280 KKU Res. J. 2016; 21(2)

KKU Res.j. 2016; 21(2) : 280-290 http://resjournal.kku.ac.th

Effects of rhizobacteria on seed germination of water ( aquatica)

Nguyen Thi Huynh Nhu1* and Nuntavun Riddech2

1Graduate school of Biological Science program, Faculty of Science, Khon Kaen University, . 40002 2Department of Microbiology, Faculty of Science, Khon Kaen University, Thailand. 40002 *Corresponding author: [email protected]

Abstract

Rhizobacteria are considered as the most useful microorganism based on their properties in nitrogen fixing, phosphate and solubilizing, IAA and other hormones which improve the germination of seed. However, seed germination varied depending on stimulator such as bacterial strains, bacterial growing media. This investigation focused on testing the germination of Ipomoea aquatica by using three rhizobacteria isolates including H1-702, C1-112, P1-5071 and their growing media. Percentage of seed germination, shoot length, root length and number of root hairs of I. aquatica were lowest found in nutrient media treatments without bacteria (50%, 1.67 cm, 1.47 cm, and 1.47, respectively) while the highest numbers were obtained from the treatment of soaked seed with bacterial suspension which gave rise 90%, 4.88 cm, 3.01 cm and 19.26, respectively. Among all treatments, percentage of seed germination was highest (83%) in the single inoculums treatments C1-112 and H1-702. In day 5, the highest root length (2.4 cm) and root hair number (22.05) was showed in H1-702 treatment while C1-112 treatment revealed the highest shoot length (4.52 cm). The combination of two bacterial isolates did not stimulate the germination of I. aquatica, especially, P-H treatment which got the lowest number in all observation data. Keywords : Rhizobacteria, Ipomoea aquatica, seed germination. KKU Res. J. 2016; 21(2) 281

1. Introduction Furthermore, overusing chemical fertilizers in developing the crop of Ipomoea aquatica, or water spinach, I. aquatica has been causing ecosystem is an , fast-growing, economic change, soil nutrient decrease, human health and high nutrient (Khamwan et al, 2013), it effect and numerous money lost, so provides calcium, potassium, iron, chemical fertilizers should be reduced. and proteins with high levels. Wargovich Currently, there are few reports (2000) reported that play an documented on the roles of rhizobacteria important role in human health and applying in Ipomoea aquatica vegetable, the nutrition, particularly as sources of new strategy in agricultural habitats should C, thiamine, niacin, pyridoxine, folic acid, be applied for good qualities ofwater minerals and . According to spinach and sustainable agriculture. Jampeetong (2012), water spinach had been In this research,bacterial growing widely applied to purify eutrophic water and media and the three rhizobacteria isolates microorganisms played a main duty in the were chosen for testing plant growth removal mechanism of nitrate, nitrogen and promoting properties percentage of ammonia nitrogen in water spinach (Zhang germination (PG) in I. aquatica. et al., 2104). It is cultivated in the old world tropics and role as a weed of more than 20 2. Materials and methods crops in many regions of 60 countries, called by people in the as 2.1 Materials water cabbage or water spinach. According Three rhizobacterial strains to the advantage of I. aquatica as mention H1-702, P1-5071, C1-112 (Sritongon, at above, there were very necessary to 2015) were received from Microbial improve the I. aquatica production, Fertilizer Laboratory group, Department of especially, the germination of seed. Microbiology, Faculty of Science, However, according to Vrbničanin (2011), KhonKaen University, Thailand. Nutrient seed germination varied depending on agar media, Gram stain chemicals, fitter bacterial media and germination was paper. inhibited by bacterial treatments. 2.2 Characterization of the In addition, one of the most useful rhizobacteria microorganism in agricultural systems is The cell biological properties and Plant Growth Promoting Rhizobacteria Gram staining were observed under (PGPR). Rhizobacteria can be able to microscope at magnification 1000 X. excrete plant growth promoting substances These isolates were grown in Nutrient such as vitamins, kinetin and gibberellins agar medium for 2 days and determined and will also improve the vigor and colony morphology on the basis of shape, productivity of the crop (Karthikeyan et al., color and diameter of colonies. 2008). Rhizobacteria could also stimulate Three isolates were grown on nutrient the germination of Strigahermonthica medium (20% inoculation size) in incubator (Babalola, 2007), the number of root hairs with shaking (150 rpm). The optical and root laterals response to IAA production density at 600 nm was used for measuring by rhizobacteria (Shahab et al., 2009). the bacterial density in every 2 hours until 24 hours. 282 KKU Res. J. 2016; 21(2)

