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Soil Biology & Biochemistry xxx (2008) 1–9

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Soil Biology & Biochemistry

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55
1 56
2 57
3
Alternatives to peat as a carrier for rhizobia inoculants: 58
4 Solid and liquid formulations 59

F
5 60
6 61
Marta Albareda*, Dulce N. Rodrı́guez-Navarro, Marı́a Camacho, Francisco J. Temprano
62

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7
8 Q1 IFAPA, Centro Las Torres-Tomejil, Ctra. Sevilla-Cazalla de la Sierra, Km 12.2, C.P. 41200 Alcalá del Rı́o, Sevilla, Spain 63
9 64
10 65
11 a r t i c l e i n f o a b s t r a c t 66
12 67

PR
Article history: Many of the microbial inoculants all over the world are based on solid peat formulations. This has been 68
13
Received 19 March 2008 mostly true for well developed legume inoculants based on selected rhizobial strains, due to peat
14 69
Received in revised form 22 July 2008 bacterial protection properties. Six carriers (bagasse, cork compost, attapulgite, sepiolite, perlite and
15 Accepted 24 July 2008 70
amorphous silica) were evaluated as alternatives to peat. Compost from the cork industry and perlite
16 Available online xxx 71
were superior to peat in maintaining survival of different rhizospheric bacteria. Other tested materials
17 72
were discarded as potential carriers for soybean rhizobia. Also, different liquid culture media have been
18 Keywords: 73
assayed employing mannitol or glycerol as C sources. Some media maintained more than 109 cfu ml1 of
Inoculants
ED
19 Sinorhizobium (Ensifer) fredii SMH12 or Bradyrhizobium japonicum USDA110 after 3 months of storage. 74
Rhizobium
20 Rhizobial survival on pre-inoculated seeds with both solid and liquid formulations previously cured for 75
Bradyrhizobium
21 Sinorhizobium fredii 15 days led to a higher bacterial numbers in comparison with recently made inoculants. An additional 76
22 PGPR curing time of solid inoculants up to 120 days had a beneficial effect on rhizobial survival on seeds. The 77
23 Carriers performance of different formulations of two highly effective soybean rhizobia strains was assayed under 78
Soybean field conditions. Soybean inoculated with cork compost, perlite and liquid formulations produced seed
CT

24 79
Adhesives yields that were not significantly different to those produced by peat-based inoculants.
25 80
Ó 2008 Published by Elsevier Ltd. 81
26
27 82
28 83
E

29 84
30 85
31 1. Introduction Ferreira and Castro, 2005). However, these properties only indicate 86
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32 the potential for a good carrier. The final selection must be based on 87
33 Commercial legume inoculant formulations include powder or rhizobia multiplication and survival during storage (Ruiz-Argüeso 88
34 granular carriers, broth cultures or liquid formulations (Bashan, et al., 1979). 89
35 1998). Peat is the carrier of choice for agricultural applications Liquid inoculants simplify the production process as there is no 90
36 (Thompson, 1980). This substrate has been also used as carrier to need to prepare and amend a carrier and the application to seeds or 91
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37 formulate plant growth promoting rhizobacteria (PGPR) or field is easier. However, bacterial survival in this type of inoculant 92
38 biocontrol agents (Okon and Labandera-González, 1994; Vidhya- and on inoculated seed is worse because bacteria lack carrier 93
39 sekaran and Muthamilan, 1995; Kishore et al., 2005). However, protection (Singleton et al., 2002; Tittabutr et al., 2007). 94
40 some countries lack natural peat deposits (Graham-Weiss et al., Inoculants are mainly applied by adhering the product onto 95
41 1987) or the peat-mines are located in preserved wetland ecosys- pre-inoculated seeds stored before sale or at sowing. Bacterial 96
42 tems, so that its extraction is forbidden (Daza et al., 2000). Due to survival on the seed is mainly affected by three factors: desicca- 97
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43 these limitations, more readily available alternative carriers for tion, the toxic nature of seed coat exudates and high temperatures 98
44 inoculants production have been investigated (Thompson, 1980; (Deaker et al., 2004). Survival on seed directly affects the resulting 99
45 Stephens and Rask, 2000; Hungrı́a et al., 2005). legume yield (Brockwell and Bottomley, 1995). Early studies 100
46 An appropriate material for carrying microorganisms must offer demonstrated that rhizobial survival on seeds improves if the 101
47 special properties such as high water-holding capacity, chemical peat-based or vermiculite-based inoculants were applied 4 or 8 102
48 and physical uniformity, a lack of compounds toxic to microbial weeks after inoculant production, respectively (Materon and 103
49 strains and be environmentally safe. At the same time, these Weaver, 1985). 104
50 materials should have near neutral or readily adjustable pH and be The objective of this study was to determine: (1) the survival 105
51 abundant locally at a reasonable cost (Stephens and Rask, 2000; of some rhizobia strains and PGPR in different carrier materials 106
52 and liquid formulations, and (2) the survival of soybean rhizobia 107
53 * Corresponding author. Tel.: þ34 955045505; fax: þ34 955045625. inoculants on seeds and their performance under field 108
54 E-mail address: marta.albareda.ext@juntadeandalucia.es (M. Albareda). conditions. 109

0038-0717/$ – see front matter Ó 2008 Published by Elsevier Ltd.

doi:10.1016/j.soilbio.2008.07.021

Please cite this article in press as: Albareda, M., et al., Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations, Soil
Biology & Biochemistry (2008), doi:10.1016/j.soilbio.2008.07.021
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2 M. Albareda et al. / Soil Biology & Biochemistry xxx (2008) 1–9

