Isolation, molecular characterization and growth ... - Springer Link

World J Microbiol Biotechnol (2007) 23:997–1006 DOI 10.1007/s11274-006-9326-y

ORIGINAL PAPER

Isolation, molecular characterization and growth-promoting activities of endophytic sugarcane diazotroph Klebsiella sp. GR9 Munusamy Govindarajan Æ Soon-Wo Kwon Æ Hang-Yeon Weon

Received: 2 October 2006 / Accepted: 19 November 2006 / Published online: 21 December 2006 Springer Science+Business Media B.V. 2006

Abstract A group of endophytic diazotrophs were isolated from surface-sterilized roots and stems of different sugarcane varieties in the Tamilnadu region of India. From these, four isolates were selected, based on the highest acetylene reduction activity. Gene-specific PCR amplification confirmed the presence of nif-D genes in those isolates. The 16S rRNA sequence of isolates GR4 and GR7 had a 99.5% sequence similarity to the Pseudomonas sp. pDL01 (AF125317) and 16S rDNA sequence of isolate GR3 had a 100% similarity to that of Burkholderia vietnamiensis (AY973820). The 16S rDNA sequence of isolate GR9 was 99.79% similar to that of the Klebsiella pneumoniae type strain (KPY17657). Colonization by the isolates was confirmed using micropropagated sugarcane and sterile rice seedlings. Isolate GR9, identified as Klebsiella M. Govindarajan Postgraduate and Research Department of Microbiology and Botany, AVVM Sri Pushpam College (Bharathidasan University, Trichy), Tanjore 613503 Tamilnadu, India S.-W. Kwon Genetic Resources Division, National Institute of Agricultural Biotechnology, Rural Development Administration 225th, Seodun-dong, Gwonseon-gu, Suwon 441-707, Republic of Korea H.-Y. Weon Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration (RDA), Suwon 441-707, Republic of Korea Present Address: M. Govindarajan (&) Dr. Rajan Laboratories, 28/6, Ganesh Flats, Thirumangai Mannan Street, Sundaram Colony, East Tambaram, Chennai 600 059 Tamilnadu, India e-mail: [email protected]

pneumoniae, was consistently more active in reducing acetylene as compared with the other isolates. The effects of GR9 and the sugarcane diazotroph Gluconacetobacter diazotrophicus were compared in inoculated micropropagated sugarcane plantlets. The effects of K. pneumoniae GR9, and four other diazotrophs, G. diazotrophicus, Herbaspirillum seropedicae, Azospirillum lipoferum 4B, and Burkholderia vietnamiensis in inoculated rice seedlings were compared. GR9 alone or in combination with the other diazotrophs performed best under pot conditions. The combined effects of nitrogen fixation and endophytic colonization of this diazotroph may be useful for the development of bio-inoculants. Keywords Sugarcane endophytic diazotrophs AR activity IAA production nifD gene amplification 16S rDNA sequences Inoculation

Introduction The green revolution in agriculture has resulted in large increases in cereal grain production in the developing world since the 1960s, due to the development of plant genotypes that are highly responsive to chemical fertilizers, particularly nitrogen (N). Plants derive most of their nitrogen from nitrate and ammonia. The atmospheric deposition of nitrous oxides and ammonia, primarily from pollution, also contributes to the N-supply (Bockman 1996). Tropical agriculture might be expected to be more dependent on N-fertilizers than agriculture in temperate regions, since heavy rains and more rapid decomposition of organic matter cause leaching and rapid loss of N-fertilizers

123

998

(Dobereiner 1997). Nitrogenous chemicals account for as much as 30% of total crop fertilizers. However nitrogenous fertilizers are becoming more scarce and costly. N2-fixation is one of the possible biological alternatives to N-fertilizers and could lead to more productive and sustainable agriculture without harming the environment (Dobereiner and Urquiaga 1992). The identification of nitrogen-fixing bacteria and measurement of their nitrogenase activity has revealed that a number of diazotrophic species are associated with grasses and cereals, either as colonizers of plant rhizospheres or of intercellular spaces within xylem vessels of monocots (James and Olivares 1997). Many N2-fixing bacteria are associated with sugarcane (Boddey et al. 2003). Free-living N2-fixing bacteria belonging to the genera Beijerinckia, Azospirillum, Azotobacter, Bacillus, Derxia, Enterobacter, and Erwinia appear to be frequent colonizers of sugarcane (Dobereiner and Ruschel 1958; Arias et al. 1978; Hegazi et al. 1979; Purchase 1980; Rennie et al. 1982; Graciolli et al. 1983; Seldin et al. 1984). The 1988 discovery of Acetobacter diazotrophicus (Syn. Gluconacetobacter diazotrophicus), a nitrogen-fixing bacterium inhabiting the interior of roots, stems, and leaves of sugarcane, opened a new avenue of research into endophytic nitrogen fixation in sugarcane. Recently, many species of Azospirillum (Baldani et al. 1997), Herbaspirillum (Reis et al. 2000), and Burkholderia (Govindarajan et al. 2006) have been isolated. In the present investigation, previously undescribed nitrogen-fixing and phytohormone-producing bacteria from sugarcane plants were isolated and characterized using morphological and physiological studies, as well as molecular characterization of nif genes, and 16S rRNA sequence analysis. The colonization and growthpromoting activity of Klebsiella sp GR9 were evaluated in micropropagated sugarcane and rice seedlings.

