Variation in acid soil tolerance among strains of Rhizobium phaseoli

Field Crops Research, 5 (1982) 121--128 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

121

VARIATION IN ACID SOIL TOLERANCE AMONG STRAINS OF RHIZOBIUM PHASEOLI

P.H. GRAHAM, S.E. VITERI, F. MACKIE ~, A.T. VARGAS 2 and A. PALACIOS 3

Centro Internacional de Agricultura Tropical (CIA T), AA 6713, Call (Colombia) IPresent address: Laboratorio de Microbiologfa, Universidad Nacional San Cristbbal de Huamanga, Ayacucho (Peril) 2Present address: EMCAPA, Vitoria, ES (Brazil) 3Present address: UFRRJ, Km 47, Seropedica, Rio de Janeiro (Brazil) (Accepted 29 October 1981)

ABSTRACT Graham, P.H., Viteri, S.E., Mackie, F., Vargas, A.T. and Palacios, A., 1982. Variation in acid soil tolerance among strains of Rhizobium phaseoli. Field Crops Res., 5: 121--128. Response of bean (Phaseolus vulgaris L.) cultivars to inoculation with Rhizobium phaseoli under field conditions in Latin America has been inconsistent, one of the contributory factors being the low soil pH in many regions of bean production. In this study the tolerance of 55 strains of Rhizobium phaseoli to acid pH, aluminium (A1) and manganese (Mn) in nutrient medium was examined and the tolerance of these stresses related to the survival and nodulating ability of strains of R. phaseoli in acid soils. At pH 4.6 only six of the 55 strains tested produced isolated colonies on modified Keyser--Munns medium. Eight strains grew well on pH 4.7 medium to which 6 ppm AI had been added, but only six of these grew when the Al was replaced with 50 ppm Mn. When two strains differing in tolerance to pH in nutrient medium were introduced into an Ultisol from Santander de Quilichao, Colombia, which had been limed to differing pH values, the tolerant strain CIAT 899 survived better from pH 4.15 to 4.90. In a field trial with soil limed to pH values from 3.8 to 4.4, the percentage of plants nodulated and the number of nodules per plant was greater when CIAT 899 was used as inoculant, instead of the strain CIAT 632, shown sensitive to pH in both nutrient media and soil tests. In this trial granular, soil applied inoculants gave better results than those which were seed applied. In a second field trial, on a Typic Distrandept limed from pH 4.45 to 5.20, yields of P. vulgaris were enhanced by inoculation, but again CIAT 899 performed better than did CIAT 632. In this trial there was no significant difference between inoculation methods. The possible value of granular inoculation methods to the production of P. vulgaris under small-farm conditions is discussed.

INTRODUCTION

Cultivation of dry beans (Phaseolus vulgaris L.) in Latin America occurs predominantly on small, near subsistence holdings and uses minimal tech-

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122 nical inputs (De Londofio et al., 1978). Nitrogen (N) deficiency is a common production constraint (Diaz-Romeu et al., 1970; Malavolta, 1972; Bazan, 1975) but N-fertilization can be uneconomic in regions of inconsistent, and often limited rainfall. Responses to inoculation with Rhizobium phaseoli have also been extremely variable (Graham and Haliday, 1977),a contributory problem being the relatively low soil pH in many regions of bean production. Recent studies have demonstrated acid soil tolerance among strains of slow-growing rhizobia (Date and Halliday, 1977; Keyser and Munns, 1979; Munns et al., 1979). Some variation in the ability of strains of Rhizobium phaseoli to grow at acid pH has also been noted {Graham and Parker, 1964}. This study examines strains of bean rhizobia for tolerance to low pH, aluminium (A1) and manganese (Mn) in nutrient medium, and relates tolerance of these stresses to the ability of isolates to survive and induce nodulation under acid soil conditions in the field. MATERIAL AND METHODS

