Isolation and enumeration of bacteria
A total of 238 bacterial strains were isolated from different soils, and the highest numbers of bacteria were reported from the oak forest soils (surface and deep) followed by pine forest soil and agricultural land soil. The results of this study revealed that the maximum amount of total bacterial population, i.e., 44 % was isolated from oak forest soil. It was further analyzed that oak surface soil (OSS) contributed 24.78 %, followed by oak deep soil (ODS) that represented 20.16 % of total oak bacterial count.
On analyzing the contribution of pine forest in total bacterial count, it was found that only 29 % of total bacterial count was from pine forest soil. The data further exhibited that out of 29 %, the pine forest soil (PSS) contributed 16.80 %, followed by pine deep soil (PDS) 13.86 %. The least percentage of total bacterial count (23 %) was reported from agricultural soil ecosystems. The data showed that agricultural surface soil (ASS) contributed 15.54 % of bacterial count, whereas agricultural deep soil (ADS) contributed only 8.82 % of bacterial count.
Reports have shown that the size and structure of microbial populations are affected by soil type and plant species (Sah et al. 2016). Total bacterial count was analyzed for all six soil types and compared for significant differences among them through one way analysis of variance (ANOVA). It was found that the soil bacterial count differed significantly across all six soil types (F = 104.690, df = 5; P < 0.001). We also explored the data further via Tukey’s post hoc multiple comparisons for soil bacterial count between various soil groups (i.e., a pair by pair analysis). Tukey’s post hoc multiple comparisons revealed that there is a statistically significant difference (P < 0.05) in bacterial count when PSS is compared with other soil types, i.e., PDS, OSS, ODS, and ADS (Table 1).
There was a significant difference when PDS was compared with PSS, OSS, ODS, and ADS with a mean difference of −7.0, −15.0, −26.0, −15.0, and 12.0, respectively, while no significant difference was observed in PDS and ASS and a mean difference of −4.0 was ODS followed by OSS. The highest mean difference of 38.0 was observed between ODS and ADS bacteria count followed by PDS (26.0), ASS (22.0), PSS (19.0), and OSS (−11.0). A mean difference of −3.0 and 4.0 between ASS and PSS/PDS, respectively, and a value of P > 0.05 represents that there is no significant difference in their bacterial count. No significant difference is observed between PSS and ASS (P > 0.05, mean difference 3.00), and PDS and ASS (P > 0.05, mean difference = −4.00).
Screening for siderophore producing isolates
All the 238 isolates were investigated for siderophore production. Fifty-six isolates representing a 23.52 % of total bacterial population isolated were positive for siderophore production. Of the 56 siderophore producing isolates, 46.42 % of the isolates belong to the oak forest soil (OSS: 32.14 %; ODS: 14.28 %), 14.99 % from pine forest soil (PSS: 17.85 %; PDS: 7.14 %), and 28.56 % from Agriculture soil (ASS: 23.21 %; ADS: 5.35 %).
We also quantified the siderophore producing Pseudomonas and analyzed it statistically for all six soil types and compared for significant differences among them through one way analysis of variance (ANOVA). It was observed that the siderophore producing Pseudomonas differed significantly across all 6 soil types (F = 18.900, df = 5; P < 0.001). We explored the data further via Tukey’s post hoc multiple comparisons for siderophore producing Pseudomonas between various soil groups (i.e. a pair by pair analysis).
Tukey’s post hoc multiple comparisons for siderophore producing Pseudomonas reveals significant differences in different types of soil from Uttarakhand State of Indian Himalayan Region (IHR) (Table 2). A statistically significant difference was observed in number of siderophore producing Pseudomonas when PSS soil was compared to other soil types, e.g., PDS, OSS, and ADS (P < 0.05). A significant difference was also observed in bacterial count of PSS and oak surface soil, while no significant difference was observed between PSS and OSS (P > 0.05). When the total number of siderophore producing Pseudomonas in PDS was compared with various soil types, e.g., PSS, ODS, and ASS with a mean difference of −7.00, −15.00, −7.00, respectively, it exhibited a statistically significant difference (P < 0.05). No statistically significant difference was observed in siderophore producing Pseudomonas between PDS and ADS (P > 0.05), whereas a significant difference was observed in siderophore producing Pseudomonas count when OSS was compared with various soil types, e.g., PSS, PDS, ODS, ASS, and ADS with a mean difference of 8.00, 15.00, 11.00, 8.00, and 15.00, respectively.
Identification of presumptive Pseudomonas isolates using Pseudomonas specific primer
Of the 56 isolates, PCR amplification of 16S rDNA Pseudomonas specific region yielded a single amplicon of 990 bp for 51 isolates. Hence, these 51 isolates belong to Pseudomonas (sensu stricto) group. The five isolates which were not amplified may not be from within the Pseudomonas (sensu stricto) group.
