Diversity and Population Dynamics of Rhizospheric Beneficial Bacteria Associated with Maize (Zea mays)

Abstract

In the present study 32 bacterial isolates were obtained from the rhizosphere of maize and identified on the basis of 16S rRNA sequence analysis. These isolates belonged to genera Achromobacter, Azospirillum, Bacillus, Bosea, Brevibacillus, Enterobacter, Microbacterium, Pseudomonas, Rhodococcus, Stenotrophomonas and Xanthomonas. Major growth promoting mechanisms IAA production, P-solubilization and nitrogen fixation ability were studied in these bacterial strains. Sixteen bacterial strains produced IAA in the medium supplemented with tryptophan as precursor of biosynthesis. Out of these pure bacterial cultures, eight belonging to Achromobacter, Agrobacterium, Bacillus, Enterobacter, Rhodococcus and Stenotrophomonas genera showed no IAA production in the tryptophan-free medium. Azospirillum strains were the best IAA producers (20 mg IAA L-1 ) among the tested strains. Phosphate solubilization was observed in 9 bacterial strains. Among the tested strains bacilli were the most effective P-solubilizers (127 mg phosphate L-1 ) while the activity was not detected in any of the tested Azospirillum, Pseudomonas and Stenotrophomonas strains. nifH gene was amplified from all the bacterial strains belonging to genus Azospirillum confirming the isolates as nitrogen fixing strains. The bacterial strains were tested as inocula in the non-sterilized sand under controlled conditions as well as in earthen pots, micro-plots and in the field under natural conditions. All the bacterial strains promoted the growth of maize under controlled environment. The plants inoculated with Azospirillum, Bacillus and Enterobacter showed more root length, plant height and dry weight than the non-inoculated plants and the plants inoculated with the strains of Achromobacter, Pseudomonas, Rhodococcus and Stenotrophomonas. In an experiment conducted in the growth room, cotyledons were detached from the roots four days after germination to deprive the seedlings of internal nutrient source and to make them dependent on the inoculants for nutrient supply. The data of these experiments showed clear differentiation among the inoculated treatments compared with plants with intact-cotyledons. The results of a pot experiment conducted using the same strains as used in cotyledon detachment experiment showed that the sequence of effectiveness (Azospirillum ZN1> Azospirillum strain M7> Azospirillum strain M1> Achromobacter strain M25> Rhodococcus strain M28) as inoculum was comparable to that observed in growth xiii room experiment in which cotyledons were detached. Bacterial population recorded at flowering stage indicated that maximum bacteria (2.8 X 107 cfu/g dry soil) were present in the rhizosphere of plants inoculated with Azospirillum strain ZN1. In second pot experiment bacterial strains Azospirillum ZN1 and Pseudomonas KY1 were used to inoculate the plants to study effect of bacterial inoculants with reduced level (20 % of recommended) of N fertilizer applied as well as without N application. The plants inoculated with Azospirillum strain ZN1 exhibited more plant dry weight than Pseudomonas strain KY1 inoculated plants and the respective non-inoculated control in both N treatments. In third pot experiment, Bacillus strains MP5 and MP8, Enterobacter strains M18 and MP7, Microbacterium strain MP4, Pseudomonas strains M9 and M14 and Stenotrophomonas strain M27 were used as inoculum to evaluate the effect on the hybrid (FSH-810) and the Composite varieties of maize. In both maize varieties inoculation with Bacillus strain MP5 resulted in more plant dry weight and grain yield compared with respective non-inoculated treatments. In Composite variety increase in plant dry weight and grain yield was 19 % and 17 % over control and for hybrid maize plants inoculated with Bacillus strain MP5 increase recorded for plant dry weight and grain weight was 18.6 % and 19 %, respectively. Bacterial population in both the Composite and the hybrid varieties were in the range of 107 CFU/g dry soil at early growth stage and at harvesting stage. Maximum bacterial population was recorded at flowering stage (2.3X108 CFU/g dry soil) in plants inoculated with Bacillus strain MP5. In micro-plot experiments both hybrid (FSH-810) and Composite varieties were used with different plant populations to test the bacterial strains as single strain inocula. In first micro-plot experiment hybrid variety (FSH-810) was used with high (PXP 8 cm) and low (PXP 16 cm) plant populations and treated with Azospirillum strains M1, M7, ZN1 and Pseudomonas strain M14. Plots with high plant density revealed significant increase in biomass (16 %) and in grain yield (13 %) where Azospirillum strain M7 was used as inoculum compared with non-inoculated control plants. In low plant population treatments, plant biomass (20.5 %) and grain weight (22.5 %) was more in plants inoculated with Azospirillum strain ZN1 compared with non-inoculated control. Bacterial population was recorded at early, flowering and maturity stages of plant. More bacterial population was observed in the plots with high plant density compared to low plant density treatments. In this study maximum bacterial population (2.2X108 / g dry soil) xiv was recorded at flowering stage of the high plant population treatment where Azospirillum strain ZN1 was used as inoculum. In the second micro-plot experiment Azospirillum strains M1, M7, ZN1, Bacillus strain MP5, Enterobacter strain M18 and consortium of Azospirillum strain ZN1 and Bacillus strain MP5 were inoculated to Composite variety with low and high plant density. In both high and low plant density treatments