Growth on agar medium For the initial evaluation of strain tolerance to low pH and high A1 and Mn, 55 strains of R. phaseoli from the CIAT Rhizobium collection were cultured on a medium modified from that of Keyser and Munns (1979) and of pH adjusted to values between 4.5 and 7.0. The medium contained (ppm): glycerol, 5000; K2SO4, 131; sodium glutamate, 220; MgSO4" 7H20, 74; K2HPO4, 1.36; CaC12"2H20, 7; iron EDTA, 35; MnC12"4H20, 0.25; ZnSO4"5H20, 0.11; CuC12"2H~O, 0.017; Na2MoO4-2H20, 0.004; thiamineHCI, 0.4; calcium pantothenate, 0.4; biotin, 0.001, and agar 20.000. The medium was dispensed, autoclaved at 15 psi pressure for 15 min, cooled to 52°C, then the pH adjusted and plates poured. In subsequent experiments this medium was supplemented with 50 ppm Mn as MnSO4"2H20 or 6 ppm Al as A1C13"6H20. Survival o f R. phaseoli in acid soil When strains tested on the medium described above proved variable in ability to grow at pH 4.5, an experiment was undertaken to compare strains tolerant or intolerant of this pH for survival in acid soft. An Ultisol from Santander de Quilichao, Colombia varying in pH from 3.8--4.1, with 60% A1 saturation of the effective cation exchange capacity and 25 ppm Mn in 1 N KC1 (CIAT, 1977) was used. The soil was limed to pH 4.15, 4.50, 4.90 or 5.80 with dolomitic limestone 3 weeks before the survival study was initiated. The experiment compared two strains, CIAT 899 and CIAT 640, and for ease in counting used mutants of each resistant to 200 ppm streptomycin and 200 ppm spectinomycin. For each strain--pH combination four 2-kg

123 quantities of soil were prepared and milled, then the appropriate Rhizobiurn strain added to a final concentration of approximately 107 cells per gram of soft. The soils were then stored at 24°C and 10% moisture content. Rhizobium numbers surviving in soil were determined by serial dilution of two 10-g samples per treatment at time 0, and at 2, 5, 8 and 16 days thereafter Yeast extract plus congo red medium (Vincent, 1970) containing actidione, streptomycin and spectinomycin was used in the preparation of pour plates, with Rhizobium colonies counted after 3 days incubation at 28°C.

Nodulation studies To determine whether the strain differences identified in survival studies had field implications, two inoculation trials were undertaken in acid soils. In the first experiment two strains of R. phaseoli differing in tolerance to acid pH in culture medium, were evaluated for nodulating ability in the Santander de Quilichao soil. Four levels of lime (0, 2, 4 and 6 t/ha; final pH 3.8, 4.0, 4.2 and 4.6 respectively) were used, with the lime broadcast and incorporated 3 weeks before planting. Fertilizer, applied broadcast at planting, included (kg/ha); KC1, 100, MgSO4.7H20, 100; ZnSO4.5H20, 7; H3BO3, 1; and Na2MoO4, 1. Triple superphosphate at the rate of 300 kg P2Os/ha was banded alongside the seed row, and 10 cm from it. Strains CIAT 899 and CIAT 632 were used as inoculants; in one treatment each strain was suspended in 10% sucrose and applied directly to seeds of P. vulgaris (final concentration approximately 10 s rhizobia per seed); in the other, granular inoculants were prepared (Graham et al., 1980) and applied beneath the seed at the rate of I × 10 s rhizobia per m of row. Ten 5-m rows of seed were planted for each treatment in a split plot design. Nodule number per plant and percentage plants nodulated were determined from 40 ten-plant samples per treatment, 35 days after planting. The second inoculation trial was undertaken on a Typic Distrandept near Popayan, Colombia. The properties of this soil and its fertilization for bean production have been detailed by Graham and Rosas (1977). In this trial only three levels of lime (0, 2 and 4 t/ha, final pH 4.50, 4.75 and 5.20 respectively) were used, with the lime and appropriate mixed fertilizers broadcast as in the previous experiment. Four inoculation treatments were used: no inoculation; slurry inoculation of seeds, as described above; inoculation and lime pelleting of seeds as described by Date (1965); and granular, soil-applied inoculation (Graham et al., 1980). For each inoculation method duplicate, replicated plots were prepared; one series used CIAT 899 as the inoculant, the other CIAT 632. Six 5-m rows of seed were planted for each treatment in a split plot design. Details of nodulation and of the persistence of strains over time will be presented separately. This paper reports only yield data taken from a subplot 4 m 2 per replication.

