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Soil Biology & Biochemistry 40 (2008) 2771–2779

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Soil Biology & Biochemistry

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Alternatives to peat as a carrier for rhizobia inoculants:Solid and liquid formulations

Marta Albareda*, Dulce N. Rodrıguez-Navarro, Marıa Camacho, Francisco J. TempranoIFAPA, Centro Las Torres-Tomejil, Ctra. Sevilla-Cazalla de la Sierra, Km 12.2, C.P. 41200 Alcala del Rıo, Sevilla, Spain

a r t i c l e i n f o

Article history:Received 19 March 2008Received in revised form 22 July 2008Accepted 24 July 2008Available online 19 August 2008

Keywords:InoculantsRhizobiumBradyrhizobiumSinorhizobium frediiPGPRCarriersSoybeanAdhesives

* Corresponding author. Tel.: þ34 955045505; fax:E-mail address: marta.albareda@juntadeandalucia

0038-0717/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.soilbio.2008.07.021

a b s t r a c t

Many of the microbial inoculants all over the world are based on solid peat formulations. This has beenmostly true for well developed legume inoculants based on selected rhizobial strains, due to peatbacterial protection properties. Six carriers (bagasse, cork compost, attapulgite, sepiolite, perlite andamorphous silica) were evaluated as alternatives to peat. Compost from the cork industry and perlitewere superior to peat in maintaining survival of different rhizospheric bacteria. Other tested materialswere discarded as potential carriers for soybean rhizobia. Also, different liquid culture media have beenassayed employing mannitol or glycerol as C sources. Some media maintained more than 109 cfu ml�1 ofSinorhizobium (Ensifer) fredii SMH12 or Bradyrhizobium japonicum USDA110 after 3 months of storage.Rhizobial survival on pre-inoculated seeds with both solid and liquid formulations previously cured for15 days led to a higher bacterial numbers in comparison with recently made inoculants. An additionalcuring time of solid inoculants up to 120 days had a beneficial effect on rhizobial survival on seeds. Theperformance of different formulations of two highly effective soybean rhizobia strains was assayed underfield conditions. Soybean inoculated with cork compost, perlite and liquid formulations produced seedyields that were not significantly different to those produced by peat-based inoculants.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Commercial legume inoculant formulations include powder orgranular carriers, broth cultures or liquid formulations (Bashan,1998). Peat is the carrier of choice for agricultural applications(Thompson, 1980). This substrate has been also used as carrier toformulate plant growth promoting rhizobacteria (PGPR) orbiocontrol agents (Okon and Labandera-Gonzalez, 1994; Vidhya-sekaran and Muthamilan, 1995; Kishore et al., 2005). However,some countries lack natural peat deposits (Graham-Weiss et al.,1987) or the peat-mines are located in preserved wetland ecosys-tems, so that its extraction is forbidden (Daza et al., 2000). Due tothese limitations, more readily available alternative carriers forinoculants production have been investigated (Thompson, 1980;Stephens and Rask, 2000; Hungrıa et al., 2005).

An appropriate material for carrying microorganisms must offerspecial properties such as high water-holding capacity, chemicaland physical uniformity, a lack of compounds toxic to microbialstrains and be environmentally safe. At the same time, thesematerials should have near neutral or readily adjustable pH and beabundant locally at a reasonable cost (Stephens and Rask, 2000;

þ34 955045625..es (M. Albareda).

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Ferreira and Castro, 2005). However, these properties only indicatethe potential for a good carrier. The final selection must be based onrhizobia multiplication and survival during storage (Ruiz-Arguesoet al., 1979).

Liquid inoculants simplify the production process as there is noneed to prepare and amend a carrier and the application to seeds orfield is easier. However, bacterial survival in this type of inoculantand on inoculated seed is worse because bacteria lack carrierprotection (Singleton et al., 2002; Tittabutr et al., 2007).

Inoculants are mainly applied by adhering the product ontopre-inoculated seeds stored before sale or at sowing. Bacterialsurvival on the seed is mainly affected by three factors: desicca-tion, the toxic nature of seed coat exudates and high temperatures(Deaker et al., 2004). Survival on seed directly affects the resultinglegume yield (Brockwell and Bottomley, 1995). Early studiesdemonstrated that rhizobial survival on seeds improves if thepeat-based or vermiculite-based inoculants were applied 4 or 8weeks after inoculant production, respectively (Materon andWeaver, 1985).

