Selection of Rhizobium strains on their competitive ability for nodulation

So,, Lb, B,<,chrm Vol 13 pp 481 to 486. 1981 Pnnted ,n Great Brltam All rights resened

0038-0717 81 060481.0660200 0 CopyrIght 0 1981 Perpamon Prrs Ltd

SELECTION OF RHIZOBIUM STRAINS ON THEIR COMPETITIVE ABILITY FOR NODULATION N. AMARGER Institut

National

de la Recherche

Agronomique.

Laboratoire

de Microbiologic

des Sols.

B.V. 1540. 21034 Dijon Cedex. France (Acceprrd

10 April 1981)

Summary The shoot dry weighl of alfalfa inoculated with an effective strain of Rhi-_ohium mrliloti mixed with an ineffective strain in different ratios was found to be directly proportional to the log of the number of effective nodules. Consequently the comparison of the shoot dry weight of plants inoculated with a mixture of effective and ineffective strains with the shoot dry weight of plants inoculated with the effective strain should allow the estimation of the relative competitiveness of the effective strains. To check this. the competitiveness of 14 antibiotic-resistant strains of R. leyumino.surum was evaluated in this way and compared with the ability of the strains to form nodules when inoculated to seeds of Vicia /iiha planted in a soil containing indigenous R. Irgumw~~wrum. The percentage of recovery of the ‘inoculum strains in the nodules of field-grown fababeans was positively correlated with the competitlveness of the strains as estimated by the greenhouse test. This simple way of evaluating the nodulating competitiveness of strains of rhizobia being indicative of their competitive behaviour with indigenous rhizobia in the field could therefore be useful for screening a large number of strains for competitiveness.

INTRODUCTION

My aims were to ascertain whether the growth of a legume inoculated with a mixture of effective and ineffective strains of rhizobia would be correlated with the number of nodules formed by the effective strain. If this was so. it could be used to evaluate the nodulating competitiveness of strains of rhizobia. It would be necessary to know also whether or not such an evaluation made with plants grown in a controlled environment was indicative of the ability of strains to compete with indigenous rhizobia when they were inoculated into a field. Alfalfa was used to study the relation between the origin of the nodules and the growth of the plant because the origin of the nodules from test tube-grown plants could be determined easily. The evaluation of the method in the field was made using R. leguminosarum strains and Viciu fabu.,

The main criterion used in the selection of strains of Rhizohium for legume inoculant is the ability to form effective symbiosis with the hosts for which the inoculant is recommended; the potential value of these strains is fully reached only when they are inoculated into a soil free of rhizobia. However, if the soils on which a legume is to be grown contain natural populations of rhizobia specific for this legume but more or less able to form effective nodules, inoculation with a superior strain may not improve the fixation as the inoculant strain will have to compete with the indigenous strains and may not be successful in forming nodules (Johnson et al., 1965; Ham et ul., 1971; Roughley et al.. 1976; Boonkerd et d., 1978). To improve inoculant strains Brockwell rt rrl. (1968) proposed additional criteria including ability to form nodules promptly, to persist in soil and to compete with naturalized rhizobia. However the selection of strains able to compete with other strains for infection sites is not easy to undertake because of the lack of a convenient methodology. The strains to be tested should be distinguishable from natural strains and the origin of a rather large number of nodules has to be determined. this limits the number of strains which can be tested. The use of a chlorosis-inducing strain of R. ,japonicum, as described by Jqhnson and Means (1964). allows the screening of a great number of strains of rhizobia for competitiveness but the general application of this procedure is difficult because chlorosisinducing strains have to be found for each species to be studied. Ineffective strains. which can be found easily for each host. could be used in the same way as, when they are mixed with effective strains, instead of inducing a chlorosis indicative of the proportion of nodules made by the chlorosis-inducing strain, they will reduce the growth of the plants (Nicol and Thornton. 1942) which can be easily measured.