World J Microbiol Biotechnol (2007) 23:997–1006

5; mannitol, 5; malic acid, 5; ammonium chloride, 1; pH 5.8. Reference strains Gluconacetobacter diazotrophicus (Yamada et al. 1997) strain LMG7603T (ATCC49037) was isolated from sugarcane root tissues in Brazil as strain Pal5 of Acetobacter diazotrophicus (Gillis et al. 1989). Herbaspirillum seropedicae (Baldani et al. 1986) strain LMG6513T (ATCC35892) has been isolated from surface-sterilized rice roots in Brazil. Burkholderia vietnamiensis (Gillis et al. 1995) strain LMG10929T was isolated by Tran Van et al. (1996), from rice roots in Vietnam. Azospirillum lipoferum (Bally et al. 1983) strain 4B (LMG4348) was isolated from a rice rhizosphere in France. Isolation and characterization

Materials and methods

The roots of different varieties of sugarcane were sampled from farmers’ fields in different parts of Tamilnadu State, India. One gram of fresh roots was washed with running tap water, five times with sterile distilled water, with a bleach solution containing sodium hypochlorite (4%) for 5 min, and five times with sterile water. The roots were rolled on Luria-Bertani agar to verify proper root-surface sterilization and then were macerated in a blender in 10 ml of sucrose solution (1%). Ten-fold serial dilutions of the suspension were used to inoculate N-free semisolid LGIM media in triplicate (Estrada et al. 2002). After 96–120 h of incubation, vials were assayed for acetylene reduction activity (ARA) (Mascarua-Esparza et al. 1988). Bacterial growth from nitrogenase-positive vials, with a white or yellowish pellicle 1–4 mm below the surface, were streaked on LGIM agar plates and incubated for 72–96 h. A final streaking and purification was performed on Burkholderia genus-specific PCAT and Pseudomonas agar F (PAF; Difco).

Culture media

Physiological and biochemical characterization

LGIM (Estrada et al. 2002) consisted of (per liter deionized water) cane sugar, 10 g; K2HPO4, 0.2 g; KH2PO4, 0.6 g; MgSO47H2O, 0.2 g; CaCl2, 0.02 g; Na2MoO42H2O, 0.002 g; FeCl36H2O, 0.01 g; bromothymol blue (0.5% solution in 0.2 M KOH), 5 ml; pH 5.5. PCAT (Burbage and Sasser 1982) contained (per liter deionized water) azelaic acid, 2.0; KH2PO4, 4.0; K2HPO4, 4.0; MgSO4, 0.1; yeast, 0.02; tryptamine, 2; agar, 15 g; pH 5.7. CCM (Mascarua-Esparza et al. 1988) consisted of (per liter deionized water) glucose,

Pigment-production tests were carried out on King medium B (King et al. 1954). Colony morphology was examined on PCAT (Burbage and Sasser 1982) and Pseudomonas agar F (PAF; Difco) plates. Morphology and Gram type were determined using a Trinocular phase contrast fluorescent microscope (Olympus AX 80T, Japan). Bacterial motility was tested by growth in a semisolid 0.3%-mannitol motility test medium. Oxidase and catalase were determined using commercially available discs (Hi media, Bombay, India). Utilization

123


of carbon sources was examined using the Biolog identification system. All strains were tested three times with GN2 microplates (Biolog) according to the Manufacturer’s recommendations, with reactions observed after 24 or 48 h. An API 20 NE test kit was also used for classical and phenotypic tests; examination was done after 48 h.

999

evolutionary distance matrix was generated as described by Jukes and Cantor (1969). The evolutionary tree for the dataset was inferred from the neighbor-joining method of Saitou and Nei (1987) using the neighborjoining program of MEGA version 2.1 (Kumar et al. 2001). The stability of relationships was assessed using bootstrap analyses of the neighbor-joining data based on 1000 resamplings.

Isolation of total DNA Nitrogenase (acetylene reduction) activity Isolates and type strains were grown in nutrient agar medium at 28C for 24 h and centrifuged at 12,300g. The pellets were washed with TE buffer and resuspended in 10 ml of TE (1·) containing 3 ml of 5% SDS in TE (1·) and 3 ml of proteinase K (2.5 mg/ml). After incubation at 37C for 1 h, the cleaned lysates were extracted with phenol:chloroform:isoamyl alcohol (25:24:1). DNA was precipitated by adding 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ethanol to the supernatant. The dried pellets were dissolved in TE (1·) buffer. PCR amplification of the nifD genes The sequences of the nifD forward and reverse primers were, 5¢-ATSGARTWCAACTTCTTCGG-3¢ and 5¢-A RTCCCAIGAGTGCATYTGICGGAA-3¢ (Azotobacter vinelandii M20568 nucleotide between 883 and 1337), respectively (where I is inosine, R is A or G, S is C or G, W is A or T, and Y is C or T) (Ueda et al. 1995). Amplification reactions were performed in a total volume of 25 ll. The reaction mixture contained 2.5 ll 10· PCR buffer, 2.5 ll each of 2 mM dNTP, 3 ll of each forward and reverse primer (30 ng), 1 ll of template DNA (10 ng), and 0.3 ll of Taq DNA polymerase (3 units/ll), to a final volume of 25 ll with milli-Q water. The step-up PCR procedure included denaturation at 95C for 3 min, 52C for 1 min, and 72C for 1 min, followed by 30 cycles of 95C for 1 min, 54C for 30 s, and 72C for 1 min, with a final extension at 72C for 10 min. Amplification products were electrophoresed on a 1.5% agarose gel in 1· TBE buffer.

The acetylene reduction (AR) activity of inoculated plants roots was determined according to Ladha et al. (1986). Ten seedlings from each treatment were taken at panicle initiation and grain-filling stages and roots were separated and washed twice with sterile water to remove loosely associated bacteria. The roots were then transferred to fresh, N-free, liquid Jensen’s medium (Somasegaran and Hoben 1994). The tubes containing the plants were sealed with a rubber seal and 10% of the headspace volume was replaced with acetylene. Uninoculated plant roots and tubes not injected with acetylene served as controls. They were then returned to the growth chamber and incubated in the dark for 12 h at 30C. AR activity was determined using a Systronic (Systronic, Japan) gas chromatograph. Indole-3-acetic acid (IAA) production To quantify the production of IAA by the isolates, isolates were grown in CCM for 1 week and the cells were pelleted by centrifugation at 10,000g for 15 min. The pH of the supernatant was adjusted to 2.8 with HCl and then extracted three times with equal volumes of ethyl acetate (Tien et al. 1979). The extract was evaporated to dryness and resuspended in 1 ml of ethanol. The samples were analyzed by HPLC (Shimadzu SPE 10A & 10AD) using an UV detector and a Techsphere C-18 column. Pure IAA was used as the standard. Methanol:acetic acid:water (30:1:70 v/v/v) was used as the mobile phase at the rate of 1.2 ml/min. (Rasul et al. 1998).

