GROwTh PROmOTING ChaRaCTeRIsTICs Of RhIzObaCTeRIa aND am fUNGI fOR bIOmass amelIORaTION Of Zea mays

Plant growth promoting rhizobacteria (PGPR) and mycorrhiza were evaluated on the growth (biomass) and yield of Zea mays. In the present study, selective rhizospheric PGPR (Azotobacter chroococcum, Pseudomonas aeruginosa, Azospirillum brasilense and Streptomyces sp.) and a combination of six strains of arbuscular mycorrhizal fungi (AMF) (Acaulospora morrowae, Gigaspora margarita, Glomus constrictum, Glomus mossae, Glomus aggregatum and Scutellospora calospora) were isolated and identified with standard methods and 16S rRNA sequence analysis. PGPR and AMF were checked for their growth-promoting behavior under specific treatment conditions. The 30-48-day-old treated plants in all combinations showed a significantly higher mass value. The average dry weight from the shoot was in a range from 41-52% as compared to the control. This increase also translated into a higher mass value of the roots. Overall, an 82% growth rate was observed in terms of height as the consequence of biomass production, specifically in the case of AMF + rhizobacteria combination. We report an efficient, sustainable and cost-effective biofertilizer for enhanced biomass of Z. mays, one of the staple food crops worldwide.


INTRODUCTION
Maize is one of the most important crops, domesticated around the world since prehistoric times.The enormous demand for carbohydrates from maize not only stems from the ever-growing food industry, but also from poultry, pharmaceutical and textile sectors.In addition, paper, brewing and bioenergy production sectors are the potential recipients of maize feedstock production.Due to the ever-increasing world population, maize production needs to be increased significantly to fulfill the market demand (Grassini et al., 2013;Rattray, 2012).One of the contemporary ways to achieve this is seed priming with plant growth promoting rhizobacteria (Junges et al., 2013;Ashrafi and Seiedi, 2011).It has been found that rhizospherically active microorganisms carry out numerous activities important to plant growth and development.Arbuscular mycorrhizal fungi (AMF) and plant growth promoting rhizobacteria (PGPR) both play an important role in the supply of phosphorus (P) to plants.Channelization of rhizospheric P into plants through the symbiosis/interaction of plant-associated AMF and PGPR helps the continuous supply of P from the soil to the host (Bhardwaj et al., 2014).Similarly, AMF and PGPR have been found to complement each other and increase the surface absorption at nitrogen (N) level.Certain mycorrhization helper bacteria (MHB) can also found to be associated with mycorrhizal roots and mycorrhizal fungi ultimately resulting in collectively promoting the establishment of mycorrhizal symbioses (Kurth et al., 2013).These microorganisms have been found to act together, and the population dynamics of one affects the other.For example, the AMF have diverse effects on the rhizobacterial community structure in the mycorrhizosphere (Marschner and Baumann, 2003;Barea et al., 2002;Barea, 2000).For this change in the microsphere of soil, the organic compounds synthesized by mycorrhiza play a crucial role affecting soil aggregation and the supply of nutrients (Johansson et al., 2004).
Several reports that have shown that there is specific microbe-microbe interaction that modulates the effectivity of AMF on plant physiology (Cosme and Wurst, 2013).The cumulative effect of the plant-AMF-PGPR interaction has been reported earlier.
AMF and PGPR complement each other, and increase the surface absorption at N and P level.Behl et al. (2007) studied the cumulative effect of plant-AMF-PGPR interaction against avoidance of abiotic stress in wheat crops.In their study, they concluded that the dual inoculation of efficient strains of Azotobacter chroococcum and Glomus fasciculatum in responsive wheat genotypes adapted to low input stress conditions could be profitably used to maximize wheat production.Likewise, AMF, PGPR and N-fixing bacteria are used for their symbiotic growth with plants, supporting sustainable growth against heavy metals (Chaudhry andKhan, 2002, 2003).In order to make it more economical and sustainable, a standard approach to isolate and characterize PGPR from the rhizosphere of Z. mays has been developed and used to improve production (Karnwal, 2012;Marques et al., 2010).
In this report, we have characterized the effect of PGPR and AMF on Z. mays in terms of physical growth and biomass increase.Comparison of Zea mays growth rates (heights) with specific inocula after 15 to 45 days treatments.
bacteria along with AM fungal spores associated with maize plants, with the ultimate aim to isolate different inocula.