2.3 Evaluation I. aquatica seed 2.4 Evaluation I. aquatic seed germination by using bacterial growing germination using rhizobacteria lisolates media Seven treatments including: Seeds were sterilized with 1% inoculated with single of each bacterial (v/v) sodium hypochlorite solution for 10 isolateH1-702, P1-5071, and C1-112; minutes, then rinsed three times with sterile Combined two isolates: H-P (H1-702 + distilled water. Ten seeds of I. aquatica P1-5071), H-C (H1-702 + C1-112), C-P were transferred to petridish and treated (C1-112 + P1-5-71); and combined three with 3 ml of supernatant solution. Pure isolates C-H-P (C1-112 + H1-702 + sterilized water was used for the control. P1-5071) All treatments of bacteria were Three replications used for each treatment inoculated in the dark incubator with were placed in an incubator under dark shaking (150 rpm) until the number of condition (28 - 30°C) (Vrbničanin et al., bacteria reach 108 CFU/ml. 2011). Seeds were sterilized with 1% (v/v) After 5 days, the shoot length, root sodium hypochlorite solution in 10 minutes, length and number of root hairs were and then rinsed three times with distilled collected and calculated for the percentage water. of germination (PG) as described The shoot length, root length and by Kham-iam et al.(2005) using the number of root hair were collected and following equation: calculated for the percentage of germination in day 3, 4 and 5. Complete randomized design and triplicates in each treatment was designed for the experiments. Seed was grown on the following 2.5 Data analysis method treatments;C: control, M: Media, S: Data were subjected to statistical supernatant SS: soaked seed, when Control analysis by IBM SPSS Statistics 19 (C) was 3 ml pure sterilize water, Media (M) software; least significant difference test is 3 ml fresh media containing peptone and (LSD) at 5% level was use for comparing beef extract, Supernatant (S) was 3 ml means of the treatments with three bacterial supernatant after centrifuging at replications by complete randomized 600 rpm in 20 minutes, and soaked seed design. Microsoft Office Excel 2013 was (SS) was soaked the seeds in the bacterial used for data presentation. supernatant (centrifuged at 600 rpm in 20 minutes) in 60 minutes before placing in the 3. Results and discussion fitter paper in petri dish and added 3 ml of pure water.

Morphology, Gram stain and Growth curve

Figure 1. Colony of C1-112, H1-702 and P1-5071 in nutrient agar. KKU Res. J. 2016; 21(2) 283

Figure 2. Growth curve of three rhizobacterial isolates.

The colony shape of three rhizobacterial growth rate at 22 hours (figure 2) were isolates were circular, convex, and entire observed. H1-701 was rod shape and gram with white color and colony diameter was negative while others were coccus and gram varied from 0.7 to 1.5 mm) (figure 1), and positive bacteria (table 1). similar growth rate and the maximum Table 1. Properties of three rhizobacterialisolates. Colony Colony Bacterial Gram Sampling Isolates Colony Shape color diameter shape stain location H1-702 Circular Convex white 1.5 mm Rod - Pak Chong- Entire Nakhon Ratchasima- Thailand C1-112 Circular Convex white 1.2 mm Coccus + Chulapbron Entire Dam-Thailand P1-5071 Circular Convex white 0.7 mm Coccus + Muang- Nakhon Entire Ratchasima- Thailand

3.2 Seed germination method nutrient media and bacterial supernatant The length of root, shoot, and treatment. It can be concluded that number of root hairs and percentage of bacterial nutrient media inhibited the seed germination in seed soaking treatments germination of I. aquatica (Figure 3). were significantly higher than bacterial

