Water Agar Plates Research Articles

Methods of Evaluating Pecan Cultivars for Tolerance to Feeding Damage Caused by the Black Pecan Aphid, Melanocallis caryaefoliae (Davis)

Two aphid rearing methods were evaluated for their suitability of making biological observations on the black pecan aphid, Melanocallis caryaefoliae (Davis), and as rapid assessments of pecan cultivars for tolerance to aphid feeding damage. There were no significant differences in the biological and feeding damage data between the two experiments. In comparison, the agar method produced less aphid mortality, was easier to maintain, utilized less space, and would allow fewer samples to provide accurate results. ‘Melrose’ was the most tolerant of the nine cultivars evaluated for tolerance to feeding damage by the black pecan aphid using water agar plates. ‘Jubilee’, ‘Suprize’, and ‘82-17-680’ (Wichita open-pollinated) exhibited intermediate tolerance to feeding damage while ‘Salado’, ‘82-17-1316’ (Wichita open-pollinated), ‘Schley’, ‘Oconee’, and seedlings expressed little tolerance to damage caused by feeding. It has yet to be seen if there is a correlation between tolerance to aphid feeding defined by this experiment and actual resistance to the black pecan aphid in the field.

Compatibility of systemic acquired resistance and microbial biocontrol for suppression of plant disease in a laboratory assay

AbstractSystemic acquired resistance (SAR) and microbial biocontrol each hold promise as alternatives to pesticides for control of plant diseases. SAR and Bacillus cereus UW85, a microbial biocontrol agent, separately suppress seedling damping‐off diseases caused by oomycete pathogens. The purposes of this study were to investigate how expression of SAR affected the efficacy of biocontrol by UW85 and if UW85 treatment of plants induced SAR. We devised a laboratory assay in which seedling damping‐off disease, induction of SAR, and growth of UW85 could be quantified. Seedlings of Nicotiana tabacum Xanthi nc were germinated on moist filter paper and transferred after 7 days to water agar plates (40 seedlings per plate). Zoospores of oomycete pathogens (Pythium torulosum, Pythium aphanidermatum, or Phytophthora parasitica) were applied at concentrations that caused 80% seedling mortality within 10 days. Seedling mortality was dependent on zoospore inoculum concentration. The level of disease suppression caused by treatment with UW85 depended on the UW85 dose applied. SAR was induced with 0.5‐mM salicylic acid or 0.1‐mM 2,6‐dichloroisonicotinic acid. Expression of an SAR‐related gene was confirmed by northern analysis with a probe prepared from a tobacco PR‐1a cDNA. Induction of SAR suppressed disease caused by each of the oomycete pathogens, but did not alter the growth of UW85 on roots. Treatment of seedlings with UW85 did not induce the expression of PR‐1a. The combination of induction of SAR and treatment with UW85 resulted in additive suppression of disease as measured by seedling survival.

Pathogenicity of Cylindrocladium scoparium to pasture clover and grass species

Cylindrocladium scoparium was isolated from roots of pasture plant species in the Waikato, North Island, New Zealand. The extent of infection, root colonisation and pathogenicity of this fungus was assessed in a series of laboratory and pot experiments. Seedlings of four legume species and eight grass species grown on water agar plates were infected within 5 days of inoculation with C. scoparium. Emergence of the four legume and three grass species in inoculated sterile sand was significantly reduced following inoculation with C. scoparium. Inoculation of pasture plants grown in fumigated and non-fumigated soil significantly reduced shoot and root dry matter production of several legume and grass species. Root rot was most severe on legumes. These results demonstrate that C, scoparium is a soilborne pathogen of pasture plants and may decrease the yield and persistence of many species.

In vivo passage through calves of nematophagous fungi selected for biocontrol of parasitic nematodes.

The experiment was designed to test the survival and performance of stress selected nematophagous fungi after passage through the gastro-intestinal tract of cattle. Ruminating calves were fed daily with a fixed amount of fungal material grown on barley grains. The excreted dung was collected on days four and five after the start of the feeding experiment. Barley grains were washed out of the excreted dung and incoculated on water-agar plates. After incubation for one week, nine of ten fungal isolates were re-isolated from these plates. The predatory capacity of the fungi in the excreted faeces was tested in a dung pat bioassay and a faecal culture system. In the dung pat bioassay, two fungi of the genus Arthrobotrys and six of the genus Duddingtonia reduced the development of Ostertagia ostertagi third stage larvae by 85% (61%-93%), compared to the number of larvae developed from fungus-free control pats. In seven out of these eight isolates, the reduction of larvae in the faecal cultures was 92% (76%-99%).

