An efficient method for the purification and characterization of nematicidal azadirachtins A,B, and H,using MPLC and ESIMS

Azadirachtin A enriched concentrate containing 60% active ingredient (a.i.) was prepared from the methanolic extract of the de-fatted neem (Azadirachta indica A. Juss) seed kernels. Azadirachtins A, B, and H, the three major bioactive constituents of neem seed kernel, were purified from this methanolic concentrate by employing reverse phase medium-pressure liquid chromatography (MPLC), using methanol-water solvent system as an eluant. The three pure azadirachtin congeners thus obtained were characterized by their unique mass spectral fragmentation, using electrospray probe in positive ion mode (ESI). All three azadirachtins exhibited nematicidal and antifungal activities. Azadirachtin B was the most effective against the reniform nematode Rotylenchulus reniformis (EC(50) 96.6 ppm), followed by Azadirachtin A (119.1 ppm) and H (141.2 ppm). At 200-ppm concentration, the test compounds caused 50-65% mortality of Caenorhabditis elegans nematode. Azadirachtin H showed the highest activity against the phytophagous fungi Rhizoctonia solani (EC(50) 63.7 ppm) and Sclerotium rolfsii (EC(50) 43.9 ppm), followed by B and A. The isolation of pure azadirachtins A, B, and H directly by MPLC purification from its concentrate and their characterization by ESIMS are unique and less time-consuming.     

Increase in Growth,Productivity and Nutritional Status of Wheat (Triticum aestivum L.) and enrichment in Soil Microbial Population Applied with Biofertilizers Entrapped with Organic Matrix

To find effective alternatives to reduce the application of conventional urea (CU), a conventional biofertilizer (CB) preparation (charcoal mixed Azotobacter chroococcum and Bacillus subtilis) and the same biofertilizers entrapped in an organic matrix consisting of cow dung, rice bran, dried powder of neem leaves, and clay soil in 1:1:1:1 ratio and 25% (w/w) saresh (plant gum of Acacia sp.), named as super granules of biofertilizers (SGBF) were applied to cultivate wheat (Triticum aestivum L. cv. ‘WH-711’) in experimental plots. The results revealed that the efficacy of commercially available charcoal mixed biofertilizers could not prove as effective alternative to CU, whereas the same dose of biofertilizers entrapped in the organic matrix, SGBF, resulted in a significant increase in growth and productivity of wheat. It appears that SGBF prepared and applied in this study is an effective organic alternative to the urea for wheat cultivation in semi-arid subtropical agro-ecosystems.     

Interactions amongst Mycorrhiza,Azotobacter chroococcum and Root Characteristics of Wheat Varieties

A field experiment was conducted in a randomized block design with three replications over 2 years to evaluate the effect of wheat cultivar and dual inoculation of Azotobacter chroococcum (Azc) and arbuscular mycorrhiza fungi (AMF, Glomus fasciculatum) on root characters and AMF infection in three crosses of wheat. The experimental material comprised four wheat parents, WH‐147, WH‐157, WH‐542 and PBW‐175, and three F₁ crosses, WH‐147 ×WH‐157, WH‐147 × WH‐542 and WH‐147 × PBW‐175. Comparison of treatment averages, i.e. control (mineral nutrients 60 kg N + 30 kg P₂O₅ + 12.5 kg ZnSO₄ ha^?1, as in other two treatments), AMF and AMF + Azc, revealed that inoculation of Azc led to an increase in AMF infection in roots. Maximum root biomass was obtained in F₁ hybrids WH‐147 × WH‐157 in the AMF treatment and in WH‐147 × PBW‐175 receiving AMF + Azc. Total root length and AMF infection of roots was maximum in WH‐147 × PBW‐175 for all the treatments during both years. A positive association between AMF infection in roots and Azotobacter survival in the rhizosphere was apparent. Similarly, maximum A. chroococcum counts were observed 80 and 120 days after sowing in the AMF + Azc treatment in cross WH147 × PBW175.     

An Analysis of Seasonal Effects on Leaf Nitrate Reductase Activity and Nitrogen Accumulation in Maize (Zea mays L.)

The important enzyme in nitrogen (N) assimilation, nitrate reductase (NR), is an inducible enzyme influenced by many external (light, temperature, etc.) and internal (genotype) factors. The efficiency of the N assimilation system may vary with genotype and season. In the present study, the effects of season on NR activity in relation to N accumulation in maize plants were investigated. Six different cultivars of maize, namely Ganga-11, Deccan-103, Hi-starch (hybrids), Arun, Manjari and Vijay (composites), were sown during the monsoon (88-day crop duration) and in winter (150-day crop duration). In vivo NR activity in the last fully expanded leaf (LFEL), and the N contents of the whole plant and the LFEL were estimated at seven phenological growth stages. Three different states of N metabolism in maize, namely (i) low NR activity per unit leaf area per unit time coinciding with high accumulation of N, (ii) high NR activity coinciding with low N accumulation, and (iii) low NR activity coinciding with low N accumulation, were noted. These results clearly demonstrate that the relationships between N uptake and accumulation parameters change with the season and crop growth stage and are subject to a genotypic influence. Thus it is necessary to evaluate genotypes under similar environments to select a genotype with high N use efficiency. As these relationships are growth dependent, care must be taken to evaluate them at a particular phenological stage rather than on the basis of days after sowing.     

