Survival, efﬁcacy and rhizospheric effects of bacterial

136 137

Survival, efficacy and rhizospheric effects of bacterial inoculants onCajanus cajan

Richa Sharmaa, Jeny Singh Paliwala, Preeti Choprab, Deepshikha Dograa, Vijay Pooniyac,Virendra Swarup Bisariaa, Karivaradharajan Swarnalakshmib, Shilpi Sharmaa,*aDepartment of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, IndiabDivision of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, IndiacDivision of Agronomy, Indian Agricultural Research Institute, New Delhi 110012, India

A R T I C L E I N F O

Article history:Received 29 March 2016Received in revised form 14 February 2017Accepted 15 February 2017Available online xxx

Keywords:Bacillus megateriumPseudomonas fluorescensAzotobacter chroococcumRif mutantsNon-target effects

A B S T R A C T

Bioinoculants serve as a promising, eco-friendly alternative to conventional chemical fertilizers andpesticides. While their direct positive effect on plant growth is well known, non-target effects of theseagricultural amendments have so far not been extensively studied. The present study is an attempt toassess (a) the survival of Bacillus megaterium, Pseudomonas fluorescens and Azotobacter chroococcum in therhizosphere of Cajanus cajan (pigeon pea), (b) the target effects of unconventional combinations of thesebioinoculants on the crop, and (c) the non-target effects (on the resident soil microflora) of the bacterialsupplements, when applied individually and in combination. Rifampicin-resistant strains wereemployed to follow the persistence of the bioinoculants in the rhizosphere. They could be detecteduntil approximately two months after sowing. The effect of the bioinoculants in the field was assessed onvarious plant growth parameters. Triple inoculation competed well with chemical fertilizer with respectto plant growth parameters. Grain yield (kg ha�1) was 1.5- and 1.7-fold higher with mixed consortiumand chemical fertilizer, respectively, than that of the untreated control. A cultivation-dependentapproach was employed to assess important microbial groups in the plant rhizosphere. In a comparisonof the treatments with bulk soil, a clear effect on the rhizosphere was apparent. Apart from theinoculation effect, pronounced changes in the microbial diversity were observed during plantdevelopment. At the vegetative stage, the mixed consortium showed increases of 1.08-, 1.22- and4.2-fold in the abundance of nitrogen fixers, Pseudomonas and Actinomycetes, respectively, as comparedto the untreated control. Additionally, the bioinoculants were found to be compatible with other groupsof plant growth promoting rhizobacteria.

© 2017 Elsevier B.V. All rights reserved.

1. Introduction

Sustainable agriculture involves the utilization of eco-friendlytechnologies, which include the application of bioinoculants forcrop improvement. Bioinoculants are widely used in agriculturebecause of their beneficial interaction with plant roots, protectionagainst soil borne pathogens, and improvement of grain yield. Inthe past decade, a large amount of data has been generated thatsupport the efficacy of bioinoculants in enhancing plant growthand yield (Tilak et al., 2006; Niranjana et al., 2009; Kumar et al.,2010; Gupta et al., 2012). However, only limited information isavailable on the ‘non-target’ effects of bioinoculants on the

resident microbial community (Naseby et al., 2000; Björklöf et al.,2003; Pereira et al., 2009; Gupta et al., 2014). Non-target effects canbe defined as the effects caused by the introduction ofbioinoculants on organisms other than the target organisms(Winding et al., 2004). When bioinoculants are released into theenvironment, they can induce transient disturbances in residentmicrobial community, which may be either positive or negative.Aseri et al. (2008) reported enhanced activities of the enzymesdehydrogenase, alkaline phosphatase and nitrogenase in therhizosphere of Punica granatum upon inoculation with Azospirillumbrasilense, Azotobacter chroococcum, Glomus fasciculatum and G.mosseae. Increase in microbial biomass and abundance of specificmicrobial groups have also been observed upon application ofbioinoculants (Zhang et al., 2010; Trabelsi et al., 2011). These effectson the native microflora depend on various parameters, like type ofsoil, mode of application and other environmental conditions (Berg* Corresponding author.

E-mail address: [email protected] (S. Sharma).

http://dx.doi.org/10.1016/j.agee.2017.02.0180167-8809/© 2017 Elsevier B.V. All rights reserved.

Agriculture, Ecosystems and Environment 240 (2017) 244–252

Contents lists available at ScienceDirect

Agriculture, Ecosystems and Environment

journa l homepage : www.e l sev ier .com/ loca te /agee

Reproduced from Agriculture, Ecosystems and Environment 240: 244-252 (2017).Virendra S. Bisaria: Participant of the 1st UM, 1973-1974.

yamamoto

Table of Contents

yamamoto

136 137

Survival, efficacy and rhizospheric effects of bacterial inoculants onCajanus cajan

Richa Sharmaa, Jeny Singh Paliwala, Preeti Choprab, Deepshikha Dograa, Vijay Pooniyac,Virendra Swarup Bisariaa, Karivaradharajan Swarnalakshmib, Shilpi Sharmaa,*aDepartment of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, IndiabDivision of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, IndiacDivision of Agronomy, Indian Agricultural Research Institute, New Delhi 110012, India

A R T I C L E I N F O

Article history:Received 29 March 2016Received in revised form 14 February 2017Accepted 15 February 2017Available online xxx

Keywords:Bacillus megateriumPseudomonas fluorescensAzotobacter chroococcumRif mutantsNon-target effects

A B S T R A C T

Bioinoculants serve as a promising, eco-friendly alternative to conventional chemical fertilizers andpesticides. While their direct positive effect on plant growth is well known, non-target effects of theseagricultural amendments have so far not been extensively studied. The present study is an attempt toassess (a) the survival of Bacillus megaterium, Pseudomonas fluorescens and Azotobacter chroococcum in therhizosphere of Cajanus cajan (pigeon pea), (b) the target effects of unconventional combinations of thesebioinoculants on the crop, and (c) the non-target effects (on the resident soil microflora) of the bacterialsupplements, when applied individually and in combination. Rifampicin-resistant strains wereemployed to follow the persistence of the bioinoculants in the rhizosphere. They could be detecteduntil approximately two months after sowing. The effect of the bioinoculants in the field was assessed onvarious plant growth parameters. Triple inoculation competed well with chemical fertilizer with respectto plant growth parameters. Grain yield (kg ha�1) was 1.5- and 1.7-fold higher with mixed consortiumand chemical fertilizer, respectively, than that of the untreated control. A cultivation-dependentapproach was employed to assess important microbial groups in the plant rhizosphere. In a comparisonof the treatments with bulk soil, a clear effect on the rhizosphere was apparent. Apart from theinoculation effect, pronounced changes in the microbial diversity were observed during plantdevelopment. At the vegetative stage, the mixed consortium showed increases of 1.08-, 1.22- and4.2-fold in the abundance of nitrogen fixers, Pseudomonas and Actinomycetes, respectively, as comparedto the untreated control. Additionally, the bioinoculants were found to be compatible with other groupsof plant growth promoting rhizobacteria.

© 2017 Elsevier B.V. All rights reserved.

