Inoculants of Azospirillum brasilense: Biomass production, survival and growth promotion of Setaria italica and Zea mays

Soil Biol. Biochem. Vol. 28, No. 1, pp. 123-126, 1996

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INOCULANTS OF AZOSPIRILLUA4 BRASILENSE: BIOMASS PRODUCTION, SURVIVAL AND GROWTH PROMOTION OF SETARIA ITALICA AND ZEA MAYS ELAZAR

FALLIK’ and YAACOV

OKON?*

‘AR&The Volcani Center, Department of Postharvest Science of Fresh Produce, Bet Dagan 50250, Israel and ?The Hebrew University of Jerusalem, Faculty of Agriculture, Department of Plant Pathology and Microbiology, Rehovot 76100, Israel (Accepted IO June 1995) Summary-We grew Azospirillumbrusilensebiomass in fed-batch culture for 28 h using succinic acid as a C source and liquid NH> as N source. Viable cell concentration reached l-3 x lo’@CFU ml-l after 28 h. Ground or granular peat, serving as carriers for the bacteria, maintained the highest number of A. brusilensecompared to vermiculite, talcum powder, basalt granules or bentonite. A. brasilenseviable counts decli:ned from about IO’OCFU g-’ peat to 105-IO6CFU g-’ peat after 6 months. A pot system for evaluating the effect of A. brasilenseinoculants on plant growth in greenhouses demonstrated that application (of peat-carrier containing IO8CFU g-’ peat, significantly increased panicle length and dry weight of Setaria iralicaand ear and kernel weight of maize (Zea muys L). Highest growth promotion effects on maize were observed when using A. brasilensecells containing 40% polyhydroxybutyrate (PHB) and by applying the peat inoculant 2 cm below the maize seed.

INTRODUCTION

Bacteria of the genus Azospirillum colonize rhizospheres of forage and grain grasses (Dell-Gallo and Fendrik, 1994). Az’ospirilla possess physiological and morphological properties such as chemotaxis, aerotaxis, accumulation of storage materials as poly-Dhydroxybutyrate, production of plant-growth promoting substances, N2 fixation and formation of cysts (Hartmann and Zimmer, 1994). Azospirillum promotes plant growth primarily by inducing morphological and physiological changes in plant roots, and by enhancing plant water and mineral uptake (Fallik et al., 1994). The use of azospirilla as inoculants has been reviewed by Sumner (1990) and Fages (1994). Worldwide data accumulated over 20 years of field inoculation experiments with Azospiriilum concluded that these bacteria are capable of increasing yield of agriculturally important crops in different soils and climatic regions. The data indicate a 60-70% success rate with statistically significant increases in yield as much as 30% (Okon and Labandera-Gonzalez, 1994). Large-scale commercial use of Azospirillurn inoculants requires considerable bacterial biomass. Only a few studies have been devoted to studying AzospirilIum growth in fermentors (Albrecht and Okon, 1980; Fages, 1992). In an optimized biomass production process, an A. lipqferum CRT 1 culture consumed 3 1.5 g 1-l glucose in 26 h and attained a biomass of 15 g 1-l (Fages, 1992). We describe a method for *Author for correspondence.

biomass production of Azospirillum in a fed-batch fermentor, and a system for evaluating the effect of Azospirillum inoculant formulations on plants grown in the greenhouse under controlled conditions. MATERIALS AND METHODS Bacterial strain, medium and growth conditions Azospirillum brasilense ATCC 29729 (Cd) isolated from roots of the grass Cynodon dactylon was used in all experiments. A single colony was inoculated into a 2 1 liquid culture medium in baffled Erlenmeyer flasks containing in g 1-l: K2HP04, 6; KHIPOI, 4; Na-succinate, 5; NH4Cl, 1; MgS0.,.7H?O, 0.2; yeast extract, 0.2; NaOH, 3.5; trace elements as described by Albrecht and Okon (1980). Inoculated flasks were shaken overnight at 35°C with vigorous aeration (rotary shaker, 200 rev min-I). Growth was assessed by measuring optical density (OD) at 660 nm; at this an OD of 1.0 corresponded to wavelength, 1 x lo9 CFU ml-‘. Exponential-phase Erlenmeyer cultures were used to inoculate a 16 1 fermentor (New Brunswick Inc., New Jersey, U.S.A.) containing, in g 1-l: K?HPO+ 3; KH>POd, 2; MgS04.7H20, 0.2; yeast extract, 0.2; NaOH, 3.5; trace elements as described above; various C and N sources as shown in Table 1. All C and N sources were added as powder, except ammonia that was added as liquid. After sterilization, pH was adjusted to 7.0. A silicone antifoam agent (Sigma antifoam emulsion) was added. Growth temperature was maintained at 35°C. Dissolved 0 (D.O.) was maintained at 3O-50% 123

