Selective infection of maize roots by streptomycin-resistant Azospirillum lipoferum and other bacteria
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J O H A N ND AO B E R E I NAENRD VERALUCIADIVANB A L D A N I Cotrscl/~oNnt~iorrcrlde D r s r ~ i c o l ~ ~ i t r z cCir~rt;jit.o ~t~to r T~c~rlologico, Gripreso Brcrsilrrn cle Prscl~riscrAgroprcrrc~rin,Kt1147, Srropc;dic.o 23460, Rio tlr J a t ~ r i r oBrozil , Accepted July 20. 1979
D ~ B E R ~ I NJ .E, R and . V . L. D. B A L D A N I1979. . Selective infection of maize roots by streptomycin-resistant A;ospiri//rr~tilipofe~rrrrrrand otherbacteria. Can. J. Microbiol. 25: 1264- 1269. The percentage of low-level streptomycin-resistz~nt(20 pg/mL) bacteria in surface-5te1-ilizedo r washed maize roots was more than a thousand times higher than that in soil populations. There was also a higher incidence of resistant bacteria in rhizosphereas compared with non-rhizosphere soil and bacteria isolated from maize roots were relatively tolerant to several other antibiotics. A io.~prt~llrrtri - . ..' lipofirlrt~rwas predominant in sur-face-sterilized roots of field-grown maize a n d was low-level streptomycin-resistant while most soil isolates were sensitive. Inoculation with A . hro.siletr.sc, isolated from wheat roots was unsuccessful in terms of establishment even when streptomycin-resistant strains were used. Unidentified causes of specific plant-bacteria affinities therefore transcend the role of antibiotic resistance in maize root infection. D O B E R E I N EJ.. R ,et V. L. D. B A L D A N I1979. . Selective infection of maize roots by streptomycinresistant Azosl~irillrrtr~ lipofc,r~rtrrand other bacteria. Can. J. Microbiol. 25: 1264-1269. Le pourcentage de b;rcteries resistantes i de faibles niveeux de streptomycine (20 pg/mL) chez les racines de miis lavees ou sterilisees en s u ~ f a c eest plus que mille fois plus eleve que celui d e s p ~ u l a t i o n sdu sol. L'incidence des bacteries resistantes est egalement plus eleveedans un sol B rh~zospherescompare i un sol sans rhizosphtres. e t les bacteries isolees des racines de mais sont relativement tolel.antes plusieurs autres antibiotiques. Azospirillrr~nlipofirrrt~rpredomine chez les racines provenant de champs de miis et sterilisees en surface et il est resistant i~ de faibles niveaux de streptomycine, tandis que la plupart des isolats du sol sont senibles. L'inoculation avec A . brcr.silc~tr.scisole de racines de ble ne [-Cussit pas en termes d'etablissement meme si d e s souches resist antes sont employees. Des causes non identifiees d'affinites specifiques entre plantes de bacteries transcendent donc le rble de ~ e s i s t a n c eaux antibiotiques dans I'infection des racines de mai's. [Traduit par le journal]
Introduction croprotation with cel-eals has been recommended as a biological control for certain bacterial diseases (Robbs 1960) and the of actinomycetes in the rhizosphere is well known (Katznelson 1965; Krasil'nikov 1958). Nevertheless the on]y report on enl-ichment of streptomycin-resistant microorganisms in the rhizoSphere is that of B~~~~ (19611, which shows large increases i n the proportion of resistant bacteria on the r-oot of several legumes, but less pronounced effects in cereals. Streptomycin resistance has been used as a marker in many Rhizohiut?l inoculation experiments, but no selective advantage has been I-eported under field conditions (Careth-Jones and Bromfield 1978). nitrogen fixation i n maize (Biilow and Dobereiner 1975) and host-plant specificity in the infection of maize, rice, and wheat by Azospirillu~n spp. has been demonstrated (Ba]dani and Diibereiner 1979) where maize was selected for Azospirill~rn~ lipofelurn and wheat and rice for the A . bmsilense nir- group. TO confirm the specific
infection, Azospirillutn strains marked with lowlevel streptomycin resistance were used as ~ O C ulants in a field experiment with maize and wheat. In both species, in the noninoculated control plots and in the plots inoculated with heterologous strains, high proportions of homologous streptomycinresistant Azospirillurn spp. were obtained from surface-sterilized roots but not from rhizosphere soil. These observations indicated selective infection of roots by ~ t r e p t ~ m y c i n - r e s i ~ t astrains. nt In the present paper this remarkable characteristic is c o n f i ~ - ~ ~ - ~ e d fmaize o r t h e plant. Materials and Methods Fic~ldE.rpc~t.i~tret~t~ F o r counts of total numbers of streptomycin-resistant bacteria, maize cv. M-102 (Agroceres). planted in podzolic soil (field A) o r in gray hydromorphic soil (field B) fertilized with 20 kg N . 50 kg P. I20 kg K. per hectare, was used at the flowering stage. For the evaluation of streptomycin-resistant A Z O S pirillrrtri spp.. soil and plant samples were taken at various growth stages from plots of an experiment described before (Baldani and Diibereiner 1979). In this field experiment maize cv. Pirango was planted in podzolic soil fertilized with 30 kg N . 30 kg p. 100 kg K , and 40 kg FTE (fritted trace elements) per hectare. Plots were 6 m x 6 m in size and had three uninoculated
0008-4 16617911 1 1264-06$0 1 .OO/O @ 1979 National Research Council of CanadaIConseil national d e recher-ches du Canada
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DOBEREINER A N D BALDANl
bordel- lines between inoculation treatments. Inoculation with A . lipofi,rritt~ strr (resistant to 20 pg/mL of streptomycin) or A . h~.rr.siletl.sestrr nir- (spontaneously nitrite reductase negative strain) (Baldani and Diibel-einer 1979) was pelformed a t sowing with 150 mL/m2 of liquid cultures containing 101° cells per millitre. T h e most probable number (MPN) of Azospirillritr~ spp. before inoculation was 3.5 x lo7 per gram of soil with a frequency of streptomycin- (20 pg/mL) resistant mutants of 10F. T w o plants were harvested from each plot, 45.60, 75, and 95 days after planting. The experimental design was in [randomized complete blocks, with four replicates. CriI~rit~t, M~(/itr
The media used in this study were as follows. (i) Modified nitrogen-free semisolid malate medium (NFb): 5 g malic acid: 0.5 g K2HP0,; 0.2 g M g S O , 7H,O; 0. I g NaCI; 0.02 g CaCI,; 2 m L trace element solution; 2 m L alcoholic solution of bromothymol blue (5%); 4 mL Fe EDTA (1.64%); I mL vitamin solution; 4 g KOH; 1.75 g agar; 1000 mL H,O; pH adjusted to 6.8. The trace element solution was 0.2g N a , M o 0 , , 2 H 2 0 ; 0.235 g MnSO,. H,O; 0.28 g H,BO,; 0.008 g CuSO,. 5H,O; 0.024 g ZNSO,. 7H,O; 200 m L H Z O .The vitamin solution was 10 mg biotin; 20 mgpyridoxin; I00 mL H,O. (ii) Potato infusion agar (BMS): 200 g potatoes; 2.3 g malic acid; 2.0 g KOH: 2.5 g raw cane sugar: I mL vitamin solution (see above). Washed potatoes were cooked for 30 min and then filtered through cotton. Malic acid (2.5 g) was dissolved in 50 m L H,O, adding 2 drops of bl-omothymol blue (0.5% solution in ethanol). and adjusted with KOH until green (pH 7.0). This solution together with sucrose. agar, and vitamins wereadded to the potato filtrate and made to 1000 mL.
