Molecular characterization and PCR detection of a nitrogen-fixing Pseudomonas strain promoting rice growth

Nitrogen-fixing plant growth-promoting rhizobacteria (PGPR) from the genus Pseudomonas have received little attention so far. In the present study, a nitrogen-fixing phytohormone-producing bacterial isolate from kallar grass (strain K1) was identified as Pseudomonas sp. by rrs (16S ribosomal RNA gene) sequence analysis. rrs identity level was high with an uncharacterized marine bacterium (99%), Pseudomonas sp. PCP2 (98%), uncultured bacteria (98%), and Pseudomonas alcaligenes (97%). Partial nifH gene amplified from strain K1 showed 93% and 91% sequence similarities to those of Azotobacter chroococcum and Pseudomonas stutzeri, respectively. The effect of Pseudomonas strain K1 on rice varieties Super Basmati and Basmati 385 was compared with those of three non-Pseudomonas nitrogen-fixing PGPR (Azospirillum brasilense strain Wb3, Azospirillum lipoferum strain N4 and Zoogloea strain Ky1) used as single-strain inoculants. Pseudomonas sp. K1 was detected in the rhizosphere of inoculated plants by enrichment culture in nitrogen-free growth medium, which was followed by observation under the microscope as well as by PCR using a rrs-specific primer. For both rice varieties, an increase in shoot biomass and/or grain yield over that of noninoculated control plants was recorded in each inoculated treatment. The effect of Pseudomonas strain K1 on grain yield was comparable to those of A. brasilense Wb3 and Zoogloea sp. Ky1 for both rice varieties. These results show that nitrogen-fixing pseudomonads deserve attention as potential PGPR inoculants for rice.     

Tree age influences nutritional,pectin, and anatomical changes in developing ‘Kinnow’ mandarin (Citrus nobilis Lour × Citrus deliciosa Tenora) fruit

An understanding of variation in fruit quality in relation to tree age can help understand the issue of poor fruit quality in young orchards; however, limited information is available on the citrus fruit. In this study, the endogenous nutritional status in rind, rag, and leaves; pectin status in rind and rag; and anatomical fruit growth parameter in rind tissues were studied in ‘Kinnow’ mandarins fruit during their development on trees from three age (6-, 18-, and 35-year-old) groups. In older (35-year-old) trees, rind, rag, and leaf nitrogen (N), phosphorus (P), and potassium (K) concentrations were superior. In fruit from all tree age groups, total pectin and protopectin reduced; however water-soluble pectin (WSP) improved. In rind tissues harvested from young (6-year-old) trees, cell density was more while cell size was less. In all tree age groups, cell density in rind tissues correlated negatively with rind WSP.     

Molecular and biologic characterization of a phytoplasma associated with <Emphasis Type="Italic">Brassica campestris</Emphasis> phyllody disease in Punjab province,Pakistan

A phytoplasma-associated disease was identified in Brassica campestris (sarson) plants during a survey conducted in Punjab province of Pakistan in 2014–2016. The symptomatic plants showed characteristic symptoms of phyllody and witches’ broom. Phytoplasma presence was detected by polymerase chain reaction on 16S ribosomal and tuf DNAs, followed by RFLP analysis and sequence comparison of the 16S rRNA and tuf genes. The phytoplasma detected was classified in a new ribosomal subgroup designed 16SrIX-H. The phytoplasma presence in phloem tissues of symptomatic sarson samples was also confirmed through light microscopy and transmission studies to healthy plants through dodder and the leafhopper Orosius albicinctus. This is the first report of identification of 16SrIX-H subgroup phytoplasma associated with sarson and its natural vector in Pakistan.     

