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Plant Growth-Promoting Bacteria As Biofertilizer Fauzia Y

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Plant Growth-Promoting Bacteria As Biofertilizer Fauzia Y Plant growth-promoting bacteria as biofertilizer Fauzia Y. Hafeez, Sumera Yasmin, Dini Ariani, Non Renseigné, Yusuf Zafar, Kauser A. Malik To cite this version: Fauzia Y. Hafeez, Sumera Yasmin, Dini Ariani, Non Renseigné, Yusuf Zafar, et al.. Plant growth- promoting bacteria as biofertilizer. Agronomy for Sustainable Development, Springer Verlag/EDP Sciences/INRA, 2006, 26 (2), pp.143-150. hal-00886338 HAL Id: hal-00886338 https://hal.archives-ouvertes.fr/hal-00886338 Submitted on 1 Jan 2006 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Agron. Sustain. Dev. 26 (2006) 143–150 143 © INRA, EDP Sciences, 2006 DOI: 10.1051/agro:2006007 Research article Plant growth-promoting bacteria as biofertilizer Fauzia Y. HAFEEZa*, Sumera YASMINa, Dini ARIANIb, Mehboob-ur-RAHMANa, Yusuf ZAFARa, Kauser A. MALIKa a National Institute for Biotechnology and Genetic Engineering (NIBGE), PO Box 577, Jhang Road, Faisalabad 38000, Pakistan b R&D Centre for Biotechnology, The Indonesian Institute of Sciences, JI. Raya Bogor Km 46, Cibinong 16911, Indonesia (Accepted 18 May 2006) Abstract – Seventeen rhizobacteria isolated from different ecological regions, i.e. Brazil, Indonesia, Mongolia and Pakistan were studied to develop inoculants for wheat, maize and rice. Almost all the bacterial isolates were Gram-negative, fast-growing motile rods and utilized a wide range of carbon sources. These isolates produced indole-3-acetic acid at concentrations ranging from 0.8–42.1 µg/mL, irrespective of the region. Fifteen isolates fixed N at rates ranging from 20.3–556.8 nmole C2H2 reduced/h/vial. Isolate 8N-4 from Mongolia produced the highest amount of indole-3-acetic acid (42.1 µg/mL), produced siderophores (0.3 mg/mL) and was the only isolate that solubilized phosphate (188.7 µg P/mL). Inoculation of the wheat variety Orkhon with 8N-4 isolate resulted in the maximum increase in plant biomass, root length, and total N and P contents in plants. Random amplified polymorphic deoxyribonucleic acid (RAPD) analysis, conducted with 60 decamer primers, revealed a high level of polymorphism among the bacterial isolates from different geographic regions and a low level of polymorphism among isolates from the same region. The complete 16S rRNA gene sequence analysis demonstrated that 8N-4 is a Bacillus pumilus strain (Accession number AY548949). It was concluded that Bacillus pumilus 8N-4 can be used as a bio-inoculant for biofertilizer production to increase the crop yield of wheat variety Orkhon in Mongolia. Rhizobacteria / genetic diversity / RAPD 1. INTRODUCTION in soils of diverse geographical regions and screened for their ability to produce plant growth hormones, solubilize phos- Plant growth-promoting bacteria (PGPB) are of great agro- phates and fix nitrogen. This investigation was planned as a sys- nomic importance. Indeed, they produce metabolites such as tematic exploratory study to determine the occurrence of PGPB plant growth regulators that directly promote growth and facil- in soils from Indonesia, Mongolia and Pakistan. An attempt itate nutrient uptake by plants (Bai et al., 2002; Salamone et al., was made to characterize these rhizobacteria and to assess their 2001). There is widespread distribution of PGPB that flourish genetic relatedness using random amplified polymorphic DNA in different geographical habitats (Dobereiner et al., 1976; Staley, (RAPD) analysis in relation to biochemical and physiological 1999; Santos et al., 2001). variation of the isolates. The complete 16S rRNA gene of a The large genetic diversity in natural populations of PGPB highly promising strain was sequenced for identification pur- increases the scope to improve the efficacy of inoculants poses. The study will help in developing biofertilizers for cere- (Raaijmakers and Weller, 2001). Conventionally, diversity has als with potential benefit to improve the crop productivity. been assessed by exploring phenotypic and genotypic variation (Hartmann and Amarger, 1991). With the advent of nucleic acid fingerprinting and rRNA sequencing (Bull et al., 1992), it 2. MATERIALS AND METHODS has become possible to distinguish between phenotypically similar strains that are genetically different. These tools pro- 2.1. Morphological, physiological and biochemical vide a more precise way of establishing relationships between many bacterial species (Muyzer and Smalla, 1998). characterization There is great potential for use of PGPB as biofertilizing 2.1.1. Isolation of bacteria agents for a wide variety of crop plants in a wide range of cli- matic and edaphic conditions (Reed and Glick, 2004). In the Seeds of maize, wheat and