Lecithinase activity of Proteus vulgaris

Lecithinase production is described as a new biochemical property of P. vulgaris strains grown in a selective agar medium containing brilliant green, crystal violet and lecithin (BCL agar), the authors' own modification of egg-yolk culture medium. By using this BCL agar as a medium inhibiting the swarming growth of P. vulgaris cultures the authors succeeded in identifying 12 lecithinase-positive strains among the P. vulgaris isolates obtained from patients with Crohn's disease. Of 50 P. mirabilis strains tested in parallel none gave the positive test for lecithinase production in this medium.     

The effects of micro-organisms on plant growth

Summary Subterranean clover, tomato, phalaris, and radiata pine were grown with a complete plant- nutrient solution in sterile sand and agar and inoculated with soil suspensions prepared from unsterilized and from sterilized soil.The presence of micro-organisms reduced primary-root growth in all plants and total root growth in most plants. The total numbers of secondary roots were lower in non-sterile treatments but there was a tendency for an increase in the concentrations of secondary roots with the non-sterile plants. Under the test conditions only radiata pine grown in sterile sand produced significantly greater top growth than those grown in the presence of micro-organisms. Root-stunting micro-organisms were shown to occur in each of four different soil types used in the studies but the extent of stunting varied with the soil. In agar, root stunting was observed at 5 days and 9 days after planting (and inoculation) with subterranean clover and tomato respectively.Production and growth of root hairs by subterranean clover was markedly reduced by organisms from all four soils tested, the reduction varying with the soil. In contrast to root-stunting organisms, root-hair suppressing micro-organisms were abundant in soil. Root-hair suppression was apparent in sand after 3 days and is an inhibition of root-hair development rather than microbial digestion of existing root hairs. Only slight root-hair reduction was observed for tomato and phalaris.     

Utilization of phosphorus by pasture plants supplied with myo-inositol hexaphosphate is enhanced by the presence of soil micro-organisms

A range of pasture grass (Danthonia richardsonii and Phalaris aquatica) and legume (Medicago polymorpha, M. sativa, Trifolium repens and T. subterraneum) species showed limited capacity to obtain phosphorus (P) from inositol hexaphosphate (IHP), when grown in either sterile agar (pH 5.0 or 5.5) or sand-vermiculite media (pH 5.0). The total P content of shoots from IHP-supplied plants grown in agar was between 20% and 34% of that for seedlings supplied with an equivalent amount of P as inorganic phosphate (P_i), while in sand-vermiculite, the total P content of IHP-grown plants was between 5 and 10% of control plants. The poor ability of plants to utilize P from IHP resulted in significantly lower tissue P concentrations and, in general, reduced plant dry weight accumulation. In contrast, the P nutrition of plants supplied with IHP was significantly improved by inoculating media with either a cultured population of total soil micro-organisms or with a specific isolate of Pseudomonas sp., selected for its ability to release phosphate from IHP (strain CCAR59; Richardson and Hadobas, 1997 Can. J. Micro. 43, 509-516). In agar and sand-vermiculite media, respectively, the P content of IHP-grown plants increased with inoculation by up to 3.9- and 6.8-fold, such that the dry weight and P content of the plant material were equivalent to those observed for control plants supplied with P_i. However, the response to inoculation was dependent on the growth medium and the source of micro-organisms used. In sand-vermiculite, the cultured population of soil micro-organisms was effective when IHP was supplied at an equivalent level of P_i required for maximum plant growth. By comparison, inoculation of plants with the Pseudomonas strain was only effective at very high levels of IHP supply (×36), whereas in agar a response to inoculation occurred at all levels of IHP. The ability of pasture plants to acquire P from phytate was, therefore, influenced by the availability of IHP substrate, which was further affected by the presence of soil micro-organisms. Our results show that in addition to having an effect on the sorption characteristics of the growth media, soil micro-organisms also provided a source of phytase for the dephosphorylation of phytate for subsequent utilization of P_i by plants.     