soil sampling and analysis
Rhizospheric soil samples (soil adhering to the plants) were collected at different depths along with root in five replicates from maize.The upper soil at ground level (0-5 cm) was discarded to avoid the chances of collecting foreign particles and litter.The collected soil (depth 5-10 cm) and root samples were placed in ziploc bags and transported to the laboratory where they was stored at 4 o C until further use.The soil samples were properly homogenized and sieved to remove particulate material and other impurities.Part of the pure and air-dried samples was sent for physicochemical analysis at a leading soil-testing laboratory.

Isolation and identification of amf
Rhizosphere soil samples collected from the field were sieved to obtain spores using wet sieving and a decanting technique (Gerdemann and Nicolson, 1963), and the spore population was determined.
The root samples obtained from different locations were gently washed under tap water, and processed further as described by Phillips and Hayman (1970) to calculate percentage root colonization (Giovannetti and Mosse, 1980) along with a number of vesicles.
The collected spores of AM fungi were identified following Schenck and Perez (1987) and Morton (1988).

Isolation and identification of rhizobacteria
Rhizobacteria were isolated from the rhizospheric soil of Z. mays by serial dilution and appropriately diluted culture samples were spread on specific nutrient media to screen for Streptomyces (actinomycetes isolation agar media) (Porter et al., 1960), Azotobacter (Ashbays agar media) (Rubio et al., 2000), Pseudomonas (King's B media) (Jayaswal et al., 1990) and Azospirillum (Rojo Congo media) (Doroshenko et al., 2007).Identification, biochemical and molecular characterization of rhizobacteria were carried out by using various standard biochemical and taxonomic parameters.
Biochemical tests performed in the present study were: gram staining, methyl red, the Voges-Proskauer (VP) test, citrate test, starch hydrolysis, urease test, triple sugar iron agar test and casein hydrolysis.

Characterization of plant growth promoting characteristics of rhizobacteria
After identification, the rhizobacteria were tested for their plant growth promoting traits such as phosphate dissolution, siderophore production, ammonia and indole acetic acid production.To determine phosphate solubilization ability, isolates were sprayed over Pikovskaya's broth following the method described by Gaur (1990).The formation of fluorescence pigments indicated the presence of siderophore production (Yasmin et al., 2009;Da Silva and de Almeida, 2006).Ammonia and indole acetic acid (IAA) production was studied following standard protocols.The devel- opment of brown to yellow color was positive for ammonia production (Cappuccino and Sherman, 2005).

Compatibility among rhizobacterial strains
The rhizobacterial isolates were tested for compatibility (adapted from Fukui et al. 1994).The compatibility of isolates was determined on nutrient agar by streaking them parallel to each other, followed by incubation at 28+2°C for 72 h, and observed for the presence/absence of inhibition zone.

molecular characterization
The genomic DNA was isolated from bacterial samples using standard molecular techniques (Sambrook and Russel, 2001).The universal primers 27F and 1492R specific for 16S rDNA were used to perform PCR using bacterial genomic DNA as a template.A single discrete band of ~1500 bp was observed when PCR products were resolved on 1% agarose gel.The DNA was eluted and purified from gel using a gel purification column and sequenced using 27F (AGAGTTTGATC-MTGGCTCAG) and 1492R (GGTTACCTTGTTAC-GACTT) primers.The 16S rDNA gene sequence was used to conduct BLAST against the NCBI database.
Based on the maximum identity score the bacterial strain was identified using the multiple alignment software program Clustal W. A distance matrix was generated using the ribosomal database project (RDP) and the phylogenetic tree was constructed using MeGA 4.

microbial treatment preparation
Different combinations of rhizobacteria and mycorrhiza were taken into consideration in assessing their potential on growth and biomass development of Z. mays compared to a control.They were formulated as a combination of rhizobacteria: Pseudomonas + AMF; Azospirillum + AMF; Azotobacter + AMF; Streptomyces + AMF; All rhizobacteria + AMF and AMF alone, respectively.

