Effect of inoculum parameters on the specificity of secondary synthesis in cultures producing novobiocin and mycoheptin]

The impact of the vegetative inoculum parameters on specificity of the secondary synthesis in the cultures producing novobiocin and mycoheptin was studied. During the study the fermentation conditions were varied by using the vegetative inoculum differing in the respiration rate after its transfer to the fermentation medium. To show the decisive role of the inoculum parameters in regulation of the specificity of the secondary synthesis, the dynamics of accumulation of certain metabolites forming from glucose along with the main antibiotic and the activity of the key enzymes of the carbohydrate metabolism during the culture growth in the fermentation media were studied. It was found that the specificity of the secondary synthesis with respect to certain metabolites was defined by the intensity of carbohydrate metabolism, i. e. the ratio of the activity of enzymes of glycolysis and the pentosephosphate pathway. In this regard, the inoculum with the maximum respiration rate in an amount of 10 to 20 per cent promoted the highest productivity of the mycelium by the synthesis of novobiocin and mycoheptin while the rate of accumulation of fatty acids, carbohydrates and phenol compounds (for Streptomyces spheroides) and mycopentene (for Streptoverticillium mycoheptinicum) decreased.     

Emergence of distinct brome mosaic virus recombinants is determined by the polarity of the inoculum RNA

Despite overwhelming interest in the impact exerted by recombination during evolution of RNA viruses, the relative contribution of the polarity of inoculum templates remains poorly understood. Here, by agroinfiltrating Nicotiana benthamiana leaves, we show that brome mosaic virus (BMV) replicase is competent to initiate positive-strand [(+)-strand] synthesis on an ectopically expressed RNA3 negative strand [(-) strand] and faithfully complete the replication cycle. Consequently, we sought to examine the role of RNA polarity in BMV recombination by expressing a series of replication-defective mutants of BMV RNA3 in (+) or (-) polarity. Temporal analysis of progeny sequences revealed that the genetic makeup of the primary recombinant pool is determined by the polarity of the inoculum template. When the polarity of the inoculum template was (+), the recombinant pool that accumulated during early phases of replication was a mixture of nonhomologous recombinants. These are longer than the inoculum template length, and a nascent 3' untranslated region (UTR) of wild-type (WT) RNA1 or RNA2 was added to the input mutant RNA3 3' UTR due to end-to-end template switching by BMV replicase during (-)-strand synthesis. In contrast, when the polarity of the inoculum was (-), the progeny contained a pool of native-length homologous recombinants generated by template switching of BMV replicase with a nascent UTR from WT RNA1 or RNA2 during (+)-strand synthesis. Repair of a point mutation caused by polymerase error occurred only when the polarity of the inoculum template was (+). These results contribute to the explanation of the functional role of RNA polarity in recombination mediated by copy choice mechanisms.     

The effect of sodium butyrate on Tipula iridescent virus synthesis in insect suspension cell cultures

The effect of sodium butyrate on Tipula iridescent virus (TIV) synthesis in suspension-cultured cells of Estigmene acrea was investigated. Sodium butyrate reduces viral-induced cell fusion but this is reversible with the removal of butyrate. At 7 mM sodium butyrate, TIV replicates in cells within 8 hr, but does not replicate in this time with 10–20 mm butyrate in the cell medium; cells so treated contain large vesicles with inoculum. Upon removal of the inhibitor, TIV replication appears normal, but large inoculum vesicles can still be found in the cytoplasm, and many infected cells have highly condensed chromatin in their nuclei. Sodium butyrate causes a lag of at least 2 hr in viral DNA synthesis as detected by [³H]thymidine incorporation into viroplasmic centres and at 7 mm butyrate viral DNA synthesis is reduced by 50–60%. In comparison, butyrate at 7 and 10 mm concentration does not inhibit host DNA synthesis, but at 15 and 20 mm, nuclear DNA synthesis is markedly reduced.     

Ectomycorrhizal synthesis on seedlings of Afzelia quanzensis Welw. using various types of inoculum

Ectomycorrhizal synthesis on seedlings of Afzelia quanzensis was initiated in the greenhouse and in the field using basidiospores or soil inoculum originating from fungi associated with adult trees of A. quanzensis, Brachystegia microphylla, B. spiciformis, and Julbernardia globiflora. Of the spore inocula used, only a Pisolithus sp. associated with adult A. quanzensis formed mycorrhizae. Seedlings raised in contact with all soil inocula formed mycorrhizae; however, the mycorrhizal types formed differed between soil inoculum used in the greenhouse and soil inoculum directly used in the field. A. quanzensis has a low specificity for mycorrhizal association. The concepts of ectomycorrhizal succession are also applicable to African savanna ecosystems.     