The effect of phytase on the availability of P from myo-inositol hexaphosphate (phytate) for maize roots

The effect of adding phytase to the root medium of maize plants on the P-availability of added myo-inositol hexaphosphate (phytin) has been studied in pot experiments. When 40 mM phytin-P in nutrient solution was incubated in quartz-sand for 15 days in the absence of plants, 80% of it could be recovered from the solution as soluble organic P. Maize plants growing on this mixture assimilated P from phytin at rates comparable to those from inorganic phosphate (Pi). At a lower addition rate (2 mM phytin-P) only 10% was recovered in the soil solution, and plant growth was severely limited by P. At this low phytin level, the addition of phytase (10 enzyme units per kg sand) increased the plants' dry weight yield by 32%. The relative increases of the Pi concentration in the solution and of the amount of P in the plants were even higher, indicating that the observed growth stimulation was due to an increased rate of phytin hydrolysis. The enzyme-induced growth stimulation was also observed with plants growing in pots filled with soil low in P, when phytin was added. However, on three different soils the addition rates of phytin and phytase necessary for obtaining a significant phytase effect were both about 10 times higher than those required in quartzsand. It is concluded that the P-availability from organic sources can be limited by the rate of their hydrolytic cleavage.Abbreviation Pi inorganic phosphate     

Cell-wall structure and composition of Penicillium janczewskii as affected by inulin

Penicillium janczewskii, a filamentous fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae), grows rapidly on media containing either sucrose or inulin as carbon sources. Maintenance of P. janczewskii on inulin medium induces secretion of proteins with high inulinase activity but results in a mycelium that easily collapses and breaks. We evaluated the influence of inulin on fungal growth and colony morphology and on cell-wall structure and composition in comparison with growth and wall characteristics on sucrose-containing medium. P. janczewskii grown on Czapek medium with agar containing 1% (w/v) sucrose or inulin showed differences in the color and morphology of the colonies, although growth rates were similar on both carbon sources. Scanning-electron microscopy revealed that the hyphae from fungus grown on inulin-containing medium are much thinner than those from fungus cultivated on sucrose. Ultrastructural analysis of 5 d old cultures using transmission-electron microscopy indicated significant differences in the cell-wall thickness between hyphae grown on inulin or sucrose media. No differences were detected in the overall carbohydrate and protein contents of cell walls isolated from cultures grown on the two carbon sources. Glycosyl composition analyses showed glucose and galactose as the predominant neutral monosaccharides in the walls but showed no differences attributable to the carbon source. Glycosyl linkage composition analyses indicated a predominance of 3-linked glucopyranosyl in the hyphal walls when P. janczewskii was grown on inulin-containing medium. Our results suggest that growth on inulin as the sole carbon source results in structural changes in the mycelia of P. janczewskii that lead to mycelial walls with altered physical and biological properties.     

Nitrogen Metabolism of Lemna minor. I. Growth, Nitrogen Sources and Amino Acid Inhibition

Lemna minor grown in sterile culture on a minerals-sucrose medium can utilize as nitrogen source, in order of increasing growth rate: ammonia, nitrate, a mixture of glutamic and aspartic acids plus arginine, or a balanced mixture of amino acids (hydrolyzed casein). Maximum growth is found with nitrate plus hydrolyzed casein.Many synthetic mixtures of amino acids are unable to support growth. Many single amino acids are inhibitory, and when added (at 2 mm or less) to cultures, growing in the presence of nitrate, cause a decrease in growth rate or even death of the plants (e.g. with alanine, valine, methionine or leucine). Some of these inhibitory effects are also found when the amino acid is added to cultures growing on ammonia or hydrolyzed casein. Arginine was the only amino acid of those tested which gave a marked stimulation of growth when added to cultures growing with inorganic nitrogen.The rapid rate of growth, sterile nature of tissue, decreased biological variation of samples containing many plants and ability to utilize different culture media make this an attractive organism for studies on higher plant metabolism.     

The properties of a biologically formed manganese oxide,its availability to oats and its solution by root washings

Summary Manganese oxide, produced byCorynebacterium sp. in liquid medium was found to be amorphous, probably hydrated and was readily reduced by neutral quinol. Preparations of the oxide had values of n in the formula MnO_n which ranged from 1.76 to 1.88. The oxide was completely available to oats grown in sand culture but only slightly available in a manganese deficient soil. Plants grown under sterile conditions on agar slopes were able to obtain manganese from manganese oxide, indicating that the roots and not associated micro-organisms, were responsible for the solution process. Root washings of oat plants contained substances which dissolved manganese oxides and the activity of these substances increased with increasing acidity. The possible importance of these substances in making soil manganese available to plants is discussed.     

