Polyphasic Characterization of Plant Growth Promoting Cellulose Degrading Bacteria Isolated from Organic Manures.

In the present study, twenty seven cellulose-degrading bacteria (CDB) were isolated from various organic manures and their cellulolytic activities were determined. The bacterial isolate CDB-26 showed the highest cellulolytic index, released 0.507 ± 0.025 mg/ml glucose and produced 0.196 ± 0.014 IU/ml cellulase enzyme under in vitro conditions. Biochemically, all the 27 isolates showed difference in the 6 biochemical tests performed. Further, all the 27 CDB isolates were subjected to various plant growth-promoting activities, and all CDB strains were positive for IAA production, GA3 production and siderophore production, whereas 19 strains were positive for ACC deaminase activity, 21 strains showed NH3 production and 19 strains were positive for HCN production. Out of 27 CDB isolates, 18 isolates were able to solubilize phosphate, 21 isolates were able to solubilize potash and 10 CDB isolates were found positive for silica solubilization. The molecular diversity among different CDB isolates was studied through ARDRA and demonstrated very high genetic diversity among these bacteria. The in vitro cellulose-degradation potential of these CDB isolates using vegetable waste as substrate were also assessed, and the 3 CDB isolates viz. Serratia surfactantfaciens (CDB-26), Stenotrophomonas rhizophila (CDB-16) and Pseudomonas fragi (CDB-5) showed the highest cellulose-degrading potential under in vitro conditions. Hence, the cellulolytic microbes isolated in the present study could be used for effective bioconversion of plant biomasses into enriched compost.

Bacteria assisted green synthesis of copper oxide nanoparticles and their potential applications as antimicrobial agents and plant growth stimulants.

Copper oxide nanoparticles (CuO-NPs) have piqued the interest of agricultural researchers due to their potential application as fungicides, insecticides, and fertilizers. The Serratia sp. ZTB29 strain, which has the NCBI accession number MK773873, was a novel isolate used in this investigation that produced CuO-NPs. This strain can survive concentrations of copper as high as 22.5 mM and can also remove copper by synthesizing pure CuO-NPs. UV-VIS spectroscopy, DLS, Zeta potential, FTIR, TEM, and XRD techniques were used to investigate the pure form of CuO-NPs. The synthesized CuO-NPs were crystalline in nature (average size of 22 nm) with a monoclinic phase according to the XRD pattern. CuO-NPs were found to be polydisperse, spherical, and agglomeration-free. According to TEM and DLS inspection, they ranged in size from 20 to 40 nm, with a typical particle size of 28 nm. CuO-NPs were extremely stable, as demonstrated by their zeta potential of -15.4 mV. The ester (C=O), carboxyl (C=O), amine (NH), thiol (S-H), hydroxyl (OH), alkyne (C-H), and aromatic amine (C-N) groups from bacterial secretion were primarily responsible for reduction and stabilization of CuO-NPs revealed in an FTIR analysis. CuO-NPs at concentrations of 50 µg mL-1 and 200 µg mL-1 displayed antibacterial and antifungal activity against the plant pathogenic bacteria Xanthomonas sp. and pathogenic fungus Alternaria sp., respectively. The results of this investigation support the claims that CuO-NPs can be used as an efficient antimicrobial agent and nano-fertilizer, since, compared to the control and higher concentrations of CuO-NPs (100 mg L-1) considerably improved the growth characteristics of maize plants.

The polyembryo gene (OsPE) in rice.

T-DNA insertional mutagenesis is one of the most important approaches for gene discovery and cloning. A fertile polyembryo mutant generated by T-DNA/Ds insertion in Oryza sativa, cv. Basmati 370 showed twin or triple seedlings at a frequency of 15-20%. T-DNA insertion was confirmed by 950 bp hpt gene amplification in the promoter region of the candidate gene. The annotated protein corresponding to the OsPE candidate gene has been reported as a hypothetical protein in O. sativa. OsPE gene lacked functional homologs in other species. No OsPE paralog was found in rice. No conserved domains were found in the protein coded by OsPE. RT-PCR showed the expression of OsPE gene in Basmati 370 shoots. Full-length OsPE gene was cloned in Basmati 370. The combined use of Southern blot, genome walking, TAIL-PCR, RT-PCR techniques, and bioinformatics led to the identification of a candidate gene controlling the multiple embryos in rice. There is gain of function, i.e., multiple embryos in the seeds in the knockout mutant OsPE whereas its wild-type allele strictly controls single embryo per seed. The seeds with multiple embryos are distributed at random in the rice mutant panicle. The origin of multiple embryos, whether apomictic, zygotic or both is under investigation.

