Trypanosoma acomys (Wenyon, 1909): Continuous Culturing with a Mouse Fibroblast Cell-Line (A9)

The continuous culturing of Trypanosoma acomys in the presence of a murine areolar-adipose cell line (A9) was possible for the 1st time. The trypanosomes were cultured at 37° C with A9 in DMEM supplemented with 20% heat inactivated fetal bovine serum, using an initial inoculum from primary cultures of lung or blood clots from infected spiny mice. The cultures were maintained for 115 days and underwent 15 passages before termination and cryopreservation. Using this culture system T. acomys subcultures were initiated from 3 different initial inocula (3 × 10⁴, 1.5 × 10⁵ and 7.4 × 10⁵ parasites/ml) and growth curves revealed that the lowest inoculum gave the best growth pattern. This inoculum yielded a population doubling time of less than 12 h for 4 days, a high peak density of 7 × 10⁶ parasites/ml and the most gradual decline compared to the other 2 inocula. Rosetting epimastigotes and nests of amastigotes were observed in close association with the feeder layer cells. Epimastigotes were the most predominant form in culture supernatants but other morphological forms observed included trypomastigotes and sphaeromastigotes.     

Growth and Immunologic Studies on the Culture Forms of Trypanosoma lewisi*

SYNOPSIS. Culture forms of Trypanosoma lewisi grown at 27 C in a diphasic blood agar medium resemble in structure the stage found in the invertebrate host. Cultures inoculated with approximately 1 × 10⁶ trypanosomes/ml attain maximum populations of 2–7 × 10⁷ organisms/ml after 5–6 days of incubation. The stationary phase persists 6–15 days. The decline of the population is of relatively long duration with approximately 1 × 10⁶ viable organisms/ml present after 90 days. Variations in growth were attributed to the preparation of defibrinated heated rabbit blood incorporated into the culture medium. With inocula of 3.0 × 10⁵ trypanosomes/ml there was a lag in growth not observed with larger inocula. Trypanosomes incubated at elevated temperatures had altered growth curves compared to organisms at 27 C. Agitation of cultures did not affect the growth or stationary phases, but hastened the population decline. Heated and unheated 5% (v/v) normal rat serum incorporated in the liquid phase of the medium altered the growth of the organisms. Heated serum caused a decrease in the population and an extended lag phase. The effects on growth were more marked with unheated serum suggesting that both heat-stable and labile components affect growth. Antisera from rats injected with live culture forms included in the liquid phase inhibited, while antisera from rats 24 days after infection with the blood stream forms had no effect on the growth of the culture forms. Antisera from rabbits immunized with sonicates of culture forms also altered the growth of the organisms in culture. Rabbit antisera prepared by immunization with sonicates of dividing and non-dividing blood stream forms had no effect on the in vitro growth. Antisera from animals immunized with rat blood and culture medium were also without effect. The immunologic implications of the data are considered and discussed.     

Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor

Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below 32°C resulted in growth suppression. However, specific productivity of FSH, q _FSH, increased as culture temperature decreased, and the maximum q _FSH of 43.4 ng/10⁶ cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures, we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 10⁷ cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising.     

PROPERTIES OF TYROSINE HYDROXYLASE IN PERIPHERAL NERVES

Approximately 80 per cent of tyrosine hydroxylase activity in bovine mandibular nerve and rabbit sciatic nerve was soluble, and the rest of the activity was particle-bound. The soluble enzyme in bovine mandibular nerve was isolated by ammonium sulphate fractionation (25–35 per cent saturation). The enzyme had a pH optimum at 5·9 in Tris-acetate buffer, and at 6·5 in Tris-HCl or phosphate buffer. The enzyme required a tetrahydropteridine cofactor. K_m values toward various tetrahydropteridines such as l -erythro-tetrahydrobiopterin (a probable natural cofactor), 2-amino-4-hydroxy-6-methyltetrahydropteridine, and 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine were 2 × 10⁻⁵m , 5 × 10⁻⁵m and 4 × 10⁻⁴m , respectively. The K_m value for tyrosine at 1 × 10⁻³m -2-amino-4-hydroxy-6-methyltetrahydropteridine as a cofactor was 5 × 10⁻⁵m . The enzyme activity was markedly stimulated with Fe²⁺ or catalase, but Fe²⁺ gave higher activity. The activity was inhibited with α, α′-dipyridyl, l -α-methyl-p-tyrosine, and various catecholamines. Among catecholamines, dopamine was the most potent inhibitor. l -5-Hydroxytryptophan was an inhibitor as potent as dopamine. Neither d -5-hydroxytryptophan nor 5-hydroxytryptamine inhibited the enzyme. The inhibition by l -5-hydroxytryptophan was partially competitive with tetrahydrobiopterin at concentrations higher than 9 × 10⁻⁵m , and partially uncompetitive at concentrations lower than 9 × 10⁻⁵m . The addition of heparin or lysolecithin did not affect enzyme activity with tetrahydrobiopterin as cofactor.     