MATERIALS AND METHODS Strains

of R. meliloti

Two effective strains 2OllS2 and SAK4 and two ineffective strains 201lNl and SAN4 were used2OlIS2 and 201 IN1 were both derived from the same strain 2011, SAK4 and SAN4 were both spontaneous mutants of the strain SAlO. Alfalja

plant

culture.

inoculution

and nodule

identilicu-

tion Seedlings of Medicago xhcu, var. Argafric in the first experiment and var. Mireille in the second. were grown and inoculated as described by Amarger (1981). In the frist experiment the effective strain 2OlIS2 was mixed on a volumetric basis with each ineffective strain 2011Nl and SAN4 so as to give intended proportions of 0, 0.1, 0.3, 0.5, 0.7. 1. In the second experiment each effective strain 2OllS2 and SAK4. was mixed with each ineffective strain 2OllNl and SAN4 in approximate ratios of 0.2. 0.5, 0.8. The position of the nodules was mapped 14 days after 481

482

N.AMARGER

Table 1. Strains of Rhizohium

Parent

strain

FH13

FS16

FH34 FH37

FA45

Mutants Identification

FH134S’ FS162Sp’ FS163K’ FS163S’ FH341S’ FH342Sp’ FH345K’ FH373Sp’ FH374S’ FH375.S’ FH371S’ FA4511S’ FA4513S’

FHZOSl* FSSI’

_

used Effectiveness

+ + + + + + + + + + _ + + + +

leyumirmarum

used

Antibiotic resistance Streptomycin Spectinomycin

+ _ + + _ _ _ + + + + + + +

markers Kanamycin

_ + _ _ _ + _ + _ _ _ _ _ _

_ _ + _ _ + _ _ _ _ _ _

1 Amarger (1975). * Amarger (1974). inoculation in the first experiment and 17 days after inoculation in the second experiment. Ten days later these nodules were classified visually as being formed by the effective or ineffective strain. The shoots were cut 45 days after inoculation in the first experiment, 35 days after inoculation in the second. they were then oven-dried and weighed. Strains of R. leguminosarum One ineffective and 14 effective antibiotic resistant spontaneous mutants of different strains of our collection were used (Table 1). The bacteria were grown on Bergersen’s (1961) medium modified by addition of 0.2 g l-’ of yeast extract. Greenhouse experiment with V. faba Seeds of I’. fnha var. Ascott were surface sterilized by immersion’in 5% calcium hypochlorite for 45 min. After three washings in sterilized water, 4 seeds were sown per 5 1 plastic pot containing heat sterilized siliceous sand lying on a 4 cm deep bed of gravel. Each pot was inoculated 10 days after sowing with 10 ml of a suspension of agar cultures of rhizobia containing 7 k 10” bacteria of either an effective strain alone or a mixture of an effective strain with the ineffective strain FH371S. There were 3 replicates of each treatment. The pots were irrigated with a nutrient solution of the following composition: 0.14g K2HP04, 0.22g CaCl,, 0.2 g MgS04, 7Hz0, 70mg K2S04, 10 mg Fe3+ (Sequestrene B8 Fe, Geigy), 1.8 mg MnSO,, H20, 2 mg H3B03, 0.25 mg Na,Mo O,, 2H,O, 80 pg CuS04, 5H20, 2Opg ZnS04, 7 HzO, Co’+ traces, in 1 1 deionized water. The plants were grown in a greenhouse from 23 August till 8 October with a daily maximum temperature of 26°C. At harvest the roots were examined for nodulation, the shoots were excised, oven-dried and weighed. Field experiments Field work was conducted on a clay loam soil with a pH of 6.8. The number of indigenous R. leguminosarum determined by the plant infection count (Vincent, 1970) using I/. satiua with 4 replications and lo-fold dilutions was 5.8 x lo4 g-’ (95% fiducial limits 1.522 x 104) at the location of the first experi-