PCR amplification and 16S rRNA sequencing

Sugarcane plant colonization

The 16S rRNA gene sequences were determined by PCR amplification (Kwon et al. 2003), followed by direct sequencing (Hiraishi 1992). For the phylogenetic analyses, related 16S rRNA gene sequences within the genera Pseudomonas, Burkholderia, and Klebsiella were included. The 16S rDNA sequences were aligned using the MEGALIGN program of DNASTAR. An

Micropropagated sugarcane seedlings were developed from the apical meristems of cultivar CoV 92102, following the method described by Sreenivasan and Sreenivasan (1984). Both liquid and solid media were modified with respect to the concentration of growth hormones for promoting shoot initiation from apical meristems. Fully developed seedlings were obtained by

123

1000

70–75 days. Plants of uniform size were transferred to 50-ml test tubes containing 20 ml of 10-fold-diluted MS multiplication medium. Strains of novel isolates were grown in diluted MS (1/10) medium. Bacterial concentrations were adjusted using a spectrophotometer at 540 nm and 0.1 ml of suspensions containing 108 cells were inoculated aseptically. The plants were maintained in a growth chamber (27±2C; 75% relative humidity; 14-h light intensity of 60 mlux m–2; 10 h dark). Five days after inoculation, uniform-sized seedlings were planted in plastic containers with 5 kg of gravel with no detectable N and supplied with N-deficient Hoagland’s solution (100 ml) every 2 weeks. Sterile water (150 ml) was added every other day to maintain adequate moisture. Sugarcane pot experiments Micropropagated seedlings of cultivar CoV 92102 were used for pot experiments. Plastic containers (50 l) were filled with 40 kg of thoroughly homogenized upperlayer farm soil, pH 7.5. The experiment included nine treatments and six replicates in a randomized complete block design (RCBD). Novel isolate GR9 and G. diazotrophicus were inoculated individually and together into seedlings fertilized with 50% of the recommended N dose. Uninoculated seedlings fertilized with 100, 50, and 0% of the recommended N dose served as controls. Phosphorus and potassium were applied at 115 and 65 kg ha–1, respectively, in all treatments. The containers were irrigated by natural rainfall, and tap water if required. Bacterial populations from root tissues were determined by the MPN method. Leaf N was determined every month by the microKjeldahl method (Humphries 1956). After 6 months, the plants were harvested by uprooting and roots and shoots were weighed separately for comparison of biomass production.


growth chamber (27 ± 2C; 75% relative humidity; 14-h light intensity of 60 mlux m–2; 10 h dark). Five days after inoculation uniform-sized seedlings were planted in plastic containers containing 5 kg of gravel without detectable N and supplied with N-deficient Hoagland’s solution (100 ml) every two weeks. Sterile water (150 ml) was added every other day to maintain adequate moisture. Rice pot experiments Pot experiments were conducted using cultivar ADT43. Five-day-old uncontaminated seedlings were treated with a suspension of respective cultures containing 108 c.f.u. ml–. After 30 min contact, seedlings were transplanted into pots. Pots were 60 cm in diameter, 45 cm high, and contained 30 kg of soil. Experiments comprised seven levels of inoculation viz. (1) isolate GR9, (2) G. diazotrophicus, (3) A. lipoferum, (4) H. seropedicae, (5) B. vietnamiensis, (6) combination of all five strains and (7) control pots treated with an autoclaved mixture of bacterial strains. In each pot, eight hills were maintained, with three seedlings per hill. Irrigation was natural rainfall and borewell water. Nucleotide sequence accession numbers The nucleotide sequences of diazotrophs isolated in this work have been deposited under the accession numbers to NCBI: DQ100466 (strain GR3), DQ100463 (strain GR4), DQ100464 (strain GR7) and DQ100465 (strain GR9). Data analysis The data were compared by analysis of variance or t- tests.

Rice plant colonization

Results

Rice cultivar ADT-43 (length of growth cycle 110 d) was selected for axenic experiments. Dehulled seeds were surface-sterilized in 70% ethanol for 5 min, followed by 0.2% mercuric chloride for 30 s, and washed five times with sterile water. Seeds were germinated on nutrient agar plates and uncontaminated seedlings were used for axenic experiments. Uniform-sized uncontaminated seedlings were transferred to 120-ml glass tubes containing 30 ml of N-free Jensen’s medium (Somasegaran and Hoben 1994). One ml of liquid bacterial suspension containing 108 c.f.u. ml–1 was inoculated aseptically. The tubes were maintained in a

Isolation and characterization of diazotrophic bacteria

123

Eighty-one endophytic diazotrophs were isolated from surface-sterilized roots of sugarcane grown in various regions of Tamilnadu. Colonies on LGIM agar plates were large, yellow round with entire margins. Colonies on PCAT agar plates were small, white, round, smooth, and convex with entire margins. Four isolates were selected based on the higher AR activity (>100 nmol h–1 tube–1) and used for further studies. Isolates were gramnegative, motile, oxidase- and catalase-positive. The


1001

isolates were able to catabolize D-glucose and other carbohydrates with the production of acid. The data from morphological characterization and Biolog identification system biochemical tests were compared with standard species using Bergey’s Manual of Determinative Bacteriology. Based on these morphological and physiological characteristics, the isolates were identified as species of the genera Pseudomonas, Burkholderia, and Klebsiella.

