Agricultura 5: 47 - 52 (2008)
Growth and N2 fixation in Sesbania rostrata by H2O2 pretreated Azorhizobium caulinodans and it's effect as green manure on lowland rice
Manoharan Melvin JOE and Palanivel Karpagavinayaga SIVAKUMAAR
Department of Microbiology, Faculty of Agriculture, Annamalai University, Annamalainagar-608002, India
The response of Azorhizobium caulinodans to reactive oxygen species such as hydrogen peroxide (H2O2) plays an important role in nodulation and nitrogen fixation. It has been found that pretreatment of A.caulinodans with a selective oxidant viz., H2O2 (200^M) has enhanced the osmotic, thermal, and desiccation tolerence. The survivability in different carrier materials was found to be enhanced by H2O2 pretreatment as compared to untreated controls. A pot culture study was conducted to compare the efficiency of H2O2 pretreated cells for nodulation, plant dry wt and ARA activity in Sesbania rostrata, followed by the Sesbania incorporation as green manure for rice grown under pot culture condition. The crop was studied for certain plant growth and yield components, such as plant ht, plant dry wt, no of panicles, total "N" uptake. The results of our present study have showed that the plant inoculated with H2O2 pre-treated cells of A. caulinodans positively augmented an increase in the growth and yield in both Sesbania rostrata as well for rice grown under flooded conditions in the pot culture experiment.
Key words: Azorhizobium caulinodans, hydrogen peroxide, carrier material, rurvival, rice
INTRODUCTION
Nitrogen is one of the key nutrients that most frequently limit the rice production. Biological nitrogen fixation (BNF) is a fascinating phenomenon, which involves a highly specialized and intricately evolved interaction between soil microorganisms and higher plants, harnessing the atmospheric elemental nitrogen. One of the most exciting recent advances in Biological Nitrogen Fixation (BNF) is the development of an annual tropical African legume shrub, Sesbania rostrata, which grows rapidly in the wet season, producing high levels of biomass even in flooded conditions and exhibits higher rates of nitrogen accumulation, which makes this species one of the most valuable green manure (Ndoye et al. 1988, Boivin et al. 1997) Sesbania rostrata estabilishes a highly specific interaction with genus Azorhizobium caulinodans (Dreyfus et al. 1988), which induces effective nodules on the stem and root of Sesbania rostrata and also posses a unique capacity among rhizobia to fix N2 in the free living state in culture and in plants (Dreyfus and Dommergues 1981, Dreyfus et al. 1988). It has been found that for these two disparate N2 fixation processes, dissolved O2 optima may vary some orders of magnitude: 10^M in culture versus 10mM in plants (Bergersen et al. 1986, Buckmiller et al. 1991).
Biological N2 fixation is extremely O2 sensitive, since the cellular metabolism of molecular oxygen produces reactive and potentially toxic oxygen species such as superoxide, hy-droxyl radicals and hydrogen peroxide (H2O2) Halliwell and
Correspondance to: Manoharan Melvin JOE Phone: +919 894 095 443 Email : mel_vin@sify.com
Gutteridge (1989). To defend these reactive oxygen species, microorganisms produce certain antioxidants and enzymes that prevent or repair oxidative damage. One among them are catalases, which are haem-containing enzymes, disassociating H2O2 to O2 and H2O, which play a role in the reduction of the formation of the highly reactive hydroxyl radical, which arises from the degradation of H2O2 via the Fenton reaction (Halliwell and Gutteridge 1989).
In the early stage of interaction between rhizobia and legumes, reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide anion (O2-) and hydroxyl radical (OH) are generated and this ROS generation is similar to that seen when pathogens infect host plants (Lambert and Dixon 1997, Santos et al. 2000). It has been confirmed that H2O2 plays a supportive role in the nodule initiation by mediating the nod factor responses between Sesbania rostrata and Azorhizobium caulinodans (Haeze et al. 2003). Although there has been numerous reports regarding the H2O2 mediated cata-lase enzyme induction in rhizobium and its subsequent effect on the nodulation in leguminous crops (Santoz et al. 2001, Jamet et al. 2002, Carmen-Vergas et al. 2003). Little is known about the survival of H2O2 adapted symbiotic nitrogen fixing bacteria under adverse soil conditions and its role on nodulation in legumes(Graham 1992, Kitts and Ludwig 1994).
