Survival of Rhizobium leguminosarum in soil after addition as inoculant

FEMS MicrobiologyEcology45 (1987) 221-226 Published by Elsevier

221

FEC 00124

Survival of Rhizobium leguminosarum in soil after addition as inoculant E.S. J e n s e n and L.H. S o r e n s e n Agricultural Research Department, Riso National Laboratory, Roskilde, Denmark

Received 25 February 1987 Revision received27 April 1987 Accepted 27 April 1987 Key words: Rhizobium leguminosarum; Soil; Survival

1. S U M M A R Y

2. I N T R O D U C T I O N

Three streptomycin-resistant (str r) strains of the root-nodule bacteria Rhizobium leguminosarum biovar oiceae were added to a field soil harbouring an indigenous population of R. leguminosarum. Three or 4 years later more than 10 4 bacteria remained per gram of soil. The size of the str r population decreased with time, its 'half-life' ranged between 1.2 and 2.1 years. The survival was positively influenced by host-legume, nonhost-legume and non-legume crops. The highest and lowest survival rates were found in soils cropped with a host legume every year, and in soil kept fallow, respectively. The percentage of nodules on pea plants (Pisum satioum L.) occupied by the str r rhizobia decreased during the 3- or 4-year period following the introduction of the str r rhizobia to the soil.

Soil bacteria belonging to the genus Rhizobium are able to form a Nz-fixing symbiosis with leguminous plants. Rhizobium leguminosarum biovar viceae forms nodules on pea, field bean, lentil and vetch, which are commonly grown in Europe. Indigenous populations of this Rhizobium species are present in most soils in amounts of 103-10 5 b a c t e r i a . g - 1 of soil, even without cropping of host legumes for many years [1,2]. This indicates that R. leguminosarum may be able to persist saprophytically for a long time in the field. Rhizobia, like other soil bacteria, are subject to bacteriostasis, lysis, parasitism and predation, and to adverse physical or chemical factors, and it is also known that different strains of Rhizobium may differ in their ability to persist in soil [3,4]. In the presence of the host, Rhizobium is stimulated to multiply rapidly, but also non-host-legume and non-legume rhizospheres may stimulate the growth of rhizobia [5-7]. Consequently, the farming practice, especially the number of legume crops in the rotation, may influence the survival of rhizobia in soil [1]. Inoculation of legumes with rhizobia is needed

Correspondence to: E.S. Jensen, Agricultural Research Department, Rise National Laboratory, DK-4000 Roskilde, Denmark.

0168-6496/87/$03.50 © 1987 Federation of European MicrobiologicalSocieties

222 if (i) the appropriate Rhizobium species is absent from the soil, (ii) the indigenous strains are ineffective or poorly effective, or (iii) if new strains with improved effectiveness or other beneficial traits developed by genetic engineering become available. The inoculum must be able to survive for at least one growth season, and compete with indigenous populations if such are present in the soil. Strains of Bradyrhizobium japonicum, which nodulate soybeans, have been reported to survive well in the field for more than 5 years, even without cropping of the host legume [8]. In another experiment with soybean rhizobia, it was reported that the percentage of nodules formed by the introduced strain increased with time indicating a permanent establishment and a high competitive ability [9]. The aims of the present work were to utilize soil removed from a field experiment to a further study on (i) the survival of strains of R. leguminosarum biovar viceae introduced as inoculant for pea to a soil, harbouring indigenous populations of these bacteria; (ii) the effect of host-legume, non-host-legume and non-legume rhizosphere on the survival compared to soil without plants (long-term fallow) and (iii) the ability of the introduced strains and the indigenous rhizobia to form nodules on pea plants. Streptomycin-resistant (str ~) mutant strains of R. leguminosarum were selected and used for these purposes [3,4,6,10-13].

3. MATERIALS A N D M E T H O D S 3.1. Rhizobium strains Three strains of Rhizobium leguminosarum biovar uiceae were studied, Riso l a (effective strain) and SV10 (ineffective strain) both isolated from Riso soil, and R1045 (effective strain) from the Rothamsted Rhizobium Collection. Spontaneous mutants resistant to streptomycin were isolated from yeast mannitol agar media [14] containing 110 # g - m l 1 streptomycinsulfate. The mutant strains were tested for symbiotic effectiveness.