Cultivation of Trypanosoma theileri at 37 C in Partially Defined Media*

SYNOPSIS. Trypanosoma theileri has become the 1st mammalian trypanosome to be serially cultivated in a blood- and cell-free medium at 37 C. This was accomplished by progressive simplification of an initial culture medium containing whole bovine blood. Fifty-one subcultures in 4- to 5-ml quantities of monophasic liquid media were made over a period of 1 year. The final medium contained hemin and a defined modification of McCoy 5a tissueculture medium, supplemented with a peptone solution. Hemin was essential and T. theileri grew best with ～1 mg/100 ml. Counts reached 8 × 10⁶ organisms/ml in 3–4 days. Seventy-ml volumes of medium supported growth of T. theileri to 3-4 × 10⁶ organisms/ml. Early in the study trypomastigotes were the predominant nondividing form, but they were absent when the medium was being rapidly modified. Trypomastigotes reappeared when the medium was stabilized at the end of the study. Elimination of serum antigens from the medium should greatly facilitate antigenic analysis of T. theileri.     

Binding of the Bovine and Porcine Pancreatic Secretory Trypsin Inhibitor (Kazal) To Human Leukocyte Elastase: A Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTT – K_a = 6.3 × 10⁴M^?1, δ5G° = -26.9kJ/mol, δH° = +11.7kJ/mol, and δS° = +1.3 × 10² entropy units; porcine PSTI –K_a = 7.0 × 10³M^?1,δG° = -21.5kJ/mol, δH° = +13.0kJ/mol, and δS° = +1.2 × 10² entropy units (values of K_a δG° and δS° were obtained at 21.0°C; values of δH° were temperature independent over the range (between 5.0°C and 45.0°C) explored). On increasing the pH from 4.5 to 9.5, values of K_a for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from ?7.0, in the free enzyme, to ?5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

RNA-Binding Efficacy of N-Phenylbenzohydroxamic Acid: An Invitro and Insilico Approach

RNA has attracted recent attention for its key role in gene expression and hence targeting by small molecules for therapeutic intervention. This study is aimed to elucidate the specificity of RNA binding affinity of parent compound of N-arylhydroxamic acids series, N-phenylbenzohydroxamic acid trivially named as PBHA,C6H5NOH.C6H5C ? O. The binding behavior was examined by various biophysical methods such as absorption, fluorescence, and viscosity measurements. Molecular docking was also done. The value of affinity constant and overall binding constant was calculated 5.79 ± 0.03 × 10⁴ M^?1 and K’ = 1.09 ± 0.03 × 10⁵ M^?1, respectively. The Stern-Volmer constant K_sv obtained was 2.28 ± 0.04 × 10⁴ M^?1. The compound (PBHA) shows a concentration-based enhancement of fluorescence intensity with increasing RNA concentration. Fluorescence quenching of PBHA–RNA complex in presence of K₄ [Fe(CN)₆] was also observed. Viscometric studies complimented the UV results where a continuous increase in relative viscosity of the RNA solution was observed with added optimal PBHA concentration. All the experimental evidences indicate that PBHA can strongly bind to RNA through an intercalative mode.     