TABLE1. Enrichnient of streptomycin-resistant bacteria in field-grown maize roots
Sample Rhizosphere soil Rhizosphere soil treated with Distreptine 7 and 2 days before'samplingt Washed roots Surface-sterilized roots$
Total no. of bacteria per gram dry wt. x 10'
resistant t o streptomycin*
39 5 27
1.7k1.7
+
57 29 284 5 274 344 262
+
%
2.6k0.2 43 _+ 27 4356
'Percentage from colony numbers o n BMS a g a r plates with and without 20 p g / m L streptomycin. Values are means from three samples with s t a n d a r d deviations o f means. tcomrnercially available streptomycin product applied in 4000 p p m solution. $Treated for 30 min in 1Z chloramine T.
incubation at 32°C and negative cultures were discarded. Further enrichment was obtained in 24-h-old cultures in N F b medium which were streaked out on NFb agar plates containing 20 ppm yeast extl.act. After I week, typical small, white, dense, single colonies were picked and transferred into semisolid N F b medium. Pellicle formation in this medium indicated success of isolation. For final purification, these cultures were streaked o u t on potato agar (BMS) and the typical pink, often wrinkled colonies transferred for storage and identification tests. F o r this. and loop of semisolid MFb cultures was inoculated into N F b Corrt11.s of 7bltrl Nrrttrhers rrtltl of S / r e p ~ o ~ t ~ c . i t ~ - r ~ ~Bnc. v i . s ~ vials t ~ t ~ ~containing 20pg/mL of streptomycin. and into N F b medium with glucose 21s sole carbon source. Azospirillritt~ leritr Maize plants wereexcavated with most of the root system and lipc?fi,rlr~tlwas differentiated from A . hrrrsiletrse by itsgrowth o n taken to the laboratory. T h e soil adhering to the roots, after glucose (Tarrand rt trl. 1978). The same purified single-colony shaking off the loose soil was considered rhizosphere soil. Con- isolates were used for identification of species, and streptomycin resistance and growth are valuated in parallel tests. Theretrol soil was collected between rows. The roots were washed and (or) sudace-sterilized and 10 g of fresh weight proportions fore, selection of resistant mutants in the streptomycin vials is was homogenized in 100 m L of a 4% cane suga!. solution in a unlikely. blender. Roots were sulface-sterilized in I% aqueous solution of chloramine T . The cut ends were kept outside the disinfectant At~ribiolicTP.YI.S~ i . i l h"Setlsi Di.vcs" Distinct colonies of bacteria on BMS plates with the highest and removed afterwards. Washings were carried out for the or cultures of Azospirilhit?~were used to inocusame time interval as that of immersion in the disinfectant (30 or dilutions 60 min). There was one washing with 0.05 M phosphate buffer late to semisolid BMS medium, and after 48 h of incubation, solution and four washings with sterile water. All dilutions ex- 0.1 m L of the cultures was plated. without dilution, into BMS cept those of the experiment presented in Table 1 , where water agar. T h e "Sensi-Discs" (Becton. Dickinson & Co) of various was used, were performed in 4%' cane sugar solutions to avoid antibiotics were placed o n the plates and the size of the clear osmotic shock a s reported for Rllizohilitn bacteroids (Gresshoff halo starting from the margin of the discs measured after48 h. I n some cases the halo was not clear. Then the halo reading was el (11. 1977). Preliminary experiments indicated higher recovery. especially of streptomycin-resistant bacteria, when the water reduced according to visual estimates of t h e propoltion of was r e ~ l a c e dbv the sugar solution. The soil or root dilutions of growth.
lo-s and lo-', and in some cases also lo-'. were plated in potato agar (MBS) containing 60 ppm of actidione sterilized separately by filtration. Equal amounts of the same dilutions were plated into potato agar containing 20 pglmL streptomycin sterilized by filtration and added to the medium cooled to 48°C. iust before pourlng the plates The percentage of streptomycin-res~stant bacter~awas calculated from the d~fferenceIn numbers of colonles on plates with and without streptomycin. IsoItrtiotl or1~1 I~it~t~tiJjc~rtiotl of Azospirillli~?~ .~pp.
Roots were hal-vested. washed, and (or) surface-sterilized as described for bacterial counts. T h e roots were then cut into 5- to 8-mm pieces which were macerated with forceps and introduced into NFb medium (4 m~ in 6 m L serum vials). Nitrogenase activity (C2HI reduction) was checked after 40 h of
Results The applications of high levels of streptomycin ( I 0 times recommended for treatment of bacterial diseases of tomatoes) to rhizosvhere soil seemed to have little effect (Table I), probably because it is rapidly decomposed or adsorbed to the clav (Pramer 1958). Bacteria in washed and surfack-sterilized roots were more numerous and contained higher proportions streptomycinresistant forms (Table 1). Watanabe and Barraquio (1979) reported that 80% of root isolates of rice can fix low amounts of N, when supplied with low
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C A N . J. MICROBIOL. VOL. 25. 1979
levels of combined nitrogen. To see whether our isolates from su~face-sterilizedmaize roots were similar, we transferred 60 colonies from the above-mentioned pour plates. with and without streptomycin. into the medium used by the authors above. All colonies transferred into this medium grew. but none reduced C I H 2 ,not even after incucolbation for 24 h. There were no Azo.\pi~.illlr~?~ onies on the BMS plates. either with or without streptomycin. Numbers of this organism have been found to be about two orders of magnitude lower than total numbers of bacteria in soils or roots (Magalhaes et o l . 1979) and would therefore not become identifiable on dilution plates. It seems therefore that there were no N1-fixing organisms among the streptomycin-resistant bacteria contained in Table 1. Samples from two additional fields confirmed the selection of maize roots for low-level streptomycin-resistant bacteria (Table 2). As in the first experiment. variation between samples was large (see standard deviations) while the percentage of stl-eptomycin-resistant bacteria was more constant. In field A the frequency of spontaneous streptomycin-resistant bacteria was about lo-' which is in accordance with the occul-rence of st~.eptomycin-I-esistantAzo.\pi~.ill~rt?~ in a similar field and which was 10-% In soil B. which contains more organic matter, quite a different microflora should have existed and bacterial numbers in surface-sterilized roots were small compared with those in soil A. Nevertheless the striking difference in the PI-opol-tionof streptomycin-resistant bacteria was again observed between soil and roots. T o investigate resistance to othel- antibiotics. I-andomly selected bacteria from the highest dilution plates of the two former experiments were tested. The results from fields A and B are summarized in Table 3. There is general tendency towards higher incidence of resistant bacteria in sterilized roots, but differences between soil and sterilized roots were most pronounced in the I-esistance to streptomycin. The resistance to othel- antibiotics varied with soil. The data in Table 4 confirm the higher incidence and also higher mean resistance to antibiotics. especially stl-eptomycin, of bacteria isolated from within roots as compared with those from non-sterilized roots. Resistance to antibiotics and possibly associated characters therefore seem to be of advantage for multiplication in the rhizosphere, and for root infection. It may be seen from Fig. 1 that Azo.spit.ilIrrt7z infection of maize I-oots seems also to be correlated with streptomycin resistance. In the uninoculated control plots during the whole growth
TABLE2. Enrichment of streptomycin-resistant bacteria in the rhizosphere and roots of maize grown in two fields Total no. of bacteria per gram dry wt, x l o 7
% resistant t o streptomycin*
Field A (red yellow podzolic) Soil between rows Rhizosphere soil Surface-sterilized rootst
45k 36 138k87 554k 246
0.0026k0.0014 0.53k0.28 88.4k5.0
Field B (grey hydromorphic) Soil between rows Rhizosphere soil Surface-sterilized roots:
83 k 23 148k 80 3.4k0.367
Sample
0.12_+0.10 1.01+0.28 111.0k 20.1
*Percentage calculated from colony numbers on BMS agar plates with a n d without ?O k~gimLstreptomycin. Values are means of three samples with standard devlatlons of (he means. tThis value is the mean of three plates from one sample and the other two samples contained fewer than 10' bacteria (no colonies on the plates inoculated a t a dilution of' 10-5). :Sterilized fbr 30 min in 1 z chloramineT.