+ P1-5-71); and combined three isolates C-H- from 0.7 to 1.5 mm) (figure 1), and similar P (C1-112 + H1-702 + P1-5071) growth rate and the maximum growth rate at All treatments of bacteria were inoculated in 22 hours (figure 2) were observed. H1-701 the dark incubator with shaking (150 rpm) was rod shape and gram negative while others until the number of bacteria reach 108 werecoccus and gram positive bacteria (table CFU/ml. 1). Seeds were sterilized with 1% (v/v) sodium Table 1. Properties of three rhizobacterialisolates. hypochlorite solution in 10 minutes, and then Colony Colony Colony Bacterial Gram Sampling rinsed three times with distilled water. Isolates Shape color diameter shape stain location The shoot length, root length and number of root hair were collected and calculated for the Pak Chong- Circular Nakhon percentage of germination in day 3, 4 and 5. Convex Ratchasima Complete randomized design and triplicates H1-702 Entire white 1.5 mm Rod - - Thailand in each treatment was designed for the Circular Chulapbron experiments. Convex Dam- C1-112 Entire white 1.2 mm Coccus + Thailand 2.5. Data analysis method Muang- Circular Nakhon Data were subjected to statistical analysis by Convex Ratchasima IBM SPSS Statistics 19 software; least P1-5071 Entire white 0.7 mm Coccus + - Thailand significant difference test (LSD) at 5% level was use for comparing means of the treatments with three replications by complete 3.2. Seed germination method randomized design. Microsoft Office Excel 2013 was used for data presentation. The length of root, shoot, and number of root hairs and percentage of germination in seed 3. Results and discussion soaking treatments were significantly higher than bacterial nutrient media and bacterial 3.1. Morphology, Gram stain and supernatant treatment. It can be concluded Growth curve that bacterial nutrient media inhibited the 284 KKU Res. J. 2016; 21(2) treatment.Hseed germinationowever,it of I. aquaticwas twicea(Figure high 3).er than those fromthe treatment of bacterial media (with just 2.14 cm) and 1.6 times higher than the treatment supernatant (2.96 cm) (Figure 4). Figure 1. Colony of C1-112, H1-702 and P1-5071 in nutrient agar.

Figure 5. Number of root hair after 3 days germination.C: control, M: Media, S: supernatant, SS: soaked seed.

PG value was variety among the treatment, Figure 3. Seed germinationFigure 3. Seed treated germination with water, treated culture with media, water, supernatant culture and soaked in Figure 2. Growth curve of three rhizobacterial isolates. media, supernatant and soaked in bacterial supernatant. soaked seed got the highest PG with 90%, bacterial supernatant. cellcell supernatant following by water (76.7%) and supernatant The colony shape of three rhizobacterial The length Theof root length in soaked of seedroot was in soakedhigher thanseed thosewas from4.88 the treatment70 %of while the lowest PG belonged to media isolates were circular, convex, and entire4.88 cmwith comparing Figure to water 4. 4.49Root cm, and there shoot bacterial length media after (with 3 justdays 2.14 cm) and 1.6 white color and colony diameter waswas varied no significant cm differencecomparing among to waterthese 4.49times cm, higher there than was the treatment no supernatanttreatment with 50% (Figure 6). germinationsignificant . C:difference control, M: among Media, S:these supernatant two , two treatment.SS: However, soaked seedit was. twice (2.96 cm) (Figure 4).

Figure 6. Percentage of germination (PG) of I. aquatica after 3 days. C: control, M: Media, S: supernatant, SS: soaked seed.