110 2. Materials and methods The effect of the carriers on bacterial growth was evaluated as 176
111 follow: SMH12 and USDA110 strains were grown in Alvarez 177
112 2.1. Bacterial strains and culture conditions medium supplemented with 2% (w/v) of each ground substrate. The 178
113 cell counts were determined in duplicate at the end of logarithmic 179
114 Bacteria strains used in this work are listed in Table 1. Bra- phase on YMA plate supplemented with Congo red, after serial 180
115 dyrhizobium, Mesorhizobium and Sinorhizobium (Ensifer) strains dilution on buffer solution. Control treatments without substrate 181
116 were grown in Alvarez medium (Rodrı́guez-Navarro et al., 2003). and with 2% of ground peat from Padul (Granada, Spain) were 182
117 Rhizobium strains were grown in yeast extract-mannitol (YEM) included. 183
118 medium (Vincent, 1970), Bacillus megaterium Bc6 in tryptone-soy 184
119 broth (TSB, Difco) and Chryseobacterium balustinum Aur9 in tryp- 2.3. Inoculants production and quality control 185
120 tone-yeast extract (TY) medium (Beringer, 1974). 186
121 All the strains were grown at 28  C on a rotary shaker at A proper volume of saturated bacterial liquid cultures 187
122 180 rev min1 and cell counts were determined on YEM agar plate (108–1010 cfu ml1), according to the moisture retention charac- 188

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123 supplemented with Congo red (25 mg l1) for rhizobia strains or TY teristic curves (data not shown) of each substrate, was aseptically 189
124 medium for Bc6 and Aur9 strains, after serial dilutions on phos- injected into each sterilized-carrier bag. For each substrate, within Q3 190
125 phate buffer (5 mM) (Vincent, 1970). the pF [pF ¼ 3 þ log(bars)] or water potential, interval 2.5–3.5 191
126 (Roughley, 1970; Thompson, 1980), we selected the appropriate 192
127 moisture content that allowed a maximum volume of bacterial 193

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128 2.2. Carrier characteristics and amendment culture to be injected but providing a non-compacted consistency 194
129 of the inoculant. The final moisture content considered for peat, 195
130 The inorganic carriers were perlite (Spavik, S.A., Huesca, Spain), grape bagasse compost, cork compost, attapulgite, sepiolite, perlite 196
131 attapulgite (Smectagel, Tolsa S.A), sepiolite (Pansil 100, Tolsa S.A) and amorphous silica was 42.9, 43.5, 43.2, 33.8, 47.4, 58.0 and 62.8% 197
132 and amorphous silica (Enfersa, S.A.), all from Spanish commercial on a wet weight basis, respectively. 198
133
134
135
enterprises. Perlite is a volcanic stone composed of a partially
hydrated aluminium silicate. Sepiolite and attapulgite are clay
minerals. Sepiolite is a hydrated magnesium silicate and attapulgite
DP Three liquid media, YEM, Alvarez and Bergersen (Bergersen,
1961), either supplemented with mannitol or glycerol (10 g l1),
have been tested as liquid inoculants. The assay was carried out in
199
200
201
136 is a type of crystalloid hydrous magnesium–aluminium silicate 100 ml Erlenmeyer flasks containing 50 ml of each medium fitted 202
137 mineral. Compost of grape bagasse and compost of cork industry with a foam stopper (for air exchange) and bacteria were grown 203
138 residues were tested as organic substrates. until stationary phase. 204
Inoculants were stored at 25  C and periodically sampled in
TE
139 Cork and grape bagasse composting was performed through 205
140 piling the material on a cement surface to favour temperature duplicate (independent bags or Erlenmeyer flasks). Viable bacteria 206
141 increase of the material. Residues were maintained at 65–70% were estimated by plating 10-fold serial dilutions on YEM agar 207
142 moisture content (wet weight basis) by periodic watering. Weekly, plate supplemented with Congo red for rhizobia strains or TY for 208
143 the pile was turned over. The process lasted about 1 year. The Bc6 and Aur9 strains. The moisture content of the solid inoculants 209
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144 Q2 chemical compositions of cork compost (Moreno et al., 1995) and or pH of liquid inoculants was also determined at sampling. 210
145 grape bagasse (Bustamante et al., 2008) were previously 211
146 determined. 2.4. Seed inoculation and bacterial survival on seeds 212
147 Black peat from Padul (Granada, Spain) was used as reference 213
148 carrier (Ruiz-Argüeso et al., 1979). Carriers were dried to a moisture Seed lots of soybean [Glycine max (L.) Merr.] cultivar Osumi were 214
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149 content of 5% in an oven (80  C for 24 hours) and were finely inoculated with equal amounts of peat or cork-based inoculants of 215
150 ground in a hammer mill to pass a 70 mm screen. Attapulgite, B. japonicum USDA110 or Sinorhizobium fredii SMH12 and a water 216
151 sepiolite and amorphous silica were commercially dried and with solution of gum arabic (GA; 40%, Panreac) or carboxymethylcellu- 217
152 an appropriate particle size (<100 mm). Organic carriers, cork lose (CMC; 4%, medium viscosity, Sigma) to give a starting pop- 218
153 compost and grape bagasse, and amorphous silica have a pH of 7.1, ulation of 106 rhizobia/seed, approximately, taking into account the 219
154 6.5 and 6.3, respectively, while sepiolite, attapulgite and perlite number of viable rhizobia g1 of each inoculant and the average 220
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155 have pHs of 8.5, 9.5 and 8.0. Attapulgite was adjusted to near weight of the individual seed. When liquid inoculants were applied 221
156 neutral pH (7.2) with H2SO4. to seeds, a water solution of polyvinylpyrrolidone at 5% was used as 222
157 All carriers were pre-packaged in low density polyethylene bags adhesive and FeEDTA 0.2% as additive (Singleton et al., 2002). To 223
158 (0.05 mm gauge), covered with a self adhesive-label and sterilized study the effect of curing (storage) period of inoculants on rhizobia 224
159 with 50 KGy of gamma irradiation. Sterility of the carriers was survival on seeds, solid and liquid inoculants were cured for 0, 15, 225
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160 confirmed by plating several dilutions of buffer suspensions of the 30 and 120 days. 226
161 irradiated material on plate count agar medium (PCA, Scharlau) and The inoculated seeds were allowed to dry for 1 h at room 227
162 making growth observations. temperature and stored at a relative humidity of 60%. Bacterial 228
163 229
164 230
Table 1
165 231
Origin and plant host of the bacterial strains used in this work
166 232
167 Bacterial strains Species Plant host Source of reference 233
168 USDA110 Bradyrhizobium japonicum Glycine max Sadowsky et al., 1987 234
169 SMH12 Sinorhizobium (Ensifer) fredii Glycine max Rodrı́guez-Navarro et al., 1996 235
HH103 Sinorhizobium (Ensifer) fredii Glycine max Dowdle and Bohlool, 1985
170 236
ISLU16 Bradyrhizobium sp. Ornithopus sp. Temprano et al., 2002
171 ISC19 Mesorhizobium ciceri Cicer arietinum This work 237
172 IST83 Rhizobium leguminosarum bv. trifolii Trifolium sp. Sadowsky et al., 1987 238
173 ISP42 Rhizobium etli Phaseolus vulgaris Rodrı́guez-Navarro et al., 2000 239
Aur9 Chryseobacterium balustinum Lupinus albus Gutiérrez-Mañero et al., 2003
174 240
Bc6 Bacillus megaterium Lupinus albus Gutiérrez-Mañero et al., 2003
175 241

