Present address: Department of Microbiology, MacDonald Campus of McGill Univeristy, Ste-Anne de. Bellevue, P.Q. Canada HOA ICO. 439. Plant and Soil 56, ...
Ms. 4285
OXYGEN AND MANNITOL CONSUMPTION OF R H I Z O B I U M M E L I L O T I IN R E L A T I O N TO S Y M B I O T I C N I T R O G E N F I X A T I O N E F F I C I E N C Y * by L. M. B O R D E L E A U , R. L A L A N D E * * a n d H. A N T O U N Station de Recherches, Agriculture Canada, 2560 Boulevard Hochelaga, Sainte-Foy, QuObec, Canada GIV 2J6 and Dbpartement de Phytologie, Facultd des Sciences de l'Agriculture et de l'Alimentation, Universitk Laval, Sainte-Foy, Qudbec, Canada GIK 7P4
KEY WORDS
Alfalfa meliloti
Bacteria Mannitol consumption Symbiotic efficiency
Oxygen consumption
Plant
Rhizobium
SUMMARY The rate of oxygen and total mannitol consumption were studied with 48 strains ofRhizobium meliloti in relation to their symbiotic nitrogen fixation efficiencyas expressed by the plants dry weight yields. The rate of oxygen consumption is positively correlated to the total mannitol consumption and significant inverse relationship between these two physiological properties and symbiotic efficiency are apparent. The possibility of using the rate of oxygen consumption as a preselection tool is discussed. INTRODUCTION Strains of rhizobia differ widely in their symbiotic n i t r o g e n fixation efficiency. The most reliable m e t h o d available to distinguish between effective a n d ineffective strains, is the performance with the host plants, which is time c o n s u m i n g 4. In order to find a m o r e rapid a n d simple m e t h o d for the e v a l u a t i o n of the symbiotic efficiency of rhizobia, several physiological, n u t r i t i o n a l a n d cultural characteristics were studied. Effective strains express a higher dehydrogenase activity 5,9, a n d require less oxygen for growth 11. P r o d u c t i o n of organic acids is negatively correlated with efficiency 8'~4 a n d this p a r a m e t e r can be used as a preselection criterion of large n u m b e r of isolates 3. N o significant correlation were f o u n d between the symbiotic efficiency a n d nitrate reduction ~"~2, p r o d u c -
* Contributionno 159. ** Present address: Department of Microbiology,MacDonald Campus of McGill Univeristy,Ste-Annede Bellevue,P.Q. CanadaHOA ICO. 439 Plant and Soil 56, 439-443 (1980). 0032-079X/80/0563-0439500.75. 9 Martinus NijhoffPublishers, The Hague. Printed in The Netherlands.
440
L. M. BORDELEAU, R. LALANDE AND H. ANTOUN
t i o n a n d d e g r a d a t i o n o f i n d o l - a c e t i c a c i d 12, r e s i s t a n c e t o a n t i b i o t i c s 15, a n d t h e immunological
c h a r a c t e r i s t i c s o f t h e b a c t e r i a 6.
In the present paper, the oxygen and mannitol consumption
Rhizobium meliloti w e r e d e t e r m i n e d
b y 48 s t r a i n s o f
with the aim of correlating these two
p h y s i o l o g i c a l p r o p e r t i e s o f b a c t e r i a w i t h t h e i r s y m b i o t i c efficiency as i n d i c a t e d b y t h e d r y w e i g h t o f p l a n t s 4.
MATERIAL AND METHODS Strains of R. meliloti used in this study and their symbiotic efficiency were previously described 4. As the second cutting ofnodulated plants give the most necessary information for the exact evaluation of efficiency, the dry weight of plants obtained during this cutting were used in this study as an indication of the symbiotic efficiency of the strains 4. The bacteria were cultured in a medium containing per litre of distilled water: mannitol, 10.0 g; MgSO4.7H 20, 0.20 g; NaC1, 0.10 g; CaC12.2H20, 0.05 g; yeast extract (Difco), 0.20 g; K2HPO4, 0.522 g; KH2PO4, 0.408 g; COC12.6H20, 0.004 mg; H3BO3, 2.86 mg; MnC12.4H20, 1.81 mg; ZnSO4.7H20, 0.22 mg; CuSO4.5H20, 0.008 mg; H2MoO,,.H20, 0.09 mg; ferric citrate, 25.0 mg. To avoid precipitation, phosphates were prepared and sterilized in half the volume and then mixed to the other half after cooling. After sterilization, pH was 6.85. Growth was performed in 250 ml flasks containing 100 ml of medium inoculated with approximately 106 washed cells. Flasks were incubated at 26 ~ on an orbital shaker (125 rpm). Oxygen consumption was measured on 5-ml samples of log phase cultures (36-42 hrs) using a biological oxygen monitor (Yellow Spring Instrument, Ohio, Model 1353) equipped with a polarographic electrode 2. At saturation dissolved oxygen in the medium was determined according to the modified method of Winkler lO. Oxygen consumption was measured during 10 minutes and the cells from 2-ml aliquot were separated and washed twice in phosphate saline buffer (0.01 M, pH 7.2) by centrifugation at 12000 g during 10 min at 4 ~C. Cellular protein was determined in the precipitate 7. Supernatant and washings were combined and lyophilized after addition of 5 x 10 5 mole oferythritol as internal standard. The dry residues were converted to their corresponding alditol acetates before analysis by gas chromatography 13. Acetylated mannitol was measured on 0.1 pl samples injected at 225~ in a gas chromatograph (Tracor 220) equipped with glass columns (1.2 x 6.25 mm) packed with 3% ECNSS-M.on Chromasorb-Q 100-200 mesh (Regis Chemicals) maintained at 160 ~C, and dual flame ionization detector at 275 ~ (carrier nitrogen at 80 cc/min, air and H z at 500 and 60 cc/min respectively). Under these conditions, mannitol was chromatographed in 20 min, while the internal standard (erythritol) appeared after 2 min. Consumed mannitol was calculated as the difference between the original concentration in the medium and that after growth.