Figure 4. Root and shoot length after 3 days germination. C: control, M: Media,According S: to Roychowdhury (2012), supernatant,The shoot SS: soakedlength seed. and number of root hair germination percentage of floricultural was were the highest in soaked seed treatment decreased when the used plant growth with 3.01 and 19.26 cm, respectively, and the regulators increasing concentrations because lowest number was found in bacterial media the lower concentration of growth regulators treatment with shoot length of 1.67 cm and favors the increased enzymatic activity which root hair number of 1.47 (Figure 5). leads to the favorable environment for the germination as well as the growth of the radical and plumule. This result was similar to the study ofVrbničanin (2011), seed germination varied depending on bacterial media, germination was inhibited by bacterial treatments Pseudomonas fluorescensand Bacillus licheniformis. Nutrient media comprises Soya Peptone and Beef extract containing nitrogen - + in inorganic form as NO3 or NH4 and organic form as polypeptides or amino acids. Dijk and Eck (1995) reported that some species cannot use inorganic nitrogen, when KKU Res. J. 2016; 21(2) 285 The shoot length and number of root found in bacterial media treatment with hair were the highest in soaked seed shoot length of 1.67 cm and root hair treatment.H owever,it was twice hightreatmenter than with 3.01 and 19.26 cm, number of 1.47 (Figure 5). those fromthe treatment of bacterialrespectively, media and the lowest number was (with just 2.14 cm) and 1.6 times higher than thetreatment.H treatmentowever,it supernatant was (2.96 twice cm) high (Figureer than 4).those fromthe treatment of bacterial media (with just 2.14 cm) and 1.6 times higher than the treatment supernatant (2.96 cm) (Figure 4).

Figure 5. Number of root hair after 3 days germination.C: control, M: Media, S: supernatant, SS: soaked seed.

Figure 5. NumberPGFigure ofvalue root 5. Number hairwas aftervariety of root 3 days hair among germination.C:after 3 daysthe treatment, control, M: Media, S: supernatant,germination SS: soaked.C: control, seed. M: Media, S: supernatant, SS: soakedsoaked seedd. got the highest PG with 90%, PG value followingwas variety by among water the (76.7%) supernatant and supernatant70 % while the lowest PG treatment, soakedPG seed value got the was highest variety PG amongbelonged the to mediatreatment, treatment with 50% 70soaked % while seed the got lowest the highest PG belong PG edwith to media90%, Figure 4. Root and shoot length after with3 days 90%, followingtreatment by water with (76.7%) 50% and (Figure (Figure 6). 6). germination. C: control, M: Media, S: supernatant, following by water (76.7%) and supernatant SS: soaked seed. 70 % while the lowest PG belonged to media Figure 4. Root and shoot length after 3 days treatment with 50% (Figure 6). germination. C: control, M: Media, S: supernatant, SS: soaked seed.

Figure 6. PercentageFigure of 6germination. Percentage (PG) of ofgermination I. aquatica after(PG) 3 ofdays. I. C: control, M: Media,aquatica S: supernatant, after 3 SS: days soaked. C: seed. control, M: Media, S: supernatant, SS: soaked seed. According toFigure Roychowdhury 6. Percentage (2012), of environmentgermination for(PG) the germinationof I. as well as germination percentageAccordingaquatica of floriculturalafter to3 days wasRoychowdhury. C: thecontrol, growth M:of theMedia, radical(2012), S: and plumule. The shoot length and number of root hair supernatant, SS: soaked seed. decreased wheng erminationthe used plant percentage growth of Thisfloricultural result was similarwas to the study of were the highest in soaked seed treatmentregulators increasingdecreasedAccording concentrations whento theRoychowdhury Vrbničaninused plant (2011), (2012),growth seed germination varied withThe 3.01 shoot and length 19.26 andcm, respectively,number of root becauseand hairthe the lowerregulatorsg erminationconcentration increasing ofpercentage growth concentrations dependingof floricultural on because bacterialwas media, lowestwere numthe berhighest was infound soaked in bacterial seed treatmentregulators media favorsthedecreased the lower increased concentrationwhen enzymatic the germinationusedof growth plant wasregulators growth inhibited by bacterial activity which leads to the favorable treatments Pseudomonas fluorescens and treatmentwith 3.01 with and shoot19.26 cmlength, respectively, of 1.67 cm and and the favorregulatorss the increasedincreasing enzymatic concentrations activity because which rootlowest hair numbernumber ofwas 1.47 found (Figure in bacterial 5). media leadsthe lower to the concentration favorable environmentof growth regulators for the treatment with shoot length of 1.67 cm and germinationfavors the increased as well enzymatic as the growthactivity ofwhich the root hair number of 1.47 (Figure 5). radicalleads toand the plumule favorable. environment for the Thisgermination result aswas well similar as the togrowth the ofstudy the ofradicalVrbničanin and plumule (2011),. seed germination varied dependingThis result on wasbacterial similar media to, gtheermination study wasofVrbničanin inhibited (2011), by seedbacterial germination treatments varied Pseudomonasdepending on bacterialfluorescens mediaand, germinationBacillus licheniformis.was inhibited Nutrient by mediabacterial comprises treatments Soya PeptonePseudomonas and Beef fluorescensextract containingand Bacillusnitrogen licheniformis. Nutrient media -comprises+ Soya in inorganic form as NO3 or NH4 and Peptone and Beef extract containing nitrogen organic form as polypeptides- or amino+ acids. Dijkin i norganicand Eck form (1995) as NOreported3 or NHthat4 someand speciesorganic cannotform as use polypeptides inorganic ornitrogen amino, acids.when Dijk and Eck (1995) reported that some species cannot use inorganic nitrogen, when

