Fuctional and Genomic Diversity of Agriculturaly Important Bacillus from Rhizosphere of Wheat and Maize

Abstract

Rhizosphere of plant is the zone which is directly under the impact of plant roots. The application of microorganisms in the field of agriculture for improving the availability of nutrients has become an important practice nowadays. Different bacterial strains that present in rhizosphere play significant role in crop improvements. These are known as plant growth promoting rhizobacteria (PGPR). These bacteria strains belongs to genus Bacillus, Pseudomonas, Ochrobacterum, Azotobacter, Rhizobium, Stenotrophomonas, Serratia, Azosperillum and Enterobacteria etc. It has already reported by many scientists that genus Bacillus is dominant in the rhizospheric zone because of having external protection of hard cell wall, firm cell membrane, internal system of enzymes and endospore formation, which guards it against physical and chemical risks and having plant growth promoting characteristics. Present study deals with 64 Bacillus strains isolated from different areas of Punjab, Pakistan as 32 strains from Corn rhizosphere and 32 Bacillus strains from Wheat rhizosphere. These strains are identified upto species level by 16S rRNA sequencing. The Corn rhizospheric strains are identified as Bacillus sonorensis-CSH27, Bacillus sp.-CSH23, Bacillus thuringiensis-Cu47, Bacillus axarquiensis-CF18, Bacillus safensis-CU35, Bacillus mojavensis-CU4, Bacillus safensis-CN17, Bacillus subtilis-CN2, Bacillus endophyticus CU33, Bacillus cereus-CMS17, Bacillus pumilus-CU9, Bacillus cereus-CMS7, Bacillus pumilus-CSH4, Bacillus toyonensis-Cu48, Bacillus safensis-CF14, Bacillus tequilensis-CN7, Bacillus anthracis-CMS14, Bacillus cereus-CSH2, Bacillus anthracis-CL13, Bacillus safensis-CF4, Bacillus mycoides-CL2, Bacillus thuringiensis-CMS20, Bacillus pseudomycoides-CN9, Bacillus anthracis-CN13, Bacillus sonorensis-CSH1, Bacillus anthracis-CSH26, Bacillus anthracis-CU7, Bacillus mojavensis-CU14, Bacillus cereus CU46, Bacillus subtilis-CU31, Bacillus anthracis-CL11, Bacillus cereus-CN12. Similarly 32 Bacillus strains isolated from Wheat rhizosphere and identified as Bacillus subtilis-WRY23, Bacillus pumilus-WM3, Bacillus thuringiensis-WSK9, Bacillus thuringiensis-WG5, Bacillus anthracis-WFS6, Bacillus axarquiensis-WSH8, Bacillus anthracis-WUS2, Bacillus thuringiensis-WG1, Bacillus sp.-WUS16, Bacillus sp.-WSH5, Bacillus anthracis-WRY1, Bacillus subtilis-WRY11, Bacillus cereus-WFS20, Bacillus subtilis-WL22, Bacillus anthracis-WSH13, Bacillus mojavensis-WSK11, Bacillus pseudomycoides-WSK6, Bacillus flexus-WSH3, Bacillus sonorensis-WG3, Bacillus flexus-WG14, Bacillus cereus-WM13, Bacillus megaterium-WSH10, Bacillus cereus-WSK13, Bacillus thuringiensis-WSK16, Bacillus vallismortis-WUS4, Bacillus anthracis-WM14, Bacillus thuringiensis-WG20, Bacillus pumilus-WM2, Bacillus velezensis-WG4, Bacillus amyloliquefaciens-WG8, Bacillus vallismortis-WG21, Bacillus safensis-WM20. Out of all 32 Bacillus isolates from Corn rhizosphere, most abundant species are Bacillus cereus, Bacillus anthracis and Bacillus subtilis. Similarly isolates from Wheat rhizosphere are Bacillus subtilis, Bacillus cereus and Bacillus thuringiensis. Phylogenetic analysis showed 99-100% homology of all isolated strains to Bacillus genera and all of them showed common evolutionary lineage in their respective phylogenetic trees. These sixty-four strains are gram positive and motile rods with spore forming ability. Majority of the isolates having growth temperature 37ºC and pH range 7, respectively. Similarly majority of Bacillus strains have ability to grow at high concentration of salts (NaCl) provided in the medium. Majority of bacterial isolates are resistant to high concentrations of heavy metals and are involved in catalase production, hydrolysis of starch, lipid and gelatin and use citrate as carbon source and also involved in the nitrification and denitrification process. Plant growth promoting bacteria play important role in nitrogen cycle and acts as ammonifiers, nitrifiers and denitrifiers and plays very important role in transformation of nitrogen and make it available for the plant by the processes of ammonification, nitrification and denitrification, respectively. There is great functional diversity found among these Corn and Wheat Bacillus isolates. All of them are involved in production of auxin. Some strains produces auxin in less quantity whereas some produce more. About 21.8% Corn isolates are phosphate solubilizers and similarly about 21% Wheat isolates are involved in phosphate solubilization. Siderophores are also important contributing factor in plant growth promotion. About 12.5% Corn isolates and 15.6% Wheat isolates are siderophore producers. Majority of Bacillus strains involved in the production of ACC deaminase. 