GR4- and GR9-treated plants produced more ethylene than plants inoculated with other isolates. The bacterial populations in roots, as estimated by ARA-based MPN counts, also were higher at the grain-filling stage than at the panicle-initiation stage. In sugarcane, AR activity was determined at 30 and 60 days after inoculation (DAI). Higher AR activity was recorded at 60 DAI as compared with 30 DAI. Isolates GR4 and GR9 produced the more ethylene at both time points (Table 1).

NifD gene amplification IAA production NifD gene PCR amplification showed that isolates GR3, GR4, GR7, GR9 and type strains G. diazotrophicus, H. seropedicae, A. lipoferum and B. vietnamiensis produced the expected 450-bp amplification product (Fig. 1). The acetylene reduction activity and nifD gene amplification proved that these isolates were nitrogen-fixing bacteria. 16S rRNA sequence analysis Isolates GR4 (DQ100463) and GR7 (DQ100464) had the highest similarity (99.5%) to Pseudomonas sp. pDL01 (AF125317). The 16S rDNA sequence of isolate GR3 (DQ100466) was 100% similar to that of Burkholderia vietnamiensis (AY973820). The 16S rDNA sequence of isolate GR9 (DQ100465) had the highest similarity (99.79%) to that of the Klebsiella pneumoniae type strain (KPY17657) (Fig. 2). Nitrogenase activity AR activity was determined at the rice panicle initiation and grain-filling stages. The AR activity was approximately three times higher at the grain-filling stage as compared with the panicle-initiation stage. M 1

2

3

4

5

6

7

8

9 10 11 M

450 bp

Fig. 1 Agarose gel electrophoresis of PCR amplification products obtained with the nifD gene specific primers. Lanes, M: 100 bp DNA ladder; 1: Burkholderia vietnamiensis type strain LMG10929T 2: Burkholderia cepacia type strain, 3: GR1, 4: GR2, 5: GR3, 6: GR4, 7: GR5, 8: GR6, 9: GR7, 10: GR8, 11: GR9, M: Marker

Growth media from all of the isolates contained substantial amounts of the phytohormone indoleacetic acid. Considerably higher amounts of IAA were produced in the presence of tryptophan (100 mg/l) than in its absence. Isolate GR9 produced the highest amount of IAA with added tryptophan (23.2 lg/ml), GR3 produced more IAA (16.0 lg/ml) whereas type strain G. diazotrophicus produced 10.2 lg ml–1 and A. lipoferum produced 21.62 lg ml–1, B. vietnamiensis 14.57 lg ml–1, and least IAA produced by H. seropedicae 8.32 lg ml–1. Sugarcane plant colonization Seedlings inoculated with GR9 and fertilized with 50% of the recommended N dose yielded more biomass (3166 g/plant) than the control plants fertilized with 100% of the recommended N (3105 g/plant). Plants inoculated with G. diazotrophicus and fertilized with 50% of the recommended N yielded a comparable biomass (3131 g/plant). Plants inoculated with both G. diazotrophicus and G9 and fertilized with 50% of the recommended N yielded less biomass (3121 g/plant) than plants inoculated with either bacterium alone. The mean leaf-N content of GR9-inoculated plants fertilized without N was 4.86 mg g–1 dry wt, compared with a mean leaf-N content of 4.81 mg g–1 dry wt of uninoculated plants treated with 100% of recommended N. The highest mean leaf-N contents, 4.87 and 4.80 mg g–1 dry wt, were obtained with G. diazotrophicus alone and combined with isolate GR9, respectively, and fertilized with 50% of the recommended N. Control plants without inoculation or N yielded the lowest amount of leaf-N (4.16 mg g/dry wt) and biomass (2548 g/plant) (Table 2). Rice plant colonization The 1000-grain weights from rice plants with different treatments showed that inoculation with GR9 or a

123

1002

World J Microbiol Biotechnol (2007) 23:997–1006 64 GR-4 99 Pseudomonas aeruginosa LMG 1242T (Z76651) 78 GR-7

50

59 99

Pseudomonas resinovorans LMG 2274T (Z76668) Pseudomonas citronellolis ATCC 13674T (AB021396) Pseudomonas stutzeri CCUG 11256T (U26262) Pseudomonas alcaligenes IAM 12411T (D84006) Pseudomonas indica IMT37T (AF302795) 68 Pseudomonas monteilii CIP 104883T (AB021409) Pseudomonas putida DSM 291T (Z76667) Pseudomonas fluorescens IAM 12022T (D84013) 97 Pseudomonas chlororaphis ATCC 9446T (AF094723) 97 Pseudomonas syringae ATCC 19310T (D84026) 55 Pseudomonas pertucinogena IFO 14163T (AB021380) Pseudomonas thermotolerans CM3T (AJ311980) Klebsiella oxytoca JCM 1665T (AB004754) GR-9 67 Klebsiella pneumoniae subsp. rhinoscleromatis ATCC 13884T (AF13098 100 T 66 Klebsiella pneumoniae subsp. pneumoniae DSM 30104 (X87276) 61 Klebsiella variicola F2R9T (AJ783916) Klebsiella singaporensis LX3T (AF250285) 59 Klebsiella granulomatis KH34 (AF010253) 90 Klebsiella pneumoniae subsp. ozaenae ATCC 11296T (Y17654) Burkholderia glathei LMG 14190 T(Y17052) 69 Burkholderia graminis LMG 18924T (U96939) Burkholderia andropogonis LMG 2129T (X67037) 100 Burkholderia gladioli ATCC 10248T (X67038) 53 Burkholderia glumae LMG 2196 T (U96931) Burkholderia mallei ATCC 23344 T (AF110188) 98 Burkholderia ambifaria LMG 19182T (AF043302) 58 78 Burkholderia stabilis LMG 14294T (AF148554) Burkholderia cepacia ATCC 25416T (AF097530) 71 Burkholderia ubonensis EY 3383T (AB030584) GR-3 50 Burkholderia cenocepacia LMG 16656T (AF148556) 96 Burkholderia vietnamiensis TVV75T (U96928)