The potential transmission of clover rot, Sclerotinia trifoliorum Erikss., by slugs

AbstractSlugs of three species, Deroceras reticulatum, Arion fasciatus and A. subfuscus, which had fed on sclerotia or apothecia of Sclerotinia trifoliorum, transmitted the disease to water agar plates. In the case of Arion spp., transfer of excreta also produced infection.After feeding on apothecia D. reticulatum transmitted the disease to white clover plants, although the direct transfer of apothecia produced higher levels of infection. The visible symptoms of the disease were reduced when slugs were allowed to remain on the plants, apparently owing to a feeding preference of D. reticulatum for diseased leaf tissue.

Conidial traps — a new survival structure of the nematode-trapping fungus Arthrobotrys oligospora

Conidia of A. oligospora germinated directly into adhesive traps when applied close to cow faeces on water agar plates. With preincubation periods of 2–9 days before inoculation, around 90% of the conidia germinated as conidial traps close to the faeces and as normal germ-tubes further away from the faeces. Conidial traps appear to develop in response to diffusing substances from cow faeces, rather than in response to living nematodes. The conidial trap is considered a survival structure enabling the fungus to overcome fungistasis. Traps adhere to the surface of passing nematodes, thus facilitating the spread of the fungus, before penetration of the nematode cuticle and immobilization of the nematode take place.

Survival and Germination of Urediniospores of Puccinia recondita and P. graminis

Reproducible, uniform inoculum distribution was obtained using a settling tower and transfer paper technique to measure effect of temperature, moisture, light intensity, and host : parasite combination on viability and survival of urediniospores of Puccinia recondita and P. graminis. Significant differences were observed on water agar plates at the P = 0.05 level in percentage of germination among cultures of both species. Significant differences were observed on host leaves at the P = 0.10 level in percentage germination among host : parasite combinations. Germination percent decreased with age of the urediniospore in both species. Increasing light intensity from levels found in the canopy on cloudy days to the intensity found in full sunlight at the top of the canopy significantly increased germination of spores. Survival of urediniospores suspended in the atmosphere was significantly longer during summer conditions than during winter conditions. Wheat leaf and stem rust, incited by Puccinia recondita Rob. ex Desm. f. sp. tritici and P. graminis Pers. f. sp. tritici, respectively, have reduced wheat (Triticum aestivum L.) yields by as much as 50% in the Great Plains region of the USA (Samborski and Peterson, 1960; Eversmeyer and Bender, 1974). Viable urediniospores and dormant mycelia are the principal inoculum sources (Burleigh et al., 1969) that contribute to the early spring establishment of the disease and the eventual development of an epidemic in the Central Great Plains. Given the early occurrence of the disease (Eversmeyer et al., 1988), favorable environments and compatible host: parasite inter

Concave rot of melon fruit caused by two Phomopsis fungi.

Many isolates belonging to the genus Phomopsis were obtained from the concave rot of melon fruit, Cucumis melo, a new postharvest disease. They were divided into four groups of A, B, C and D according to their cultural characters on PSA. Several representative isolates selected from each type of isolates showed clear pathogenicity to melon fruits. Morphologically, isolates were divided into two groups; one having shorter β-conidia and containing colony groups of A and B, and the other having longer β-conidia and containing colony groups C and D. Some isolates of the former group produced perithecial stage on PSA and also on water agar plate containing a fragment of melon fruit. A new variety of Diaporthe melonis Beraha et O'Brien, D. melonis var, brevistylospora Kobayashi et Ohsawa was proposed for the fungus. Although the isolates of the latter did not produce any perithecia on artificial media mentioned above, these were identified as the conidial stage of Diaporthe melonis Beraha et O'Brien var. melonis, Phomopsis cucurbitae McKeen sensu Behara et O'Brien.