38% of Corn and 35% of Wheat isolates were involved in production of hydrogen cyanide. Majority of isolates showed antibacterial activity against pathogens as among Corn isolates, about 50% showed activity against S. aureus, 15.6% against E. coli, 62.5% against Klebsiella, 56.3% against Pseudomonas, 50% against Proteus, 21.8% against Enterobacter and 78% showed activity against Acinetobacter. Similarly among Wheat isolates 18.8% showed activity against S.aureus, 3% against E.coli, 21.8% against Klebsiella, 21.8% against Pseudomonas, 12.5% against Proteus 6.25% against Enterobacter, 12.5% against Acinetobacter. These Corn rhizospheric strains were used for the growth promotion of Corn in the field experiments. It was observed that majority of bacterial strains induced positive growth promoting attributes in vegetative growth of Corn. Strain CU9 showed significant, (49%) increase in shoot length of plants. Inoculation with bacterial isolates significantly increased the cob length in all Corn plants except CSH23 inoculated plant as compared to control plant. Inoculated plants also exhibited increment in dry weight of plant except CN17 and CSH4. Among all inoculated plants strain CU47 showed significantly high peroxidase activity (163%) whereas CSH23, Comb 4A inoculated plants also showed increase of about 107% and 94%, respectively, as compared to control plants. Comb 4B showed remarkable high content of acid phosphatase (811%) as compared to control plants. Bacteria isolated from Wheat rhizosphere were also used for growth promotion of Wheat plant both in laboratory as well as in field trials. In laboratory trials, there is increment in shoot length and root length of Wheat seedling by majority of strains. Comb 5A and MIX inoculated seedlings exhibited increase in shoot length, root length and number of lateral roots. In pot experiments, there is an increase in plant height by all strains whereas combinations of strains (5A and 5B) and mixture of strains (MIX) also produced good results. About 18% to 19% increase in grain yield is observed in case of Comb 5A and Comb 5B inoculated plants as compared to control. WG1 showed about 71% increase in peroxidase activity than control whereas WUS2 showed remarkable increase of about 172% in acid phosphatase content. In another field trial where the impact of bacterial strains along with chemical fertilizer was applied, four different treatments were given (a) Bacterial inoculation and its respective uninoculated control (b) Bacterial inoculation along with DAP and its respective uninoculated control (c) Bacterial inoculation along with Urea and its respective uninoculated control (d) Bacterial inoculation along with Urea and DAP and its respective uninoculated control. Comb 5B showed maximum enhancement (27%) in shoot length without addition of any chemical fertilizer. The significant increase in spike length is also observed in inoculated plants supplemented with DAP, Urea and mixture of these two fertilizers in comparison to their respective uninoculated plants used as control. MIX inoculated plants showed 29% increase in spike length of Wheat with DAP supplementation and 18% in case of Urea addition. Similarly increase in weight of spikes in WSK11 is 81% in Urea supplemented plant. There is an increase in weight of 100 grains per plant in all inoculated plants but Urea supplemented WRY23 and Comb 5B showed 70% increase in weight of grains. Majority of inoculated plants supplemented with Urea showed increase in peroxidase activity whereas Comb 5B inoculated plant treated with Urea and DAP showed increment of 846%. DAP treated WM3 showed 356% and MIX showed 842% increase in acid phosphatase activity. Some species of Bacillus (Bacillus thuringiensis, Bacillus safensis, Bacillus pumilus, Bacillus subtilis, Bacillus axarquiensis, Bacillus anthracis, Bacillus tequiliensis, Bacillus cereus, Bacillus sonorensis) were present in both Wheat and Corn rhizosphere whereas some species were host specific as only in Corn (Bacillus toyoensis, Bacillus endophyticus, Bacillus mycoides) and only in Wheat (Bacillus flexus, Bacillus vallismortis, Bacillus amyloliquiefaciens, Bacillus megaterium, Bacillus velezensis). The species also varies from sample to sample taken from same crop. Similarly plant growth promoting attributes also varies from strain to strain. Majority of them involved in auxin production and ACC deaminase activity. Phosphate solubilization, HCN production, Siderophore production, Phytase activity, Antibacterial activities varies in all strains. These differences may be due to several reasons which may affect the presence of bacterial community in rhizospheric soil which involves root exudation, use of fertilizers in certain fields, pH of Soil, presence of organic matter, phosphorus and potassium present in soil etc. Majority of Bacillus species have remarkable plant growth promoting attributes and showed significant effects on growth of Corn and Wheat plants. These plant growth promoting strains can helpful in future for the formulations of biofertilizer. The objective of this study is to isolate different species of Bacillus from Wheat and Corn rhizosphere to check their functional and genomic diversity. Rhizospheric bacterial strains from Corn and Wheat rhizosphere will be screened for different plant growth promoting attributes and selected strains will be used to study the growth promoting effects on Corn and Wheat plant. The interaction of chemical fertilizers and bacterial strains effect on Wheat will also be studied.