ment and 7.2 x lo4 gg ’ (95% fiducial limits 1.9-27 x 104) at the location of the experiment made the year after. The inocula were prepared by growing the different strains in liquid medium on a rotary shaker at 29 ‘C for 112 h in the first experiment and 44 h in the second. They were used as pure cultures or diluted with tapwater, the dilutions being made in the field just before use. The number of bacteria in the inocula was determined by plate counts made on the pure cultures when they were returned to the laboratory after the field work was completed (2-3 h elapsed between the use of inocula and counting). Seeds of V.faha var. Ascott were sown by hand at 5 cm spacings into previously-opened drill rows 40 cm apart. The inoculum was applied over the seeds as a liquid suspension at a rate of 30 ml m 1 and the furrows were immediately closed. Each treatment consisted of 4 rows 3 m long without replication. Nodulation measurements were made at three dates: between 70 and 77 days, between 90 and 97 days and between 105 and 112 days after planting for the experiment of the first year, and 74, 81 and 88 days after planting for the experiment in the following year. Nodules for R. leguminosarum strain identification were sampled separately from each plot: two samples of six plants were taken, from each sample 30 nodules were chosen at random and put together to be surface sterilized by immersion in 0.2% HgCl, for 3 min and rinsed 6 times in sterile water. Then 30 nodules were taken for strain identification: each nodule was crushed in 2ml sterile water and a small drop of the suspension was spotted with a multiple point inoculator on duplicate Petri dishes of agar medium, one of which was supplemented with the antibiotic the inoculant strain was resistant to. The following concentrations of antibiotic were used: streptomycin sulfate 100 ng ml- I, kanamycin sulfate 30 ,ug ml- I, spectinomycin dichlorhydrate 50 pg ml- I. RESULTS

The results for the first experiment on alfalfa inoculated with mixtures of the effective strain 2011 S2 with

Selection of competitive Rhizohium

483

Table 2. Percentage of effective nodules and shoot dry weight of alfalfa inoculated a mixture

Ineffective strain

% 2OllS2 in inoculum

201 lN1 201 IN1 2OllNl 2011Nl 2OllNl SAN4 SAN4 SAN4 SAN4 SAN4 None

0 13 36 57 76 0 9 28 47 67 100

each of the ineffective varying

of 201 IS2 with ineffective strain in different proportions cates f standard error)

28.2 21.5 22.7 20.3 16.7 26.0 22.3 27.6 20.4 22.0 17.8

+ 3.0 + 3.9 + 4.2 + 3.0 k 1.9 + 3.1 + 3.6 k 3.0 + 5.1 &- 4.4 + 1.4

centage of effective nodules increased with increasing proportions of the effective strain in the inoculum. This percentage was lower for equivalent number of effective bacteria in the inocula when the ineffective strain was SAN4 compared to 2011N1, which is not surprising as SAN4 was found to be more competitive than 2011Nl in other work (Amarger, 1981). The shoot dry weight increased with increasing number of effective nodules formed 14 days after inoculation. The mean shoot dry weight was calculated for all plants bearing the same number of effective nodules and plotted against the number of effective nodules (Fig. la). The linear regression between the mean shoot dry weights and the log of the number of the effective nodules was highly significant (P < 0.001) (Fig. lb). In the second experiment a different variety of alfalfa was inoculated with one of two effective strains mixed with an ineffective one in different ratios. Significant linear regressions were obtained between the mean shoot dry weight of plants bearing the same number of nodules and the log of the number of effective nodules produced by the strain 2OllS2 as well as by the strain SAK4 (Fig. 2). The two regression coefficients were not significantly different (P < 0.05). The shoot dry weights of V’.faba grown in the greenhouse and inoculated by the effective strains,

0’

0

3

8

8 ’

’ 5

’

3

a ”

IO

’

’

8

’

15

Number of effective nodules

0 33 39 50 80 0 6 17 28 33 100

Shoot dry weight, mg 5.7 48.9 44.5 47.4 56.7 3.4 18.4 36.9 47.7 46.5 50.0

+ + & + + + k k + k *

1.1 5.9 5.6 4.5 3.2 0.7 6.0 3.3 6.4 4.0 4.0

alone or mixed with the ineffective strain FH371S in an approximate ratio of 1 to 100, are given in Table 3. There were significant differences (P < 0.05) in symbiotic effectiveness between effective strains (column A, Table 3). The interaction strain x treatment was significant (P < 0.01) indicating that the addition of the ineffective strain to the inoculum modified the

strains, 2011Nl and SAN4, in are given in Table 2. The per-

proportions

T/, Effective nodules

Nodule number at 14 days

with (mean of 10 repli-

-.

2011s2

--4

SAK4

IO

Number

of affective

20

nodules

Fig. 2. Comparison of the dry weight of Medicago suricu. var. Mireille, inoculated with a mixed inoculum as a function of the number of effective nodules for 2 effective strains: 201 lS2, SAK4.

. 3

.J

20 Number

of effective

noduks

Mean shoot dry weight k standard error of Medicago satica, var. Argafric. inoculated with a mixture of effective 201 lS2 and ineffective strains 2OllNl and SAN4 in relation to the number (a) and to the log number (b) of effective nodules.

