Eunice J. Allan

Mycelial Growth and Branching of Streptomyces coelicolor A3(2) on Solid Medium

SUMMARY: Early growth of the filamentous actinomycete Streptomyces coelicolor A3(2) on solid medium was investigated. Growth kinetics were similar to those of filamentous fungi in that total mycelial length and the number of branches increased exponentially at the same specific rate, with attainment of a constant hyphal growth unit. However, both germ tube and branch hyphae showed initial linear, rather than exponential extension. Logarithmic relationships were found between branch order and both branch length and branch number, although the constancy of these relationships varied during growth. Length ratio, obtained from branch pattern analysis, reached a constant value as did the hyphal growth unit, but branch ratio did not become constant during the period of study. There was some evidence of mycelial development analogous to that found in filamentous fungi.

A standard procedure for the micropropagation of the neem tree (Azadirachta indica A. Juss)

Abstract Micropropagated shoots were initiated from leaf explants of the neem tree, Azadirachta indica A. Juss. Regardless of their origin, shoots were successfully produced by culturing leaf explants on Murashige and Skoog medium containing benzylaminopurine (1 mg l–1), kinetin (0.8 mg l–1) and adenine sulphate (6 mg l–1) in complete darkness. These shoots were further multiplied on Murashige and Skoog medium containing benzylaminopurine (0.1 mg l–1), kinetin (0.08 g l–l) and adenine sulphate (0.6 mg l–1). Within 32 weeks, 80 shoots could be produced from a single leaf explant (10 mm×10 mm). Fifty-five percent of these shoots rooted on Murashige and Skoog medium containing indolebutyric acid (1 mg l–1) and all of these grew on transfer to soil.

Behavioural responses of the maize weevil, Sitophilus zeamais, to host (stored-grain) and non-host plant volatiles

BACKGROUND Four-arm olfactometer bioassays were conducted to assess the behavioural responses of the adult maize weevil, Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae), to harvested seeds of host plants, i.e. white maize, yellow maize (Zea mays L.) and winter wheat (Triticum aestivum L.) (Poaceae), and non-host plant materials, i.e. alligator pepper, Aframomum melegueta (Rosk) K. Schum (Zingiberaceae), rhizomes of ginger, Zingiber officinale (Roscoe) (Zingiberaceae), and West African black pepper, Piper guineense Thonn and Schum (Piperaceae). Additional bioassays with host plant volatiles were conducted in the presence of three doses of non-host plant materials. RESULTS Both sexes of the weevil showed strong attraction to maize and wheat seed volatiles, but were significantly repelled (P < 0.001) by odours from A. melegueta, Z. officinale and P. guineense. Furthermore, S. zeamais avoided maize and wheat seeds presented in combination with the non-host plant material at 10% (w/w) and 33% (w/w) levels. CONCLUSIONS A. melegueta, Z. officinale and P. guineense have the potential for use in the protection of stored grains by resource-poor farmers with local access to these plants.

Antifeedant effects of in vitro culture extracts of the neem tree, Azadirachta indica against the desert locust (Schistocerca gregaria (Forskål))

Callus and micropropagated shoots were initiated from leaf explants of the neem tree, Azadirachta indica A. Juss. A variety of whole plant and in vitro cell cultures from neem seedlings of Ghanian origin were tested for insect antifeedant compounds using the desert locust (Schistocerca gregaria (Forskål)). Feeding suppression occurred when whole extracts of seed, leaf, callus, suspension and shoot cultures were tested in no-choice feeding bioassays. Controls of sucrose, carrot callus and the plant growth medium showed no feeding deterrence. Azadirachtin, the main known antifeedant in neem seed kernels, was quantified from a seed extract by HPLC but was not detected in any of the other extracts. Antifeedancy was determined during batch growth of a suspension culture which had been in culture for 5 months; results indicated that antifeedants were still being formed and that levels increased after maximum biomass was attained.

An ELISA for the detection of Bacillus subtilis L-form bacteria confirms their symbiosis in strawberry

Aims: To develop an ELISA for the detection of antigens derived from stable Bacillus subtilis L‐form bacteria and to detect these in plants injected with L‐form bacteria.

