Menthol tolerant clones of Mentha arvensis: approach for in vitro selection of menthol rich genotypes

In vitro raised shoots of Mentha arvensis L. were screened for menthol tolerance level by growing them in media containing 0–100 g ml^–1 menthol. A total of 2850 regenerated shoots were step wise screened for menthol tolerance at the concentrations of 50 g ml^–1 followed by 60 and 70 g ml^–1. In this screening, only 30 individual regenerated shoots were able to survive. The clones from the primary screen were inoculated into rooting medium and, after rooting, transferred to pots in the greenhouse. Ultimately, these 30 menthol tolerant clones were multiplied and grown in the field in replicated plots of 2.5×2.5 m sizes. Twigs of 30 clones from the replicated trials were rechecked for tolerant phenotypes at a concentration of 70 g ml^–1 menthol wherein, these survived even after 7 days (secondary screening). These clones were checked for oil and menthol content and were found to be better than the control plants. Out of these 30 plants, five tolerated 80 g ml^–1 menthol (tertiary level screening) and were found to contain the highest amount of menthol per g leaf biomass. Molecular analysis through RAPD showed distinct variation in the profiles of these five plants, in comparison to the control. Using this method the relationship between the primer OPT 04, menthol tolerance and high menthol content character of the genotype was established. Further, a cultivar `Saksham' was released from the selections by CIMAP for superior performance.     

Arsenic concentrations in water and fish from Chautauqua Lake,New York

Synopsis Arsenic persists in Chautauqua Lake, New York waters 13 years after cessation of herbicide (sodium arsenite) application and continues to cycle within the lake. Arsenic concentrations in lake water ranged from 22.4–114.81 g l^–1, = 49.0 ag l^–1. Well water samples generally contained less than 10 g l^–1 arsenic. Arsenic concentrations in lake water exceeded U.S. Public Health Service recommended maximum concentrations (10 g l^–1) and many samples exceeded the maximum permissible limit (50 g l^–1). Fish accumulated arsenic from water but did not magnify it. Fish to water arsenic ratios ranged from 0.4–41.6. Black crappie (Pomoxis nigromaculatus) contained the highest arsenic concentrations (0.14–2.04 g g^–1 ), X = 0.7 g g^–1) while perch (Perca flavescens), muskellunge (Esox masquinongy) and largemouth bass (Micropterus salmoides) contained the lowest concentrations (0.02–0.13 g g^–1). Arsenic concentrations in fish do not appear to pose a health hazard for human consumers.     

Quantification of the contribution of surface outgrowth to biocatalysis in sol-gels: oxytetracycline production by <Emphasis Type="Italic">Streptomyces rimosus</Emphasis>

A technique was developed for differentiating the activity of microbes solely within sol gels by using the contribution of biomass outgrowth. Streptomyces rimosus was immobilised in colloidal silica gels and biomass growth, oxytetracycline synthesis, pH and carbohydrate consumption were compared for UV surface-sterilised gels, untreated gels, and liquid cultures. Absolute and biomass specific oxytetracycline yields were higher for non-sterile gels than for liquid culture. Biomass solely within colloidal silica gels (1.7 mg ml^–1), and gels obtained from colloidal silica modified by addition of larger silica particles (1.2 mg ml^–1) yielded 27 and 21 g ml^–1 oxytetracycline compared with 97 and 104 g ml^–1 for unsterilised gels (3.6 and 5.2 mg ml^–1 biomass) displaying outgrowth. It was therefore apparent that biomass and antibiotic production within the gels was limited and that optimisation requires gel modification.     

A novel substrate for yeast alcohol dehydrogenase – p-nitroso-N,N-dimethylaniline

Yeast alcohol dehydrogenase (EC 1.1.1.1) catalyzes the novel reduction of p-nitro-so-N,N-dimethylaniline with NADH as a cofactor. Apparent kinetic constants for this enzymatic reaction are: V ₂=2.1 s^–1, K _Q=456 M, K _iQ=119 M, and K _P=1.47 mM, at pH 8.9, 25 °C. This reaction is especially useful for the quantitative determination of NAD⁺ and NADH by enzymatic cycling.     