cycle, the majority of strains isolated from I-h. sulface-sterilized roots were low-level streptomycin-resistant. In the plots inoculated with A . lipofet.lit~lstrr. most of the soil and root strains were resistant indicating competitive advantages of the inoculated resistant strains in the rhizosphere soil. These effects. however, were not equally pronounced at different sites and growth stages of the plant. In a pot experiment. 12% of the isolates from young plants. 62% from plants at the flowering stage, and none from older plants were resistant to 20 pg/mL streptomycin (data not shown here). The general spectrum of antibiotic resistance in Azo.spit.illlrt?l spp. is shown in Table 5. In both species low-level streptomycir, resistance was accompanied by a higher tolerance to chlorarnphenicol. None of the three other tested antibiotics showed any connection with streptomycin I-esistance. The antibiotics used in this test had been selected in a preliminary test with 13 other A z o .spit~illrnnstrains and 14 antibiotics ("Sensi-Disc" tests). In these tests all strains were resistant against penicillin-G (10 units), cephaloridine (30 pg), methicillin ( 5 pg), ampicillin (I0 pg), lincomycin (2 pg), coly-mycin (10 pg), oxacillin ( I pg), vancomycin (30 pg), mycostatin (100 units) (L. Vasconcellos, A. Drozdovicz, and J . Dobereiner, unpublished results), whereas most strains were sensitive to the antibiotics mentioned in Table 5. Discussion The results presented in this paper, together with those reporting host-plant specificity for A z o s pi~.illutn spp. infection in cereals (Baldani and Dobereiner 1979), throw interesting new light on
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I I O B E K E I N E R A N D BAL.IIAN1
TABLE 3. Resistance of soil, rhizosphere, and root isolates" to various antibiotics Soil
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Antibiotic
Rhizosphere
% Inhibition resistant.1 zone,: mnl
Field A (Red yellow podzolic) Streptomycin (10 pg) Tetracyclin (30 pg) Chloramphenicol (30 pg) Gentamycin (10 pg) Erythramycin (15 pg)
0 0 0 25 0
8.7k5.5 11.0k1.7 11.5k3.3 5.2k4.5 7.223.2
Field B (gray hydromorphic) Streptomycin (10 pg) Tetracyclin (30 pg) Chloramphenicol (30 pg) Gentamycin (10 pg) Erythramycin (15 pg)
25 0 0 50 0
5.5k4.4 7.2k2.2 8.Ok 1 . 4 5.7k4.3 8.7k4.3
% resistant
Surface-sterilized roots"
Inhibition zone, mm
% resistant
Inhibition zone, mnl
'Four to S I X apparently d~fferentcolonles from the h~ghcstd ~ l u t l o nplates (experiment presented In Table 1) of e a c h treatment xcre tested agalnst thc barlous a n t ~ b l o u c sw ~ t h ' S e n s ~DISCS" Values are means o f four to S I X ~solntesw ~ t hstandard de\latlons o f the means tlsolates whtcli produced a n i n h ~ b l t ~ ozone n smaller than 3 mm were considered rcslstant t l . n h ~ b ~ t l ozone n IS the distance, In m1ll1metrcs, o f the halo lrom the rnargln o f the d ~ s c llSterlllzed for 30 mln In I:;;, c h l o r a m ~ n eT
T A ~ L4.EResistance to various antibiotics of bacteria* isolated from wsshed and from surface-sterilized roots Washed roots
%
Surface-sterilized roots''
%
Antibiotic
resistant?
Inhibition zone.$ mm
resistant-t
Inhibition zone.!: nlnl
Streptonlycin (10 pg) Tetracyclin (30 pg) Chloramphenicol (30 pg) Gentan~ycin(10 11g) Erythamycin (15 pg)
12 0 12 0 37
5.6k3.3 11.0f 6 . 0 7.7k6.4 4.6k2.2 3.5k3.0
78 44 33 55 44
0 . 7 k 1.4 6.2k5.2 7.4k6.1 2.8k3.3 4.2k3.7
-
-
---
*Eight apparently different colonies from the highest dilution plates (cxperimcnt presented in Table I) were tested against thc various antibiotics by the Sensi-Disc test. Values represent means o f eight isolates with standard deviations of the means. tlsolarcs which produced an inhibition zone smaller than 3 m m were considered resistant. :Inhibition zone is the distance, in millimetres, of the halo from the margin o f the disc. ISterilized for 30 min in IZ, chloramine T .
grass-Azo~pirill~rrllassociations. Streptomycin resistance seems to confer advantages in the infection of roots. but host-~lants~ecificitvmust have other reasons independent of antibiotic resistance. Baldani and Dobereiner (1979) have demonstrated that maize was infected predominantly by A. lipofircrm and the recovery of A. lipofifirrn strrfi-om sur-facesterilized roots in all plots, even those inoculated with A. brasilensc nir- strr, is shown in Fig. 1. The introduction of A. bt.risilet7se, therefore, even if it was made streptomycin-resistant, did not help to establish this organism in the roots although the large majority of the rhizosphere isolates were from the inoculated strain. This confirms host-plant specificity as an important factor- controlling maize root infection by Azospir.ill~rmspp. A field kxperiment with wheat gave similar results except the
instead of A. wheat selected for A. brc~.~iletzsc~ lip(?fc)t.~rtn(Baldani and Dobereiner 1979). The enrichment of antibiotic-resistant. e s ~ e c i ally streptomycin-resistant, bacteria in the rhizosphere is not unknown. Brown (1961) comparing root-surface soil with control soil without plants found similarly striking rhizosphere effects on the proportion of streptomycin-resistant bacteria in several legumes, wheat, and vegetables. From her data it can be calculated that 7 and 5% of the bacteria from control pots and 12 and 48% of the rootsurface bacteria of pea plants were resistant to str-eptomycin in two experiments, respectively. Bacteria which have no specific growth requirements (basal medium) contained 0.05% resistant forms in control soil and 2% in root-surface soil. Amino acid requiring bacteria contained about 2%
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C A N . J . MICROBIOL. VOL. 25. 1979
TABLE5. Resistance t o various antibiotics of Azospirillli~r~spp. Reaction to 20 pg/mL streptomycin
N o . of strains
Cm
str*
Tc
Ge
Er
-
-~
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A. lipoferum
Sensitive Resistant, selected in media Resistant upon isolation
4 5 1
13+2t 0 0
14+5 6+ 1 3
15+2 16+ 2 17
Ilk2 lo+ 2 10
13+2 14+3 13
2
12+ 1 0 0
12+3 4+2 4f l
9_+2 14+ 2 1Ok 1
8+0 Ilk3 11+1
12+ 3 15+3 17+3
A . brasiletise
Sensitive Resistant, selected in media Resistant upon isolation
5
3
*"Sensi-Discs": Str, streptomycin (10 pg); Crn, chloramphenicol (30 pg); Tc, tctracyclin (30 ~ l g ) ; Ge, genlamycin (10 118); Er, erithromycin (15 pg). ?Inhibition zones, distance in rnillirnetres, from disc with standard deviations of Incnns.
O .
ROOTS
' O
UN INOCULATED
1,.-;- -. ,/A.
.-aI
2 n
100-
SOIL
.
N m A e 4
4
-A
1
@ @ P O -\ \
u
$ 503
5q
INOC. WlTH A. lipoferum s t r r
4. brosilense
INOC. WlTH a
\
.
.
strr
SOIL r
50
60 70 80 90 DAYS AFTER PLANTING
i
100
FIG. I . lncidence of spontaneous low-level streptomycinresistant Azospir-illror~ li~~of~~r.rrrr~ in rhizosphere soil and surface-sterilized ( I h) roots of maize grown in the field, and effect of inoculation. Each point was calculated from six to eight isolates. T h e soil contained about spontaneous streptomycin resistant Aro?;pirill~irrl sp. before planting. Inoculz~tionconsisted of application of 150 mL/m2 of a liquid culture of A . lipoJi,r~itris t r r o r A . brcisilcri.sc strrstrains isolated from surfacesterilized maize o r wheat roots. respectively, and selected for low-level streptomycin resistance (20 pg/mL).
