Inoculation With Azospirillum spp. Acts as the Liming Source for Improving Growth and Nitrogen Use Efficiency of Potato.

Nitrogen (N) is one of the limiting factors for plant growth, and it is mainly supplied exogenously by fertilizer application. It is well documented that diazotrophic rhizobacteria improve plant growth by fixing atmospheric N in the soil. The present study investigates the nitrogen-fixing potential of two Azospirillum spp. strains using the 15N isotope-dilution method. The two diazotrophic strains (TN03 and TN09) native to the rhizosphere of potato belong to the genus Azospirillum (16S rRNA gene accession numbers LN833443 and LN833448, respectively). Both strains were able to grow on an N-free medium with N-fixation potential (138-143 nmol mg-1 protein h-1) and contained the nifH gene. Strain TN03 showed highest indole acetic acid (IAA) production (30.43 µg/mL), while TN09 showed highest phosphate solubilization activity (249.38 µg/mL) while both diazotrophs showed the production of organic acids. A 15N dilution experiment was conducted with different fertilizer inputs to evaluate the N-fixing potential of both diazotrophs in pots. The results showed that plant growth parameters and N contents increased significantly by the inoculations. Moreover, reduced 15N enrichment was found compared to uninoculated controls that received similar N fertilizer levels. This validates the occurrence of N-fixation through isotopic dilution. Strain TN09 showed higher N-fixing potential than TN03 and the uninoculated controls. Inoculation with either strain also showed a remarkable increase in plant growth under field conditions. Thus, there were remarkable increases in N use efficiency, N uptake and N utilization levels. Confocal laser scanning and transmission electron microscopy showed that TN03 is an ectophyte, i.e., present outside root cells or within the grooves of root hairs, while TN09 is an endophyte, i.e., present within root cells, forming a strong association withroot it. This study confirms that diazotrophic Azospirillum spp. added to potato systems can improve plant growth and N use efficiency, opening avenues for improvement of potato crop growth with reduced input of N fertilizer.

Diazotrophs for Lowering Nitrogen Pollution Crises: Looking Deep Into the Roots.

During and after the green revolution in the last century, agrochemicals especially nitrogen (N) were extensively used. However, it resulted in a remarkable increase in crop yield but drastically reduced soil fertility; increased the production cost, food prices, and carbon footprints; and depleted the fossil reserves with huge penalties to the environment and ecological sustainability. The groundwater, rivers, and oceans are loaded with N excess which is an environmental catastrophe. Nitrogen emissions (e.g., ammonia, nitrogen oxide, nitrous oxide) play an important role in global climate change and contribute to particulate matter and acid rain causing respiratory problems, cancers, and damage to forests and buildings. Therefore, the nitrogen-polluted planet Earth needs concerted global efforts to avoid the disaster. Improved agricultural N management focuses on the synchronization of crop N demand and N supply along with improving the N-use efficiency of the crops. However, there is very little focus on the natural sources of N available for plants in the form of diazotrophic bacteria present inside or on the root surface and the rhizosphere. These diazotrophs are the mini-nitrogen factories that convert available (78%) atmospheric N2 to ammonia through a process known as "biological nitrogen fixation" which is then taken up by the plants for its metabolic functioning. Diazotrophs also stimulate root architecture by producing plant hormones and hence improve the plant's overall ability to uptake nutrients and water. In recent years, nanotechnology has revolutionized the whole agri-industry by introducing nano-fertilizers and coated/slow-releasing fertilizers. With this in mind, we tried to explore the following questions: To what extent can the crop N requirements be met by diazotroph inoculation? Can N input to agriculture be managed in a way leading to environmental benefits and farmers saving money? Can nanotechnology help in technological advancement of diazotroph application? The review suggests that an integrated technology based on slow-releasing nano-fertilizer combined with diazotrophs should be adopted to decrease nitrogen inputs to the agricultural system. This integrated technology would minimize N pollution and N losses to much extent.

First report of diazotrophic Brevundimonas spp. as growth enhancer and root colonizer of potato.

