2004 红树林来源的细菌作为海洋生物肥料

Nitrogen-fixing azotobacters from mangrove habitat and their utility as marine biofertilizersS.Ravikumar a,*,K.Kathiresan b ,S.Thadedus Maria Ignatiammal c ,M.Babu Selvam a ,S.Shanthy aaDepartment of Marine Microbiology,Centre for Marine Science and Technology,Manonmaniam Sundaranar University,Rajakkamangalam-629502,Tamil Nadu,India bCentre of Advanced Study in Marine Biology,Parangipettai-608502,Tamil Nadu,IndiacDepartment of Botany,Holy Cross College,Nagercoil-629004,Kanyakumari District,Tamil Nadu,IndiaReceived 30December 2003;received in revised form 2April 2004;accepted 29May 2004AbstractA dearth of information is available for nitrogen-fixing bacteria in coastal mangroves,and hence,the present study has been undertaken to analyse 44root and associated soil samples,derived from a mangrove habitat of southeast coast of India.The root samples exhibit high counts of total heterotrophic bacteria and azotobacters along with high rates of nitrogen fixation,as compared to the rhizosphere soil samples.Among the plant species,Bruguiera cylindrica records high microbial counts and nitrogen fixation.From the samples analysed,three species of Azotobacter ,viz.,A.chroococcum,A.virelandii and A.beijerinckii were isolated,purified and identified.These species exhibit high growth,nitrogen fixation and in vitro production of phytohormone (Indole Acetic Acid,IAA)at NaCl salinity of 30g l À1.The azotobacters,which were inoculated with Rhizophora seedlings,increased significantly the average root biomass up to by 98.2%,the root length by 48.45%,the leaf area by 277.86%,the shoot biomass by 29.49%as compared to controls and they also increased the levels of total chlorophylls and carotenoids up to by 151.0%and 158.73%,respectively.Thus,azotobacterisation is beneficial in raising vigorous seedlings of mangroves in coastal wetlands.D 2004Elsevier B.V .All rights reserved.Keywords:Azotobacter ;Biofertilizer;Mangroves;Nitrogen fixation;Rhizophora0022-0981/$-see front matter D 2004Elsevier B.V .All rights reserved.doi:10.1016/j.jembe.2004.05.020*Corresponding author.Tel.:+91-4652-253078(O),+91-4652-261320(R);fax:+91-4652-221214.E-mail address:ravibiotech201320@ (S.Ravikumar)/locate/jembeJournal of Experimental Marine Biology and Ecology312(2004)5–171.IntroductionMangroves represent a unique and ecologically important coastal habitat in the tropical and sub-tropical belts (Chapman,1984).A number of static and dynamic biological,physical and chemical factors are known to influence the development and stability of the mangrove community.These factors and their interactions play a significant role in the nutrient flows in the system and it is necessary to understand various processes interacting with them (Kathiresan and Bingham,2001).Nitrogen is one of the most important limiting factors affecting the development of mangrove vegetation (Chandramohan,1988).The nitrogen is present in low concentration in particulate matter,mainly in the mangrove plant detritus,which is exported from the ecosystem through tidal action.Despite the export of large quantities of the particulate matter,loss of nitrogen is relatively small (in the order of 3.7g N À1year À1)which is equivalent to 13%of average annual net primary production of mangrove forests (Botto and Bunts,1982;Botto and Robertson,1990;Lakshmanaperumalsamy,1987).In view of this,there is a need to determine the microorganisms involved with such nitrogen transformations and the rates at which these transformations occur in situ.In India,azotobacters have been isolated from the roots of several plants (Sadasivam,1963;Thomas,1991),but only a few from saline soil (Lakshmanaperumalsamy,1987;Ravikumar et al.,2002).No such studies are available with salt tolerant azotobacters in mangroves.Generally,one would not expect high abundance of azotobacters in the mangrove sediment as the tannin-rich conditions in that sediment