3.2. The soil The soil was a loamy sand (26% coarse sand,

43% fine sand, 15% silt, 14% clay and 2% organic matter in 0-20 cm depth). The p H . 2 o was 8.0. The indigenous populations of R. leguminosarum biovar viceae were 8.0 x 103 and 3.0 x 103 cells • g- 1dry soil as determined by plant infection ( Vicia hirsuta L.) and MPN estimation [14] in 1982 and 1983, respectively, when the field experiments were started [13]. The following inoculum rates were used: 3.7 × 10 9, 3.8 × 10 6 and 2.9 × 10 6 cells. cm l row (row distance 15 cm) of Riso la str r, SV10 str r and R1045 str r, respectively. After harvest of pea plants soil was sampled from the upper 10 cm layer within the inoculated plots, sieved, homogenized and placed in PVC-pots (diameter 23 cm, height 26 cm) provided with bottom holes for free drainage of excess water. The soil was removed from the field because the stV rhizobia were not considered to be homogeneously distributed in the soil. The pots were buried in the field with the soil at the same level as the field soil. A 5-cm rim was above the soil surface to avoid contamination from surrounding soil. All precautions were taken to avoid cross-contamination between samples. Each pot contained soil corresponding to 12.5 kg air-dry soil. Two pots were also included with soil not inoculated with str r rhizobia.

3. 3. Plant material and experimental layout Pots in Experiment 1 (see Table 1) were cropped with pea (Pisum sativum L. cv. 'Bodil') (10 plants per pot) or spring barley (Hordeum vulgare L.) (20 plants per pot) or kept free from plant growth. In Experiment 2 (see Table 2) pots were cropped with pea (cv. 'Bodil'), perennial ryegrass (Lolium perenne L.) (50 plants per pot at germination), lupin (Lupinus luteus L. or Lupinus albus L.) (10 plants per pot) or kept free from plant growth. Pots cropped with barley or perennial ryegrass were fertilized with 1 g N O ~ - N • pot-1 ~ 240 kg N - h a 1 per growth season. Pots were cropped during June to August, and the treatments were imposed on the same experimental unit during 4 (Exp. 1) or 3 (Exp. 2) years. All pots were watered during the growing season, and weeds were removed from the fallowed pots. Crops were harvested by cutting the plants at the soil surface. The soil was left undisturbed

223 Table 1

Table 2

Survival of Rhizobium leguminosarum biovar viceae strain Riso la str r in field soil kept fallow or cropped with pea (host) or barley

Survival of Rhizobium leguminosarum biovar viceae strains SV10 str r and R1045 str r in field soil kept fallow or cropped with pea (host) or lupin or perennial ryegrass

Experiment 1.

Experiment 2.

Day

Day

Log10 no. of cells, g- 1 dry soil

0 70 265 325 ;' 421 553 714 ~ 905 I 051 1305 1 444 ~ a

Pea

Barley

Fallow

LSDoo5

5.41 5.36 5.21 5.82 5.27 4.98 4.98 4.87 4.78 4.70 4.38

5.10 4.96 4.89 4.81 4.76 4.63 4.72 4.61

-

-

5.11 4.93 4.75 4.54 4.63 4.28 4.53 4.34

0.25 0.28 NS b 0.31 0.15 NS NS NS

Sample time after cropping of pea or barley. NS = differences not significant at the 5% level of significance.

u n t i l n e x t s p r i n g w h e n t h e s u r f a c e was l i g h t l y tilled b e f o r e p l a n t i n g o f the n e w crop. P o t s w i t h r y e g r a s s w e r e n o t t r e a t e d a n d c a r r i e d the s a m e p l a n t s d u r i n g the e n t i r e e x p e r i m e n t . T h e e x p e r i mental designs were randomized blocks with three r e p l i c a t e s for e a c h strain. 3.4. P l a t e c o u n t s o f str r r h i z o b i a in soil Soil was s a m p l e d 2 o r 3 t i m e s e a c h y e a r to e n u m e r a t e the str r r h i z o b i a . S a m p l e s ( 1 5 - 2 0 g) w e r e t a k e n w i t h a soil a u g e r ( d i a m e t e r 8 r a m ) at 4 - 6 p o i n t s to the b o t t o m o f the pot. P o r t i o n s of 10 g of n o n - d r i e d soil w e r e s u s p e n d e d in 90 m l o f sterile w a t e r for 10 rain o n a m i x e r , a n d d i l u t i o n series w e r e p r e p a r e d . Y e a s t m a n n i t o l a g a r [14] c o n t a i n i n g 110 / ~ g . m 1 - 1 s t r e p t o m y c i n , 40 /~g. m l - ~ c y c l o h e x i m i d e (to c o n t r o l c o n t a m i n a n t g r o w t h ) a n d 2.5 ~ g . ml ~ c o n g o red was p o u r e d i n t o petri d i s h e s c o n t a i n i n g 1 m l of d i l u t e d soil s u s p e n s i o n . T h e p l a t e s (o.d. 9 cm, f o u r r e p l i c a t e s ) w e r e i n c u b a t e d for 4 - 6 d a y s at 28 o C a n d c o u n t e d . T h e c o e f f i c i e n t of v a r i a t i o n o n r e p l i c a t e p l a t e c o u n t s was n o r m a l l y less t h a n 15%. C o u n t s are e x p r e s s e d as logt0 no. o f c e l l s - g - 1 d r y soil (105 ° C in 24 h). T h e m e t h o d was t e s t e d b y m e a n s of soil s a m p l e s w i t h a n d w i t h o u t a k n o w n a m o u n t o f str r