The symbiosis of Bacillus subtilis L-forms with Chinese cabbage seedlings inhibits conidial germination of Botrytis cinerea

Aims: To establish whether germination of Botrytis cinerea was affected by the symbiosis of Bacillus subtilis L‐form bacteria with Chinese cabbage.

Induction, growth and antibiotic production of Streptomyces viridifaciens L-form bacteria.

Aims: To induce, cultivate and investigate the characteristics of L‐form bacteria derived from the filamentous actinomycete Streptomyces viridifaciens.

A kinetic study of the colony growth of Streptomyces coelicolor A3(2) and J802 on solid medium

Summary: Colony growth kinetics of Streptomyces coelicolor A3(2), the wild-type strain, and J802, a mutant incapable of utilizing agar as the sole carbon and energy source, have been studied on solid medium. General features comparable to colony growth of filamentous fungi were observed in both streptomycete strains. Hyphal frequency at the margins of colonies was assessed as the number of hyphae crossing an arc of defined length and increased in an exponential manner with distance from the margin, reaching a maximum at approximately 200 μm. Aerial hyphal initials were formed approximately 350 μm from the margin. Colony radial extension was linear, but in the wild-type strain grown at low medium depths, growth was sometimes multiphasic, with successive phases of linear growth each exhibiting a slower radial growth rate than that of the preceding phase. Hyphal frequency at the colony margin decreased as colony diameter increased, indicating significant changes in environmental conditions in the peripheral growth zone during colony growth. In the primary phase of linear growth, the colony radial growth rate of both strains was independent of medium depth and in the absence of a cellophane membrane was independent of glucose concentration. Colony radial growth rate of strain J802 growing on a cellophane underlay increased with glucose concentration. Hyphal frequency of strain A3(2) increased gradually with medium depth but was independent of glucose concentration. In strain J802, hyphal frequency exhibited a strong dependence on both medium depth and glucose concentration. A threshold glucose concentration for growth was not found and inhibition of strain A3(2) occurred above 1 g 1−1. The results agree with many aspects of accepted theories for colony growth of both unicells and filamentous fungi, but suggest production of staling products and/or secondary metabolites to be of equal significance to nutrient limitation in controlling colony development.

Azadirachta indica A. Juss. (Neem Tree): In Vitro Culture, Micropropagation, and the Production of Azadirachtin and Other Secondary Metabolites

Neem (Azadirachta indica A. Juss.) is a multi-purpose tree whose products have been used traditionally for centuries for insecticidal, antiseptic, contraceptive, antipyretic and antiparasitic purposes. In addition, it is used for reforestation and as a source of wood and provider of shade. The fruit produces oil which is used in soaps and detergents while other by-products are used for fertiliser and soil amendments. Over the last two decades, there has been a burgeoning of interest in neem, mainly due to the use of its extracts for insect control (Schmutterer et al. 1981; Schmutterer and Ascher 1984,1987; National Research Council 1992). The recent book by Schmutterer (1995) provides a wealth of information on neem. Plant cell and tissue culture provides scope for improvement of the tree, an opportunity for understanding the biosynthesis of important neem metabolites and the possibility of standardised, year round production of biologically active materials.

Cell suspension culture of Picrasma quassioides: the development of a rapidly growing, shear resistant cell line capable of quassin formation

Summary Picrasma quassioides Bennett ( Simaroubaceae ) produces the tetracyclic triterpenoid quassin. The development of a suspension culture of P. quassioides capable of growing in bioreactors and which produces low levels of quassin is described. The suspension culture was initially highly aggregated and slow growing (doubling time 12 days) but after 2 years cultivation and a number of changes in subculture methods a less aggregated and faster growing (doubling time, 2.9 days) suspension has been achieved. In addition to an increased growth rate the cultures have begun to produce low levels of quassin (30 – 150 μg . g dry weight -1 ). The growth of P. quassioide s suspension cultures in volumes above 100 ml was initially difficult to establish, but after a year growth in 1 litre volumes was achieved followed a year later by growth in a stirred-tank bioreactor. This cell line has been shown to have developed shear resistance in this time.