Agar In Petri Dishes Research Articles

Influence of environmental conditions on dispersal of Botrytis cinerea conidia and on post‐harvest infection of gerbera flowers grown under glass

Dispersal of Botrytis cinerea in a gerbera crop grown in two glasshouses 30 km apart was studied over a period of 18 months, in 1988 and 1989. Conidia were caught in spore traps consisting of agar in petri dishes exposed at different heights in the crop in each glasshouse. No seasonal patterns could be identified in the spore catches, assessed as colonies on the agar traps after incubation. The number of lesions caused by conidial infection of gerbera flowers following incubation, however, showed a distinct pattern. In spring and early summer few lesions were recorded whereas at other times of the year many lesions appeared. In linear regression analysis, variation in numbers of colonies (spore catches) could not be explained by environmental factors recorded during the experiments. Linear regression accounted for 77% and 81% of the variation in the number of lesions on flowers in the two glasshouses, in terms of relative humidity (postively correlated), global radiation outside the glasshouse (negatively correlated) and age of the crop (positively correlated). Despite differences in the systems by which the gerbera crop was produced and in the spore catches, the numbers of lesions on gerbera flowers in the two glasshouses were significantly correlated though not significantly different from each other.

Horizontal and vertical distribution of airborne conidia of Botrytis cinerea in a gerbera crop grown under glass

The horizontal and vertical distribution of airborne conidia ofBotrytis cinerea in a gerbera crop in two glasshouses (100 m2 and 350 m2) was studied during 18 months in 1988 and 1989. Conidia ofB. cinerea were caught in simple spore traps consisting of agar in Petri dishes placed in a regular pattern at three different heights in the glasshouse and counted as colonies, after incubation. Lesions due to conidial infection were counted on gerbera petals. The horizontal and vertical distribution of conidia ofB. cinerea in a gerbera crop grown under glass was fairly uniform in both distinct glass-houses. Conidia ofB. cinerea trapped in a glasshouse can originate from sources inside and outside the glasshouse. No significant interaction was found between location and time for the colony counts and for the log transformed (ln(N+1)) lesion counts. The results of this study suggest that spore trapping at one height and at a limited number of locations and dates is sufficient for efficient monitoring ofB. cinerea in a glasshouse.

Resynthesis in Pure Culture of a Common Subalpine Fungus-Root Association Using Phialocephala fortinii and Menziesia ferruginea (Ericaceae)

A strain of Phialocephala fortinii isolated from Luetkea pectinata was used to inoculate axenically grown 1-wk-old Menziesia ferruginea (Ericaceae) seedlings in cellulose agar petri dish cultures. After 3 mo, P. fortinii was observed in cortical cells of whole root mounts, but not in the stele. The fungus produced dark, simple septate hyphae forming extensive wefts on the root surface and intracortical sclerotia consisting of compact masses of darkly pigmented and irregularly lobed, thick-walled hyphae. Isolates of P. fortinii also formed numerous sclerotia in the cellulose agar medium. Presence of P. fortinii caused a ten-fold increase in seedling mortality over controls. Growth rates, based on whole plant fresh weight, were not affected by the presence of P. fortinii. Cenococcum geophilum, Phialocephala dimorphospora, and Phialophorafinlandia, which were also used as inoculum for comparative purposes, did not form sclerotia in M. ferruginea. This is the first report to show that the distinct fungus-root associations formed between P. fortinii and subalpine plants can be reproduced consistently in pure culture under laboratory conditions.

Studies of the life cycle of Gremmeniella abietina on Scots pine in southern Sweden

AbstractAspects of the life cycle of Gremmeniella abietina (Lagerb.) Morelet were studied from 1988 to 1990 in stands of Pinus sylvestris L., 16–32 years old, in southern Sweden, initiated in 1988 with a widespread outbreak of the disease. Pycnidia started to release conidia in late spring and apothecia began to release ascospores in summer. Latent infections could still be detected one year after their establishment by cultivation of healthy looking shoots on agar petri dishes. G. abietina appeared to have a mainly biennal life cycle, as most spores were released two years after infection of the shoot.

