inoculation of alfalfa seed for increased yield on

INOCULATION OF ALFALFA SEED FOR INCREASED YIELD ON MODERATELY ACID SOIL W. A. RICE and P. E. OLSEN Research Station, Agriculture Canada, Beaverlodge, Alberta T)H 0C0. Contribution no. NRG 82-14, received l5 Feb. 1983, accepted 23 Mar. 1983.

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RrcE, W. A. eNo Or-spN, P. E. 1983. Inoculation of alfalfa seed for increased yield on moderateiy acid soil. Can. J. Soil Sci. 63:541-545. Methods currently available for inoculating alfalfa (Medicago sativa L.) seed with Rizobium meliLoti were evaluated to determine their ability to produce effective nodulation and to increase forage yield under moderately acid (pH 5.8-5.9) soil conditions. Five inoculation treatments were applied in a field experiment: no inoculant,5.5 g inoculant/kg seed applied without inoculant sticker,5.5 g inoculant/kg seed applied with inoculant sitcker, 50.7 g inoculant/kg seed applied with inoculant sticker, and 5.4 g inoculant/kg seed applied with a commerical seed-coating process. Alfalfa yields were not increased relative to the uninoculated control when inoculant was applied without sticker at 5.5 g inoculant/kg seed. However, when the inoculant was applied at50.'7 g/kg seed using a sticking agent alfalfa yields were increased 857o during the first harvest year and 47Vo in the second harvest year. Lower, but significant yield increases were obtained with 5.5 g inoculant/kg seed applied with a sticking agent and 5.4 g inoculant/kg seed applied with the commerical seed-coating process. In an assay conducted in the greenhouse, double antibiotic (streptomycin, rifampicin) resistant strains of R. meliloti were used to determine the proportion of nodules formed by the inoculant Rhizobium. Between 92 and lO0% of the nodules contained antibiotic resistant strains when the inoculant was applied with a sticking agent or with the seedcoating process, while only 38Vo of the nodules in the treatment without sticker contained antibiotic iesistant strains. The results demonstrate the importance of applying inoculants with a sticking agent or in conjunction with seed coating to obtain maximum yields of alfalfa on moderately acid soils. Key words: Acid soil, Rhizobium meliloti, Medicago satluzT, inoculation, antibiotic reslstanl mutants

[Inoculation de la semence de luzerne pour accroitre les rendements en sol mod6r6ment acide.l Titre abr6g6: M6thodes d'inoculation de la semence de luzerne en sols acides. On a 6vaiu6 les m6thodes actuellement disponibles d'inoculation de la semence de luzerne (.Medicago sativa L.) avec Rhizobium meliloti pour ddterminer son aptitude d produire une bonne nodulation et pour accroitre le rendement fourrager en sols mod€r6ment acides (pH de 5,8 d 5,9). On a appliqu6 cinq traitements d'inoculation en plein champ, soit absence d'inoculant,5,5 g d'inoculant/kg de semence sans adh6sif, 5,5 g/kg avec adhdsif, 5,5 g/kg avec adh6sif, 50,7 g/kg avec adh6sif et 5,4 g/kg par un proc6dd commercial d'enrobage. Les rendements n'augmentent pas par rapport au t6moin non trait6 lorsque I'inoculant est appliqu6 sans adh6sif d raison de 5,5 g/kg. Mais la dose de 50,7 g/kg avec adh6sif relbve les rendements de 85o/o la premidre annde de r6colte et de 47Va la seconde ann6e. On observe 6galement des hausses de rendement plus faibles, mais significatives, aux doses de 5,5 g/kg avec adh6sif et de 5,4 g/kg par procddd commerciai d'enrobage. Dans un essai en serre, des souches r6sistantes aux deux antibiotiques streptomycine et rif ampicine de R. meLiloti ont servi ir d6terminer la proportion de nodositds form6es Can. J. Soil Sci. 63: 541-545 (Aug. 1983) 541

542

CANADIAN JOURNAL OF SOIL SCIENCE

par I'inoculant Rhizobium. Entre 92 eI lOOTo des nodosit6s contenaient des souches r6sistantes lorsque l'inoculant 6tait appliqu6 avec adhdsif ou par le proc6d6 d'enrobage, contre seulement 3SVa avec application sans adh6sif. Les r6sultats prouvent l'importance d'appliquer les inoculants avec un adh6sif ou par enrobage de la semence pour maximiser les rendements de la luzerne en sols mod€rdment acides.