Phosphatase activity ofPenicillium citrinum submerged batch cultures and its relationship to fungal activity

Acid and alkaline phosphatase activities were evaluated using batch fermenter cultues ofPenicillium citrinum, an organism used in studies of fungal functioning in soil. Fungal activity was assessed by monitoring rates of O₂ utilization, glucose utilization, dry weight changes over time, and lengths of FDA-stained hyphae. At low growth rates (7 g dry wt increases·h^–1·ml^–1) and low culture activity, phosphatase activity at both pH 8.5 and 5.5 tended to decrease with culture age, with the exception that phosphatase activity at pH 8.5 peaked during early stationary phase. At higher growth rates (25 g dry wt increase·h^–1·ml^–1) and high culture activity, phosphatase activity tended to remain constant throughout the course of the experiment. The relationship between phosphatase activity and other measures of fungal activity was consistent only at low growth rates for acid phosphatase. These results suggest that phosphatase measurements will be of limited utility in assessing activity, except at low growth rates.     

Phenotypic expression of Vogesella indigofera upon exposure to hexavalent chromium,Cr6+

A eubacterium producing a blue pigment was isolated from a drinking water filter, and subsequently identified as Vogesella indigofera. This bacterium was further investigated for its morphological and biochemical characteristics after exposure to hexavalent chromium, Cr⁶⁺. The threshold Cr⁶⁺ concentration inhibiting the pigment production by V. indigofera was 200–300 g ml^–1 in liquid cultures of nutrient broth and 100–150 g ml^–1 on nutrient agar plates. The Cr⁶⁺ concentration preventing V. indigofera growth was 300–400 g ml^–1 in liquid cultures, but greater than 150 g ml^–1 on agar plates. Moreover, rugose colonies without the blue pigmentation were observed on agar plates amended with 150 (g Cr⁶⁺) ml^–1. The biochemical utilization profiles of the colonies without pigmentation did not differ from the original pigment-producing ones, indicating phenotypic plasticity of this bacterium. The difference of phenotypic expression of V. indigofera under various Cr⁶⁺ concentrations might have potential application as a pollution bioindicator for heavy metals.     

Accumulation of cadmium by immobilizedZoogloea ramigera 115

Summary Zoogloea ramigera 115 was immobilized into beads of calcium-alginate and placed into batch air-bubbled column reactors. In the absence of any added nutrients the immobilized bacterium adsorbed Cd from solutions containing levels of 2 and 20 g ml^–1 per day, over a period of 21 and 20 days, respectively. Adsorption of Cd from solutions containing 20 g ml^–1 Cd was better than 90% for 16 days. Beads treated with Cd at 2 g ml^–1 never adsorbed less than 95% of the metal. Alginate adsorbed Cd as well, but inclusion of cells changed the effectiveness of adsorption. Of a 250 g ml^–1 Cd solution, alginate adsorbed 70.4% Cd in 60 min whereas alginate plus cells adsorbed 90.5% in the same time span. Temperature had no effect on adsorption by immobilized cells at levels of 2 and 10 g ml^–1 Cd. However at higher concentrations, binding was enhanced as temperature increased.Z. ramigera beads were stable during all treatments and for prolonged periods of time (21 days).     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Insecticidal activity of avidin and streptavidin against four species of pest Lepidoptera

Two biotin-binding proteins, avidin and streptavidin, were found to be insecticidal to the larvae of four species of Lepidoptera – light brown apple moth, Epiphyas postvittana (Walker) (fam. Tortricidae), green-headed leaf-roller, Planotortrix octo (Dugdale) (fam. Tortricidae), brown-headed leaf-roller, Ctenopseustis obliquana (Walker) (fam. Tortricidae) and potato tuber moth, Phthorimaea operculella (Zeller) (fam. Gelechiidae). Mortality occurred in all species, but there was a wide range in susceptibility. P. operculella larvae were the most susceptible with an LC₅₀ of respectively, 2.3 g ml^–1 for avidin and 1.4 g ml^–1 for streptavidin after 9 days. E. postvittana larvae had an LC₅₀ of 43.4 g ml^–1 for avidin after 21 days, and C. obliquana larvae of 45.7 g ml^–1 for avidin after 28 days. Although significant mortality occurred in P.octo at the highest doses of avidin, there was no sufficient dose-mortality response to calculate an LC₅₀ for this species. For all species mortality curves were steep over a close range of doses followed by a plateau where mortality did not increase significantly with dose and did not reach 100%. Mortality was significantly affected by the amount of biotin in the diet on which the parental generation had been reared. Where this was rich in biotin, significant mortality of the offspring was much lower: larval offspring of a colony of E. postvittana, reared for five generations on a biotin-free diet had an LC₅₀ of 5.1 g ml^–1 after 14 days compared with 76.7 g ml^–1 for larvae from a colony reared on general purpose diet. The implications for use of these proteins to confer insect resistance on transgenic plants are discussed.     