Our previous study (Joe and Sivakumaar 2007) has showed that pre-treatment of A. caulinodans cells with 200^M H2O2 has imparted a higher resistance to further H2O2 exposure i.e., up to 3mM H2O2 treatment as compared to the untreated control. It has been also demonstrated that these pretreated exponential cultures also typically showed a moderate increase in catalase activity as compared to the untreated control. This fascinated us to undertake the present study to explore the ability of pretreated H2O2 cells with enhanced
catalase activity for survival under different created stress conditions and also in different carrier materials. Additionally we assessed the ability of the H2O2 preated cells of Azorhizobium caulinodans for its nodulation, plant dry wt and ARA activity in Sesbania rostrata, followed by incorporation of the same as green manure for rice grown under pot culture condition.
MATERIALS AND METHODS
Culture and Growth conditions
Azorhizobium caulinodans ORS-571 obtained from IM-TECH Chandigarh, India was used in this work and grown in YEM(Yeast Extract Manitol) broth(Vincent, 1970) consisting of (g1-1) yeast extract (0.4), K2HPO4 (0.5) , NaCl (0.1) MgSO4.7H20 (0.2) mannitol (10) , or TY medium comprising (g1-1) bacterialtryptone (5), yeast extract (3) , CaCl2.6H2O (1-3) for solid medium.
H2O2 pretreatment of Azorhizobium caulimo-dans for adaptation experiments
Azorbizobium caulinodans was inoculated into YEM broth and grown with shaking at 30° C to a value of 0.2 at OD 600 (4x10 6 cfu. ml-1) aliquots (5ml) of the culture were transferred into sterile tubes and H2O2 was added to the desired final concentration of 300^M and incubated as before. Samples were taken immediately prior to and periodically after H2O2 addition, diluted in YEM minus mannitol and plated into TY agar to monitor the cell viability. The surviving colonies after 2 days incubation at 30°C were used for the further study. The protocol was repeated about three times and the surviving cells were used for further study.
Desiccation resistance and thermal tolerance
The desiccation resistance of H2O2 pretreated cells was determined as per the methods as determined by Bleaky et al. 1998 with a slight modification.. The cells of Azorhizobium (1 X 109 were suspended in YEM (Yeast extract manitol) medium placed in a desiccator oven for 24 h at 25°C and then subjected to an 8-day old incubation at 30°C.
Initial cell numbers, before and after the drying treatment were determined by plate count. For enumeration of the cells surviving the desiccation 2.0ml of minimal salts solution was added to each plate, and the cells from the dried film were suspended with scraping and mixing and then the enumeration of the bacteria was done. For thermal tolerance experiment 1ml cell suspension (1 x 10 9 cfu/ml) of culture was kept in a water bath 45° for 60 min and the survival per cent of bacteria was determined.
Osmotic tolerance and Osmotic shock
The sensitivity of the cells to osmotic tolerance and osmotic shock were done as per the methods as determined by Kadouri et al.(2003) in which 25-ml portion of 4M glucose solutions were added of bacterial suspensions, The final glucose concentration were adjusted to 2M. The bacteria were incubated at 30° for 24 h. Sensitivity to osmotic shock was
determined by adding 25ml of Tris-glycerol solution(0.05M tris, 4M glycerol; pH 7.6) to 25 ml of cell suspension and incubating the preparation for 30 min at 30°C. The cells were then centrifuged (4000 x g, 10 min) and then re-suspended in 50ml of distilled water. Bacterial viability was determined by plate count.
