Comparative Studies of Culturable Soil Bacterial Flora and Their Characterization from bt and Non-Bt Cotton Varieties

Abstract

The current study was conducted to investigate and characterize the culturable bacterial flora in the rhizosphere of Bt and non-Bt cotton varieties (cultivars) cultivated at four locations of Multan, Punjab (Pakistan); I) Chak 1-Faiz, Lar, II) Central Cotton Research Institute (CCRI), III) Cotton Research Station (CRS) and IV) Bahauddin Zakariya University College of Textile Engineering, Multan. Rhizosphere soil samples were collected for the determination of soil texture, pH, electrical conductivity, water holding capacity, organic matter contents, total nitrogen (N), extractable phosphorus (P), extractable potassium (K) and for the isolation, identification and characterization of bacterial flora. Soils of selected locations showed some variability in textural classes. The Location-I was classified as silty clay loam owing to 35% clay content. The Locations-II and-III each had silt loam texture with clay contents of 26% and 24%, respectively. The Location-IV was sandy loam in texture with 10% clay content. The pH values of Bt cotton rhizosphere soils at Location-I ranged from 8.65 to 8.80 and those of non-Bt from 8.64 to 8.85. The pH values at Location-II were noted from 8.50 to 8.85 in Bt while in non-Bt samples, pH ranged from 8.38 to 9.00. Similarly, the rhizosphere soil pH values at Location-III ranged from 8.58 to 9.00 in Bt and 8.56 to 9.00 in non-Bt. The Location-IV, which had the lowest clay contents, had soil pH values in the range of 8.70 to 9.00 in Bt and from 8.72 to 9.00 in non-Bt cotton rhizospheres. Among the selected sites, pH of Location-IV was the highest while Location-II had the lowest pH values. Overall average, rhizosphere soil pH of non-Bt cotton was slightly higher than Bt cotton. A comparison of means of the locations, for rhizosphere soil pH values, revealed non-significant differences in non-Bt (F-value, 2.821; p-value, 0.064) while significant differences in Bt (F-value, 3.708; p-value, 0.028) cotton. Electrical conductivity (EC) at Location-I was 690 to 740 µS cm-1 in Bt cotton rhizosphere and 680 to 730 µS cm-1 in non-Bt rhizosphere. Similarly, the EC values, in Bt cotton rhizospheres, ranged from 770 to 825 µS cm-1 at Location-II, from 670 to 700 µS cm-1 at Location-III and from 460 to 520 µS cm-1 at Location-IV while in non-Bt cotton rhizospheres, the EC values varied from 765 to 800 µS cm-1 at Location-II, from 641 to 694 µS cm-1 at Location-III and from 435 to 500 µS cm-1 at Location-IV. Among different locations, EC of Location-II had the highest and that of Location-IV had the viii lowest values. A comparison showed that Bt cotton rhizospheres had higher average EC values than non-Bt rhizospheres in three out of the four locations tested. Mean EC values of the locations differed significantly (p<0.05) in both Bt (F-value, 328.757; p-value, 0.000) and non-Bt (F-value, 347.196; p-value, 0.000) cotton rhizospheres. The water holding capacity (WHC) of Bt and non-Bt cotton rhizospheres at Location-I ranged from 42% to 46% and from 42% to 44%, respectively. Similarly, WHC remained at 39% to 42% in Bt and 37% to 39% in non-Bt cotton at Location-II, while at Location- III, WHC did not differ in Bt and non-Bt cotton rhizospheres and it remained in the range of 36% to 39%. The Location-IV showed considerably different WHC than the other locations, where the WHC ranged from 26% to 30% in Bt and 25% to 28% in non-Bt cotton rhizospheres. Among the four locations, WHC of Location-I was the highest and that of Location-IV was the lowest, owing to its sandy loam soil texture. A comparison of means of the locations revealed significant (p<0.05) differences in WHC of Bt (F-value, 197.704; p-value, 0.000) and non-Bt (F-value, 387.746; p-value, 0.000) cotton. Organic matter (OM) content at all the locations was on lower side (<1.0%). At Location-I in Bt rhizosphere OM ranged from 0.85% to 0.86% while it was 0.83% to 0.85% in non-Bt rhizosphere. Similarly, OM at Location-II varied from 0.84% to 0.85% in Bt and from 0.79% to 0.83% in non-Bt; at Location-III from 0.80% to 0.82% in Bt and from 0.78% to 0.82% in non-Bt. At Location-IV, OM was the lowest among the selected locations where it ranged from 0.59% to 0.67% in Bt and from 0.58% to 0.65% in non-Bt cotton. Overall, OM content in rhizospheres of Bt cotton was slightly higher as compared to non-Bt cotton soils at all four locations. The mean OM values of locations varied significantly (p<0.05) both in Bt (F-value, 191.2; p-value, 0.00) and non-Bt (F-value, 245.3; p-value, 0.00) cotton rhizospheres. Total nitrogen (N) at Location-I in Bt cotton rhizosphere ranged from 0.095% to 0.120% and that of non-Bt from 0.092% to 0.115%. The total N ranged from 0.098% to 0.120% in Bt and from 0.096% to 0.116% in non-Bt at Location-II; from 0.086% to 0.098% in Bt and from 0.086% to 0.094% in non-Bt at Location-III. The Location-IV had the lowest total N, where the rhizosphere soil content varied from 0.060% to 0.076% in Bt and from 0.056% to 0.073% in non-Bt cotton. Total N in Bt rhizosphere soils was ix comparatively more than that found in the non-Bt rhizosphere soil samples. A comparison of means of the locations revealed significant (p<0.05) differences in total N of Bt (F-value, 38.5; p-value, 0.00) and non-Bt (F-value, 45.7; p-value, 0.00) cotton rhizosphere soils. Extractable phosphorus (P) at Location-I in Bt cotton rhizosphere was measured to be in the range 14.5 to 16 mg kg-1 soil while it was 13.6 to 15.8 mg kg-1 soil in non-Bt cotton. The extractable-P ranged from 14.0 to 16.2 mg kg-1 soil in Bt rhizosphere and 13.6 to 15.6 mg kg-1 in non-Bt rhizosphere at Location-II; from 12.5 to 14.5 mg kg-1 soil in Bt and from 12 to 14 mg kg-1 soil in non-Bt at Location-III. The Location-IV had the lowest extractable-P levels in the soil where values ranged from 7.20 to 8.23 mg kg-1 in Bt cotton and from 7.08 to 8.32 mg kg-1 in non-Bt cotton rhizosphere. The extractable-P in rhizosphere soil of Bt cotton was comparatively higher as compared to non-Bt cotton rhizosphere soils. The extractable-P of Location-IV was the lowest among the locations owing to sandy loam soil texture of the site. A comparison of the mean values of locations revealed that extractable-P of the soils differed significantly (p<0.05) both in Bt (F-value, 236.5; p-value, 0.00) and non-Bt (F-value, 191.6; p-value, 0.00) cotton rhizospheres. Extractable soil potassium (K) at Location-I in Bt cotton was found in the range of 112 to 124 mg kg-1 and in non-Bt rhizosphere it was measured to be from 110 to 118 mg kg-1 . The extractable-K was observed from 126 to 136 mg kg-1 in Bt and from 124 to 134 mg kg-1 soil in non-Bt cotton rhizospheres at Location-II; from 104 to 116 mg kg-1 soil in Bt and from 105 to 113 mg kg-1 soil in non-Bt at Location-III. The Location-IV had the lowest extractable-K values which ranged from 38 to 48 mg kg-1 in Bt and from 39 to 46 mg kg-1 soil in non-Bt rhizospheres. The Bt cotton rhizospheres, on overall average basis, had the highest extractable-K values as compared to non-Bt cotton rhizospheres. A comparison of the locations revealed that mean values of extractable-K differed significantly (p<0.305) in Bt (F-value, 630.2; p-value, 0.00) and non-Bt (F-value, 841.9; p-value, 0.00) cotton rhizospheres. In the rhizospheres of both Bt and non-Bt cotton, pH had negative Pearson’s correlations