124

Elazar

Fallik and Yaacov Okon

Table 1, The effect of carbon and nitrogen sources, added to the fermentor at various concentrations (in parentheses), on yield of Azxpirillum hrasiknse Cell yield

( x lo9 cfu ml - ‘) I - ‘)

Nitrogen source (g

Ammonium nitrate (2.0) Car/m

Ammonium (26.5%) (2.6)*

Ammonium chloride (1.5)

saurct= (gI - ‘)

DL-Malic acid (5.0) Succinic acid (5.0) Fumaric acid (5.0) Glycerol (5.0) Lactic acid (5.0) *ml l-

Ammonium acetate (2.0)

6+ 5f 2* 5; 2+

1.5 0.9 0.5 1.3 0.8

3 f 1.9 4 f 0.9 3 k 0.6 5 ?r 0.7 3 f 0.5

20 f 24 f 3+ 3+ 3*

8.5 6.0 1.0 I.1 1.1

6+ 5+ 2* 6-1 5*

1.0 1.2 1.0 1.2 2.0

’of liquid NHx.

saturation by adjusting both agitation and sparging. The first stage of fermentor growth required 24 h and terminated when D.O. began to rise. A sterilized feeding medium (pH 4.0) containing a concentrated C source, N source and trace elements as described above was continually fed into the fermentor through the acid pump activated by the pH controller. Exponential growth continued until all the N in the fermentor was exhausted, and the D.O. began to rise again. The total absence of N was confirmed by performing the Nessler test on a sample culture supernatant. Enrichment for poly-fl-hydroxybutyrate

(PHB)

Growth of the culture proceeded as described above until the dissolved O2 was reduced to near 0% (equilibrium between cell respiration and O2 dissolution). The pH controller maintained the flow of the C source without any N source for 34 h. The C source in the cell was converted to PHB in the absence of N. PHB content of cells was determined as described by Tal and Okon (1985). At the end of the growth cycle viable counts were estimated by a most probable number (MPN) procedure involving growth in a semi-solid medium selective for A. brasilense Cd described by Albrecht and Okon (1980). Carriers and analysis of survival

After harvesting, samples of bacteria were mixed with one of six types of gamma-irradiated carriers: Ground peat (45 pm, Nitragin Co. [Lyphatec], U.S.A.) mixed with 8% CaCOl (w/w); Granular peat (1 mm dia, Nitragin Co. [Lyphatec], U.S.A.) mixed with 5% CaC03 (w/w); Vermiculite (2 mm) (Agrikal, Israel); Talcum powder (45 pm) (Machteshim Co., Israel); Basalt granules (l-2 mm) (Hagolan Co., Israel); Bentonite (45 pm) (Machteshim Co., Israel). An electric concrete mixer was used to mix the bacteria with the carriers. A batch of 6 1 bacterial suspension was mixed with about 13 kg of carrier for 1 min. Inoculants were stored in sterile polyethylene bags at 20 + 2°C in the dark. After 1 day, 6 days, 1 month, 2 months and 6 months, inoculants were sampled in order to assess A. brasifense viable counts. Two 10 g samples of inoculant were obtained by a sample splitter (Ari Ltd, Bene Brak, Israel) and were

stirred for 2 h at 35°C and 200 rev min-’ in phosphate buffer pH 6.1. After serial dilutions, viable counts were estimated by a most probable number (MPN) procedure involving growth in a semi-solid medium selective for A. brasilense Cd in g I-‘: yeast extract 0.2 g; MgS04.7H20, 0.2 g; Na succinate, 10.0; K2HP04, 3.0; KH2P04, 2.0; agar, 0.75; bromthymol blue, 0.02; trimethoprim, 0.2; streptomycin, 0.2; cycloheximide, 0.2. The last three ingredients were added after autoclaving. Plant growth, pot experiments and inoculation

Commercial maize seeds Zea mays cv. “Hazera 224” and Setaria italica foxtail millet (Hazera Company, Haifa) were used. Depending on the experiment, three seeds of S. italica or one seed of maize were sown 2.5 cm deep in a 10 or a 25 1 bucket containing sandy soil. The plants were grown in the greenhouse at 30 k 3°C during the day and 15 f 3°C at night with an 1l-13 h light period. The plants were watered with tap water. S. italica was grown for 5&60 days and plants were fertilized with a total of 30 mg N as NH,NO, plant-‘. Maize plants were grown to maturity for 110 days and during the growth season the plants were fertilized with a total of 2 g N as NH,NO, plant-‘. Granular peat (1 g) containing lo* A. brasilense was used as an inoculant and was put together with the seed, 2 cm under or above the seed, 2 cm beside of both sides of the seed, or at all locations described above. Peat (1 g) mixed with autoclaved bacteria was used as the control. Three experiments were conducted with 10 (S. italica) or 25 (maize) replicates for each treatment. Results were analyzed using T-test or Duncan’s range test at P = 0.05. RESULTS