New Culture Medium for Maintenance of Tsetse Tissues and Growth of Trypanosomatids*

SYNOPSIS. A new culture medium (SM), based on the amino-acid composition of tsetse hemolymph and containing fetal bovine serum, was designed for the maintenance of tsetse organs and the cultivation of various trypanosomatids. For optimum growth 20% (v/v) serum was required. The medium supported prolonged peristalsis of the alimentary tract and salivary glands of pre-emerged Glossina morsitans morsitans. In established cultures, derived from bloodstream forms of pleomorphic Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense strains, inocula of ～ 10⁶ procyclics/ml yielded 4–5 × 10⁷ organisms/ml after 4 or 5 days of incubation at 28 C. Bloodstream forms of a cloned monomorphic T. b. brucei strain were also able to transform into procyclics, which, however, multiplied at a lower rate, with maximum yields of ～ 2 × 10⁷ after 5 days. Cultures of Trypanosoma congolense and of a nearly monomorphic Trypanosoma brucei gambiense strains could be established in SM medium only in the presence of tsetse alimentary tract. The procyclic trypomastigotes of these species, adapted to SM medium and able to grow in it without Glossina organs, gave maximum populations of ～ 4.5 × 10⁷ cells/ml. Promastigotes of Leishmania donovani, cultivated routinely in a diphasic Table's medium, multiplied actively upon being transferred into SM medium, producing yields of ～ 4 × 10⁷ cells/ml.     

Occurrence in vivo of "hidden breaks" at specific sites of 26 S ribosomal RNA of Musca carnaria.

A fragmentation process occurs in 26 S ribosomal RNA of mature cytoplasmic ribosomes of Musca carnaria. It consists of the sequential appearance of three “hidden breaks” that fragment 26 S rRNA (M_r = 1.42 × 10⁶) into four pieces with approximate molecular weights of 0.68 × 10⁶, 0.35 × 10⁶, 0.29 × 10⁶ and 0.096 × 10⁶, respectively. This fragmentation was not observed in 17 S rRNA (M_r = 0.74 × 10⁶).Extremely mild treatment of newly assembled ribosomes with pancreatic RNAase reproduces the 26 S rRNA fragmentation phenomenon in vitro in the same way as it occurs in vivo.This evidence is discussed in relation to the secondary structure of 26 S rRNA and its binding with specific ribosomal proteins.     

Nosema whitei,a microsporidan pathogen of some species ofTribolium

The pathogenicity ofNosema whitei was studied using a dose-mortality technique; larvae ofTribolium castaneum were reared for the duration of each experiment in flour mixed with known numbers of spores. The susceptibility of each of the first 5 larval instars was compared. The LD₅₀ (for mortality after 20 days) increased consistently from the first instar (1.8×10⁶ spores/g) to the fifth instar (1.0×10¹⁰ spores/g). The slopes of the probit lines increased consistently as age increased (from b=1.1 to b=3.9). Two factors which reduce the development time ofT. castaneum, high temperature and high humidity, both reduced the pathogenicity ofN. whitei. Thus pathogenicity decreased as the temperature was increased fram 25°C (LD₅₀=4.2×10⁶) through 30°C (LD₅₀=1.3×10⁷) to 35°C (LD₅₀=3.2×10⁶), also pathogenicity decreased consistently as humidity was increased fram 10%, through 30, 50, 70% to 90% R.H. Adults, emerging fromNosema free larvae, became infected only when exposed to a very high dose (2×10¹⁰ spores/g for 14 days from the day of emergence). Infected larvae were treated for 1 hr. at 45°C in an attempt to cure the infection. The infected larvae were not cured, rather the treatment had an adverse alfect on their survival.

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Résumé La pathogénicité deNosema whitei a été étudiée en élevant des larves deT. castaneum dans de la farine mélangée à des quantités connues de spores. La sensibilité des larves diminue uniformément en fonction de l'age; La DL₅₀ varie de 1,8×10⁶/g (1^er stade) à 1,0×10¹⁰ spores/g (5^e stade). Deux facteurs, qui accélèrent le développement deT. castaneum, des températures et des humidités élevées, réduisent tous les deux la pathogénicité deN. whitei. Les adultes ne peuvent être infectés qu'en les exposant à la dose extrêmement élevée de 2×10¹⁰ spores/g. Un traitement par la chaleur (45°C pendant une heure) n'a pas réussi à guérir les larves.

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This work financed by a Science Research Council (U.K.) studentship is based on a thesis submitted for a degree of Ph. D. at the University of Newcastle-upon-Tyne.     