The objective of this study was to determine: (1) the survivalof some rhizobia strains and PGPR in different carrier materialsand liquid formulations, and (2) the survival of soybean rhizobiainoculants on seeds and their performance under fieldconditions.

M. Albareda et al. / Soil Biology & Biochemistry 40 (2008) 2771–27792772

2. Materials and methods

2.1. Bacterial strains and culture conditions

Bacteria strains used in this work are listed in Table 1. Bra-dyrhizobium, Mesorhizobium and Sinorhizobium (Ensifer) strainswere grown in Alvarez medium (Rodrıguez-Navarro et al., 2003).Rhizobium strains were grown in yeast extract-mannitol (YEM)medium (Vincent, 1970), Bacillus megaterium Bc6 in tryptone-soybroth (TSB, Difco) and Chryseobacterium balustinum Aur9 in tryp-tone-yeast extract (TY) medium (Beringer, 1974).

All the strains were grown at 28 �C on a rotary shaker at180 rev min�1 and cell counts were determined on YEM agar platesupplemented with Congo red (25 mg l�1) for rhizobia strains or TYmedium for Bc6 and Aur9 strains, after serial dilutions on phos-phate buffer (5 mM) (Vincent, 1970).

2.2. Carrier characteristics and amendment

The inorganic carriers were perlite (Spavik, S.A., Huesca, Spain),attapulgite (Smectagel, Tolsa S.A), sepiolite (Pansil 100, Tolsa S.A)and amorphous silica (Enfersa, S.A.), all from Spanish commercialenterprises. Perlite is a volcanic stone composed of a partiallyhydrated aluminium silicate. Sepiolite and attapulgite are clayminerals. Sepiolite is a hydrated magnesium silicate and attapulgiteis a type of crystalloid hydrous magnesium–aluminium silicatemineral. Compost of grape bagasse and compost of cork industryresidues were tested as organic substrates.

Cork and grape bagasse composting was performed throughpiling the material on a cement surface to favour temperatureincrease of the material. Residues were maintained at 65–70%moisture content (wet weight basis) by periodic watering. Weekly,the pile was turned over. The process lasted about 1 year. Thechemical compositions of cork compost (Moreno et al., 1995)and grape bagasse (Bustamante et al., 2008) were previouslydetermined.

Black peat from Padul (Granada, Spain) was used as referencecarrier (Ruiz-Argueso et al., 1979). Carriers were dried to a moisturecontent of 5% in an oven (80 �C for 24 hours) and were finelyground in a hammer mill to pass a 70 mm screen. Attapulgite,sepiolite and amorphous silica were commercially dried and withan appropriate particle size (<100 mm). Organic carriers, corkcompost and grape bagasse, and amorphous silica have a pH of 7.1,6.5 and 6.3, respectively, while sepiolite, attapulgite and perlitehave pHs of 8.5, 9.5 and 8.0. Attapulgite was adjusted to nearneutral pH (7.2) with H2SO4.

All carriers were pre-packaged in low density polyethylene bags(0.05 mm gauge), covered with a self adhesive-label and sterilizedwith 50 KGy of gamma irradiation. Sterility of the carriers wasconfirmed by plating several dilutions of buffer suspensions of theirradiated material on plate count agar medium (PCA, Scharlau) andmaking growth observations.

Table 1Origin and plant host of the bacterial strains used in this work

Bacterial strains Species

USDA110 Bradyrhizobium japonicumSMH12 Sinorhizobium (Ensifer) frediiHH103 Sinorhizobium (Ensifer) frediiISLU16 Bradyrhizobium sp.ISC19 Mesorhizobium ciceriIST83 Rhizobium leguminosarum bv. trifoliiISP42 Rhizobium etliAur9 Chryseobacterium balustinumBc6 Bacillus megaterium

The effect of the carriers on bacterial growth was evaluated asfollow: SMH12 and USDA110 strains were grown in Alvarezmedium supplemented with 2% (w/v) of each ground substrate. Thecell counts were determined in duplicate at the end of logarithmicphase on YMA plate supplemented with Congo red, after serialdilution on buffer solution. Control treatments without substrateand with 2% of ground peat from Padul (Granada, Spain) wereincluded.

