Effects of water activity, leucine and thiamine on production of aroma compounds by Ceratocystis fimbriata

Ceratocystis fimbriata was grown in a standard liquid medium to determine the production of aroma compounds as affected by thiamine addition to the inoculum, thiamine or leucine addition to the medium, and the effect of water availability. Ethanol constituted more than half of the total volatiles production in the headspace, followed by ethyl acetate (22.6%), ethyl butyrate (10.8%), isobutanol (7.6%), amyl alcohol (1.6%), isoamyl acetate (1.5%), acetaldehyde (1.2%), ethyl propionate (0.9%), isobutyl acetate (0.4%), diacetyl (0.6%) and isoamyl alcohol (0.3%). Although significant two-way interactions were observed (P < 0.05), production of volatile compounds tended to be higher in inocula prepared with thiamine (T+) than in inocula without thiamine (T–), and in the standard medium with thiamine (SMT) as compared to the standard medium alone (SM) and the SM with leucine (SML). Also, the reduction of water activity (a _w) resulted in lower quantities of volatiles being produced.     

The fine structure of ascospore shape and development inCeratocystis fimbriata

Ascospore development inCeratocystis fimbriata Ell. & Halst. commenced in an eight-nucleate ascus. A single vesicle formed along the periphery of the ascus from fragments of ascospore delimiting membranes, surrounded all eight nuclei and eventually invaginated, first forming pouches with open ends, then finally enclosing each of the eight nuclei in a separate sac, thus delimiting ascospores. Pairing of the ascospores followed and brim formation occurred at the contact area between two ascospores. Osmiophilic bodies contributed to the formation of brim-like appendages by fusing to the ascospore walls. Additional brims were observed at opposite ends of the ascospores giving them a double-brimmed appearance.Abbreviations AV ascus vesicle - DM delimiting membrane - EV electron translucent bodies - G granules - M mitochondria - N nucleus - OB osmiophilic bodies - PMV plasmamembrane vesicles - PW primary wall - SW secondary wall     

The life-cycle of Apatania fimbriata Pictet in the Breitenbach

The life-cycle of Apatania fimbriata (Pictet, 1843) (Trichoptera: Limnephilidae) was studied in the Breitenbach (Hesse, Federal Republic of Germany) and in the laboratory at 6 °C, 10 °C and 14 °C at 14 h day length. Embryogenesis was mainly temperature dependent in the 4 °C–18 °C range. At a constant 22 °C embryos developed to well-differentiated larvae which did not hatch. In the laboratory experiments, developmental rates increased with increasing temperature (6 °C, 10 °C and 14 °C); larvae went through a dormancy in the third and fourth larval instars; in the field this occurred between November and February. The dormancy synchronized the larval development. Laboratory studies led to the conclusion that the dormancy was not temperature dependent but endogenously determined. The emergence pattern of adults showed a clear diurnal pattern, with a maximum emergence approximately two hours after sunset. A comparison of several years emergence studies showed a decreasing number of females with increasing distance from the spring. A comparison of the dry weight of females between two traps at the stream showed a significantly higher weight from the downstream site. For males no between-site differences were observed.     

In vitro culture of shoots of Pinus caribaea on a liquid medium

Shoot apices of a clone of Pinus caribaea Morelet were cultured and multiplied in vitro by supporting them with their basal cut ends immersed in a liquid nutrient medium.The initial heights of explants and their initial numbers of leaves were positively correlated with the numbers of buds and shoots produced by the explants after a bud induction phase and after a shoot elongation phase. The final numbers of buds and shoots were positively correlated with reductions in the quantities of phosphorus detected in the media and negatively correlated with the numbers of brown leaves produced on the explants.In a comparison between the growth of shoot explants on liquid and solid media, shoots incubated on the liquid medium showed significantly greater increases in length in a four-week period than those cultured on solid medium.This technique, using liquid media, provides a system in which both the nutrient utilization and the growth rates of isolated pine tissues can be readily assessed. Furthermore, the multiplication rate of the tissue can be predicted following the observation of correlated characters early in the micropropagation cycle.     

Exopolysaccharide and extracellular metabolite production by Lactobacillus delbrueckii subsp. bulgaricus, grown on lactose in continuous culture

Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483, when grown on lactose in continuous culture, showed increasing specific yields and volumetric productivities of exopolysaccharide (EPS) with increasing dilution rate. Specific and volumetric productivities of lactate and galactose, as extracellular metabolites, increased in response to the incremental changes in the dilution rate up to 0.4 h^–1. Elevated Y_p/s values determined for EPS (0.025 g EPSg lactose^–1) at the dilution rates of 0.3 h^–1–0.4 h^–1, relative to those determined at lower dilution rates, suggest a diversion of carbon flux towards EPS being associated with the higher rates of growth.     