Mots cl6s: Sol acide, Rhizobiu.m meliloti, Me.dicago sativa, inoculation, mutants

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reslstants aux antrblotloues

Expanded forage production in northwest-

This study was conducted to examine the

will depend heavily on the abil-

effect of various inoculation methods on nodulation and growth of alfalfa in mod-

ern Canada

ity of the legume-Rhizobium symbiosis to function satisfactorily under various stress conditions encountered on marginally arable land. Soil acidity has been identified as a major factor limiting alfalfa production in Alberta and northern British Columbia (Penney et al. 1977; Rice et al. 1971). Poor viability of Rhizobium meliloti in acid soil is a major factor contributing to inadequate nodulation of alfalfa (Rice et al. 1977). Although liming satisfactorily alleviates the problem (Penney et al. 1977), this is not always economically feasible. Low-pH-tolerant strains of R. melilotihave been selected which substantially improved yield of alfalfa on moderately acid soil (Rice 1982). However, even with these strains maximum yield potential was not achieved unless the inoculant was applied at rates considerably higher than the minimum standards established for commercial inoculant products. In addition, Burton (1975\ has observed that the method of inoculation affects the number of Rhizobium cells adhering to the seed as well as the number remaining viable until the seed is planted.

erately acid soil.

MATERIALS AND METHODS A field experiment was conducted on a Black Solod soil (Landry CL) with pH 5.8-5.9 water:soil, 2.5:l). Plant infection counts (Vincent 1970) showed that this soil contained 3.3 x 106 ineffective R. melilotl per gram, and less than 1O'zeffective R. meliloti per gram. Alfalfa (Medicago sativa L. 'Angus') was planted in the spring of 1978 with five inoculation treatments (Table l). The inoculant contained three strains of Rhizobium meLiloti (NRG-43, NRG61 and NRG-118) in equal numbers in a peat base. The strains were selected for their ability

to

perform under low-pH conditions (Rice 1982). The dry, sticker (Nitra-coat), and sticker

x l0

(Pelgel) treatments were applied to the of planting. Nitra-coat and Pel-

seed on the day

gel contain protective and sticking agents for peat-base inoculants, and were manufactured by

the Nitragin Company, Inc., Clearwater, Fla. The inoculant stickers were used according to the manufacturer's instructions. Prill-On coated seed was prepared by Canadian Seed Coaters Ltd., Brampton, Ont. The coating process incorporates peat-base inoculant, nutrients for the rhizobia, trace elements and lime into the seed

Table 1. Inoculation rates and methods of applying inoculant to alfalfa seed in a field experiment Inoculation rate

Inoculation method

g inoculant/ Description

Designation

No inocuiant

Dry Sticker

Sticker Coated

x

10

No inoculant. no sticker Inoculant without sticker Inoculant with Nitra-coat sticker lnoculant with Pelgel sticker Inoculant in Prill-On seed coat

kg seed

Rhizobia/seed 0

0

:).) 5.5 50.7 5.4

5.2x104

5.2x

104

x

10s

x

101

4.8 5.1

RICE AND OLSEN

-

ALFALFA INOCULATION METHODS FOR AC]D SOILS

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coat. The coated treatment was applied approximately 8 wk prior to planting. The coated seed was stored at 4oC except for the 14 days required for shipping from the seed-coating plant in Ontario to the Beaverlodge Research Station. The plot design was a randomized biock with six replicates. Each plot consisted of four rows, 5.5 m in length, spaced 0.30 m apart. All trear ments were planted at a rate of 1.65 g seed/row. Forage harvests were taken in 1979 and 1980, with two cuts at the 107o bloom stage in each year. The two center rows with 0.25 m discarded from each end were used for obtainins

yield samples. Total-N analysis by the semi micro-Kjeldahl method (Bremner 1965) was conducted on 500-9 subsamples taken from the yield samples. The increase in N yield in the forage of the inoculated treatments over the uninoculated treatments was calculated and expressed as AN, fixed (kg N/ha). A greenhouse assay was conducted to determine the effect of inoculation method on nodule occupancy by the inoculant strains. Plastic pots (15 cm diam. x 15 cm depth) with bottom drainage were filled to within 3 cm of the top with soil obtained from the field plot prior to planting and stored at 4"C. Angus alfatfa seed (from the same lot as used in the field exoeriment) was inoculated with the same treatments described above for the field experiment (Table I ). Inoculant was prepared from doubly tabelled streptomycin and rifampicin resistant (Str., Rif.)

mutants isolated from the three strains of R. meliloti used in the field experiment. The mutants were labelled in the streptomycin-rifamp-

icin sequence with resistance to 500 pg

strep-

tomycrn + 100 pg rifampicin/ml.