the requirements for amino compounds are shoot length belonged to C1-112 with 4.12 most probably satisfied by transport of these cm and 4.52 cm, respestively, following by substances with a symbiotic fungus. H1- 702 (4.35 cm in day five). The length of Moreover, most amino acids act as inhibitor shoot ofthe control treatment in day five was or do not increase the growth of seedling, 4.34 cm and there were not significantly 286 only arginine and aspartic acid supportKKU Res. J. diference2016; 21(2) among all treatments, except P-H growth (Mitra, 1989). Therefore I. aquatic which the lowest shoot length found from Bacillus licheniformismust be. Nutrient soaked media 60 minutes or do notin increasethe bacterial the growth ofthree seedling, germination days, 1.9 cm, 2.6 cm, and comprises Soya supernatantPeptone and Beef t oextract absorb only thearginine sti andmulation aspartic acid2.8 support cm in day three, four and five, containing nitrogenhormone in inorganic such form as as auxingrowth from(Mitra, 1989).bacteria Therefore respectively I. aquatic (Table 2). NO - or NH + and organic form as must be soaked 60 minutes in the bacterial 3 4 secretion. Table 2. Seed germination of I.aquatica. polypeptides or amino acids. Dijk and Eck supernatant to absorb the stimulation (1995) reported 3.3.that some Seed species germination cannot hormone of I.aquatic such asa auxin from bacteria use inorganic nitrogen, when the secretion. requirements for amino compounds are 3.3. Seed germination of I. aquatica most probably satisfiedThe resultsby transport showed of these thatThe threeresults rhizosphereshowed that three rhizosphere substances withbacteria a symbioticl isolates fungus. could bacterialstimulate isolates the couldgrowth stimulate the growth Moreover, most aminoof I.aquatic acids acta as (Figure inhibitor 7). of I. aquatica (Figure 7).

Note. The everage values with the same letter in each column were not significantly different at the 95% confidence level

The number of root hairs increased sharply from day two to day three and increased slower in day five, one typical example, the root hair number in treament H1-702 was 16.03, 21.19 and 22.05 in three continuous observation days. In day five, the root hair numbers of all treatments were significantly higher than the control treament which had