resistant forms independent of rhizosphere effects. It is very surprising that the interesting data presented by Brown (1961) were not further explored. Rhizobiutn is generally more tolerant to streptomycin than common bacteria including E. coli, Azorobncrrt-, and Azospirillum (Strzelczykowa and
Strzelczyk 1958; Baldani and Diibereiner 1979), which are inhibited by concentrations below I pglmL. Levels of 5 to 10 pglmL were reported to permit normal growth of various fast-growing strains of Rhizobi~lt?7(Schwinghammer 1967; Josey r t (11. 1979) and levels between 50 and 150 pg/mL are used to select Rhizohirltu for I-esistance to this antibiotic. This could indicate that legume root infection also requires cei-tain streptomycin resistance. Although labeled strains have been widely used, Rhizobir~minfection seems not generally enhanced by increased streptomycin resistance (I00 rrglmL). Souto (S. M. Souto. 1976. M.Sc. Thesis. Univ. Fed. Rural do Rio de Janeiro), however, reported that one Styloscrnthrs gi!jltrtletl.si.s cultivar, which has inherent nodulation problems, exhibited increased nodule numbers from 69 to 206. nodule weights from 12 to 44 mg per pot, and nitrogenase activities from 3 14 to 2040 nmol C2H41hper pot when inoculated with a streptomycin-resistant Rhizobium mutant. This author used 150 pmollmL streptomycin to select this mutant and it seems worth while to examine streptomycin accumulation in roots of this Sry1o.rotzrhe.s cultivar because it could explain the nodulation difficulties. Interesting inten-elations of in cilro nitrogenase activity of fast-growing Rhizobiion strains with spectinomycin (Skotnicki et (11. 1979) and streptomycin (M. P. Nuti, personal communication) resistance have recently been reported. A possible explanation for the relative enrichment of streptomycin-resistant bacteria in the rhizosphere, and especially in the roots, is the stimulation of actinomycetes in the rhizosphere and on the root surface of certain plants a s observed by many authors (Katznelson 1965; Krasil'nikov 1958). Pramer (1955) reported that streptomycin is actively assimilated into plant cells (algae) by energy-requiring, ion-binding processes, whereas chloramphenicol enters the cells by diffusion, and penicillin is not assimilated at all. Also Krasil'nikov (1958) found that antibiotic concentrations in wheat
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D~BEREINERAND BAL-DANI
I-ootswere much higher than those available in soil. This author suggested that actinomycetes growing in the rhizosphere produce antibiotics which are assimilated into plant I-oots. streptomycin can persist within plant tissue for 8 weeks (Pramer 1959); however it is rapidly inactivated in soil, partly resulting from decomposition, and partly resulting from adsorbtion by clay. From 10 000 ppm streptomycin applied to soil, an antibiotic activity of only I ppm was recovered (Pramer 1958). This also explains why the application of two doses of 5 L per plant of a 4000-ppm streptomycin solution, 8 and 2 days before sampling maize plants, did not affect the percentage of resistant bacteria in the rhizosphere soil (Table I). An alternative explanation would be that genes coding for streptomycin resistance are spread among microbial populations within plant roots observed in other environments (Kleckner et ol. 1 977). The findings presented in this paper. together with the observation on host-plant specificity in associations (Baldani and the grass-Azo.spi~~illrr~n Diibereiner 1979) open a large field of research possibilities. Introduction of selected 01.manipulated Azospirilluln strains 01- other N1-fixing bacteria into roots of field-grown plants now seems possible and therefore their ]-ole can be studied without the a]-tifacts of sterile test tube cultures. In view of these findings we re-emphasize our earlier claims for better understanding of these associations before field inoculation trials are started (Neyra and Diiber-einer 1977). Even though we have shown now that low-level resistance to antibiotics, especially to streptomycin, confers competitive advantages. specific plant-bacteria affinities transcend them. We know neither the mechanism of specificity nor the role of other str' bacteria which infect roots and occur there more than 100 times greater (Tables 1 and 2) than the Nz-fixing Azospirillu~nspp. Extensive studies will be required to identify host-plant specificity and antibiotic-resistant groups. But they are an essential basis for inoculation and may avoid future confusion like the insoluble problem of classification in the "cowpea Rlzizobilr~nmiscellany ." Acknowledgements We thank Charles Robbs and Charles Rosenberg for helpful discussions. We also acknowledge the efficient assistance of vanderlei de Oliveira Andl-ade and Marcio Velotao da Silva.
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B A L D A N IV. . L. D., and J. D B N ~ R L I N E1979. R . Host plant specificity in the infection of cereals with Azospirillrrt)7 spp. Soil Biol. Biochem. (In press). BROWN,M . E. 1961. Stimulation of streptomycin-resistant bacteria in the rhizosphere of leguminous plants. J. Gen. Microbiol. 24: 369-377. B i i ~ o wJ. . F. W. v o ~ and . J. DOBEREINER. 1975. Potential for nitrogen fixation in maize genotypes in Brazil. P1.o~.Natl. Acad. Sci. U.S.A. 72: 2389-2393. GARETH-JONES, D., end E. S. P. B R O ~ I F I E L1978. D . A study of the competitive ability of streptomycin and spectinomycin mutants of R1iizohirrt)i trifolii using various marker techniques. Ann. Appl. Biol. 88: 448-456. GRESSHOFF. P. M.. M . L. S K O T N I C KJ .I .F. E A D I Eand , B. G. ROLFE.1977. Viability of Rhizobirrt)~trifblii bacteroids from clover root nodules. Plant Sci. Lett. 10: 299-304. J ~ S E YD.. P.. J. L. B E Y N O NA. , W. B. JOHNSTON, and J . E. BERINGER 1979. . Strain identification in R11izohirrt)i using intrinsic antibiotic resistance. J . Appl. Bacterial. 46: 343-350. KATZNELSON. H. 1965. Natul-e and importance of the rhizosphere. h i Ecology of soil borne plant pathogens. Etlitpcl hy Baker. K. F. and W. C . Snyder. Univ. California Press. Davis, CA. pp. 187-209. K L E C K N E N.. R , J. ROTH.and D. BOTSTEIN.1977. Genetic engineering it7 ciro using transloc;ttable drug-resistance elements. J . Mol. Biol. 116: 125-160. KRASIL'NIKOV. N. A. 1958. Soil microorganisms and higher plants. Israel Program for Scientific Translations, Jerusalem. pp. 372-386. M A C A L H ~ ~F. E SM. . M., D. P A - ~ R I Q Uand I N . J . DBBEREINER. 1979. Infection of field gt-own maize with Azospirillrrtti spp. Rev. Bras. Biol. 39: 587-596. N C Y R AC. . A , . and J . D O B E R E I N E1977. R . Nitrogen fixation in grasses. Adv. Agron. 29: 1-38. P R A M E RD.. 1955. Absorbtion ofantibiotics by plant cells. Science, 121: 507-508. The persistence and biological effects of antibiotics in soil. Appl. Microbiol. 6: 22 1-224. 1959. Absorbtion ofantibiotics by plant cells. V. Penicillin. Antibiot. Chemother. (Washington. D.C.). 9: 501-504. Roses. C . F. 1960. Estudos sobre o controle d o cancro bacteria ~~tti com no d o tomateiro ( C o r : v r ~ ~ b t r ~ - r o . i1)7ic~lrigcrrrc~r1scs) especial referencia a estroptomicina. 111 Bacterioses Fitopatogenicas no Brasil. Editctl by C . F. Robbs. Ser. Divulgasio Pesquisas No 2. Univ. Fed. Rulxl d o Rio de Janeiro, Rio de Janeiro. pp. 47-63. S C H W I N G H A M ~E,. IA. ~ R1967. . Effectiveness of Rl~izobirrrnas modified by mutation for resistance to antibiotics. Antonie van Leeuwenhoek. J. Microbiol. Set-01. 33: 121-136. S K O T N I C KM. I , L.. B. G . ROLFE,and M. REPORTER. 1979. Nitrogenase activity in pure culture of spectinumycinresistant fast and slow-gl-owing Rhi:ol)i~rr)~. Bioch. Biophys. Res. Commun. 86: 968-975. STRZELCZYKOWA. A,. and E. STRZELCZYK. 1958. The influence of antigonistic sctinomycetes on some soil bacteria. Acta Microbiol. Pol. 7: 283-297. T A R R A N DJ ., J., N. R K R I E C ,and J. D O B E R E I N E 1978. R. A taxonomic study of the Spirillrrt)7 lipoj2rrrr)~group. with descriptions of a new genus Azospirillurn gen. nov. and Azos~irillurnbrasilense S P . nov. Can. J . Microbiol. 24: 967-980. WATANABE. I . , and W. L . BARRAQUIO. 1979. Low levels of nitrogen required for isolation of free living N2-fixing organisms from rice roots. Nature (London). 277: 565-566.