Development of a leukocytopenic baby guinea pig model with azathioprine and cyclophosphamide

Immunosuppressive agents were given to baby guinea pigs to develop a leukocytopenic baby guinea pig model. Azathioprine in different doses, and cyclophosamide and methylprednisolone combinations did not result in predictable leukocytopenia without significant mortality. A combination of azathioprine 20mg and cyclophosphamide 20mg administered once daily for 5 days to 42 baby guinea pigs resulted in a mean white blood cell count of 2531/mm³ ±1198 (a reduction of 3931 ± 1413 from pretreatment values). The mean counts for polymorphonuclear leukocytes and lymphocytes on day 6 were 398/mm³ and 2035/mm³, respectively. Only 7% mortality was seen in these animals. This model can be utilized to study a variety of bacterial and viral infections in “immunocompromised hosts”.     

Neuromuscular pedicle graft for restoration of arytenoid abductor function in dogs with experimentally induced laryngeal hemiplegia

Left laryngeal hemiplegia was induced by resection of the left recurrent laryngeal nerve in 12 dogs. A neuromuscular pedicle graft formed from the first cervical nerve and sternothyroideus muscle was transplanted after 1 week to the denervated cricoarytenoideus dorsalis muscle in 8 dogs. The remaining 4 dogs served as controls. Left arytenoid abduction was blindly evaluated by laryngoscopy with video photography at time 0, at 1 week, and at 19 weeks in all dogs. At 19 weeks, biopsy specimens of the left cricoarytenoideus dorsalis muscle and the neuromuscular pedicle were taken from 4 of the treatment dogs, and biopsy specimens of the left cricoarytenoideus dorsalis muscle were taken from the 4 control dogs. All biopsy specimens were blindly evaluated by histologic and histochemical examination. At 36 to 44 weeks, the remaining 4 treatment dogs, from which biopsy specimens had not been taken, were reevaluated by use of laryngoscopy with video photography. Complications and difficulties encountered during surgery included hemorrhage in the area of the cricoarytenoideus dorsalis muscle, location of a branch of the first cervical nerve that was long enough to prevent tension at the graft site, orientation of the muscle pedicle in the cricoarytenoideus dorsalis muscle without the use of an operating microscope, and preservation of the terminal portion of the first cervical nerve while forming the neuromuscular pedicle. Results of the arytenoid movement evaluations revealed improvement in arytenoid abductor function in the treatment group, compared with that in the control group at 19 weeks. Arytenoid abduction in the treatment group at this time, however, was still significantly decreased (P less than 0.05), compared with presurgical movement evaluations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of bovine neutrophils in Pasteurella haemolytica mediated damage to pulmonary endothelial cells

The purpose of these studies was to determine mechanisms of pulmonary tissue damage mediated by Pasteurella haemolytica and interaction with bovine neutrophils. Bovine pulmonary artery endothelial cell monolayers were treated with various combinations of P. haemolytica factors including bacterial culture supernatant (CS) and purified LPS, with and without bovine neutrophils. Damage to endothelial cells was monitored by 51Cr release, cell detachment rate, and morphological changes. At 5 h post-treatment (PT) bacterial factors produced very little toxic change in cells, however, by 22 h PT both crude leukotoxin and LPS caused high levels of cytotoxicity and detachment. Neutrophils did not augment toxicity mediated by LPS, but actually protected endothelial cells from low levels of LPS. When the LPS component of CS was neutralized with polymyxin B, leukotoxin mediated neutrophil killing resulted in extensive endothelial cell damage. These results suggest that LPS may directly injure endothelial cells and this toxic effect may be reduced by neutrophils. However, neutrophil killing by leukotoxin may also contribute to endothelial cell damage in the absence of LPS.     

Effect of Benzyladenine on Transpiration, Water Potential and its Components in Genotypes of Wheat Contrasting in Drought Tolerance

Drought tolerant and susceptible cultivars of wheat, C-306 and Kalyan sona, growing under non-stressed and water-stressed conditions, were sprayed with benzyladenine (BA) at 70 days after sowing (DAS). Observations recorded at 5, 10, 15, 20 and 25 days after spraying revealed that BA increased the rate of transpiration (TR) in C-306 under non-stressed conditions. However, under water stress, the increase was significant only after 5 days of BA spraying. In Kalyan sona BA treatment either decreased TR or did not exhibit significant increase under non-stressed conditions, but caused significant increase in TR under water stress. The increase in TR was elicited through enhanced stomatal opening. Water potential, osmotic potential and pressure potential of both genotypes decreased on account of water stress. The effect of BA was not perceptible on restoration of leaf water potential (LWP) or its components. The effect of BA was possibly confined to stomatal behaviour and transpiration.     

Serodiagnosis of porcine cysticercosis by enzyme-linked immunosorbent assay (ELISA) using fractionated antigens

The sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Taenia solium cysticercosis was evaluated in experimentally and naturally infected pigs, using T. solium larval scoleces and its fractionated 1st and 2nd peaks on Sephadex G-200 as antigens. First peak antigen gave maximum sensitivity and highest antibody titres. The overall sensitivity of this test was found to be 91.5, 95.8 and 70.8% with scolex, 1st and 2nd peak antigens, respectively. False positive reactions occurred in 9.09% of uninfected pigs with scolex and 1st peak antigens and cross-reactions occurred in 25% of Taenia hydatigena-infected animals using scolex and 2nd peak antigens. No cross-reaction was observed using 1st peak antigen. The specificity of the test was 92.3, 96.2 and 92.3% with scolex, 1st and 2nd peak antigens, respectively.