The growth and phosphorus utilisation of plants in sterile media when supplied with inositol hexaphosphate, glucose 1-phosphate or inorganic phosphate

Seedlings of six temperate pasture species, three grasses and three legumes, were grown for 19–24 days in sterile agar or sand-vermiculite media, in the presence of inorganic phosphate (P_i), glucose 1-phosphate (G1P) or inositol hexaphosphate (IHP). Agar (pH 5.0) had a low IHP-sorbing capacity while IHP was almost completely sorbed in sand-vermiculite. P_i and G1P were relatively available in both media. Growth of each species was measured in relation to phosphorus (P) supply and levels of P_i supply at which shoot yields reached 90% of maximum yield (P_crit) were determined. P_crit values were generally higher for the legume species than for the grasses, and were six-fold higher for Trifolium subterraneum L. seedlings when grown in sand-vermiculite relative to agar. When supplied with G1P, seedlings of the six species grew as well as plants supplied with P_i. By contrast, IHP was a poor source of P for plant growth, even when supplied in agar at levels up to 40-fold greater than P_crit. Using the growth of T. subterraneum in the presence of IHP, it was calculated that roots released approximately 0.09 nkat phytase g^-1 root dry wt per day, over 20 days of growth. By supplementing agar containing IHP with phytase from Aspergillus niger (E.C. 3.1.3.8; 0.012 nkat plant^-1, or _∼1.3 nkat g^-1 root dry wt), sufficient P became available to enable T. subterraneum seedlings to grow as well as P_i-supplied plants. These results indicate that while pasture plants can quite effectively use P from some organic P sources (e.g. G1P), the acquisition of phytate-P is limited both by availability of substrate and the capacity of plant roots to hydrolyse available IHP. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biological Control of Damping-Off of Alfalfa Seedlings with Bacillus cereus UW85

We explored the potential of biological control of alfalfa (Medicago sativa L.) seedling damping-off caused by Phytophthora megasperma f. sp. medicaginis by screening root-associated bacteria for disease suppression activity in a laboratory bioassay. A total of 700 bacterial strains were isolated from the roots of field-grown alfalfa plants by using Trypticase soy agar. A simple, rapid assay was developed to screen the bacteria for the ability to reduce the mortality of Iroquois alfalfa seedlings that were inoculated with P. megasperma f. sp. medicaginis zoospores. Two-day-old seedlings were planted in culture tubes containing moist vermiculite, and each tube was inoculated with a different bacterial culture. Sufficient P. megasperma f. sp. medicaginis zoospores were added to each tube to result in 100% mortality of control seedlings. Of the 700 bacterial isolates tested, only 1, which was identified as Bacillus cereus and designated UW85, reduced seedling mortality to 0% in the initial screen and in two secondary screens. Both fully sporulated cultures containing predominantly released spores and sterile filtrates of these cultures of UW85 were effective in protecting seedlings from damping-off; filtrates of cultures containing predominantly vegetative cells or endospores inside the parent cell had low biocontrol activity. Cultures grown in two semidefined media had significantly greater biocontrol activities than cultures grown in the complex tryptic soy medium. In a small-scale trial in a field infested with P. megasperma f. sp. medicaginis, coating seeds with UW85 significantly increased the emergence of alfalfa. The results suggest that UW85 may have potential as a biocontrol agent for alfalfa damping-off, thus providing an alternative to current disease control strategies.     

Reducing carbon: phosphorus ratio can enhance microbial phytin mineralization and lessen competition with maize for phosphorus

We tested the hypothesis that reducing the carbon (C):Phosphorus (P) ratio in rhizosphere soil would reduce bacterial competition with the plant for P from phytin, which would then increase phytin use efficiency for the plant. A three-factor pot experiment was carried out to study the effect of inoculation with a phytin-mineralizing bacterium, Pseudomonas alcaligenes (PA), on maize P uptake from phytin. Two levels of organic P, two levels of inorganic P, and three different PA inoculation treatments were used. When maize plants were grown in low available P soil with phytin, PA transformed soil P into microbial biomass P, which caused competition for available P with plant and inhibited plant uptake. When 5 mg P kg^?1 as KH₂PO₄ was added, inoculation with PA increased soil acid phosphatase activity which enhanced the mineralization rate of phytin. PA mobilized more P than it immobilized in microbial pool and enhanced plant P uptake. We conclude that the decreased C:P ratio by adding small amount of inorganic P in the rhizosphere could drive phytin mineralization by the bacteria and improve plant P nutrition.     