Suitability of Immobilized Pseudomonas fluorescens SM1 Strain for Remediation of Phenols, Heavy Metals, and Pesticides from Water

Immobilized microbial cells for the biological treatment have the potential to degrade toxic chemicals faster than conventional wastewater treatment systems. In the present study, suitability of immobilized Pseudomonas fluorescens SM1 strain in calcium alginate beads for remediation of the major toxicants in Indian water bodies was tested by means of GC/HPLC and AAS techniques. Roughly 80% reduction in the concentration of phenols was observed by immobilized SM1 cells compared with 60% by the free cells. Also, in the case of the bioremediation of heavy metals, immobilized SM1 cells were found to be more efficient compared with the free cells. Suspension of P. fluorescens SM1 cells in the test model water for 24 h brought down the concentrations of Cu⁺⁺, Cd⁺⁺, Ni⁺⁺, and Pb⁺⁺ by more than 75% under free cell state and 7?C9% better efficiency under the immobilized conditions. However, Cr(VI) could show only 44% removal by the cell immobilized system, whereas a mere 35% reduction in the Cr(VI) levels was shown in the test model water by the free SM1 cells under the same conditions. Moreover, a model water containing 2,000 ppb of BHC, 1,248 ppb mancozeb, and 312 ppb 2,4-D passed through the cell immobilized column resulted in the decline in their concentrations up to 362 ppb, 750 ppb, and 126 ppb, respectively. Generally, AAS, HPLC, and GC analyses of treated test model waters with the free and immobilized SM1 cells exhibited high potential of immobilized SM1 in detoxification of test water. From the results, we conclude that immobilized cells of P. fluorescens SM1 strain were quite effective in bioremediation of major toxicants present in Indian water bodies, and we also recommend the use of immobilized bacterial cells rather than the free cells for the bioremediation/detoxification process.     

In vitro activity of isothiocyanates against Fusarium graminearum

Isothiocyanates are biotoxic degradation products formed as a result of enzymatic hydrolysis of glucosinolates present in Brassica species. The application of biofumigant Brassica crops, as an alternative crop protection method for soilborne pathogens and pests is increasingly gaining interest. However, little is known of the potential of biofumigation to reduce the inoculum of Fusarium species affecting cereals. The aim of this study was to evaluate the antifungal activity of five isothiocyanates, namely allyl, benzyl, ethyl, 2-phenylethyl and methyl isothiocyanates, against germination and growth of Fusarium graminearum under in vitro conditions. Aromatic isothiocyanates were more inhibitory than the aliphatic isothiocyanates against mycelial growth, whereas the reverse was observed for conidial germination. Among the tested isothiocyanates, allyl and methyl isothiocyanates were more efficient overall, showing lower ED₅₀ values (35–150 mg/L) for conidial germination and mycelial radial growth. The findings suggest that Brassica plants containing allyl and methyl glucosinolates could have a suppressive effect, reducing the inoculum of F. graminearum in soil prior to cereal production.     

Direct detection of chicken genomic DNA for gender determination by thymine-DNA glycosylase

1. Birds, especially nestlings, are generally difficult to sex by morphology and early detection of chick gender in ovo in the hatchery would facilitate removal of unwanted chicks and diminish welfare objections regarding culling after hatch. 2. We describe a method to determine chicken gender without the need for PCR via use of Thymine-DNA Glycosylase (TDG). TDG restores thymine (T)/guanine (G) mismatches to cytosine (C)/G. We show here, that like DNA Polymerase, TDG can recognise, bind and function on a primer hybridised to chicken genomic DNA. 3. The primer contained a T to mismatch a G in a chicken genomic template and the T/G was cleaved with high fidelity by TDG. Thus, the chicken genomic DNA can be identified without PCR amplification via direct and linear detection. Sensitivity was increased using gender specific sequences from the chicken genome. 4. Currently, these are laboratory results, but we anticipate that further development will allow this method to be used in non-laboratory settings, where PCR cannot be employed.     