Conditions for cultivation of the fungus Penicillium melinii UzLM-4 and its biosynthesis of lipases

Cultivation of the fungus Penicillium melinii UzLM-4 on a Raistrick's medium of our own modification made it possible to increase the biosynthesis of lipases three to four times. The following conditions ensured a high rate of synthesis of the extracellular lipase: age of the inoculum, 15 days; concentration of the inoculum, 15 x 10(6) conidia per 100 ml medium; initial pH of the nutrient medium, 8.0; and cultivation in a shaker at 150 rpm (25 degrees C).     

Temperature-sensitive Mutants of Sindbis Virus: Biochemical Correlates of Complementation

Temperature-sensitive mutants of Sindbis virus fail to grow at a temperature that permits growth of the wild type, but when certain pairs of these mutants, mixed together, infect cells at that temperature, viral growth (i.e., complementation) occurs. The yield from this complementation, however, is of the same order of magnitude as the infectivity in the inoculum. Since in animal virus infections the protein components of the virion probably enter the cell with the viral nucleic acid, it was necessary to demonstrate that the observed complementation required synthesis of new viral protein and nucleic acid rather than some sort of rearrangement of the structural components of the inoculum. To demonstrate that complementation does require new biosynthesis, three biochemical events of normal virus growth have been observed during complementation and correlated with the efficiency of viral growth seen in complementation. These events include: (i) entrance of parental viral ribonucleic acid (RNA) into a double-stranded form; (ii) subsequent synthesis of viral RNA; and (iii) synthesis and subsequent incorporation of viral protein(s) into cell membranes where they were detected by hemadsorption. Although the infecting single-stranded RNA genome of the wild type was converted to a ribonuclease-resistant form, the genome of a mutant (ts-11) incapable of RNA synthesis at a nonpermissive temperature was not so converted. However, during complementation with another mutant also defective in viral RNA synthesis, some of the RNA of mutant ts-11 was converted to a ribonuclease-resistant form, and total synthesis of virus-specific RNA was markedly enhanced. The virus-specific alteration of the cell surface, detected by hemadsorption, was also extensively increased during complementation. These observations support the view that complementation between temperature-sensitive mutants and replication of wild-type virus are similar processes.     

Macromolecular Synthesis During Microcycle Sporogenesis of Bacillus cereus T

Microcycle sporogenesis induced in Bacillus cereus T by phosphate limitation occurs over a narrow range of phosphate to spore inoculum ratios. Sufficient phosphate is required to satisfy the demands for a twofold increase in deoxyribonucleic acid; net ribonucleic acid synthesis is not required. The total ribonucleic acid content of the culture was variable, and deoxyribonucleic acid synthesis was restricted to a twofold increase. Developmental changes during outgrowth occurred synchronously, whereas enzyme synthesis was periodic. The timing of the synthesis of tricarboxylic cycle enzymes, extracellular protease, arginase, histidase, and alkaline phosphatase was measured. Histidase could be induced after 2.5 hr throughout microcycle sporogenesis. Several other features of macromolecular synthesis during microcycle sporogenesis are described. Differences between this pattern and those observed during outgrowth leading to cell division are discussed. A technique for accurately estimating the levels and time of synthesis of incompletely extractable, labile enzymes is also presented.     

Conditions for Cultivation of the Fungus Penicillium melinii UzLM-4 and Its Biosynthesis of Lipases

Cultivation of the fungus Penicillium melinii UzLM-4 on a Raistrick's medium of our own modification made it possible to increase the biosynthesis of lipases by three to four times. The following conditions ensured a high rate of synthesis of the extracellular lipase: age of the inoculum, 15 days; concentration of the inoculum, 15 × 10⁶ conidia per 100 ml medium; initial pH of the nutrient medium, 8.0; and cultivation in a shaker at 150 rpm and 25°C.     

怀牛膝细胞悬浮培养条件的优化

为进一步优化怀牛膝(Achyranthes bidentata)细胞悬浮培养条件,对接种量、继代周期、pH、光照及Cu2+等多种影响因子的作用效果进行了研究,以提高怀牛膝细胞生长量及牛膝多糖含量。结果显示,接种量50 g·L^–1、继代周期14天,pH5–6和光照培养可以使细胞保持良好的生长状态及多糖合成能力;添加50μmol·L^–1 Cu^2+,细胞的干重最大,可达44.63 g·L^–1,多糖含量也最高,为4.02 mg·g^–1。     

Production of pectolytic enzymes by Aspergillus niger: effect of inoculum size and potassium hexacyanoferrate II-trihydrate