Loglo no. of cells, g-1 dry soil Pea

Lupin

Perennial ryegrass

Fallow

LSDo.o5

Strain S V10 str r 0 5.53 97 5.62 240 5.47 385 a 5.45 578 5.46 722 a 5.36 983 5.06 1 115 a 5.10

5.73 5.33 5.62 5.46 5.51

5.35 5.56 5.15 5.25 5.20

5.33 5.27 4.66 4.76 4.92

0.27 NS 0.35 0.35 0.36

Strain R1045 str ~ 0 6.52 97 5.29 240 5.14 385 a 5.14 578 4.89 722 ~ 4.47 983 4.81 1 115 a 4.78

5.17 4.67 5.16 5.24 5.12

5.18 4.92 4.63 4.66 4.36

5.14 4.68 4.24 4.48 4.39

NS NS 0.36 0.56 0.27

a Sample time after cropping of pea, lupin and perennial ryegrass. NS = differences not significant at the 5% level of significance.

r h i z o b i a a d d e d to the soil u n d e r l a b o r a t o r y c o n d i t i o n s b e f o r e i n i t i a t i n g the e x p e r i m e n t s . 3.5. N o d u l e o c c u p a n c y b y str r r h i z o b i a P o t s w i t h R h i z o b i u m - f r e e p e a p l a n t s (10 p e r p o t ) in g r a v e l w e r e i n o c u l a t e d w i t h 1 - m l a l i q u o t s o f the soil s u s p e n s i o n s p r e p a r e d for the p l a t e c o u n t s . A f t e r a s u i t a b l e t i m e ( 3 - 4 weeks) t h e p r o p o r t i o n o f n o d u l e s o n the p l a n t s o c c u p i e d b y str r r h i z o b i a was d e t e r m i n e d ( f o r d e t a i l s see [13]).

4. R E S U L T S

AND

DISCUSSION

4.1. E v a l u a t i o n o f the p l a t e c o u n t i n g m e t h o d T h e c o n c e n t r a t i o n s o f the i n h i b i t o r s in the m e d i a did n o t c o m p l e t e l y e l i m i n a t e g r o w t h o f the o r d i n a r y soil o r g a n i s m s , w h i c h a p p e a r e d o n the

224 plates after 3 days of incubation as tiny colonies in a number of about 100 × 10 4 per g of soil. The growth of these colonies was inhibited, the diameter did not exceed 0.5 mm even after prolonged incubation; their congo red-dependent colour indicated Gram-positiveness. On plates inoculated with suspensions of soil containing str ~ rhizobia, colonies resembling typical rhizobia appeared after 3 days of incubation. The growth of these colonies continued, and after 5 days they were circular, convex, shining, translucent to slightly whitish, with distinct borders and a diameter of 5-6 mm. Microscopic examination and tests for ability to form root nodules on pea plants demonstrated that these colonies were formed by rhizobia. A few of the atypical colonies mentioned above continued to grow, but they were clearly distinguishable from the typical ones as the colonies were flat, non-shining and with irregular indistinct borders. Isolates from a variety of such colonies were never found to form nodules on pea plants. When the experiments were terminated these tests were performed again with the same results. This indicated that the method was suitable for enumerating the str r rhizobia in soil, as also quoted by Bushby [12]. 4.2. Surc, i~'~al o f str r rhizobia added to the soil as inoculum The strains Riso l a str r, SV10 str r and R1045 occupied on average 87, 58 and 75% of the nodules, respectively, on the main root of field-grown pea plants 3 weeks after seedling emergence [13]. These figures indicate that the strains survived well during the 2-3-week period from sowing and inoculation to seedling emergence and infection, but it is unknown whether the number of bacteria in the soil decreased from the time of inoculation to soil sampling. The figures for day zero in Tables 1 and 2 indicate the number on the day when the soils were sampled, approximately 4 months after sowing and inoculation. The number of Riso la str r, SV10 str ~ and R1045 str ~, respectively, decreased from 2.6 × 105, 3.4 x 105 and 3.3 x 106cells- g 1 soil to 2.2 X 10 4, 8.3 X 10 4 and 2.5 × 104 cells • g 1 soil after 4, 3 or 3 years in fallow soil, respectively (Tables 1 and 2). This indicates that the strains survived well in