Mycorrhiza formed by basidiospores of Tomentella crinalis on Pinus sylvestris

The basidiospores of Tomentella crinalis have been used for synthesizing mycorrhiza with Pinus sylvestris under sterile conditions. The basidiospores germinated in two flasks of four and mycorrhiza developed which resembled, morphologically as well as anatomically, ectendomycorrhiza. The basidiocarps, with toothed hymenophore typical of T. crinalis , also formed on the substrate. Isolates of T. crinalis from characteristic plumose colonies on modified Melin-Norkrans nutrient agar in Petri dishes.

The dispersal of bacteria from leaf surfaces by water splash

J. BUTTERWORTH AND H.A. McCARTNEY. 1991. Advances in the techniques of genetic engineering have made possible the use of genetically manipulated micro‐organisms (GMOs) for the control of pests and diseases. Before GMOs can be widely used in agriculture, however, their fate after release must be understood. Dispersal of released GMOs will have an important influence on their action or survival under field conditions. The object of this study was to quantify the efficiency of rain as a means of removing and disseminating such micro‐organisms from foliar crop surfaces.Spontaneous mutants of three species of bacteria (Pseudomonas syringae, Klebsiella planticola and Bacillus subtilis), resistant to the antibiotic rifampicin, were sprayed on two plant species, french bean, Phaseolus vulgaris, and oilseed rape, Brassica napus. The leaves from the plants were then exposed to artificial rain and splash droplets generated by the impact on the leaves were collected on selective nutrient agar in Petri dishes and in sterile glass 28 ml screw‐capped bottles. The bacterial content of the splashed drops was assessed, as was the content of the water which ran off the leaf surfaces. Comparisons of the numbers of bacteria removed from the leaves with the numbers applied prior to splashing show that rainfall can be a very efficient means of removing bacteria from foliar surfaces, but that most of the removed bacteria run off to the soil. Only a small proportion was splashed relatively short distances from the source.

An Improved Technique for Determining Net Blotch Resistance in Barley

An agar leaf disk technique is described for determining net blotch resistance in barley. Leaves of barley were collected and disinfested with different methods using alcohol and sodium hypochlorite. Twelve disinfested leaf disks were placed on 1.5% water agar in petri dishes with 0, 80, or 120 μg/ml of benzimidazole. Disinfestation of leaf disks with 5% sodium hypochlorite for 1 min, followed by three rinses (5 min) in sterile distilled water and placement on 1.5% water agar containing 80 μg/ml of benzimidazole (...)

Cloning of Primary Human Tumors in Capillary Tube versus Petri Dish: A Head-to-Head Comparison

Plating efficiencies (PEs) of primary human tumors cloned in a capillary assay system were compared to those derived from the conventional two-layer agar Petri dish assay system. A total of 143 consecutively received primary human tumors of 24 different pathologies were simultaneously tested in both assay systems. The successful clonal growth rate in the capillary assay was 82.7%, while in the Petri dish it was 64.7% (p less than 0.001). The median PE was 0.017% in the capillary assay demonstrating a 4.25-fold increase over the 0.004% PE of the Petri dish system. The data confirmed previous results showing that cancer cells of ovarian, breast, and lung origins clone with higher PE in capillary tubes. In contrast, we confirmed that stomach carcinoma cells were the only tumor type that showed a higher PE with the Petri dish method. In addition, this study shows for the first time that lymphomas and renal cell carcinoma, when they survive in vitro, clone equally well in both methods. However, the capillary cloning method resulted in a 66% success rate for lymphoma cell cloning, but Petri dish cloning resulted in only a 33% success rate. Thus, for some types of cancers (i.e., lymphoma), capillary cloning may be advantageous because it improves the probability of obtaining evaluable results. In other cases, the advantage of capillary cloning may be only the decreased amount of specimen and reagents needed for the assay.