1. Introduction

Sustainable agriculture involves the utilization of eco-friendlytechnologies, which include the application of bioinoculants forcrop improvement. Bioinoculants are widely used in agriculturebecause of their beneficial interaction with plant roots, protectionagainst soil borne pathogens, and improvement of grain yield. Inthe past decade, a large amount of data has been generated thatsupport the efficacy of bioinoculants in enhancing plant growthand yield (Tilak et al., 2006; Niranjana et al., 2009; Kumar et al.,2010; Gupta et al., 2012). However, only limited information isavailable on the ‘non-target’ effects of bioinoculants on the

resident microbial community (Naseby et al., 2000; Björklöf et al.,2003; Pereira et al., 2009; Gupta et al., 2014). Non-target effects canbe defined as the effects caused by the introduction ofbioinoculants on organisms other than the target organisms(Winding et al., 2004). When bioinoculants are released into theenvironment, they can induce transient disturbances in residentmicrobial community, which may be either positive or negative.Aseri et al. (2008) reported enhanced activities of the enzymesdehydrogenase, alkaline phosphatase and nitrogenase in therhizosphere of Punica granatum upon inoculation with Azospirillumbrasilense, Azotobacter chroococcum, Glomus fasciculatum and G.mosseae. Increase in microbial biomass and abundance of specificmicrobial groups have also been observed upon application ofbioinoculants (Zhang et al., 2010; Trabelsi et al., 2011). These effectson the native microflora depend on various parameters, like type ofsoil, mode of application and other environmental conditions (Berg* Corresponding author.

E-mail address: [email protected] (S. Sharma).

http://dx.doi.org/10.1016/j.agee.2017.02.0180167-8809/© 2017 Elsevier B.V. All rights reserved.

Agriculture, Ecosystems and Environment 240 (2017) 244–252

Contents lists available at ScienceDirect

Agriculture, Ecosystems and Environment

journa l homepage : www.e l sev ier .com/ loca te /agee

and Smalla, 2009). On the other hand, López-Valdez et al. (2011)could not see any positive effect of Bacillus subtilis at the harveststage of Helianthus annuus. Costa et al. (2006) did not observe anysignificant rhizospheric effects on actinobacterial population in therhizosphere of oilseed rape in the early growth season.

Besides the crucial aspect of non-target effects of bioinoculants,it is essential to know their persistence in the rhizosphere beforeapplication. This is a relatively under explored area, as tracking ofbioinoculants at strain level is cumbersome due to a limitedavailability of tools. To monitor a bioinoculant, the specific strainmust have a selective characteristic that does not interfere with itscolonization of the rhizosphere and survival (Turco et al., 1986).Selection of strains with spontaneous antibiotic-resistances hasprovided a simple and effective method to monitor bioinoculantsafter their introduction into soil. Rifampicin resistance in bacteriais due to a rare mutation in the RNA polymerase gene. Due to itschromosomal nature, such a mutation is stable and non-transfer-able, in contrast to plasmid-borne markers (Sippel and Hartmann,1968).

India is the largest producer and consumer of Cajanus cajan,accounting for about 70% of the global production (Odeny, 2007).After chickpea (Cicer arietinum) and field pea (Pisum sativum),pigeon pea is the third most important legume crop in India(Sharma et al., 2012). It also has high nitrogen fixation ability(España et al., 2006). As shown by Saxena and Nadarajan (2010), inIndia, the average productivity of pigeon pea of around 700 kg ha�1

has not changed in the last five decades. Given the immense overalltechnological improvement of agricultural productivity, thisstagnation is a serious concern. The present study aims to attaina deeper understanding of the interactions between non-conventional combinations of bioinoculants with pigeon pea,and with its resident rhizospheric microflora with respect toenhancement of grain yield. The bioinoculants selected for thestudy were Azotobacter chroococcum A-41, Bacillus megateriumMTCC 453 and Pseudomonas fluorescens MTCC 9768. A. chroococ-cum is a free-living diazotroph, B. megaterium is a potential agentfor the biocontrol of plant diseases, and P. fluorescens has multipleplant growth promoting properties like production of side-rophores, hydrogen cyanide and indole acetic acid (Gupta et al.,2016). We hypothesized that the positive impact of bioinoculantson plant growth and yield is a cumulative effect of their directcontributions, as well as their effects (both positive as well asnegative) on the resident microbial community of the plant. Thiscould explain that the bioinoculants can have a lasting effect on theplant, despite their relatively short-term persistence in the soil.This would in turn have an impact on plant growth. Our objectiveswere thus (i) to monitor the persistence of plant growth-promoting A. chroococcum, B. megaterium and P. fluorescens inthe rhizosphere of C. cajan, (ii) to quantify the target effect of thebioinoculants on plant growth parameters in the field, and (iii) toassess the non-target effects of the bioinoculants on the culturablerhizospheric microbiome.

2. Materials and methods

2.1. Plant system, microbial strains and compatibility assay

Seeds of pigeon pea (C. cajan) cultivar UPAS-120 were obtainedfrom the National Seed Corporation, Pusa, New Delhi, India.Bioinoculants used were B. megaterium MTCC 453, P. fluorescensMTCC 9768 and A. chroococcum A-41. B. megaterium MTCC 453 andP. fluorescens MTCC 9768 were procured from the Institute ofMicrobial Technology, Chandigarh. A. chroococcum A-41 andBradyrhizobium sp. (rhizobium recommended for the crop) wereobtained from the Division of Microbiology, Indian AgriculturalResearch Institute (IARI), New Delhi, India. The three strains of

bioinoculants, and rhizobium, were assessed for compatibilityusing standard cross streak assay method (Anandaraj and LeemaRose Delapierre, 2010).

2.2. Isolation of spontaneous rifampicin resistant bacterial strains

Spontaneous rifampicin resistant mutants of the three bio-inoculant strains were isolated by plating overnight-cultures onthe respective agar medium containing 100 mg mL�1 rifampicin(Nautiyal, 1996). Cultures of rifampicin resistant mutants withplant growth promoting properties comparable to those of therespective wild types (data not shown), were serially diluted andplated on agar plates containing 0, 5, 25, 50, and 100 mgrifampicin mL�1. Colonies were observed on the plates after24–36 h. The mutants were stored as glycerol stocks at �80 �Cin antibiotic-supplemented (100 mg mL�1) Luria broth.

2.3. Preparation of formulation, seed surface sterilization andbacterization

Culture broths containing 1.0 � 1010 cfu (colony forming unit)mL�1 of each bacterial strain were used for preparation offormulations. For approximately 100 g formulation, 80 g auto-clave-sterilized talcum powder (inorganic carrier) was mixed with18 mL of bacterial culture broth, 1 mL of 50% autoclaved glyceroland 1.0 mL of 0.1% filter sterilized carboxy methyl cellulose. Theformulation was dried in an incubator at 27 �C to reduce themoisture content, packed in sterile polythene bags and sealed. Forthe combination of two or three bioinoculants (as specified inSection 2.4), individual cultures were mixed in equal quantities,resulting in formulations containing �2.0 � 109 cfu g�1 of eachbioinoculant. Seeds of C. cajan were surface-sterilized using 70%ethanol for 30 s, followed by soaking in 0.01% NaClO for 2 min. Theseeds were then washed with 0.01 N HCl to remove excess NaClO(Abdul-Baki, 1974), and finally rinsed eight times with steriledistilled water. The sterilized seeds were then soaked in autoclavedwater and kept overnight. Seeds of similar size and shape (visualobservation) were selected for bacterization. Standard dilution andplate counting techniques were used to determine the cfu of theformulations prior to sowing of the seeds. The average cfu per seedwas calculated for all treatments and were found to be in the rangeof 4–8 � 108.

2.4. Pot experiment for tracking bioinoculants in the rhizosphere ofCajanus cajan

The soil used in this study was collected from an agriculturalfield in Delhi, India. It had the following properties: clay loam (40%clay, 35% sand and 25% silt), 0.42% organic matter content, 7.2 pH(1:2.5 soil and water ratio), electrical conductivity of 0.04 dS m�1,and moisture content of 14%. Nine different formulations wereprepared: (1) B. megaterium (B), (2) B. megaterium rifampicinresistant mutant (Br), (3) P. fluorescens (P), (4) P. fluorescensrifampicin resistant mutant (Pr), (5) A. chroococcum (A), (6) A.chroococcum rifampicin resistant mutant (Ar), and three combi-nations of rifampicin resistant mutants: (7) A. chroococcum and B.megaterium (Ar + Br), (8) B. megaterium and P. fluorescens (Br + Pr),(9) A. chroococcum and P. fluorescens (Ar + Pr). Pots of 40.6 cmdiameter were filled with soil described above, and three seeds perpot were sown at a depth of approximately 4–5 cm. The set-up wascompletely randomized (CRD). Seeds without inoculation (C) andbulk soil (S) served as control.