with EC, WHC, OM, TN, EK, EP, clay contents of soil and colony forming unit (CFU) of bacteria. EC had positive relationship with WHC, OM, TN, EK, EP, clay x and CFU. The WHC had positive correlations with OM, TN, EK, EP, clay and CFU. OM had positive relationship with TN, EK, EP, clay and CFU. Similarly, TN showed positive relationship with EK, EP, clay and CFU and EK had positive relationship with EP, clay and CFU. EP had positive correlation with clay and CFU and clay also showed positive relationship with CFU. For isolation of bacteria, the rhizosphere samples were processed within 24 hours after sampling under sterile conditions. Bacterial isolates were identified by detailed morphological and biochemical methods. Further, for the confirmation of identification of bacterial strains, 16S rRNA gene sequence analysis was carried out of commonly identified bacterial isolates from four locations. The bacterial strains from Bt cotton showed 95-99% and from non-Bt cotton, they showed 92-99% similarity with their nearest relatives available on GenBank data through BLAST analysis. Fifty-one strains of sixteen different bacterial species were identified in the rhizosphere of Bt cotton i.e. Bacillus cereus (33.3%), Bacillus subtilis (23.5%), Bacillus safensis (7.84%) and Serratia marcescens (5.88%). Each of Bacillus endophyticus, Bacillus licheniformis and Bacillus pumilus were 3.92%. Bacillus toyonensis, Bacillus anthrasis, Bacillus marisflavi, Bacillus pseudomycoides, Bacillus vietnamensis, Bacillus drentensis, Bacillus stratosphericus, Lysinibacillus marcoides and Pseudomonas monteilii each of them was populated up to 1%. In the rhizosphere soil of non-Bt cotton fifteen bacterial species of forty-nine strains were isolated and identified as Bacillus cereus (38.8%), Bacillus subtilis (20.4%), Serratia marcescens (10.2%), Bacillus safensis (6.12%) and Bacillus pumilus (4.08%). Each of Bacillus licheniformis, Bacillus endophyticus, Bacillus marisflavi, Bacillus aryabhattai, Bacillus tequilensis, Bacillus pseudomycoides, Bacillus toyonensis, Bacillus flexus, Bacillus stratosphericus and Pseudomonas plecoqlossicida was populated up to 1%. In the rhizosphere of non-Bt cotton Bacillus cereus, Serratia marcescens and Bacillus pumilus were found to be in great abundance however, Bacillus subtilis, Bacillus licheniformis, Bacillus safensis and Bacillus endophyticus were identified more populated in the rhizosphere of Bt cotton. The bacterial spp. i.e. Pseudomonas monteilii, Lysinibacillus marcoides, Bacillus anthrasis, Bacillus vietnamensis and Bacillus drentensis were identified in the rhizosphere of Bt cotton and Bacillus aryabhattai, Bacillus tequilensis, Bacillus flexus and Pseudomonas xi plecoqlossicida were present in the rhizosphere of non-Bt cotton. On serial dilution, it was observed that the bacterial population in the rhizosphere soils of Bt cotton was comparatively less than non-Bt cotton in all locations that indicated that Bt toxins had negative impact on the microbial population. Maximum bacterial population was observed in rhizosphere soils of Location-I where the average OM was noted at 0.86% in Bt and 0.85% in non-Bt cotton and soil texture was silty clay loam having clay contents up to 35%. Minimum bacterial population was noted in the rhizosphere soils of Location IV, where OM was 0.62% in Bt and 0.60% in non-Bt cotton with sandy loam soil texture having clay contents up to 10%. Bacillus species exhibited to be the predominant group in the study areas. Further, the results suggested that rhizosphere of Bt and non-Bt cotton of these areas is an affluent resource of Bacillus species which have a prospective to be used as inoculants of PGP in future to improve the cotton crop.