Pattern of pore morphogenesis in the resupinate basidiome of Phellinus contiguus

On malt agar in Petri dishes in the light, basidiome development of the resupinate polypore Phellinus contiguus proceeded centrifugally after a minimum but variable colony diameter had been attained. Dissepiments were initiated as more or less separate islands of aerial hyphae approximately 3 mm behind the mostly submerged mycelial margin. Localized aerial fascicles of apically-extending hyphae grew from these island initials both horizontally, where they coalesced with fascicles from adjacent initials and delimited pores, and vertically, resulting in lengthening of dissepiments. On dilute (0.2%) malt agar and towards the edge of dishes containing more concentrated malt agar, initials tended to fail to unite laterally and grow as vertical spines. Horizontal growth of branching and interconnecting aerial fascicles also occurred at the margins of more mature poroid basidiome patches, increasing the poroid area. Time lapse studies of development from widely spaced initials transferred to fresh medium weekly showed that on 0.2% malt agar, areas with basidia and lacking extending aerial hyphae remained as the bases of developing primary tubes. On 2 % malt agar secondary tubes were formed at a later stage by differential vertical growth within dissepiment areas. Where groups of initials were removed poroid basidiome tissue was regenerated mainly by lateral fascicle development from the surrounding basidiome tissue.

Circumventing the diapause of potato cyst nematodes

The diapause of potato cyst nematodes was bypassed by avoiding desiccation of the cysts. Larvae were artificially hatched by cutting the cysts in halves and subsequent incubation in potato root diffusate. Approximately 40% of the cyst content hatched. These treatments had no influence on viability and fecundity as ascertained by rearing nematodes in pots and on roots of sprouts grown on water agar in Petri dishes. With the artificial hatching procedure it is possible to produce five to six generations a year in Petri dishes and three to five generations in pots.

Atividade antifúngica de Bidens pilosa L.

The authors observed the Bidens pilosa L. antifungic activity which was collected from Sao Paulo city. Alcoholic extracts of fresh green plants were prepared for testing. Discs paper were impregnated by the alcoholic concentrate. The antifungic activity was tested in Sabourand dextrose agar in petri dishes against 47 strains of fungi. Sporothrix schenckii, Aureobasidium sp, Aspergillus niger, A. flavus and Rhizopus had not their growth inhibited.

Chemotaxis of a Cyanobacterium on Concentration Gradients of Carbon Dioxide, Bicarbonate and Oxygen

Summary: Trichomes of an Oscillatoria sp. suspended in soft agar in Petri dishes showed active movement away from areas where gaseous exchange had been prevented by placing glass coverslips on the surface. This clearance response occurred only in the light and it was related to gradients of inorganic carbon that formed in the agar layer. In plates incubated in the dark trichomes accumulated in the central areas below glass coverslips but the substance eliciting this response could not be identified. Gradients of diffusible substances were established within lawns of cyanobacteria suspended in soft agar and it was demonstrated that trichomes of Oscillatoria sp. moved towards CO2. HCO3 and O2 in light-dependent chemotactic reactions. No chemotaxis occurred in response to these substances in the dark. The chemotactic responses were detected after 2 h but became increasingly distinct up to 8 h. The chemotactically active concentration of CO2 was greater than the atmospheric concentration since trichomes moved from air towards a source of CO2. Using diffusivity coefficients it was calculated that trichomes of Oscillatoria sp. accumulated at a CO2 partial pressure of 0.02 bar (equivalent to 0.83 mm). With O2 as the chemoattractant the value was 0.35 bar (14.56 mm). These results are discussed with reference to the roles of inorganic carbon and O2 in cyanobacterial metabolism and it is concluded that chemotactic behaviour may be important in movements within the photic zone of sediment environments.