A New Species of Helicocephalum from Iowa

During a survey of nematode-destroying fungi in woodland soils from Johnson County, Iowa (Kitz and Embree, 1979) a previously undescribed fungus was isolated on several 2% water agar plates seeded with a pinch of leaves and other plant debris from a riparian woodland composed mainly of Betula nigra, Acer saccharinum and Acer negundo. Sporangiophores typical of the genus Helicocephalum, but not fitting the description for any of the known species in this genus, were observed.

Quantification of parasitism of the soil-borne nematode Criconemella xenoplax by the nematophagous fungus Hirsutella rhossiliensis

A “dilution plate assay” was developed to quantify parasitism of C. xenoplax by H. rhossiliensis. Aliquots of C. xenoplax in suspension (treated with 0.5% NaOCl after extraction from soil by wet sieving and centrifugation) were spread onto 1.5% water agar plates supplemented with 200 parts 10 −6 streptomycin sulfate. After 5 days at 22 ± 2°C, the numbers of C. xenoplax with or without sporulating H. rhossiliensis were determined using a dissecting microscope. The assay was used to measure the rate of decay (disappearance) of parasitized C. xenoplax in field samples incubated in the laboratory. Time required for 50% reduction in number of parasitized nematodes (averaged across all life stages) incubated at −33 kPa soil water potential and at 10,15 or 20°C was 61, 26 or 16 days, respectively. Rate of decay was much faster for parasitized second- and third-stage juveniles than for parasitized fourth-stage juveniles and adults. Rate of infection can be estimated if the number of parasitized nematodes and rate of decay of such nematodes are known.

A procedure for preservation of the mosquito pathogen Culicinomyces clavisporus

An effective storage procedure has been demonstrated for the mosquito pathogen Culicinomyces clavisporus. A mycelial preparation was harvested by filtration, sprayed with a sucrose solution and air dried at 20°C in a laminar flow cabinet until the mycelial mat became crisp. This material was then ground in a hammer mill and particles of less than 355 μm were sieved out. Viability of the particles was assessed by studying mycelial growth and conidial formation when particles were added to water agar plates or to water. The particles retained 100% viability after 9 weeks storage at-20°C or 6 days at 4°C. Preparations lost activity rapidly if stored at 20°C. Conidia produced by this method were pathogenic to mosquito larvae.

Quantitative assay of sporangia and zoospore production by Pythium aphanidermatum with a soaking plain water agar culture method.

A method was devised to assay sporangia and zoospore production of Pythium aphanidermatum quantitatively. One-day old 4-mm mycelial disc from PDA culture was placed centrally on a 2% water agar plate in a Petri dish, and flooded with 4ml of an incubation solution. With this method, sporangium formation and zoospore discharge were more stimulated by Petri's salt solution than Wills's salt solution, soil extract or distilled water as incubation solution. Sporangia and zoospore discharge were more abundant at 30C than at 25 or 20C.

Induction of tumors on Solanum tuberosum L. by Agrobacterium: quantitative analysis, inhibition by carbohydrates, and virulence of selected strains

The Agrobacterium tumorigenesis bioassay of Anand & Heberlein [ 1 ] was modified such that potato ( Solanum tuberosum L. cultivar Red LaSoda or Red LaRouge) tuber discs were submersed in bacterial suspensions for 10 min and then placed on water agar plates. The relationship between log cells ml −1 of inoculum and log tumors per disc was linear from 10 3 to 10 6 cells ml −1 and was maintained from day 6 (when tumors first appeared) to day 16. For A. tumefaciens strain B6: (i) one tumor was produced per approximately 2500 bacterial cells in the initial inoculum, and (ii) the infectivity curve was shallower than that predicted for simple 1-particle or 2-particle infection events. Although at 10 7 cells ml −1 A. rubi, A. rhizogenes , and strains of A. tumefaciens known to harbor the Ti plasmid all produced tumors, the relative tumorigenicity of the strains differed considerably. When supplied at 10 mg ml −1 , uronic acid-containing carbohydrates, including several thought to competitively inhibit bacterial attachment to the host, greatly inhibited tumorigenesis. A crude pectin preparation which could serve as starting material for purification of the host attachment sites for Agrobacterium , was isolated from potato tubers and shown to inhibit tumorigenesis.