Please cite this article in press as: Albareda, M., et al., Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations, Soil
Biology & Biochemistry (2008), doi:10.1016/j.soilbio.2008.07.021
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242 survival on seeds was periodically determined by transferring 20 stationary phase. Negligible differences were observed for cork 308
243 inoculated seeds to 100 ml buffer solution and plating 10-fold serial compost and amorphous silica for SMH12 strain. 309
244 dilutions on Congo red-YEM agar supplemented with cyclohexi- Survival of S. fredii SMH12 and B. japonicum USDA110 was fol- 310
245 mide (50 mg l1) (Vincent, 1970). lowed in solid inoculants kept at 25  C for 360 days (Fig. 1). The 311
246 carriers showed a different capacity to maintain an adequate 312
247 survival of the rhizobia strains studied. Cork substrate was as 313
2.5. Field experiments
248 effective as peat in maintaining high populations of rhizobia. The 314
249 initial population was 1010 bacteria g1 and remained unchanged 315
All field experiments were carried out in the Agricultural
250 through 90–120 days of incubation. At the end of the period of 316
Research Station IFAPA, Las Torres-Tomejil (Seville SW-Spain). All
251 storage, the viable numbers of SMH12 and USDA110 strains were 317
were conducted in a loam soil (alluvial soil, Xerofluvent, pH 7.8,
252 greater than 109 and 5  108 cells g1, respectively. Bacterial 318
0.89% organic matter, 12.5 mg P kg1, 194 mg K kg1,
253 1 þ 1 densities obtained with perlite-based inoculants remained higher 319
12 mg NO3-N kg , 6 mg NH4 -N kg and 20.5% CaCO3) free of
254 than 108 cells g1, and viable USDA110 cells were not significantly 320

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soybean-nodulating bacteria. The experimental field was laid out in
255 different (P < 0.05) from those obtained with peat and cork-based 321
a randomized complete block design with four replicates. Within
256 inoculants. 322
each block, there were plots (7  2 m2) divided into four rows,
257 SMH12 inoculants based on attapulgite and sepiolite main- 323
spaced 0.5 m apart. A space of 1 m was allowed between plots and
258 tained a viable population higher than 108 bacteria g1 for more 324
3 m between blocks. Each plot was sown with 150 g of soybean cv.
259 than 5 months of storage, then slowly declined till a final counts of 325

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Osumi. Soybean seeds were inoculated with solid or liquid inocu-
260 around 107 bacteria g1. In the case of bradyrhizobial USDA110 326
lants of B. japonicum USDA110 or S. fredii SMH12 at a rate to get
261 inoculants prepared with these inorganic carriers the decline was 327
106 rhizobia/seed. Two uninoculated controls with or without N
262 faster at the beginning of the assay and the final viable counts were 328
fertilizer were included. The N fertilized plots received two doses of
263 similar to those raised by SMH12. Amorphous silica was not able to 329
100 kg N ha1 as ammonium nitrate, 30 and 60 days after plant
264 maintain high bacterial densities during the storage. Number of 330
emergence. Forty five to fifty days after sowing, 12 plants per plot
viable rhizobia was below 108 bacteria g1 after 90–120 days of
265
266
267
268
were dug out to estimate the number and dry weight of nodules. At
the end of the growing season, plants were harvested to evaluate
seed yield. We determined seed N concentration by the Kjeldahl
DP storage. The viable population of SMH12 and USDA110 strains in
grape bagasse inoculants sharply declined after preparing the
inoculants and no viable cells were detected after 15–30 days
331
332
333
334
method (Vincent, 1970) and calculated seed N content by multi-
269 (Fig. 1). 335
plying concentration (%) by seed yield (kg ha1). Harvest index was
270 Some water loss occurred during the incubation period for all 336
calculated by the ratio of seed weight to total weight of harvested
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271 the carriers. At the end of the storage the moisture percentage of 337
plants.
272 the inoculants decreased 2–10 units below the original values. 338
273 339
274 2.6. Statistics 3.2. Survival of SMH12 and USDA110 strains in liquid inoculants 340
275 341
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276 Statistical analysis was done by means of the Analysis of Vari- Survival of SMH12 and USDA110 strains was also studied in 342
277 ance (ANOVA) linear model, following either a completely random different liquid media, with mannitol or glycerol as carbon source, 343
278 (rhizobia growth experiments) or a randomized block (field stored at 25  C. The results showed that Alvarez medium with 344
279 experiments) design. Multiple comparisons of treatment means mannitol supported more than 5  109 bacteria ml1 of SMH12 345
280 were done by Fisher’s protected L.S.D. method. A 5% probability strain after 90 days of storage (Fig. 2). Die-off of SMH12 strain 346
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281 level was use for rejecting the null hypothesis in all cases. The quickly occurred in Vincent and Alvarez media when glycerol was 347
282 analysis was performed using Statistix software (NH Analytical added as carbon source. Thus no viable cells were detected after 30 348
283 Software, USA). Data on nodulation and yield for USDA110 and days in these media. A decrease of pH during the bacteria growth, 349
284 SMH12 were independently analysed because S. fredii strains below 5.0, must account for the lack of viability. The remainder 350
285 produce higher numbers of nodules than B. japonicum (Daza et al., media did not greatly show changes in the pH (data not shown). 351
286 2000). 352
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All the liquid formulations tested supported an adequate