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J O H A N ND AO B E R E I NAENRD VERALUCIADIVANB A L D A N I Cotrscl/~oNnt~iorrcrlde D r s r ~ i c o l ~ ~ i t r z cCir~rt;jit.o ~t~to r T~c~rlologico, Gripreso Brcrsilrrn cle Prscl~riscrAgroprcrrc~rin,Kt1147, Srropc;dic.o 23460, Rio tlr J a t ~ r i r oBrozil , Accepted July 20. 1979
D ~ B E R ~ I NJ .E, R and . V . L. D. B A L D A N I1979. . Selective infection of maize roots by streptomycin-resistant A;ospiri//rr~tilipofe~rrrrrrand otherbacteria. Can. J. Microbiol. 25: 1264- 1269. The percentage of low-level streptomycin-resistz~nt(20 pg/mL) bacteria in surface-5te1-ilizedo r washed maize roots was more than a thousand times higher than that in soil populations. There was also a higher incidence of resistant bacteria in rhizosphereas compared with non-rhizosphere soil and bacteria isolated from maize roots were relatively tolerant to several other antibiotics. A io.~prt~llrrtri - . ..' lipofirlrt~rwas predominant in sur-face-sterilized roots of field-grown maize a n d was low-level streptomycin-resistant while most soil isolates were sensitive. Inoculation with A . hro.siletr.sc, isolated from wheat roots was unsuccessful in terms of establishment even when streptomycin-resistant strains were used. Unidentified causes of specific plant-bacteria affinities therefore transcend the role of antibiotic resistance in maize root infection. D O B E R E I N EJ.. R ,et V. L. D. B A L D A N I1979. . Selective infection of maize roots by streptomycinresistant Azosl~irillrrtr~ lipofc,r~rtrrand other bacteria. Can. J. Microbiol. 25: 1264-1269. Le pourcentage de b;rcteries resistantes i de faibles niveeux de streptomycine (20 pg/mL) chez les racines de miis lavees ou sterilisees en s u ~ f a c eest plus que mille fois plus eleve que celui d e s p ~ u l a t i o n sdu sol. L'incidence des bacteries resistantes est egalement plus eleveedans un sol B rh~zospherescompare i un sol sans rhizosphtres. e t les bacteries isolees des racines de mais sont relativement tolel.antes plusieurs autres antibiotiques. Azospirillrr~nlipofirrrt~rpredomine chez les racines provenant de champs de miis et sterilisees en surface et il est resistant i~ de faibles niveaux de streptomycine, tandis que la plupart des isolats du sol sont senibles. L'inoculation avec A . brcr.silc~tr.scisole de racines de ble ne [-Cussit pas en termes d'etablissement meme si d e s souches resist antes sont employees. Des causes non identifiees d'affinites specifiques entre plantes de bacteries transcendent donc le rble de ~ e s i s t a n c eaux antibiotiques dans I'infection des racines de mai's. [Traduit par le journal]
Introduction croprotation with cel-eals has been recommended as a biological control for certain bacterial diseases (Robbs 1960) and the of actinomycetes in the rhizosphere is well known (Katznelson 1965; Krasil'nikov 1958). Nevertheless the on]y report on enl-ichment of streptomycin-resistant microorganisms in the rhizoSphere is that of B~~~~ (19611, which shows large increases i n the proportion of resistant bacteria on the r-oot of several legumes, but less pronounced effects in cereals. Streptomycin resistance has been used as a marker in many Rhizohiut?l inoculation experiments, but no selective advantage has been I-eported under field conditions (Careth-Jones and Bromfield 1978). nitrogen fixation i n maize (Biilow and Dobereiner 1975) and host-plant specificity in the infection of maize, rice, and wheat by Azospirillu~n spp. has been demonstrated (Ba]dani and Diibereiner 1979) where maize was selected for Azospirill~rn~ lipofelurn and wheat and rice for the A . bmsilense nir- group. TO confirm the specific
infection, Azospirillutn strains marked with lowlevel streptomycin resistance were used as ~ O C ulants in a field experiment with maize and wheat. In both species, in the noninoculated control plots and in the plots inoculated with heterologous strains, high proportions of homologous streptomycinresistant Azospirillurn spp. were obtained from surface-sterilized roots but not from rhizosphere soil. These observations indicated selective infection of roots by ~ t r e p t ~ m y c i n - r e s i ~ t astrains. nt In the present paper this remarkable characteristic is c o n f i ~ - ~ ~ - ~ e d fmaize o r t h e plant. Materials and Methods Fic~ldE.rpc~t.i~tret~t~ F o r counts of total numbers of streptomycin-resistant bacteria, maize cv. M-102 (Agroceres). planted in podzolic soil (field A) o r in gray hydromorphic soil (field B) fertilized with 20 kg N . 50 kg P. I20 kg K. per hectare, was used at the flowering stage. For the evaluation of streptomycin-resistant A Z O S pirillrrtri spp.. soil and plant samples were taken at various growth stages from plots of an experiment described before (Baldani and Diibereiner 1979). In this field experiment maize cv. Pirango was planted in podzolic soil fertilized with 30 kg N . 30 kg p. 100 kg K , and 40 kg FTE (fritted trace elements) per hectare. Plots were 6 m x 6 m in size and had three uninoculated
0008-4 16617911 1 1264-06$0 1 .OO/O @ 1979 National Research Council of CanadaIConseil national d e recher-ches du Canada
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DOBEREINER A N D BALDANl
bordel- lines between inoculation treatments. Inoculation with A . lipofi,rritt~ strr (resistant to 20 pg/mL of streptomycin) or A . h~.rr.siletl.sestrr nir- (spontaneously nitrite reductase negative strain) (Baldani and Diibel-einer 1979) was pelformed a t sowing with 150 mL/m2 of liquid cultures containing 101° cells per millitre. T h e most probable number (MPN) of Azospirillritr~ spp. before inoculation was 3.5 x lo7 per gram of soil with a frequency of streptomycin- (20 pg/mL) resistant mutants of 10F. T w o plants were harvested from each plot, 45.60, 75, and 95 days after planting. The experimental design was in [randomized complete blocks, with four replicates. CriI~rit~t, M~(/itr
The media used in this study were as follows. (i) Modified nitrogen-free semisolid malate medium (NFb): 5 g malic acid: 0.5 g K2HP0,; 0.2 g M g S O , 7H,O; 0. I g NaCI; 0.02 g CaCI,; 2 m L trace element solution; 2 m L alcoholic solution of bromothymol blue (5%); 4 mL Fe EDTA (1.64%); I mL vitamin solution; 4 g KOH; 1.75 g agar; 1000 mL H,O; pH adjusted to 6.8. The trace element solution was 0.2g N a , M o 0 , , 2 H 2 0 ; 0.235 g MnSO,. H,O; 0.28 g H,BO,; 0.008 g CuSO,. 5H,O; 0.024 g ZNSO,. 7H,O; 200 m L H Z O .The vitamin solution was 10 mg biotin; 20 mgpyridoxin; I00 mL H,O. (ii) Potato infusion agar (BMS): 200 g potatoes; 2.3 g malic acid; 2.0 g KOH: 2.5 g raw cane sugar: I mL vitamin solution (see above). Washed potatoes were cooked for 30 min and then filtered through cotton. Malic acid (2.5 g) was dissolved in 50 m L H,O, adding 2 drops of bl-omothymol blue (0.5% solution in ethanol). and adjusted with KOH until green (pH 7.0). This solution together with sucrose. agar, and vitamins wereadded to the potato filtrate and made to 1000 mL.