Production of Pseudomonas Cells Having a High Fumarate Hydratase Activity

An extracellular 45 kDa endochitosanase was purified and characterized from the culture supernatant of Bacillus sp. P16. The purified enzyme showed an optimum pH of 5.5 and optimum temperature of 60°C, and was stable between pH 4.5-10.0 and under 50°C. The K _m and V _max were measured with a chitosan of a D.A. of 20.2% as 0.52 mg/ml and 7.71×10^?6 mol/sec/mg protein, respectively. The enzyme did not degrade chitin, cellulose, or starch. The chitosanase digested partially N-acetylated chitosans, with maximum activity for 15-30% and lesser activity for 0-15% acetylated chitosan. The chitosanase rapidly reduced the viscosity of chitosan solutions at a very early stage of reaction, suggesting the endotype of cleavage in polymeric chitosan chains. The chitosanase hydrolyzed (GlcN)₇ in an endo-splitting manner producing a mixture of (GlcN)_2-5. Time course studies showed a decrease in the rate of substrate degradation from (GlcN)₇ to (GlcN)₆ to (GlcN)₅, as indicated by the apparent first order rate constants, k ₁ values, of 4.98×10^?4, 2.3×10^?4, and 9.3×10^?6 sec^?1, respectively. The enzyme hardly catalyzed degradation of chitooligomers smaller than the pentamer.     

Diffusion of bovine serum albumin in a neutral polymer solution

The diffusion coefficient D of bovine serum albumin through various solutions (pH 7.0, 0.5M NaCl) of polythylene oxide (M_w ～ 1 × 10⁵, 3 × 10⁵) was studied with quasielastic light scattering. In solutions of the 1 × 10⁵ polymer solution at polymer concentrations above 0.5 g/L, D is considerably greater than would have been expected from the viscosity of water:polymer mixtures, the deviations being larger at low protein concentration that at high protein concentration. With either polymer, D falls with increasing protein concentration.     

Determination on the binding of chlortetracycline to bovine serum albumin using spectroscopic methods

In this work, the interaction of chlortetracycline with bovine serum albumin (BSA) was investigated by fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking. Results indicated that chlortetracycline quenches BSA fluorescence mainly by a static quenching mechanism. The quenching constants (K_SV) were obtained as 5.64 × 10⁴, 4.49 × 10^4/, and 3.44 × 10^4/ M_?1 at 283, 295, and 307 K, respectively. The thermodynamic parameters of enthalpy change Δ H°, entropy change Δ S°, and free energy change Δ G° were ?5.12 × 10^4/ J mol?1, ?97.6 J mol?1 K?1, and ?2.24 × 10^4/ J mol?1 (295 K), respectively. The association constant (K_A) and the number of binding sites (n) were 9.41 × 10^3/ M?1 and 0.86, respectively. The analysis results suggested that the interaction was spontaneous, and van der Waals force and hydrogen‐bonding interactions played key roles in the reaction process. In addition, CD spectra proved secondary structure alteration of BSA in the presence of chlortetracycline. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:331–336, 2012; View this article online at wileyonlinelibrary.com . DOI 10:1002/jbt.21424     

Capillary electrophoresis coupled with end‐column electrochemiluminescence for the determination of ephedrine in human urine,and a study of its interactions with three proteins

A tris(2,2‐bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺)‐based electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE) method has been established for the sensitive determination of ephedrine for the first time. Under the optimized conditions [ECL detection at 1.15 V, 25 mmol/L phosphate buffer solution (PBS), pH 8.0, as running buffer, separation voltage 12.5 kV, 5 mmol/L Ru(bpy)₃²⁺ with 60 mmol/L PBS, pH 8.5, in the detection cell] linear correlation (r = 0.9987) between ECL intensity and ephedrine concentration was obtained in the range 6.0 × 10^–8–6.0 × 10^–6 g/mL. The detection limit was 4.5 × 10^–9 g/mL (S:N = 3). The developed method was successfully applied to the analysis of ephedrine in human urine and the investigation of its interactions with three proteins, including bovine serum albumin (BSA), cytochrome C (Cyt‐C) and myoglobin (Mb). The number of binding sites and the binding constants between ephedrine and BSA, Cyt‐C and Mb were 8.52, 12.60, 10.66 and 1.55 × 10⁴ mol/L, 6.58 × 10³ mol/L and 1.59 × 10⁴ mol/L, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Noradrenaline, a Transmitter Candidate in the Retina