Five samplings points were chosen: pre-vegetative [9 and 16days after sowing (DAS)], vegetative (25 DAS) and pre-flowering(45 and 59 DAS). At the time of each sampling, rhizospheric soil(soil tightly adhering to the roots) was collected. These samples

R. Sharma et al. / Agriculture, Ecosystems and Environment 240 (2017) 244–252 245

138 139

were kept at 4 �C. The plant parameters, root length, shoot lengthand dry weight, were also measured at the time of each sampling.Root length was measured from the tip of the tap root to the rootcrown. Shoot length was measured from the root crown to the tipof the main shoot. Dry weight was measured by keeping the plantand soil samples in a hot air oven at 60 �C until a constant weightwas obtained. An additional sampling of rhizospheric soil was doneat 75 DAS for the determination of bacterial counts on specificmedia.

2.5. Field experiment

A field experiment was conducted to evaluate the impact ofdifferent treatments of bioinoculants on growth and productivityof C. cajan. The experiment was laid out in randomized blockdesign (RBD), with plot size of 10 m2 at a research farm of IARI, NewDelhi. The sandy loam soil of the experimental field had 100.4 kgha�1 alkaline permanganate oxidizable N (Subbiah and Asija,1956), 10.8 kg ha�1available P (Olsen et al., 1954), 319.3 kgha�1ammonium acetate exchangeable K (Hanway and Heidel,1952), and 0.27% organic carbon (Walkley and Black, 1934). The pHof the soil was 8.15 (1:2.5 soil and water ratio), electrical

conductivity was 0.62 dS m�1 and Fe, Mn, Zn and Cu content(Lindsay and Norvell, 1978) were 6.02, 9.47, 1.59 and 2.19 mg kg�1

of soil, respectively. The previous crop in the experimental site waswheat, followed by fallow, during the summer season. Theperformance of the bioinoculants was monitored as singleinoculants [T1-A. chroococcum (A), T2-B. megaterium (B) andT3-P. fluorescens (P)], dual [T4�A+B, T5�B+P, T6�A+P] and triplecombination [T7�A+B+P]. Treatment with rhizobium (Bradyrhi-zobium sp.) (T8) alone was also included to compare effects of itsinoculation. Control plot without any amendment (of fertilizer orbioinoculants) served as a negative control (T9), while treatmentwith 100% recommended dose of fertilizer (RDF: 100 kgdi–ammonium phosphate ha�1) without inoculants served aspositive control (T10). Formulations of the bioinoculants wereprepared as described previously (Sarma et al., 2009), and seedinoculation was performed as per the standard method (Swarna-lakshmi et al., 2011).

The experiment was carried out during Kharif (June–November)season. The mean daily maximum temperature during the growingmonths ranged from 27 to 42 �C (with the average temperaturedeclining as the experiment progressed), while the relativehumidity was between 55% and 80%. During the period, maximum

Fig. 1. Plant growth parameters of Cajanus cajan (a) root length (b) shoot length (c) dry weight, at four time points. Significantly different values (P < 0.05) between differentsampling time points for the same treatment have been marked by lowercase letters, and significantly different values between treatments for the same time points havebeen marked by uppercase letters below the columns. Error bars represent the standard deviation for n = 3. Abbreviations: Ar = rifampicin resistant mutant of Azotobacterchroococcum, Br = rifampicin resistant mutant of Bacillus megaterium, Pr = rifampicin resistant mutant of Pseudomonas fluorescens, Ar + Pr = rifampicin resistant mutants ofA. chroococcum and P. fluorescens, Br + Pr = rifampicin resistant mutants of B. megaterium and P. fluorescens, Ar + Br = rifampicin resistant mutants of A. chroococcum andB. megaterium, C = seeds without inoculation, DAS = days after sowing.

246 R. Sharma et al. / Agriculture, Ecosystems and Environment 240 (2017) 244–252

138 139

were kept at 4 �C. The plant parameters, root length, shoot lengthand dry weight, were also measured at the time of each sampling.Root length was measured from the tip of the tap root to the rootcrown. Shoot length was measured from the root crown to the tipof the main shoot. Dry weight was measured by keeping the plantand soil samples in a hot air oven at 60 �C until a constant weightwas obtained. An additional sampling of rhizospheric soil was doneat 75 DAS for the determination of bacterial counts on specificmedia.


A field experiment was conducted to evaluate the impact ofdifferent treatments of bioinoculants on growth and productivityof C. cajan. The experiment was laid out in randomized blockdesign (RBD), with plot size of 10 m2 at a research farm of IARI, NewDelhi. The sandy loam soil of the experimental field had 100.4 kgha�1 alkaline permanganate oxidizable N (Subbiah and Asija,1956), 10.8 kg ha�1available P (Olsen et al., 1954), 319.3 kgha�1ammonium acetate exchangeable K (Hanway and Heidel,1952), and 0.27% organic carbon (Walkley and Black, 1934). The pHof the soil was 8.15 (1:2.5 soil and water ratio), electrical

conductivity was 0.62 dS m�1 and Fe, Mn, Zn and Cu content(Lindsay and Norvell, 1978) were 6.02, 9.47, 1.59 and 2.19 mg kg�1

of soil, respectively. The previous crop in the experimental site waswheat, followed by fallow, during the summer season. Theperformance of the bioinoculants was monitored as singleinoculants [T1-A. chroococcum (A), T2-B. megaterium (B) andT3-P. fluorescens (P)], dual [T4�A+B, T5�B+P, T6�A+P] and triplecombination [T7�A+B+P]. Treatment with rhizobium (Bradyrhi-zobium sp.) (T8) alone was also included to compare effects of itsinoculation. Control plot without any amendment (of fertilizer orbioinoculants) served as a negative control (T9), while treatmentwith 100% recommended dose of fertilizer (RDF: 100 kgdi–ammonium phosphate ha�1) without inoculants served aspositive control (T10). Formulations of the bioinoculants wereprepared as described previously (Sarma et al., 2009), and seedinoculation was performed as per the standard method (Swarna-lakshmi et al., 2011).

The experiment was carried out during Kharif (June–November)season. The mean daily maximum temperature during the growingmonths ranged from 27 to 42 �C (with the average temperaturedeclining as the experiment progressed), while the relativehumidity was between 55% and 80%. During the period, maximum

Fig. 1. Plant growth parameters of Cajanus cajan (a) root length (b) shoot length (c) dry weight, at four time points. Significantly different values (P < 0.05) between differentsampling time points for the same treatment have been marked by lowercase letters, and significantly different values between treatments for the same time points havebeen marked by uppercase letters below the columns. Error bars represent the standard deviation for n = 3. Abbreviations: Ar = rifampicin resistant mutant of Azotobacterchroococcum, Br = rifampicin resistant mutant of Bacillus megaterium, Pr = rifampicin resistant mutant of Pseudomonas fluorescens, Ar + Pr = rifampicin resistant mutants ofA. chroococcum and P. fluorescens, Br + Pr = rifampicin resistant mutants of B. megaterium and P. fluorescens, Ar + Br = rifampicin resistant mutants of A. chroococcum andB. megaterium, C = seeds without inoculation, DAS = days after sowing.


rainfall occurred in the month of July (approx. 228 mm), withOctober and November being dry months. Five irrigations,excluding pre-sowing irrigation, were applied at critical growthstages, including flower initiation and pod filling.

