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  Vol. 52, No. 4 PPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1986, p. 807-811 0099-2240/86/100807-05 02.00/0 Copyright C 1986, American Society for Microbiology Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization byRhizobium meliloti DE MING LI AND MARTIN ALEXANDER Laboratoryof Soil Microbiology, Departmentof Agronomy, Cornell University, Ithaca, New York 14853 Received 2 December 1985/Accepted 30 June 1986 The additionofstreptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.)for several days, resulted in the enhancedgrowth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers ofnodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. melioti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and KlebsieUa pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression ofnodulation being statistically significant for the first three species. Bradyrhizobium sp. and  Sarcina lutea did not suppress root colonization nor nodulationby R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and  S.lutea were greater than the doubling time ofR. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reducesnodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria. For members of the genus Rhizobium to induce nodulation on their leguminous hosts, thebacteria probably must grow in the rhizosphere aswell as at the site on theroot hair where invasion of the plant begins. In these environments, the rhizobia are probably growing at the expense of organic products excreted by the roots, but these bacteria mustcompete for the organic nutrients withother heterotrophicinhabitants of the rhizosphere. In1941, Nicol and Thornton (9) studied the role of competition among strains of Rhizobium spp.for nodule sites and the impact ofsuch intraspecific competition on nodulation. Since that time, many investigations have dealt with intraspecific competition among strains used for inocu- lation or between inoculum strains and nativerhizobia insoil (1, 3). In therhizosphere, however, the dominant species are not members of the genus Rhizobium,and because the dominant bacteria in that environment are often able to grow rapidly (13), they are probably able to compete effectively with Rhizobium spp. and to keep the Rhizobium population small. The finding that R. trifolii, R. phaseoli, and R. japonicum (syn. Bradyrhizobium japonicum) multiplied in soil when other bacteria weresuppressed but not when other genera were not inhibited suggests that competition forthe supplyof available organic nutrients is important, at leastin soil apart from theroots (10). Nevertheless,such inter- generic competition has scarcely been explored. Hely et al.  6), however, suggested that thenative microbial communi- ties of certain Australian soils prevented the nodulationof Trifolium subterraneumbecause these microorganisms sup- pressed the colonization of the plant roots by R. trifolii. Several investigators have demonstrated that individual iso- lates of bacteria or fungi decrease the nodulationof asepti-cally grown subterranean and white clovers by R. trifolii 2, 5, 11) or the rate and extent ofnodulationof aseptically grown alfalfa by R. meliloti  4). In two of these studies, it was shown that the organisms causing the suppression did not produce toxins against R. trifolii in culture  2, 11), * Corresponding author. indicating that the suppression ofnoduleformation resulted from competition with theroot nodule bacteria; neverthe- less, theinhibition could have resulted from a microbiologi- cally induced change in the physiology of the host. The present study wasdesigned to determine whether competition between Rhizobium spp. and species from other bacterial genera occurs insoil and to establishthe charac- teristics of bacteria that are effective competitors with rhizobia in the rhizosphere. MATTRIALS AND METHODS Samples ofEel silt loam were collected to a depth of 15 cm. The soil was air dried andpassed through a sieve (pore diameter, 2 mm). A K2HPO4 solution was thoroughly mixed with the sieved soil to increase the P concentration to 500 mg/kg of dry soil and to bring the pH to about 7.2. Soil samples (20 g each wereplaced in tubes (diameter, 