The occurrence, metabolism, uptake, and release of noradrenaline were studied in the bovine retina with the following results. (1) Small amounts of noradrenaline occur in the retina and are restricted to the area corresponding to the inner nuclear and plexiform layers. (2) Retinal tissue can metabolise [¹⁴C]dopamine to form quantities of [¹⁴C]noradrenaline. (3) [¹⁴C]Noradrenaline can also be partly metabolised to form [¹⁴C]normetanephrine. (4) When bovine retinas were incubated with 5 × 10^-7 M-[³H]noradrenaline for 20 min and processed for autoradiography, most of the label was associated with apparent nerve processes in the inner plexiform layer. Biochemical analysis showed that more than 95% of the label was noradrenaline. (5) [¹⁴C]Noradrenaline uptake saturated with increasing noradrenaline concentrations and followed Michaelis-Menten kinetics. This uptake could be accounted for by two processes, a high-affinity system with a K_m1 of 5 × 10^-8 M and a V_max1 of 0.193 pmol/mg/10 min and a low-affinity system with a K_m2 of 6.3 × 10^-5 M and a V_max2 of 0.109 nmol/mg/10 min. (6) Noradrenaline uptake was strongly dependent on temperature and sodium, less dependent on potassium, and independent of calcium and magnesium ions. (7) Centrally acting drugs, such as desipramine, imipramine, desmethylimipramine, and amitriptyline, inhibited noradrenaline uptake by more than 55% at the concentration of 5 × 10^-5 M. These drugs at the same concentration diminished dopamine uptake by less than 30%. (8) Noradrenaline uptake is stereospecific, the (-) isomer having a greater affinity for the uptake sites than the (+) isomer. (9) [¹⁴C]Noradrenaline in the retina could be released by increasing the external potassium concentration. This release was calcium-dependent and was blocked by 20 mM-cobalt chloride. The present studies could be interpreted as supporting the idea that noradrenaline acts as a transmitter in the retina.     

Activity of Various Growth Substances in the Avena First Internode Test

The elongation growth of the Avena first internode segments was studied in the presence of one or several of the following growth substances: indoleacetic acid (IAA), 6-fur-furylamino purine (FAP, kinetin), 6-benzylamino purine (BAP), gibberellin A₃ (GA₃) and A₄₊₇ (GA₄₊₇), and abscisic acid (ABA). The cytokinins at concentrations of 10^?7 to 10^?6M stimulated growth with 4 to 6 per cent but this effect was not statistically significant. Concentrations higher than 5 × 10^?6M inhibited growth. FAP and BAP (from 10^?8M to 10^?6M) had no significant interaction with any other growth substance used. The two-factor interactions of IAA × ABA, IAA × GA₃, and GA₃× ABA, as well as the three-factor interaction IAA × ABA × GA₃ were significant. However, the IAA × ABA interaction was significant only when high concentration (10^?6M) of ABA was used. The growth inhibition produced by 10^?7 and 10^?6M ABA was overcome by about equimolar concentrations of IAA. The stimulation of growth by GA₃ and GA₄₊₇ (10^?9 to 10^?7M) was prevented by simultaneous application of ABA, and it was reduced significantly by application of IAA (10^?7 to 10^?8M). GA3 at 10^?8M combined with different concentrations of IAA gave slightly higher elongation than IAA alone but the observed values were significantly lower than expected assuming independent additive action.     

Purification and Properties of Formyltetrahydrofolate Synthetase from Spinach

Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was found in fresh spinach leaves and purified about 60-fold by treatments of ammonium sulfate, protamine sulfate, dialysis, and DEAE-cellulose column chromatography. Some properties of the enzyme were investigated. Optimum pH was found to be 7.5, and optimum temperature was observed to be at 37°C. In the enzyme reaction, FAH₄ and formate were required specifically as the substrates, and Mg⁺⁺ and ATP were essential components. The Michaelis constants for dl-FAH₄, formate, ATP and magnesium chloride were 1.7×10^?3 m, 1.7×10^?2 m, 4.1×10^?4 m and 3.3×10^?3 m, respectively. The primary product formed in the reaction catalyzed by the enzyme was suggested as N¹⁰-formyl-FAH₄ spectrophotometrically. It was observed that the enzyme also catalyzed the reverse reaction. The possible role of the enzyme in plants was discussed.     