TABLE1. Enrichnient of streptomycin-resistant bacteria in field-grown maize roots
Sample Rhizosphere soil Rhizosphere soil treated with Distreptine 7 and 2 days before'samplingt Washed roots Surface-sterilized roots$
Total no. of bacteria per gram dry wt. x 10'
resistant t o streptomycin*
39 5 27
1.7k1.7
+
57 29 284 5 274 344 262
+
%
2.6k0.2 43 _+ 27 4356
'Percentage from colony numbers o n BMS a g a r plates with and without 20 p g / m L streptomycin. Values are means from three samples with s t a n d a r d deviations o f means. tcomrnercially available streptomycin product applied in 4000 p p m solution. $Treated for 30 min in 1Z chloramine T.
incubation at 32°C and negative cultures were discarded. Further enrichment was obtained in 24-h-old cultures in N F b medium which were streaked out on NFb agar plates containing 20 ppm yeast extl.act. After I week, typical small, white, dense, single colonies were picked and transferred into semisolid N F b medium. Pellicle formation in this medium indicated success of isolation. For final purification, these cultures were streaked o u t on potato agar (BMS) and the typical pink, often wrinkled colonies transferred for storage and identification tests. F o r this. and loop of semisolid MFb cultures was inoculated into N F b Corrt11.s of 7bltrl Nrrttrhers rrtltl of S / r e p ~ o ~ t ~ c . i t ~ - r ~ ~Bnc. v i . s ~ vials t ~ t ~ ~containing 20pg/mL of streptomycin. and into N F b medium with glucose 21s sole carbon source. Azospirillritt~ leritr Maize plants wereexcavated with most of the root system and lipc?fi,rlr~tlwas differentiated from A . hrrrsiletrse by itsgrowth o n taken to the laboratory. T h e soil adhering to the roots, after glucose (Tarrand rt trl. 1978). The same purified single-colony shaking off the loose soil was considered rhizosphere soil. Con- isolates were used for identification of species, and streptomycin resistance and growth are valuated in parallel tests. Theretrol soil was collected between rows. The roots were washed and (or) sudace-sterilized and 10 g of fresh weight proportions fore, selection of resistant mutants in the streptomycin vials is was homogenized in 100 m L of a 4% cane suga!. solution in a unlikely. blender. Roots were sulface-sterilized in I% aqueous solution of chloramine T . The cut ends were kept outside the disinfectant At~ribiolicTP.YI.S~ i . i l h"Setlsi Di.vcs" Distinct colonies of bacteria on BMS plates with the highest and removed afterwards. Washings were carried out for the or cultures of Azospirilhit?~were used to inocusame time interval as that of immersion in the disinfectant (30 or dilutions 60 min). There was one washing with 0.05 M phosphate buffer late to semisolid BMS medium, and after 48 h of incubation, solution and four washings with sterile water. All dilutions ex- 0.1 m L of the cultures was plated. without dilution, into BMS cept those of the experiment presented in Table 1 , where water agar. T h e "Sensi-Discs" (Becton. Dickinson & Co) of various was used, were performed in 4%' cane sugar solutions to avoid antibiotics were placed o n the plates and the size of the clear osmotic shock a s reported for Rllizohilitn bacteroids (Gresshoff halo starting from the margin of the discs measured after48 h. I n some cases the halo was not clear. Then the halo reading was el (11. 1977). Preliminary experiments indicated higher recovery. especially of streptomycin-resistant bacteria, when the water reduced according to visual estimates of t h e propoltion of was r e ~ l a c e dbv the sugar solution. The soil or root dilutions of growth.
lo-s and lo-', and in some cases also lo-'. were plated in potato agar (MBS) containing 60 ppm of actidione sterilized separately by filtration. Equal amounts of the same dilutions were plated into potato agar containing 20 pglmL streptomycin sterilized by filtration and added to the medium cooled to 48°C. iust before pourlng the plates The percentage of streptomycin-res~stant bacter~awas calculated from the d~fferenceIn numbers of colonles on plates with and without streptomycin. IsoItrtiotl or1~1 I~it~t~tiJjc~rtiotl of Azospirillli~?~ .~pp.
Roots were hal-vested. washed, and (or) surface-sterilized as described for bacterial counts. T h e roots were then cut into 5- to 8-mm pieces which were macerated with forceps and introduced into NFb medium (4 m~ in 6 m L serum vials). Nitrogenase activity (C2HI reduction) was checked after 40 h of
Results The applications of high levels of streptomycin ( I 0 times recommended for treatment of bacterial diseases of tomatoes) to rhizosvhere soil seemed to have little effect (Table I), probably because it is rapidly decomposed or adsorbed to the clav (Pramer 1958). Bacteria in washed and surfack-sterilized roots were more numerous and contained higher proportions streptomycinresistant forms (Table 1). Watanabe and Barraquio (1979) reported that 80% of root isolates of rice can fix low amounts of N, when supplied with low
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C A N . J. MICROBIOL. VOL. 25. 1979
levels of combined nitrogen. To see whether our isolates from su~face-sterilizedmaize roots were similar, we transferred 60 colonies from the above-mentioned pour plates. with and without streptomycin. into the medium used by the authors above. All colonies transferred into this medium grew. but none reduced C I H 2 ,not even after incucolbation for 24 h. There were no Azo.\pi~.illlr~?~ onies on the BMS plates. either with or without streptomycin. Numbers of this organism have been found to be about two orders of magnitude lower than total numbers of bacteria in soils or roots (Magalhaes et o l . 1979) and would therefore not become identifiable on dilution plates. It seems therefore that there were no N1-fixing organisms among the streptomycin-resistant bacteria contained in Table 1. Samples from two additional fields confirmed the selection of maize roots for low-level streptomycin-resistant bacteria (Table 2). As in the first experiment. variation between samples was large (see standard deviations) while the percentage of stl-eptomycin-resistant bacteria was more constant. In field A the frequency of spontaneous streptomycin-resistant bacteria was about lo-' which is in accordance with the occul-rence of st~.eptomycin-I-esistantAzo.\pi~.ill~rt?~ in a similar field and which was 10-% In soil B. which contains more organic matter, quite a different microflora should have existed and bacterial numbers in surface-sterilized roots were small compared with those in soil A. Nevertheless the striking difference in the PI-opol-tionof streptomycin-resistant bacteria was again observed between soil and roots. T o investigate resistance to othel- antibiotics. I-andomly selected bacteria from the highest dilution plates of the two former experiments were tested. The results from fields A and B are summarized in Table 3. There is general tendency towards higher incidence of resistant bacteria in sterilized roots, but differences between soil and sterilized roots were most pronounced in the I-esistance to streptomycin. The resistance to othel- antibiotics varied with soil. The data in Table 4 confirm the higher incidence and also higher mean resistance to antibiotics. especially stl-eptomycin, of bacteria isolated from within roots as compared with those from non-sterilized roots. Resistance to antibiotics and possibly associated characters therefore seem to be of advantage for multiplication in the rhizosphere, and for root infection. It may be seen from Fig. 1 that Azo.spit.ilIrrt7z infection of maize I-oots seems also to be correlated with streptomycin resistance. In the uninoculated control plots during the whole growth
TABLE2. Enrichment of streptomycin-resistant bacteria in the rhizosphere and roots of maize grown in two fields Total no. of bacteria per gram dry wt, x l o 7
% resistant t o streptomycin*
Field A (red yellow podzolic) Soil between rows Rhizosphere soil Surface-sterilized rootst
45k 36 138k87 554k 246
0.0026k0.0014 0.53k0.28 88.4k5.0
Field B (grey hydromorphic) Soil between rows Rhizosphere soil Surface-sterilized roots:
83 k 23 148k 80 3.4k0.367
Sample
0.12_+0.10 1.01+0.28 111.0k 20.1
*Percentage calculated from colony numbers on BMS agar plates with a n d without ?O k~gimLstreptomycin. Values are means of three samples with standard devlatlons of (he means. tThis value is the mean of three plates from one sample and the other two samples contained fewer than 10' bacteria (no colonies on the plates inoculated a t a dilution of' 10-5). :Sterilized fbr 30 min in 1 z chloramineT.