A sampling was performed at the start of the experiment tocharacterize the resident soil microflora at the time of sowing.Fungi were detected on Rose Bengal Chloramphenicol medium,Actinomycetes on Kenknight and Munaier's medium, gram negativeenterobacteria on Eosin methylene blue agar, nitrogen fixers onJensen's medium, Azotobacter species on Burk's medium, Pseudo-monas on King's B medium, and phosphate solubilizers onPikovskaya's medium. The abundance of these different micro-organisms was in the order of 106 cfu g�1 dry soil, except for fungi,for which it was in the order of 104 cfu g�1 dry soil. Furthersampling was done at three time points during the growth of theplant, the vegetative (45 DAS), flowering (90 DAS) and harveststages (190 DAS). Soil samples were taken from a depth of �15 cm.The plants were uprooted at each sampling point and biometricmeasurements (shoot and root height) were recorded. The dryweight of the plants was measured by keeping the plants in hot airoven at 60 �C until constant weight was obtained. Plant develop-ment was expressed in terms of shoot biomass per plot and yieldattributes (branch numbers per plant, pod numbers per plant,grains per pod, and grain yield per hectare).

2.6. Enumeration of specific rhizospheric microbial groups

The rhizosphere soil samples collected in the pot experimentwere subjected to cultivation-dependent analysis for tracking ofbioinoculants (both wild type and mutants) on appropriateselective media. The abundance of A. chroococcum, B. megateriumand P. fluorescens was observed on Burk's, Sperber and tryptoneglucose yeast extract (TGY) media, respectively. Plates containing100 mg rifampicin mL�1, an amount sufficient to inhibit the growthof other microorganisms found in non-sterilized soil, were used torecover the respective resistant strains from the samples.

Cultivation-dependent analysis of the field experiment wasdone to quantify specific groups of rhizospheric microorganisms.The specific microbial groups have been described in Section 2.5.After serial dilution, samples were plated on the appropriateplates. The inoculated plates were incubated at 30 �C for 48 h.Plates on which 30–300 colonies appeared were counted. Theresults were expressed as cfu g�1 dry soil.

2.7. Statistical analysis

Analysis of variance (ANOVA) was performed on the data, usingSPSS Statistics 16.0 for Windows (SPSS Inc., Chicago, Ill., USA). One-way ANOVA was performed with treatment as the independentvariable, and CFU count or plant growth parameter as thedependent variable. Except for plant growth parameters fromfield experiment, all data sets were additionally subjected to asecond set of analysis with CFU count and plant growth parameters(from pot experiment) as dependent variable and time point asindependent variable. Tukey's HSD post hoc test was employed tocompare means when ANOVA yielded P < 0.05 (Gomez and Gomez,1984).

3. Results

3.1. Effect of treatment of rif mutants on plant growth parameters ofCajanus cajan

The three strains of bioinoculants were found to be compatiblewith each other and with rhizobium, as no growth inhibition zoneformed around the colonies of any bioinoculant (data not shown).

The effects of the treatments with rif mutants, individually and incombinations of two, on growth of C. cajan (Fig. 1a–c) weremonitored. At 59 DAS (pre-flowering stage of the plant develop-ment), the dual inoculations, except Ar + Pr, surpassed the resultsof individual inoculations in terms of dry weight, with the Ar + Brtreatment exhibiting the best result (1.55-fold enhancement).

3.2. Quantification of specific rhizospheric groups and tracking ofrifampicin resistant mutants in the rhizosphere of Cajanus cajan

To estimate the abundance of all the three bioinoculants,enumeration was performed with or without rifampicin in therespective media. A steady decline with time, of the populations ofrifampicin-resistant mutants of B. megaterium, P. fluorescens and A.chroococcum was observed (Fig. 2). No mutants were detected after59 DAS (soil samples collected at 75 DAS; data not shown). Intreatments with dual inoculation (except Ar + Pr) at 9 DAS, therhizospheric population of resident Bacillus sp., Pseudomonas sp.and Azotobacter sp. (monitored without antibiotic supplementa-tion) was almost 3-, 10- and 30-fold higher, respectively, comparedto the individual treatments. This trend was same for the mutants;the dual inoculations (except for Ar + Br in case of B. megaterium)resulted in higher abundance of the mutants compared toindividual treatments at 9 DAS.

On rifampicin-supplemented media, the rhizospheric popula-tion at 9 DAS of the mutants of B. megaterium in Br + Pr inoculationwas 3.27-fold higher than in Br inoculation alone (Fig. 2a). Therhizospheric level of the mutant of P. fluorescens in the dualinoculation was about 20-fold and 4.12-fold higher in Br + Pr andAr + Pr, respectively, as compared to single inoculation (Fig. 2b).Thereafter, the populations of dual inoculations decreasedcontinuously. In the case of A. chroococcum, Ar + Br was the bestcombination in terms of its abundance on both rifampicinsupplemented and non-supplemented media (Fig. 2c). At 9 DAS,the rhizospheric level of the mutant of A. chroococcum was, for dualinoculations, higher than for the single inoculation Ar (9.2-fold forAr + Br, and 4.34-fold for Ar + Pr). By 25 DAS, the level of themutants in the dual inoculation was 19.34-fold higher for Ar + Brthan for single inoculations.


3.3.1. Plant parametersAt the first sampling, performed during the crop's vegetative

stage (45 DAS), shoot length ranged from 65.84 cm to 77.20 cm,with maximum values obtained for treatment with chemicalfertilizer (100% RDF), followed by dual inoculations involvingPseudomonas (Table 1). The root length ranged from 10.39 cm to12.54 cm, with the maximum value observed with T9(un-inoculated control), followed by dual T4 (B + P) and triple T7(A + B + P) inoculations. Shoot weight per plant varied from 4.83 gto 6.01 g. The highest plant weight was observed with treatmentT10 (chemical fertilizer), followed by T7 and T4.

During flowering stage (90 DAS), the shoot length (which variedfrom 129.00 cm to 160.28 cm) and shoot weight per plant (53.91 gto 107.01 g) were highest for the chemical fertilizer treatment. Thiswas followed by T7 (A + B + P) in case of shoot weight.

At 190 DAS (harvest stage), additional plant parameters wereassessed, namely biomass, which ranged from between 94 gplant�1 to 138 g plant�1, the average number of branches per plant,which varied from 19 to 25, the number of pods per plant (in therange of 94–133), and grain yield (in the range of 1150 kg ha�1 to2050 kg ha�1) (Table 2). The grain yield (kg ha�1) was 1.5- and1.7-fold higher for the mixed consortium A + B + P and the chemicalfertilizer, respectively, than the uninoculated control. Overall, thehighest values of all these parameters were observed for the


140 141

chemical fertilizer, followed by the triple inoculation. Theinoculation effect was more pronounced at early stages of plantgrowth, which seemed to influence the productivity of the plant atthe harvest stage.

3.3.2. Enumeration of specific rhizospheric microbial groupsThe fungal population was highest at 45 DAS and decreased

thereafter with time for all treatments, except for P, A + P, A + B + Pand bulk soil (Fig. 3a). The three treatments and bulk soil had lowfungal abundance even at 45 DAS. At 45 DAS, inoculation with

Azotobacter enhanced the fungal abundance by 3.75-fold, ascompared to bulk soil and 1.55-fold compared to the uninoculatedcontrol. However, towards the harvest stage, the triple inoculationexhibited the maximum abundance of fungi compared to thecontrol (3.11-fold) and chemical fertilizer samples (2.33-fold).Similar to the dynamics of fungal populations, gram-negativeenteric bacteria generally exhibited a decline with time, A + B, B + Pand bulk soil being exceptions (Fig. 3b). At 45 DAS, the maximumabundance was observed for the control and the chemical fertilizertreatment, where the values were approximately 2.8- fold higher

Table 1Effect of bioinoculants on plant parameters of C. cajan at vegetative stage (45 DAS) and flowering stage (90 DAS).