The

antibiotic-resistant mutants were shown to be

as

infective and effective as the wild-tvDe strains

543

in growth chamber tests. The coated treatment was prepared in the laboratory according to the procedure of Vincent (1970). Each pot was planted with 12 seeds. After emergence the alfalfa seedlings were thinned to five per pot. The experiment was conducted in a randomized block design with six replicates. The alfalfa was grown in a greenhouse with supplemental light providing 550 pE/sec.m, for 16 h. The temperature was maintained at 2l'C during the day and varied from 15 to 2l"C during the night. The pots wpre watered daily with distilled water. Eight weeks after planting, the alfalfa roots

were removed from the soil, washed and the nodules were counted. Nodules larger than 0.5 mm diameter were excised and surface was sterilized by immersion for I min in 95Vo ethyl al-

cohol, followed by 1 min in 0.2Vo acidified HgCl, and several washings in sterile HrO. Each nodule was squashed with sterile tweezers and replicate-plated on yeast-extract mannitol agar (YEMA) and str, rif YEMA contain-

ing 350 pg streptomycin/ml * 35 pg rifampicin/ml. The plates were observed for growth after incubation at 26'C for 5 days.

RESULTS AND DISCUSSION

Yields

of alfalfa were significantly

in-

creased by the sticker, sticker x 10 and coated inoculant treatments (Table 2). The average yield of these three treatments was 70Vo greater than the uninoculated control

during the first harvest year and

39Vo

greater than the uninoculated control during the second harvest year. Application of the inoculant without sticker did not sie-

Table 2. The effect of different methods of inoculation of alfalfa seed on forage vield and N, fixation in the field

Yield (kg dry matter/ha)

AN2 fixed (kg N/ha)f

Inoculation

First

Second

First

Second

method

year

year

year

year

2076b 2130b 3328a

1691 b

2l75ab

3842a 3388c

No inoculant D.y Sticker

Sticker Coated

x

10

1733b

Total

2

I

3

14

2490a

38 53

24

52 77

2406a

40

2l

61

fANz fixed (kg N/ha) : (N in inoculated treatments) - (N in uninoculated treatment). a,b Any two means within a column followed by the same letter are not significantly different (Duncan's multiple range test, P < 0.05).

CANADIAN JOURNAL OF SOIL SCIENCE

544

1972), yet

were low, varying from I .7 to 4.4 nodules/ plant for the inoculated treatments (Table 3). Similar nodule numbers have been observed on alfalfa inoculated with the same strains of R. melilotl and grown in soil with pH 5.5-6.0. Even though nodule numbers

products.

ences between treatments. Nodule numbers

nificantly increase alfalfa yields above those of the uninoculated control. These results support the contention that application of inoculant without a sticker is not

the most satisfactory method

(Burton

it is often recommended on the package label of commercial inoculant

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Application of the inoculant with a stick-

ing agent or in conjunction with a

seed-

coating process resulted in increases in N,

fixation of 52-11 kg N/ha in the forage over 2 yr (Table 2). This represents a substantial amount of N in relation to the fertilizer N requirements for nonlegume crop production on this soil (Hennig and Rice 1911).

Although the number of rhizobia applied per seed was in the range 5 . 1 x lOa to 4. 8 x 105 (Table 1), the actual number of viable rhizobia on the seed when planted

probably was considerably less. Burton (1972) has reported a reduction by a factor

of 10 within I h in the number of viable cells of R. meliloti when applied as a peatbase inoculant to alfalfa seed. In addition, physical losses of 907o of the inoculant material readily occur with the dry application method. Stickers not only greatly increase the amount of inoculant material adhering to the seed, but provide improved viability of the rhizobia particularly if the

sticking agent contains

a

disaccharide

(Burton 1915).

In the greenhouse test, nodule numbers

were low, there were significant differwere significantly higher with the sticker, sticker x 10 and coated inoculation treatments than in the no inoculant and dry inoculation treatment. The dry inoculation

treatment

did not significantly

increase

nodule numbers over those found in the no inoculation treatment. The sticker, sticker

x l0

and coated treatments resulted in

greater thang\Vo of the nodules containing Str', Rif' strains, while the dry treatment resulted in only 38.l%o of the nodules con-

taining Str', Rif' strains. Little information is available on the nodulation success of R' meliloti inoculant applied to alfalfa seed, but occupancy of clover nodules by inoculant R. trifulii has been reported at90Vo or greater (Gibson et al. 1916; Roughley et al. 1916; Brockwell et al. 1917).In this study, the inoculant R. meliloti was readily able to compete for nodulation sites on alfalfa grown in a moderately acid soil pro-

vided the inoculant was applied with a sticker or in a seed coat. In the dry inoculation treatment, onethird of the nodules contained rhizobia of inoculant origin. However, this treatment did not have a significant effect on nodule

Table 3. The effect of several methods of inoculation of alfalfa seed on nodule numbers and the proportron of nodules formed by the inoculant strains in a greenhouse test Nodules

No. of Nodules per plant

Inoculation method

nodules tested

No inoculant Dry

l.lb

Sticker

3.2a 4.4a

62

62

90

90

3.6a

74

'71

Sticker

x

10

Coated

a,b Any two

P < 0.05).