rice were surface-sterilized with present study, rhizobacteria were isolated from Triticum aesti- 0.1% mercuric chloride for five minutes and then washed three vum (wheat), Zea mays (maize) and Oryza sativa (rice) grown times with sterile water. The sterilized seeds were germinated * Corresponding author: [email protected], [email protected] Article published by EDP Sciences and available at http://www.edpsciences.org/agro or http://dx.doi.org/10.1051/agro:2006007 144 F.Y. Hafeez et al. Table I. Indole acetic acid (IAA) production, acetylene reduction assay (ARA) and phosphate solubilization (P) activities of PGPB strains iso- lated from soils obtained from geographically different regions. Strains Host Region Tentative IAAB ARAB PB identificationA (µg/mL) (nmol/h/vial) (µg/mL) Bt.J No.8 Maize Indonesia Azospirillum sp. 19.1 ± 1 49.5 ± 0.5 -E LS-1 Maize Indonesia Azospirillum sp. 0.3 ± 0.6 49.5 ± 0.5 – 07-K Maize Indonesia Azospirillum sp. 1.9 ± 0.2 49.2 ± 0.5 – AZ-B Rice Indonesia Azospirillum sp. 1.4 ± 0.2 0 ± 0.1 – AZ-S Rice Indonesia Azospirillum sp. 0.8 ± 0.2 0 ± 0.1 – 8N-4 Wheat Mongolia Bacillus sp. 42.1 ± 4 20.3 ± 0.7 188.7 ± 3 8N-2 Wheat Mongolia Azotobacter sp. 28.3 ± 1.6 116.9 ± 1 – 24-N2 Wheat Mongolia Azotobacter sp. 19.8 ± 2.1 47.3 ± 0.6 – MST-6.1 Wheat Mongolia Azotobacter sp. 1.9 ± 0.1 25.4 ± 0.5 – MST-4.1 Wheat Mongolia Pseudomonas sp. 37.7 ± 2.3 23.4 ± 0.5 – M-4 Maize Pakistan Azospirillum sp. 8.9 ± 0.9 125 ± 1 – M-8 Maize Pakistan Azospirillum sp. 3.2±0.4 49±1 – W-1 Wheat Pakistan Azospirillum sp. 3.5 ± 0.1 63.2 ± 0.5 – Wb-3D Wheat Pakistan A. brasilense 16.1 ± 1.7 215.2 ± 15 – N-4D Rice Pakistan A. lipoferum 6.3 ± 1 237.4 ± 2.5 – JCM-1224D Maize Brazil A. brasilense 18.5 ± 1.6 210.3 ± 0.6 – JCM-1270D GrassC Brazil A. lipoferum 15.2 ± 0.8 556.8 ± 8.7 – A Tentative identification based on morphological characteristics. B The results of IAA, ARA and P are an average of three replicates ± standard deviation. C Digitaria decumbens. D Reference strains of Azospirillum brasilense and Azospirillum lipoferum. E No clear zone formed around bacterial colonies on Pikovskaia’s medium containing tricalcium phosphate. on water-agar (1.5% agar) plates and one-week-old contami- 2.1.3. Phosphate solubilization nation-free wheat seedlings were transplanted into soil samples collected from Indonesia, Mongolia and Pakistan. The plants A single colony of bacterial culture grown on Luria Bertani were harvested after three weeks and roots were thoroughly medium was streaked onto Pikovskaia’s medium containing washed with sterile water to remove adhering soil. One-gram tricalcium phosphate (Pikovskaia, 1948) and incubated at root pieces were homogenized in 10 mL of sterile water and 30 ± 1 °C for 7–10 days. The plates were observed for Clear serial dilutions were prepared. These dilutions were used to P-zone formation around the colonies. Quantification of avail- inoculate N-free combined carbon medium (Rennie, 1981) and able phosphorus solubilized by the bacterial isolate was quan- N-free malate medium (Okon et al., 1977) and incubated at tified by the phospho-molybdate blue color method (Gull et al., 30 ± 1 °C. The vials showing bacterial growth and acetylene 2004). Fresh bacterial culture was grown in Pikovskaia broth reduction activity were used to inoculate plates of the same on a rotary shaker for 12 days at 24 ± 1 °C. The suspension was g solid media to obtain pure colonies. centrifuged at 6000 × for 15 minutes. The supernatant was decanted and filtered, and the pH of the sample was analyzed. The bacterial isolates were grown in Luria Bertani medium The available phosphorus was determined at 882 nm using a with shaking at 30 ± 1 °C (Yasmin et al., 2004) and studied for spectrophotometer and calibrated with a standard phosphate mean generation time (Hafeez et al., 1995) and Gram reaction curve. (Vincent, 1970). The morphological and cultural characteris- tics of the bacterial strains were studied by light microscopy 2.1.4. Acetylene reduction assay (Gopala, 1967). 2.1.2. Bacterial strains Nitrogenase activity was measured by acetylene reduction assay (Hardy et al., 1968). Pure bacterial colonies were inocu- Out of the seventeen bacterial strains used in the present lated onto N-free combined carbon medium (Rennie, 1981) in study, five were isolated from Indonesian soils, five from Mon- vials and incubated at 30 ± 1 °C for 2–3 days. Acetylene (10% golian soils and three from Pakistani soils. Bacterial strains v/v) was injected into the vials. After incubation for 1 h at room Azospirillum brasilense JCM-1224 (ATCC 29145) and A. temperature, gas samples (100 µL) were analyzed on a gas chro- lipoferum JCM-1270 (ATCC 29709) were obtained from Bra- matograph (Thermoquest, Trace G.C, Model K, Rodono, zil (Tarrand et al., 1978)., and A.
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