124 RESULTS AND DISCUSSION While 51 of the 55 tested strains grew well on modified Keyser--Munns medium at pH 5.0 only six strains (CIAT Nos. 1 4 4 , 3 0 9 , 895, 8 9 6 , 8 9 8 , and 889) produced isolated colonies on this medium at 4.5. Most strains showed visible growth only in the region of the heaviest inoculation (see Fig. 1). At pH 4.7, CIAT strains 57 and 255 also grew and all eight strains grew well at this pH on medium containing 6 ppm Al. Strains 309 and 898 did n o t grow on medium of pH 4.7 to which 50 ppm Mn had been added. Dobereiner (1966) reported that levels of Mn as low as 25 ppm could affect the nodulation of P. vulgaris in acid soils.

Fig. 1. Growth o f two strains of R. phaseoli on modified Keyser--Munns medium at pH 4.5, 5.0 and 7.0.

When the strains CIAT 899 and 640 (the former producing isolated colonies on modified K e y s e r - M u n n s medium at pH 4.5, the latter unable to grow at this pH) were compared for ability to survive in acid soil, marked strain--pH interaction was evident (see Fig. 2). Thus, in soil of pH 4.15, counts of strain 640 fell from more than 107 cells to less than 102 cells per gram of soil in only 5 days, while more than 103 cells of strain CIAT 899 per gram of soft could be recovered even 15 days after inoculation. CIAT 899 also performed better in both field inoculation studies. At Santander de Quilichao plots inoculated with CIAT 899 showed a higher percentage of plants nodulated, and more nodules per plant than plots in-

125

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8 12 Days o f t e r inoculotlon

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Fig. 2. Survival o f two strains of R. phaseoli in an acid soil limed to pH values from pH 4.15 to 5.80. Legend: CIAT 899; . . . . . . CIAT 640. • pH 4.15; o pH 4.50; pH 4.90; • pH 5.80. TABLE I

Influence of inoculant method and strain of Rhizobium phaseoli used on percentage plants nodulated in an acid soil with differing lime amendments a,b Strain used

CIAT CIAT CIAT CIAT

Inoculation method

899 899 632 632

Granulated Seed applied Granulated Seed applied

Lime applied (t/ha) 0 pH 3.8

2 pH 4.0

4 pH 4.2

6 pH 4.6

80.00 30.25 37.25 6.25

87.50 74.75 64.75 17.50

79.00 42.25 28.50 13.00

83.25 25.75 65.50 19.50

a b b c

a ab bc d

a b bc c

a b a b

aAverage for 40 ten-plant samples per treatment. bValues for each level o f lime not followed by the same letter are significantly different at the 5% level.

oculated with strain 632 in all treatments except those receiving 6 t/ha of lime (see Tables I and II). Some nutrient imbalances were evident in the 4 and 6 t/ha lime treatments, with nodule number per plant in these plots less than when only 2 t/ha of lime was applied. In this trial, and for both strains, granular soil applied inoculants proved better than those applied directly to the seed. A striking response to inoculation was obtained in the Popayan trial; plots inoculated with CIAT 899 outyielded the uninoculated control plots by more than 500 kg/ha at each lime level (Table III). CIAT 632 again appeared

126 TABLE II Influence of inoculant m e t h o d and strain of Rhizobium phaseoli used on nodule number per plant in an acid soil with differing lime amendments a.b Strain used

CIAT CIAT CIAT CIAT

Inoculation method

899 899 632 632

Granulated Seed applied Granulated Seed applied

0 pH 3.8

2 pH 4.0

4 pH 4.2

6 pH 4.6

14.43 3.62 2.08 0.64

16.91 8.48 6.85 1.37

11.58 4.25 1.39 0.60

11.88 2.25 5.18 0.71

a b bc c

a b b c

a b c c

a b ab b

aAverage for 40 ten-plant samples per treatment. bValues at each level of lime not followed by the same letter are significantly different at the 5% level. TABLE III Influence of strain and m e t h o d of inoculation used, and lime applied, on the yield (kg/ha) of P. vulgaris cv. BAT 76 in Popayan Strain and inoculation method used