Role of Extracellular Phosphatases in the Phosphorus-Nutrition of Clover

This paper examines the relationship between the ability ofsubterranean clover to use P-esters as sources of P for growth,and the enzymatic hydrolysis of those P-esters at the root surface.Trifolium subterraneum (cv. Mt. Barker) was grown under sterileconditions in porous agar containing either KH₂PO₄ (P₁), 2',3'-cyclicadenosine monophosphate (cAMP) or inositol hexaphosphate (IHP)as the source of P in the medium. Subterranean clover used cAMPas well as P¹ as a source of P for growth, but made little useof IHP. This preference in the use of P-esters was associatedwith differences in the substrate specificities of the externallyaccessible root phosphatases; roots of P-deficient clover grownunder sterile conditions had high hydrolytic activity againstcAMP but not IHP. These results are discussed in terms of anhypothesis on the function of the externally accessible phosphatases,i.e. that the phosphatases are present to recapture P from organicP compounds leaked from the cells. Key words: Organic P, extracellular phosphatase, roots, P uptake, clover     

Photosynthetic Rates of Sporophytes and Gametophytes of the Fern, Todea barbara

The photosynthetic rates of intact sporophytes or gametophytes of the fern Todea barbara grown in sterile culture were measured using an infrared gas analyzer. Sporophytes consisted of single whole plants with roots and leaves grown in tubes of agar. Gametophytes were grown as several plants covering the surface of the agar. Sporophytes had photosynthetic rates at light saturation of 8.50 microliters CO₂ per hour per milligram dry weight and 1,300 microliters CO₂ per hour per milligram chlorophyll, whereas rates for gametophytes were lower, 2.36 microliters CO₂ per hour per milligram dry weight and 236 microliters CO₂ per hour per milligram chlorophyll.     

The influence of medium aeration on in vitro rooting of Australian plant microcuttings

Media with different air filled porosity were compared with standard agar medium for root induction and root elongation for two Australian plants Grevillea thelemanniana and Verticordia plumosa×Chamelaucium uncinatum. Microcuttings from shoot cultures were pulsed for 7 days on a high auxin (40 M IBA), agar-solidified medium in the dark. The rooting of the microcuttings was then compared on standard agar medium (M1, 1/2 MS, no hormones) and on three experimental treatments: – porous-agar medium (1/2 MS, no hormones, 30 g agar l^–1, solidified then blended to provide aeration); – white sand, or white sand wet with M1 medium; and – a sterile propagation mix. The protocol using the propagation mix is referred to as IVS (In Vitro Soil). A separate experiment involved flushing the IVS soil profile with low or normal oxygen. The controls on M1 medium showed low and variable rooting percentages. The rate of root induction and the average total root length per microcutting at final harvest was significantly higher using the IVS protocol, porous-agar or white sand, while addition of agar medium to sand suppressed the percentage rooting and elongation as did flushing the air space in the IVS rooting medium with low oxygen. Other species tested on M1 medium and IVS including Pimelea physodes, Conospermum eatoniae, Verticordia grandis, and a Chamelaucium megalopetalum×C. uncinatum hybrid all showed a significant improvement on the IVS system. The IVS culture technique reduces plant-handling costs.     

Characteristic symptoms of photosynthesis inhibition by herbicides are expressed in photomixotrophic tissue cultures of Nicotiana