2.3. Inoculants production and quality control

A proper volume of saturated bacterial liquid cultures(108–1010 cfu ml�1), according to the moisture retention charac-teristic curves (data not shown) of each substrate, was asepticallyinjected into each sterilized-carrier bag. For each substrate, withinthe pF [pF¼ 3þ log(�bars)] or water potential interval 2.5–3.5(Roughley, 1970; Thompson, 1980), we selected the appropriatemoisture content that allowed a maximum volume of bacterialculture to be injected but providing a non-compacted consistencyof the inoculant. The final moisture content considered for peat,grape bagasse compost, cork compost, attapulgite, sepiolite, perliteand amorphous silica was 42.9, 43.5, 43.2, 33.8, 47.4, 58.0 and 62.8%on a wet weight basis, respectively.

Three liquid media, YEM, Alvarez and Bergersen (Bergersen,1961), either supplemented with mannitol or glycerol (10 g l�1),have been tested as liquid inoculants. The assay was carried out in100 ml Erlenmeyer flasks containing 50 ml of each medium fittedwith a foam stopper (for air exchange) and bacteria were grownuntil stationary phase.

Inoculants were stored at 25 �C and periodically sampled induplicate (independent bags or Erlenmeyer flasks). Viable bacteriawere estimated by plating 10-fold serial dilutions on YEM agarplate supplemented with Congo red for rhizobia strains or TY forBc6 and Aur9 strains. The moisture content of the solid inoculantsor pH of liquid inoculants was also determined at sampling.

2.4. Seed inoculation and bacterial survival on seeds

Seed lots of soybean [Glycine max (L.) Merr.] cultivar Osumi wereinoculated with equal amounts of peat or cork-based inoculants ofB. japonicum USDA110 or Sinorhizobium fredii SMH12 and a watersolution of gum arabic (GA; 40%, Panreac) or carboxymethylcellu-lose (CMC; 4%, medium viscosity, Sigma) to give a starting pop-ulation of 106 rhizobia/seed, approximately, taking into account thenumber of viable rhizobia g�1 of each inoculant and the averageweight of the individual seed. When liquid inoculants were appliedto seeds, a water solution of polyvinylpyrrolidone at 5% was used asadhesive and FeEDTA 0.2% as additive (Singleton et al., 2002). Tostudy the effect of curing (storage) period of inoculants on rhizobiasurvival on seeds, solid and liquid inoculants were cured for 0, 15,30 and 120 days.

The inoculated seeds were allowed to dry for 1 h at roomtemperature and stored at a relative humidity of 60%. Bacterial

Plant host Source of reference

Glycine max Sadowsky et al., 1987Glycine max Rodrıguez-Navarro et al., 1996Glycine max Dowdle and Bohlool, 1985Ornithopus sp. Temprano et al., 2002Cicer arietinum This workTrifolium sp. Sadowsky et al., 1987Phaseolus vulgaris Rodrıguez-Navarro et al., 2000Lupinus albus Gutierrez-Manero et al., 2003Lupinus albus Gutierrez-Manero et al., 2003

M. Albareda et al. / Soil Biology & Biochemistry 40 (2008) 2771–2779 2773

survival on seeds was periodically determined by transferring 20inoculated seeds to 100 ml buffer solution and plating 10-fold serialdilutions on Congo red-YEM agar supplemented with cyclohexi-mide (50 mg l�1) (Vincent, 1970).