Growth assay of maize
The experiment for studying the effect of different PGPR along with AMF on the growth of Z. mays was conducted in pots (25 cm in diameter) containing 300 g of autoclaved loamy soil.Physicochemical properties, except pH, organic carbon and eC of the soil were analyzed by ICP-AeS technique.The analytical values were pH 7.9, organic carbon 21 g kg -1 , eC 1.13 dS m -1 , available N 132 kg ha -1 , available P 9.4 kg ha -1 , available K 164 mg kg ha -1 and available zn 0.44 mg kg ha -1 .Seeds of Z. mays were surfacesterilized with a mixture of ethanol and 30% H 2 O 2 (1:1).Three pots as replicates were used for each treatment.Five surface-sterilized seeds were sown in each pot at a 1.0-cm depth.For inoculation, all rhizobacteria were maintained in the exponential phase and collected by centrifugation at 12000 rpm for 10 min, washed with sterile distilled water, and recentrifuged.Bacterial inoculum was prepared by resuspending sediment cells in sterile distilled water to get an inoculum density of ca. 10 8 CFU ml -1 .Bacterial suspensions (10 ml pot -1 ) were sprayed on the soil surface one week after seedling emergence.For mycorrhizal inocula, twenty grams of soil containing chlamydospores and roots of S. bicolor was used in each pot containing sterilized soil.Non-inoculated plants were taken as a control.The pots were placed outdoors.During cultivation, minimal and maximal temperature ranged from 13 to 17 o C and from 22 to 32 o C, respectively.The soil was moistened with water and maintained at 60% of its holding capacity.Growth of the plants in the respective pots was recorded and a log register was maintained.

biomass measurement
Thirty-to 48-day-old treated and controlled plants were cut into different segments (root and shoot) for biomass measurement.The dry weight of the plant samples was measured after oven drying at 72°C for 48 h (Isaac and Johnson, 1985).

statistical analysis
The data on plant height, root dry weight and shoot dry weight were subjected to one-way analysis of variance (ANOVA) to evaluate the differences in means

ResUlTs aND DIsCUssION
The physicochemical characteristics of soil samples collected from rhizosphere of Z. mays at different areas in the Jalandhar region are listed in Table 1.All the rhizosphere soil samples were found to be slightly alkaline in pH (7.9-8.2).The organic carbon and moisture of these soils ranged between 0.79-0.92%and 37.2-47.2%,respectively.The concentrations of N, P and K ranged from 17.1-19.1 kg/hectare, 12.9-14.5kg/hectare and 133.6-148.1 kg/hectare, respectively.elements such as copper, zinc, manganese and iron were present in ranges of 1.69-2.88,7.10-9.06,4.01-5.65 and 7.10-9.06ppm of soil when subjected to ICP-AeS analysis, respectively.
Selected rhizobacteria were all gram negative except Streptomyces.Azotobacter, Streptomyces and Pseudomonas showed positive results for starch hydrolysis, indicating production of amylase, whereas all isolates showed catalase activity with nitrate reducing ability.Azotobacter, Streptomyces and Azospirillum were identified as urease-producing rhizobacteria.Pseudomonas, Azospirillum and Streptomyces were identified as proteolytic bacteria.Azotobacter, Azospirillum and Pseudomonas were found to be oxidase positive, indicating the presence of cytochrome c oxidase and use oxygen for energy production in an electron transfer chain.All isolates were methyl red negative, while Azotobacter and Azospirillum were VP test positive, confirming the production of non-acidic products.
The phosphate solubilization activity of isolated rhizobacteria was marked by the presence of a halo zone around the inoculated area.All selected rhizobacteria showed positive results in a range of 1.07-1.95cm of the calcium phosphate zone.The production of siderophore was noticed in the form of distinct yellow-green fluorescent pigment produced by the bacteria.