0.02

Fig. 2 Phylogenetic positions of strains GR3, GR4, GR7, and GR9 within the genera Burkholderia, Pseudomonas, and Klebsiella on the basis of 16S rDNA gene sequences. The phylogenetic tree was constructed by the neighbor-joining

method: The numbers at nodes indicate the levels of the bootstrap support based on a neighbor-joining analysis of 1000 resampled data sets. The bootstrap values below 50% were not indicated. Bar, 2 nucleotide substitutions per 100 nucleotides

Table 1 Acetylene reduction activity during panicle-initiation and grain-filling stages after inoculation of rice Cv.ADT-43 with novel isolates and acetylene reduction activity in micropropa-

gated sugarcane seedlings (Cv.CoV 92102) inoculated with novel isolates, at 30 and 60 days after inoculation (DAI)

Isolate code

GR1 GR2 GR3 GR4 GR5 GR6 GR7 GR8 GR9 F-value Coeff. of variation

Paddy (nmol C2H4 g–1 d. wt)*

Sugarcane (nmol C2H4 g–1 d. wt)*

Panicle MPN · 104 g–1 initiation root

Grain filling

17.36b 15.26b 13.28b 24.32a 21.32a 19.32a 23.14a 19.56a 24.32a 40.41 17.09

56.43c 4.50b 62.45b 11.5a 58.53b 11.5a 95.51a 11.5a 67.35b 1.50c 58.95b 4.50b 52.14c 11.5a 46.32c 11.5a 101.24a 5.50a 45.51 127.11 20.23 32.11

1.50c 1.50c 4.50b 11.5a 4.50b 1.50c 4.50b 2.10c 5.15a 404.03 76.90

MPN · 105 g–1 root

Different letters indicate treatments differing at the 5% level (Tukey test) * Mean of 10 tubes

123

30 DAI


60 DAI


10.13c 12.13b 10.15c 18.13a 16.32a 13.62b 15.65a 14.52b 16.25a 29.29 18.30

11.5a 1.50c 4.50b 11.5a 1.50c 4.50b 4.50b 11.5a 5.60a 416.72 62.32

20.21c 4.50b 21.28b 1.50c 19.16c 1.50c 42.29a 11.5a 35.62a 4.50b 25.21b 11.5a 23.12b 11.5a 18.96c 11.5a 37.32a 12.50a 17.42 217.42 11.37 36.34

World J Microbiol Biotechnol (2007) 23:997–1006 Table 2 Effects of inoculation with the novel isolate Klebsiella sp.GR9 on total leaf-N content and biomass of micropropagated sugarcane cultivar CoV 92102, grown for 6 months with 65 and 115 kg ha–1 of P and K, respectively

Different letters indicate treatments differing at the 5% level (Tukey test) * Mean of 12 hills a Mixture of autoclaved cells

1003 Leaf-N content (mg g–1 dry wt)* Days after planting

Treatments

30 kg N ha–1 control a kg N ha–1 + Klebsiella sp. GR9 kg N ha–1 + G. diazotrophicus kg N ha–1 + Klebsiella sp. GR9 + G. diazotrophicus 140 kg N ha–1 controla 140 kg N ha–1 + Klebsiella sp. GR9 140 kg N ha–1 + G. diazotrophicus 140 kg N ha–1 + Klebsiella sp. GR9 + G. diazotrophicus 280 kg N ha–1 controla F-value Coeff. of variation

0 0 0 0

combination of all five strains yielded a 4.3% increase in grain weight as compared with the autoclaved control. Inoculation with A. lipoferum yielded a 4.2% increase and inoculation with G. diazotrophicus yielded a 4.1% increase. Inoculation with H. seropedicae and B. vietnamiensis yielded 3.9% increases in the 1000-grain weights. Pots inoculated with the combined diazotrophs produced more panicles (66%) than the autoclaved control and more panicles than the individually inoculated plants. Plants inoculated with isolate GR9 or A. lipoferum produced 49% more panicles. Those inoculated with G. diazotrophicus or B. vietnamiensis produced 33% more panicles. Combined inoculation increased the number of filled grains per plant by 62%. Inoculation with GR9 increased the number of grains per plant by 44%. Inoculation with A. lipoferum increased the number of filled grains per plant by 42% (Table 3).

Discussion We have undertaken to isolate new nitrogen-fixing endophytes from different sugarcane cultivars from Tamilnadu State, India. The concept of biological nitrogen fixation (BNF) by endophytes (Dobereiner 1992) has led to investigations on the potential uses of endophytic nitrogen-fixing bacteria that colonize graminaceous plants. It has been suggested that these bacteria express their nitrogen-fixing potential better when inside plant tissues, due to lower competition for nutrients and protection from high levels of O2 that are present on the root surface (Boddey and Dobereiner 1995). PCR amplification of nifD confirmed the presence of this structural nitrogenase gene in all of the isolates (Fig. 1). Since the PCR primers were designed from

60

90

120

150

180

Total Biomass (g/ Mean plant)*

4.58a 5.60a 5.46a 5.21a

4.41b 5.43a 5.32a 5.10a

4.42b 5.13a 5.20a 5.12a

3.92 4.66 4.63 4.13

3.89 4.22 4.14 4.03

3.77 4.17 4.04 4.06

4.16 4.86 4.79 4.59

2548 3043 3012 3022

5.26a 5.52a 5.41a 5.28a

5.0a 5.32a 5.38a 5.23a

4.96a 5.24a 5.27a 5.11a

4.31 5.23 4.61 4.63

4.16 4.16 4.24 4.32

4.12 4.21 4.26 4.23

4.63 4.78 4.87 4.80

2801 3166 3131 3121

5.28a 5.21a 5.03a 4.72 4.31 4.32 4.81 12.35 19.73 15.23 16.21 13.36 12.74 2.70 14.31 11.32 10.25 13.21 12.05 14.81 4.84