Treatment Shoot length(cm)(45 DAS)

Root length(cm)(45 DAS)

Shoot weight (gplant�1)(45 DAS)

Root weight (gplant�1)(45 DAS)

Shoot length(cm)(90 DAS)



Azotobacter chroococcum(A)

T1 65.84 11.17 4.83d 0.44 136.09 56.02c 5.04

Bacillus megaterium (B) T2 69.96 11.54 5.28bcd 0.49 140.11 59.28c 5.12Pseudomonas fluorescens(P)

T3 69.91 10.89 5.13cd 0.46 129.84 54.13c 4.93

B + P T4 74.92 12.14 5.51bc 0.53 129.00 56.48c 5.79A + B T5 69.96 10.72 5.29bcd 0.47 144.77 58.74c 5.92A + P T6 74.99 11.02 5.34bcd 0.50 130.44 54.41c 5.01A + B + P T7 74.90 11.59 5.77ab 0.52 143.53 74.84b 6.74Only rhizobium T8 70.91 10.39 4.94d 0.39 144.48 53.91c 5.63Uninoculated T9 71.40 12.54 4.92d 0.53 139.22 57.12c 4.70Chemical fertilizer (100%RDF)

T10 77.20 10.94 6.01a 0.51 160.28 107.01a 7.95

Significantly different values (P < 0.05) between treatments have been marked by lower case letters.

Fig. 2. Survival of (a) Bacillus megaterium (b) Pseudomonas fluorescens and (c) Azotobacter chroococcum in the rhizosphere of Cajanus cajan over five different time points,assayed by plating samples on (i) selective media without rifampicin, and (ii) selective media with rifampicin. Significantly different values (P < 0.05) between differentsampling time points for the same treatment have been marked by lowercase letters, and significantly different values between treatments for the same time points havebeen marked by uppercase letters below the columns. Error bars represent standard deviation for n = 3. Abbreviations: B = Bacillus megaterium, Br = rifampicin resistant mutantof B. megaterium, P = Pseudomonas fluorescens, Pr = rifampicin resistant mutant of P. fluorescens, A = Azotobacter chroococcum, Ar = rifampicin resistant mutant ofA. chroococcum, Ar + Br = rifampicin resistant mutants of A. chroococcum and B. megaterium, Br + Pr = rifampicin resistant mutants of B. megaterium and P. fluorescens,Ar + Pr = rifampicin resistant mutants of A. chroococcum and P. fluorescens, C = seeds without inoculation, S = bulk soil, DAS = days after sowing.


140 141

chemical fertilizer, followed by the triple inoculation. Theinoculation effect was more pronounced at early stages of plantgrowth, which seemed to influence the productivity of the plant atthe harvest stage.

3.3.2. Enumeration of specific rhizospheric microbial groupsThe fungal population was highest at 45 DAS and decreased

thereafter with time for all treatments, except for P, A + P, A + B + Pand bulk soil (Fig. 3a). The three treatments and bulk soil had lowfungal abundance even at 45 DAS. At 45 DAS, inoculation with

Azotobacter enhanced the fungal abundance by 3.75-fold, ascompared to bulk soil and 1.55-fold compared to the uninoculatedcontrol. However, towards the harvest stage, the triple inoculationexhibited the maximum abundance of fungi compared to thecontrol (3.11-fold) and chemical fertilizer samples (2.33-fold).Similar to the dynamics of fungal populations, gram-negativeenteric bacteria generally exhibited a decline with time, A + B, B + Pand bulk soil being exceptions (Fig. 3b). At 45 DAS, the maximumabundance was observed for the control and the chemical fertilizertreatment, where the values were approximately 2.8- fold higher

Table 1Effect of bioinoculants on plant parameters of C. cajan at vegetative stage (45 DAS) and flowering stage (90 DAS).

Treatment Shoot length(cm)(45 DAS)

Root length(cm)(45 DAS)



Shoot length(cm)(90 DAS)



Azotobacter chroococcum(A)

T1 65.84 11.17 4.83d 0.44 136.09 56.02c 5.04

Bacillus megaterium (B) T2 69.96 11.54 5.28bcd 0.49 140.11 59.28c 5.12Pseudomonas fluorescens(P)

T3 69.91 10.89 5.13cd 0.46 129.84 54.13c 4.93

B + P T4 74.92 12.14 5.51bc 0.53 129.00 56.48c 5.79A + B T5 69.96 10.72 5.29bcd 0.47 144.77 58.74c 5.92A + P T6 74.99 11.02 5.34bcd 0.50 130.44 54.41c 5.01A + B + P T7 74.90 11.59 5.77ab 0.52 143.53 74.84b 6.74Only rhizobium T8 70.91 10.39 4.94d 0.39 144.48 53.91c 5.63Uninoculated T9 71.40 12.54 4.92d 0.53 139.22 57.12c 4.70Chemical fertilizer (100%RDF)

T10 77.20 10.94 6.01a 0.51 160.28 107.01a 7.95


Fig. 2. Survival of (a) Bacillus megaterium (b) Pseudomonas fluorescens and (c) Azotobacter chroococcum in the rhizosphere of Cajanus cajan over five different time points,assayed by plating samples on (i) selective media without rifampicin, and (ii) selective media with rifampicin. Significantly different values (P < 0.05) between differentsampling time points for the same treatment have been marked by lowercase letters, and significantly different values between treatments for the same time points havebeen marked by uppercase letters below the columns. Error bars represent standard deviation for n = 3. Abbreviations: B = Bacillus megaterium, Br = rifampicin resistant mutantof B. megaterium, P = Pseudomonas fluorescens, Pr = rifampicin resistant mutant of P. fluorescens, A = Azotobacter chroococcum, Ar = rifampicin resistant mutant ofA. chroococcum, Ar + Br = rifampicin resistant mutants of A. chroococcum and B. megaterium, Br + Pr = rifampicin resistant mutants of B. megaterium and P. fluorescens,Ar + Pr = rifampicin resistant mutants of A. chroococcum and P. fluorescens, C = seeds without inoculation, S = bulk soil, DAS = days after sowing.


than in bulk soil. Treatment with chemical fertilizer led to a1.33-fold enhancement in enteric bacteria compared to the mixedconsortium, indicating that the latter kept a check on this group.

The Actinomycetes population was at its maximum at 45 DAS.Subsequently, it showed a decline (at 90 DAS) and then a slightincrease at 190 DAS in case of mono-inoculations (Fig. 3c). In thetreatments with chemical fertilizer and the “no inoculation”control, there was no apparent decline with time, similar to thetrend in bulk soil. The triple inoculation exerted a positiveinfluence on the abundance of the Actinomycetes population overthe control at the vegetative stage (45 DAS), showing anenhancement of 4.2-fold. At 190 DAS, a higher abundance inActinomycetes was observed with the individual treatmentscompared to the chemical fertilizer and the controls (bulk and“no inoculation”). Pseudomonas numbers were highest at 45 DASfor all treatments, except individual treatments with Azotobacterand Pseudomonas, and the mixed consortium (Fig. 3d). The mixedconsortium exerted a positive influence on the abundance ofPseudomonas species over the control at maturity stage (190 DAS),showing an increase of 1.73-fold.

The abundance of Azotobacter species was found to besignificantly enhanced by the mixed consortium with respect tothe control only at the last time point (Fig. 3e). For phosphatesolubilizers, there was a decrease in the microbial count with timefor mono-inoculations, while for dual (except B + P) and tripleinoculations, the maximum count was observed at 90 DAS (Fig. 3f).For nitrogen fixers, significant increases (except for singleinoculation with B. megaterium and the Rhizobium treatment)were observed at 45 DAS, compared to bulk soil (Fig. 3g).