A candidate gene OsAPC6 of anaphase-promoting complex of rice identified through T-DNA insertion.

A dwarf mutant (Oryza sativa anaphase-promoting complex 6 (OsAPC6)) of rice cultivar Basmati 370 with 50% reduced plant height as compared to the wild type was isolated by Agrobacterium tumefaciens-mediated transformation using Hm(R) Ds cassette. This mutant was found to be insensitive to exogenous gibberellic acid (GA(3)) application. Homozygous mutant plants showed incomplete penetrance and variable expressivity for plant height and pleiotropic effects including gibberellic acid insensitivity, reduced seed size, panicle length, and female fertility. Single copy insertion of T-DNA and its association with OsAPC6 was confirmed by Southern hybridization, germination on hygromycin, and 3:1 segregation of HPT gene in F(2) from OsAPC6 x Basmati 370 cross. The T-DNA flanking region sequenced through thermal asymmetric interlaced polymerase chain reaction showed a single hit on chromosome 3 of japonica rice cultivar Nipponbare in the second exonic region of a gene which encodes for sixth subunit of anaphase-promoting complex/cyclosome. The candidate gene of 8.6-kb length encodes a 728-amino acid protein containing a conserved tetratricopeptide repeat (TPR) domain and has only a paralog, isopenicillin N-synthase family protein on the same chromosome without the TPR domain. There was no expression of the gene in the mutant while in Basmati 370, it was equal in both roots and shoots. The knockout mutant OsAPC6 interferes with the gibberellic acid signaling pathway leading to reduced height and cell size probably through ubiquitin-mediated proteolysis. Further functional validation of the gene through RNAi is in progress.

Zinc tolerant plant growth promoting bacteria alleviates phytotoxic effects of zinc on maize through zinc immobilization.

The increasing heavy metal contamination in agricultural soils has become a serious concern across the globe. The present study envisages developing microbial inoculant approach for agriculture in Zn contaminated soils. Potential zinc tolerant bacteria (ZTB) were isolated from zinc (Zn) contaminated soils of southern Rajasthan, India. Isolates were further screened based on their efficiency towards Zn tolerance and plant growth promoting activities. Four strains viz. ZTB15, ZTB24, ZTB28 and ZTB29 exhibited high degree of tolerance to Zn up to 62.5 mM. The Zn accumulation by these bacterial strains was also evidenced by AAS and SEM-EDS studies. Assessment of various plant growth promotion traits viz., IAA, GA3, NH3, HCN, siderophores, ACC deaminase, phytase production and P, K, Si solubilization studies revealed that these ZTB strains may serve as an efficient plant growth promoter under in vitro conditions. Gluconic acid secreted by ZTB strains owing to mineral solubilization was therefore confirmed using high performance liquid chromatography. A pot experiment under Zn stress conditions was performed using maize (Zea mays) variety (FEM-2) as a test crop. Zn toxicity reduced various plant growth parameters; however, inoculation of ZTB strains alleviated the Zn toxicity and enhanced the plant growth parameters. The effects of Zn stress on antioxidant enzyme activities in maize under in vitro conditions were also investigated. An increase in superoxide dismutase, peroxidase, phenylalanine ammonia lyase, catalase and polyphenol oxidase activity was observed on inoculation of ZTB strains. Further, ZIP gene expression studies revealed high expression in the ZIP metal transporter genes which were declined in the ZTB treated maize plantlets. The findings from the present study revealed that ZTB could play an important role in bioremediation in Zn contaminated soils.