C o n t r o l M y c o r r h iz a A ll r h iz o b a c t e r ia M y c o r r h iz a + A ll r h iz o b a c t e r ia P s e u d o m o n a s + M y c o r r h iz a A z o s p ir illu m + M y c o r r h iz a A z o t o b a c t e r + M y c o r r h iz a A c t in o m y c e t e s + M y c o r r h iz a
Fig. 3.

fig. 3.
Rate of biomass production (root dry weight) in response to specific inocula and Zea mays interaction for 32-48 days.
Three of the rhizobacterial isolates, Azotobacter chroococcum, Pseudomonas aeruginosa and Azospirillum brasilense, were able to produce siderophores.Fluorescent pigment resulted due to low Fe in the medium and a high affinity for chelating Fe 3+ .The production of ammonia indirectly influences plant growth and is considered an important trait of PGPR.
Ammonia production was detected in all isolates.Rhizobacterial isolates were further screened for their ability to produce IAA.In the presence of L-tryptophan, P. aeruginosa produced significantly higher concentrations of IAA compared to Azotobacter chroococcum, Azospirillum brasilense and Streptomyces sp. in the order of 3.6, 3.0, 2.3 and 1.9 mg/l, respectively.The biochemical results of the various isolates are summarized in Table 2.
For reliable identification of rhizobacterial isolates, molecular characterization was done by amplifying the 16S rRNA gene sequence.The sequences of 16S rRNA were subjected to BLAST analysis, and we found that the partial sequences of the isolated strains belong to the genera Pseudomonas, Streptomyces, Azotobacter and Azospirillum, respectively (Table 3).
To explore the positive interaction among the selected rhizobacteria, the compatibility test was per-formed in triplicate.All selected rhizobacteria showed positive interactions and promoted growth of other associations, confirms the formation of a healthy rhizobacterial consortium which was used for inoculation.
An extensive field investigation was carried out to evaluate the interaction and colonization of AMF species present in the rhizosphere of Z. mays and to study the effects of edaphic factors on AMF populations in the rhizosphere (Table 4).Rhizosphere soil samples collected from various localities revealed the presence of different AM species, namely Acaulospora morrowae, Gigaspora margarita, Glomus constrictum, Glomus mossae, Glomus aggregatum and Scutellospora calospora.
During this investigation, rhizobacteria and AMF were explored from the root zone of Z. mays plants from cultivated as well as uncultivated sites.AMF association was totally absent in plants collected from cultivated land, while Z. mays plants collected from uncultivated areas showed a significant presence of AMF and a great variety of rhizobacteria.This might be due to an excessive use of chemical fertilizers in the cultivated soil by farmers that can retarded the proliferation of mycorrhiza and rhizobacteria (Lopez-Arredondo et al., 2014).The ICP-AeS analysis for nutrient and metal presence in the soil samples of agri- cultural and uncultivated areas revealed nearly equal amounts of all the metals.Thus, the rhizobacteria and AMF isolated from the uncultivated soil can be used for the experimental inocula for Z. mays plants.
Different isolates were studied to determine their effect on seed germination.The maize seed inoculated with rhizobacteria noticeably improved the germination process and seedling vigor, but the pace of development showed a variable pattern with the treatment of AMF and rhizobacterial strains.All AMF + rhizobacteria combinations, except Streptomyces + AMF, increased the biomass and productivity up to twofold as compared to the control (Fig. 1).The highest growth rate was obtained in AMF and all rhizobacteria and P. aeruginosa + AMF, which recorded 115% and 57%, respectively, as compared to the control, followed by Azotobacter chroococcum + AMF; Azospirillum brasilense + AMF; Streptomyces + AMF and AMF at 54%; 47%; 42% and 26%, respectively.The interaction of PGPR was considered in terms of a healthy response.A revitalization step was not required during experimentation with PGPR which was maintained for 48 days during one treatment.