2.5. Field experiments

All field experiments were carried out in the AgriculturalResearch Station IFAPA, Las Torres-Tomejil (Seville SW-Spain).All were conducted in a loam soil (alluvial soil, Xerofluvent,pH 7.8, 0.89% organic matter, 12.5 mg P kg�1, 194 mg K kg�1,12 mg NO3-N kg�1, 6 mg NH4

þ-N kg�1 and 20.5% CaCO3) free ofsoybean-nodulating bacteria. The experimental field was laid out ina randomized complete block design with four replicates. Withineach block, there were plots (7�2 m2) divided into four rows,spaced 0.5 m apart. A space of 1 m was allowed between plots and3 m between blocks. Each plot was sown with 150 g of soybean cv.Osumi. Soybean seeds were inoculated with solid or liquid inocu-lants of B. japonicum USDA110 or S. fredii SMH12 at a rate to get106 rhizobia/seed. Two uninoculated controls with or without Nfertilizer were included. The N fertilized plots received two doses of100 kg N ha�1 as ammonium nitrate, 30 and 60 days after plantemergence. Forty five to fifty days after sowing, 12 plants per plotwere dug out to estimate the number and dry weight of nodules. Atthe end of the growing season, plants were harvested to evaluateseed yield. We determined seed N concentration by the Kjeldahlmethod (Vincent, 1970) and calculated seed N content by multi-plying concentration (%) by seed yield (kg ha�1). Harvest index wascalculated by the ratio of seed weight to total weight of harvestedplants.

2.6. Statistics

Statistical analysis was done by means of the Analysis of Vari-ance (ANOVA) linear model, following either a completely random(rhizobia growth experiments) or a randomized block (fieldexperiments) design. Multiple comparisons of treatment meanswere done by Fisher’s protected L.S.D. method. A 5% probabilitylevel was use for rejecting the null hypothesis in all cases. Theanalysis was performed using Statistix software (NH AnalyticalSoftware, USA). Data on nodulation and yield for USDA110 andSMH12 were independently analysed because S. fredii strainsproduce higher numbers of nodules than B. japonicum (Daza et al.,2000).

3. Results

3.1. Survival of soybean rhizobia SMH12 and USDA110 strainson different solid inoculants

An assay to evaluate the possible negative effect of differentcarriers on bacterial growth was carried out by adding eachsubstrate to the Alvarez medium. A decrease of SMH12 or USDA110viability in relation to the control was not observed (Table 2). Allcultures reached about 1010 cfu ml�1 at the end of the growth

Table 2Growth of Sinorhizobium (Ensifer) fredii SMH12 and Bradyrhizobium japonicum USDA110

Bacterial strains Treatments

Control Peat Grape bagasse compost Cork

SMH12 10.15 ab 10.15 ab 10.05 abc 9.80USDA110 10.04 ab 10.01 ab 10.09 a 10.01

Decimal logarithmic of viable cells g�1 inoculant. Data are mean values of four replicates.P< 0.05.

stationary phase. Negligible differences were observed for corkcompost and amorphous silica for SMH12 strain.

Survival of S. fredii SMH12 and B. japonicum USDA110 was fol-lowed in solid inoculants kept at 25 �C for 360 days (Fig. 1). Thecarriers showed a different capacity to maintain an adequatesurvival of the rhizobia strains studied. Cork substrate was aseffective as peat in maintaining high populations of rhizobia. Theinitial population was 1010 bacteria g�1 and remained unchangedthrough 90–120 days of incubation. At the end of the period ofstorage, the viable numbers of SMH12 and USDA110 strains weregreater than 109 and 5�108 cells g�1, respectively. Bacterialdensities obtained with perlite-based inoculants remained higherthan 108 cells g�1, and viable USDA110 cells were not significantlydifferent (P< 0.05) from those obtained with peat and cork-basedinoculants.

SMH12 inoculants based on attapulgite and sepiolite main-tained a viable population higher than 108 bacteria g�1 for morethan 5 months of storage, then slowly declined till a final counts ofaround 107 bacteria g�1. In the case of bradyrhizobial USDA110inoculants prepared with these inorganic carriers the decline wasfaster at the beginning of the assay and the final viable counts weresimilar to those raised by SMH12. Amorphous silica was not able tomaintain high bacterial densities during the storage. Number ofviable rhizobia was below 108 bacteria g�1 after 90–120 days ofstorage. The viable population of SMH12 and USDA110 strains ingrape bagasse inoculants sharply declined after preparing theinoculants and no viable cells were detected after 15–30 days(Fig. 1).

Some water loss occurred during the incubation period for allthe carriers. At the end of the storage the moisture percentage ofthe inoculants decreased 2–10 units below the original values.