SELENIUM STIMULATES GROWTH OF MARINE MACROALGAE IN AXENIC CULTURE1

The marine brown alga Fucus spiralis L. and the red alga Goniotrichum alsidii (Zanard) increase their growth upon the, addition of SeO₃^2- or SeO₄^2- when cultivated axenically in the artificial seawater ASP6 F2. In the concentration range 1 · 10^?10-1 · 10^?7 M there are two optima, one at 3.3 · 10^?10 M and another at 3.3 · 10^?8 M. α-To-copherol, often administered together with selenium to mammals suffering from selenium deficiency, gives no additive effect with selenium, but α-tocopherol in the concentration range 1 × 10^?7-1 × 10^?6 M does influence the morphology of the Fucus plants. Organically bound selenium has no effect.     

Susceptibility of Phthorimaea operculella Zeller (Lepidoptera; Gelechiidae) to its granulovirus PoGV with larval age

1 Granulovirus PoGV is a strong candidate to substitute for chemical insecticides in integrated pest management (IPM) of the potato tuber moth Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae). Generally, susceptibility to baculoviruses decreases with increasing larval age. For effective field applications, knowledge of the age‐related change in susceptibility is crucial. 2 The relative susceptibility of various instars of P. operculella larvae to PoGV was studied by leaf‐disc bioassays in the laboratory at 26 °C incubation temperature. The LC₅₀ increased from 4 × 10⁶ granules/mL for neonate larvae to 1.2 × 10⁷, 1.1 × 10⁸, 4.4 × 10⁸, and 4.2 × 10⁹ granules/mL, signifying resistance ratios of 3, 29, 110, and 1051, for 5, 6, 7, and 9‐day‐old larvae, respectively. 3 The relationship between log‐LC₅₀ values and log‐larval weight was significantly linear. A logistic function described significantly the relation between larval weights and physiological age measured in degree‐days (DD > 13.4 °C). Both functions allowed prediction of the activity ratio of PoGV for different larval ages by using temperature summation to express physiological age. 4 PoGV was found to be highly active against P. operculella larvae up to a physiological age of approximately 50 DD (>13.4 °C) (i.e. the first third of the total larval development time). Thereafter, the virus rapidly lost its activity against older larvae. Prospects for applying this knowledge in the field are discussed.     

Development of emulsion from rhizobial fermented starch industry wastewater for application as Medicago sativa seed coat

Starch industry wastewater was efficiently employed for the production of Sinorhizobium meliloti and the concentrated culture was used for the development of a biofertilizer formulation. Tween‐80 (0.02 g/L) acted as the best emulsifier for a Sinorhizobium–canola oil emulsion. The stability of the emulsion and survival of the organism was enhanced by supplementation of xanthan gum at pH 8. The refrigerated condition was most favorable for stability and survival of the microorganism. The survival of microorganism at 4±1°C was 2.78×10¹⁰ and 2.01×10¹⁰ CFU (colony forming unit)/mL on storage for 1 and 2 months, respectively. The values were higher than the prescribed cell count (×10³ CFU/mL) for field application. At 40°C, the survival of bacteria reduced from 3×10¹⁰ CFU/mL to 8.1×10⁹ and 8.8×10⁶ CFU/mL in 1 and 2 months, respectively. Emulsion‐coated seed was incubated at different temperatures and a cell count of 10⁵ CFU/seed was observed after 2 months of storage at 4°C, which was equal to the highest level of the described requirement (10³–10⁵ CFU/seed). Emulsion supplemented with xanthan gum improved the shelf‐life under optimized conditions (Sinorhizobium concentrate – canola oil (1:1) emulsion with 0.02 g/L Tween‐80; storage at pH 8 and temperature 4±1°C) and this emulsion with the required cell count and prolonged viability was used for the pre‐inoculation of seed or for in situ soil application.