Summary The effect of inoculum size and potassium hexacyanoferrate II-trihydrate, K₄[Fe(CN)₆]·3 H₂O (KHCF), on pectinase synthesis by Aspergillus niger in submerged conditions were studied. Experiments were performed in shake flasks and in a 10-1 stirred bioreactor. Spore concentrations in the range 10²–10⁸ spores/1 of substrate were tested. Enzyme activity measured by the Apple Juice Depectinizing Assay (AJDA) showed the highest values using the smallest inoculum. Higher spore concentrations led to a 25% or even up to a 50% reduction of activity. Polygalacturonase (PG) activity decreased similarly to AJDA activity with higher inoculum concentration. Pectinlyase (PL) showed the opposite relationship, while pectin esterase (PE) did not show any correlation with inoculum concentration. Experiments in the fermentor using a reduced inoculum of 10² spores/1 showed that the whole process was prolonged in comparison to 10⁸ spores/1 inoculum. A pronounced effect of KHCF on fungal morphology as well as on enzymatic activity was observed. With increased concentration the morphology gradually changed from loose pellets to smaller compact ones. The enzymatic activity was markedly improved. In the bioreactor the amount of biomass was reduced from about g/l to 8 g/l. The activities were improved in comparison to fermentations without KHCF as follows: AJDA from 68 to 112 units (U)/ml, viscosity reduction from 83% to 90%, PG from 0.8 to 3.3 U/ml, PE from 32 to 49 U/ml and PL from 0.05 to 0.12 U/ml. The fermentation time was reduced from 96 to 68 h. Offprint requests to: J. Friedrich     

Microbial production of gallic acid by modified solid state fermentation

Bioconversion of tannin to gallic acid from powder of teri pod (Caesalpinia digyna) cover was achieved by the locally isolated fungus, Rhizopus oryzae, in a bioreactor with a perforated float for carrying solid substrate and induced inoculum. Modified Czapek-Dox medium, put beneath the perforated float, with 2% tannic acid at pH 4.5, temperature 32°C, 93% relative humidity, incubated for 3 days with 3-day-old inoculum was optimum for the synthesis of tannase vis-à-vis gallic acid production. Conversion of tannin to gallic acid was 90.9%. Diethyl ether was used as the solvent for extraction of gallic acid from the fermented biomass. Received 14 December 1998/ Accepted in revised form 17 June 1999     

Synthesis of the tobacco mosaic virus in blocked spreading of infection

The possibility of infection of tobacco upper leaves with tobacco mosaic virus (TMV) was examined in experiments where the inoculum was imbibed through the cut stem. The inoculum used were: a) a preparation of a virus-specific informosome-like ribonucleoproteins (vRNP) isolated from TMV-infected plants; b) a TMV preparation; or c) a mixture of TMV and vRNP. Multiplication of TMV in upper leaves was observed in neither of the variants; nevertheless in the vascular tissue and/or probably in adjoining parenchymal cells, two kinds of RNA were synthesized: of mol. w. (1.1--1.3) X 10(6) and (0.6--0.8) X 10(6). These RNA were not found in healthy plants in the presence of actinomycin D. The synthesis of genomic TMV RNA is suppressed under these conditions. Thus, some kind of abortive TMV infection takes place under the condition of experimental inoculation of plants through a cut stem. Molecular hybridization with the DNA of recombinant plasmid containing a nucleotide sequence complementary to the 3'-portion of genomic TMV RNA proves that short RNAs synthesized under the abortive infection conditions are TMV-specific. The experiments with differential temperature treatment of N-gene-containing plants under abortive infection conditions suggest that necrotization is not necessarily induced by genomic TMV RNA synthesis.     

Modelling and Estimation of the Relative Potential for Infection of Winter Wheat by Inoculum of Gaeumannomyces graminis Derived from Propagules and Infected Roots

Abstract A model was developed to estimate the mean number of infections of seminal roots of wheat exposed to two sources of inoculum of the take-all fungus Gaeumannomyces graminis var. tritici , in an experimental system. The sources comprise discrete propagules of initial, soil inoculum and infected roots of volunteer plants that had been infected by the initial inoculum, prior to the growth of crop plants. The model was based on the probability of escape from infection by individual roots ofthe crop plants. Parameter estimation was by maximum likelihood. A model was first fitted to data for infection of roots from the soil inoculum. This yielded estimates for the efficiency of soil inoculum to cause infection in the absence of volunteer plants. The parameter for efficiency of infection by soil inoculum was resolved into components for inoculum density, survival of inoculum and the probability of success of individual propagules. The model was extended to include simultaneous exposure of crop roots to soil inoculum and to root inoculum on the volunteer plants. The presence of volunteer seedlings prior to sowing of crop plants resulted in an increase in the effectiveness of inoculum to cause disease. Sowing date and soil condition, as affected by addition of sand, were shown to have significant effects on the efficiency of both sources of inoculum.     