inoculated with bacterial consortium Azospirillum strain ZN1 with Bacillus strain MP5 resulted in increased dry weight of plants. In plots with high plant density, inoculation with Azospirillum strain ZN1 with Bacillus strain MP5 resulted in more plant dry weight (17 %) than non-inoculated control plants. Population dynamics of bacteria was high in case of high plant density treatments than low plant density plots. In high plant population treatments, bacterial population was maximum (6.8X 107 CFU/g dry soil) at flowering stage. Selected bacterial strains were used under field conditions to evaluate the inoculation effect on Hybrid and Composite varieties grown with low and high plant populations. Bacterial strains belonging to genera Achromobacter, Azospirillum, Bacillus, Pseudomonas, Rhodococcus and Stenotrophomonas were used as inoculant. The data on hybrid maize variety FSH-810 indicated that Azospirillum strain ZN1 increased the fresh weight (9.8 %), dry weight (12.8 %) and grain yield (13.5 %) compared to non-inoculated control. Maximum bacterial population (2.6X106 ) was calculated at flowering stage of plants inoculated with Azospirillum strain ZN1. In second field experiment the strains were tested on the hybrid (FSH-810) and the Composite varieties of maize. In hybrid variety (FSH 810) maximum increase in plant fresh weight (14 %) and dry weight (15 %) over non inoculated control was calculated in the plots inoculated with Azospirillum strain M7. In Composite variety maximum increase (20.3 %) in fresh weight and 21 % increase in dry weight was recorded in plots inoculated with Azospirillum strain ZN1. Population dynamics of bacteria was estimated and maximum bacterial population 7.7 CFU/g dry soil (log value) was present at flowering stage of the maize variety Composite inoculated with Azospirillum strain ZN1 and the log value of CFU/g dry soil was 7.6 in case of hybrid maize variety FSH-810. In third field experiment different plant densities of hybrid maize variety FSH-810 were maintained. Achromobacter strain M25, Azospirillum strain ZN1, Bacillus strain MP5, Enterobacter strain M18 and Microbacterium strain MP4 were used as inoculum. Maximum increase (19 %) in plant biomass was observed in plots treated with xv Bacillus strain MP5 while 18 % increase in grain yield was recorded where Azospirillum strain ZN1 was used as inoculum in high plant density plots. In case of low plant density treatments, 17.6 % increase in biomass and 19 % increase in grain yield over non-inoculated plants was recorded where Bacillus strain MP5 was applied. Data of bacterial population revealed that more colony forming units were present in the rhizosphere of Composite variety as compared to Hybrid variety. Plant population was maximum i.e., 3X107 at flowering stage in plants inoculated with Azospirillum strain ZN1. Culture-independent technique was employed to study bacterial diversity in the rhizosphere soil samples of non-inoculated plants grown with recommended plant population (i.e., low plant population) from the field experiment on Hybrid FSH-810. Two clone libraries of 16S rRNA gene were constructed using soil DNA for PCR amplification. 16S rRNA sequences related to Firmicutes, Acidobacteria, Actinobacteria, Proteobacteria, Chloroflexales, Planctomycetes, Gemmatamonedales, Nitrospirae and uncultured bacteria were obtained from the clone libraries. The sequence data demonstrated that in clone library C1, 59 % sequences were related to non-cultured bacteria and 41 % showed similarity with the culturable bacterial fraction. Among the sequences related to culturable bacteria, sequences related to Bacillus sp., Rhodospirillales, Syntrophaceae, Kaistobacter sp. Sphingomonadales, and Streptomyces sp. were obtained that are known for plant growth promotion. The sequences also included Terribacillus sp. and Acidobacteria-related sequences but no PGPR activity has been reported in these two groups. In clone library C2, 56 % sequences were related to non-cultured bacteria and 44 % were related to culturable bacteria. The culturable fraction of bacterial clone sequences related to Janibacter sp., Nocardioides sp., Azospirillum sp., Sphingomonas sp., Sphingomonadales, Bacillus sp., Rubrobacteraceae and Nonomuraea were detected. All these genera with one or more plant beneficial traits have been reported. Sequence of some clones in this library had similarity with culturable bacteria for which no plant beneficial traits have been reported. These include Gemmata sp. Kouleothrix sp., Gemmatimonas sp. Chloroflexi and Actinobacterium. Comparing the data of clone libraries with the culturable fraction reported in the present study, Azospirillum, Achromobacter, Rhodococcus and Bacillus were present in the clone libraries but no sequence related to Pseudomonas was present. Functional gene nifH was PCR amplified from the soil DNA extracted from rhizosphere of maize. Amplified products were cloned and 40 xvi randomly selected clones were sequenced among which 18 clones revealed good quality sequence of sufficient length for comparison with databank. Among the 18 clone sequences, 7 showed similarity with the sequence of uncultured bacterial nifH while 5 clones showed sequences similarity with Azoarcus sp. The remaining clones showed maximum sequence similarity with Pseudomonas stutzeri (2 clones), Aeromonas (2 clones), Azotobacter (1 clone), Agrobacterium (1 clone), Zoogloea oryzae (1 clone) and Bacillus (1 clone). Unlike 16S rRNA sequences obtained from soil, nifH sequences in soil DNA related to those of Pseudomonas were also detected. These results indicated that diverse diazotrophs exist in the rhizosphere of maize that can be isolated and used as inoculants.