3105 22.76 14.82

the conserved regions of nifD genes, there was no possibility of amplifying other nif genes (Ueda et al. 1995). The nif genes only occur in nitrogen-fixing microorganisms and have been used to monitor the presence of these diazotrophs in pure cultures (Franke et al. 1998), as well as in plants (Lovell et al. 2000). The nif genes also have been detected in arbuscular mycorrhizal fungal spores (Minerdi et al. 2001), in marine environments (Zehr et al. 1998), and in termite guts (Kudo et al. 1998). 16S rDNA sequence of isolates GR4 (DQ100463) and GR7 (DQ100464) have 99.5% similar to Pseudomonas sp. pDL01 (AF125317). The 16S rDNA sequence of isolate GR3 (DQ100466) had 100% similarity to Burkholderia vietnamiensis (AY973820). The 16S rDNA sequence of isolate GR9 (DQ100465) was 99.79% similar to that of the Klebsiella pneumoniae type strain (KPY17657) (Fig. 2). At least three times higher AR activity was observed in our isolates at the grain-filling stage as compared with the panicle-initiation stage (Table 1). Likewise Watanabe et al. (1979) reported that in two varieties, IR36 and IR26, maximum ARA was detected at the grain-filling stage. Higher AR activity at a particular growth stage may be due to a reduction in inhibitory nitrogen concentrations in the soil or overproduction of root exudates that are conducive to diazotroph growth and activity (Jagnow 1983). The phytohormone indoleacetic acid was found in the growth medium of all of our isolates. The amount of IAA produced varied among the strains, as previously reported (Mehnaz et al. 2001). Pot trials with sugarcane demonstrated that plants inoculated with the novel isolate Klebsiella sp GR9 and fertilized with 50% of the recommended N dose produced more biomass (3166 g/plant) than the uninoculated control plants fertilized with 100% of the

123

1004


Table 3 Effects of inoculation of the novel isolate Klebsiella sp. GR9 and other diazotrophs on the yields of Cv.ADT-43* Treatments

Control Klebsiella sp. GR9 G. diazotrophicus A. lipoferum H. seropedicae B. vietnamiensis Combined inoculation with all five strains F-value Coeff. of variation

1000-grain wt (g)

% increase above control

19.3b 22.1a 21.3a 21.6a 20.4a 20.3a 22.3a

– +4.3 +4.1 +4.2 +3.9 +3.9 +4.3

141.13 6.4

– –

Panicles Plant–1 6.34c 9.21a 8.16b 9.65a 7.38b 8.42b 10.23a 330.16 26.16


Filled grains Plant–1


– +49 +33 +49 +16 +33 +66

482.56c 697.64b 627.71b 686.68b 603.57b 638.61b 784.73a

– +44 +30 +42 +25 +32 +62

356.22 15.13

– –

– –

Grain yield g/pot 66.10b 71.60a 70.35a 71.80a 70.80a 70.95a 74.30a 133.85 21.40

% increase above control – 8.4 5.9 8.7 7.2 7.4 12.57 – –

Different letters indicate treatments differing at the 5% level (Tukey test) * Mean of 24 hills

recommended N dose (3105 g/plant). Plants inoculated with both G. diazotrophicus and isolate GR9 and fertilized with 50% of the recommended N yielded less biomass (3121 g/plant) than those inoculated with either of the strains alone. The mean leaf-N content of plants inoculated with isolate GR9 and fertilized without N (4.86 mg/g dry wt) was higher than the mean leaf-N content of uninoculated plants with 100% of the recommended N (4.81 mg/g dry wt). Plants inoculated with G. diazotrophicus alone or in combination with novel isolate GR9 and fertilized with 50% of the recommended N had the highest mean leaf-N content (4.87 and 4.80 mg/g dry wt) (Table 2). Dobereiner and Urquiaga (1992) reported that inoculation of micropropagated sugarcane seedlings could increase plant growth and encourage efficient nitrogen fixation under field conditions. A possible reason for the enhanced yield of the inoculated plants over the uninoculated controls may be that N2-fixation contributes N whenever the plants require it and that this N may be important during critical stages of plant development, as suggested by Bashan et al. (1989) for Azospirillum inoculation. The contribution of BNF to Brazilian sugarcane varieties ranged from 170 to 210 kg N ha–1 for varieties CB-45-3 and SP-70-1143 and from 240 to 270 kg N ha–1 for the variety krakatau (Boddey and Dobereiner 1995). Pot experiments with rice were performed to evaluate isolate GR9 and compare it to the type strains of B. vietnamiensis, H. seropedicae, A. lipoferum, and G. diazotrophicus, and to a mixture of all these strains. In terms of overall effects on 1000-grain weight, number of panicles per plant, and number of filled grains per plant, the inoculates ranked as follows: (1) combined inoculum, (2) Klebsiella sp. GR9,

123

(3) A. lipoferum 4B, (4) G. diazotrophicus, (5) B. vietnamiensis, and (6) H. seropedicae. The effect on the weight of 1000 grains roughly paralleled the effect on final yield, suggesting that the effects of inoculated bacteria persist throughout the plant growth cycle (Table 3). The local isolate GR9 of Klebsiella sp. has the most potential for improving rice production. Omar et al. (1992) demonstrated that inoculation of rice seeds with a N-fixing bacterium worked better with the local strain Azospirillum brasilense then with the strain A. brasilense Sp7, which significantly decreased the yield (Heulin et al. 1991). This could be the result of bypassing the initial competition phase in the rhizosphere colonization by inoculating the bestadapted strain. Soil inoculation could boost a local component of the soil microflora, ensuring that the plant is colonized readily by the ‘‘best’’ bacterium, rather than wasting exudates on a diversity of soil bacteria with varying levels of N-fixing efficiency (Heulin et al. 1989). The widespread use of synthetically compounded fertilizers has resulted in environmental degradation, decline of beneficial micro- and macro-organisms and accumulation of chemical residues in the food web. For a sustainable agriculture, use of biologically derived fertilizers would be ecologically sound and economically viable alternatives. These crop-associated indigenous nitrogen fixers may be agronomically important because they could supply part of the nitrogen that the crop requires. Acknowledgements We sincerely thank to Prof. P. Vandamme and Dr. Tom Coenye, LMG-BCCM, University of Gent, Belgium, for providing B. vietnamiensis and B. cepacia reference cultures and Prof. J. Balandreau, Microbial Ecology, France, for his encouragement and support.