fallow soil together with an indigenous population of rhizobia. Nutmann and Hearne [1] found that in soil fallowed continuously for 18 years, the population of R. leguminosarum biovar uiceae was reduced to about 20 per g. This was presumably due to the effect of fallow. In fallow soil carbon available to rhizobia is scanty and therefore they will gradually disappear. However, rhizobia can survive for a long time in soil. It has been reported that cells remained viable in soil which was kept dry for more than 30 years [15]. The survival of the three strains was higher in the plant rhizospheres, whether belonging to barley, ryegrass or legumes, than in fallow soil. Differences between fallow and pea were significant only in the case of Riso la str r during the first 3 years (Table 1). The pea plant improved the survival of strains SV10 str r and R1045 str r compared to fallow soil, but the improved survival was only at a few sampling dates statistically significant (Table 2). The non-host-legume lupin improved significantly the survival of both strains except for a few cases when the lupin was poorly established (Table 2). It is generally accepted that the growth of rhizobia in the rhizosphere is a response to nutrients (energy source, amino acids and vitamins, etc.) [16]. It is well-known that cropping of a legume stimulates the growth of its homologous rhizobia [16], as also the present experiments indicate. We found that the non-host-legume lupin was able to improve the survival of the introduced strains even more efficiently than the host-legume (Table 2). This may be because the lupin crop, being later in maturity, was able to excrete carbon substrates during a longer period. Peraa-Cabriales and Alexander [6] and Robert and Schmidt [7] among others, similarly reported that the growth of non-host-legumes can improve the growth and survival of R. leguminosarum biovar phaseoli. It has been reported [6,16] that non-legume rhizospheres stimulate rhizobial growth, but the effect was generally found to be smaller than the stimulation caused by legume rhizospheres as also found by us. The means of all treatments were regressed on time. The regressions fit well (r2: 0.77, 0.88 and 0.91 for strains R1045 str r, SV10 str r and Riso la

225

s t r r respectively) except for the high death rate of strain R1045 str r during the first 100 days, indicating a double exponential pattern of death. 'Half-life' values were calculated tentatively from the estimated slopes of the regression lines; the values were found to be 1.2, 2.1 and 1.4 years, respectively, for Rise l a str r, SV10 str ~ and R1045 s t r r.

4.3. Nodule formation by str r strains When the experiments were initiated Riso l a str r and R1045 str r occupied 69 and 43%, respectively, of the root nodules on pea plants after 3 weeks of growth, whereas the ineffective strain SV10 str r occupied only 16% of the nodules (Table 3). Nodule occupancy by the two efficient strains (la and 1045) was markedly reduced by pea cropping as compared to fallow (Table 3). The cause of this is unknown since we do not have any counts of the total number of R. leguminosarum in the soil, but it is speculated that the indigenous strains may have proliferated more on the pea root exudates than the inoculant strains. Their larger number in the soil would consequently result in a larger nodule occupancy. In contrast to the present observations, Dunigan et al. [9] reported that, once established, an introduced strain of Bradyrhizobium became competitive with the indigenous rhizobia in the soil, and each year formed a higher percentage of the nodules on the soybean root. Table 3 Percentage of nodules on pea plants occupied by streptomycinresistant rhizobia Rhizobium strain

Treatment

% of nodules occupied by str r-rhizobia Experiment start

Last sampling

Riso la str r

Pea cropping Fallow soil

69 (410) a ND

2 (230) a 30 (280)

SV10 str r

Pea cropping Fallow soil

16 (160) ND

12 (260) 2 (300)

R1045 str r

Pea cropping Fallow soil

43 (160) ND

14 (260) 29 (260)

a Number of nodules checked. N D = not determined.