The effect of C and N sources on the yield of A. brasilense

Two C sources, DL-malate and Na-succinate, together with liquid NH3, increased the bacterial yield by almost lo-fold as compared to other C and N sources added to the fermentor (Table 1). At the end of the growth cycle after induction of PHB accumulation, the viable count obtained with

Inoculants of A. brasdense

125

Table 2. Surviving mean numbers of viable cell of A:ospiri//umin various carriers after various storage times (in days)

Carrier (size)

1d

37 37 40 34 30 33

1500 1750 570 180 60 3

Granular peat (1 mm) Ground p-cat (45 pm) Vermiculite (2 mm) Basalt granules (2 mm) Talcum powder (45 pm) Bentonite (45 pm) ND-not

Viable counts

Final humidity (%)

( x 10’cfu g - ’carrier)

Survival of A. bra.silense

The survival of A. brasilense on different carriers was examined during a 6 month period (Table 2). Azospirillum bradense was applied to the carriers as a suspension containing l-3 x 10” CFU ml-‘. After l-6 days from application, only ground and granular peat maintained the bacterial concentration at lOlo CFU g-l carrier; bacterial concentrations had dropped 10-1000 fold in the other carriers (Table 2). After 2 months, the numbers of A. brasilense counted in the ground and granular peat was 2.7 x 10’ and respectively. 6.7 x 10’bacteria g-’ carrier, Six months after application, only the granular peat still contained 4 x lo* CFU bacteria inoculant g-’ carrier (Table 2). Greenhouse experiments Setaria italica plants inoculated with A. brasilense had panicles with lsignificantly increased dry weight, as compared to unmoculated controls (7.5 and 5.1 g, respectively). Inoculation of maize with A. brasilense resulted in significant increases in ear and kernel weight, compared to uninoculated maize (185 and 162 g plant-‘, and 170 and 140 g plant-‘, respectively). No significant differences were observed between uninoculated plants and plants inoculated with A. brasilense (in peat-based carrier), which did not accumulate PHB during growth (Table 3). High PHB-containing inoculants caused a non significant increase in ear and kernel weight as compared to low-PHB inoculani. However, inoculant containing A. brasilense having 40% PHB increased significantly both ear and kernel weight as compared to uninoculated controls (Table 3). Table 3. The effect of PHB content of bacterial cells on ear and kernel weight of maize cv. Hazera 224.

Control (-) PHB (3%) (+) PHB (40%)

30d

60d

180d

62 25 5.5 3 0.04 0.04

7.7 2.7 0.05 0.01 ND ND

0.4 0.03 ND ND ND ND

detectable.

DL-malate or Na-succinate, together with liquid NH3 was 1-3 x 10” cell ml-’ (Table 1).

Treatment (PHB cont.)

6d 1500 1500 150 37.5 3.5 0.35

Ear weight (g plant - ‘)

Kernel weight (g plant - ‘)

182b 207ab 225a

154c 176cd l!Xd

In each column means followed by different letters are signilicanl.ly different according to Duncan’s range test (P = 0.05).

EfSect of application site of inoculum on ear weight of maize

Only a slight increase in ear weight was observed when A. brasilense inoculants were applied next to or above seeds at a distance of 2 cm (Table 4). An application of inoculant 2 cm below seeds, or at four locations both above and beside the seed, or together with the seed, significantly increased ear weight as compared to uninoculated controls (Table 4). DISCUSSION

Our results and those of Fages (1992) demonstrate that it is possible to grow cultures of A. brasilense with counts of l-3 x 1O’OCFU ml-l. Media commonly employed for growth of Azospirillum contain an organic acid as a C source combined with NHKl (Albrecht and Okon, 1980). In such media, however, utilization of the C source results in high pH which inhibits further growth (Albrecht and Okon, 1980). We solved this problem by using a fed-batch culture in which succinic acid was constantly fed to the fermentor by monitoring the changes in pH with a pH controller. When fed DL-malate, the bacteria only utilized the L-malate (50% waste of C source). Liquid NH, was the preferred N-source in the fermentation process; its use did not result in the buildup of Cl when NH4Cl is used. By maintaining adequate D.O. during growth, it was possible to obtain fast-growing cultures with replication rates of about 0.4 h-l resulting in a high concentration of cells (l3 x 10” CFU ml-‘) after 24 h. At the end of the growth phase, from 24 to 28 h, high C-to-N ratios and low D.O. induced 40% PHB accumulation. Cells with high PHB content are more resistant to desiccation, UV and starvation stresses, and PHB serves in A. brasilense as an internal C and Table 4. The effect of A:ospirillumbrasiltwse inoculum site on the ear weight of maize (Zea mays cv. Hazera 224) (seed was planted 2.5cm deep) Location I. 2. 3. 4. 5. 6.