cycle, the majority of strains isolated from I-h. sulface-sterilized roots were low-level streptomycin-resistant. In the plots inoculated with A . lipofet.lit~lstrr. most of the soil and root strains were resistant indicating competitive advantages of the inoculated resistant strains in the rhizosphere soil. These effects. however, were not equally pronounced at different sites and growth stages of the plant. In a pot experiment. 12% of the isolates from young plants. 62% from plants at the flowering stage, and none from older plants were resistant to 20 pg/mL streptomycin (data not shown here). The general spectrum of antibiotic resistance in Azo.spit.illlrt?l spp. is shown in Table 5. In both species low-level streptomycir, resistance was accompanied by a higher tolerance to chlorarnphenicol. None of the three other tested antibiotics showed any connection with streptomycin I-esistance. The antibiotics used in this test had been selected in a preliminary test with 13 other A z o .spit~illrnnstrains and 14 antibiotics ("Sensi-Disc" tests). In these tests all strains were resistant against penicillin-G (10 units), cephaloridine (30 pg), methicillin ( 5 pg), ampicillin (I0 pg), lincomycin (2 pg), coly-mycin (10 pg), oxacillin ( I pg), vancomycin (30 pg), mycostatin (100 units) (L. Vasconcellos, A. Drozdovicz, and J . Dobereiner, unpublished results), whereas most strains were sensitive to the antibiotics mentioned in Table 5. Discussion The results presented in this paper, together with those reporting host-plant specificity for A z o s pi~.illutn spp. infection in cereals (Baldani and Dobereiner 1979), throw interesting new light on
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I I O B E K E I N E R A N D BAL.IIAN1
TABLE 3. Resistance of soil, rhizosphere, and root isolates" to various antibiotics Soil
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Antibiotic
Rhizosphere
% Inhibition resistant.1 zone,: mnl
Field A (Red yellow podzolic) Streptomycin (10 pg) Tetracyclin (30 pg) Chloramphenicol (30 pg) Gentamycin (10 pg) Erythramycin (15 pg)
0 0 0 25 0
8.7k5.5 11.0k1.7 11.5k3.3 5.2k4.5 7.223.2
Field B (gray hydromorphic) Streptomycin (10 pg) Tetracyclin (30 pg) Chloramphenicol (30 pg) Gentamycin (10 pg) Erythramycin (15 pg)
25 0 0 50 0
5.5k4.4 7.2k2.2 8.Ok 1 . 4 5.7k4.3 8.7k4.3
% resistant
Surface-sterilized roots"
Inhibition zone, mm
% resistant
Inhibition zone, mnl
'Four to S I X apparently d~fferentcolonles from the h~ghcstd ~ l u t l o nplates (experiment presented In Table 1) of e a c h treatment xcre tested agalnst thc barlous a n t ~ b l o u c sw ~ t h ' S e n s ~DISCS" Values are means o f four to S I X ~solntesw ~ t hstandard de\latlons o f the means tlsolates whtcli produced a n i n h ~ b l t ~ ozone n smaller than 3 mm were considered rcslstant t l . n h ~ b ~ t l ozone n IS the distance, In m1ll1metrcs, o f the halo lrom the rnargln o f the d ~ s c llSterlllzed for 30 mln In I:;;, c h l o r a m ~ n eT
T A ~ L4.EResistance to various antibiotics of bacteria* isolated from wsshed and from surface-sterilized roots Washed roots
%
Surface-sterilized roots''
%
Antibiotic
resistant?
Inhibition zone.$ mm
resistant-t
Inhibition zone.!: nlnl
Streptonlycin (10 pg) Tetracyclin (30 pg) Chloramphenicol (30 pg) Gentan~ycin(10 11g) Erythamycin (15 pg)
12 0 12 0 37
5.6k3.3 11.0f 6 . 0 7.7k6.4 4.6k2.2 3.5k3.0
78 44 33 55 44
0 . 7 k 1.4 6.2k5.2 7.4k6.1 2.8k3.3 4.2k3.7
-
-
---
*Eight apparently different colonies from the highest dilution plates (cxperimcnt presented in Table I) were tested against thc various antibiotics by the Sensi-Disc test. Values represent means o f eight isolates with standard deviations of the means. tlsolarcs which produced an inhibition zone smaller than 3 m m were considered resistant. :Inhibition zone is the distance, in millimetres, of the halo from the margin o f the disc. ISterilized for 30 min in IZ, chloramine T .
grass-Azo~pirill~rrllassociations. Streptomycin resistance seems to confer advantages in the infection of roots. but host-~lants~ecificitvmust have other reasons independent of antibiotic resistance. Baldani and Dobereiner (1979) have demonstrated that maize was infected predominantly by A. lipofircrm and the recovery of A. lipofifirrn strrfi-om sur-facesterilized roots in all plots, even those inoculated with A. brasilensc nir- strr, is shown in Fig. 1. The introduction of A. bt.risilet7se, therefore, even if it was made streptomycin-resistant, did not help to establish this organism in the roots although the large majority of the rhizosphere isolates were from the inoculated strain. This confirms host-plant specificity as an important factor- controlling maize root infection by Azospir.ill~rmspp. A field kxperiment with wheat gave similar results except the
instead of A. wheat selected for A. brc~.~iletzsc~ lip(?fc)t.~rtn(Baldani and Dobereiner 1979). The enrichment of antibiotic-resistant. e s ~ e c i ally streptomycin-resistant, bacteria in the rhizosphere is not unknown. Brown (1961) comparing root-surface soil with control soil without plants found similarly striking rhizosphere effects on the proportion of streptomycin-resistant bacteria in several legumes, wheat, and vegetables. From her data it can be calculated that 7 and 5% of the bacteria from control pots and 12 and 48% of the rootsurface bacteria of pea plants were resistant to str-eptomycin in two experiments, respectively. Bacteria which have no specific growth requirements (basal medium) contained 0.05% resistant forms in control soil and 2% in root-surface soil. Amino acid requiring bacteria contained about 2%
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C A N . J . MICROBIOL. VOL. 25. 1979
TABLE5. Resistance t o various antibiotics of Azospirillli~r~spp. Reaction to 20 pg/mL streptomycin
N o . of strains
Cm
str*
Tc
Ge
Er
-
-~
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A. lipoferum
Sensitive Resistant, selected in media Resistant upon isolation
4 5 1
13+2t 0 0
14+5 6+ 1 3
15+2 16+ 2 17
Ilk2 lo+ 2 10
13+2 14+3 13
2
12+ 1 0 0
12+3 4+2 4f l
9_+2 14+ 2 1Ok 1
8+0 Ilk3 11+1
12+ 3 15+3 17+3
A . brasiletise
Sensitive Resistant, selected in media Resistant upon isolation
5
3
*"Sensi-Discs": Str, streptomycin (10 pg); Crn, chloramphenicol (30 pg); Tc, tctracyclin (30 ~ l g ) ; Ge, genlamycin (10 118); Er, erithromycin (15 pg). ?Inhibition zones, distance in rnillirnetres, from disc with standard deviations of Incnns.
O .
ROOTS
' O
UN INOCULATED
1,.-;- -. ,/A.
.-aI
2 n
100-
SOIL
.
N m A e 4
4
-A
1
@ @ P O -\ \
u
$ 503
5q
INOC. WlTH A. lipoferum s t r r
4. brosilense
INOC. WlTH a
\
.
.
strr
SOIL r
50
60 70 80 90 DAYS AFTER PLANTING
i
100
FIG. I . lncidence of spontaneous low-level streptomycinresistant Azospir-illror~ li~~of~~r.rrrr~ in rhizosphere soil and surface-sterilized ( I h) roots of maize grown in the field, and effect of inoculation. Each point was calculated from six to eight isolates. T h e soil contained about spontaneous streptomycin resistant Aro?;pirill~irrl sp. before planting. Inoculz~tionconsisted of application of 150 mL/m2 of a liquid culture of A . lipoJi,r~itris t r r o r A . brcisilcri.sc strrstrains isolated from surfacesterilized maize o r wheat roots. respectively, and selected for low-level streptomycin resistance (20 pg/mL).