30 mm), and the soil was brought to a moisture content of 23 (wt/wt). When sterilesoil was needed, 20-g portions in test tubes were adjusted to 23 (wt/wt) moisture and autoclaved for 1 h. Thesamples wereincubated for 1 day at 30°C. The samples were then again autoclaved for 1 h. Portions of the autoclaved samples were plated on nutrient agar, and no colonies appeared after 5 days. Seeds of alfalfa (Medicago sativa var. Iroquois) were surface sterilized by soaking them in a solution of 70 ethanol for 1 min, washing them with sterile distilled water, soaking them for 5 min in 50 sodium hypochlorite, and then washing them again insterile water. The seeds were allowed to germinate in petridishes containing water agar, and the seedlings were transferred to tubes (30 by 180 mm; three seedlings per tube) containing either soil or agar medium.The plants were grown in tubesof soil or agar. Each tube of agar contained 25 ml of modifiedFahraeus medium (15). The agar medium was sterilized by autoclav- ing, inoculated when molten at 45°C with 105 bacterial cells per ml, and allowed to cool into slants, and then the seedlings wereintroduced into the tubes. The tubes contain- ing soil or agarwere placed randomly in a Biotronette Mark 807   onM ar  c h  8  ,2  0 1  8  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   808 LI AND ALEXANDER TABLE 1. Populationsof R. meliloti and other bacteria in the rhizosphereof alfalfa grown in sterile soil after inoculation No. of cells (106) per g of roots and adhering soil after inoculation with: Day after inoculation Bacterial R. meliloti R. meliloti and bacterial mixture mixture aln alone aloneBacterial mixture' R. meliloti 7 117116 90.5 0.3014 148156 1382.80 21170 167145 1.00 a Numbers of thespecified microorganisms in the rhizosphere. III environmental chamber (Lab-Line Instruments, Inc., Melrose Park, Ill.) maintained at 27°C, and theplants were exposed to 14 h of light each day. Five replicate tubes were used for each time interval, each cell density, and each species in studies ofpopulation changes, and three replicates were used in investigations of nodule number. The dry weights of the plants were obtained by shaking the roots and washing them to remove soil particles, retrieving small roots that hadbeen broken,drying the plants at 105°C for 24 h, and then weighing them. To prepare inocula, Arthrobacter citreus 837, Enterobac- ter aerogenes,  Sarcina lutea, Acinetobacter sp., Klebsi- ella pneumoniae, and Pseudomonas marginalis were grown in nutrientbroth. A strain of R. meliloti that was resistant to 1.0 mg of streptomycin(Sigma Chemical Co., St. Louis, Mo.)and 50 ,ug of erythromycin (Sigma) per mland a strain of Bradyrhizobium sp. that nodulates cowpeas weregrown in yeast extract-mannitol  YEM) broth (15). After soil was inoculated, it was brought to 23 (wt/wt) moisture and mixed, and then seedlings were planted in the tubes. When the soil was amended with antibiotics, 10 mg of streptomycin sulfate was mixed with 10 g of nonsterile soil before the inoculumand water were added. To count microorganisms in the rhizosphere, the plants were removed from the soil, shaken gently, and placed inlOO ml of sterile distilled water in a bottle containing glass beads. The bottle was shaken 100times by hand, dilutions were made,and cell numbers were counted by the pour plate method. R. meliloti was distinguished fromBradyrhizobium sp., Arthrobacter citreus, and  S.lutea, because only R. meliloti formed colonies on YEM agar containing 1.0 mg ofstreptomycin and 50 ,ug of erythromycin per ml. R. meliloti colonies on agar were distinguished from thoseof P. marginalis because only the formerproduced pink colonies on YEM agar containing 0.5 congo red (Calbiochem- Behring, La Jolla,Calif.), whereas only the latter produced red colonies on nutrient agar containing 0.1 rose bengal (Calbiochem-Behring). In samples containing R. meliloti and E. aerogenes, Acinetobacter sp., or K. pneumoniae, counts were made on YEM agar plates incubated at 27°C and plates of nutrient agar incubated at 40°C because the latter three species but not R. meliloti grew at 40°C and all four grew at 27°C;the counts on nutrient agar gave the numbers of one of the species in