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Perlite as a carrier for bacterial inoculants

A. Daza*, C. SantamarõÂa, D.N. RodrõÂguez-Navarro, M. Camacho, R. Orive,

F. Temprano

C.I.F.A. ``Las Torres y Tomejil'', Aptdo. O®cial, 41200 AlcalaÂ del Rio (Sevilla), Spain

Accepted 28 September 1999

Abstract

Growth and survival of Rhizobium leguminosarumbv.phaseoli, R. tropici,Bradyrhizobium japonicum andBacillus megaterium in peat and perlite-based inoculants were evaluated. In general, survival was similar for all strains in both carriers. Better survival was observed when inoculants were maintained at 48C compared to 288C. Studies with two dierent stickers suggested the existence of interactions between carriers and adhesives and showed that combination of a sucrose adhesive with the perlite carrier gave better survival of bacteria on seeds. Bean and soybean ®eld experiments indicated that perlite-based inoculants produced similar number of nodules, nodule dry weight, crop yield and nitrogen content, as peat-based inoculants. 7 2000 Published by Elsevier Science Ltd. All rights reserved.

Keywords:Bacterial inoculants; Peat-and perlite-based inoculants; Bacterial survival; Symbiotic properties of inoculants; Field experiments

1. Introduction

Inoculation of seeds with rhizobia dates from the end of 19th century (Voelcker, 1896). Finely ground peat is the most commonly used carrier in convention-al legume inoculant production (Burton, 1967; Rough-ley, 1970). Other microorganisms of potential value as biological control agents or as plant growth promoting bacteria have also been used to produce inoculants (LoÂpez et al., 1981; Subba Rao, 1982; Fallik and Okon, 1996). However, peat is not always available (Strijdom and Deschodt, 1976). It is also well known that some peats may inhibit growth of someRhizobium strains (Brockwell, 1985). Furthermore autoclaved or gamma-irradiated peat may be dicult to obtain. The high temperature during steam sterilization or the high dosage needed for irradiation might generate toxic substances for bacteria (Strijdom and Rensburg, 1981; Mulligan and Cooper, 1985). In addition, the need to preserve wet-land ecosystems make the extraction of peat unadvisable in some countries. Thus, other

poten-tial materials able to support good growth and survi-val of bacteria are needed. Many materials have been evaluated, including dierent coals (Crawford and Ber-ryhill, 1983) bentonite, corn oil (Kremer and Peterson, 1983), mineral soils (Chao and Alexander, 1984), or vermiculite (Sparrow and Ham, 1983; Graham-Weiss et al., 1987). Perlite is a volcanic stone composed of a little-hydrated aluminium silicate. For commercial use it is exposed to an exfoliation process at high tempera-tures that kills microorganisms. However, if necessary, it can be easily sterilized with no risk of producing toxic substances.

Our objective was to study the feasibility of using perlite as a carrier for bacterial inoculants following the commom production and application procedures used for most peat-based inoculants. Peat was used as a reference carrier.

2. Materials and methods

2.1. Bacterial strains and growth conditions

Bacterial strains used in this work were Bradyrhizo-bium japonicum USDA110 (Sadowsky et al., 1987),

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* Corresponding author. Fax: +34-95-565-0373.

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Rhizobium fredii SMH12 (RodrõÂguez-Navarro et al., 1996),R. leguminosarum bv. phaseoli ISP23 and ISP42 (strains from our Culture Collection), R. tropici CIAT899 (Graham et al., 1994) and Bacillus megater-ium ATCC33085. B. japonicum was grown in a liquid medium containing (lÿ1_{): 0.4 g K}

2HPO4, 0.6 g KH2PO4, 0.1 g CaCl2, 0.7 g KNO3, 0.3 g (NH4)2HPO4, 5 mg MnSO4.7H2O, 1 g yeast extract, 10 ml glycerol, pH 6.8; Rhizobium strains in yeast extract mannitol (YEM) medium (Vincent, 1970) and B. megaterium in tryptone soy broth (TSB, Oxoid). Stock cultures were maintained in 20% glycerol at

ÿ208C.