Genetic variation among the isolates was analyzed by RFLP of PCR product with three restriction endonucleases (AluI, MspI, and RsaI). Different profiles having four to seven fragments with variation in size were observed after digestion and dendrogram was constructed by Dendro UPGMA method (Fig. 1). The unique digestion patterns observed segregated the isolates into different groups and subgroups. The representatives from various groups were sequenced for conformation.
The dendrogram represented eight different clusters and six outlying branches based on Jaccard’s similarity coefficient. Isolates showing less than 75 % similarity were considered as outlying branches. The isolates representing >75 to 99 % similarity were considered as one cluster, and isolates representing <75 to 99 % were considered as different cluster or an outlying branch. The branches of dendrogram signify that there is heterogeneity among the isolates
Cluster I included the highest number of isolates. This was split into two, split 1(a) includes four isolates and split 1(b) includes six isolates. The sequencing results revealed the identity of isolates belonging to cluster 1(a) and 1(b) as P. aeruginosa. Cluster II, the second largest cluster includes seven isolates. This cluster is split into two groups. 2(a) includes a single isolate, identified as Pseudomonas. sp., while 2(b) includes six isolates. All the isolates in cluster 2(b) are 100 % similar. Clusters I and II share 50 % similarity among them. Cluster III is split into 3(a) and 3(b). Isolates belonging to cluster 3(b) represent 100 % similarity among them. Cluster 3(a) represented by one isolate is 60 % similar to the split 3(b). Cluster III represents 55 % similarity to the outlying branch (OB1) with a single isolate. The second outlying branch (OB2) includes two isolates 20 and 30 representing 100 % similarity among them. Cluster IV includes two isolates representing 50 % similarity among them. Cluster V is split into 5(a) that includes two isolates 39 and 42 having 100 % similarity among them. Isolate 42 is identified as P. tolassi, isolate 39 representing 100 % similarity with 42 may also be P. tolassi. 5(b) includes isolate 50 identified as P. fluorescens and shares 100 % similarity with isolates 15, 47, 48, and 49. Isolates 15, 47, 48, and 49 may also be P. fluorescens. Cluster VI is split into two 6 (a) and 6 (b), having 75 % similarity among them. Cluster 6(a) includes a single isolate and cluster 6(b) includes four isolates having 100 % similarity among them. Isolate 46 forms an outlying branch (OB 3). OB3 is less than 60 % similar to Cluster VI. Cluster VII is split into two as 7(a) and 7(b). 7(a) includes a single isolate which is 70 % similar with 7(b). Isolate 35 identified as P. putida is 100 % similar with 37 and 38. Hence, it can be said that the isolates belonging to cluster 7(b) may be P. putida. Cluster VIII is split into 8(a) and 8(b) with 40 % similarity among them. Isolate belonging to 8(b) represent 100 % similarity among them. Isolate 7 forms an outlying baranch (OB4). Isolates 45 and 51 having less than 15 % similarity among them form two outlying branch OB5 and OB6.
Since the digestion site for the enzymes AluI (AG↓CT) and RsaI (GT↓AC) was different, different restriction patterns were observed. The isolates representing 100 % similarity on dendrogram may be same or with a difference in their subspecies. The sequencing results support the study that the isolates belong to different subgroups of Pseudomonas (sensu stricto). The isolates were segregated into different groups as P. aeruginosa, P. spp, P. tolassi, P. fluorescens, P. putida, and P. korensis, representing the heterogeneity among the isolates. Isolates from lesser or undisturbed lands of oak and pine forests were placed in all the clusters as the isolates from agricultural land or a disturbed land. In some of the clusters, isolates from forest and agricultural land are 100 % similar. All of the Pseudomonas isolates were further explored for their PGPR properties.
All the 51 Pseudomonas strains were investigated for the plant growth promotory (PGP) traits that include production of siderophore, P-solubilization, HCN production, and IAA production. Results indicate that all the 51 isolates were siderophore producers and IAA producers. It was found 68.62 % isolates were phosphate solubilizers and 62.74 % were HCN producers. It was observed that 50 % (23 strains) of the isolates were positive for all the PGP traits. Out of these 23 isolates 12 belonged to oak forest, 6 to pine forest and 5 to the agricultural land. These 23 isolates belong to different phylogenetic groups formed according to the RFLP results. Pseudomonas strains are beneficial and possess PGP traits and hence are explored the most. Among the more important attributes are, the ability to colonize roots successfully (colonization potential), sustain competition through release of bioactive molecules, build up large populations during the growth stage of a plant in soil (Table 3).