the mixturesof two species, and the numbers ofR. meliloti were obtained by subtracting the counts of the second species from the total counts. If the only bacterium present was R. meliloti, counts were made on YEM agar, and colonies were counted after 3 to 5 days of incubation at 30°C. In samples containing other bacteria, R. meliloti was enumerated on YEM agar supplemented with 1.0 mg of streptomycinand 50 ,ug of erythromycin per ml after incu- bation at 30°C for 5 to 7days. Counts of indigenous soil bacteria were made on YEM agar, and the plates were incubated for 5 days at 30°C. The numbers of bacteria other than R. meliloti were thenobtained by subtracting the counts on YEM agar containing the antibiotics. In samples without R. meliloti,the numbers of bacteria werecounted on YEM agar after incubation for 1 to2 days at 30°C. To obtain a mixture of soil bacteria,dilutions ofa soil suspension were plated on nutrientagar. After 3 days of incubation at 30°C single colonies were picked. Eleven of thesebacteria were grown in nutrient broth for 1 to 2 daysand then mixed together to give final densities of each isolate of ca. 105 cells per ml. (This inoculum is hereinafter referred toas thebacterial mixture.) To show that theindividual isolates did not produce toxins affecting R. meliloti, thebacteria were grown in nutrient broth for 2 days, thecultures were centrifuged at 7,700 x g for 10 min, and equal volumes of the supernatant fluid were mixed with distilled water to prepare YEM agar. Counts made after3 days of R. meliloti grown in YEM broth were the same when YEM agar either with or without the bacterial excretions was used. The doublingtimeof bacteria was measured during the logarithmic growth phase. For this purpose, thebacterial community on roots and adhering agar was counted by using nutrient agar. Formeasuringgrowth ratesin culture, thebacteria were grown at 30°C in 200-ml Erlenmeyer flasks containing 100 ml of nutrientbroth, except that the root nodule bacteria were grown in YEM broth, andchanges in optical density weremeasured at 660 nm. RESULTS Sterile soil was inoculated with a mixtureof soil bacteria, R. meliloti alone, or a mixture of soil bacteria and R. meliloti. The initial number of cells ofeach species used was 4 x 105 cells pergofdry soil. Alfalfa was grown in the soil. The data show that the bacterial mixture markedly sup- pressed the density of R. meliloti (Table 1). The countsof cells in the bacterial mixture were not significantlydifferent statisticallyat7, 14, or21 dayswhether R. meliloti was added or not. On the other hand, the counts of R. meliloti were significantly less (P   0.05) at 7, 14, and 21 days in the presence of the other bacteria than intheir absence. At 21 days, an averageof 27nodules was present per threeplants if the soil receivedonly R. meliloti, but anaverage ofonly 12 nodules was present per threeplants if R. meliloti was inoculated alongwith the other bacteria. This difference was statistically significant (P < 0.01). Determinations of plant dryweights showed no significant differences among the three treatments at 7 and 14 days; however, the dry weights at 21 days were significantly less (P < 0.05) if the inoculum was the bacterial mixture without R. meliloti(8.70 mg per three plants) or was R. meliloti plus the bacterial mixture (7.77 mg) than if it was just R. meliloti (11.1 mg),although the differences between the first two treatments were not statistically significant. Thus, the bacterial mixture inhibited both nodulation and alfalfa growth. Alfalfa was grown for 21 days in nonsterile soil either with 1 mg ofstreptomycin pergof dry soil or without streptomy- cin. The soil was inoculated with 1.1 x 106 R. meliloti cells pergofdry soil, andwater was added to bringthe moisture level to 23 . The seedlings were soaked in a suspension containing 1.1 x 106 cells of R. meliloti per mland then planted in the soil. The changes in population in theseveral treatments are shown in Table 2. The value for total bacteria at 2 days in soil withoutstreptomycin was high and not significantlydifferent from the R. meliloti count in soil with APPL. ENVIRON. MICROBIOL.   