are unfavourable for the growth of bacteria (Ravikumar,1995).Therefore,a thorough study is necessary on the occurrence,distribution and activity of halophilic azotobacters from the mangrove environment.Strains of azotobacters have been reported to secrete growth promoting hormones such as auxins,gibberellins and cytokinin into their culture media (Brown and Burlingham,1968;Azeon and Barea,1975).By virtue of this attribute,pre-treatment of seeds with a suspension of azotobacters has been shown to improve seed germination and plant growth (Brown and Burlingham,1968;Barea and Brown,1974).These experiments have been done with crop plants,but not with mangrove seedlings which pose a serious problem of poor growth (Kathiresan and Veera Ravi,1990)due to high salinity and anaerobic soil conditions (McKee,1993).The present study has also made an attempt to improve the growth of mangroves by using azotobacters derived from mangrove habitat.2.Materials and methods2.1.Isolation of bacteria and analysis of chemical constituentsRoot and adjoining soil samples were collected from 16plant species viz.Acanthus illicifolius L.(Acanthaceae),Aegiceras corniculatum L.Blanco (Myrsinaceae),Avicen-nia marina (Frosk.)Vierh.(Avicenniaceae),Avicennia officinalis L.,Bruguiera cylin-drica L.Bl.(Rhizophoraceae),Ceriops decandra (Griff.)Ding Hou (Rhizophoraceae),S.Ravikumar et al./J.Exp.Mar.Biol.Ecol.312(2004)5–176S.Ravikumar et al./J.Exp.Mar.Biol.Ecol.312(2004)5–177 Excoecaria agallocha L.(Euphorbiaceae),Lumnitzera racemosa Willd,(Combretaceae), Rhizophora mucronata Poir.(Rhizophoraceae),Rhizophora apiculata Blume(Rhizo-phoraceae),Rhizophora annamalayana Kathir.(Rhizophoraceae),Sesuvium portulacas-trum L.(Aizoaceae),Sonneratia apetala J.Smith(Sonneratiaceae),Salicornia brachiata Roxb.(Chenopodiaceae),Suaeda maritima(L.)Dumort(Chenopodiaceae), Xylocarpus granatum Koen.(Meliaceae)from a mangrove forest of Pichavaram (11j27V N;79j47V E)on the southeast coast of India.All the samples were transferred to clean and previously unused polythene bags and brought to the laboratory.Total counts of heterotrophic bacteria and azotobacters from collected samples were enu-merated by serial dilution and plating(Holt et al.,1994).About5g of the rhizosphere soil and root samples was separately inoculated into the nitrogen-free Winogradsky’s medium as mentioned in Bergey’s Manual(Holt et al.,1994). The inoculated samples were incubated at28F2j C for a week.After attaining visible growth,azotobacters were isolated by repeated streaking on nitrogen-free agar plates and maintained on nitrogen-free agar slants.The isolates were identification at species level (Holt et al.,1994).Various parameters viz.pH,salinity,nitrogen,phosphorus,potassium (Anon,1975),total amino acids and total sugars(Ravikumar,1995)from the rhizosphere soils were analysed.All the determinations were carried out in triplicate and the results are expressed as counts per gram dry weight of soil or root sample.2.2.In vitro nitrogen fixation by azotobactersThe effect of salinity on the rate of nitrogen fixation was examined in the following manner.The salinity regimes of0,5,10,15,20,25,30and35g lÀ1NaCl were attained by adjusting the salinity in50ml nitrogen-free broth in different flasks.Liquid cell suspensions of Azotobacter chroococcum,A.beijerinckii and A.vinelandii at cell density of108cells/ml were inoculated separately into65ml of sterile glass vials containing25 ml of sterilized liquid Winogradksy’s broth.Once visible growth was observed,the cotton plugs,which allowed air exchange,were removed and the bottles were sealed with rubber stoppers.From these bottles,10%of the gaseous phase was removed and the same volume of acetylene gas was injected.After12h of incubation,the amount of ethylene was analysed in the gas chromatograph(GC)(Chemito,3800,flame ionising detector,Poropak-T column3and 3.1-mm stainless steel,80–100mesh,ignition temperature150j C,flow rate of gas45.16in.