Rhizobacteria contain various plant-beneficial traits and their inoculation can sustainably increase crop yield and productivity. The present study describes the growth-promoting potential of Brevundimonas spp. isolated from rhizospheric soil of potato from Sahiwal, Pakistan. Four different putative strains TN37, TN39, TN40, and TN44 were isolated by enrichment on nitrogen-free malate medium and identified as Brevundimonas spp. based on their morphology, 16S rRNA gene sequence, and phylogenetic analyses. All strains contained nifH gene except TN39 and exhibited nitrogen fixation potential through acetylene reduction assay (ARA) except TN40. Among all, the Brevundimonas sp. TN37 showed maximum ARA and phosphate solubilization potential but none of them exhibited the ability to produce indole acetic acid. Root colonization studies using transmission electron microscopy and confocal laser scanning microscopy showed that Brevundimonas sp. TN37 was resident over the root surface of potato; forming sheets in the grooves in the rhizoplane. TN37, being the best among all was further evaluated in pot experiment using potato cultivar Kuroda in sterilized sand. Results showed that Brevundimonas sp. TN37 increased growth parameters and nitrogen uptake as compared to non-inoculated controls. Based on the results obtained in this study, it can be suggested that Brevundimonas spp. (especially TN37) possess the potential to improve potato growth and stimulate nitrogen uptake. This study is the first report of Brevundimonas spp. as an effective PGPR in potato.

Growth stimulatory effect of AHL producing Serratia spp. from potato on homologous and non-homologous host plants.

Plant growth promoting rhizobacteria are known to improve plant performance by developing healthy and productive interactions with the host plants. These associations may be symbiotic or asymbiotic depending upon the genetic potential of the resident microbe and promiscuity of the host. Present study describes the potential of two Serratia spp. strains for promotion of plant growth in homologous as well as non-homologous hosts. The strains KPS-10 and KPS-14; native to potato rhizosphere belong to genus Serratia based on 16S rRNA gene sequences (accession no. LN831934 and LN831937 respectively) and contain multiple plant growth promoting properties along-with the production of quorum sensing acyl homoserine lactone (AHL) molecules. Both Serratia spp. strains showed solubilization of inorganic tri-calcium phosphate while KPS-14 also exhibited phytase activity (1.98 10-10 kcat). KPS-10 showed higher P-solubilization activity (128.5 µg/mL), IAA production (8.84 µg/mL), antifungal activity and also showed the production of two organic acids i.e., gluconic acid and lactic acid. Both strains produced three common AHLs: C6-HSL, 3oxo-C10-HSL, 3oxo-C12-HSL while some strain-specific AHLs (3OH-C5-HSL, 3OH-C6-HSL, C10-HSL specific to KPS-10 and 3OH-C6-HSL, C8-HSL, 3oxo-C9-HSL, 3OH-C9-HSL specific to KPS-14). Strains showed roots and rhizosphere colonization of potato and other non-homologous hosts up to one month. In planta AHLs-detection confirmed a likely role of AHLs during seedling growth and development where both extracted AHLs or bacteria inoculated roots showed extensive root hair. A significant increase in root/shoot lengths, root/ shoot fresh weights, root/shoot dry weights was observed by inoculation in different hosts. PGP-characteristics along with the AHLs-production signify the potential of both strains as candidate for the development of bio-inoculum for potato crop in specific and other crops in general. This inoculum will not only reduce the input of chemical fertilizer to the environment but also improve soil quality and plant growth.

A comparative study of bacterial diversity based on culturable and culture-independent techniques in the rhizosphere of maize (Zea mays L.).

OBJECTIVE: Maize is an important crop for fodder, food and feed industry. The present study explores the plant-microbe interactions as alternative eco-friendly sustainable strategies to enhance the crop yield. METHODOLOGY: Bacterial diversity was studied in the rhizosphere of maize by culture-dependent and culture-independent techniques by soil sampling, extraction of DNA, amplification of gene of interest, cloning of desired fragment and library construction. RESULTS: Culturable bacteria were identified as Achromobacter, Agrobacterium, Azospirillum, Bacillus, Brevibacillus, Bosea, Enterobacter, Microbacterium, Pseudomonas, Rhodococcus, Stenotrophomonas and Xanthomonas genera. For culture-independent approach, clone library of 16S ribosomal RNA gene was assembled and 100 randomly selected clones were sequenced. Majority of the sequences were related to Firmicutes (17%), Acidobacteria (16%), Actinobacteria (17%), Alpha-Proteobacteria (7%), Delta-proteobacteria (4.2%) and Gemmatimonadetes (4.2%) However, some of the sequences (30%) were novel that showed no homologies to phyla of cultured bacteria in the database. Diversity of diazotrophic bacteria in the rhizosphere investigated by analysis of PCR-amplified nifH gene sequence that revealed abundance of sequences belonging to genera Azoarcus (25%), Aeromonas (10%), Pseudomonas (10%). The diazotrophic genera Azotobacter, Agrobacterium and Zoogloea related nifH sequences were also detected but no sequence related to Azospirillum was found showing biasness of the growth medium rather than relative abundance of diazotrophs in the rhizosphere. CONCLUSION: The study provides a foundation for future research on focussed isolation of the Azoarcus and other diazotrophs found in higher abundance in the rhizosphere.