17.63 root hairs (Table 2). Figure 7. I. aquaticaFigure seeds 7.I. in aquatica all treatmentsseeds inafter all treatments5 days germination. after 5 days germination. In germination percentage, the results showed that three treatments accounting for 83% were The overallThe strend overal was lthat strend the root was that theThere root shoot length length ofwas no significantly length of all treaments incresed slighly from different in the treatment H1-702H1 -at702, day C1-112 and C-P which were higher all treaments incresed slighly from day three day three to day five of germination. In day three with 3.23 cm comparing thanto control the control (80%). Germination to day five of germination. In day three, the three, the root length of P-H (1.40 cm) and (3.11 cm). However, in day fourpercentage and day of P1-6071 was also 80%. The root length of P-H (1.40 cm) and C-P (1.50 C-P (1.50 cm) lower than the control (1.59 five, the higest shoot length belongedhighest PGto belonged to P-H treatment (86%). cm) while highestcm) root lower length than was inthe the control C1-112 (1.59 with cm) 4.12 while cm and 4.52 cm, Comparing to control treatment, C-H and C- treatment C-H-P (2.1highest cm) and root there length were nowas inrespestively, the treamentC following-H by-P H1- 702 (4.35 cm H-P had lower PG, 76% and 70%, significant difference(2.1 cm) among and otherthere werein day five).no significant The length of shoot of the respectively (Figure 8). treaments. However,difference the highest among root length other controltreaments. treatment However, in day five was 4.34 cm in day five was in H1-702 (2.40 cm), and there were not significantly diference following by C1-112the highest (2.27 cm) root and length the inamong day fiveall treatments, was in H1 excepted- P-H which control treament,702 1.72 cm.(2.40 The cm), root lengthfollowing the lowestby C1 shoot-112 length (2.27 found from three inceased slighly incm) H1-702 and fromthe control1.8 cm in treament, germination 1.72 days, cm. 1.9 The cm, 2.6 cm, and 2.8 day three to 2.40root cm in length day five inceased (Table 2). slighlycm in inday H1 three,-702 four from and five, respectively 1.8 cm in day three to (Table2.40 2).cm in day five (Table 2). There shoot length was no significantly different in the treatment H1-702 at day three with 3.23 cm comparing to control (3.11 cm). However, in day four and day five, the higest e eees s e s e ee 1112 .12 s sse s ese .2 esese sses s s. 1 02 (. e). e e ee s s s s e ee e s ese e see . ee ee s e s s eee ees ee ( 1). eee I. aquaticKKU Res. J. 2016; 22(1) e es s e 287 s e se 60 es e e ee e s 1. 2.6 among all treatments, except P-H which the cm in day three, four and five, respectively se s e slowest shoot2. length found from threeee (Table 2). e e s s eKKUgermination Res. J.esee 2016; days, 21(2) 1.9 cm, 2.6(e cm, and 2). 2.8 287 see. TableTable 2. 2. Seed SeedTable germination germination 2. ee of eof I.I.aquatica. aquatica I.aquatica.

3.3. Seed germination of I.aquatica

e ess se ee see e ses se e I.aquatica (e ).