À1hydrogen16lb in.À1oxygen25lb in.À1).Control bottles were maintained without the bacterial samples.Triplicate samples were maintained for each treatment for each species of azotobacter.The rate of nitrogen fixation was calculated by using the following formula(Ravikumar et al.,2002). Nitrogenase activity(nM ethylene produces hÀ1)=peak length(mm)Âattenua-tionÂrangeÂ0.0000006Âvolume of gas inside the flask/h of incubationÂvolume of gas sample injected into the GC.2.3.In situ acetylene reduction assay for root and soilFive grams of the root and soil samples was transferred separately into120ml of sterilized serum bottles each containing50ml of filtered mangrove water and wassealed.Triplicates were maintained for each sample.Two control samples were maintained,one without acetylene and another one with mangrove water alone.The rates of nitrogen fixation in each of the samples were analysed by following the above method (Ravikumar et al.,2002).Triplicates were maintained for each treatment.2.4.In vitro production of IAA by azotobactersThe effect of salinity on Indole Acetic Acid (IAA)production was examined in the following manner.The salinity regimes of 0,5,10,15,20,25,30and 35g l À1NaCl were attained by adjusting the salinity in 50ml phosphate buffer (pH 7.0)withTable 1Counts of total heterotrophic bacteria and azotobacters in root and rhizosphere soil samples of mangrove habitat Name of plant speciesCounts of THB Counts of azotobacters Root (107)Rhizosphere soil (Â106)Root (Â102)Rhizosphere soil (Â101)AcanthaceaeAcanthus Illicifolius 52.1222.3834.9550.00MyrsinaceaeAegiceras corniculatum 71.2432.2522.3510.67Avicenniaceae Avicennia marina82.2643.3732.7818.26Avicennia marina (pneumatophore)96.2452.2435.2521.33Avicennia officinalis94.2239.2613.237.66Avicennia officinalis pneumatophore 100.2648.5822.389.30RhizophoraceaeBruguiera cylindrica 150.2568.6566.3536.67Rhizophora mucronata25.2411.36 3.01 1.24Rhizophora mucronata (stilt root)30.1413.72 5.5 2.00Rhizophora apiculata36.3813.2313.7 4.03Rhizophora apiculata (stilt root)46.2615.2515.72 6.37Rhizophora annamalayana48.4215.7222.38 6.67Rhizophora annamalayana (Stilt root)50.2618.4818.128.24Ceriops decandra 72.2234.9521.2212.33EuphorbiaceaeExcoecaria agallocha 102.7255.2542.6724.66CombretaceaeLumnitzera racemosa 69.3434.2825.2210.66AizoaceaeSesuvium portulacastrum 18.46 5.5010.00 2.56SonneratiaceaeSonneratia apetala39.2618.2815.25 6.24Sonneratia apetala (pneumatophore)74.7535.7818.227.00ChenopodiaceaeSalicornia brachiata 10.02 3.010.480.32Suaeda maritima 12.73 2.240.460.22MeliaceaeXylocarpus granatum66.3830.0925.2618.33Values are average of three samples.All standard errors values are less than 10%of mean values.Values between the samples are significant at 5%level.S.Ravikumar et al./J.Exp.Mar.Biol.Ecol.312(2004)5–1780.005M DL tryptophan and5.0g sucrose.Five millilitres of cell suspension(108cells mlÀ1)of A.chroococcum,A.beijerinckii and A.vinelandii were added separately into each flask and incubated in dark at28F2j C for24h.Triplicates were maintained for each treatment.After incubation,the culture was filtered through Whatman No.42 filter paper and50ml of filtrate was adjusted to pH3.0using2N HCl.The filtrate was evaporated in a vaccum flask evaporator and the residue was dissolved in2ml methanol.Indole acetic acid in the methanol fraction was determined by employing salper reagent(Gorden and Paleg,1957).To1.5ml of distilled water in a test tube, 0.5ml of methanol residue was mixed.Four millilitres of fresh Salper reagent was rapidly added and kept in complete darkness for1h and read the optical density at 535nm in a spectrophotometer(220s,HITACHI,Japan).A standard graph was Table2Chemical properties of rhizosphere soils from16mangrove plant speciesName of plant species Total phosphate(mg gÀ1)Total nitrogen(mg gÀ1)Total organiccarbon(mg gÀ1)Salinity(x)pHAcanthaceaeAcanthus