The presence of inocula noticeably influenced seed germination.A comparative study of growth rate in combination and stand-alone are shown in Supplement Fig. 1.Different age groups of plants have shown a progressive growth rate that has been evidenced by the higher biomass production rate as compared to the control.Series of combinations (AMF, rhizobacteria and AMF + rhizobacteria) were taken into consideration to measure the mean value of growth rate and biomass interrelationship between 30 to 48 days (Figs. 2 and 3).30-to 48-day-old treated plants in all combinations showed significant increase in mass value, whereas the average dry weight of shoots was in a range of 670.7-1419.1 g as compared to the control, which was in the range of 345.7-590.4g.Similarly, the root was also found to be higher in mass value as the tested biomass production value of root was in the range of 611.5-991.5 g, while the control plant root was measured as 286.3-490 g.Overall, an 82% growth rate was observed in height as the consequence of biomass production specifically in case of the AMF + rhizobacteria combination.The involvement of microbial inocula in the native condition was noted as per the report of soil characteristics.Growth efficacy was measured by the growth rate, which is directly proportional to the biomass production (dry weight measurement).The phenotypic behavior of the tested plants was normal; however, the length and an overall growth rate enhanced significantly as compared to control conditions.
The interaction between AMF, PGPR and plant results in increase of growth in maize.PGPR seems to enhance the benefits of mycorrhizal effect.The most evident effects were observed in the presence of all rhizobacteria where the activities of PGPR were dependent on their associations with other rhizosphere organisms, especially the commonly found AMF (Hrynkiewicz and Baum, 2012;Chiquito-Contreras et al., 2012).earlier reports have discussed the symbiotic associations of plants with AMF, which is also influenced by a number of rhizosphere organisms, including PGPR (McNear, 2013;Khan, 2006).Dual inoculation with both soil microorganisms (rhizobacteria and AMF) also induced higher biomass yield and even higher nutrient uptake (Mader et al., 2011;Adesemoye and Kloepper, 2009).
The results reported in this work are concomitant to earlier reports.The beneficial interactions observed in our results may be due to physicochemical parameters that are regulated by AMF and PGPR that may affect plant growth, as confirmed by Richardson et al. (2009).PGPR secrete some phytohormones that promote not only AMF colonization, but also increase the root surface area and susceptibility of a plant through their hyphal penetration.experiments have been designed to visualize the individual response of AMF and its combination with rhizobacteria (Martinez-Viveros et al., 2010;Yang et al., 2009;Barea et al., 2009;Kohler et al., 2007).A higher vegetative growth (biomass) was registered in plants inoculated with a combination of AMF and rhizobacteria.earlier reports also elucidated similar behavior of rhizosphere microbial interaction with different hosts and their native conditions (Berg and Smalla, 2009;Morgan et al., 2005;Wu et al., 2005).This may be due to the intrinsic property of AMF to act as a bridge between phosphate solubilization by PGPR and the host plant (Vafadar et al., 2014).Positive augmentation and better biomass may also be amplified due to certain specific properties of PGPR such as their phosphate solubilizing capability (Guinazu et al., 2010;Barea et al., 2005) or their roles in aggregate stabilization.AMF also provides habitable pore space for bacteria (Arora et al., 2011), including PGPR, thereby enhancing the growth promoting characteristics of PGPR.In combination, AMF and PGPR modify soil nutrient concentration ratios and nutrient mobility to facilitate nutrient retention in the plant tissue (Singh et al., 2011;Jeffries et al., 2003).Many reports (Nihorimbere et al., 2011;Roesti et al., 2006;Khan, 2005) on the influence of microbial communities in the rhizospheric zone and the progressive nature of controlling the growth and development of host plants also confirm our experimental design.
In conclusion, both AMF and PGPR could be used to promote the desired growth of plants.The rhizobacteria and mycorrhiza studied here could also be utilized commercially as biofertilizers.The optimized pot conditions, specific inocula and host compatibility improve our understanding of the interactive behavior.
Fig. 2.fig.2.Rate of biomass production (shoot dry weight) in response to specific inocula and Zea mays interaction for 32-48 days.

Table 1 .
Physical characteristics and micronutrient status of the rhizosphere soil at various localities of Jalandhar, India.

Table 2 .
Biochemical and PGP traits of rhizobacteria performed for identification of different rhizobacteria collected from Zea mays rhizosphere. s.

Table 3 .
Blast analysis of rhizobacterial strains collected from rhizosphere of Zea mays.

Table 4 .
Different attributes of AM fungal colonization in the rhizosphere soil collected from Zea mays