e.Note.Note. e The The eee everage everage es values values with with e the the se same same ee letter letter in ine each each column column ee were were notnot s significantly significantly ee differentdifferent e atat the the 95%95% ee confidenceconfidence ee levellevel TheThe numbernumber ofof rootroot hairshairs increasedincreased InIn germination germination percentage, percentage, the the results results sharplysharply fromfrome dayday e twotwo toto day day three three andand s showedshowed ese that that three three treatments treatmentss accounting accounting for for increasedincreased slowerslower inin dayday five,five, oneone typical typical 83% ee83% were were H1-702, H1-702, ese C1-112 C1-112 and and C-P C-P which which example,example, the sethe root root hair hair number number in ine treament treament e were were higherhigher ee thanthan thethe controlecontrol (80%).(80%). H1-702H1-702 was was 16.03, 16.03, 21.19 21.19 and and 22.05 22.05 in in three three GerminationGermination percentagepercentage ofof P1-6071P1-6071 waswas continuouscontinuous observationobservation days.days.e InIn dayday five,five, ee alsoalso 80%. 80%. The The1 highest highest02 PG PGs belonged belonged to to P-H P-H thethe root root hair hair16.0 numbers numbers 21.1of of all all treatments treatments 22.0were were treatmenttreatment ee (86%).(86%). ComparingComparings toto controlcontrol significantlysignificantly higherhigher thanthan thethe controlcontrol treatment,treatment, C-HC-H andand C-H-PC-H-P hadhad lowerlower PG,PG, treamenttreament se whichwhich hadhad 17.6317.63 s. rootroot hairs hairs 76%76% e and and 70%, 70%,e respectively respectively (Figure (Figure 8). 8). (Table(Table 2). 2).es ees ee s e e ee Moree studiesses shoulds bee donee to identifye 1.6 s (e 2). rhizobacteriae usings assnalysis of 16s16s rR RNA Figure 7.I. aquaticasees ees e e eee e ess segene ee,and thee potentiale of applying theseese s e. ses s e ee e eee ee ees eeisolates as the biofertilizer are needed for e e se s e e futuree study s in pot experiment. eee. 102 1112 ee e 5. Acknowledgement ees ese s ee 5. Acknowledgement e (0). eWe e would likee to thank Faculty of Scienceee e e. ee e ee eee 1601 s s 0. eand Department of Microbiology and Faculty e (1.0 ) (1.0 of Scienceee, KhonK KaenKe University,Ues Thailand es ee ee (6). s s. ) e e (1. ) e for supporting our study. ee 6.6. ReferencesReferences es e s e ee Figure 8. eee e () I. FigureFigure 8. 8. PercentagePercentage Figure ofofgermination germination e8. Percentage (PG)(PG) inI.inofgermination I .aquatic aquatica6 seedsat seed(PG) atday in dayI. 5. 05. 1.1. Babalola, O.O.,.. Berneree D.K.,.K. and Amusa,s (2.1 ) ee ee s aquaticaquatic seseedsates day 5 . . esee (e ). N..A.. 2007.200. Evaluation of somese bacteriale eee e ees. ee These results was similar to the results tsignificantly increase in the treatmentinoc- Theseese resultsess wass similars to thee resultsess of isolatesses ass germinationstimulantsess of e es e e s 1of Sachevet et al. (2009), the root and shoot ulated with Klebsiella supernatantStrigahermonthica J length of wheat seedlingsSacheve wereete al. .significan (2009),(200)- theecomparing root and shoottos the control. lengthe Strigahermonthica African Journal of 02 (2.0 ) 1112 (2.2 of wheate seedlingssees wereee significantslyincreaseese Agricultural ResearchRese Vol.. 2(1), 2(1) pp 02702- ) e ee 1.2 . e in thee treatmentinoculatedeee with 0302.2. KlebsiallaKlebsiallasupernatantsecomparing to thee 2.2. Davies,es P.J..J. 1987.1. Thee plant hormones:es e ese s 102 control.. theire nature, e occurrence, ee and functions. s. In: 1. ee 2.0 e Babalola (2007)(200) alsos demonstratedese that thereee PlantPlant hormoneshormones andand theirtheir rolerole inin plantplant (e 2). wereee some se potentiale in certaine growthgrowth andand development.development.MartinusNijhoffs rehizobacteriacould stimulatese thee germinatione Publishes,ses Dordrecht,e pp.. 11-11e11,Springer ee s e s s of SStrigahermonthicatrigahermonthicainin vitrovitroexperiment.eee. Netherlands,ees New e York. . ee e ee 102 ee Furthermore,ee all isolatedse bacteriae could 3.. Dijk, E.,. Eck, N.. 1995.1. Ammonium .2 (.11 ). producee IAA and Gibberellicee acid which thee toxicity and nitratee responseesse ofaxenically e Dactylorhizaincarnate ee e e es keye regulatorses to seedsee germinatione (Davies,(es grown Dactylorhizaincarnate 1987)1) during their e growth. . Seedlings.ees.eNew Phytot 131, 11 pp 361 616-367 4.. Hari, M.,. Perumale K.,K. 2010.2010. Biofertilizer.ee. e Rese 4.4. Conclusion Conclusion and and suggestion suggestion Shri AMM MurugappaChettiar Research Centreee Taramani. . Chiennai e600011-6000113, 4 .-5. 4.14.1 Conclusion Conclusion 5.. Jampeetong,Jee A.,. Brix, H.,. Kantawanichkul,K S.. 22012.012. Effectses of Threeeereseshizobacterialisolatesass singlese inoculum inorganic nitrogene formss on growth, (H1(1-702,02 C11-112112 and P11-5071)01) which morphology, nitrogene uptakee capacity and producede IAA and plant hormoneses could nutriente allocation of four tropical aquatic stimulatese thee germinatione of II. . aquataquaticaicaseed,see macrophyteses (Salviniacucullata,( IpomoeaIpomoea increasedese thee lengthe of root and shoots and thee aquaticaaquatica, Cyperusinvolucratusess and numbere of root hairs.s. Thee treatmenteeco- Vetiveriazizaeenioides).es). Aquatic Botany inoculum of two isolatesses wereee low effectee on 97,pp 10 1016.-16. seedsee germination,e especiallyese P-H treatmentee 6.6. Karthikeyan,Ke B.,. Abdul J.C.,J.. Lakshmanans had thee lowestes among all investigated ese data.. G.M.A.,... M.. Deiveekasundaram,eees M.. 2008.200. Seedee germinatione wass inhibitede by thee Studieses on rhizospheresee microbial diversityes mediume whilee thee soaks seedsee with bactereial of somese commerciallye important suspensionsses treatmentsees wereee betteree choicee medicinale plants.s. Colloidss and Surfaceses becauseese of itss highestes stimulations in I.I. B: Biointerfaces ees 62 62, pp 143 11.-145. aquaticaquaticgermination.e. 7.. Khamwan,K K.,K. Akaracharanya, A.,. Chareonpornwattana,e S.,. Choi, Y.E.,.. 4.2.4.2. Suggestion Suggestion Nakamura, T.,. Yamaguchi, Y.,. Sano, H.,. Shinmyo, A.. 2003.200. Geneticee 288 KKU Res. J. 2016; 21(2)