References Arias OE, Gatti IM, Silva DM, Ruschel AP, Vose PB (1978) Primeiras observaciones al microscopio eletronico de bacterias fijadoras de N2 en la raiz de la cana de azucar (Saccharum officinarum L.). Tarrialba 28:203–207 Baldani JI, Baldani VLD, Seldin L, Dobereiner J (1986) Characterization of Herbaspirillum seropedicae gen.nov.sp. nov., a root associated nitrogen fixing bacterium. Int J Syst Bacteriol 36:86–93. Baldani JI, Caruso L, Baldani VLD, Goi SR, Dobereiner J (1997) Recent advances in BNF with non-legume plants. Soil Biol Biochem 29:911–922 Bally I, Thomas-Bauzon D, Heulin T, Balandreau J, Richard C, De Ley J (1983) Determination of the most frequent N2fixing bacteria in a rice rhizosphere. Can J Microbiol 29:881–887 Bashan Y, Levanony H, Mitiku G (1989) Changes in proton efflux of intact wheat roots induced by Azospirillum brasilense Cd. Can J Microbiol 35:691–697 Bockman OC (1996) Fertilizers and biological nitrogen fixation as sources of plant nutrients: perspectives for future agricultures. Forskningssenteret, Porsgunn, Norsko Hydro, Norway Boddey RM, Dobereiner J (1995) Nitrogen fixation associated with grasses and cereals; recent progress and perspectives for the future. Fertil Res 42:241–250 Boddey RM, Urquiaga S, Alves BJR, Veronica R (2003) Endophytic nitrogen fixation in sugarcane: present knowledge and future applications. Plant Soil 252:139–149 Burbage DA, Sasser M (1982) A medium selective for Pseudomonas cepacia. Phytopathology 72:706 Dobereiner J, Ruschel AP (1958) Uma nova especie de Beijerinkia. Rev Biol 1:261–272 Dobereiner J (1992) History and new perspectives of diazotrophs in association with nonleguminous plants. Symbiosis 13:1–13 Dobereiner J, Urquiaga S (1992) Soil biology and sustainable agriculture. An Acad Bras Sci 84:127–133 Dobereiner J (1997) Biological nitrogen fixation in the tropics: social and economic contributions. Soil Biol Biochem 29:771–774 Estrada P, Mavingui P, Cournoyer B, Fontaine F, Balndreau J, Cabellero-Mellado J (2002) A N2-fixing endophytic Burkholderia sp. associated with maize plants cultivated in Mexico. Can J Microbiol 48:285–294 Franke IH, Fegan M, Hayward AC, Sly LI (1998) Nucleotide sequence of the nifH gene coding for nitrogen reductase in the acetic acid bacterium Acetobacter diazotrophicus. Lett Appl Microbiol 26:12–16 Govindarajan M, Balandreau J, Muthukumarasamy R, Revathi G, Lakshminarasimhan C (2006) Improved yield of micropropagated Sugarcane following inoculation by endophytic Burkholderia vietnamiensis. Plant Soil 280:239–252 Gillis M, Kresters K, Hoste B, Janssens D, Kropenstedt RM, Stephen MP, Teixeira KRS, Dobereiner J, De Ley J (1989) Acetobacter diazotrophicus sp. nov., a nitrogen-fixing acetic acid bacterium associated with sugarcane. Int J Systemat Bacteriol 39:361–364 Gillis M, Tran Van V, Bardin R, Goor M, Hebar P, Willems A, Segers P, Kersters K, Heulin T, Fernandez MP (1995) Polyphasic taxonomy in the genus Burkholderia leading to an emended description of the genus Burkholderia and transposition of Burkholderia vietnamiensis sp. Nov. for N2fixing isolates from rice in Vietnam. Int J Systemat Bacteriol 45:274–289