N. AMARGER

484

Table 3. Shoot dry weights in g per 4 plants of V. fuha inoculated with effective strains alone (A), or with effective strain mixed with the ineffective strain FH371 S (B). Mean of triplicates + standard error Strain FH134S FS 162s~

FS163S FS163K FH341 S FH342Sp FH345K FH373Sp FH374S FH375S FA451 IS FA45 13s FSSl FH2OSl

A 7.78 i_ 0.60’ a 9.66 + 0.54 bed

B 7.68 + 0.47

5.25 + 5.28 + 8.06 + 0.69 a 5.59 f 8.67 k 0.37 ab 9.69 + 8.81 F 0.24 ab 10.22 + 10.90 F 0.32 d 9.57 I 0.56 bed 10.17 + 8.59 f 9.43 F 0.32 bc 9.82 f 0.39 bed 10.00 + 9.64 i 10.25 f 0.52 cd 9.88 f 0.07 bed 9.00 i 8.47 + 0.24ab 7.70 f 0.90 a 9.22 + 0.79 ab

Ratio B:A

0.38 0.59 0.41 0.70 0.25 0.43 0.40 0.22 0.39

0.84 5.98 + 0.49 5.68 i 0.85 8.36 + 0.69

I Numbers in the column not sharing differ significantly (P < 0.05).

0.98

0.54 0.66 0.64 1.10 0.94 I .06 0.9 1 I .02 0.94 0.91 0.71 0.74 0.91

a common

letter

shoot dry weights differently for the various strains. The differences between strains in terms of nodulating competitiveness with an ineffective strain were not related to the differences in symbiotic effectiveness. In order to have the estimation of competitiveness independent of the effectiveness, the ratio of the shoot dry weight of plants inoculated with the effective-ineffective strain mixture to the shoot dry weight of plants

inoculated with the effective strain alone was calculated; it varies from 0.54 to 1.10 (Table 3). The proportion of nodules formed by the strains of R. /egumino.scrrum when inoculated at two different levels onto I/. ,ftihu in the field experiments, are presented in Table 4. The inoculant strains differed in their ability to compete with the indigenous strains for the formation of nodules. Decreasing the number of applied bacteria decreased their percentage recovery in nodules but the ranking of strains according to their competitiveness was not different (P < 0.05) for the two inoculation levels. For the X strains which were tested twice in successive years on the same soil type, there was no significant difference (P cc 0.01) between experiments in the distribution of inoculant strain. If the ranking of strains of R. Irguminosurum for competitiveness as it was found in the greenhouse test, is compared to the performances of the same strains in the fields. we observe a significant correlation between the results of the greenhouse test and those of the field experiment at the two levels of inoculation (dose 1 r = 0.890. dose 2 r = 0.748); this correlation for the dose 1 is shown m Fig. 3. DISCUSSION

When effective strains differ in their competitiveness the number of nodules they will form when they are mixed with a similar but ineffective strain and inoculated onto plants, will be dependent on their

Table 4. Percentage of nodules formed by the inoculum strain applied at two different rates to V. ,faba planted in a soil containing effective nodulating bacteria (30 nodules identified for each treatment on 3 sampling dates:pooled data)

Inoculum strain FH134S FS 162s~ FS163S FS163K FH341S FH342Sp FH345K FH373Sp FH374S FH375S FA45llS FA4513S FSSl FHZOSl

Experiment 1 Log rhizobia “/b Nodules applied per 5 cm of inoculum of row strain 9.2 7.2 8.8 6.8 9.2 7.2 8.7 6.7 9.3 7.3 9.8 7.8 9.3 7.3 8.0 6.0 9.3 7.3 8.1 6.1 8.1 6.1 9.4 6.4 9.1 7.1 9.5 7.5

40 12 3 0 29 9 1 0 67 24 77 36 90 46 50 30 88 51 64 27 42 33 37 9 20 2 67 53

Experiment

2

Log rhizobia

“<,Nodules

applied per 5 cm of row

of inoculum strain

9.2 7.2 9.1 7.1

42 IO 16 8

9.3 7.3

93 65

9.5 7.5

94 66

9.3 7.3 9.2 7.2 8.4 6.4 9.2 7.2

19 6 25 15 9 2 89 37

Selection

of competitive

345 374,s

R=0.870

342

.