onM ar  c h  8  ,2  0 1  8  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   R. MELILOTI NODULATION AND ROOT COLONIZATION 809 streptomycin because the count in one replicate w s  nom alously high and resulted in a largestandard deviation. Inthe soil receiving streptomycin, the population of soil bacteria was significantly smaller than in the untreated soil at 2 and 4 days. However, the size of the bacterial population in the streptomycin-amended soil then increased, and the popula- tion sizes in the streptomycin-amended and unamended soils were statistically indistinguishable at 7, 14, and 21days. The number of R. meliloti cellsin soil treated with streptomycin was significantly greater than that in the unamended soil after all but one time period. It is interesting thatthe marked risein the populationof bacteria after4 days in the presence of streptomycin was not correlated with a change in the size of the populationof R. meliloti. Measurements of the weights of plants grown in strepto- mycin-amended and unamended soils revealed no significant difference at 2,4,7,14, and 21days. These data suggest that the additionofstreptomycin did not influence the growth of alfalfa, although it is not clear how long the streptomycin remained active. On the other hand, theplants grown in soil with and without streptomycin had 29 and 13 nodules, respectively, on 9 plants after 21 days, a difference that is statistically significant (P < 0.01). Experiments were conducted to determine the effects ofseveralbacteria individually on the multiplication of R. meliloti and on the nodulationof alfalfa grown on agar. Each tubewith three plants was inoculatedwith the test bacterium only, R. meliloti102, or both. In experiments in which plant weight was determined,uninoculated plants were also in- cluded. The inoculum was 5.0 x 106, 4.3 x 106, 7.5 x 106, 7.5   106, 15 x 106, 13 x 106, or 7.5 x 106 cells of E. aerogenes, P. marginalis, Acinetobacter sp., K. pneumoniae, Arthrobacter citreus, Bradyrhizobium sp., or  S. lutea, respectively, per tube. In theseexperiments, the R. meliloti inoculum was 20 x 106, 5.0 x 106, 7.5 x 106, 10 x 106, 13 x 106, or 75 x 106 cells per tube, respectively. Except for Arthrobacter citreus, the numbers of the test bacteria at each sampling date were the same in the absence or presence of R. meliloti; i.e., the latter had no statisticallysignificanteffects on six of the seven species of bacteria (Table 3).  S.lutea was apparently partially suppressed at two of the sampling dates. Because of variability in counts among replicates of Bradyrhizobium sp., large differences in num- bers were not statisticallysignificant. However, appreciable and statistically significant effects were exerted by some of the test bacteria on R. meliloti. E. aerogenes, P. marginalis,Acinetobacter sp., K. pneumoniae, and Arthrobacter citreus significantly reduced the R. meliloti populations, except for the first sampling date for fourof the five test bacteria and the secondsampling date for Acinetobacter sp. However, Bradyrhizobium sp. and  S. Iutea had no detrimental effect TABLE 2. Populations of R. meliloti and total bacteria in the rhizosphere of alfalfa grown in nonsterile soil No. of cells (106) per g of roots and adhering soila inoculation Soil withstreptomycin Soil without streptomycin Total bacteria R. meliloti Totalbacteria R. meliloti 2 3.11 B 18.5 A 85.7 A 3.69 B 4 7.52C 20.7B 149A 8.11C 7 130 A 29.3 B 111 A 8.53 C 14 132 A 25.7 B 142 A 11.3 B 21182 A 26.1 B 154 A 8.37 C Values in a row followed by different letters are significantly different  P < 0.05). TABLE 3. Populations ofR. meliloti and test bacteria in the rhizosphere of alfalfa grownon agar No. of cells (106) pergof roots and adheringagarinoculated with': Test bacterium and day after inoculation Test Mixed inoculum bacterium R. meliloti Test bacterium E. aerogenes 3 33.5 A 0.70 B 33.4 A 0.44 B 7 44.7 A 32.8 A 41.7 A 3.51 B 1462.0 A 73.4 A 58.2 A 5.81 B 21 54.7 A 31.5 B 52.4 AB 6.00 C P. marginalis 3 21.7 A 2.91 B 19.9 A 0.26 C 7 23.6 A 21.1 A 22.7 A 8.71 B 14 36.7 A 39.7 A 34.5 A 10.1 B 2140.7 B 52.3 A 40.9 AB 13.6 C Acinetobacter sp. 3 18.9 A 0.20 B 19.2 A 0.14 B 7 28.3 A 0.51 B 23.1 A 0.52 B 1456.7 A 47.9 A 50.8 A 2.90 B 2197.9 A 92.8 A 76.0 A 7.81 B K. pneumoniae 3 3.60 A 0.80 