A photomixotrophic tissue culture system for Nicotiana plumbaginifolia and N. tabacum has been developed in which a primary symptom (bleching) of the inhibition of photosynthetic electron transport by herbicides can be observed. Photomixotrophic cultures were initiated and maintained in the light on medium containing 0.2–0.3% sucrose or glucose (low-sugar medium) as sole source of respirable carbohydrate. The usual medium for growing heterotrophic cultures contains 2–3% sucrose or glucose (high-sugar medium). Callus grown on low-sugar medium achieved a fresh weight three to four times greater in the light than in the dark and reached about half that of callus grown on high-sugar medium. Carbon-dioxide fixation rates were an order of magnitude higher in cultures grown on low-sugar medium in the light than in those grown on high-sugar medium or in any of the dark-grown cultures. The lightdependent growth and CO₂-fixation rates of cultures grown on low-sugar medium indicated that a major proportion of the weight increase resulted from photosynthesis. Under these photomixotrophic conditions it was found that a number of photosystem-II herbicides, at concentrations which inhibit photosynthetic electron transport, also inhibited the light-dependent component of callus growth, and caused bleaching. These effects could not be demonstrated on high-sugar medium.Abbreviations PSII photosystem II For common names of the herbicides the reader is referred to Weed Res. 19, 401–406 (1979)     

Cytochrome P-450 and the activation of promutagens in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the cellular content of cytochrome P-450 was investigated and shown to be related to the growth phase of aerobic cultures when glucose was the carbon source. When grown on glucose medium the log-phase cells of the diploid strain D5 contained about 9× more cytochrome P-450 than log-phase cells of the diploid strain D4. The D4 cells grown on medium containing glucose contained about 10× more cytochrome P-450 than D4 cell grown on medium containing galactose as carbon source. Cells of strain D4, harvested from log-phase cultures grown on glucose, were capable of metabolizing aflatoxin B₁, dimethylnitrosamine, β-naphthylamine, ethyl carbamate, cyclophosphamide and dimethylsulphoxide to products active genetically in the same cells. The metabolism of the compounds tested was attributed to cyctochrome P-450-dependent mixed-function oxidation since genetic activity was high in log cells grown on medium containing glucose but negligible in log cells grown on medium containing galactose. However, aflatoxin B₁ differed from the other promutagens tested since the genetic activity of this compound in cells grown on galactose medium was similar to the activity in cells grown on glucose medium. This result is discussed in relation to enzyme systems which could metabolize aflatoxin B₁. The results of treating log-phase cells of the strain D5, grown on medium containing glucose, with aflatoxin B₁ and dimethylnitrosamine are presented and compared with the results from the strain D4.     

The effects of soil sterilization, mycorrhizal inoculation, and rates of phosphorus on growth and survival of Kalopanax septemlobus microplants during the acclimatization period

In order to establish some cultural practices that can improve growth and survival of somatic embryo (SE)-derived microplants during the acclimatization period, Kalopanax septemlobus was uninoculated or inoculated with mycorrhizal fungi coded as AMM6 (a mixture of unidentified species of Glomus and Acaulospora collected in a closed mine tailing site in Bonghwa, Korea) during ex vitro and grown in oven-sterilized peat vermiculite medium. After 2 months, treated microplants were transferred into pots filled with the same medium amended with phosphorus fertilizer {0, 2, 4, 8, 16 and 32 mg P [as Ca(H₂PO₄)₂·H₂O] kg medium^?1 coded as P0, P2, P4, P8, P16 and P32, respectively}. At this stage, inoculated plants were greener, with broader leaves and well-developed root systems and had higher survival than the uninoculated ones. After 6 months, inoculated plants were 54 % heavier than the uninoculated counterpart. In sterile medium, total dry weight of uninoculated plants was promoted at P8 and highest at P16. Total dry weight at P16 by uninoculated plants was attained at P4 by the mycorrhiza-inoculated counterpart. In non-sterile medium, total dry weight of inoculated plants was increased at P8. By contrast, uninoculated plants did not respond to the applied P rates. In conclusion, more SE-derived microplants survived and grew better in sterile medium. Maximum benefits from AMM6 was attained with applied 4 and 8 mg P kg medium^?1 (P4–P8) in sterile and non-sterile medium, respectively.     

Fungicide seed treatments increase growth of perennial ryegrass

Field, laboratory and glasshouse experiments were carried out to measure effects of seed treatments with captan or thiram on growth of perennial ryegrass (Lolium perenne L.). Field-sown captan- or thiram-treated seed gave twice as many seedlings as untreated seed. Spaced plants growing from fungicide-treated seed produced almost 6 times more dry matter 16 weeks after sowing than those from untreated seed. This effect, though diminishing with time, was still apparent more than a year after sowing. Fungicides in sterile agar growth medium were phytotoxic to seedlings at concentrations of 10μg/ml and greater. Seedlings grown from treated seeds sown from 5 to 15 mm away from developing colonies of the virulent seedling pathogenFusarium oxysporum Schlecht. were more than 4 times larger than those grown from untreated seeds. Captan-treated seed sown into pots containing field soil produced more and larger seedlings than untreated seed. Methyl bromide fumigation of the same soil also increased both number and size of seedlings. Fungicidal, rather than direct chemical effects, at early stages of seedling growth, account for increased growth of plants from fungicide-treated seed.     