2.2. Carriers and inoculant production

Carriers used were peat and perlite. Black peat was obtained from Padul, Granada, Spain. This peat has been used since 1975 as a carrier for commercial legume inoculant production in Spain (RuõÂz-ArguÈeso et al., 1979; RodrõÂguez-Navarro et al., 1991). The peat has a pH of 7 and does not require CaCO3 for pH adjustment. Perlite (Spavik, S.A., Huesca, Spain) con-tained (%) 70±80 SiO2, 12±16 Al2O3, 2±5 Na2O, 2±5 K2O, 0±2 CaO, 0±1 MgO, 0±1 FeO3, 0±1.5 S and SO3, pH 6.6±7. Both carriers were ®nely ground in a ham-mer mill to pass a 70 mm screen, autoclaved at 1208C for 40 min and mixed with saturated bacteria liquid cultures (1±4109cell ml±1) to obtain a uniform

mix-ture with a ®nal moismix-ture of 36±38%. After packaging in heat-sealed polyethylene bags, inoculants were stored either at 48C or 288C. Duplicate samples were made for each inoculant. Periodically, inoculants were sampled and viable bacteria were estimated by plating 10-fold serial dilutions on Congo-red-YEM agar. Numbers of B. megaterium are referred to colony forming units.

2.3. Seed inoculation and bacterial survival

Bean and soybean seed lots of 50 g were mixed with 0.5 g of inoculant and 0.5 ml of a water solution of adhesive (20% gum arabic or 10% sucrose). Survival of perlite-based inoculants on seeds was also measured using other stickers including 2% carboxymethycellu-lose, 25% glycerol and 50% polyethyleneglycol (Mol. Wt. 1450). The inoculated seeds were allowed to dry for 1 h at room temperature (48±50% air relative moisture) and stored in glass vessels at room tempera-ture (18 to 208C). Bacterial survival on seeds was periodically determined by transferring 20 inoculated seeds to 100 ml sterile saline buer and plating 10-fold serial dilutions on Congo-red-YEM agar.

2.4. Symbiotic properties of inoculants

Inoculants were tested for eectiveness on their re-spective host plants in 2.5 l pots containing sand (4 replicates per treatment). Five inoculated seeds were sown in each pot and, after germination, thinned to three seedlings per pot. Plants were grown at 18±288C (night/day) in a greenhouse with a photoperiod of about 14 h for 45 d and were irrigated with a N-free solution (Rigaud and Puppo, 1975). After harvest, shoot dry weight, number of nodules and nodule dry weight were determined.

2.5. Field experiments

Two ®eld experiments, with soybean [Glycine max (L.)Merr] and with fresh green bean (Phaseolus vul-garis), were carried out in Seville (Southern Spain). Both experiments were placed in a loam soil of pH 8.1 (2.2% organic matter, 14 mg P kgÿ1

3). The experimental ®eld was laid out in a randomized complete block design with four replicates. Plots (7 2.5 m) were divided into

rows, spaced 0.5 m. A space of 1 m was allowed between plots and 3 m between blocks. Each plot was sown with 220 g of soybean cv. Kure seeds or with 190 g of bean cv. Mutin seeds (Asgrow). Soybean seeds (1 kg) were inoculated with 10 g of peat or per-lite-based inoculant (ca. 106 bacteria seedÿ1_{) of} _B.

japonicum USDA110 or R. fredii SMH12. Bean seeds (1 kg) were inoculated with 10 g of peat or perlite-based inoculant of R. leguminosarum bv. phaseoli ISP23 (ca. 106bacteria seedÿ1

). In both trials, uninocu-lated controls with or without N fertilizer were included. The N fertilized plots received a single appli-cation of 200 kg N haÿ1

as ammoniun nitrate. Forty ®ve to 50 d after sowing, 10 to 15 plants per plot were dug out to estimate number and dry weight of nodules. At the end of the growing season, plants were har-vested to evaluate soybean seed yield or fresh bean pod yield. The soybean seeds N content was deter-mined by the Kjeldahl method (Vincent, 1970). In the soybean experiment, data on number and dry weight of nodules for B. japonicum USDA110 and R. fredii SMH12 were independently analysed because both greatly dier in their capacity to nodulate soybean cul-tivars, being greater for R. fredii in alkaline soils (BuendõÂa-ClaverõÂa et al., 1994). However, the lower rate of nodulation in B. japonicum is overcome by its greater nitrogen ®xation eectiveness, so a single analysis was performed on seed yield and N content.