4. Discussion

Bioinoculants have been observed to compete well withchemical fertilizers and have helped in reducing the use ofchemical fertilizers (Adesemoye and Kloepper, 2009). Althoughbioinoculants have been widely promoted for increased plantgrowth and better grain yield, more information is required tojudge their risks, which include their effect not only on the cropunder study, but also on the resident microflora. Also, to optimizetheir application, an understanding of their persistence in a givenplant-soil system is of great value.

A study conducted on the same crop as investigated here, byGupta et al. (2014), had focused on the effect of bioinoculants onthe resident microflora in the plant rhizosphere, but did notaddress the question of their persistence in the rhizosphere. Also,the non-target effect was assessed on fewer groups of soilmicroorganisms in pot experiments, which limits the extrapola-tion to real-life scenario in fields. The present study was conductedto fill the knowledge gap in this area using cultivation-dependentanalysis.

Growth comparison of mutants and their parental strains isconsidered a suitable assay for competitiveness (Nautiyal, 1996,Fischer et al., 2010). In the present study in addition to growth, theecological fitness of the different mutants was compared in termsof their different plant growth promoting properties, which havealso been reported by others (Compeau et al., 1988; Carroll et al.,1995).

A study conducted by Fischer et al. (2010) documented a declinein the introduced bacterial population upon inoculation in soil.Similar results were found in our study, where the mutantpopulation (in dual inoculations) was observed to be in the order of>104 cfu g�1 dry soil for about 10 days after sowing and suffered asteady decline, reaching undetectable levels after about 60 days.Other studies have documented detection of mutants in therhizosphere for different time periods, depending on theexperimental conditions. While Bolstridge et al. (2009) and Gulatiet al. (2009) could detect the mutant population for up toapproximately four weeks, Liu and Sinclair (1992) and Stockwellet al. (1993) found mutant populations to be stable for six monthsto approximately two years. The persistence of introduced strainsappears to depend on a variety of abiotic and biotic factors.

The inoculation effect of the consortium consisting ofB. megaterium MTCC 453, P. fluorescens MTCC 9768 andA. chroococcum A-41 on C. cajan in the field was promising, as itwas on par with that of the chemical fertilizer. Comparisonbetween treatments with bioinoculants and bulk soil indicated thegeneralized response to inoculation in C. cajan. This can be relatedto the cumulative effect of N and P nutrition, phyto-stimulation byplant hormone synthesis, and protection against soil-bornediseases (Glick, 1995). An inoculation effect was conspicuous onlyat the early stages of plant growth, while at later stages, it seemedto be of lesser importance. The same is also supported by survivalstudies in the pot experiment. Our time-course analyses ofantibiotic resistant bacterial inoculants using culture-dependentmethods reflected the re-shaping of soil bacterial communities byinoculation at early stage and alteration of the rhizosphericmicrobiota at later stages, which correlated well with our fieldresult. In fact, compared to the changes observed in mediasupplemented with rifampicin, relatively lesser differences inpopulation of specific microorganisms were observed when theenumeration was done without antibiotic. Such a masking of effectcould be because the abundance of the introduced strainconstituted only a fraction of the total population of the targetedmicroorganism. The attachment of inoculants to plant roots andthe establishment of populations are the key determinants ofsuccessful inoculation (Brudman et al., 2000). High densities ofviable and competent microbes can induce a transient shift in therhizospheric microbial community. However, the presence of alarge diversity of microbial species in the soil reduces changes tothe ecosystem balance due to inoculation (McCann, 2000).

Table 2Effect of bioinoculants on plant parameters of C. cajan at harvest stage (190 DAS).

Treatment No. of branches/plants No of pods/plant Plant height (cm) Biomass/plant (g) Grain yield (kg ha�1)

Azotobacter chroococcum (A) T1 19.11d 114.00c 182.11 108.89d 1400.00cde

Bacillus megaterium (B) T2 19.89d 119.78bc 194.89 106.67de 1433.33cd

Pseudomonas fluorescens (P) T3 20.67cd 119.33bc 165.22 95.56f 1466.67c

B + P T4 22.67b 119.33bc 178.00 127.78ab 1433.33cd

A + B T5 23.11b 118.67bc 179.89 121.11bc 1733.33b

A + P T6 22.78b 127.22ab 178.78 121.11bc 1566.67bc

A + B + P T7 23.78ab 130.89a 213.00 135.56ab 1816.67ab

Only rhizobium T8 22.00bc 114.78c 171.11 94.44f 1150.00e

Uninoculated T9 19.22d 94.89d 194.78 97.78ef 1183.33de

Chemical fertilizer (100% RDF) T10 24.89a 133.00a 199.22 138.89a 2050.00a



142 143

Fig. 3. Abundance of non-target microorganisms in the rhizosphere of Cajanus cajan at three different time points. (a) Fungus, (b) gram-negative enteric bacteria, (c)Actinomycetes species, (d) Pseudomonas species, (e) Azotobacter species, (f) phosphate solubilizers and (g) nitrogen fixers. Significantly different values (P < 0.05) betweendifferent sampling time points for the same treatment have been marked by lowercase letters, and significantly different values between treatments for the same time pointshave been marked by uppercase letters below the columns. Error bars represent standard deviation for n = 4. Abbreviations: A = Azotobacter chroococcum, B = Bacillus


142 143

Fig. 3. Abundance of non-target microorganisms in the rhizosphere of Cajanus cajan at three different time points. (a) Fungus, (b) gram-negative enteric bacteria, (c)Actinomycetes species, (d) Pseudomonas species, (e) Azotobacter species, (f) phosphate solubilizers and (g) nitrogen fixers. Significantly different values (P < 0.05) betweendifferent sampling time points for the same treatment have been marked by lowercase letters, and significantly different values between treatments for the same time pointshave been marked by uppercase letters below the columns. Error bars represent standard deviation for n = 4. Abbreviations: A = Azotobacter chroococcum, B = Bacillus


Inoculations also cause changes in the profiles of carbon sourceutilization (Naiman et al., 2009). Thus, analyzing the impact ofinoculation on ecosystem functioning is crucial for manipulation ofrhizospheric microbial communities relevant to sustainable cropproduction.

Increase in fungal abundance as a consequence of individualtreatment with Azotobacter can be attributed to the positiveinteraction between Azotobacter and fungi, as shown by Azcón(1989). The gram-negative enteric bacterial population declinedafter 45 DAS. These bacteria are r-strategic, as they grow faster anddecline in later stages for the benefit of k-strategic species likegram-positive bacterial and fungal populations (Peacock et al.,2001; Marschner and Baumann, 2003; Feng and Simpson, 2009;Lazcano et al., 2012). In treatments with bioinoculants, theirabundance was low compared to treatments with chemicalfertilizer and control samples. Gram-negative enteric bacterialspecies can be plant pathogens (Heeb and Haas, 2001). Thissupports our hypothesis that bioinoculants can exert effects onnon-target organisms. In mono-inoculations, the abundance ofActinomycetes initially decreased, followed by an increase. For allthe other treatments, nugatory effects were observed on theabundance of Actinomycetes, as also shown by Gupta et al. (2013).

A healthy association of our agricultural amendments with theresident microflora resulted in a remarkable increase of soilmicrobiota, except for gram-negative enteric bacteria. Thisobservation can be explained by fixation/solubilization of certainsubstances, which in turn act as substrates for the residentmicrobial community (Gyaneshwar et al., 2002; Richardson et al.,2009). Specifically, the positive effect of bioinoculants on microbialabundance can be explained by the increased mobilization ofnutrients such as N, P, etc. (Abd-Alla et al., 2014), that possiblyserve as nutrients for the resident microflora, and thus lead to theirproliferation. Another possible mechanism could be the bioino-culants serving as biocontrol agents, inhibiting the growth ofpathogenic microorganisms, thereby indirectly promoting growthand development of the resident microbial population (Glick et al.,2007; Martínez-Viveros et al., 2010; Saharan and Nehra, 2011).