Effect of liquid culture media on morphology,growth, propagule production,and pathogenic activity of the Hyphomycete,Metarhizium flavoviride

Two isolates of Metarhizium spp. were studied for propagule production, because of their pathogenic activity towards locusts and grasshoppers (Mf189 = M. flavoviride (or M. anisopliae var. acridum) strain IMI 330189, and Mf324 = M. flavoviride strain ARSEF324). Both isolates were grown in seven different liquid media, which have been developed for mass production of various Hyphomycetes, considered as candidates for microbial control of noxious insects. Shake-flask experiments were carried out at 28 °C in the dark. Production was quantified for 72 h and the effects of the tested media were evaluated on propagule concentration, morphology and pathogenicity. Based on preliminary experiments, all tested media were supplemented with 0.4% Tween 80 to avoid the formation of pellets and to produce unicellular propagules. Submerged propagule yields were higher withMf189 than with Mf324 in all seven media. While high concentrations of propagules (1.4 to 2.4 × 10⁸ propagules ml^-1 for MF189 and1.4 to 8.3 × 10⁷ propagules ml^-1 for Mf324) were produced in four media (Adamek, Catroux, Jackson, and Jenkins–Prior media), production of propagules was lower in the three other media (Goral, Kondryatiev, and Paris media). Both isolates produced oblong blastospore-like propagules, except in Kondryatiev medium in which they provided ovoid propagules. In this case, Mf189 submerged propagules looked like aerial conidia, but scanning observations did not demonstrate a typical conidiogenesis via phialides. In Kondryatiev medium, Mf324 submerged propagules were significantly smaller than aerial conidia. Infection potential of submerged propagules was assayed on Schistocerca gregaria. Second-instar larvae fed for 48 h on fresh wheat previously contaminated by a spraying suspension of each inoculum titrated at 10⁷ propagules ml^-1. All seven media produced submerged propagules that were highly infectious for S. gregaria larvae. Shake flask culture assays permitted us to select three low-costmedia, Adamek, Jenkins–Prior, and Catroux for improving scale-up of liquid fermentation focused on mass-production of Metarhizium propagules for mycoinsecticides devoted to locust control. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differential characterization of two leucine aminopeptidases in Drosophila melanogaster

Two leucine aminopeptidases from Drosophila melanogaster larvae have been partially purified. The LAP A and D enzymes have similar biochemical characteristics including molecular weights of 280,000 daltons, Michaelis-Menten constants of 0.05 mM, associations with metal cofactors, and specificities toward natural and chromogenic substrates. They differ in their pH optima and spatial distributions. If the closely linked genes that code for these enzymes have resulted from a tandem gene duplication event, it is suggested that there has been subsequent evolutionary divergence. This would provide Drosophila larvae with two related, but functionally distinct enzymes.Virginia K. Walker was supported by an NRC Predoctoral Scholarship and a Killam Merit Scholarship.     

The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture

Spore yields were measured for various fungal entomopathogens grown in six nutritionally different liquid media with low and high carbon concentrations (8 and 36 g l^–1, respectively) at carbon-to-nitrogen (C:N) ratios of 10:1, 30:1 and 50:1. Six fungi were tested: two Beauveria bassiana strains, three Paecilomyces fumosoroseus strains and one Metarhizium anisopliae strain. Spore yields were examined after 2, 4 or 7 days growth. In general, highest spore yields were obtained in media containing 36 g/l and a C:N ratio of 10:1. After 4 days growth, highest spore yields were measured in the three Paecilomyces isolates (6.9–9.7 × 10⁸ spores ml^–1). Spore production by the B. bassiana isolates was variable with one isolate producing high spore yields (12.2 × 10⁸ spores ml^–1) after 7 days growth. The M. anisopliae isolate produced low spore concentrations under all conditions tested. Using a commercial production protocol, a comparison of spore yields for the coffee berry borer P. fumosoroseus and a commercial B. bassiana isolate showed that highest spore concentrations (7.2 × 10⁸ spores ml^–1) were obtained with the P. fumosoroseus isolate 2-days post-inoculation. The ability of the P. fumosoroseus strain isolated from the coffee berry borer to rapidly produce high concentrations of spores prompted further testing to determine the desiccation tolerance of these spores. Desiccation studies showed that ca. 80% of the liquid culture produced P. fumosoroseus spores survived the air-drying process. The virulence of freshly produced, air-dried and freeze-dried coffee berry borer P. fumosoroseus blastospores preparations were tested against silverleaf whiteflies (Bemisia argentifolii). While all preparations infected and killed B. argentifolii, fresh and air-dried preparations were significantly more effective. These results suggest that screening potential fungal biopesticides for amenability to liquid culture spore production can aid in the identification of commercially viable isolates. In this study, P. fumosoroseus was shown to possess the production and stabilization attributes required for commercial development.     