Metabolic responses of microbiota to diesel fuel addition in vegetated soil

The effects of trees and contamination on microbial metabolic activity, especially that of hydrocarbon degrading bacteria, were compared during phytoremediation to find which conditions increase diesel fuel removal. Diesel fuel utilisation, microbial extracellular enzyme activities and utilisation of Biolog ECO plate carbon sources by soil bacteria were determined during phytoremediation experiments consisting of two separate diesel applications. Diesel fuel removal after 28 days of second diesel application was 20–30% more than after the first application 1 year earlier. Soil microbiota utilised 26–31 of the 31 Biolog ECO plate carbon sources. Carbon source utilisation profiles indicated minor differences in microbiota in soil vegetated with pine compared to microbiota in soil vegetated with poplar. The potential maximum rates of aminopeptidase activity were 10–10² M AMC/h/g dry soil prior to and after second diesel application, except 14days after the second diesel addition, where the rates were at the scale of 10³M AMC/h/g dry soil. The potential maximum rates of esterase activity were 10³–10⁴M MUF/h/g dry soil. The presence of plants did not influence the activity of esterases. The utilisation of diesel by soil bacteria in Biolog MT2 plate assay was higher in contaminated soil, especially when vegetated, than in uncontaminated soil, measured both as lag times and maximum specific utilisation rates. MT2 plate assay detected the biological response after diesel fuel addition better than general activity methods.     

The effects of aflatoxin B1 on immature germinating maize (Zea mays) embryos

Immature maize (Zea mays L.) embryos were treated with aflatoxin B₁ concentrations, ranging from 0.1 g ml^–1 to 25 g ml^–1. Below 5 g ml^–1 aflatoxin B₁, root and shoot elongation was not significantly inhibited. Ultrastructurally, root tip cells showed little deterioration, except a possible diffused clearing in mitochondria and plastids. As the toxin concentration was increased above 5 gml^–1, shoot, and particularly root elongation, was progressively inhibited. Associated with this, there was an apparent decrease in the ribosome population. Furthermore, membranes, particularly the vacuolar membrane, became abnormal and vacuolar distension occurred. At 20 and 25 g ml^–1, these effects were exacerbated, and mitochondria and plastid structure was disrupted. At these concentrations, there was evidence of a disruption in lipid metabolism. The results are discussed in the context of known aflatoxin effects on cellular control mechanisms and ultrastructure in animal systems.     

Reactivation of photosynthesis in the photoinhibited green alga Chlamydomonas reinhardtii: Role of dark respiration and of light

Effect of quality, quantity and minimum duration of light on the process of recovery was investigated in the photoinhibited cells of the green alga Chlamydomonas reinhardtii. Complete and rapid reactivation of photosynthesis took place in diffuse white light of 25 mol m^–2 s^–1. The recovery was partial (< 10%) in the dark. Far red (725 nm), red (660 nm) and blue light (480 nm) in the range of 10 to 75 mol m^–2 s^–1 did not enhance the process of reactivation. Photoinhibited cells incubated in dark for 15 min when exposed for 5 min to diffuse light (25 mol m^–2 s^–1) showed complete reactivation. Even exposure of 15 min dark incubated photoinhibited cells to photoinhibitory light (2500 mol m^–2 s^–1) for 5 s fully regained the photosynthesis. The study indicated a very precise and triggering effect of light in the process of reactivation. The dark respiratory inhibitor KCN and uncouplers FCCP and CCCP increased the susceptibility of C. reinhardtii to photoinhibition and also prevented photoinhibited cells to reactivate fully even after longer period of incubation under suitable reactivating conditions. Of the various possibilities envisaged to assign the role of dark respiration in recovery process, supply of ATP by mitochondrial respiration appeared sound and pertinent.Abbreviations CCCP- carbonyl cyanide m-chlorophenylhydrazone - D1- 32 kDa protein of PS II reaction center - FCCP- carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone - KCN- potassium cyanide - PBQ- phenyl-p-benzoquinone - PFD- photon flux density - SHAM- salicylhydroxamic acid NBRI Research Publication No. 431.     