Lime applied (t/ha) 0 pH 4.50

2 pH 4.75

4 pH 5.20

N ot inoculated

1791

1881

2325

Inoculation with CIAT 632 a. Slurry inoculated b. Lime pelleted c. Granular, soil applied

1475 1969 1863

2385 1904 2163

2875 3147 2135

Average

1769

2150

2719

Inoculation with CIAT 899 a. Slurry inoculated b. Lime pelleted c. Granular, soil applied

2575 1912 2442

2508 2674 2160

2530 2865 3083

Average

2309

2447

2826

F (inoculation), 12.14"*; F (lime level), 10.39"*; F (inoculant strain), 6.19"; F (method of inoculation) and all interaction terms, ns at 5% level.

suspect at low pH: no response to inoculation was obtained at pH 4.50, but at pH 5.20, yields in plots inoculated with CIAT 632 were n o t significantly different from those in which CIAT 899 inoculant was used. In this trial there was no difference between the methods of inoculation employed. It is evident from these results that variation in tolerance to acid soil fac-

127

tots does exist in R. phaseoli and can contribute to strain survival in soil and subsequent nodulation. Given the acid soil conditions in many regions of bean production, acid soil tolerance should be taken into consideration in selecting strains of R. phaseoli for use in inoculants. Preselection of strains by their growth on modified Keyser--Munns medium at pH 4.5--5.0 appears justified. Longer term studies to evaluate population shifts among inoculant strains in acid soils, and the effects of pH on strain competition for nodulation sites also seem warranted. Yield increases in excess of 500 kg/ha were obtained following inoculation at Popayan, and clearly justify continued studies to adapt inoculant technology for use by small scale bean farmers. While the slurry inoculation and lime pelleting methods popular in developed countries could be accepted, the relative complexity of these methods could cause problems at the small farm level. Granular inoculants applied in the seed hole or furrow could be more readily accepted. While not yet extensively tested in the tropics initial reports with granular inoculants applied under acid soil conditions appear promising. Thus Graham et al. (1980) reported excellent nodulation of P. vulgaris using granular inoculants in moderately acid soil while Brockwell et al. (1980) also found granular inoculants as good as, and frequently better than, seed applied inoculants. These authors provided no pH data, but 12 of the 16 trials they reported were on soils described as Podzolic. The results obtained in the present study support these observations, the granular inoculants again performing as well as, or better than the other inoculation methods tested. Further studies at the small farm level are, however, needed.

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128 Graham, P.H. and Halliday, J., 1977. Inoculation and nitrogen fixation in the genus Phaseolus. In: J.M. Vincent, A.S. Whitney and J. Bose (Editors), Exploiting the Legume Rhizobium Symbiosis in Tropical Agriculture. Univ. Hawaii College Trop. Agric. Misc. Publ. 145, pp. 313--334. Graham, P.H. and Parker, C.A., 1964. Diagnostic features in the characterization of the root-nodule bacteria of legumes. Plant Soil, 20 : 383--396. Graham, P.H., Ocampo, G., Ruiz, L. and Duque, A., 1980. Survival of Rhizobium phaseoli in contact with chemical seed protectants. Agron. J., 72: 625--627. Keyser, H.H. and Munns, D.N., 1979. Tolerance of rhizobia to acidity, aluminium and phosphate. Soil Sci. Soc. Am. J., 43: 519--523. Malavolta, E., 1972. Nutri~o e aduba~'o. In: Anais do I Simp6sio Brasileiro de Feij~o. Universidade Federal de Vi~osa I, pp. 211--242. Munns, D.N., Keyser, H.H., Fogle, V.W., Hohenberg, J.S., Righetti, T.L., Lauter, D.L., Zaroug, M.G., Clarkin, K.L. and Whitacre, K.W., 1979. Tolerance of soil acidity in symbioses of mung bean with rhizobia. Agron. J., 71: 256--260. Vincent, J.M., 1970. A Mannual for the Practical Study of Root-Nodule Bacteria. IBP Handbook No. 15. Blackwell Scientific Publications, Oxford, 164 pp.