Change in corneal endothelial cell density after phacoemulsification in patients with type II diabetes mellitus.

OBJECTIVE: To compare the mean change in Corneal Endothelial cell Density (CED), from baseline (pre-operative value), two months after phacoemulsification cataract surgery between type II diabetic patients and non-diabetic patients. METHODS: This prospective stratified controlled study was conducted at PNS Shifa Hospital, Karachi. 80 eyes of 72 type II diabetic patients and 80 eyes of 77 non diabetic controls, having Nuclear Opalescence (NO) grades 2 and 3 on slit lamp examination underwent phacoemulsification surgery. CED was measured in cells/mm2, of concerned eye of each subject preoperatively and 2 months post operatively using specular microscope. The difference in mean CED change between the two groups after surgery was analyzed. RESULTS: Mean age of study population was 61.41± 6.76 years. Out of study population, 92 (57.5%) were males and 68 (42.5%) were females. There was a statistically significant difference between both groups in terms of mean post-operative CED, mean change in CED and mean frequency change in CED (p <0.05). There was no statistically significant difference between both groups in age, gender, laterality of eyes and mean pre-operative CED, (p >0.05). Difference of pre-operative CED from post-operative CED in each group was statistically significant. CONCLUSION: There is a significant difference between diabetic population and normal population in terms of corneal endothelial loss after uneventful phacoemulsification cataract surgery.

Pseudomonas sp. AF-54 containing multiple plant beneficial traits acts as growth enhancer of Helianthus annuus L. under reduced fertilizer input.

Plant growth promoting rhizobacteria (PGPR) are capable to increase the growth and yield of crops in eco-friendly and sustainable manner. To evaluate the response of sunflower towards inoculation with PGPR, a sunflower root associated bacterium AF-54 isolated from Diyar Gali Himalayan Mountain region, Azad Jammu and Kashmir (AJK), identified as Pseudomonas sp. by 16S rRNA sequence analysis and was characterized using polyphasic approach. The bacterium produced 23.9 µgmL-1 indole-3-acetic acid in tryptophan-supplemented medium, showed 44.28 nmoles mg-1 protein h-1 nitrogenase activity through acetylene reduction assay and released 48.80 µg mL-1 insoluble phosphorus in Pikovskaya's broth. During P-solubilization, the pH of the Pikovskaya's medium decreased from 7 to 3.04 due to the production of acetic acid, malic acid and gluconic acid. Pseudomonas sp. AF-54 showed metabolic versatility by utilizing 79 carbon sources from BIOLOG GN2 plates and resistance to many antibiotics. Furthermore, it inhibited the growth of Fusarium oxysporum in dual culture assay. To evaluate the plant-inoculation response, series of experiments conducted in hydroponic, sterilized soil and fields at AJK, and Faisalabad where inoculated plants with reduced fertilizer showed a significant increase in growth, yield, oil contents and achene NP uptake as compared to non-inoculated control. AF-54 showed extensive root colonization in sterilized and non-sterile conditions documented through yfp-labeling and fluorescent in situ hybridization coupled with confocal laser scanning microscopy. This study concludes that the Pseudomonas sp. strain AF-54 containing multiple plant growth promoting traits can be a potential candidate for biofertilizer production to enhance sunflower crop yield with reduced application of chemical (NP) fertilizers.

Frequency of Systemic Risk Factors in Central Serous Chorioretinopathy.