RESULTS AND DISCUSSION Rates of oxygen consumption
and total mannitol consumption
b y s t r a i n s o f R.
meliloti a r e s h o w n i n T a b l e 1. T h e i n e f f e c t i v e s t r a i n 11 h a d t h e h i g h e s t r a t e o f oxygen consumption
a n d t h e e f f e c t i v e s t r a i n A 5 h a d t h e l o w e s t r a t e . All t h e R.
meliloti s t r a i n s t e s t e d u s e d m a n n i t o l d u r i n g g r o w t h . T h e h i g h e s t c o n s u m p t i o n o f m a n n i t o l w a s r e c o r d e d w i t h t h e i n e f f e c t i v e s t r a i n A1, w h i l e t h e e f f e c t i v e s t r a i n $7 s h o w e d t h e l o w e s t c o n s u m p t i o n . T h e r a t e o f o x y g e n u s e d b y R. meliloti d u r i n g
Table 1. Oxygen and mannitol consumption of 48 strains of R . meliloti Strain
Symbiotic* efficiency
Rate of 0 2 consumption Picomoles x m i n - 1 x pg protein 1
Total mannitol consumption nanomoles x /~g p r o t e i n -
$1, V3 A2 A3 A4 3Doa8
VE VE VE VE VE VE
687 578 541 521 488 335
475 339 430 432 440 420
St $2 S3 $4 S5 S6 $7 Ss S9 $10 Sll Sl2 $13 Sis S~6 Sl9 $2o S2L Sa2 V1 V2 44 Vs Vv D2 D3 A5 13 R~ E2 3Doa2oa 54032 54033 23A
E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E
772 613 669 456 696 549 398 746 554 830 495 524 629 386 405 409 474 700 511 592 480 664 355 590 276 580 250 743 732 617 342 707 362 332
297 288 321 454 296 434 245 321 309 307 254 382 296 283 302 280 366 307 246 314 322 296 280 312 277 332 391 294 330 440 284 486 279 407
V6 D1 A1 11 12 I4 E1 A1N1ND
I I I I I I I I
536 575 945 1605 569 500 1486 712
328 327 2250 1385 276 260 2000 314
* Symbi•ticef•ciencybased•nthedrymatteryie•d•btainedinthesec•ndcuttingasrep•rtedbyB•rde•eauetal.
VE - very effective, E - effective, ! - ineffective,
4
442
L . M . BORDELEAU, R. L A L A N D E A N D H. A N T O U N
T a b l e 2. C o r r e l a t i o n s between o x y g e n a n d m a n n i t o l c o n s u m p t i o n a n d dry weight o f p l a n t s (syrfibiotic efficiency) in R. melioti Mannitol
Efficiency
0.691 ** (47.8)
- 0.495** (24.46)
Oxygen
Mannitol
- 0.421 ** (17.73)
** Significant at 1% level. Values in parenthesis are the determination coefficients.
growth is positively correlated with mannitol utilization, and these two physiological parameters are negatively correlated with the symbiotic efficiency (Table 2). The present study show an apparent significant and negative correlation between the symbiotic efficiency, as expressed by the dry weight of plants, and oxygen and mannitol utilization by the free living bacteria, during growth. It is then expected that a very effective strain of R. meliloti will use less oxygen and mannitol to produce the same amount of growth (protein), than an ineffective strain. The rate of oxygen consumption appear to be a more useful tool than mannitol. In fact the determination coefficients are 24?/0 and 17~o for oxygen and mannitol consumption respectively. It was previously showed that the final pH of R. meliloti cultures, was significantly correlated to the symbiotic efficiency 3 and this physiological parameter could be used as a preselection tool. This.study also show that the rate of oxygen consumption can be used as a preselection criterion for a very large number of isolates. For example, if one consider a rate of oxygen consumption of 550 picomoles x m i n - 1 x ug protein- x as the highest acceptable rate, of the 48 strains tested, 23 strains will be retained. This step will reduce to 50~o a large population of isolates. The initial population of 48 strains contained 12.5~o very effective, 70.8~o effective and 16.7~ ineffective strains. The 23 selected strains will include 17.4~o very effective, 7 4 ~ effective, 74~o effective and 8.6~o ineffective strains. By using the rate of oxygen consumption as a criterion for preselection of a large number of isolates it is also possible to reduce almost by half the incidence of ineffective strains while slightly increasing the very effective and effective strains in the new selected population. Some physiological characteristics of R. meliloti can offer promising tools to develop a more rapid and simple method for the in vitro selection of symbiotically effective strains.
OXYGEN AND MANNITOL CONSUMPTION OF R. MELILOTI
443
ACKNOWLEDGEMENT The authors thank Dr. A. Alarie and Dr. R. A. Lachance for helpful discussions and comments. R. Lalande was supported by a grant from 'Le Conseil des Recherches et Services agricoles du Qu6bec'. Received 11 December 1979. Revised April 1980.
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