Molecular characterization and the effect of salinity on cyanobacterial diversity in the rice fields of Eastern Uttar Pradesh, India

Abstract

Background: Salinity is known to affect almost half of the world's irrigated lands, especially rice fields. Furthermore, cyanobacteria, one of the critical inhabitants of rice fields have been characterized at molecular level from many different geographical locations. This study, for the first time, has examined the molecular diversity of cyanobacteria inhabiting Indian rice fields which experience various levels of salinity.

Results: Ten physicochemical parameters were analyzed for samples collected from twenty experimental sites. Electrical conductivity data were used to classify the soils and to investigate relationship between soil salinity and cyanobacterial diversity. The cyanobacterial communities were analyzed using semi-nested 16S rRNA gene PCR and denaturing gradient gel electrophoresis. Out of 51 DGGE bands selected for sequencing only 31 which showed difference in sequences were subjected to further analysis. BLAST analysis revealed highest similarity for twenty nine of the sequences with cyanobacteria, and the other two to plant plastids. Clusters obtained based on morphological and molecular attributes of cyanobacteria were correlated to soil salinity. Among six different clades, clades 1, 2, 4 and 6 contained cyanobacteria inhabiting normal or low saline (having EC < 4.0 ds m(-1)) to (high) saline soils (having EC > 4.0 ds m(-1)), however, clade 5 represented the cyanobacteria inhabiting only saline soils. Whilst, clade 3 contained cyanobacteria from normal soils. The presence of DGGE band corresponding to Aulosira strains were present in large number of soil indicating its wide distribution over a range of salinities, as were Nostoc, Anabaena, and Hapalosiphon although to a lesser extent in the sites studied.

Conclusion: Low salinity favored the presence of heterocystous cyanobacteria, while very high salinity mainly supported the growth of non-heterocystous genera. High nitrogen content in the low salt soils is proposed to be a result of reduced ammonia volatilization compared to the high salt soils. Although many environmental factors could potentially determine the microbial community present in these multidimensional ecosystems, changes in the diversity of cyanobacteria in rice fields was correlated to salinity.

Figure 1

Statistical analysis of the data of soil samples. (A) The principal component analysis of the physicochemical properties of soil, and (B) the regression analysis between Na⁺ concentration and electrical conductivity showing distribution of experimental sites across the regression line.

Figure 2

Regression analysis between number of cyanobacteria (DGGE bands) and (A) electrical conductivity, (B) total nitrogen. Analyses depict the effects of these parameters on cyanobacterial abundance in selected rice fields.

Figure 3

Community of cyanobacteria collected from different rice fields as seen in microscope (resolution 40×). Some of the cyanobacterial genera that constituted the community were Anabaena (A, C and F), Aulosira (B), Gloeotrichia (D), Aphanothece (E), Nostoc (G and H) and Hapalosiphon (I). Bars, 10 μm.

Figure 4

DGGE band profile of the selected rice fields of (A) Azamgarh (1 – 4) and Chandauli (5 – 8), (B) Jaunpur (9 – 11), Mirzapur (12 – 14) and Sant Ravi Das Nagar (15 and 16), and (C) Varanasi (17 – 20). Numbered bands had similarity with the corresponding cyanobacteria in Table 3. Band marked "*" designate that the band present is at exact position on the gel as compared to corresponding band number. Only sections of the gels containing bands are shown. For details of the experimental sites see Table 2.

Figure 5

Neighbor joining tree showing phylogenetic relationship of the sequenced DGGE bands. Total 1000 bootstraps were performed and only more than 50% bootstrap support values are mentioned. All the phylogenetic analysis was performed using MEGA4 software. Numbers designate the clades. Values in parentheses indicate the range of soil salinities (in ds m^-1) corresponding to the clade of cyanobacteria. For details of methodology refer materials and method section.

Figure 6

Distribution of different cyanobacteria in the selected rice fields. The arrow denotes the increasing level of salinity measured in terms of electrical conductivity. The cyanobacterial genus name includes all the DGGE bands showed similarity with the corresponding organism. Sings * and ^# represent the heterocystous cyanobacteria and unknown cyanobacteria respectively.

Figure 7

Map of experimental site. The map of India showing location of Uttar Pradesh and map of Uttar Pradesh (23°52' N and 31°28' N latitude and 77°3' E and 84°39' E longitude) showing the experimental sites in six districts (the location of each district is given in the material and methods section).