3.2. Survival of SMH12 and USDA110 strains in liquid inoculants

Survival of SMH12 and USDA110 strains was also studied indifferent liquid media, with mannitol or glycerol as carbon source,stored at 25 �C. The results showed that Alvarez medium withmannitol supported more than 5�109 bacteria ml�1 of SMH12strain after 90 days of storage (Fig. 2). Die-off of SMH12 strainquickly occurred in Vincent and Alvarez media when glycerol wasadded as carbon source. Thus no viable cells were detected after 30days in these media. A decrease of pH during the bacteria growth,below 5.0, must account for the lack of viability. The remaindermedia did not greatly show changes in the pH (data not shown).

All the liquid formulations tested supported an adequatesurvival of USDA110 strain, providing more than 108 bacteria ml�1

at the end of the assay. During the first 30 days Alvarez medium(mannitol or glycerol) had the highest number of viable USDA110cells but, at the end of the assay, Bergersen medium was the bestinoculant in maintaining high populations of this strain, with morethan 109 bacteria ml�1.

3.3. Survival of other rhizobia and PGPR strains on selected carriers

Survival of C. balustinum Aur9, R. leguminosarum bv trifolii IST83,B. megaterium Bc6, R. etli ISP42, M. ciceri ISC19 and Bradyrhizobium

in Alvarez medium supplemented with 2% (w/v) of each substrate

compost Attapulgite Sepiolite Perlite Amorphous silica

e 10.08 ab 10.23 a 10.03 abc 9.87 cdeab 9.97 ab 9.86 b 9.91 ab 10.01 ab

Values followed by the same letter, within each file, are not significantly different at

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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 twoindependent counts� standard error.

M. Albareda et al. / Soil Biology & Biochemistry 40 (2008) 2771–27792774

sp. (Lupinus) ISLU16 was determined in peat, cork compost andperlite-based inoculants kept at 25 �C for 6 months (Fig. 3).Bacterial populations maintained in cork-based inoculants weresimilar or significantly (P< 0.05) higher than those in peat inocu-lants during 6 months of storage. In general, organic carrierspromoted the inocula growth during the first 15–30 days of storage.The number of viable bacteria supported by perlite carrier wassmaller than those maintained by the other carriers, except forISLU16 strain, where there were no significant differences amongthe three types of inoculants.

3.4. Survival of SMH12 and USDA110 strains on soybean seeds

The effect of inoculant storage (at 25 �C) from 0 to 120 days onstrain survival of pre-inoculated soybean seeds was followed up to30 days (Fig. 4). Cork-based (using GA as adhesive) and liquidinoculants of SMH12 and USDA110 strains were evaluated. Liquidinoculants consist of the culture media that gave the best results inprevious experiment. A curing period of 15 days was necessary forimproving the survival of strains on seeds. Increasing the curingperiod of solid inoculants from 15 up to 120 days had an additional

Vincent-mannitol

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Fig. 2. Survival of Sinorhizobium (Ensifer) fredii SMH12 and Bradyrhizobium japonicum USDAmean values of two independent counts. Standard error of the mean was lower than 5%.

positive effect on the survival of both strains. In contrast, for liquidinoculants an extension of the curing time from 30 to 120 days didnot increase the survival of the strains on seeds. In general, thesurvival of USDA110 was better than SMH12.

In addition, the survival of SMH12 and USDA110 strains wasstudied on soybean seeds using peat and cork-based inoculants,previously cured for 4 months. For this purpose, GA or CMC wasemployed as adhesive solutions (Fig. 5). Cork was less effective thanpeat in maintaining the survival of SMH12 on seeds when GA wasused as adhesive; however, the survival was similar when CMC wasused with both carriers. Survival of USDA110 was similar in bothcarriers with either of the adhesives used. SMH12 showed a lowersurvival than USDA110 under all the tested conditions.

3.5. Field experiments

The performance of different inoculant formulations of twohighly effective soybean-nodulating strains was evaluated in thefield. All the inoculation treatments effectively nodulated soybeanOsumi (Table 3), since they produced seed yields and a seed Ncontent that were higher than those in the untreated control plots

Alvarez-glycerol

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Fig. 3. Survival of Rhizobium leguminosarum bv. trifolii IST83, R. etli ISP42, Mesorhizobium ciceri ISC19, Bradyrhizobium sp. (Lupinus) ISLU16, Chryseobacterium balustinum Aur9 andBacillus 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 waslower than 5%.