287 survival of USDA110 strain, providing more than 108 bacteria ml1 353
288 at the end of the assay. During the first 30 days Alvarez medium 354
3. Results
289 (mannitol or glycerol) had the highest number of viable USDA110 355
290 cells but, at the end of the assay, Bergersen medium was the best 356
3.1. Survival of soybean rhizobia SMH12 and USDA110 strains
291 inoculant in maintaining high populations of this strain, with more 357
on different solid inoculants
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292 than 109 bacteria ml1. 358

293 359
An assay to evaluate the possible negative effect of different
294 360
carriers on bacterial growth was carried out by adding each 3.3. Survival of other rhizobia and PGPR strains on selected carriers
295 361
substrate to the Alvarez medium. A decrease of SMH12 or USDA110
296 362
viability in relation to the control was not observed (Table 2). All Survival of C. balustinum Aur9, Rhizobium leguminosarum bv
297 363
cultures reached about 1010 cfu ml1 at the end of the growth trifolii IST83, B. megaterium Bc6, Rhizobium etli ISP42,
298 364
299 365
Table 2
300 366
Growth of Sinorhizobium (Ensifer) fredii SMH12 and Bradyrhizobium japonicum USDA110 in Alvarez medium supplemented with 2% (w/v) of each substrate
301 367
302 Bacterial strains Treatments 368
303 Control Peat Grape bagasse compost Cork compost Attapulgite Sepiolite Perlite Amorphous silica 369
304 SMH12 10.15 ab 10.15 ab 10.05 abc 9.80 e 10.08 ab 10.23 a 10.03 abc 9.87 cde 370
305 USDA110 10.04 ab 10.01 ab 10.09 a 10.01 ab 9.97 ab 9.86 b 9.91 ab 10.01 ab 371
306 Decimal logarithmic of viable cells g1 inoculant. Data are mean values of four replicates. Values followed by the same letter, within each file, are not significantly different at 372
307 P < 0.05. 373

Please cite this article in press as: Albareda, M., et al., Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations, Soil
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374 440
Peat Sepiolite
375 441
Grape bagasse compost Perlite
376 442
Cork compost Amorphous silica
377 443
Attapulgite
378 444
379 445
SMH12 USDA110
380 11 11 446

Log No. bacteria g-1 inoculant

Log No. bacteria g-1 inoculant

381 10 10 447
382 9 9 448
383 8 8 449
384 7 7 450
385 6 6 451
5 5
386 452

OF
4 4
387 3 3 453
388 2 2 454
389 1 1 455
390 0 0 456
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
391 457

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392 Time (days) Time (days) 458
393 Fig. 1. Survival of Sinorhizobium (Ensifer) fredii SMH12 and Bradyrhizobium japonicum USDA110 in different carrier materials at 25  C. Each point represents the mean values of two
459
394 independent counts  standard error. 460
395 461
396 462
397 Mesorhizobium ciceri ISC19 and Bradyrhizobium sp. (Lupinus) ISLU16 DP positive effect on the survival of both strains. In contrast, for liquid 463
398 was determined in peat, cork compost and perlite-based inoculants inoculants an extension of the curing time from 30 to 120 days did 464
399 kept at 25  C for 6 months (Fig. 3). Bacterial populations maintained not increase the survival of the strains on seeds. In general, the 465
400 in cork-based inoculants were similar or significantly (P < 0.05) survival of USDA110 was better than SMH12. 466
401 higher than those in peat inoculants during 6 months of storage. In In addition, the survival of SMH12 and USDA110 strains was 467
402 general, organic carriers promoted the inocula growth during the studied on soybean seeds using peat and cork-based inoculants, 468
first 15–30 days of storage. The number of viable bacteria supported previously cured for 4 months. For this purpose, GA or CMC was
TE
403 469
404 by perlite carrier was smaller than those maintained by the other employed as adhesive solutions (Fig. 5). Cork was less effective than 470
405 carriers, except for ISLU16 strain, where there were no significant peat in maintaining the survival of SMH12 on seeds when GA was 471
406 differences among the three types of inoculants. used as adhesive; however, the survival was similar when CMC was 472
407 used with both carriers. Survival of USDA110 was similar in both 473
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408 3.4. Survival of SMH12 and USDA110 strains on soybean seeds carriers with either of the adhesives used. SMH12 showed a lower 474
409 survival than USDA110 under all the tested conditions. 475
410 The effect of inoculant storage (at 25  C) from 0 to 120 days on 476
411 strain survival of pre-inoculated soybean seeds was followed up to 3.5. Field experiments 477
412 30 days (Fig. 4). Cork-based (using GA as adhesive) and liquid 478
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413 inoculants of SMH12 and USDA110 strains were evaluated. Liquid The performance of different inoculant formulations of two 479
414 inoculants consist of the culture media that gave the best results in highly effective soybean-nodulating strains was evaluated in the 480
415 previous experiment. A curing period of 15 days was necessary for field. All the inoculation treatments effectively nodulated soybean 481
416 improving the survival of strains on seeds. Increasing the curing Osumi (Table 3), since they produced seed yields and a seed N 482
417 period of solid inoculants from 15 up to 120 days had an additional content that were higher than those in the untreated control plots 483
418 484
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419 485
420 Vincent-mannitol Alvarez-glycerol 486
421 Vincent-glycerol Bergersen-mannitol 487
422 Alvarez-mannitol Bergersen-glycerol 488
423 489
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424 SMH12 USDA110 490