Survivability in different Inoculant carriers
The survivability in different carier materials were tested as per the methods of Falik and Okon (1996). One ml bacterial suspension was mixed with one of the following autoclaved carriers, such as vermiculte, lignite and peat. Inoculants were stored in sterile flasks at 30°C. After 30 days incubation, the carriers have undergone desiccation to different extends. One gram of each sample was added to potassium phosphate buffer (0.06M, pH 6.8) and stirred at 200 rpm for 2 h at 30°C and their bacterial viability was determined.
Pot culture experiment
The experiment was conducted in the Department of Microbiology, Faculty of Agriculture, Annamalai University in the period of Oct to Dec-2006.
The soil were sieved through a 20-mesh, thoroughly mixed, and placed in clay pots (15cm diameter), which were kept continuously flooded. Each pot was given a basal dose of triple super phosphate (37.5mg P2O5), murrate of potash (25mg K2O) and ammonium molybdate (0.625mg), before S.rostrata crop.
Treatment
Two cycles of alternate S. rostrata and rice were grown under continuously flooded condition during Sep - Dec 2006. In both the experiments the H2O2 pre treated cells were compared for their efficiency against the vegetative cells of A. caulinodans six replications were maintained for each treatment.
The treatments were maintained in two sets to meet the above two objectives of the study. In the first set the treatments were compared in order to investigate the effects of H2O2 pretreated cells on nodulation, acetylene reduction activity and plant biomass of S. rostrata. In the second set of the treatment the effect of the pretreated cells were evaluated for their efficiency in rice for yield and N balance.
Pot culture and green manure incorporation experiment
Seed priming with A. caulinodans
Seeds of S. rostrata coated with either vegetative cells of S. rostrata or H2O2 pretreated cells of A. caulinodans were sown. The plants were thinned to three per pot after 5 days of planting. The shoots of S. rostrata 45 days after incorporated in to the soil were cut into small pieces (3-6 cm) and mixed thoroughly along with roots into the soil. Fifteen days old seedlings(excluding 15 days in seed bed) of rice were transplanted into each pot 5-10 days after incorporation of green
manure, the rice plants were grown for an average of 90 days
Azorhizobium inoculation
For seed treatment the seeds were treated with either H2O2 pretreated or vegetative cells at 10 ml per pot (Minimum inoculation load of 1 x 109) mixed with lignite, 5 ml of rice gruel to enhance the adhesiveness.
Acetylene Reduction Activity, Dry wt and N determination
The acetylene reduction activity, dry wt of stem and root nodules, dry wt and N content of the plant were measured 45 days after emergence. Four replicates pots of each treatment were randomly uprooted and the soil carefully washed off. The plant were excised at about 2-3 cm above the crown and the aerial stem portions of three plants from each pot were separately pooled in a plastic bag (15 x 30 cm) and the acetylene reduction activity was carried out as per the methods as determined by Ladha et al. (1992).
The plant dry wt, the number ofroot and stem nodules of the Sesbania plant used for assay and the rice growth and yield parameters such as plant ht, number of tillers, number of panicles, grain and straw yield were determined using the standard methods. The total N content of the plant was determined using the Microkheldal assay
Statistical analysis
Data were analyzed by analysis of variance (ANOVA) and the treatment means were compared relative to control following a post hoc test or least significant difference (LSD) test. Unless indicated otherwise, differences were only considered when significant at P<0.05.
RESULTS AND DISCUSSION
It has been clearly evident from the Table 1 that H2O2 preated Azorhobium caulinodans cells exhibited a higher thermal tolerance, desiccation tolerence, osmotic tolerance and shock as compared to the untreated vegetative cells ofA caulinodans. The results of our present study has clearly demonstrated that, pretreated cultures ofA. caulinodans adopts to normal levels of created stress, this adaptive response is similar to those previously described for enteric bacteria Christman et al. (1989), in B. subtilus (Dowds et al. 1987) and in yeast (Jamieson 1992).