M. Albareda et al. / Soil Biology & Biochemistry 40 (2008) 2771–2779 2775

(uninoculated and non-fertilized). The nodulation rate of anyformulation based on USDA110 strain was lower than that ofSMH12 inoculants and, for both strains, liquid formulations led toa lower number of nodules than the corresponding solid inoculants.However, the superior nodulation capacity of solid formulations ofS. fredii SMH12 was not associated with a different nodule dry mass.In fact the nodules formed by liquid inoculants had more mass thanthose induced by solid inoculants. In the case of B. japonicumUSDA110 the reduction in nodule numbers formed by perlite andliquid inoculants was related with a reduction in the correspondingnodule dry mass.

Seed yields and N content using different soybean inoculantswere not significantly different (P< 0.05) among the formulationstested. In addition, all the inoculated treatments produced soybeanyields that were not significantly different than those obtained withthe nitrogen-fertilized treatment (200 kg N ha�1), and the harvest

index of the inoculated treatments was always superior to that ofuninoculated controls.

4. Discussion

4.1. Carriers and survival of bacterial strains on differentinoculants formulations

Chemical and physical properties are indicative of the feasibilityof employing a given substrate in inoculant technology (Stephensand Rask, 2000). We characterized properties of six inoculantcarriers to determine their suitability as alternatives to peat, whichis a well-characterized legume inoculant carrier. The peat we usedas a reference carrier in this work has been used since 1975 assubstrate for commercial legume inoculant production in Spain(Ruiz-Argueso et al., 1979; Rodrıguez-Navarro et al., 1991).

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Fig. 4. Survival of Sinorhizobium (Ensifer) fredii SMH12 and Bradyrhizobium japonicum USDA110 on soybean cv. Osumi seeds using cork compost-based inoculants and liquidformulations 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%.

M. Albareda et al. / Soil Biology & Biochemistry 40 (2008) 2771–27792776

Most of the evaluated carriers had a pH of about 7.0 or it wasadjusted to near neutrality. All had a high water retention capacityfor the optimal moisture potential for growth and survival ofrhizobia in inoculants (Roughley, 1970; Thompson, 1980). Thevolume of liquid added to adjust the carriers to the correspondingoptimal moisture potential range (pF 2.5–3.5) was higher thantheir dry weight. In addition, preliminary studies of growth ofrhizobia have shown that these carriers were non-toxic forbacterial growth.

Bacterial survival data after storage indicated that cork compostis the best alternative carrier to peat for inoculant technology. This

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Fig. 5. Survival of Sinorhizobium (Ensifer) fredii SMH12 and Bradyrhizobium japonicum USDAarabic (GA) or 4% carboxymethylcellulose (CMC) as adhesive solutions. Each point represen

substrate could maintain densities of rhizobia and PGPR similar orhigher than those obtain with peat. Ferreira and Castro (2005) useddifferent manufactured residues from cork industry mixed withcalcareous soil. They obtained viable cell counts from 108 and109 bacteria g�1 in inoculants of R. leguminosarum bv. trifolii and M.ciceri after 1 year of storage.

Perlite, as has been described (Ronchi et al., 1997; Daza et al.,2000; Khavazi et al., 2007), is also a suitable substrate. However,our results for most of the strains tested, except for bradyrhizobia,showed that bacterial densities in perlite were lower than thosesupported by peat and cork substrates.

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110 peat and cork compost inoculants on Glycine max cv. Osumi seeds using 40% gumts the mean of two replicates. Standard error of the mean was lower than 5%.