2.6. Statistics

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sofware (NH Analytical Sofware, USA). Results were expressed at theP< 0.05 level of signi®cance.

3. Results and discussion

3.1. Survival of bacterial strains on peat and perlite based inoculants

Survival of R. leguminosarum bv. phaseoli (strains ISP23 and ISP42), R. tropici and Bacillus megaterium

was followed in peat and perlite inoculants kept at 4 and 288C for 6 months (Fig. 1). The results showed that perlite was as eective as peat in maintaining high populations of rhizobia andB. megaterium.In general, survival was greater when the inoculants were kept at 48C. However bacterial survival was higher at 288C on perlite. Numbers of R. tropiciCIAT899 declined more rapidly than other strains in both inoculants. Perlite was also an adequate substrate for growth and survival of B. japonicum USDA110 and R. fredii SMH12 (A. Daza, unpublished results).

Fig. 1. Survival of R. leguminosarum bv. phaseoli ISP23, R. leguminosarum bv. phaseoli ISP42, R. tropici CIAT899 and B. megaterium

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3.2. Survival of R. phaseoli, R. tropici and B. japonicum on seeds

Perlite was less eective than peat in promoting rhi-zobial survival on seeds when gum arabic was used as the adhesive agent, but survival was similar with both carriers when sucrose was used, providing more than 104 rhizobia per seed after 3 weeks (Fig. 2). Results obtained using perlite-based inoculants combined with 2% carboxymethylcellulose, 25% glycerol or 50% polyethyleneglycol (Mol.Wt.1450) as adhesives indi-cated that none of them was better than 10% sucrose (data not shown). Fig. 3 shows survival of R. tropici CIAT899 and B. japonicum USDA110 on seeds (bean and soybean, respectively) inoculated with peat and perlite-based inoculants using sucrose as adhesive. Sur-vival was similar for both strains during the ®rst 16 d but CIAT899 showed better survival (P< 0.05) in per-lite than in peat-based inoculants 32 d after seed in-oculation.R. tropici CIAT899 showed a lower survival on seeds when compared with other strains tested.

3.3. Eectiveness of R. tropici CIAT899 in peat and perlite-based inoculants

One assay was performed under greenhouse con-ditions using non-stored inoculated-seeds (ca. 106 rhi-zobia seedÿ1_{) and inoculated seeds of Canellini bean}

seeds stored at room temperature for 16 d (ca. 104 rhi-zobia seedÿ1_{) using} _{R. tropiciCIAT 899 in peat- and}

perlite-based inoculants. Shoot dry matter, number and dry weight of nodules were determined 45 d after planting. No signi®cant dierences were obtained either on nodules or shoot dry weight of Canellini bean seeds inoculated with both types of inoculants (Table 1). The nodulation response obtained with inoculated seeds maintained for 16 d at room tempera-ture before sowing was similar to that obtained with non-stored inoculated seeds, probably due to the lack of speci®c rhizobia in the sand used in the growth sys-tem, as reported by Brockwell et al. (1980). Similar eectiveness was also obtained with Mutin bean seeds inoculated with peat- and perlite-based inoculants of R. leguminosarumbv.phaseoliISP42 (data not shown).

Fig. 3. Survival of R. tropiciCIAT899 and B. japonicumUSDA110 from peat and perlite-based inoculants on inoculated seeds using 10% sucrose as adhesive. Each point represents the mean of two replicates. Variability for each point was lower than 10%. LSD values are only indicated when there are signi®cant dierences. Fig. 2. Survival of R. leguminosarum bv.phaseoli ISP42 from peat