Illicifolius0.38270.813.0618.237.2 MyrsinaceaeAegiceras corniculatum0.383278.213.03513.447.3 AvicenniaceaeAvicennia marina0.408306.214.00227.727.5 Avicennia marina(pneumatophore)0.408312.814.01529.007.2 Avicennia officinalis0.401297.613.05326.207.6 Avicennia officinalis pneumatophore0.405301.513.09227.807.5 RhizophoraceaeBruguiera cylindrica0.415320.814.03716.647.6 Rhizophora mucronata0.358264.613.6515.127.6 Rhizophora mucronata(stilt root)0.372270.413.6915.208.2 Rhizophora apiculata0.374264.213.6821.767.7 Rhizophora apiculata(stilt root)0.382269.113.6920.267.3 Rhizophora annamalayana0.365269.213.6412.147.4 Rhizophora annamalayana(Stilt root)0.379267.413.7012.287.8 Ceriops decandra0.390285.213.38426.247.6 EuphorbiaceaeExcoecaria agallocha0.413314.614.0258.327.1 CombretaceaeLumnitzera racemosa0.385278.512.7024.267.2 AizoaceaeSesuvium portulacastrum0.352269.212.6324.567.1 SonneratiaceaeSonneratia apetala0.302282.213.0719.608.0 Sonneratia apetala(pneumatophore)0.397291.813.07516.648.8 ChenopodiaceaeSalicornia brachiata0.377260.212.6227.527.1 Suaeda maritima0.301257.812.5823.687.2 MeliaceaeXylocarpus granatum0.381272.412.7816.647.8 Values are average of three samples.All standard errors values are less than10%of mean values.Values between the samples are significant at5%level.S.Ravikumar et al./J.Exp.Mar.Biol.Ecol.312(2004)5–179prepared for IAA (Sigma,USA)for its use in calculating the IAA level in the culture filtrates.2.5.Biofertilizer treatmentThe bacterial species of A.chroococcum ,A.vinelandii and A.beijerinckii (which were isolated from mangrove environments)were inoculated separately into 200ml of Winogradsky’s broth and were cultured at 28F 2j C for 3days in a shaker.The liquid culture was centrifuged at 12,000rpm for 15min in a centrifuge (Hewlett-Packard,USA).The pellet was suspended in phosphate buffer (pH 7.0)and washed repeatedly with the buffer and were resuspended in the same buffer solution.To study the influence of bacterial species on the growth of Rhizophora seedlings,100ml of suspended culture (108cells ml À1)of A.chroococcum ,A.beijerinckii and A.vinelandii were mixed separately with 1kg of sterilized soil and were kept in sterilized polybags.Propagules of mangrove plants species [R.apiculata (27F 2cm);R.mucronata (35F 2cm)]were planted into the soil and were irrigated with sterile water (100ml per bag per kg of soil with 30g À1NaCl salinity).The propagules without bacterial treatments were maintained as control.Ten propagules were maintained for each treatment.After 45days,the growth characteristics of mangrove seedlings such as average root length,root biomass,shoot biomass and leaf area were recorded.Levels of total chlorophylls and carotenoids,which were extracted in 80%ice cold acetone from leaves,were measured by following respectively the methods of Arnon (1949)and Ridely (1977).The results were statistically analysed by following the two-way analysis of variance for significance at 99%and 95%confidence levels.3.Results3.1.Bacterial counts and chemical parametersTotal heterotrophic bacterial (THB)counts ranged from 2.24to 68.65Â106g À1in rhizosphere soil and they varied from 10.02to 150.25Â107g À1in the root samples.Azotobacter counts varied from 0.22to 36.67Â101g À1in rhizosphere soil samples and they ranged from 0.46to 66.35Â102g À1in root samples.In general,counts of THB and azotobacters were higher in root samples than those in their corresponding soil samples.Among the plant species,the bacterial counts were the highest in B.cylindrica and lowest in S.maritima (Table 1).The chemical parameters varied with the rhizosphere soil samples (Table 2).Salinity in the rhizosphere soils ranged from 8.32to 29g kg À1.The pH varied from 7.1to 8.9.Total nitrogen ranged from 260to 320.8A g g À1,total phosphate from 0.301to 0.415mg g À1and total organic carbon from 12.58to 14.03mg g À1.The maximum pH (8.8)was recorded in S.apetala (Pneumatophore)and the highest levels of total nitrogen (320.8A g g À1),total phosphate (0.415mg g À1),total organic carbon (14.037mg g À1)wereS.Ravikumar et al./J.Exp.Mar.Biol.Ecol.312(2004)5–1710S.Ravikumar et al./J.Exp.Mar.Biol.Ecol.312(2004)5–1711recorded in B.cylindrica(Table2).Level of total amino acids ranged from2.215to20.214 mg gÀ1.The level of total sugars varied from1.47to7.268mg gÀ1(Table3).The total sugars(7.268mg gÀ1)in R.mucronata(Stilt root)and the total amino acid(18.06mg gÀ1)in rhizosphere soil samples of B.cylindrica were found to be the maximum among the soil samples.Among the chemical parameters,the content of nitrogen has a highly significant positive correlation with the counts of THB(r=0.95)and azotobacters (r=0.80).3.2.In situ nitrogen fixationNitrogen fixation in44roots and associated soil samples was analysed.Of the22root samples tested,B.cylindrica showed the highest nitrogenase activity(72.64nM hÀ1gÀ1) Table3Levels of total amino acids and total sugars in rhizosphere soils from16mangrove plant speciesName of plant species Total amino acids(mg gÀ1)Total sugars(mg gÀ1) AcanthaceaeAcanthus illicifolius12.82 3.42MyrsinaceaeAegiceras corniculatum14.48 2.97 AvicenniaceaeAvicennia marina17.26 2.83Avicennia marina(pneumatophore)16.52 1.94Avicennia officinalis16.23 2.63Avicennia officinalis pneumatophore17.21 1.83 RhizophoraceaeBruguiera cylindrica18.06 6.12Rhizophora mucronata16.22 6.53Rhizophora mucronata(stilt root)17.257.27Rhizophora apiculata 4.24 2.64Rhizophora apiculata(stilt root) 4.62 3.15Rhizophora annamalayana7.26 3.28Rhizophora annamalayana(Stilt root)Ceriops decandra 2.21 2.13 EuphorbiaceaeExcoecaria agallocha17.63 1.47 CombretaceaeLumnitzera racemosa 2.21 2.13AizoaceaeSesuvium portulacastrum10.83 5.12 SonneratiaceaeSonneratia apetala7.83 1.49Sonneratia apetala(pneumatophore)7.84 5.38 ChenopodiaceaeSalicornia brachiata 4.63 1.98Suaeda maritima 4.09 4.47MeliaceaeXylocarpus granatum14.53 2.163Values are average of three samples.All standard errors values are less than10%of mean values.Values between the samples are significant at5%level.followed by Excoeceria agallocha (63.3nM h À1g À1).A.corniculatum (57.21nM h À1g À1)and A.officinalis (Pneumatophore)(49.6nM h À1g À1)(Table 4).However,the activity was minimum in S.brachiata ,S.maritima and Sesuviam portulacastrum .In the soil samples,B.cylindrica showed the maximum nitrogenase activity (19.5nM h À1g À1)followed by E.agallocha (12.48nM h À1g À1),A.corniculatum (10.46nM h À1g À1)and A.officinalis (8.28nM h À1g À1).However,nitrogenase activity was not recorded in rhizosphere soil samples of R.mucronata ,R.annamalayana ,S.maritima ,S.portulacas-trum ,S.brachiata and X.granatum (Table 4).The variation in the rate of nitrogen fixation among the plant species seems to be due to the variation in the nitrogen-fixing bacterial counts (Table 1).There is also a significant correlation between azotobacter counts andTable 4Rates of nitrogen fixation in root and rhizosphere samples of mangrove plant species Name of plant speciesRate of nitrogen fixation (nM ethylene h À1g À1dry weight)Root Rhizosphere soil AcanthaceaeAcanthus illicifolius 26.24 1.40MyrsinaceaeAegiceras corniculatum 57.2110.46Avicenniaceae Avicennia marina37.90 2.60Avicennia marina (pneumatophore)39.30 4.26Avicennia officinalis42.50 5.80Avicennia officinalis pneumatophore 49.608.28RhizophoraceaeBruguiera cylindrica 72.6419.50Rhizophora mucronata20.49NA Rhizophora mucronata (stilt root)22.84NA Rhizophora apiculata43.99 2.91Rhizophora apiculata (stilt root)29.90 1.34Rhizophora annamalayana 22.63NA R .annamalayana (Stilt root)24.26NA Ceriops decandra 37.90 2.24EuphorbiaceaeExcoecaria agallocha 63.3012.48CombretaceaeLumnitzera racemosa 31.60 2.60AizoaceaeSesuvium portulacastrum 1.95NA SonneratiaceaeSonneratia apetala16.90NA Sonneratia apetala (pneumatophore)30.10NA ChenopodiaceaeSalicornia brachiata 2.10NA Suaeda maritima 1.23NA