHAN,2* A. M. M. HAMMAD1 and Y. AHARCHI2 1

Department of Agricultural Microbiology, Faculty of Agriculture, El-Minia University, El-Minia, Egypt and 2Department of Agronomy, Iowa State University, Ames, IA 50011, U.S.A. (Accepted 21 January 1998)

Bacteriophages having broad host ranges may be important in horizontal gene transfer and their occurrence in natural environments needs to be better understood. Ninety rhizobiophages, collected from the rhizosphere of ®eld-grown soybean (Glycine max L. Merr.), were isolated against three fast-growing strains of Sinorhizobium fredii, three stock strains of Bradyrhizobium japonicum (USDA 110, 123 and 135) and six ®eld strains of Iowa B. japonicum belonging to serocluster 123. Although S. fredii was not present in the soil, phages virulent to S. fredii were isolated. Based on host ranges when challenged by 24 strains of Rhizobium, Bradyrhizobium and Sinorhizobium, the 90 phage isolates were classi®ed into 32 phage groups. All but one of the isolated phages were polyvirulent. Bacteriophages are frequently reported to be rather narrow in their host range (Coetzee, 1987), but exceptions have been noted, and phage polyvalency appears to be fairly common in Enterobacteriaceae (Bradley, 1967). Werquin et al. (1988) studied 33 S. meliloti rhizobiophages, some isolated from ®eld soils and some from laboratory cultures. The soil phages had a broad host range, whereas phages isolated from bacterial cultures in the laboratory showed a narrow host range. This suggests that phage particles repeatedly cultured on a speci®c host may lose their ability to infect more diverse hosts. Brom®eld et al. (1986) found variation in rhizobiophage sensitivity among strains of S. meliloti in the nodules of Medicago sativa at two ®eld sites. They analyzed 1920 nodules at each site and found 65 and 55 di€erent rhizobiophage types present. Thurman and Brom®eld (1988) also showed great diversity among the indigenous S. meliloti popu*Author for correspondence.

lations among and within the legume species of Medicago when a sensitivity pattern was developed from typed nodules obtained from cross-inoculation groups. To study rhizobiophage polyvalency in a soil environment, we isolated phages against Rhizobium, Bradyrhizobium and Sinorhizobium from the rhizosphere of soybean growing in a Webster (coarseloamy, mixed, mesic Aquic Hapldolls; organic matter 52 g kgÿ1; pH 5.90) clay loam soil when plants were at the early reproductive (R1) growth stage. The soil associated with ®ve randomly selected plants, chosen from a single row at 3 m intervals, was sampled using the procedure described by Hicks and Loynachan (1989) and combined into a composite sample. Of the 12 root-nodule bacteria used for initial phage isolation (Table 1), three were fast-growing S. fredii strains (HH 003, HH 102 and HH 103 described by Manjanatha et al., 1992) and nine were B. japonicum strains infective on soybean. Three stock strains (USDA 110, 123 and 135) were obtained from Dr. van Berkum, Curator, National Rhizobium Culture Collection, Soybean and Alfalfa Research Laboratory, Agricultural Research Center, Beltsville, MD, and six ®eld strains were Iowa isolates of B. japonicum serocluster 123 (G1-6, G3-2, H1-5, H2-2, S1-1 and S2-10) described by Berg and Loynachan (1985). For phage isolation, rhizosphere soil (5 g) was incubated overnight with 10 ml of YMA (yeast extract mannitol ``medium 79'' (Allen, 1959)) broth at 30±338C. One ml of chloroform was added, and the sample was shaken for 10 min followed by centrifugation at 2,000 g to remove soil and bacteria. An aliquot of the supernatant was added to 5 ml of liquid culture (age 16± 18 h) of each strain used for isolation. After multi-

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Table 1. The Sinorhizobium and Bradyrhizobium strains used for phage enrichment and the number of phages isolated on each strain Host strain HH 003 HH 102 HH 103 USDA 110 USDA 123 USDA 135 G 1±6 G 3±2 H 1±1 H 2±2 S 1±1 S 2±10

No. isolated phages* Sinorhizobium

Bradyrhizobium

5 18 5 7 10 12 7 3 7 6 3 7

*A total of 90 phages were isolated, each having di€erent appearing plaque morphologies with the indicated host strains.

plication of phages (16±20 h at 30±338C), bacteria were killed by shaking with 3 ml of chloroform for 10 min. Samples were clari®ed by centrifugation at 11,000 g for 30 min. Phage detection was by the double-layer technique (Adams, 1959). After dilution and incubation, clear-zone contents were picked and transferred separately into Eppendorf tubes containing 1 ml of SM medium (Maniatis et al., 1982). Single plaque inoculum from clear or turbid zones was diluted 10ÿ4 to 10ÿ6 to separate viral particles and plated again on double-layer medium. This procedure was repeated once more for virus puri®cation. Single plaques with di€erent morphological characteristics were picked at random by using sterile Pasteur pipettes and transferred into Eppendorf tubes containing 500 ml of SM medium and 200 ml of chloroform and stored at 48C. To con®rm culture purity and evaluate phage morphology, each isolate was examined by electron microscopy as described by Hammad (1993). Infectivity was evaluated against diverse rhizobial isolates obtained from Dr. van Berkum and homologous with several legumes: Glycine max L. (USDA 138, 191 and 201), Medicago sativa (USDA 1011 and 1021), Trifolium pratense (USDA 2046), Trifolium repens (USDA 2063), Phaseolus vulgaris (USDA 2669 and 2674), Crotalaria paulina (USDA 3384), Macrolyloma africanum (USDA 3451) and Vigna ungiuculata (USDA 3456). The soil at the study site had been maintained in a corn±soybean rotation for at least 20 y before sampling. No record of recent inoculation exists. When soybean was ®rst introduced to the site, however, the soil presumably was inoculated since soybean rhizobia were not native to Iowa. Earlier studies (Manjanatha et al., 1992) indicated the soil contained only slow-growing bradyrhizobia. From the diverse group of rhizobia tested, 32 phage