OBJECTIVE: To determine the frequency of various systemic risk factors associated with acute central serous chorioretinopathy (CSCR) in our setup. STUDY DESIGN: Descriptive case series. PLACE AND DURATION OF STUDY: Armed Forces Institute of Ophthalmology (AFIO), Rawalpindi, from July 2011 to June 2014. METHODOLOGY: All consecutive patients with acute CSCR who presented in the outpatient department during the study period were recruited. Clinical findings were endorsed on a pre-devised proforma with special emphasis on inquiring about known systemic risk factors for CSCR in detail from each patient. Patients were managed conservatively with control of modifiable risk factors and topical 0.1% Nepafenac eye drops. Analysis of data was done using SPSS version 13.0. RESULTS: Forty-four eyes of 42 patients were eligible for final analysis. The mean age of study population was 37.38 ±6.31 years with 38 (90.47%) male patients. Elevated serum cortisol and serum testosterone levels were found in 3 and 2 patients, respectively. Known systemic risk factors for CSCR were present in 36 (85.71%) patients with emotional stress/psychiatric disorder 15 (35.71%), Type Apersonality 11 (26.19%), smoking 10 (19.04%), hypertension 5 (11.90%), and acid peptic disease 4 (9.52%) were the most frequently found risk factors. CONCLUSION: Emotional stress/psychiatric illness, hypertension, acid peptic disease and use of exogenous steroids and other medicines are the established risk factors for CSCR that can be modified/withdrawn to reduce the morbidity related to CSCR.

Differential Response of Potato Toward Inoculation with Taxonomically Diverse Plant Growth Promoting Rhizobacteria.

Rhizosphere engineering with beneficial plant growth promoting bacteria offers great promise for sustainable crop yield. Potato is an important food commodity that needs large inputs of nitrogen and phosphorus fertilizers. To overcome high fertilizer demand (especially nitrogen), five bacteria, i.e., Azospirillum sp. TN10, Agrobacterium sp. TN14, Pseudomonas sp. TN36, Enterobacter sp. TN38 and Rhizobium sp. TN42 were isolated from the potato rhizosphere on nitrogen-free malate medium and identified based on their 16S rRNA gene sequences. Three strains, i.e., TN10, TN38, and TN42 showed nitrogen fixation (92.67-134.54 nmol h(-1)mg(-1) protein), while all showed the production of indole-3-acetic acid (IAA), which was significantly increased by the addition of L-tryptophan. Azospirillum sp. TN10 produced the highest amount of IAA, as measured by spectrophotometry (312.14 µg mL(-1)) and HPLC (18.3 µg mL(-1)). Inoculation with these bacteria under axenic conditions resulted in differential growth responses of potato. Azospirillum sp. TN10 incited the highest increase in potato fresh and dry weight over control plants, along with increased N contents of shoot and roots. All strains were able to colonize and maintain their population densities in the potato rhizosphere for up to 60 days, with Azospirillum sp. and Rhizobium sp. showing the highest survival. Plant root colonization potential was analyzed by transmission electron microscopy of root sections inoculated with Azospirillum sp. TN10. Of the five test strains, Azospirillum sp. TN10 has the greatest potential to increase the growth and nitrogen uptake of potato. Hence, it is suggested as a good candidate for the production of potato biofertilizer for integrated nutrient management.

Isolation and characterization of a ß-propeller gene containing phosphobacterium Bacillus subtilis strain KPS-11 for growth promotion of potato (Solanum tuberosum L.).

Phosphate-solubilizing and phytate-mineralizing bacteria collectively termed as phosphobacteria provide a sustainable approach for managing P-deficiency in agricultural soils by supplying inexpensive phosphate to plants. A phosphobacterium Bacillus subtilis strain KPS-11 (Genbank accession no. KP006655) was isolated from potato (Solanum tuberosum L.) rhizosphere and characterized for potato plant growth promoting potential. The strain utilized both Ca-phosphate and Na-phytate in vitro and produced 6.48 µg mL(-1) indole-3-acetic acid in tryptophan supplemented medium. P-solubilization after 240 h was 66.4 µg mL(-1) alongwith the production of 19.3 µg mL(-1) gluconic acid and 5.3 µg mL(-1) malic acid. The extracellular phytase activity was higher (4.3 × 10(-10) kat mg(-1) protein) than the cell-associated phytase activity (1.6 × 10(-10) kat mg(-1) protein). B. subtilis strain KPS-11 utilized 40 carbon sources and showed resistance against 20 chemicals in GENIII micro-plate system demonstrating its metabolic potential. Phytase-encoding gene ß-propeller (BPP) showed 92% amino acid similarity to BPP from B. subtilis (accession no.WP_014114128.1) and 83% structural similarity to BPP from B. subtilis (accession no 3AMR_A). Potato inoculation with B. subtilis strain KPS-11 increased the root/shoot length and root/shoot weight of potato as compared to non-inoculated control plants. Moreover, rifampicin-resistant derivative of KPS-11 were able to survive in the rhizosphere and on the roots of potato up to 60 days showing its colonization potential. The study indicates that B. subtilis strain KPS-11 can be a potential candidate for development of potato inoculum in P-deficient soils.