Effect of zearalenone (F-2) on pea stem,maize root,and rat liver mitochondria

At 5 and 10 g ml^-1 concentration, zearalenone (F-2), a mycotoxin produced by a number of species of the genus Fusarium, causes an inhibition of the oxidative phosphorylation of isolated plant mitochondria, while at 20 and 40 g ml^-1 it causes uncoupling. However, when the mitochondria are pre-incubated for 20 min with F-2, the uncoupling appears to be the prevailing effect. F-2 is also able to inhibit the mitochondrial ATPase activity (Mg²⁺-dependent). Conversely, F-2 (40 g ml^-1) does not alter the ATP level of maize roots and only slightly affects the ATPase activity of pea stem and maize root microsomal fractions. In addition, F-2 (10–40 g ml^-1) inhibits ATP synthesis catalyzed by rat liver mitochondria. It is suggested that the phytotoxicity of F-2, also known for its ability to collapse the transmembrane electric potential of maize roots, may be mainly linked to its ability to increase the proton permeability of the cell, similar to the common uncouplers.Abbreviations F-2 zearalenone - DCCD N,N-dicyclohexylcarbodiimide - PCCP carbonyl cyanide, p-trifluoromethoxiphenylhydrazone - CBT Cerospora beticola toxin     

Utilisation of soil organic P by agroforestry and crop species in the field,western Kenya

A field experiment in western Kenya assessed whether the agroforestry species Tithonia diversifolia (Hemsley) A. Gray, Tephrosia vogelii Hook f., Crotalaria grahamiana Wight & Arn. and Sesbania sesban (L) Merill. had access to forms of soil P unavailable to maize, and the consequences of this for sustainable management of biomass transfer. The species were grown in rows at high planting density to ensure the soil under rows was thoroughly permeated by roots. Soil samples taken from beneath rows were compared to controls, which included a bulk soil monolith enclosed by iron sheets within the tithonia plot, continuous maize, and bare fallow plots. Three separate plant biomass samples and soil samples were taken at 6-month intervals, over a period of 18 months. The agroforestry species produced mainly leaf biomass in the first 6 months but stem growth dominated thereafter. Consequently, litterfall was greatest early in the experiment (0–6 months) and declined with continued growth. Soil pH increased by up to 1 unit (from pH 4.85) and available P increased by up to 38% (1 g P g^–1) in agroforestry plots where biomass was conserved on the field. In contrast, in plots where biomass was removed, P availability decreased by up to 15%. Coincident with the declines in litterfall, pH decreased by up to 0.26 pH units, plant available P decreased by between 0.27 and 0.72 g g^–1 and P_o concentration decreased by between 8 and 35 g g^–1 in the agroforestry plots. Declines in P_o were related to phosphatase activity (R²=0.65, P<0.05), which was greater under agroforestry species (0.40–0.50 nmol MUB s^–1 g^–1) than maize (0.28 nmol MUB s^–1 g^–1) or the bare fallow (0.25 nmol MUB s^–1 g^–1). Management of tithonia for biomass transfer, decreased available soil P by 0.70 g g^–1 and P_o by 22.82 g g^–1. In this study, tithonia acquired P_o that was unavailable to maize. However, it is apparent that continuous cutting and removal of biomass would lead to rapid depletion of P stored in organic forms.     

Effect of phytotoxic metabolites ofFusarium culmorum on barley root and root-hair development

Summary A mixture of gluconic acid and two unidentified organic acids, isolated from the culture filtrate ofFusarium culmorum, grown in glucose-limited continuous culture, were found to be highly toxic to growth of barley seedlings. A concentration of 900 g ml^–1 of the acid totally inhibited germination of barley seeds, with lower concentrations greatly inhibiting growth and development of germinated seeds. Root extension and root-hair production were inhibited at a concentration of 450 g ml^–1 of the acids, but the number of roots per seedling was increased. Toxicity of the acids was promoted at low pH.     