Artificial infection of grasses with endophytes

SUMMARYThe endophytic fungi Acremonium loliae and a Gliocladium‐like sp. were isolated from Lolium perenne; A. coenophialum and a Phialophora‐like sp. from Festuca arundinacea; and Epichloe typhina from F. rubra. All five fungi infected endophyte‐free seedlings of the host grasses and F. arundinacea after artificial inoculation. All fungi except A. coenophialum were able to infect L. perenne. The inoculation technique involved placing endophyte mycelium into the coleoptile tissue of sterile seedlings growing on water agar in Petri dishes. Infection of mature plants with endophytes was not achieved. The presence of some endophytes in grasses can be beneficial to plant growth and persistence but deleterious to the health of animals which graze them. The desirability of infecting cultivars of grasses with endophytes is discussed.

Fruiting and Genetic Analysis ofHeterobasidion Annosum

Several methods for fruiting Heterobasidion annosum are presented. The methods are simple, reproducible, and can be applied to heterokaryotic mycelia isolated from nature as well as the mycelia resulting from crosses of homokaryotic strains. Methods for mutagenesis and recovery of auxotrophic and morphological mutants are discussed. Instances of fruiting in common-^ heterokaryons and in homokaryons are analyzed. The importance of meeting genetic criteria for successful fruiting is discussed. Heterobasidion annosum (Fr.) Bref. [-Fomes annosus (Fr.) Karst.] causes root decay in coniferous trees. A voluminous literature describes the fungus (see Koenigs, 1960, and Hodges, 1969, for reviews). Few reports consider the genetics of H. annosum. This is understandable because of the scarcity of requisite infor? mation on the mating system and reliable methods to promote sexual fruiting. Recent reports (Korhonen, 1978; Chase and Ullrich, 1983) satisfy essential needs for rudimentary information on the mating system. Heterobasidion an? nosum is heterothallic, multiallelic and unifactorial except for homothallic isolates from Australia (Chase, unpubl. data). Reports on the fruiting of H. annosum in laboratory culture are infrequent. Reynolds (1973), Miller (1960), and Boyce (1962) developed nonaxenic methods of fruiting H. annosum. These involve the use of logs which contain H. annosum incubated under moist but nonsterile conditions. These methods are not wellsuited to genetic analysis. There have been few reports of fruiting of cultures of H. annosum under axenic conditions. Liese (1931) obtained fruiting of H. an? nosum on malt extract agar following exposure of cultures to temperatures less than -26 C. Sychev and Negrutskii (1978) obtained fruiting on White's medium, wort agar, and on various types of wood. Cartwright and Findlay (1958) observed occasional fruiting on sterilized wood blocks. Siepmann (1970) induced H. an? nosum to fruit on sterile wood under controlled environmental conditions. Most recently Holt et al (1983) developed a method for fruiting cultures isolated from infected roots and wood, as well as from mycelia resulting from crosses of ho? mokaryotic strains in the laboratory. This method involves a complex medium and long (up to six months) incubation periods. Except for the report of Holt et al (1983) the earlier papers have not included data to permit assessment of the reproducibility of their particular methods. None has utilized a large, diverse group of H. annosum isolates and fruiting has usually occurred only on very complex media. During the course of our genetic studies (Chase and Ullrich, 1983) we observed that many independent heterokaryons of H. annosum fruited when inoculated to standard 1.25% malt extract agar in Petri dishes. We report improvements in this procedure that permit routine genetic analysis in H. annosum.