Toxic activity from liquid culture of Colletotrichum acutatum

Colletotrichum acutatum has become an increasingly important plant pathogen worldwide. With this background, a study was carried out to characterize the toxicity of liquid culture media from different isolates and to identify some properties of the toxic principles. Liquid culture media from all isolates were toxic to rubber leaves and induced the anthracnose symptoms. Toxicity of the culture filtrate was not host specific and toxic substances were thermostable. Acetone soluble fraction of the culture filtrates retained the toxic activity and it was effective even at a concentration of 700 μg dry mycelium mass/ml. This revised version was published online in June 2006 with corrections to the Cover Date.     

Karyotypic variability in plants of Solanum melongena regenerated from callus grown in presence of culture filtrate of Verticillium dahliae

Summary Plantlets were regenerated from calli derived from leaf expiants of three genotypes of Solanum melongena (two parental genotypes and their hybrid). The cytological analysis showed that a) plants regenerated were all mixoploid, b) toxic medium (basal medium added with filtrate culture of Verticillium dahliae) was able to evidence karyotypic differences between genotypes not displayed by plants regenerated from callus grown on control medium, c) chromosomal mosaicism persists up to plant maturity and also in the selfed progeny. The results are discussed in terms of a selective process involving genes controlling chromosome number and/or a direct effect of toxic medium on the activity of the same genes.This research is supported by a grant from ERSO (Ente per la Ricerca e Sperimentazione in Ortoflorifrutticoltura e Sementi) — Regione Emilia Romagna     

Purification and properties of an intracellular leucine aminopeptidase from the fungus,Penicillium citrinum strain IFO 6352

An intracellular leucine aminopeptidase (LAP) fromPenicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 45–50°C whenl-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60°C. The Michaelis-Menten constants forl-Leu-pNA andl-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co²⁺ ions. The activity of EDTA-treated enzyme was restored by addition of Zn²⁺, but reconstitution with Ca²⁺, Mg²⁺ or Mn²⁺ restored some enzyme activity. It is likely that Co²⁺ ions play an important role in the catalysis or stability of thePenicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.     

Production of single-cell oil from prickly-pear juice fermentation by Cryptococcus curvatus grown in batch culture

The biomass of Cryptococcus curvatus, an oleaginous yeast, reached 11 g/l and accumulated 46% (w/w) lipid when grown for 35 h in batch culture on diluted (25%) prickly-pear juice. The C:N ratio of the juice was about 50 g/g. The efficiency of substrate conversion was 0.48 g/g for biomass and 0.22 g/g for lipids. The extracted lipids were mainly oleic (18:1) and palmitic (16:0) acids and the quality of pipid approached that of palm oil.M. Hassan was and P.J. Blanc, A. Pareilleux and G. Goma are with the Département de Génie Biochimique et Alimentaire, URA-CNRS 544, Institut National des Sciences Appliquées. Complexe Scientifique de Rangueil, F-31077 Toulouse Cedex France; M. Hassan is now with the Institute of Nutrition and Food Science, University of Dhaka, Dhaka-1000, Bangladesh.     