1005 Graciolli LA, Freitas JR, Ruschel AP (1983) Bacterias fix adoras de nitrogenio nas raizes, caules e folhas de cana-de-acucar (Saccharum sp.). Rev Microbiol 14:191–196 Hegazi NA, Eid M, Farag RS, Monib M (1979) Asymbiotic N2fixation in the rhizosphere of sugarcane planted under semiarid conditions of Egypt. Rev Ecol Biol Soil 16:23–37 Heulin T, Rahman M, Omar AMN, Rafidison Z, Pierrat JC, Balandreau J (1989) Experimental and mathematical procedures for comparing efficiencies of rhizosphere N2 fixing bacteria. J Microbiol Methods 9:163–173 Heulin T, Omar N, Rahman M, Balandreau J (1991) Some principles for inoculation of rice by Nitrogen-fixing bacteria under field conditions. In: Dutta SK, Sloger C (eds) Biological Nitrogen fixation associated with rice production. Oxford and IBH publishing Co., New Delhi, pp 221–227, ISBN 8120405609 Hiraishi A (1992) Direct automated sequencing of 16S rDNA amplified by polymerase chain reaction from bacterial cultures without DNA purification. Lett Appl Microbiol 15:210–213 Humphries EC (1956) Mineral components and ash analysis. In: Peach K, Tracged MV (eds) Modern methods of plant analysis, Springer-Verlag, Berlin, pp 468–502 Jagnow GC (1983) Nitrogenase (C2 H2) activity in non-cultivated and cereal plants; influence of nitrogen fertilizer on population and activity of nitrogen fixing bacteria. Z Pflanzenkr Pflanzenschutz 146:217–227 James EK, Olivares FL (1997) Infection and colonization of sugar cane and other graminaceous plants by endophytic diazotrophs. Crit Rev Plant Sci 17:77–119 Jukes TH, Cantor CR (1969) Evolution of protein molecules. In: Munro HN (ed) Mammalian protein metabolism, vol. 3. Academic Press, New York, pp 21–132, ISBN 0125106033 King ED, Ward MK, Raney DE (1954) Two simple media for the demonstration of pyocyanin and fluorescein. J Lab Clin Med 44:301–307 Kudo TM, Ohkuma M, Moriya S, Noda S, Ohtokak (1998) Molecular phylogenetic identification of the intestinal anaerobic microbial community in the hindgut of the termite, Reticulitermes speratus, without cultivation. Extremophiles 2:155–161 Kumar S, Tamur K, Jakobsen IB, Nei M (2001) MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244–1245 Kwon SW, Kim JS, Park IC, Yoon SH, Park DH, Lim CK, Go SJ (2003) Pseudomonas koreensis sp. nov., Pseudomonas umsongensis sp. nov., and Pseudomonas jinjuensis sp. nov., novel species from farm soils in Korea. Int J Systemat Evol Microbiol 53:21–27 Ladha JK, Triol AC, Daroy LG, Caldo G, Ventura W, Watanabe I (1986) Plant-associated N2 fixation (C2H2) reduction by five rice varieties, and relationship with plant growth characters as affected by straw incorporation. Soil Sci Plant Nutr 32:91–106 Lovell CR, Piceno YM, Quattro JM, Baagwell CE (2000) Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass, Spartina alterniflora. Appl Environ Microbiol 66:3814–3822 Mascarua-Esparza MA, Villa-Gonzallez R, Caballero-Melado J (1988) Acetylene reduction and indolacetic acid production by Azospirillum isolates from Cactaceous plants. Plant Soil 106:91–95 Mehnaz S, Mirza MS Haurat J, Bally R, Normand P, Bano A, Malik KA (2001) Isolation and 16S rRNA sequence analysis of the beneficial bacteria from the rhizosphere of rice. Can J Microbiol 47:110–117

123

1006 Minerdi D, Fani R, Gallo R, Boarino A & Bonfante P (2001) Nitrogen fixation genes in an endosymbiotic Burkholderia strain. Appl Environ Microbiol 67:725–732 Omar N, Berge O, Shalaan SN, Hubert JL, Heulin T, Balandreau J (1992) Inoculation of rice with Azospirillum brasilense in Egypt. Results of five different trials between 1985 and 1990. Symbiosis 13:281–289 Purchase BS (1980) Nitrogen fixation associated with sugarcane. In: Proceedings of the South African Sugar Technologists Association, pp 173–176 Rasul G, Mirza MS, Latif F, Malik KA (1998) Identification of plant growth hormones produced by bacterial isolates from rice, wheat and kallar grass. In: Malik KA, Mirza MS, Ladha JK (eds) Nitrogen fixation with non-legumes, Kluwer Academic Publishers, Netherlands, ISBN 0792348737 Reis VM, Baldani JI, Baldani VLD, Dobereiner J (2000) Biological dinitrogen fixation in Gramineae and palm trees. Crit Rev Plant Sci 206:205–211 Rennie RJ, Freitas JRD, Ruschel AP, Vose PB (1982) Isolation and identification of N2-fixing bacteria associated with sugarcane (Saccharum sp.). Can J Microbiol 28:462–467 Saitou N, Nei M 1987 The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Seldin L, Van Elsas JD, Penido EGC (1984) Bacillus azotofixans sp.nov., a nitrogen fixing species from Brazilian soils and grass roots. Int J Systemat Bacteriol 34:451–456 Somasekaran P, Huben HJ (1994) Handbook for Rhizobia. Springer-Verlag, pp 340, ISBN 0387941347

123

World J Microbiol Biotechnol (2007) 23:997–1006 Sreenivasan J, Sreenivasan TV (1984) In vitro propagation of Saccharum officinarum and Sclerostachya fusca hybrid. Theor Appl Genet 67:171–174 Tien TM, Gaskins MH, Hubbel DH (1979) Plant growth substances produced by Azospirillum brasilense and their effect on growth of pearl millet (Pennisetum americanum L). Appl Environ Microbiol 37:1016–1024 Tran Van V, Berge O, Balandreau J, Ngo Keˆ S, Heulin T (1996) Isolement et activite´ nitroge´nasique de Burkholderia vietnamiensis, bacte´rie fixatrice d’azote associe´e au riz (Oryza sativa L) cultive´ sur un sol sulfate´ acide du Vieˆt-nam. Agronomie 16:479–491 Ueda T, Suga Y, Yahiro N, Mat Suguchi T (1995) Genetic diversity of N2 fixing bacteria associated with rice roots by molecular evolutionary analysis of a NifD library. Can J Microbiol 41:235–240 Watanabe I, Barraquio WI, de Guzman MR, Cabera DA (1979) Nitrogen fixing (C2H2 reduction) activity and population of aerobic heterotrophic nitrogen fixing bacteria associated with wetland rice. Appl Environ Microbiol 37:813–819 Yamada Y, Hoshino K, Ishikawa T (1997) The phylogeny of acetic acid bacteria based on the partial sequences of 16S ribosomal RNA: the elevation of the subgenus Gluconacetobacter to the generic level. Biosci Biotechnol Biochem 61:1244–1251 Zehr JP, Mellon MT, Zani S (1998) New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of nitrogenase (nif-H) genes. Appl Environ Microbiol 64:3444–3450