The phytotoxicity and degradation of diesel hydrocarbons in soil

Hydrocarbon contamination in soils may be toxic to plants and soil microorganisms and act as a source of groundwater contamination. The objective of the study was to evaluate the fate of adding diesel in soil with no previous history of hydrocarbon contamination. Particular aspects examined were soil respiration, changes in microbial population, breakdown of diesel hydrocarbon and phytotoxicity to germination of ryegrass. Soil respiration was measured as evolved CO₂. Bacterial population was determined as Colony Forming Units (CFUs) in dilution plates, and fungal activity was measured as hyphal length. The fate of individual hydrocarbons was determined by GC‐MS after extraction with dichloromethane. When 0.64 % (w/w) of diesel was added to soil, the respiration response showed a lag phase of 2 days and maximum respiration occurred at day 7. The lag phase was 5 days and maximum respiration occurred at day 11 in soil after adding 1.6 %, 4.0 %, and 13.6 % of diesel (w/w). After the peak, respiration decreased up to 20 days, in each of the four levels of diesel addition to the soil. Thereafter, respiration becomes more or less constant but substantially greater than the control. Diesel addition up to 4.0 % (w/w) increased the bacterial population to 10 fold but fungal hyphal length did not increase. However, bacterial population did not increase after adding 13.6 % (w/w) of diesel and fungal hyphal length was significantly less than the control and other three levels of diesel. Removal of inhibition to germination of perennial ryegrass was linked to the decomposition of n‐C₁₀ and n‐C₁₁ hydrocarbons and took from 11 to 30 days depending on the levels of diesel added to the soil. Contamination with 13.6 % (w/w) of diesel inhibited the germination of perennial ryegrass until 24 weeks of incubation.     

First report on molecular surveillance based on duplex detection of Anaplasma marginale and Theileria annulata in dairy cattle from Punjab,Pakistan

The study aimed to investigate if vegetable-based high-energy mash diets supplemented with NaHCO₃, l-arginine?+?vitamin C, and vegetable oils were effective against tachycardia and polycythemia in the broiler chicken. A total of 256 Ross-308 day-old male broiler chicks were randomly distributed into eight dietary treatment groups in a three-way ANOVA with 2?×?2?×?2 factorial arrangement (three factors, i.e., NaHCO₃, l-arginine?+?vitamin C, and vegetable oil each with two levels, e.g., 0 and 0.1% of NaHCO₃ and l-arginine?+?vitamin C; 3 and 4% of vegetable oil supplemented with basal diet) for a period of 35 days. Iso-caloric and iso-nitrogenous diets were formulated and supplied ad libitum. The final live weight (FLW), average daily feed intake (ADFI), average daily gain (ADG), feed efficiency (FE), carcass traits, cardio-pulmonary morphometry, total protein (TP), hemoglobin (Hb), triiodothyronine (T₃), incidence of tachycardia, and polycythemia were examined. Supplementation of NaHCO₃ increased 2.2% ADFI, 5.5% FE, and 23.2% TP. The l-arginine?+?vitamin C increased 2.4% FLW and decreased 1.9% heart rate. Vegetable oil increased 1.3% ADFI, 4.2% ADG, 8.6% FE, 23.1% Hb, and 15.5% PCV. The NaHCO₃, l-arginine?+?vitamin C, and vegetable oil additively interacted to increase 31.5% T₃ at the expense of 21.1% of the weight of the right ventricle (RV). The RV:TV, carcass traits, and hemato-biochemical indices remained within normal range irrespective of the levels of the supplementations of the test ingredients. It was concluded that vegetable-based high-energy mash diets were not susceptible to tachycardia and polycythemia. The addition of NaHCO₃ and l-arginine?+?vitamin C ameliorated the propensity of tachycardia and polycythemia without deteriorating performance, carcass traits, and hemato-biochemical indices of the broiler chicken in a dose-dependent manner.

     

Isolation of nematicidal compounds from Tagetes patula L. yellow flowers: structure-activity relationship studies against cyst nematode Heterodera zeae infective stage larvae

Bioassay-guided isolation studies on the extracts of yellow flowers of Tagetes patula L. against the Heterodera zeae were carried out to identify phytochemicals lethal to this economically important cyst nematode. In vitro investigation of a polar extract and fractions showing activity led to the isolation of phenolic compounds (flavonoids and phenolic acids). In the nonpolar extract, a few fatty acids, their methyl esters, and thiophenes (including α-terthienyl) were detected. In studies of compounds obtained commercially, α-terthienyl and gallic and linoleic acids showed 100% mortality at concentrations of 0.125% after 24 h. Assessment of structure-activity relationships revealed that an increase in the number of hydroxyl groups in phenolic acids increased the activity; with fatty acids, activity depended on chain length and the number and position of double bonds. Crude extracts of the flowers of different colors also have promising activity.