Quantification of Aspergillus flavus Growth on Inoculated Excised Kernels of Corn Genotypes1

abstractCorn (Zea mays L.) genotypes that resist invasion by Aspergillus flavusor inhibit toxin production would be one major approach to controlling aflatoxin contamination. The primary objective of this study was to determine if a particular laboratory technique could be used to detect differences in responses to A. flavusgrowth among maize genotypes. In addition, several other aspects were considered including a) effect of pollen source, b) kernel sample size, c) incubation period, d) surface sterilization of kernels, and e) stage of kernel development when sampled. Evaluation of corn genotypes involved the removal of kernels from ears of field‐grown hybrids at various times throughout the kernel development period. Kernels were placed on NaCl amended agar in petri dishes and inoculated with a suspension of A. flavusconidia. After incubation, samples were evaluated by recording the extent of A. flavusgrowth on each kernel. Significant genotypic differences were detected at all stages of kernel development sampled. However, these differences were not always consistent over sampling dates nor were they always consistent over years for a given sample date. Visual ratings of kernels sampled early in the kernel development period were higher than the ratings of those kernels sampled toward the end of the maturation process. Surface sterilized kernels promoted more A. flavusgrowth than did unsterilized kernels. Evaluation of F1, seed produced on inbreds revealed significant differences between reciprocal crosses. When a resistant inbred was used as the female parent, the seed gave a resistant response. However, when the susceptible parent was used as a female, A. flavusgrowth on inoculated kernels was markedly greater.

Methanogenic Bacteria from the Bondyuzhskoe Oil Field: General Characterization and Analysis of Stable-Carbon Isotopic Fractionation

Selective enrichment culture techniques were employed to obtain mixed cultures of methanogenic rods and sarcina from surface flooding waters and deep subsurface ( approximately 1650 m) oil-bearing sedimentary rocks and formation waters sampled from an old oil field in the U.S.S.R. previously reported to display active biological methanogenesis. The methanogens were selectively isolated as colonies on agar petri dishes that were incubated in a novel container. The general cellular and growth features of three Methanobacterium isolates were determined. These strains grew optimally at 37 to 45 degrees C in anaerobic pressure tube cultures with a doubling time of 16 to 18 h on H(2)-CO(2) and proliferated as autotrophs. Acetate addition significantly enhanced the final cell yield. Growth of these strains was completely inhibited by either 0.6 g of sodium sulfide per liter or 31.0 of sodium chloride per liter, but growth was not inhibited by either 0.3 g of sodium sulfide per liter or 1.0 g of sodium sulfate per liter. One novel isolate, Methanobacterium sp. strain ivanov, was grown on H(2)-CO(2), and the stable-carbon isotopic fractionations that occurred during synthesis of methane, cell carbon, and lipids were determined. The results of this study were used to examine the anomalous relationship between the isotopic and chemical compositions of natural gas occurring in the deep subsurface environment of the oil field.

Technique for single spore infection by Plasmodiophora brassicae

An improved technique using agar in Petri dishes has been developed for single-spore inoculation of brassicas by Plasmodiophora brassicae. The agar dish technique gave a higher percentage infection than the paraffin drop method. When single spore isolations were made from two populations, ECD 16/31/31 and ECD 22/31/31, they gave rise to a number of different physiologic races, probably indicating that the original populations were mixed. When these single spore isolates were re-tested by successive multisporous transfer through ECD hosts, they continued to show the same host specificity.

Anticancer drug testing [formula omitted]: Use of an activating system with the human tumor stem cell assay

The soft agar tumor colony assay has been adapted for measurement of cytotoxicity of drugs such as cyclophosphamide whose antitumor activity depends upon biotransformation to active metabolites. The S-9 fraction of rat liver, MgCl 2, KCl, glucose-6-phosphate and NADP in phosphate buffer was added to medium containing cells from various continuous human tumor cell lines in the presence and in the absence of drug. After incubation for 1 hour at 37°, cells were washed twice with medium, seeded into 0.3% agar, and plated onto 0.5% agar in petri dishes. Colonies were counted 7 to 21 days later under phase contrast microscopy. Incubation of cells from human lines with cyclophosphamide or heliotrine, an experimental antitumor agent, in the absence of complete activating system caused no or minimal inhibition of colony formation. Incubation of cell lines with cyclophosphamide or heliotrine in the presence of complete activating system markedly reduced colony formation. The cytotoxic effects of both drugs were NADP dependent. This simple technique extends the usefulness of the soft agar stem cell assay to drugs requiring microsomal activation.