425 491
Log No. bacteria ml-1 inoculant
Log No. bacteria ml-1 inoculant

11 11
426 10 10 492
427 9 9 493
428 8 8 494
429 7 7 495
6 6
430 496
5 5
431 4 4 497
432 3 3 498
433 2 2 499
434 1 1 500
435 0 0 501
0 20 40 60 80 100 0 20 40 60 80 100
436 502
Time (days) Time (days)
437 503
438 Fig. 2. Survival of Sinorhizobium (Ensifer) fredii SMH12 and Bradyrhizobium japonicum USDA110 in different liquid inoculant formulations stored at 25  C. Each point represents the 504
439 mean values of two independent counts. Standard error of the mean was lower than 5%. 505

Please cite this article in press as: Albareda, M., et al., Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations, Soil
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506 Peat
572
Cork compost Perlite
507 573
508 IST83 ISP42 574
509 11 11 575
Log No. bacteria g-1 inoculant

Log No. bacteria g-1 inoculant

510 10
576
10
511 577
9 9
512 578
513 8 8 579
514 580
7 7
515 581
516 6 6 582
517 5 5 583
518 584

OF
519 4 4 585
0 30 60 90 120 150 180 0 30 60 90 120 150 180
520 586
Time (days) Time (days)
521 587
522 588
ISC19 ISLU16
523 589

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11 11
Log No. bacteria g-1 inoculant

Log No. bacteria g-1 inoculant

524 590
525 10 10 591
526 9 9
592
527 593
8 8
528 594
529
530
531
532
7

5
DP 7

5
595
596
597
598
533 4 4 599
534 0 30 60 90 120 150 180 0 30 60 90 120 150 180 600
Time (days)
TE
535 Time (days) 601
536 602
537 Aur9 Bc6 603
538 11 11 604
Log No. bacteria g-1 inoculant

Log No. bacteria g-1 inoculant

539 10 10 605
EC

540 606
9 9
541 607
542 8 8 608
543 7 7
609
544 610
RR

545 6 6 611
546 5 5 612
547 4
613
4
548 0 30 60 90 120 150 180 0 30 60 90 120 150 180 614
549 Time (days) Time (days) 615
550 616
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551 Fig. 3. Survival of Rhizobium leguminosarum bv. trifolii IST83, R. etli ISP42, Mesorhizobium ciceri ISC19, Bradyrhizobium sp. (Lupinus) ISLU16, Chryseobacterium balustinum Aur9 and 617
552 Bacillus megaterium Bc6 in peat, perlite and cork-based inoculants stored at 25  C. Each point represents the mean values of two independent counts. Standard error of the mean was 618
lower than 5%.
553 619
554 620
555 621
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556 (uninoculated and non-fertilized). The nodulation rate of any index of the inoculated treatments was always superior to that of 622
557 formulation based on USDA110 strain was lower than that of uninoculated controls. 623
558 SMH12 inoculants and, for both strains, liquid formulations led to 624
559 a lower number of nodules than the corresponding solid inoculants. 4. Discussion 625
560 However, the superior nodulation capacity of solid formulations of 626
561 S. fredii SMH12 was not associated with a different nodule dry mass. 4.1. Carriers and survival of bacterial strains on different 627
562 In fact the nodules formed by liquid inoculants had more mass than inoculants formulations 628
563 those induced by solid inoculants. In the case of B. japonicum 629
564 USDA110 the reduction in nodule numbers formed by perlite and Chemical and physical properties are indicative of the feasibility 630
565 liquid inoculants was related with a reduction in the corresponding of employing a given substrate in inoculant technology (Stephens 631
566 nodule dry mass. and Rask, 2000). We characterized properties of six inoculant 632
567 Seed yields and N content using different soybean inoculants carriers to determine their suitability as alternatives to peat, which 633
568 were not significantly different (P < 0.05) among the formulations is a well-characterized legume inoculant carrier. The peat we used 634
569 tested. In addition, all the inoculated treatments produced soybean as a reference carrier in this work has been used since 1975 as 635
570 yields that were not significantly different than those obtained with substrate for commercial legume inoculant production in Spain 636
571 the nitrogen-fertilized treatment (200 kg N ha1), and the harvest (Ruiz-Argüeso et al., 1979; Rodrı́guez-Navarro et al., 1991). 637

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6 M. Albareda et al. / Soil Biology & Biochemistry xxx (2008) 1–9

638 704
Cork/SMH12 Liquid/SMH12
639 705
Cork/USDA110 Liquid/USDA110
640 706
641 707
642 Maturity 0 days Maturity 15 days 708
7 7
643 709
644 710

Log No. bacteria seed-1

Log No. bacteria seed-1

6 6
645 5
711
5
646 712
647 4 4 713
648 3 3 714
649 715
2 2
650 716

OF
651 1 1 717
652 0 0 718
653 0 5 10 15 20 25 30 0 5 10 15 20 25 30 719
654 Time (days) Time (days) 720
655 721