To our knowledge, this is the first report of evaluation of directand indirect effects of a combination of bacterial inoculants onC. cajan grown in the field. The field experiment was preceded byan assessment of the inoculants’ survival and persistence in potculture, using a cultivation-dependent approach.

5. Conclusions

The present study highlights the target as well as non-targeteffects of three bioinoculants (B. megaterium MTCC 453,P. fluorescens MTCC 9768 and A. chroococcum A-41) uponintroduction into the rhizosphere of C. cajan in both pot and fieldconditions. Tracking rifampicin-resistant mutants revealed a steepdecline in abundance of the bioinoculants with time. However, thebioinoculants exerted their positive effects on various plant growthparameters even at harvest stage, as observed in field experiments.These eco-friendly alternatives compared well with chemicalfertilization. Mixed triple consortium performed better than mostof the dual and individual treatments, indicating synergistic effectsof the three bioinoculants. The bioinoculants not only significantlyenhanced plant growth, but also boosted the natural rhizosphericmicrobial community in a manner that may be beneficial to theplant. These bioinoculants can therefore serve as an eco-friendlyand safe alternative to chemical fertilizers.

Acknowledgements

RS acknowledges the Council of Scientific and IndustrialResearch, India, for award of fellowship. The authors thank theDepartment of Biotechnology, Govt. of India, for funding the study(Grant No. BT/PR5499/AGR/21/355/2012). The authors wish tothank Stefan Oehler for his help with proofreading the manuscript.

References

Abd-Alla, M.H., El-Enany, A.W.E., Nafady, N.A., Khalaf, D.M., Morsy, F.M., 2014.Synergistic interaction of Rhizobium leguminosarum bv. viciae and arbuscularmycorrhizal fungi as a plant growth promoting biofertilizers for faba bean (Viciafaba L.) in alkaline soil. Microbiol. Res. 169, 49–58.

Abdul-Baki, A.A., 1974. Pitfalls in using sodium hypochlorite as a seed disinfectant in14C incorporation studies. Plant Physiol. 53, 768–771.

Adesemoye, A.O., Kloepper, J.W., 2009. Plant–microbes interactions in enhancedfertilizer-use efficiency. Appl. Microbiol. Biotechnol. 85, 1–12.

Anandaraj, B., Leema Rose Delapierre, A., 2010. Studies on influence of bioinoculants(Pseudomonas fluorescens, Rhizobium sp., Bacillus megaterium) in green gram. J.Biosci. Technol. 1, 95–99.

Aseri, G.K., Jain, N., Panwar, J., Rao, A.V., Meghwal, P.R., 2008. Biofertilizers improveplant growth, fruit yield, nutrition, metabolism and rhizosphere enzymeactivities of pomegranate (Punica granatum L.) in Indian Thar Desert. Sci. Hortic.(Amsterdam) 117, 130–135.

Azcón, R., 1989. Selective interaction between free-living rhizosphere bacteria andvesicular arbuscular mycorrhizal fungi. Soil Biol. Biochem. 21, 639–644.

Berg, G., Smalla, K., 2009. Plant species and soil type cooperatively shape thestructure and function of microbial communities in the rhizosphere. FEMSMicrobiol. Ecol. 68, 1–13.

Björklöf, K., Sen, R., Jørgensen, K.S., 2003. Maintenance and impacts of an inoculatedmer/luc-tagged Pseudomonas fluorescens on microbial communities in birchrhizospheres developed on humus and peat. Microb. Ecol. 45, 39–52.

Bolstridge, N., Card, S., Stewart, A., Jones, E.E., 2009. Use of rifampicin-resistantbacterial biocontrol strains for monitoring survival in soil and colonization ofpea seedling roots. N. Z. Plant Prot. 62, 34–40.

Brudman, S., Okon, Y., Jurkevitch, E., 2000. Surface characteristics of Azospirillumbrasilense in relation to cell aggregation and attachment to plant roots. Crit. Rev.Microbiol. 26, 91–110.

Carroll, H., Moënne-Loccoz, Y., Dowling, D.N., O’gara, F., 1995. Mutational disruptionof the biosynthesis genes coding for the antifungal metabolite 2,4-diacetylphloroglucinol does not influence the ecological fitness of Pseudomonasfluorescens F113 in the rhizosphere of sugarbeets. Appl. Environ. Microbiol. 61,3002–3007.

Compeau, G., Al-Achi, B.J., Platsouka, E., Levy, S.B., 1988. Survival of rifampin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soilsystems. Appl. Environ. Microbiol. 54, 2432–2438.

Costa, R., Götz, M., Mrotzek, N., Lottmann, J., Berg, G., Smalla, K., 2006. Effects of siteand plant species on rhizosphere community structure as revealed bymolecular analysis of microbial guilds. FEMS Microbiol. Ecol. 56, 236–249.

España, M., Cabrera-Bisbal, E., López, M., 2006. Study of nitrogen fixation by tropicallegumes in acid soil from Venezuelan Savannas using 15N. Interciencia 31, 197–201.

Feng, X., Simpson, M.J., 2009. Temperature and substrate controls on microbialphospholipid fatty acid composition during incubation of grassland soilscontrasting in organic matter quality. Soil Biol. Biochem. 41, 804–812.

Fischer, S.E., Jofré, E.C., Cordero, P.V., Gutiérrez, M.F.J., Mori, G.B., 2010. Survival ofnative Pseudomonas in soil and wheat rhizosphere and antagonist activityagainst plant pathogenic fungi. Antonie Van Leeuw. J. Microb. 97, 241–251.

Glick, B.R., 1995. The enhancement of plant growth by free-living bacteria. Can. J.Microbiol. 41, 109–117.

Glick, B.R., Todorovic, B., Czarny, J., Cheng, Z., Duan, J., McConkey, B., 2007. Promotionof plant growth by bacterial ACC deaminase. CRC. Crit. Rev. Plant Sci. 26, 227–242.

Gomez, K.A., Gomez, A.A., 1984. Statistical Procedures for Agricultural Research.John Wiley and Sons, New York.

Gulati, A., Vyas, P., Rahi, P., Kasana, R.C., 2009. Plant growth-promoting andrhizosphere-competent Acinetobacter rhizosphaerae strain BIHB 723 from thecold deserts of the Himalayas. Curr. Microbiol. 58, 371–377.

Gupta, R., Bisaria, V.S., Sharma, S., 2013. Bioinoculants: more than just plant growthpromoting agents. Endocytobiosis Cell Res. 8–11.

Gupta, R., Bisaria, V.S., Sharma, S., 2016. Response of rhizospheric bacterialcommunities of Cajanus cajan to application of bioinoculants and chemicalfertilizers: a comparative study. Eur. J. Soil Biol. 75, 107–114.

Gupta, R., Bru, D., Bisaria, V.S., Philippot, L., Sharma, S., 2012. Responses of Cajanuscajan and rhizospheric N-cycling communities to bioinoculants. Plant Soil 358,143–154.

megaterium, P = Pseudomonas fluorescens, B + P = Bacillus megaterium and Pseudomonas fluorescens A + B = Azotobacter chroococcum and Bacillus megaterium, A + P = Azotobacterchroococcum and Pseudomonas fluorescens, A + B + P = Azotobacter chroococcum, Bacillus megaterium and Pseudomonas fluorescens, Rhi: rhizobium, Chem fert = chemicalfertilizer, C = seeds without inoculation, S = bulk soil, DAS = days after sowing.


144 145

Gupta, R., Mathimaran, N., Wiemken, A., Boller, T., Bisaria, V.S., Sharma, S., 2014.Non-target effects of bioinoculants on rhizospheric microbial communities ofCajanus cajan. Appl. Soil Ecol. 76, 26–33.