Table 3Nodulation, 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(Ensifer) fredii SMH12 and Bradyrhizobium japonicum USDA110

Treatment Nodulation Yield Harvest index (%)

Number Dry weigh (mg) Seed yield (kg ha�1) Seed N content (kg ha�1)

T 2.5 c 14.3 b 3719 b 178.2 b 47.6 dTN 0.1 c 0.0 b 4878 ab 261.2 a 50.0 c

SMH12Peat 115.8 a 174.3 a 5275 a 304.2 a 52.2 bCork compost 121.2 a 216.8 a 5048 a 280.5 a 55.1 aPerlite 107.4 a 193.4 a 5765 a 310.5 a 55.1 aLiquid 57.3 b 156.3 a 4868 ab 258.2 a 54.0 a

CV (%) 37.0 41.1 16.4 16.9 1.8

T 2.5 c 14.3 d 3719 b 178.3 c 47.6 cTN 0.1 c 0.0 d 4878 a 261.2 b 50.0 b

USDA110Peat 82.0 a 222.6 ab 5365 a 320.7 a 52.1 bCork compost 64.6 ab 224.5 a 5216 a 302.8 ab 54.7 aPerlite 45.2 b 147.1 bc 5153 a 302.5 ab 55.7 aLiquid 40.1 b 120.0 c 5173 a 297.1 ab 55.8 a

CV (%) 52.6 41.2 10.5 10.6 2.9

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:uninoculated seeds and non-fertilized treatment; TN: uninoculated seeds and nitrogen-fertilized treatment.

M. Albareda et al. / Soil Biology & Biochemistry 40 (2008) 2771–2779 2777

Attapulgite, sepiolite and amorphous silica were not able tomaintain high viable cell counts of SMH12 and USDA110 strainsafter 1 year of storage. Sepiolite was better than the others, but thedensity of rhizobia strains was lower than 108 rhizobia g�1 at theend of the assay. Although some mineral substrates, such asvermiculite and other clays, have been described as adequate forthe production of bacterial inoculants (Sparrow and Ham, 1983;Graham-Weiss et al., 1987; Presenti-Barili et al., 1991; Figueiredoet al., 1992; Choi et al., 1998) as well as mineral soils (Chao andAlexander, 1984; Beck, 1991). Grape bagasse cannot be used asbacterial carrier. The viable cells of rhizobia strains sharply declinedafter 1 month of storage, probably due to its high content ofphenolic substances (Moreno, M.T. Universidad de Sevilla).

The cost of these substrates, including peat, and that of theprocess (composting, drying, milling, etc.) to use them adequatelyas inoculant carriers, are similar. This cost, which does not includesterilization and subsequent manipulation, represents only about10% of the final price of the inoculant. In addition, the use of organiccarriers (cork or vinery industry residues) involves a commitmentto reducing the production of waste products and local supply. So,cork compost and perlite are real alternatives to peat as carriers forinoculant production.

Our results showed that, with the best liquid formulations, itwas possible to maintain a population higher than 109 viablecells ml�1 of SMH12 and USDA110 strains at least during 3 monthsof storage. These densities are sufficient to inoculate soybean seedsand meet the recommended doses of 105–106 rhizobia seed�1

(Catroux, 1991; Lupyawi et al., 2000). Other authors, working withvarious liquid formulations amended with different polymericadditives, have obtained bacterial densities higher than108 cells ml�1 of rhizobia strains after 6 months of storage (Titta-butr et al., 2007). Other formulations supported bacterial densitiesof higher than 109 ml�1 of B. japonicum (Singleton et al., 2002) andabout 108 ml�1 of S. fredii (Videira et al., 2002). In the same way, ithas been demonstrated that commercial liquid inoculants of R.leguminosarum bv. viciae (Hynes et al., 1995) and B. japonicum(Maurice et al., 2001) strains maintained an appropriate rhizobiadensity (higher than 108 rhizobia ml�1) after 8 or more months ofstorage at 20–25 �C.

In general fast-growing rhizobia strains produce acids fromsugars after their growth in yeast extract-mannitol media, but it canvary depending on the strain and the composition of the culturemedium (Vincent, 1970; Stowers and Eaglesham, 1984; Videira

et al., 2002). In this work pH of culture media after SMH12 growthwas lower when glycerol was used as C source and viability of thisstrain decreased in those media in which pH was below 5. Thisdemonstrates that media pH, after bacterial growth and its laterevolution during the storage, has a great influence on bacterialsurvival (Glenn et al., 1999).