RO
656 Maturity 30 days Maturity 120 days 722
657 7 7 723
658 724

Log No. bacteria seed-1

Log No. bacteria seed-1

6 6
659 725
5 5
660 726
661
662
663
4

3
DP 4

3
727
728
729
2 2
664 730
665 1 1 731
666 0 0 732
TE
667 0 5 10 15 20 25 30 0 5 10 15 20 25 30 733
668 Time (days) Time (days) 734
669 735
670 Fig. 4. Survival of Sinorhizobium (Ensifer) fredii SMH12 and Bradyrhizobium japonicum USDA110 on soybean cv. Osumi seeds using cork compost-based inoculants and liquid 736
formulations previously kept for an storage period of 0, 15, 30 and 120 days. Each point represents the mean of two replicates. Standard error of the mean was lower than 5%.
671 737
EC

672 738
Most of the evaluated carriers had a pH of about 7.0 or it was substrate could maintain densities of rhizobia and PGPR similar or
673 739
adjusted to near neutrality. All had a high water retention capacity higher than those obtain with peat. Ferreira and Castro (2005) used
674 740
for the optimal moisture potential for growth and survival of different manufactured residues from cork industry mixed with
675 741
rhizobia in inoculants (Roughley, 1970; Thompson, 1980). The calcareous soil. They obtained viable cell counts from 108 and
676 742
volume of liquid added to adjust the carriers to the corresponding 109 bacteria g1 in inoculants of R. leguminosarum bv. trifolii and M.
RR

677 743
optimal moisture potential range (pF 2.5–3.5) was higher than ciceri after 1 year of storage.
678 744
their dry weight. In addition, preliminary studies of growth of Perlite, as has been described (Ronchi et al., 1997; Daza et al.,
679 745
rhizobia have shown that these carriers were non-toxic for 2000; Khavazi et al., 2007), is also a suitable substrate. However,
680 746
bacterial growth. our results for most of the strains tested, except for bradyrhizobia,
681 747
Bacterial survival data after storage indicated that cork compost showed that bacterial densities in perlite were lower than those
682 748
CO

is the best alternative carrier to peat for inoculant technology. This supported by peat and cork substrates.
683 749
684 750
685 751
686 Peat/GA Cork/GA 752
687 Peat/CMC Cork/CMC 753
UN

688 SMH12 USDA110 754

689 7 755
7
690 756
Log No. bacteria seed-1
Log No. bacteria seed-1

691 6
757
6
692 758
693 759
694 5 5 760
695 761
696 4 762
4
697 763
698 764
699 3 3 765
0 5 10 15 20 25 30 0 5 10 15 20 25 30
700 766
Time (days) Time (days)
701 767
702 Fig. 5. Survival of Sinorhizobium (Ensifer) fredii SMH12 and Bradyrhizobium japonicum USDA110 peat and cork compost inoculants on Glycine max cv. Osumi seeds using 40% gum 768
703 arabic (GA) or 4% carboxymethylcellulose (CMC) as adhesive solutions. Each point represents the mean of two replicates. Standard error of the mean was lower than 5%. 769

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770 Table 3 836

771 Nodulation, seed yield and seed nitrogen content of soybean Glycine max cv. Osumi using solid, with peat, cork compost and perlite, and liquid formulations of Sinorhizobium 837
(Ensifer) fredii SMH12 and Bradyrhizobium japonicum USDA110
772 838
773 Treatment Nodulation Yield Harvest index (%) 839
774 Number Dry weigh (mg) Seed yield (kg ha1) Seed N content (kg ha1) 840
775 T 2.5 c 14.3 b 3719 b 178.2 b 47.6 d 841
776 TN 0.1 c 0.0 b 4878 ab 261.2 a 50.0 c 842
777 SMH12 843
778 Peat 115.8 a 174.3 a 5275 a 304.2 a 52.2 b 844
Cork compost 121.2 a 216.8 a 5048 a 280.5 a 55.1 a
779 Perlite 107.4 a 193.4 a 5765 a 310.5 a 55.1 a
845
780 Liquid 57.3 b 156.3 a 4868 ab 258.2 a 54.0 a 846
781 CV (%) 37.0 41.1 16.4 16.9 1.8 847
782 848

OF
T 2.5 c 14.3 d 3719 b 178.3 c 47.6 c
783 TN 0.1 c 0.0 d 4878 a 261.2 b 50.0 b 849
784 USDA110 850
785 Peat 82.0 a 222.6 ab 5365 a 320.7 a 52.1 b 851
Cork compost 64.6 ab 224.5 a 5216 a 302.8 ab 54.7 a
786 852
Perlite 45.2 b 147.1 bc 5153 a 302.5 ab 55.7 a
787 Liquid 40.1 b 120.0 c 5173 a 297.1 ab 55.8 a 853

RO
788 CV (%) 52.6 41.2 10.5 10.6 2.9 854
789 Data are means of four replicates. Values followed by the same letter, within each column and strain, are not significantly different at P < 0.05. CV: coefficient of variation; T: 855
790 uninoculated seeds and non-fertilized treatment; TN: uninoculated seeds and nitrogen-fertilized treatment. 856
791 857
792 858
793
794
795
796
Attapulgite, sepiolite and amorphous silica were not able to
maintain high viable cell counts of SMH12 and USDA110 strains
after 1 year of storage. Sepiolite was better than the others, but the
density of rhizobia strains was lower than 108 rhizobia g1 at the
DP et al., 2002). In this work pH of culture media after SMH12 growth
was lower when glycerol was used as C source and viability of this
strain decreased in those media in which pH was below 5. This
demonstrates that media pH, after bacterial growth and its later
859
860
861
862
797 end of the assay. Although some mineral substrates, such as evolution during the storage, has a great influence on bacterial 863
798 vermiculite and other clays, have been described as adequate for survival (Glenn et al., 1999). 864
TE
799 the production of bacterial inoculants (Sparrow and Ham, 1983; 865
800 Graham-Weiss et al., 1987; Presenti-Barili et al., 1991; Figueiredo 4.2. Survival of soybean rhizobia on inoculated seeds 866
801 et al., 1992; Choi et al., 1998) as well as mineral soils (Chao and 867
802 Alexander, 1984; Beck, 1991). Grape bagasse cannot be used as Survival of R. leguminosarum bv. trifolii strains can be improved 868
803 bacterial carrier. The viable cells of rhizobia strains sharply declined when solid inoculants are previously cured. For example, Materon 869
EC