The effect of biofungicide Mycostop on Ceratocystis radicicola, the causal agent of black scorch on date palm

In vitro studies were conducted to assess the efficacy of the biofungicide Mycostop on the fungus Ceratocystis radicicola (= Ceratostomella radicicola), which causes black scorch on date palm (Phoenix dactylifera) and the effect salinity on the growth of Streptomyces griseoviridis, the activecomponent of Mycostop. C. radicicola wasisolated from roots of diseased date palm inlandscape settings, nurseries, andplantations. In vitro, the effectiveconcentration (EC₅₀) of Mycostop thatreduced the mycelial growth by 50% was 0.35 g/l of PDA. Mycostop at the rate of 0.35 g/lor greater reduced spore germination,plasmolyzed germlings and reduced sporulationof C. radicicola. In essence it reducedthe inoculum potential of C. radicicola. In sterile soil treated with Mycostop, rootpieces of date palm inoculated with C.radicicola were less necrotic than those inuntreated soil. Streptomycesgriseoviridis in Mycostop did not grow on PDAamended with 5.0% NaCl (–4.28 MPa), but thenumber of colony formed units on PDA amendedwith 1.0 to 4.0% NaCl was not significantlydifferent from a factor that was considered ifMycostop is to be used to manage black scorchin Kuwait. The observations showed the effectsof Mycostop on C. radicicola inlaboratory studies but its efficacy on blackscorch disease of date palms in nurseries andplantations is yet to be determined.     

RFLP tagging of a gene for aroma in rice

Summary We report here the identification of a DNA marker closely linked to a gene for aroma in rice. The DNA marker was identified by testing 126 mapped rice genomic, cDNA, and oat cDNA, clones as hybridization probes against Southern blots, consisting of DNA from a pair of nearly isogenic lines (NILs) with or without the aroma gene. Chromosomal segments introgressed from the donor genome were distinguished by RFLPs between the NILs. Linkage association of the clone with the gene was verified using an F₃ segregating for aroma. Cosegregation of the scented phenotype and donor-derived allele indicated the presence of linkage between the DNA marker and the gene. RFLP analysis showed that the gene is linked to a single-copy DNA clone, RG28, on chromosome 8, at a distance of 4.5 cM. The availability of a linked DNA marker may facilitate early selection for the aroma gene in rice breeding programs.     

马尾松不同部位挥发物对松墨天牛触角电位和行为反应的影响

【目的】探究马尾松健康植株不同位置挥发性物质,比较其不同部位的挥发物的种类差异及含量,分析松墨天牛成虫对健康马尾松各部位挥发物的触角电位反应和嗅觉反应,为开展松墨天牛的行为调控技术提供依据。【方法】采用静态顶空法和气相色谱-质谱联用技术分析了马尾松松针、1 cm枝条、5 cm枝条、20 cm树干、30 cm树干、松油脂6个部位的挥发物,并对松墨天牛雌雄成虫进行触角电位测定和行为反应观测。【结果】6个部位的挥发物提取物中,共检测出36种挥发性物质,各部位挥发物种类相近,相对含量差异较大,但其主要挥发物均为萜烯类物质,主要包括月桂烯、蒎烯、左旋-beta-蒎烯、莰烯等。在触角电位试验中,5 cm枝条挥发物提取物的触角电位值最高,松油脂挥发物提取物的触角电位值最高;在行为反应试验中,各部位挥发物对松墨天牛成虫均有一定的引诱作用,其中雌成虫对5 cm枝条挥发物提取物的趋向性最高,雄成虫对30 cm树干和松油脂挥发物提取物的趋向性最高。【结论】健康状态下,马尾松5 cm枝条及30 cm树干和松油脂中萜烯类挥发物制成的植物源引诱剂对松墨天牛两性成虫具有更好的引诱作用,这将为提高现有的诱捕剂效果提供参考,同时为松墨天牛虫害防治提供科学依据。     

Plasmid instability in an industrial strain ofBacillus subtilis grown in chemostat culture

Summary A pUB110-derived plasmid/Bacillus subtilis host combination was segregationally unstable when grown in chemostat culture with complex or minimal medium and under starch, glucose or magnesium limitation. The kinetics of plasmid loss were described in terms of the difference in growth rates between plasmid-containing and plasmid-free cells (d) and the rate at which plasmid-free cells were generated from plasmid-containing cells (R). Loss of plasmid-containing cells from the population was d dominated. Changes in medium composition and the nature of growth limitation caused variations in both d and R. The plasmid was most stable in glucose-limited chemostat cultures with minimal medium and least stable under starch limitation with complex complex medium. R and d were smaller for cultures in complex media than those in minimal media. Limitation by starch induced expression of the plasmid-encoded HT amylase gene and was associated with increased values of R and d. Magnesium limitation in minimal medium caused a significant increase in d and a decrease in R.Abbreviations Cm chloramphenicol - Kan kanamycin - Cm^r cells resistant to chloramphenicol (5 mg L^–1) - Kan^r cells resistant to kanamycin (5 mg L^–1) - Cm^sKan^s cells sensitive to chloramphenicol and kanamycin     