resistant forms independent of rhizosphere effects. It is very surprising that the interesting data presented by Brown (1961) were not further explored. Rhizobiutn is generally more tolerant to streptomycin than common bacteria including E. coli, Azorobncrrt-, and Azospirillum (Strzelczykowa and
Strzelczyk 1958; Baldani and Diibereiner 1979), which are inhibited by concentrations below I pglmL. Levels of 5 to 10 pglmL were reported to permit normal growth of various fast-growing strains of Rhizobi~lt?7(Schwinghammer 1967; Josey r t (11. 1979) and levels between 50 and 150 pg/mL are used to select Rhizohirltu for I-esistance to this antibiotic. This could indicate that legume root infection also requires cei-tain streptomycin resistance. Although labeled strains have been widely used, Rhizobir~minfection seems not generally enhanced by increased streptomycin resistance (I00 rrglmL). Souto (S. M. Souto. 1976. M.Sc. Thesis. Univ. Fed. Rural do Rio de Janeiro), however, reported that one Styloscrnthrs gi!jltrtletl.si.s cultivar, which has inherent nodulation problems, exhibited increased nodule numbers from 69 to 206. nodule weights from 12 to 44 mg per pot, and nitrogenase activities from 3 14 to 2040 nmol C2H41hper pot when inoculated with a streptomycin-resistant Rhizobium mutant. This author used 150 pmollmL streptomycin to select this mutant and it seems worth while to examine streptomycin accumulation in roots of this Sry1o.rotzrhe.s cultivar because it could explain the nodulation difficulties. Interesting inten-elations of in cilro nitrogenase activity of fast-growing Rhizobiion strains with spectinomycin (Skotnicki et (11. 1979) and streptomycin (M. P. Nuti, personal communication) resistance have recently been reported. A possible explanation for the relative enrichment of streptomycin-resistant bacteria in the rhizosphere, and especially in the roots, is the stimulation of actinomycetes in the rhizosphere and on the root surface of certain plants a s observed by many authors (Katznelson 1965; Krasil'nikov 1958). Pramer (1955) reported that streptomycin is actively assimilated into plant cells (algae) by energy-requiring, ion-binding processes, whereas chloramphenicol enters the cells by diffusion, and penicillin is not assimilated at all. Also Krasil'nikov (1958) found that antibiotic concentrations in wheat
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D~BEREINERAND BAL-DANI
I-ootswere much higher than those available in soil. This author suggested that actinomycetes growing in the rhizosphere produce antibiotics which are assimilated into plant I-oots. streptomycin can persist within plant tissue for 8 weeks (Pramer 1959); however it is rapidly inactivated in soil, partly resulting from decomposition, and partly resulting from adsorbtion by clay. From 10 000 ppm streptomycin applied to soil, an antibiotic activity of only I ppm was recovered (Pramer 1958). This also explains why the application of two doses of 5 L per plant of a 4000-ppm streptomycin solution, 8 and 2 days before sampling maize plants, did not affect the percentage of resistant bacteria in the rhizosphere soil (Table I). An alternative explanation would be that genes coding for streptomycin resistance are spread among microbial populations within plant roots observed in other environments (Kleckner et ol. 1 977). The findings presented in this paper. together with the observation on host-plant specificity in associations (Baldani and the grass-Azo.spi~~illrr~n Diibereiner 1979) open a large field of research possibilities. Introduction of selected 01.manipulated Azospirilluln strains 01- other N1-fixing bacteria into roots of field-grown plants now seems possible and therefore their ]-ole can be studied without the a]-tifacts of sterile test tube cultures. In view of these findings we re-emphasize our earlier claims for better understanding of these associations before field inoculation trials are started (Neyra and Diiber-einer 1977). Even though we have shown now that low-level resistance to antibiotics, especially to streptomycin, confers competitive advantages. specific plant-bacteria affinities transcend them. We know neither the mechanism of specificity nor the role of other str' bacteria which infect roots and occur there more than 100 times greater (Tables 1 and 2) than the Nz-fixing Azospirillu~nspp. Extensive studies will be required to identify host-plant specificity and antibiotic-resistant groups. But they are an essential basis for inoculation and may avoid future confusion like the insoluble problem of classification in the "cowpea Rlzizobilr~nmiscellany ." Acknowledgements We thank Charles Robbs and Charles Rosenberg for helpful discussions. We also acknowledge the efficient assistance of vanderlei de Oliveira Andl-ade and Marcio Velotao da Silva.
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B A L D A N IV. . L. D., and J. D B N ~ R L I N E1979. R . Host plant specificity in the infection of cereals with Azospirillrrt)7 spp. Soil Biol. Biochem. (In press). BROWN,M . E. 1961. Stimulation of streptomycin-resistant bacteria in the rhizosphere of leguminous plants. J. Gen. Microbiol. 24: 369-377. B i i ~ o wJ. . F. W. v o ~ and . J. DOBEREINER. 1975. Potential for nitrogen fixation in maize genotypes in Brazil. P1.o~.Natl. Acad. Sci. U.S.A. 72: 2389-2393. GARETH-JONES, D., end E. S. P. B R O ~ I F I E L1978. D . A study of the competitive ability of streptomycin and spectinomycin mutants of R1iizohirrt)i trifolii using various marker techniques. Ann. Appl. Biol. 88: 448-456. GRESSHOFF. P. M.. M . L. S K O T N I C KJ .I .F. E A D I Eand , B. G. ROLFE.1977. Viability of Rhizobirrt)~trifblii bacteroids from clover root nodules. Plant Sci. Lett. 10: 299-304. J ~ S E YD.. P.. J. L. B E Y N O NA. , W. B. JOHNSTON, and J . E. BERINGER 1979. . Strain identification in R11izohirrt)i using intrinsic antibiotic resistance. J . Appl. Bacterial. 46: 343-350. KATZNELSON. H. 1965. Natul-e and importance of the rhizosphere. h i Ecology of soil borne plant pathogens. Etlitpcl hy Baker. K. F. and W. C . Snyder. Univ. California Press. Davis, CA. pp. 187-209. K L E C K N E N.. R , J. ROTH.and D. BOTSTEIN.1977. Genetic engineering it7 ciro using transloc;ttable drug-resistance elements. J . Mol. Biol. 116: 125-160. KRASIL'NIKOV. N. A. 1958. Soil microorganisms and higher plants. Israel Program for Scientific Translations, Jerusalem. pp. 372-386. M A C A L H ~ ~F. E SM. . M., D. P A - ~ R I Q Uand I N . J . DBBEREINER. 1979. Infection of field gt-own maize with Azospirillrrtti spp. Rev. Bras. Biol. 39: 587-596. N C Y R AC. . A , . and J . D O B E R E I N E1977. R . Nitrogen fixation in grasses. Adv. Agron. 29: 1-38. P R A M E RD.. 1955. Absorbtion ofantibiotics by plant cells. Science, 121: 507-508. The persistence and biological effects of antibiotics in soil. Appl. Microbiol. 6: 22 1-224. 1959. Absorbtion ofantibiotics by plant cells. V. Penicillin. Antibiot. Chemother. (Washington. D.C.). 9: 501-504. Roses. C . F. 1960. Estudos sobre o controle d o cancro bacteria ~~tti com no d o tomateiro ( C o r : v r ~ ~ b t r ~ - r o . i1)7ic~lrigcrrrc~r1scs) especial referencia a estroptomicina. 111 Bacterioses Fitopatogenicas no Brasil. Editctl by C . F. Robbs. Ser. Divulgasio Pesquisas No 2. Univ. Fed. Rulxl d o Rio de Janeiro, Rio de Janeiro. pp. 47-63. S C H W I N G H A M ~E,. IA. ~ R1967. . Effectiveness of Rl~izobirrrnas modified by mutation for resistance to antibiotics. Antonie van Leeuwenhoek. J. Microbiol. Set-01. 33: 121-136. S K O T N I C KM. I , L.. B. G . ROLFE,and M. REPORTER. 1979. Nitrogenase activity in pure culture of spectinumycinresistant fast and slow-gl-owing Rhi:ol)i~rr)~. Bioch. Biophys. Res. Commun. 86: 968-975. STRZELCZYKOWA. A,. and E. STRZELCZYK. 1958. The influence of antigonistic sctinomycetes on some soil bacteria. Acta Microbiol. Pol. 7: 283-297. T A R R A N DJ ., J., N. R K R I E C ,and J. D O B E R E I N E 1978. R. A taxonomic study of the Spirillrrt)7 lipoj2rrrr)~group. with descriptions of a new genus Azospirillurn gen. nov. and Azos~irillurnbrasilense S P . nov. Can. J . Microbiol. 24: 967-980. WATANABE. I . , and W. L . BARRAQUIO. 1979. Low levels of nitrogen required for isolation of free living N2-fixing organisms from rice roots. Nature (London). 277: 565-566.