4.2. Survival of soybean rhizobia on inoculated seeds

Survival of R. leguminosarum bv. trifolii strains can be improvedwhen solid inoculants are previously cured. For example, Materonand Weaver (1985) found that survival on seeds increased 10 times.Burton (1976) obtained the same results with peat-based inocu-lants of Sinorhizobium meliloti strains. We observed that a storagetime of at least 15 days at 25 �C is necessary to improve rhizobiasurvival on seeds. An additional curing time of solid inoculantsfrom 15 to 120 days has a positive effect on bacterial survival.However, in the case of liquid formulations, the effect of a pro-longed curing period up to 15 days on bacterial survival was not asevident.

A period of inoculant storage before use would favour theadaptation of rhizobia to the carrier and tolerance to drying.Morphological changes, as periplasmic space occlusion, cell wallthickening and losses of PHB granules, were described in rhizobiacells from storage peat inoculant (Feng et al., 2002); these authorsassociated these morphological alterations to an enhanced survivalon seeds. On the other hand, several authors have demonstratedthat carbon or nutrient starvation in liquid cultures of Pseudomonasfluorescens (van Overbeek et al., 1995) or R. leguminosarum bv.phaseoli (Thorne and Williams, 1997) increased their tolerance tostress.

Survival of SMH12 and USDA110 strains on seeds, using cork-based inoculants and CMC as adhesive was similar to that obtainedwhen GA or CMC was employed with peat. These results areimportant because CMC is lower-cost than GA and also it is appliedto a lower concentration. Moreover, GA has a variable quality and itis less available.

B. japonicum USDA110 strain survived on soybean seeds betterthan S. fredii SMH12, probably due to a higher tolerance to desic-cation. Other authors obtained the same results working with slow-and fast-growing rhizobia strains (Daza et al., 2000; Tempranoet al., 2002). In addition, it has been demonstrated that slow-growing strains were more tolerant to desiccation in soils

M. Albareda et al. / Soil Biology & Biochemistry 40 (2008) 2771–27792778

(Marshall, 1964; Bushby and Marshall, 1977) or membrane filters(Mary et al., 1994).

4.3. Soybean inoculant evaluation under field conditions

Field studies demonstrated that soybean cultivar Osumi inocu-lated with SMH12 and USDA110 using different inoculant formu-lations produced seed yields and seed N content that were similaramong them and significantly higher than those obtained in theuninoculated and non-fertilized treatment. Soybeans inoculatedwith liquid inoculant of S. fredii SMH12 produced a seed yield lowerthan that achieved with the corresponding solid inoculants,although the reduction was not significant. Perlite carrier has beenused successfully with B. japonicum and S. meliloti in greenhouseconditions (Ronchi et al., 1997) or R. leguminosarum bv. phaseoli, S.fredii and B. japonicum under field conditions (Daza et al., 2000).Our results corroborate the possibility of employing perlite asalternative to peat. Compost residues from the cork industry hasonly been used before in survival studies of R. leguminosarum bv.trifolii and M. ciceri (Ferreira and Castro, 2005). We have demon-strated that this substrate can support and maintain a similar orhigher population of microorganisms than peat during the storageand can be employed as alternative to peat for soybeans inoculantsunder field conditions. Several studies have shown that liquidinoculants applied on seed can produce seed yields similar to thoseobtained with peat-based inoculants (Kremer and Peterson, 1983;Hynes et al., 1995; Hynes et al., 2001; Singleton et al., 2002; Thaoet al., 2002; Tittabutr et al., 2007). However, other authors did notobtain as good results with liquid formulations in comparison withthe solid ones (Rice et al., 2000; Kyei-Boahen et al., 2002; Claytonet al., 2004a; Clayton et al., 2004b).

5. Conclusions

Alternative substrates to peat can be used as carriers for legumebacteria. We tested six organic and inorganic materials with severalbacteria. Compost cork and perlite gave as good results as peat interms of support bacterial growth and maintain a long survival ofinoculated strains, as well as survival on soybean seeds. Some liquidformulations are adequate for growth and survival of soybeanrhizobia strains for at least 3 months of storage. Under fieldconditions those formulations led to soybean yield componentssimilar to those using traditional peat-based inoculants.

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