Carlo Leifert

Dietary nitrate in man: friend or foe?

Based on the premise that dietary nitrate is detrimental to human health, increasingly stringent regulations are being instituted to lower nitrate levels in food and water. Not only does this pose a financial challenge to water boards and a threat to vegetable production in Northern Europe, but also may be eliminating an important non-immune mechanism for host defence. Until recently nitrate was perceived as a purely harmful dietary component which causes infantile methaemoglobinaemia, carcinogenesis and possibly even teratogenesis. Epidemiological studies have failed to substantiate this. It has been shown that dietary nitrate undergoes enterosalivary circulation. It is recirculated in the blood, concentrated by the salivary glands, secreted in the saliva and reduced to nitrite by facultative Gram-positive anaerobes (Staphylococcus sciuri and S. intermedius) on the tongue. Salivary nitrite is swallowed into the acidic stomach where it is reduced to large quantities of NO and other oxides of N and, conceivably, also contributes to the formation of systemic S-nitrosothiols. NO and solutions of acidified nitrite, mimicking gastric conditions, have been shown to have antimicrobial activity against a wide range of organisms. In particular, acidified nitrite is bactericidal for a variety of gastrointestinal pathogens such as Yersinia and Salmonella. NO is known to have vasodilator properties and to modulate platelet function, as are S-nitrosothiols. Thus, nitrate in the diet, which determines reactive nitrogen oxide species production in the stomach (McKnight et al. 1997), is emerging as an effective host defence against gastrointestinal pathogens, as a modulator of platelet activity and possibly even of gastrointestinal motility and microcirculation. Therefore dietary nitrate may have an important therapeutic role to play, not least in the immunocompromised and in refugees who are at particular risk of contracting gastroenteritides.

Investigations into the inhibitory effects of microcystins on plant growth, and the toxicity of plant tissues following exposure.

The cyanobacterial toxins microcystins are known to affect a number of processes in plant tissues, and their presence in water used for irrigation may have considerable impact on the growth and development of crop plants. In this study, two plant bioassays were employed to investigate the phytotoxic effects of microcystins. A plant tissue culture assay revealed that the growth and chlorophyll content of Solanum tuberosum L. cultures was inhibited at microcystin-LR concentrations of 0.005 and 0.05 microg x cm(-3), respectively. A previously developed bioassay was also employed to determine the effects of three commonly occurring microcystin variants on the growth of Synapis alba L. seedlings. Microcystins-LR, -RR, and -LF inhibited the growth of seedlings, with GI50 values of 1.9, 1.6 and 7.7 microg x ml(-1), respectively. The growth of Phaseolus vulgaris was also examined in the presence of microcystin-LR. The toxin was found to have little effect on growth for up to 18 days, but impaired the development of the roots of exposed plants, causing them to take up approximately 30% less growth medium than those grown in the absence of toxin. Microcystin was also detected in the tissues of exposed plants using a commercially available ELISA kit, suggesting that the uptake of these toxins by edible plants may have significant implications for human health.

Ecology of Microbial Saprophytes and Pathogens in Tissue Culture and Field-Grown Plants: Reasons for Contamination Problems In Vitro

Abstract This review compares published surveys of microbial populations in plant tissue and cell cultures with the microbial saprophytes and pathogens found on field grown plants and microbial populations in the laboratory environment. From this comparison and the measured reduction in contamination after improvements in working practices in the laboratory, conclusions can be drawn about the importance of the explant and the laboratory as sources of contamination. Mechanisms of pathogenicity in vitro are described to explain why bacteria, fungi, and yeasts that are not pathogenic to plants in the field become pathogens in plant tissue cultures. Conversely, plant metabolism and its effect on the tissue culture environment are described to explain why prokaryotes, viruses, and viroids that cause disease in the field can stay latent in vitro. Detection methods for latent contaminants in plant tissue cultures are summarized, and the strategies and methods for prevention or treatment of contamination are disc...

Mineral and Carbohydrate Nutrition of Plant Cell and Tissue Cultures

Abstract Recent advances in our understanding of the mineral (and carbohydrate) nutrition of cultured plant cells and tissues are reviewed. The methods used for empirical selection of nutrient composition of culture media for different plant genera/species and types of culture are critically evaluated. The acquisition of nutrients is discussed in terms of their physical availability in the culture medium and uptake from the medium. The effect on uptake of factors such as pH and water potential and the relationship with growth rates and medium depletion are examined in detail. Finally, some effects of nutrients on morphogenesis of plants are reviewed.

Laboratory Contamination Management; the Requirement for Microbiological Quality Assurance

Microbial contamination is the single most important cause of losses in commercial and scientific plant tissue culture laboratories [1,2]. However, even in commercial companies the severity and implications of the problem are not recognised or admitted. Many scientific laboratories fail to record contamination losses, and the micropropagation industry often only recognises the sources of contamination after severe losses have occurred. Following a rapid increase in the production of micropropagated plants in the 1980s [3], there has been a steady decline in the number of micropropagation laboratories in the 1990s, which was at least partially caused by the inability of laboratories to reduce contamination losses to a level which allows a predictable production output and quality of micropropagated plants.