804 after 1 month of storage, probably due to its high content of and Weaver (1985) found that survival on seeds increased 10 times. 870
805 phenolic substances (Moreno, M.T. Universidad de Sevilla). Burton (1976) obtained the same results with peat-based inocu- 871
806 The cost of these substrates, including peat, and that of the lants of Sinorhizobium meliloti strains. We observed that a storage 872
807 process (composting, drying, milling, etc.) to use them adequately time of at least 15 days at 25  C is necessary to improve rhizobia 873
808 as inoculant carriers, are similar. This cost, which does not include survival on seeds. An additional curing time of solid inoculants 874
RR

809 sterilization and subsequent manipulation, represents only about from 15 to 120 days has a positive effect on bacterial survival. 875
810 10% of the final price of the inoculant. In addition, the use of organic However, in the case of liquid formulations, the effect of a pro- 876
811 carriers (cork or vinery industry residues) involves a commitment longed curing period up to 15 days on bacterial survival was not as 877
812 to reducing the production of waste products and local supply. So, evident. 878
813 cork compost and perlite are real alternatives to peat as carriers for A period of inoculant storage before use would favour the 879
814 inoculant production. adaptation of rhizobia to the carrier and tolerance to drying. 880
CO

815 Our results showed that, with the best liquid formulations, it Morphological changes, as periplasmic space occlusion, cell wall 881
816 was possible to maintain a population higher than 109 viable thickening and losses of PHB granules, were described in rhizobia 882
817 cells ml1 of SMH12 and USDA110 strains at least during 3 months cells from storage peat inoculant (Feng et al., 2002); these authors 883
818 of storage. These densities are sufficient to inoculate soybean seeds associated these morphological alterations to an enhanced survival 884
819 and meet the recommended doses of 105–106 rhizobia seed1 on seeds. On the other hand, several authors have demonstrated 885
UN

820 (Catroux, 1991; Lupyawi et al., 2000). Other authors, working with that carbon or nutrient starvation in liquid cultures of Pseudomonas 886
821 various liquid formulations amended with different polymeric fluorescens (van Overbeek et al., 1995) or R. leguminosarum bv. 887
822 additives, have obtained bacterial densities higher than phaseoli (Thorne and Williams, 1997) increased their tolerance to 888
823 108 cells ml1 of rhizobia strains after 6 months of storage (Titta- stress. 889
824 butr et al., 2007). Other formulations supported bacterial densities Survival of SMH12 and USDA110 strains on seeds, using cork- 890
825 of higher than 109 ml1 of B. japonicum (Singleton et al., 2002) and based inoculants and CMC as adhesive was similar to that obtained 891
826 about 108 ml1 of S. fredii (Videira et al., 2002). In the same way, it when GA or CMC was employed with peat. These results are 892
827 has been demonstrated that commercial liquid inoculants of R. important because CMC is lower-cost than GA and also it is applied 893
828 leguminosarum bv. viciae (Hynes et al., 1995) and B. japonicum to a lower concentration. Moreover, GA has a variable quality and it 894
829 (Maurice et al., 2001) strains maintained an appropriate rhizobia is less available. 895
830 density (higher than 108 rhizobia ml1) after 8 or more months of B. japonicum USDA110 strain survived on soybean seeds better 896
831 storage at 20–25  C. than S. fredii SMH12, probably due to a higher tolerance to desic- 897
832 In general fast-growing rhizobia strains produce acids from cation. Other authors obtained the same results working with slow- 898
833 sugars after their growth in yeast extract-mannitol media, but it can and fast-growing rhizobia strains (Daza et al., 2000; Temprano 899
834 vary depending on the strain and the composition of the culture et al., 2002). In addition, it has been demonstrated that slow- 900
835 medium (Vincent, 1970; Stowers and Eaglesham, 1984; Videira growing strains were more tolerant to desiccation in soils 901

Please cite this article in press as: Albareda, M., et al., Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations, Soil
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8 M. Albareda et al. / Soil Biology & Biochemistry xxx (2008) 1–9

902 (Marshall, 1964; Bushby and Marshall, 1977) or membrane filters Clayton, G.W., Rice, W.A., Lupwayi, N.Z., Johnston, A.M., Lafond, G.P., Grant, C.A., 968
Walley, F., 2004a. Inoculant formulation and fertilizer nitrogen effects on field
903 (Mary et al., 1994). 969
pea: nodulation, N2 fixation and nitrogen partitioning. Canadian Journal of
904 Plant Science 84, 79–88. 970
905 4.3. Soybean inoculant evaluation under field conditions Clayton, G.W., Rice, W.A., Lupwayi, N.Z., Johnston, A.M., Lafond, G.P., Grant, C.A., 971
906 Walley, F., 2004b. Inoculant formulation and fertilizer nitrogen effects on field 972
pea: crop yield and seed quality. Canadian Journal of Plant Science 84, 89–96.
907 Field studies demonstrated that soybean cultivar Osumi inocu- Daza, A., Santamarı́a, C., Rodrı́guez-Navarro, D.N., Camacho, M., Orive, R., 973
908 lated with SMH12 and USDA110 using different inoculant formu- Temprano, F., 2000. Perlite as a carrier for bacterial inoculants. Soil Biology and 974
909 lations produced seed yields and seed N content that were similar Biochemistry 32, 567–572. 975
Deaker, R., Roughley, R.J., Kennedy, I.R., 2004. Legume seed inoculation
910 among them and significantly higher than those obtained in the technologyda review. Soil Biology and Biochemistry 36, 1275–1288.
976
911 uninoculated and non-fertilized treatment. Soybeans inoculated Dowdle, S.F., Bohlool, B.B., 1985. Predominance of fast growing Rhizobium japonicum 977
912 with liquid inoculant of S. fredii SMH12 produced a seed yield lower in soybean field in the People’s Republic of China. Applied and Environmental 978
Microbiology 50, 1171–1176.
913 than that achieved with the corresponding solid inoculants, Feng, L., Roughley, R.J., Copeland, L., 2002. Morphological changes of rhizobia in
979
914 although the reduction was not significant. Perlite carrier has been peat cultures. Applied and Environmental Microbiology 68, 1064–1070. 980