Gyaneshwar, P., Kumar, G.N., Parekh, L.J., Poole, P.S., 2002. Role of soilmicroorganisms in improving P nutrition of plants. Plant Soil 245, 83–93.

Hanway, J.J., Heidel, H., 1952. Soil analysis methods as used in Iowa State College,Soil Testing Laboratory. Iowa State Coll. Bull. 57, 1–131.

Heeb, S., Haas, D., 2001. Regulatory roles of the GacS/GacA two-component systemin plant-associated and other gram-negative bacteria. Mol. Plant Microbe.Interact. 14, 1351–1363.

Kumar, H., Bajpai, V.K., Dubey, R.C., Maheshwari, D.K., Kang, S.C., 2010. Wilt diseasemanagement and enhancement of growth and yield of Cajanus cajan (L) var.Manak by bacterial combinations amended with chemical fertilizer. Crop Prot.29, 591–598.

Lazcano, C., Gómez-Brandón, M., Revilla, P., Domínguez, J., 2012. Short-term effectsof organic and inorganic fertilizers on soil microbial community structure andfunction. Biol. Fertil. Soils 49, 723–733.

Lindsay, W.L., Norvell, W.A., 1978. Development of a DTPA soil test for zinc, iron,manganese, and copper. Soil Sci. Soc. Am. J. 42, 421–428.

Liu, Z.L., Sinclair, J.B., 1992. Population dynamics of Bacillus megaterium strain B153-2-2 in the rhizosphere of soybean. Ecol. Epidemiol. 82, 1297–1301.

López-Valdez, F., Fernández-Luqueño, F., Ceballos-Ramírez, J.M., Marsch, R., Olalde-Portugal, V., Dendooven, L., 2011. A strain of Bacillus subtilis stimulatessunflower growth (Helianthus annuus L.) temporarily. Sci. Hortic. (Amsterdam)128, 499–505.

Marschner, P., Baumann, K., 2003. Changes in bacterial community structureinduced by mycorrhizal colonisation in split-root maize. Plant Soil 251, 279–289.

Martínez-Viveros, O., Orquera, M.A., Crowley, D.E., Gajardo, G., Mora, M.L., 2010.Mechanisms and practical considerations involved in plant growth promotionby rhizobacteria. J. Soil Sci. Plant Nutr. 10, 293–319.

McCann, K.H., 2000. The diversity–stability debate. Nature 405, 228–233.Naiman, A.D., Latrónico, A., García de Salamone, I.E., 2009. Inoculation of wheat

with Azospirillum brasilense and Pseudomonas fluorescens: impact on theproduction and culturable rhizosphere microflora. Eur. J. Soil Biol. 45, 44–51.

Naseby, D.C., Pascual, J.A., Lynch, J.M., 2000. Effect of biocontrol strains ofTrichoderma on plant growth, Pythium ultimum populations, soil microbialcommunities and soil enzyme activities. J. Appl. Microbiol. 88, 161–169.

Nautiyal, C.S., 1996. A method for selection and characterization of rhizosphere-competent bacteria of chickpea. Curr. Microbiol. 34, 12–17.

Niranjana, S.R., Lalitha, S., Hariprasad, P., 2009. Mass multiplication andformulations of biocontrol agents for use against fusarium wilt of pigeonpeathrough seed treatment. Int. J. Pest Manag. 55, 317–324.

Odeny, D.A., 2007. The potential of pigeonpea (Cajanus cajan (L.) Millsp.) in Africa.Nat. Resour. Forum 31, 297–305.

Olsen, S., Cole, C., Watanabe, F., Dean, L., 1954. Estimation of Available Phosphorus inSoils by Extraction With Sodium Bicarbonate. United States Department ofAgriculture Circular No. 939, 19.

Peacock, A.D., Mullen, M.D., Ringelberg, D.B., Tyler, D.D., Hedrick, D.B., Gale, P.M.,White, D.C., 2001. Soil microbial community responses to dairy manure orammonium nitrate applications. Soil Biol. Biochem. 33, 1011–1019.

Pereira, P., Nesci, A., Etcheverry, M., 2009. Impact of two bacterial biocontrol agentson bacterial and fungal culturable groups associated with the roots of field-grown maize. Lett. Appl. Microbiol. 48, 493–499.

Richardson, A.E., Barea, J.M., McNeill, A.M., Prigent-Combaret, C., 2009. Acquisitionof phosphorus and nitrogen in the rhizosphere and plant growth promotion bymicroorganisms. Plant Soil 321, 305–339.

Saharan, B.S., Nehra, V., 2011. Plant growth promoting rhizobacteria: a criticalreview. Life Sci. Med. Res. 21, 1–30.

Sarma, M.V.R.K., Sahai, V., Bisaria, V.S., 2009. Genetic algorithm based mediumoptimization for enhanced production of fluorescent pseudomonad R81 andsiderophores. Biochem. Eng. J. 47, 100–108.

Saxena, K.B., Nadarajan, N., 2010. Prospects of pigeon pea hybrids in Indianagriculture. Electron. J. Plant Breed. 1, 1107–1117.

Sharma, M., Ghosh, R., Mangala, U.N., Saxena, K.B., Pande, S., 2012. Alternariatenuissima causing Alternaria blight on pigeonpea [Cajanus cajan (L.) Millsp.] inIndia. Plant Dis. 96, 907.

Sippel, A.E., Hartmann, G.R., 1968. Mode of action of rifampicin on the RNApolymerase reaction. Biochim. Biophys. Acta 157, 218–219.

Stockwell, V.O., Moore, L.W., Loper, J.E., 1993. Fate of Agrobacterium radiobacter K84in the environment. Appl. Environ. Microbiol. 59, 2112–2120.

Subbiah, B., Asija, G.L., 1956. A rapid procedure for estimation of available nitrogenin soils. Curr. Sci. 25, 259.

Swarnalakshmi, K., Singh, M., Chaudhary, R.G., 2011. Comparison of adhesive agentson Rhizobium survival as well as nodulation and growth of urdbean under fieldconditions. J. Food Leg. 24, 332–334.

Tilak, K.V.B., Ranganayaki, N., Manoharachari, C., 2006. Synergistic effects of plant-growth promoting rhizobacteria and Rhizobium on nodulation and nitrogenfixation by pigeonpea (Cajanus cajan). Eur. J. Soil Sci. 57, 67–71.

Trabelsi, D., Mengoni, A., Ben Ammar, H., Mhamdi, R., 2011. Effect of on-fieldinoculation of Phaseolus vulgaris with rhizobia on soil bacterial communities.FEMS Microbiol. Ecol. 77, 211–222.

Turco, R.F., Moorman, T.B., Bezdicek, D.F.,1986. Effectiveness and competitiveness ofspontaneous antibiotic resistant mutants of Rhizobium leguminosarum andRhizobium japonicum. Soil Biol. Biochem. 18, 259–262.

Walkley, A., Black, I.A., 1934. An examination of Degtjareff method for determiningsoil organic matter and a proposed modification of the chromic acid titrationmethod. Soil Sci. 37, 29–37.

Winding, A., Binnerup, S.J., Pritchard, H., 2004. Non-target effects of bacterialbiological control agents suppressing root pathogenic fungi. FEMS Microbiol.Ecol. 47, 129–141.

Zhang, H.-H., Tang, M., Chen, H., Zheng, C.-L., 2010. Effects of inoculation withectomycorrhizal fungi on microbial biomass and bacterial functional diversityin the rhizosphere of Pinus tabulaeformis seedlings. Eur. J. Soil Biol. 46, 55–61.


yamamoto

Table of Contents

yamamoto

Documents

Survival, efﬁcacy and rhizospheric effects of bacterial