Batch cultivation and astaxanthin production by a mutant of the red yeast,Phaffia rhodozyma NCHU-FS501

Phaffia rhodozyma cells were treated with the mutagenic agent NTG several times and plated on yeast-malt agar containing -ionone as a selective medium. This mutagenesis of the yeast yielded a mutant (NCHU-FS501) with a total carotenoid content of 1454 g g^–1 dry biomass. Temperature and pH had only a slight effect on the volumetric pigment production by the red yeast, however astaxanthin yield and specific growth rate were influenced more significantly by temperature and pH. The optimum inoculum size, temperature and air flow rate for astaxanthin formation by the mutant in a bench-top fermentor were 7.5% (v/v), 22.5°C and 3.6 vvm, respectively. Glucose (1%, w/v) as carbon source yielded the highest volumetric astaxanthin production (6.72 g ml^–1). Peptone (15.8% total nitrogen) was the best nitrogen source for astaxanthin production (6.72 g ml^–1). Pigment formation by the mutant was further improved by increasing the glucose concentration to 3.5%, where the astaxanthin concentration was 16.33 m ml^–1. At 4.5% glucose or above astaxanthin formation was inhibited. Control of the pH of the fermentation broth did not improved pigment production.     

Changes in phosphorus concentrations and pH in the rhizosphere of some agroforestry and crop species

The aim of this work was to assess whether agroforestry species have the ability to acquire P from pools unavailable to maize. Tithonia diversifolia(Hemsley) A. Gray, Tephrosia vogelii Hook f., Zea mays and Lupinus albusL. were grown in rhizopots and pH change and depletion of inorganic and organic P pools measured in the rhizosphere. Plants were harvested at the same growth stage, after 56 days for maize and white lupin and 70 days for tithonia and tephrosia, and the rhizosphere sampled. The rhizosphere was acidified by tithonia (pH change –0.3 units to pH 4.8) and lupins (–0.2 units to 4.9), alkalinised by tephrosia (+0.4 units to pH 5.4), and remained unchanged with maize growth. Concurrent with acidification in the rhizosphere of tithonia there was a decline in resin-P (0.8 g P g^–1). However, there was also a decline in NaOH extractable inorganic P (NaOH-P_i) (5.6 g P g^–1 at the root surface) and organic P pools (NaOH-P_o) (15.4 g P g^–1 at 1.5 mm from the root), which would not be expected without specific P acquisition mechanisms. Alkalinisation of tephrosia rhizosphere was accompanied by changes in all measured pools, although the large depletion of organic P (21.6 g P g^–1 at 5 mm from the root) suggests that mineralisation, as well as desorption of organic P, was stimulated. The size of changes of both pH and P pools varied with distance away from the rhizoplane. Decline of more recalcitrant P pools with the growth of the agroforestry species contrasted with the effect of maize growth, which was negligible on resin-P and NaOH-P_i, but led to an accumulation of P as NaOH-P_o (14.2 g P g^–1 at 5 mm from the root). Overall the depletion of recalcitrant P pools, particularly P_o, suggests that the growth of tithonia and tephrosia enhance desorption and dissolution of P, while also enhancing organic P mineralisation. Both species appear to have potential for agroforestry technologies designed to enhance the availability of P to crops, at least in the short term.     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

Chitinase production byTalaromyces emersonii

Summary Talaromyces emersonii, when grown on medium containing chitin, yielded extracellular chitinase and chitobiase activities of 0.45 mol.h^–1.ml^–1 culture fluid and 1.4 mol. min^–1.ml^–1, respectively, after 2–4 days of growth under pH-controlled conditions. The enzyme system was optimally active at pH 5.0–5.5, c. 65°C and the least stable components had half-lives of 20 min at 76°C, pH 5.0.     

Pathogenicity of Sporotrichum pruinosum and Cladosporium oxysporum,isolated from the bronchial secretions of a patient,for laboratory mice

In this study we have demonstrated the occurrence of Sporotrichum pruinosum and Cladosporium oxysporum in the bronchial secretions of a patient with a presumptive diagnosis of tuberculosis. This observation coupled with the ability of both fungi to cause infection and elicit tissue responses in experimentally infected mice supported a probable etiologic relationship with the patient which could not be confirmed in the absence of histologic evidence. In vitro some antimycotics were tested against S. pruinosum and C. oxysporum by the agar dilution method. Oxiconazole with a minimum inhibitory concentration of 0.1 g/ml^–1 after 72 h and amorolfine at a concentration of 0.001 g/ml^–1 after 72 h were the most active ones against S. pruinosum and C. oxysporum respectively. It is suggested that the isolation of S. pruinosum and C. oxysporum from patients with bronchopulmonary disorders should be viewed with caution. Clinical and laboratory evaluation of such patients should be done critically before arriving at a firm diagnosis.