The H2O2 pretreated Azorhobium caulinodans cells have exhibited a higher survival percentage as compared to the untreated vegetative cells in different carrier materials. The fact that H2O2 pretreated Azorhobium caulinodans cells exhibited increased stress endurance is of greater importance for commercial bacterial inoculants. Stress endurance varies according to the inoculant preparation and the storage conditions (Ka-douri et al. 2003). The survivability of inoculated bacteri has been significantly reduced after a six months storage period (Falik and Okon 1996). This reduction in the inoculated population of bacteria may probably due to the stress that developed during storage under suboptimal conditions, such as lack of moisture, stress and available nutrients. The role of catalase
in enhancing the survival during starvation periods has been reported in various bacteria (Jamieson 1992, Christman et al. 1989).
Table 1. Studies on the desiccation, thermal and osmotic tolerance of H2O2 pretreated cells of A. caulinodans
Treatment
Number of viable cells/ ml after different stress challenge*
Desiccation Thermal Osmotic Osmotic tolerence** tolerance** tolerance*** shock***
Control	(4.2+1.02) x 106 b (2.2± 0.84) x 105 b (3.9 ±1.21) x 104 b (4.1+1.10) x 105 b H O
„7'	(7.9 ±1.53) x 108 a(3.5±0.75) x 108 a (7.2±1.42) x 107 a	(4.3±1.24) x 108 a Pretreated
Initial inoculation load 1.0 x 109 ** assayed according to Bleaky et al. (1998) *** assayed according to Kadouri et al. (2003)
Values are Mean ±SD of three replicates from one representative experiment and each experiment was carried out 3 times, and similar results were obtained each time, within a column different letters after values indicate that there is a significant difference at p value of 0.05, as determined by one way analysis ofvariance followed by a post hoc test.
This higher degree of tolerance exhibited by H2O2 pretreated A. caulinodans may be attributed to the fact that a number of proteins and their genes, which form a part of the oxidative stress response have been identified including the superoxide dismutase as well as DNA repair enzymes such as exonuclease IV, DNA polymerase, Rec B nuclease, and Rec A, which are important in repairing the DNA damage under adverse conditions (Demple 1991, Farr and Kogoma 1991). Genes involved in the control of the oxidative stress response have also been identified for example the induction of nine proteins to H2O2 stress is under the positive control of Oxy R gene product (Nystrom 1993) H2O2 adapted enteric bacteria become resistant to heat and osmotic challenge and there has been overlap between the proteins synthesized during the oxi-dative stress and that induced by carbon or nitrogen starvation in E. coli (Demple and Brook 1983).
Table 2. Survival of H2O2 pretreated Azorhizobium cells in different carrier materials
A.Caulinodans	Vermiculite Lignite	Lignite	Peat
isolates	Per cent survivability** in different carriers		
Control (wild strain)	44 ± 2.0 b	32 ± 1.5 b	38.89 ± 1.81 b
H2O2 Pretreated	74.32 ± 3.42 a	64.66 ± 3.42 a	68.34 ± 3.84 a
Initial inoculation load 1.0 x 109
** Survival population after 30 days of incubation at 30 ± 2°C
Values are mean +SD of three replicates from one representative experiment and each experiment was carried out 3 times, and similar results were obtained each time, within a column different letters after values indicate that there is a significant difference at p value of 0.05, as determined by one way analysis ofvariance followed by a post hoc test.
In our present study the H2O2 pretreated cells were also tested for its efficiency to form effective nodules in Sesbania rostrata plants and also for its ARA activity , dry wt and "N" content in Sesbania rostrata plants. The pretreated Sesbania plants were also incorporated as green manure for the rice crop. It has been clearly evident from the Table that the H2O2 pretreatment has positively augmented the increase in the number of nodules, plant dry wt, ARA activity (Table 3) the data regarding the role of H2O2 pretreated cells of Azorhizobium caulinodans in Sesbania has been scarce.