Effect of feeding yeast culture (Yea-sacc1026) on rumen fermentation in vitro and production performance in crossbred dairy cows

The effect of supplementing diet with yeast culture (Yea-sacc¹⁰²⁶) on dairy cattle was studied by rumen in vitro studies and a feeding trial. Using rumen inoculum from a cow, yeast adapted (YA) and yeast unadapted (YU), incubations were carried out with finger millet (Eleusine coracana) straw (FMS) and a commercial cattle feed (CCF). The 24 h cumulative gas production and digestibility of neutral detergent fibre and acid detergent fibre were not different with YU and YA rumen inoculum for both FMS and CCF. Microbial nitrogen synthesis (mg N ml⁻¹ gas produced) with rumen inoculum from YA feeding regime was higher (P < 0.01) than YU with starch (0.1068 vs. 0.1008) and cellulose (0.0900 vs. 0.0859).A feeding trial was conducted using twelve multiparous cows in midlactation in a switch over design. The cows were divided into two groups of six cows in each group. The duration of the trial was fourteen weeks, each period lasting for 7 weeks. There were no differences in dry matter intake, body weight gain and milk yield. Milk composition for the two groups were also similar.     

培养基和培养条件对栀子悬浮细胞合成多糖的影响

对栀子悬浮细胞合成多糖的调控因子研究表明 :B5为最适培养基 ;5～10d继代周期的细胞可以保持良好的生长状态和多糖的合成能力 ;80g L的鲜细胞的接种量有利于栀子细胞的生长和多糖的合成 ;使用单一碳源时 ,葡萄糖比蔗糖对细胞生长更有益 ,但葡萄糖成本高 ,因而混合碳源 45g L(葡萄糖 :蔗糖 =1∶1)是最佳配方 ;氮源种类对细胞生长和多糖合成没有明显的影响 ,但氮源浓度是主要因素 ,40～50mmol L是最佳浓度 ,同时运用悬浮细胞生产栀子多糖可以通过在不同时间收获的细胞来避免提取时黄色素的干扰 ,具有很好的实际意义。     

Opposite effects of dextran sulfate 500, the polyene antibiotic MS-8209, and Congo red on accumulation of the protease-resistant isoform of PrP in the spleens of mice inoculated intraperitoneally with the scrapie agent

The mode and the site of action of the major antiscrapie drugs have been studied by investigating their effects on the abnormal protease-resistant isoform of PrP (PrPres) and on its accumulation in mouse spleen. Day-by-day PrPres accumulation in the spleen and in other peripheral organs was first monitored to describe the early steps of scrapie pathogenesis. Three phases were identified: the detection of scrapie inoculum on the day of scrapie infection, a clearance phase, and then the peripheral accumulation of PrPres. In a second step, the effects of the polyene antibiotic MS-8209, the polyanion dextran sulfate 500 (DS500), and Congo red were assessed on these phases, after the drugs were coincubated with scrapie inoculum. Highly different mechanisms and sites of action were apparent. MS-8209 had a weak effect on the accumulation of PrPres in spleen, suggesting another site of intervention for this drug. DS500 delayed the beginning of the clearance phase but then blocked PrPres synthesis for a long period of time, probably because of its immunological effects on the spleen. Surprisingly, Congo red suppressed the clearance phase of scrapie inoculum and then increased transiently accumulation of PrPres in spleen. We showed in vitro that this effect was related to a direct enhancement of the protease resistance of PrPres by the drug.     

Methods for inoculating field crops with mycorrhizal fungi

Four methods were compared for inoculating red clover with selected mycorrhizal fungi when sown in a field containing an indigenous mycorrhizal population. The largest amount of mycorrhizal infection (around 65% of root length infected) was obtained by placing inoculum with the seeds in furrows. The inoculum used was standard soil inoculum from stock plant cultures spread by hand or the same inoculum concentrated to about one seventh by wet-sieving, and then fluid-drilled. The effectiveness of multiseeded pellets (seeds stuck onto pellets of soil inoculum) applied broadcast was more variable, infection ranging widely around an average of 30%. Applying both soil inoculum and seeds broadcast produced just under 10% infection, similar to that in the controls given autoclaved inoculum. Seedling establishment, in contrast, was-better where seeds were applied broadcast than in furrows. It seemed therefore that multiseeded pellets might be the best compromise for achieving reasonable infection in most plants, but fluid drilling had the advantages of greatly reducing the amount of inoculum needed and of readily combining seeds and inoculum in a single carrier.