B 4.61 A 0.30 B 7 11.5 A 9.70 A 13.7 A 3.30 B 14 23.7 A 21.7 A 13.8 A 8.71 B 21 48.4 A 24.5 A 24.5 A 6.33 B Arthrobacter citreus 3 0.81 A 0.50 A 0.60 A 0.21 A 7 6.52 A 1.11 B 2.71 B 0.64 C 14 16.3 A 6.04 B 6.71 B 1.43 C 21 23.5 B 33.1 A 18.6 B 8.53 C Bradyrhizobium sp. 3 0.49 C 0.72 B 0.48 C 0.91 A 10 1.81 A 27.0 A 12.2 A 41.0 A 1530.1 A 67.7 A 14.5 A 58.7 A 2347.6 A 81.0 A 17.6 A 66.1 A  S. lutea 3 1.70 A 1.24 A 2.14 A 1.32 A 77.80 A 11.8 A 8.93 A 12.0 A 14 19.2 A 15.1 A 14.1 A 16.3 A 21 10.1 A 29.4 A 9.61 A 31.1 A aValues in a row followed by different letters are significantly different (P < 0.05). on R. meliloti at any sampling date after 3 days. Moreover, the cell density of R. meliloti was higher in the presence of these two species than when the rhizobium cells were grown in association with any of the other five species. Nodulation in the preceding experiment was determinedon the 21-day-old plants, except that plants inoculated with Bradyrhizobium sp. were examined after 23 days. Because the experiments were conducted at different times and presumably under slightly different conditions, the numbers of nodules on plants inoculated with only R. meliloti varied in different experiments. Plantsinoculated with test bacteriaalone bore no nodules. Some of the test bacteria reduced nodulation, and others had no effect (Table 4). Thus, E. aerogenes, P. marginalis, and Acinetobacter sp. each re- duced nodulation by R. meliloti, the effect being statistically significant. K. pneumoniae appeared to have a smaller VOL. 52, 1986   onM ar  c h  8  ,2  0 1  8  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   810LI ANDALEXANDER TABLE 4. Number ofnodules on alfalfa inoculated with R. meliloti and several test bacteria and growth rates of bacteria in axenic culture on surfaces of alfalfa roots grown on agar Nodule no. on nineplantsinoculated with:Ratio GenerationTest species R. meliloti R. meliloti and of B/A time (h) test bacterium only (A) (B) E. aerogenes 29 15 0.524.05 P.marginalis152 92a0.614.13 Acinetobacter sp. 47 30b 0.644.40 K. pneumoniae 52 41C 0.79 6.34 Bradyrhizobium sp. 36 38c 1.0613.6  S.lutea 52 56C 1.0810.6 R. meliloti NAd NA NA 8.38 a Significantly different (P < 0.01) from number on plants inoculated withR. melilotialone. b Significantly different (P < 0.05). c Not significantly different. d NA, Not applicable. effect, although it was not statisticallysignificant. No influ- ence was exerted byBradyrhizobium sp. and  S. lutea. The doubling times of E. aerogenes, P. marginalis, Aci- netobacter sp., K. pneumoniae, Arthrobacter citreus,  S. lutea, Bradyrhizobium sp., and R. meliloti in nutrient broth were 4.42, 4.47,5.24, 6.19,7.30, 12.6, 14.0, and 7.72 h, respectively. Growth rates were also determined in the rhizosphere of alfalfa grown on agar. At the times that the bacteria were growing logarithmically, the seedlings were 3 to 10 days old. A logarithmic rate of increase was noted when the cell densities wereabout 106 to 107 cells per g of agar. The doublingtimes of the bacteria varied from 4.05 to 13.6 h (Table 4). E. aerogenes, P. marginalis, and Acineto- bacter sp. grew most readily in the rhizosphere, and  S. lutea andBradyrhizobium sp. grew most slowly. R. meliloti grew at a rate between the two extremes. The growth rates of individual bacteria were then com-paredwith the effects of the bacteria on nodulation. For this purpose, the data are presented as nodulation ratios, the nodulation ratio being the ratio of the number ofnoduleson plants inoculated withboth R. meliloti and the test bacteria to the number ofnodules on alfalfa inoculated withonly R. meliloti. The two species that grew more slowly than R. meliloti ( S. lutea andBradyrhizobium sp.) had little or no effect on nodulation (Table 4). Incontrast, four of the five species of bacteria that grew more quicklythanR. meliloti suppressed nodulation. The sole anomalywas Arthrobacter citreus, which had a nodulation ratio of 0.10 and a doubling timeof 8.36 h (data not shown). Testsof Arthrobacter citreus revealed that culture filtrates made after its axenic growthhad no effect on R. trifolii. However, because more than half of theroots had turned brown 8 days after transplanting the seedlings to