MeliaceaeXylocarpus granatum16.92NANA –No activity;Values are average of 3samples;All standard errors values are less than 10%of mean values.Values between the samples are significant at 5%level.S.Ravikumar et al./J.Exp.Mar.Biol.Ecol.312(2004)5–1712S.Ravikumar et al./J.Exp.Mar.Biol.Ecol.312(2004)5–1713 Table5Effect of salinity on in vitro Indole Acetic Acid(IAA)production by Azotobacter speciesConcentration of NaCl(g lÀ1)Level of IAA production(A g mlÀ1)A.chroococcum A.beijerinckii A.vinelandii 012.2(35)12(30)11.0(22) 513.0(58)12.4(40)12.2(40) 1013.8(61)12.8(50)12.4(48) 1514.0(82)13.0(70)13.1(55) 2018.5(94)16.5(88)13.8(70) 2517.3(98)16.0(88)12.5(88) 3016.3(123)15.0(98)12.3(102) 3515.8(106)14.3(92)11.5(98) Values in parenthesis are colony forming unitsÂ103mlÀ1.Values are average of three samples and significant at 1%level between species and at5%level between salinities.nitrogen fixation in root samples(r=0.88;Y=8.06+67.44x)as well as in rhizosphere soil samples(r=0.72;Y=À231+13.33x).3.3.Effect of salinity on in vitro IAA production and nitrogenase activity by azotobactersThe effect of NaCl on the level of IAA production by Azotobacter species(Table5) revealed that A.chroococcum exhibited maximum IAA production(18.5A g mlÀ1)followed by A.beijerinckii(16.5A g mlÀ1)and A.vinelandii(13.8A g mlÀ1).At the salinity of20g lÀ1,the maximum production of IAA was recorded.The IAA production was maintained relatively higher at salinities above than below20g lÀ1.However,the rates of nitrogen fixation by A.chroococcum decreased at20and25g lÀ1NaCl.Further nitrogenease activity was arrested at30g lÀ1NaCl(Table6)in A.chroococcum and at20g lÀ1in A.beijerinckii and A.vinelandii.The rate of nitrogen fixation by A.chroococcum was the highest(9.4nM C2H4hÀ1mlÀ1)followed by A.beijerinckii(4.2nM C2H4hÀ1mlÀ1)at salinity of15g1À1 (Table6).Table6Effect of salinity on the rates of nitrogen fixation(nM C2H4Â108cells mlÀ1)by three species of Azotobacter at different salinity regimesConcentration of NaCl(g lÀ1)Rate of nitrogen fixation(nM C2H4Â108cells mlÀ1)A.chroococcum A.beijerinckii A.vinelandii0 6.2(35) 3.3(30) 2.5(22)57.6(88) 4.0(40) 2.8(40)107.9(61) 4.0(50) 3.0(48)159.4(82) 4.2(20) 3.0(55)207.5(94)Nil(88)Nil(70)257.5(98)Nil(88)Nil(88)30Nil(123)Nil(98)Nil(102)35Nil(106)Nil(92)Nil(98) Values in parenthesis are colony forming unitsÂ103mlÀ1.Values are average of triplicate samples and are significant between species at1%and between salinities at5%level.3.4.Biofertilizer effectThe bacterial inoculation increased the root growth in R .mucronata treated with A.vinelandii by enhancing average root biomass by 98.27%and root length by 98.57%(Table 7).The azotobacters stimulated the leaf area in both species of Rhizophora treated with all species of Azotobacter .For instance,the leaf area was higher by 277.86%in R.mucronata treated with Azotobacter beijerinckii and by 72.18%in R.apiculata treated with A.chroococcum .The shoot biomass was higher by 29.06%in R.mucronata treated with A.vinelandii and by 29.9%in R.apiculata inoculated with A.chroococcum (Table 7).The levels of photosynthetic pigment were higher in both the species of Rhizophora treated with A.chroococcum .The total chlorophyll content was 38.28%more in R.Table 8Effect of azotobacterisation on the levels of leaf pigments in 45-day-old seedlings of Rhizophora species Bacterialspecies treated Content of chl-a (mg g À1dry tissue)Content of chl-b (mg g À1dry tissue)Content of total chlorophyll(mg g À1dry tissue)Content of carotenoids (mg g À1dry tissue)R.mucronata A.chroococcum 0.990.6523.250.49A .beijerinckii 0.730.189.260.22A .vinelandii 1.250.6218.390.49Control 0.840.299.260.19R.apiculata A.chroococcum 1.820.7325.490.41A .beijerinckii 1.600.7223.240.40A .vinelandii 1.390.7020.830.36Control1.320.5218.430.32Values are average of 10samples and are significant at 1%level between treatments for total chlorophyll,chl-b and at 5%level for