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3.4. Field experiments

The eect of inoculation with peat and perlite-based inoculants on nodulation and crop yield are presented in Tables 2 and 3. The number and the dry weight of nodules were not signi®cantly dierent (P < 0.05) using inoculants of R. leguminosarum bv. phaseoli ISP23 in both carriers (Table 2). Although the Rhizo-bium±common bean symbiosis is reported as non eec-tive when compared with other legume±rhizobia symbiosis (Isoi and Yosida, 1991), the strain increased the yield of the uninoculated control in more than 50% and reached 66% of the mineral nitrogen control. Soybean [Glycine max(L.) Merr.] plants are nodulated by the slow-growing bacterium B. japonicum and the fast-growing bacterium R. fredii. B. japonicum USDA110 andR. fredii SMH12 were used to compare perlite and peat as carriers. The results indicated that

perlite was as eective as peat in ®eld conditions (Table 3). Number of nodules, nodule mass and seed production showed no dierences for both carriers. N content of inoculated treatments were signi®cantly greater as compared with the uninoculated mineral nitrogen fertilized treatment. Recently perlite has been used as a carrier for B. japonicum and R. meliloti inoculants but only in greenhouse experiments with lucerne and soybean (Ronchi et al., 1997). We think that our paper has a greater scope as it deals with more strains of inoculum and our work was carried out in ®eld experiments. In summary, although perlite was not superior to peat as carrier, all results obtained in our work indicate that perlite can be successfully used as an alternative carrier for R. tropici, R. legumi-nosarum bv.phaseoli, B. japonicumand B. megaterium, providing the inoculant is used with arabic gum or sucrose to inoculate the seeds. These results are im-portant because the use of a sucrose solution as ad-hesive is a common pratice for farmers in Spain and other countries. Our results demonstrate that perlite inoculants can maintain a higher population of micro-organisms than peat inoculants at room temperature during 6 months at least. Better survival of bacteria under refrigerated conditions agreed with most pre-vious reports using other carriers. Strain variability in terms of survival is an important characteristic that determines performance of an inoculant and should be evaluated separately.

Acknowledgements

We thank Dr C. Labandera for a review of the manuscript. We are grateful to I. Antonio and M. Table 1

Nodulation and shoot dry weight of Canellini bean seeds using peat and perlite-based inoculants ofR. tropiciCIAT899a

Treatment Shoot dry wt

Sowing immediately after seed-coatingb

Uninoculated 3.31b 0 0

Peat 7.34a ₇₉₀a ₅₃₀a

Perlite 7.69a ₇₄₃a ₅₂₇a

LSD (0.05) 1.09 196 141.8

Sowing 16 d after seed-coatingc

Uninoculated 3.29b 0 0

Peat 7.55a 695a 531a

Perlite 7.72a 705a 492a

LSD (0.05) 1.19 279 56.95

a_{Values are means of four replicates, with three plants in each}

pot. Means followed by the same letter are not signi®cantly dierent atP< 0.05. Plants were grown for 45 d in the greenhouse.

b_{ca. 10}6_{rhizobia seed}ÿ1_. c_{ca. 10}4_{rhizobia seed}ÿ1_.

Table 2

Phaseolus vulgaris(cv. Mutin) ®eld experiment using peat and per-lite-based inoculants ofR. leguminosarumbv.phaseoliISP23

Treatment Nodulesa_{(15 plants)} _{Pod yield (kg ha}ÿ1₎

Number Dry wt (mg)

T 12.2 5.1 13,570c

TN 7.0 6.1 40,410a

Peat 188.5a _579.7a _22,040b

Perlite 260.0a _518.3a _26,820b

LSD (0.05) 89.4 379.9 5,415

a

Data represent mean values of four replicates and were estimated 45 d after sowing. T, uninoculated seeds non-fertilized treatment. TN, uninoculated seeds and mineral nitrogen fertilized treatment. Values followed by the same letter are not signi®cantly dierent atP

< 0.05.

Table 3

Glycine max (cv. Kure) ®eld assay using peat and perlite-based inoculants ofB. japonicumUSDA110 andR. frediiSMH12

Treatment Nodulesa

Peat/SMH12 947.5a _2.96a ₄₆₄₅a _296.6a

Perlite/SMH12 843.0a _2.98a ₄₄₅₀a _278.1a

LSD (0.05) 363.3 1.44

Peat/USDA110 433.0a _1.85a ₄₃₂₇a _284.0a

Perlite/USDA110 335.0a _1.34a ₄₄₂₅a _287.4a

LSD (0.05) 330.0 0.96 651.0 19.51

a

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Andra for technical assistance. Funding for this research were provided by the INIA (Ministry of Agri-culture), DGIFA (Junta de AndalucõÂa) and CICYT (Grant BIO96-1469 C03-03), Spain.

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