Table 3. Effect of H2O2 pretreated A. caulinodans inoculation on nodulation, ARA and total plant dry wt of Sesbania rostrata 30 days after emergence**
Treatment	No of nodules plant1	Dry wt of Nodules g plant-1	ARA* activity of nodule ftjml C^h-1)	ARA* of Plant ftjml C^h-1)	Total Dry wt g plant-1
Control	17 ± 2c	0.18 ± 2c	38 ± 2c	5 ± 1c	5.8 ± 1.2c
Untreated	23 ± 3'	0.28 ± 2'	74 ± 4'	11 ± 2'	7.0 ± 2.0'
H2O2 Pretreated	29 ± 3a	0.36 ± 3a	96 ± 4a	16 ± 2a	8.4 ± 2.4a
LSD (<0.05)	3.12	0.07	4.02	2.04	1.04
*ARA is expressed as n moles of C2H4 reduced h^g-1 nodule/plant fresh wt **Observations at 60 days
Values are a mean of six replications. Each experiment is repeated three times and similar results are obtained each time. Means followed by different letters are differed significantly according to least significant difference test (P<0.05)
But there were numerous reports on the H2O2 pretreated cells of Rhizobium on Legumes. During early legume nodule development, invading rhizobia grow rapidly and proliferate extensively. As they migrate to the interior of the developing cortex, and as nodule bacterial counts mount exponentially, the rhizobia experience different O2 environments (Carmen-Varges et al. 2003). In addition Luo et al. (2003) reported that in the early stages of interaction between rhizobia and legumes, hydrogen perioxide (H2O2), one among the reactive oxygen species is generated. Sigaud et al. (1996), reported that H2O2 has been found to inhibit infection thread formation and nodule development. Earlier reports by Crockford et al. (1995)
has showed an increase in HO was correlated with a two
22
fold increase in the catalase activity in the Rhizobium sp, when treated with a non-lethal dose of H2O2. Our previous studies have showed a similar trend in A. caulinodans. It has well been documented that these enzymes play a major role in reducing the formation of these highly reactive hydroxyl radicals, which arise from the degradation of H2O2 via fenton reaction (Hal-liwell and Guttendrige 1986, D Haeze et al. 2000) In soyabean Bradyrhizobium japonicum symbiosis, the oxygen protective enzymes in the nodule tissue is positively correlated with the increase in the nitrogenase activity and leghaemoglobin content (Dalton et al. 1986)
Dalton et al. (1986) reported that soyabean Brady rhizobium japonicum symbiosis of oxidative protection enzymes in nodule tissue is positively correlated with the increase in nitro-
genase activity and leghaemoglobin content. The data on the yield of rice variety ADT-42 incorporated with green manuring using the H2O2 preatreated A. caulinodans were summarized on the Table 4.
Table 4. Phyto-stimulatory effect on the incorporation of H2O2 pretreated A. caulinodans inoculated Sesbania rostrata on the yield components and N uptake of rice*
Treatment	Plant height Grain yield (cm) (g plant-1)		Panicles ( no plant-1)	Productive Total Dry wt tillers (%) (g plant:1)		Total "N" uptake** (mg plant:1)
Control	73±3c	4.8±0.4c	7+1c	70+4c	17.6+1.2c	16.0+2.0c
Untreated	85+2'	11.1+1.1'	13+2'	80+5'	37.4+2.4'	29.1+1.1'
H2O2 Pretreated	91±3a	16.2+2.2a	17+3a	94+4a	46.4+2.4a	38.5+2.5a
LSD (<0.05)	4.12	1.24	2.0	4.14	3.10	3.05
*Observations at 90 day
** "N" uptake assayed according to Microkheldhel assay
Values are a mean of six replications. Each experiment is repeated three times and similar results are obtained each time. Means followed by different letters are differed significantly according to least significant difference test (P<0.05)
It has been found that incorporation of green manure of S. rostrata inoculated with H2O2 preatreated A. caulinodans has positively augmented the plant growth and yield components of rice such as plant height, grain yield, no of panicles, etc.
Meelu and Moris (1980) has previously reported a significant increase in the total N accumulation, grain, straw yield and total plant growth of rice by incorporation with green manuring by S. rostrata.
These findings open up the possibilities for investigation of genetic basis of effective association and colonization of H2O2 preatreated A. caulinodans with S. rostrata and other mechanisms involved.
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Received: December 29, 2007
Accepted in final form: April 11, 2008