chl-a and carotenoids.Table 7Effect of azotobacterisation on root and shoot characteristics of 45-day-old seedlings of Rhizophora species Bacterialspecies treated Average length of primary root (cm seedling À1)Root biomass (g seedling À1)Leaf area(cm 2seedling À1)Shoot biomass (g seedling À1)R.mucronata A.chroococcum 8.860.3921.10 1.08A .beijerinckii 5.590.3928.00 1.72A .vinelandii 11.980.6619.02 2.28Control 8.070.347.41 1.77R.apiculata A.chroococcum 9.160.3854.27 2.56A .beijerinckii 7.380.3852.34 1.09A .vinelandii 11.260.3145.53 1.37Control10.280.5531.52 1.98Values are average of 10samples and are significant between treatments at 5%level for root length,root biomass and shoot biomass and at 1%level for leaf area.S.Ravikumar et al./J.Exp.Mar.Biol.Ecol.312(2004)5–1714S.Ravikumar et al./J.Exp.Mar.Biol.Ecol.312(2004)5–1715 apiculata and151%in R.mucronata over control.The levels of carotenoid pigment were higher by158.73%in R.mucronata and by26.77%in R.apiculata than their respective controls(Table8).4.DiscussionGenerally,the root samples exhibit higher rates of nitrogen fixation than rhizosphere soil.This nitrogen fixation among plant species was positively correlated with bacterial counts.There were about10-fold higher bacterial counts and nitrogen fixation of the root surface than those of rhizosphere soil(Tables1and4).Similar observations have been made in crop plants,attributing the reason to the growth promoting substances exuded from the roots,which in turn influence microbial counts(Parthasarathy and Mahadevan, 1985).Total counts of nitrogen-fixing Azospirillum enumerated from the samples of rhizosphere soil and root have been shown to have10-fold higher microbial counts in root than in the soil samples(Ravikumar et al.,2002).This may be attributed to the quantity and quality of root-derived carbon,which supports the biomass expansion.As the root grows through the soil,it releases photosynthetically derived carbon into the soil in a variety of soluble and insoluble forms,the totality of which is referred to as‘rhizodepo-sition’(Hiltner,1904;Kathiresan and Ravikumar,1995).Although there is a positive correlation between bacterial counts and nitrogen fixation,it is not a direct evidence of cause effect.The differences in nitrogen fixation rates observed among different species of mangroves may be due to the different species composition of bacteria,which are associated with nitrogen fixation and the nitrogen fixation is also controlled by light, temperature and seasonal variations,i.e.low in winter and high in the summer(Mann and Steinke,1993).Sengupta and Choudhuri(1991)studied nitrogen-fixing bacteria in a Ganges River mangrove community.They have found high numbers in the rhizospheres of plants in inundated areas,but plants on occasionally inundated ridges and in degraded areas have fewer rhizosphere bacteria.Ogan(1990)found similar distinct differences in nodulation and nitrogenase activity among sites and among species in a Nigerian mangal.Azotobacters have positive effects on the growth characteristics and pigments of mangroves(Tables7and8).This promotory effect may be attributed to ability of the azotobacters to fix atmospheric nitrogen and making it available to the growing seedlings of mangroves.In the present study,all of the three species of Azotobacter were capable of fixing nitrogen and also synthesising the phytohormone(Indole Acetic Acid—IAA), which are required for better growth and pigment production of mangrove seedlings (Kathiresan and Veera Ravi,1990;Ravikumar,1995).Similar findings have already been reported with azotobacter isolated from terrestrial soils which also produce IAA for promoting growth of crop plants(Azeon and Barea,1975;Ravikumar et al.,2002).5.ConclusionAmong the species,A.chroococcum has been found to be the most efficient in fixing nitrogen and in producing phytohormone(IAA).The same species is very effective in。