JL

1.1b

on str,rif YEMA

Growth

Growth on YEMA

containing

Str', Rifl strains (Vo)

6.7

30

2

^a

l6

38.

57 90 68

9r.9

1

100.0 95.8

means followed by the same letter are not significantly different (Duncan's multiple range test,

RICE AND OLSEN

-

ALFALFA INOCULATION METHODS FOR ACID SOILS

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numbers in the greenhouse test or on yield of alfalfa in the field experiment. Where indigenous ineffective or partially effective strains of rhizobia are involved. an inoculum level threshold may be required to provide adequate numbers of rhizobia for ben-

eficial nodulation. For example. Holland (1970) found that applicarions of recommended and 2 x recommended rates of commercial inoculant did not increase yields of Trifolium subterraneum L. grown on soils in California containing ineffective indigenous R. trifolii. However, application of 4 x the recommended rate or more increased the yield of T. subteruaneum by tenfold. The low level (.6.7Vo) of nodules containing rhizobia of apparent inoculant origin in the no inoculant control (Table 3) could have originated from two sources. These may have been naturally occurring Str', Rif. mutants, although the spontaneous mutation rate for even a single highlevel antibiotic resistance is generally about l0-8 (Schwinghamer 1964). A more likely source is from cross-contamination in the greenhouse assay since such occurrences are extremely difficult to prevent.

ACKNOWLEDGMENTS The authors express appreciation to Alberta Agriculture for financial assistance in carrying out this work under the Farming for the Future Program, and to M. R. Lunam and L. L. Foster for technical assistance.

M. 1965. Total nirrogen. Pages 1149-1178 In C. A. Black er al., eds. Methods

BREMNER, J.

of soil analysis. Part 2. Chemical and microbiological properties. Am. Soc. Agron., Madison, Wis.

BROCKWELL, J., SCHWINGHAMER, E. A. and GAULT, R. R. 1977. Ecological studies of

root-nodule bacteria introduced into field envlronments. V. A critical examination of the

stability

of

antigenic and streptomycin-resisof strains of

tance markers for identifications

Rhizobium triJolii. Soil Biol Biochem. 9: t9-24. BURTON, l. C. 1972. Nodulation and symbiotic nitrogen fixation. Pages 229-246 in C. H. Hanson, ed. Alfalfa science and technology. Am. Soc. Agron., Inc., Madison, Wis. BURTON, J. C. l9'75. Methods of inoculating seeds and their effect on survival of rhizobia. Pages 175-189 in P. S. Nutman, ed. Symbiotic nitrogen fixation in plants. Cambridge University Press, Cambridge, U.K. GIBSON, A. H., DATE, R. A.,IRELAND, J. A. and BROCKWELL, l. 1976. A comparison of competitiveness and persistence amongst five

strains

of

Rhizobium

trfolii. Soil

Biol.

Biochem. 8: 395-401. HENNIG, A. M. F. and RICE, W. A. 1977. Effects of date of sod breaking on nitrogen requirements and yield of barley following fescue in rotation. Can. J. Soil Sci. 57: 477485. HOLLAND, A. A. 1970. Competition between soil- and seed-borne Rhizobium trifolii in nodulation of introduced Trifolium subteruaneum. Plant Soil. 32: 293-302.

PENNEY, D. C., NYBORG, M., HOYT, P. B., RICE, W. A., SIEMENS, B. and LAVERTY. D. H. 1977. An assessment of the soil acidity problem in Alberta and northeastern

British Columbia. Can. J. Soil Sci.57:

151 -164. RICE. W. A. 1982. Performance of Rhizobium meliloti strains selected for low-pH tolerance. Can. J. Plant Sci. 62:941-948. RICE, W. A., PENNEY, D. C. and NYBORG, M. 1977. Effects of soil acidity on rhizobia numbers, nodulation and nitrogen fixation by alfalfa and red clover. Can. J. Soil Sci. 57:

191-203.

ROUGHLEY, R. J., BLOWES, W. M. and HERRIDGE. D. F. 1976. Nodulation of Trifolium subterraneum by introduced rhizobia in competition with naturalized strains. Soil Biol. Biochem. 8: 40140'7. SCHWINGHAMER, E. A. 1964. Association between antibiotic resistance and ineffective-

in mutant strains of Rhizobium spp. Can. J. Microbiol. l0 221-233. VINCENT, J. M. 1970. A manual for the practical study of root-nodule bacteria. Blackwell Scientific Publications, Oxford, U.K. ness