agarinoculatedwith Arthrobacter citreus, the anomalous nodulation ratio may have resulted from root injury. Ignoring the value for Arthrobacter citreus, the suppressionofnodulation was directlyrelated to growth rates of the bacteria that had shorter generation timesthanR. trifolii. DISCUSSION Bacteria other than rhizobia and bradyrhizobia increase in numbers more markedlyand attain greater densities in the rhizosphere of legumes than do theroot nodule bacteria (7, 8). This suggests thatthe former are good competitors withthe latter and preventthe rhizobia from obtaining a large part of the excreted carbon to support replication. Abundant evidence existsfor competition among strains of Rhizobium spp. (9, 12, 14), but these bacteria grow slowly and do notaccount for the large increase in the size of the bacterial community asroots emerge from the seed and extendthrough the soil. An effect of bacteria otherthan Rhizobium spp. on the nodulationof legumes has been demonstrated by a number of investigators. Thus, Anderson (2) reported in 1957 that the addition of several species of bacteria onto theroots of white clover grown on agar reduced the number of nodules andsometimes prevented nodulation by R. trifolii. More recent studies confirmed these observations, the later investiga-tions involving suppression by Azospirillum spp. ofnodula- tion ofwhite and subterranean clovers by R. trifolii (11) and inhibition by Erwinia herbicola of the nodulationof alfalfa by R. meliloti (4). In several instances, thebacteria suppressing nodulation on these plants did not produce toxins activeagainstthe rhizobia (2, 11), so the effect was one associated either with competition or a physiological change induced in the plant. Fungi may also decrease nodulation (2,5), but they may not be as important as bacteria because of their lowerfrequency in the rhizosphere. The present study shows that growth rates greatly affected the outcome of competition between R. meliloti and other bacteria. Bacteria with slow growth rates didnot signifi- cantly affect R. meliloti, whereas those that multiplied rapidly were good competitors. A relationship was also noted between the growth rate of bacteria and the number of nodules formed on alfalfa by R. meliloti. Bacteria that grew faster thanR. meliloti reduced the number of nodules, and those that grewmore slowly hadno such effect. The results from the tests with sterile and nonsterile soil further support the view of the importance of competition. Thus, a mixture of soil bacteria significantly reduced the density ofR. trifolii when added to sterilesoil. Similarly, suppressing bacteria in nonsterile soil for at least 4 days by the addition of streptomycinallowed a streptomycin- resistant strain of R. melilotito reach higher densities than it would have attained in the absence of the antibiotic, and the amendment alsoledto an increase in the number of nodules. Although the antibacterial effect of streptomycin had dissi- pated after 4 days possibly because of destruction of the antibiotic orthe growth of resistant bacteria, nodulation was still promoted. If competition is important under field conditions, it may be alleviated to some extent by usingrapidly growing strains of Rhizobium spp. as inoculants. Competition may also be alleviated by use of a mixedinoculum containing bacteria that produce antibacterialantibiotics and Rhizobium spp. that areresistant to the antibiotics or by additions of inhibitors to which the inoculum strain, but no other rhizosphere bacteria, is resistant. LITERATURE CITED 1. Amarger, N. 1981. Competition for nodule formation between effective and ineffectivestrains of Rhizobium meliloti. Soil Biol. Biochem. 13:475-480. 2. Anderson, K. J. 1957. The effect of soil micro-organismson the plant-rhizobia association. Phyton (Rev. Int. Bot. Exp.) 8:59-73. 3. Barnet, Y. M. 1980. The effect of rhizobiophages on the popu- lation of Rhizobium trifolii in theroot zone of clover plants. Can. J. Microbiol. 26:572-576. 4. Handelsman, J., and W. J. Brill. 1985. Erwinia herbicola iso- APPL. ENVIRON.MICROBIOL.   onM ar  c h  8  ,2  0 1  8  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om 
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