Improved method of regeneration of black gram (Vigna mungo L.) through liquid culture

Summary A very rapid and efficient regeneration method of Vigna mungo L. has been established using liquid culture. A highly regenerable explant, viz., young multiple shoots obtained by germinating the seeds in 2 mgl⁻¹ (8.9μM) N⁶-benzyladenine-supplemented Murashige and Skoog (MS) medium, was used as a source of tissue to initiate the liquid culture. The liquid medium consisted of half-strength B5 or MS salts supplemented with MS organics, α-naphthaleneacetic acid (0.1 mgl⁻¹, 0.54μM) and N⁶-benzyladenine (0.5mgl⁻¹, 2.2μM). Transferring the growing tissues to fresh medium every third day resulted in ca. 142% increase in the number of shoot buds produced after 24d. Shoot buds elongated on one-third-strength MS (MS_1/3) semisolid medium and plantlets were obtained by transferring the shoots onto MS_1/3 semisolid medium supplemented with indolebutyric acid (1 mgl⁻¹, 4.9 μM).     

Binding of colchicine to a soluble fraction of carrot cells grown in suspension culture

The binding of [³H]colchicine to soluble component prepared from carrot (Daucus carota L.) cells in suspension culture was assayed by the diethylaminoethyl(DEAE)-cellulose powder method. The binding activity was very labile and the time course of the binding indicated that the colchicine-bound complex was also unstable. The reaction was enhanced by vinblastine but lumicolchicine had no effect. The optimum temperature for the reaction was 30° C, and the colchicine binding constant was calculated to be 3.5·10⁴ l mol^–1 at 30° C.     

特基拉芽孢杆菌XK29挥发物2-甲基丁酸对甘薯长喙壳菌的抑制作用研究

【目的】研究特基拉芽孢杆菌挥发物2-甲基丁酸对甘薯长喙壳菌的抑制作用,评价2-甲基丁酸对甘薯黑斑病的防治效果。【方法】采用I-分隔平皿和气相抑菌体系,研究不同剂量的2-甲基丁酸对甘薯长喙壳菌菌丝生长和孢子萌发的抑制作用;使用乳酸酚棉蓝染色观察2-甲基丁酸对甘薯长喙壳菌显微形态的影响;利用荧光探针钙荧光白和溴化丙锭检测2-甲基丁酸对甘薯长喙壳菌细胞壁结构与细胞膜通透性的影响;使用荧光探针2,7-二氯荧光素二乙酸酯检测甘薯长喙壳菌胞内活性氧含量变化;测定谷胱甘肽含量分析病原菌应对氧化损伤能力的改变;通过线粒体脱氢酶活力和丙酮酸含量的检测,分析2-甲基丁酸对甘薯长喙壳菌线粒体功能和能量代谢的影响;评价2-甲基丁酸在甘薯黑斑病防治中的使用效果。【结果】2-甲基丁酸显著抑制甘薯长喙壳菌菌丝生长和孢子萌发,降低其产孢能力,导致菌丝折叠弯曲并形成不连续的空腔。2-甲基丁酸使甘薯长喙壳菌的细胞壁结构改变,细胞膜通透性增加,胞内活性氧含量升高,谷胱甘肽含量显著降低,使病原菌应对氧化损伤的能力下降,线粒体脱氢酶活力和丙酮酸含量显著降低,诱发线粒体功能障碍,干扰细胞能量代谢,最终导致细胞死亡。此外,2-甲基丁酸对甘薯黑斑病也具有良好的防治作用。【结论】2-甲基丁酸对甘薯长喙壳菌具有显著的抑制作用,可作为安全高效的气相抑菌材料用于新型熏蒸制剂的研发。     

Influence of culture conditions on the production of milk-clotting enzyme from Rhizomucor

The ratio of milk-clotting activity to proteolytic activity (MC/PA) was used as an index to determine the quality of milk-clotting enzyme. Solid-state fermentation on wheat bran for 5 days at room temperature gave optimal production for enzyme by Rhizomucor miehei and R. pusillus. A ratio of wheat bran to moisture of 1:0.6 (w/v) gave best results. Adding skim milk powder to the media of R. miehei did not improve the MC/PA ratio but 4% (w/w) of the powder did improve the ratio with R. pusillus. Co-cultivation of R. miehei with R. pusillus did not change the MC/PA ratio.The authors are with the Department of Biotechnogy, Bharathiar University. Coimbatore-641 046 India