Babalola (2007) also demonstrated 6. References that there were some potential in certain rhizobacteria could stimulate the 1. Babalola, O.O., Berner D.K., and germination of Strigahermonthica in vitro Amusa, N.A. 2007. Evaluation of experiment. Furthermore, all isolated some bacterial isolates as bacteria could produce IAA and Gibberellic germination stimulants of acid which the key regulators to seed Strigahermonthica African Journal germination (Davies, 1987) during their of Agricultural Research Vol. 2(1), growth. pp 027-032. 2. Davies, P.J. 1987. The plant 4. Conclusion and suggestion hormones: their nature, occurrence, and functions. In: Plant hormones 4.1 Conclusion and their role in plant growth and Three rhizobacterial isolate as development.MartinusNijhoff single inoculum (H1-702, C1-112 and Publishes, Dordrecht, pp. P1-5071) which produced IAA and plant 1-11,Springer Netherlands, New hormones could stimulate the germination York. of I. aquatica seed, increased the length of 3. Dijk, E., Eck, N. 1995. Ammonium root and shoot and the number of root hairs. toxicity and nitrate response The treatment co-inoculum of two isolates ofaxenically grown Dactylorhizain- were low effect on seed germination, carnate seedling. New Phytot 131, especially P-H treatment had the lowest pp 361-367 among all investigated data. Seed 4. Hari, M., Perumal K., 2010. germination was inhibited by the medium Biofertilizer. Shri AMM while the soak seed with bacterial MurugappaChettiar Research suspension treatments were better choice Centre Taramani. Chiennai- because of its highest stimulation in I. 6000113, 4-5. aquaticgermination. 5. Jampeetong, A., Brix, H., 4.2. Suggestion Kantawanichkul, S. 2012. Effects More studies should be done to of inorganic nitrogen forms on identify rhizobacteria using analysis of 16s growth, morphology, nitrogen rRNA gene,and the potential of applying uptake capacity and nutrient these isolates as the biofertilizer are needed allocation of four tropical aquatic for future study in pot experiment. macrophytes (Salviniacucullata, 5. Acknowledgement Ipomoea aquatica, Cyperusinvolu- cratus and Vetiveriazizanioides). We would like to thank Faculty of Aquatic Botany 97,pp 10-16. Science and Department of Microbiology and Faculty of Science, Khon Kaen University, Thailand for supporting our study. KKU Res. J. 2016; 21(2) 289

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