Jensen [13] demonstrated that it is possible to add selected strains of R. leguminosarum to the soil in such a way that they can compete with the indigenous population for nodulation of a pea crop. The present study shows that the strains introduced to the soil were able to survive for a long time, and that plant growth improves the survival of the introduced strains. The results indicate that much attention should be given to the competitive ability of strains, when selecting or developing strains of Rhizobium for inoculation purposes.

ACKNOWLEDGEMENTS We wish to thank Merete Brink and Hanne Egerup for able technical assistance, Leif Skot for most useful criticism and Anni Sorensen for typing the manuscript. REFERENCES [1] Nutman, P.S. and Hearne, R. (1980) Persistence of nodule bacteria in soil under long-term cereal cultivation. In: Rothamsted Experimental Station, Annual Report 1979, Part 2, pp. 77-90. [2] Jensen, E.S., Engvild, K., Skot, L. and Sorensen, L.H. (1985) Nitrogen supply of crops by biological nitrogen fixation, V. Occurrence and efficiency with respect to N 2 fixation of the root-nodule bacteria Rhizobium leguminosarum, pp. 11 14. Riso Report M-2477 (in Danish). [3] Gaur, Y.D. and Lowther, W.L. (1982) Competitiveness and persistence of introduced rhizobia on oversown clover: Influence of strain, inoculation rate and lime pelleting. Soil Biol. Biochem. 14, 99 102. [4] Materon, L.A. and Hagedorn, D. (1983) Competitiveness and symbiotic effectiveness of five strains of Rhizobium trifolii on red clover. Soil Sci. Soc. Am. J. 47, 491-495. [5] Alexander, M. (1984) Ecology of Rhizobium. In: Biological Nitrogen Fixation - Ecology, Technology, and Physiology (Alexander, M., Ed.), pp. 39-50. Plenum Press, New York. [6] Pe~a-Cabriales, J.J. and Alexander, M. (1983) Growth of Rhizobium in unamended soil. Soil Sci. Soc. Am. J. 47, 81-84. [7] Robert, F.M. and Schmidt, E.L. (1983) Population changes and persistence of Rhizobium phaseoli in soil and rhizospheres. Appl. Environ. Microbiol. 45, 550-556. [8] Crozat, Y., Cleyet-Marel, J.C., Giraud, J.J. and Obaton, M. (1982) Survival rates of Rhizobium japonicum populations introduced in different soils. Soil Biol. Biochem. 14, 401-405.

226 [9] Dunigan, E.P., Bollich, P.K., Hutchinson, R.L., Hicks, P.M., Zaunbrecher, F.C., Scott, S.G. and Mowers, R.P. (1984) Introduction and survival of an inoculant strain of Rhizobiumjaponicurn in soil. Agron. J. 76, 463-466. [10] Danso, S.K.A. and Alexander, M. (1974) Survival of two strains of Rhizobium in soil. Soil Sci. Soc. Am. Proc. 38, 86-89. [11] Brockwell, J., Schwinghamer, E.A. and Gault, R.R. (1977) Ecological studies of root-nodule bacteria introduced into field environments, V. A critical examination of the stability of antigenic and streptomycin-resistance markers for identifications of strains of Rhizobium trifolii. Soil Biol. Biochem. 9, 19-24. [12] Bushby, H.V.A. (1981) Quantitative estimation of rhizobia

[13] [14]

[15] [16]

in non-sterile soil using antibiotics and fungicides. Soil Biol. Biochem. 13, 237-239. Jensen, E.S. (1987) Inoculation of pea by application of Rhizobium in the planting furrow. Plant Soil 97, 63-70. Vincent, J.M. (1970) A Manual for the Practical Study of the Root-Nodule Bacteria (IBP Handbook, No. 15). Blackwell, Oxford. Jensen, H.L. (1961) Survival of Rhizobiurn meliloti in soil culture. Nature 192, 682-683. Date, R.A. and Brockwell, J. (1978) Rhizobium strain competition and host interaction for nodulation. In: Plant Relations in Pastures (Wilson, J.R., Ed.), pp. 202-216. CSIRO, East Melbourne.