Growth Medium and Phosphorus Supply Affect Cluster Root Formation and Citrate Exudation by Lupinus albus Grown in a Sand/Solution Split-Root System

We examined cluster root formation and root exudation by white lupin (Lupinus albus L. cv. Kiev Mutant) in response to growth medium and phosphorus supply in a sand/solution split-root system. The split-root system consisted of a nutrient solution compartment and a siliceous sand compartment. Phosphorus was applied at 1 (low-P plants) or 50 (high-P plants) μM as KH₂PO₄ to the solution compartment and at 10, 50 or 250 mg P kg⁻¹ as hydroxyapatite (Ca-P) to the sand compartment. In contrast to the high-P plants, P concentration and P uptake in the low-P plants increased with increasing P supply to the sand compartment. The NaHCO₃-extractable P was lower in the rhizosphere of the low-P plants than the high-P ones. The proton extrusion rate by the solution-grown roots of the low-P plants was higher than that of the high-P plants at the early growth stage. For the low-P plants, the proportion of dry root biomass allocated to cluster roots was higher in the solution compartment than that in the sand compartment. The citrate exudation increased in the sand compartment and decreased in the solution compartment with time, showing a lack of synchronization in citrate exudation by two root halves grown in different media. The cluster root proportion and citrate exudation in both compartments decreased with increasing shoot P concentration. An additional experiment with no P added to either root compartment showed that the proportion of cluster roots was about 9% lower in the sand than solution compartments. The results suggest that cluster root formation and citrate exudation can be significantly affected by the root growth medium in addition to being regulated by shoot P status. More P can be exploited from sparingly available Ca-P by the low-P plants than the high-P ones due to greater citrate exudation under P deficiency.     

Attraction of Mexican fruit flies (Diptera: Tephritidae) to bacteria: effects of culturing medium on odour volatiles

Effects of culturing medium on production and emission of volatiles by Pantoea agglomerans (Beijerinck 1888) Gavini et al. 1989 preparations and on attractiveness of the preparations to the Mexican fruit fly, Anastrepha ludens Loew, were investigated. Bacterial cultures in each of four biochemically different types of liquid media emitted different volatiles. Cultures in a medium containing uric acid as its primary nitrogen source emitted more ammonia and 2‐nonanone than the other media. We postulate that the high production of ammonia was because of uricase activity by this uricase (+) strain. Regardless of media type, supernatants emitted more volatiles than preparations containing cells that had been removed from whole cultures and put into distilled water. Attractiveness varied little with biochemical make‐up of the culturing medium although the uric acid and carbohydrate preparations were as a group more attractive than preparations made from the other two media. Supernatants and whole cultures generally were more attractive than cell preparations and non‐inoculated media. Bacteria grown in aqueous uric acid‐based media emitted volatiles similar but not identical to those emitted by bacteria grown on gel (agar or solid) uric acid‐based media in Petri plates.     

Production of proteinase and phospholipase by Paracoccidioides brasiliensis

We have investigated the production of proteinase and phospholipase by 20 different isolates of Paracoccidioides brasiliensis. Isolates were grown in Bacto-peptone, Dextrose, pH 5.5, agar slants, at 27 °C for 30 days, and cultures were transferred onto Petri dishes containing basis medium and bovine serum albumin fraction V and sterile egg yolk as substrates for enzyme production, and incubated at 27 °C. After 30 days net enzyme activity was visualized and quantitavely evaluated, measuring a ratio between colony diameter and diameter of the transparent (proteinase) or white (phospholipase) ring zone surrounding it. Results demonstrated that all isolates had the ability to produce proteinase and phospholipase, even though variability in enzyme production was noted among different isolates of P. brasiliensis. This revised version was published online in June 2006 with corrections to the Cover Date.