OF
915 used successfully with B. japonicum and S. meliloti in greenhouse Ferreira, E.M., Castro, I.V., 2005. Residues of the cork industry as carriers for the 981
production of legumes inoculants. Silva Lusitana 13, 159–167.
916 conditions (Ronchi et al., 1997) or R. leguminosarum bv. phaseoli, S. 982
Figueiredo, M.D.V.B., Stamford, N.P., Vidor, C., Vilar, J.J., Filho, E.C.O., 1992. Sobrevi-
917 fredii and B. japonicum under field conditions (Daza et al., 2000). vencia do Bradyrhizobium sp. em substratos alternativos. Pesquisa 983
918 Our results corroborate the possibility of employing perlite as Agropecuaria Brasileira Brasilia 27, 1497–1506. 984
Glenn, A.R., Reeve, W.G., Tiwari, R.P., Dilworth, M.J., 1999. Acid tolerance in root
919 alternative to peat. Compost residues from the cork industry has 985

RO
nodule bacteria. In: Bacterial Responses to pH. Wiley, J. & Sons, New York, pp.
920 only been used before in survival studies of R. leguminosarum bv. 112–130. 986
921 trifolii and M. ciceri (Ferreira and Castro, 2005). We have demon- Graham-Weiss, L., Bennett, M.L., Paau, A.S., 1987. Production of bacterial inoculants 987
922 by direct fermentation on nutrient-supplemented vermiculite. Applied and 988
strated that this substrate can support and maintain a similar or
Environmental Microbiology 53, 2138–2140.
923 higher population of microorganisms than peat during the storage Gutiérrez-Mañero, F.J., Probanza, A., Ramos, B., Colón-Flores, J.J., Lucas-Garcı́a, J.A., 989
924 and can be employed as alternative to peat for soybeans inoculants 2003. Effects of culture filtrates of rhizobacteria isolated from wild lupine on 990
925
926
927
under field conditions. Several studies have shown that liquid
inoculants applied on seed can produce seed yields similar to those
obtained with peat-based inoculants (Kremer and Peterson, 1983;
DP germination, growth and biological nitrogen fixation of lupine seedlings.
Journal of Plant Nutrition 26, 1101–1115.
Hungrı́a, M., Loureiro, M.F., Mendes, I.C., Campo, R.J., Graham, P.H., 2005. Inoculant
preparation, production and application. In: Werner, D., Newton, W.E. (Eds.),
991
992
993
928 Hynes et al., 1995; Hynes et al., 2001; Singleton et al., 2002; Thao Nitrogen Fixation in Agriculture, Forestry, Ecology and the Environment. 994
Springer, Netherlands, pp. 223–253.
929 et al., 2002; Tittabutr et al., 2007). However, other authors did not Hynes, R.K., Craig, K.A., Covert, D., Smith, C.R., Rennie, R.J., 1995. Liquid rhizobial
995
930 obtain as good results with liquid formulations in comparison with inoculants for lentil and field pea. Journal of Production Agriculture 8, 547–552. 996
Hynes, R.K., Jans, D.C., Bremer, E., Lupwayi, N.Z., Rice, W.A., Clayton, G.W.,
TE
931 the solid ones (Rice et al., 2000; Kyei-Boahen et al., 2002; Clayton 997
Collins, M.M., 2001. Rhizobium population dynamics in the pea rhizosphere of
932 et al., 2004a; Clayton et al., 2004b). 998
rhizobial inoculant strain applied in different formulations. Canadian Journal of
933 Microbiology 47, 595–600. 999
934 5. Conclusions Khavazi, K., Rejali, F., Seguin, P., Miransari, M., 2007. Effects of carrier, sterilisation 1000
method, and incubation on survival of Bradyrhizobium japonicum in soybean
935 1001
(Glycine max L.) inoculants. Enzyme and Microbial Technology 41, 780–784.
EC

936 Alternative substrates to peat can be used as carriers for legume Kishore, G.K., Pande, S., Podile, A.R., 2005. Phylloplane bacteria increase seedling 1002
937 bacteria. We tested six organic and inorganic materials with several emergence growth and yield of field-grown groundnut (Arachis hypogea L.). 1003
938 Letters in Applied Microbiology 40, 260–268. 1004
bacteria. Compost cork and perlite gave as good results as peat in
Kremer, R.J., Peterson, H.L., 1983. Field evaluation of selected Rhizobium in an
939 terms of support bacterial growth and maintain a long survival of improved legume inoculant. Agronomy Journal 75, 139–143. 1005
940 inoculated strains, as well as survival on soybean seeds. Some liquid Kyei-Boahen, S., Slinkard, A.E., Walley, F.L., 2002. Evaluation of rhizobial inoculation 1006
RR

941 methods for chickpea. Agronomy Journal 94, 851–859. 1007

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943 conditions those formulations led to soybean yield components 259–270. 1009
944 similar to those using traditional peat-based inoculants. Marshall, K.C., 1964. Survival of root-nodule bacteria in dry soils exposed to high 1010
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1046 bium fredii to other rhizobia and to the bradyrhizobia. Applied and Environ- Growth, survival and field performance of bradyrhizobial liquid inoculant 1068

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Please cite this article in press as: Albareda, M., et al., Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations, Soil
Biology & Biochemistry (2008), doi:10.1016/j.soilbio.2008.07.021