20 SI ,3p

K

.

SP 341 .

/

s

1

0.6

0.7 Greenhouse

0.6 test:

0.9

1.0

I.1

B/A

Fig. 3. Relationship between the percent recovery of inoculant strain in nodules of V. firh~~planted in a soil containing indigenous R. Ir!/ltn,i,lo.~u,lr,,l and the proportion of the dry weiiht of V. /U/XI inoculated with an effective strain mixed with the ineffective strain in a 1 to 100 ratio (B). to the dry weight of plants inoculated with the effective strain (A).

relative competitiveness. The experiments with alfalfa show that the shoot dry weights of plants receiving a mixed inoculum of effective and ineffective strains were directly proportional to the log of the number of effective nodules. This was verified with two effective strains and two varieties of alfalfa. In these conditions. the shoot dry weights should be indicative of the competitive ability for nodule formation of the effective strains as the more competitive an effective strain will be. the greater the number of nodules it will form and the heavier the dry weight of the plants will be. The shoot dry weight being proportional to the log of the number of effective nodules, this number should be kept rather low in order to be not too imprecise. which means that the ratio of effective strain to ineffective strain should be less than l-10 (l-100 has been proved to be satisfactory in most cases). Strains of identical effectiveness may form a different number of nodules with a same host but this characteristic did not correlate with the competitiveness of the strains (Marques Pinto YI ~11..1974). When the competitiveness is evaluated by the number of nodules the strain formed on a host inoculated with a mixture of strains. a sparsely nodulating strain might appear to be less competitive than an abundantly nodulating strain. Sparse nodules are generally larger and fix nore nitrogen. The same number of effective nodules on plants inoculated with effective-ineffective mixtures of strains would produce more dry matter with a sparsely-modulating effective strain than with an abundantly-nodulating effective strain; the slopes of the regression line as presented in Fig. 2 should then be different. Evaluating competitiveness by comparing shoot dry weight of such plants might therefore be more reliable than counting nodules as the differences between nodulating ability of the strains would be taken into account. This of course needs to be checked with combinations of host and bacterial strains having different nodulating abilities. Using this method differences in competitiveness were found among I4 strains of R. /eyumino.swrum.

Rhizohium

485

These strains being different in their effectiveness, in order to eliminate these differences which could alter the results of the test, the strains were rated according to the ratio of the shoot dry weight of plants receiving mixed inoculum to the shoot dry weight of plants receiving only the effective strain. When the same strains were used as inoculant in a field trial on a soil containing indigenous R. Irguminosarunz, they were more or less successful in forming nodules, The estimation of competitiveness by the measurement of the dry weight of plants inoculated with a mixture of effective and ineffective strains was in good agreement with the ability of the strains to compete successfully with indigenous rhizobia in the complex environment in the field. If the selection of strains had been made according to the results of the test with ineffective strains, it would have given a good idea of the strains which would have the best chances of success in forming nodules when applied to soil containing indigenous rhizobia. It should also be noted that the more competitive strains were the strains derived from FH34 and FH37 whereas the less competitive were mutants of FS16 which suggests that the wild type strains were different in their competitiveness although very similar in terms of effectiveness (Amarger, 1975). The fact that there was a good correlation between the greenhouse test where the strains had to compete only with ineffective rhizobia for nodule sites and the field test where they had to survive in a soil environment before competing with other rhizobia to form nodules, indicates that in the overall phenomenon of competition the ability to be selected for by the plant host plays an important part. This finding needs to be confirmed with different strains of Rhizohium and different species of legumes. If is proves to be right with other combinations the evaluation of competitiveness for nodulation could therefore be useful when there is a need to select strains able to nodulate plants when inoculated onto seeds to be planted in soils containing established rhizobia. This evaluation by the test proposed here is very easy to carry out: the effective strains do not have to be marked. it just requires a strain forming ineffective nodules with the considered host, such a strain can be found in collections. Although being not very precise as it just permits the ranking of strains with reference to others. it should allow for comparison to be made of a great number of strains as to their respective competitive ability in nodule formation and thus allow a selection on this criterion. Ackncj~~/edyr,nent.~-;-I thank for technical assistance.

M. Gaudry

and J. P. Lobreau

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486

N. AMARGER

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Selection

of

competitive Agronomy

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