groups were identi®ed (Table 2). Several of the USDA strains used in the screening were not homologous with soybean. All but one of the detected phage groups were polyvirulent. Only phage group ùR 23 lysed a single bacterial host G1-6, which was an Iowa B. japonicum isolate. No phage group lysed USDA 1011, 3384 or 3451, and only one phage group each lysed USDA 2046 and 3456. Conversely, a Phaseolus rhizobia (USDA 2674) was lysed by 11 phage groups, and two soybean rhizobia (USDA 138 and 201) were lysed by 10 phage groups. Phages that lysed fast-growing sinorhizobia were readily isolated, and phages were readily isolated against other members of serogroup 123 not present in this soil. The resistance of the three USDA strains (1011, 3384 and 3451) to the isolated phages may be due to the presence of a lysogenic relationship between these strains and the isolated phages; i.e., some of the hosts may be lysogenized by particular phages and hence resistant to superinfection (Schwinghamer and Reinhardt, 1963; Abebe et al., 1992). With the polyvirulent nature of the isolated phages, the same phage (i.e., belonging to the same phage group) may have been isolated using di€erent hosts. Individual plaques in our study varied from 1 to 4 mm dia and in appearance from turbid to clear. Results from electron microscopy indicated that phages of each phage group had similar size and morphology (all 90 were viewed). A representative micrograph for phages of each group is presented in Fig. 1. As shown, the isolated phages all were tailed morphotypes (Bradley, 1967). Phages having host ranges across di€erent genera of root-nodule bacteria may be important in horizontal gene transfer in soil and should be further explored. The popular notion of a high degree of speci®city of phages may be a laboratory artifact caused by repeated culture with a speci®c host. In nature, polyvirulent phages may be more common than previously thought. Does soil contain thousands of di€erent phages awaiting a very speci®c bacterium, or do phages have mechanisms of transferring genetic material to a variety of bacteria, maybe having common or similar receptor sites or by other means? Hewitt (1953) speculated that it is unlikely that a phage exactly matching the host strain will occur with mutant bacteria, a phage particle suciently similar to the required structure may be present to enable the phage to attack a bacterial cell, and, having once gained entry to the cell and multiplied, its progeny may tend to conform more closely to the pattern of the bacterial cells present. As previously cited, Werquin et al. (1988) reported that soil phages have a broader host range than cultured phages. Our study supports the broad host-range concept for soil rhizobiophages.

+ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

HH

ÿ ÿ + + + + + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

102

ÿ ÿ ÿ ÿ ÿ ÿ ÿ + + + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

103

ÿ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + + + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

110 ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + + + + ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

123 ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + + + + ÿ ÿ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

135 ÿ + ÿ ÿ + ÿ ÿ ÿ ÿ + + ÿ + + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + + + ÿ + ÿ ÿ ÿ

138 ÿ ÿ ÿ ÿ ÿ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ ÿ ÿ + ÿ ÿ ÿ + ÿ +

191 ÿ ÿ ÿ + ÿ ÿ + ÿ ÿ + + ÿ ÿ ÿ ÿ + + ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ ÿ + ÿ + +

201 ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

1011 ÿ ÿ ÿ ÿ + ÿ ÿ ÿ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ +

1021a

USDA

ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

2046 ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ ÿ ÿ ÿ

2063 ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ

2669 ÿ + + ÿ ÿ + ÿ ÿ ÿ + ÿ ÿ + ÿ ÿ ÿ + ÿ + + ÿ + ÿ ÿ ÿ + ÿ + ÿ ÿ ÿ ÿ

2674 ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

3384 ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

3451 ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ ÿ ÿ

3456 + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

1±6

G

ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ +

3±2 ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + + + ÿ ÿ ÿ ÿ ÿ

1±1

H

ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + + ÿ ÿ ÿ

2±2

ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ

1±1

S

ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + +

2±10

*Sinorhizobium, Bradyrhizobium and Rhizobium host strains (across top) are identi®ed in the text. A total of 90 bacteriophage isolates were evaluated ®tting into 32 phage groups; `` + '' = lysis, `` ÿ '' = no lysis.

4 3 3 4 2 5 4 1 1 2 3 2 2 3 3 3 4 3 4 3 3 3 2 2 2 3 2 3 3 1 3 4

ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR ùR

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

003

Phage group No. isolates

Table 2. Host range of the isolated bacteriophages against Sinorhizobium, Bradyrhizobium and Rhizobium strains*

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Fig. 1. Electron micrographs of uranyl acetate negatively-stained bacteriophage groups ùR 1 to ùR 32 AcknowledgementsÐThe research study by the senior scientist at Iowa State University was sponsored by a scholarship from the Egyptian Government.

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