Effects of increased nitrate availability on the control of plant pathogenic fungi by the soil bacterium Bacillus subtilis

In wet soils, low oxygen conditions often develop that favour disease development by many soil-borne plant pathogens. The introduction of a biocontrol agent, to suppress disease development, would require that the agent remains metabolically active under such conditions. Denitrifying bacteria can maintain this metabolic activity by switching to nitrate respiration. In the rhizosphere, plant roots not only supply carbon as an electron donor, but also cause a localised lowering of oxygen concentrations, conditions favourable for nitrate respiration. Two strains of Bacillus subtilis, showing strong inhibition of a number of pathogenic fungi on agar plates, and the capacity to grow under anoxic and anaerobic conditions when provided with nitrate, were used to study the possible involvement of nitrate respiration in fungal disease control. The effect of the addition of nitrate on the activity of these antagonistic strains was studied under anoxic conditions using the sealed plate method of Fiddaman and Rossal [Fiddaman, P.J., Rossal, S., 1995. Plant Pathol. 44, 695‐703]. The assay tests the activity, measured as a reduction in fungal growth, of antifungal volatiles (AFV) produced by the bacteria. The in vitro experiments showed that antagonism by the B. subtilis strains towards Fusarium oxysporumvaried under anoxic conditions, depending on the nitrate availability and agar used as a growth medium. AFV activity was increased by the presence of nitrate in the medium at concentrations of 10 mM or more. Nitrate respiration may therefore have an important role in the control of fungal root diseases by allowing denitrifying soil-borne bacteria to remain metabolically active in wet soils with low oxygen concentrations.

Biological control of Botrytis, Phytophthora and Pythium by Bacillus subtilis Cot1 and CL27 of micropropagated plants in high-humidity fogging glasshouses

Fogging glasshouses are used to acclimatise micropropagated plants for up to 6 weeks after planting [1]. However, plants grown in these high humidity environments are very susceptible to soil-borne damping-off byPhytophthoraand Pythium and attack by air-borne grey mould Botrytisand need to be sprayed frequently with fungicides. This has resulted in the development of resistance within the pathogen population. Biocontrol of fungal diseases by an applied bacterial inoculum is now seen as a viable method of controlling some diseases and several commercial biocontrol agents are available [2]. The often unpredictable/variable activity of the biocontrol agent compared with the fungicide is one of the reasons why biocontrol has not gained a large market share in comparison to fungicides. There are several mechanisms by which biocontrol is thought to work including the production of antifungal antibiotics, competition for nutrients and rhizosphere colonisation [3]. Antibiotic activity by potential biocontrol strains is often determined by in vitro plate assays on artificial media. However, there is as yet no direct evidence that in vitro activity corresponds within vivo activity. The aim of this paper is to compare the biocontrol activity of several Bacillus strains with microplants commonly grown under fogging glasshouse conditions and to determine possible constraints to the biocontrol.

Ventilation of culture vessels. II. Increased water movement rather than reduced concentrations of ethylene and CO2 is responsible for improved growth and development of Delphinium in vitro.

Summary Delphinium plantlets were cultured during Stage III in vessels modified to give different gas exchange rates. Modifications were achieved by the fitting of a covered aperture, or by the sealing of the lids. The concentrations of CO2 and ethylene were measured in conjunction with measurements of relative humidity, water loss, the uptake of mineral nutrients and growth. Both CO2 and ethylene concentrations were low in intact vessels, and significantly higher in vessels sealed with parafilm. Water loss was similar in intact and in sealed vessels, indicating that the vessel lid acted as a good barrier to water but allowed high rates of gas exchange. Plant growth rate was not affected by the level of ventilation of the vessels although leaf area was greater in vessels having lower gas exchange rates. After one week of culture, tissue concentrations of calcium and magnesium were highest in the vessels having the highest gas exchange rates. Relative humidity within the culture vessels was not affected by the level of ventilation. Improved stomatal performance, growth and survival of Delphinium from ventilated vessels appears to be due to an increased flow of water, and not to amelioration of the gaseous environment.

Yeast populations on the tropical timber tree species Milicia excelsa

H. LI, E. VEENENDAAL, N.A. AB SHUKOR, J.R. COBBINAH AND C. LEIFERT. 1995. Yeast populations found on the tropical timber tree species Milicia excelsa showed very little diversity at the genus and species level. Of 62 isolates, 87% were Cryptococcus laurentii, 5%Candida humicola, 3%Candida curvata, 1.5%Candida membranaefaciens, 1.5%Rhodotorula minuta and 1.5%Rhodotorula rubra. Approximately half of the Crypt. laurentii strains had unusual metabolic profiles when compared with the Crypt. laurentii strains in the profile library of the APILAB yeast identification software. All isolated strains were non‐pathogenic and did not show antagonism against Botrytis cinerea in an in vitro plate assay. However, three strains of Crypt. laurentii suppressed disease development of B. cinerea in a leaf disk bio‐assay. This indicates that protection of leaves against opportunistic fungal diseases may be part of the ecological function of Crypt. laurentii populations on Milicia leaves and the potential of this yeast species for biological control.

Effect of medlium acidification on filamentous fungi, yeasts and bacterial contaminats inDelphinium tissue cultures

The persistence of accidentally introduced bacterial contaminants inDelphinium tissue cultures can be prevented by acidification of the tissue culture medium. Using this preservation method the contamination rate ofDelphinium cultures could be reduced by more than 50%.