Bacteriological Examination of Ice‐cream in The Netherlands: Comparative Studies on Methods

Samples (351) of ice‐cream were examined for ‘total aerobic’ colony count, Enterobacteriaceae, coliforms and Escherichia coli. Different methods for the indicator groups were compared. A number of the samples were also examined for Staphylococcus aureus, Bacillus cereus and Salmonella spp. In addition samples were tested microbiologically to detect fraudulently added inhibitory substances. A percentage (31.1) of the samples showed a total count ≤ 103/ml; 11.0% contained > 105/ml. which is the limit laid down in the Dutch Food Law. The MPN of coliforms (without pre‐enrichment) was ≤1/ml in 46.7% of the samples; 7.4% showed MPNs > 103 ml. Corresponding figures for Enterobacteriaceae (with pre‐enrichment) were 25.4% and 16.8% respectively. A percentage (33.0) did not meet the present Dutch standard for coliforms. The figures for samples that did not meet the standards of the Food Law are somewhat higher than those found by the Food Inspection Departments, probably because the latter generally investigate more samples from large factories. Staph. aureus and B. cereus were found only sporadically. None of 36 samples, selected because they contained appreciable numbers of indicator organisms, was found to contain salmonellas. Of 100 samples 86% showed inhibitory properties to one or more test micro‐organisms. There was no correlation between a positive test and microbial quality. Sometimes, however, the flavouring agent (lemon and chocolate) seemed to exert an inhibitory activity. The hygienic quality of ice‐cream prepared in large factories was better than that of the other samples. The poor quality sometimes reported for ‘soft’ ice‐cream was not confirmed in our investigations. A typically favourable influence of flavour on bacteriological quality could be demonstrated only for lemon ice‐cream. The same values for MPNs of Enterobacteriaceae were found when overnight pre‐enrichment in buffered peptone water was replaced by 2 h resuscitation in tryptone soya broth. Resuscitation for only 45 min in peptone saline yielded lower results. When followed by an Enterobacteriaceae colony count, overnight pre‐enrichment, 2 h resuscitation in tryptone soya broth or 1 h resuscitation on tryptone soya agar in Petri dishes gave almost the same results. No differences were found in favour of MPNs when compared with colony counts of Enterobacteriaceae. The method found most efficient for the detection of Esch. coli in ice‐cream relies on resuscitation followed by enrichment in brilliant green bile lactose broth at 44°C.

Wet-Sieving Floatation Technique for Isolation of Sclerotia ofSclerotium cepivorumfrom Muck Soil

Utkhede, R. S., and J. E. Rahe. 1979. Wet-sieving floatation technique for isolation of sclerotia of Sclerotium cepivorum from muck soil. Phytopathology 69: 295-297. A wet-sieving floatation technique that facilitates the rapid isolation of forceps, surface sterilized in 0.25% sodium hypochlorite for 2.5 min, washed sclerotia of Sclerotium cepivorum from muck soils was developed. Soil in distilled water, and cut in half. The two halves were placed on potatosamples (20 g) were washed through two stacked sieves (0.595 mm openings dextrose agar in petri dishes and kept at room temperature (22-25 C) for 2 over 0.210 mm openings) for at least 5 min, and the residues on the 0.210- wk to allow identification of S. ceptiorum. Approximately 82% of sclerotia mm sieves were transferred to columns containing 2.5 M sucrose solution recovered from naturrall&-infested soils were confirmed to be S. cepivorun. ( 1.330 sp gr). After 2 hr, the soil fractions suspended in the upper portions of The specific gravity of laboratory-reared sclerotia was greater than that of the columns were collected, washed with water on 0.210-mm sieves, and sclerotia produced in the field. examined with a dissecting microscope. Sclerotia were removed with