Uninoculated control Together with the seed 2cm under the seed 2 cm above the seed 2 cm beside the seed (both sides) All four locations

Kernel weight (g plant - ‘) 107c 128b 137a 114c I1 Ic 138a

Means followed by different letters are significantly according to Duncan’s range test (P = 0.05).

different

Elazar Fallik and Yaacov Okon

126 energy

source

for growth

and Nr-fixation

(Tal and

Okon, 1985). Although ground or granular peat were the best carriers after 6 months, few cells of A. brasilense survived; viable counts decreased from about 10” to 10b CFU g-’ peat. Similar survival for Azospirillum has been observed when using peat-clay carriers or soil bovine-manure (Favilli et al., 1987) and when using peat that had been successfully used for Rhizobium inoculants in Uruguay (P. Dutto, pers. comm.). Apparently peat is suitable as a carrier for rhizobia, but is less suitable for Azospirillum; it is possible that heat-and gamma-sterilization of peat would have released toxic compounds. Better survival of Azospirillum has been observed in liquid formulations such as Zea-Nit (Heligenetics, Gaiba, Italy) (G. Castro-Videla, pers. comm.), in vermiculite formulations and with micro-encapsulated bacterial cells dehydrated in alginate matrices (Fages, 1992; Fages, 1994). Inoculation experiments in greenhouses and in the field with peat-based Azospirillum inoculants have been carried out extensively (Sumner, 1990; Okon and Labandera-Gonzalez, 1994). In most cases, as was the case in our work, only freshly-prepared peat inoculants containing lOa-lo9 CFU g-’ peat were used. Similarly, plant-growth promoting effects (plant yield and yield components) were obtained in our work for S. italica and maize. The effects in maize were significant when the inoculant was placed 2 cm below the seed or together with the seeds. This may suggest that an optimal number of azospirilla colonized emerging seedling roots. Another interesting observation was that plant growth-promoting effects were more consistent when using inoculants containing A. brasilense cells that had accumulated the storage polymer PHB (40% of cell dry wt). The above observation needs further confirmation under field conditions. In field experiments carried out in

Mexico with maize and wheat, better and consistent results have been obtained when using peat inoculants prepared with PHB-rich Azospirillum cells (J. Caballero-Mellado, pers. comm.). Acknowledgements-The work described was performed in collaboration with Bio-Technology General (Israel) Ltd. We thank Meir Fischer and Dov Kanner of Bio-Technology General for reviewing the manuscript.

REFERENCES

Albrecht S. L. and Okon Y. (1980) Cultures of Azospiriiium. In Photosynthesis and Nitrogen Fixation. Methods of Enzymology (A. San Pietro, Ed.), Vol. 69, pp. 140-749. Academic Press, New York. Dell-Gallo M. and Fendrik I. (1994) The rhizosphere and Azospirillum. In A~ospirillumlPlant Associations (Y. Okon, Ed.), pp. 57-76. CRC Press, Boca Raton, FL. Fages J. (1992) An industrial view. of Azospirillum inoculants: formulation and application technology. Symbiosis 13, 15-26.

Fages J. (1994) Azospirillum inoculants and field experiments. In Azospirillum/Plant Associations (Y. Okon, Ed.), pp. 87-110. CRC Press, Boca Raton, FL. Fallik E., Sarig S. and Okon Y. (1994) Morphology and physiology of plant roots associated with Azospirillum. In Azospirillum/Plant Associations (Y. Okon, Ed.), pp. 1786. CRC Press, Boca Raton, FL. Favilli F., Balloni W., Cappellini A., Granchi L. and Savoini G. (1987) Esperienze pluriennali di batterizzazione campo con A:ospiriNum spp. di coltore cerealiCole. Annals of Microbiology 37, 169-181. Hartmann A. and Zimmer W. (1994) Physiology of Azospirillum. In A:ospirillum/Plant Associations (Y. Okon, Ed.), pp. 15-39. CRC Press, Boca Raton, FL. Okon Y. and Labandera-Gonzalez C. A. (1994) Agronomic applications of Azospirillum: an evaluation of-20 years world-wide field inoculation. Soil Biolonv & Biochemistrv 26, 1591-1602. Sumner M. E. (1990) Crop responses to A:ospirillum inoculation. Advances in Soil Sciences 12, 53-123. Tal S. and Okon Y. (1985) Production of the reserve material poly-/l-hydroxybutyrate and its function in Azospirillum brasilense Cd. Canadian Journal of MicroII

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