腸炎ビブリオ寄生溶菌性Ｂｄｅｌｌｏｖｉｂｒｉｏの生理生態に関する研究

A host sea water(HS) agar plate method for the isolation and enumeration of marine Bdellovibrio, parasitic and lytic to Vibrio parahaemolyticus, was developed in this work. A few Bdellovibrio strains were isolated from coastal sea water and the physiological multiplication phase, similar to that of common bacteria, was observed. The raw sea water samples were inoculated with about107/ml cells of V. parahaemolyticus (Biotype 1) and the related organisms (Biotype 2 and 3), respectively, and incubated at 25°C. After 2-4 days of incubation, population of these bacteria decreased approximately to 104/ml, whereas temporal multiplication of the Bdellovibrio was observed. Using HS agar plate prepared with the Biotype 1, 2 or 3, respectively, 26 Bdellovibrio strains were isolated from coastal waters. These isolates showed highest lytic activity to Vibrio and Pseudomonas organisms among the marine bacterial flora. Lytic action of the isolates to the various terrestrial bacteria varied with the Bdellovibrio strains. The occurrence of some Bdellovibrio resistant strains was observed among the Kanagawa phenomenon negative V. perahaemolyticus.

Physiological and biochemical characterization of aggregation-deficient mutants of Dictyostelium discoideum: detection and response to exogenous cyclic AMP

Forty independently isolated aggregation-deficient (Agg−) mutants of Dictyostelium discoideum, which have been partially characterized genetically, were examined for the ability to respond chemotactically to exogenous cAMP and for certain cellular and extracellular activities thought to be involved in this process. Only five of the mutants failed to produce a statistically significant chemotactic response to cAMP. When shaken in buffer, most of the Agg− mutants produced reduced levels of the cAMP-binding sites and the cell-bound cAMP phosphodiesterase reported to be components of a cell-surface cAMP receptor involved in the chemotactic response. In addition, most of the mutants failed to form contact sites A and were unable to develop on water agar plates. When the mutants were pulsed with 100 nM cAMP, 36 of 40 mutants produced detectable contact sites A and three-quarters of these strains underwent further development when placed on a solid substratum. Treatment with cAMP pulses also stimulated certain mutants to form higher levels of cAMP receptors. These observations suggest that the impaired differentiation of the plasma membrane of many Agg− mutants is due, at least in part, to the abnormal synthesis and (or) secretion of cAMP by these strains. When grown in bacterial suspension, all 40 of the mutants produced extracellular phosphodiesterase (ePD) activity, and all but 8 of the mutants secreted detectable amounts of the ePD inhibitor. In general, Agg− strains carrying mutations at the same aggregation locus (aggF–J) exhibited very similar physiological and biochemical properties.

Recovery of Gloeocercospora sorghi from sorghum seed and soil, and its significance in transmission.

Gloeocercospora sorghi was recovered from 68% of sorghum seeds collected from the severely-diseased field at Ebina (Kanagawa prefecture), the emergence rate of these seeds grown in autoclaved soil was as low as 20%, and 80% of the emerged seedlings were diseased with lesions on their leaves. The fungus was recovered from stems near the ground from 2 out of 39 young seedlings originated from pathogen-free seeds and grown in the diseased soil collected from Ebina, but no fungus was recovered from seedlings grown in autoclaved soil. The conidia, which were variable in size and number of septation in media tested, germinated 100% within 4hr on water agar (WA) plates at 20-30C. Sclerotia prepared from 30-day-old culture also germinated 96%, and 22% of them formed sporodochia directly on them on WA in 4 days after seeding. The fungus might penetrate within 2hr and symptoms developed within 24hr after inoculation. Results indicate that the fungus is transmitted through infected seed, and also survives in soil with a potentiality causing infection.

CROWN GALL TUMORIGENESIS IN POTATO TUBER TISSUE

Potato tuber discs were examined as a possible quantitative bioassay for studying tumor induction by Agrobacterium tumefaciens. Discs from two potato varieties, Pontiac and Russett Burbank, were inoculated and cultured on water agar plates. Tumors appeared within 10 days. Both the number and weight of tumors per disc increased linearly for inoculum concentration between 1 × 107–1 × 109 cells per ml. Polarity, position of the disc relative to tuber epidermis, potato variety and the light conditions did not influence the tumor formation. The simplicity of the procedure and the homogeneity of the tissue, together with the fact that it is a quantitative bioassay, makes the potato tuber disc an ideal system for the investigation of biochemical step(s) associated with the transformation process.

A simple method for inducing sporulation in seed-borne fungi

A simple technique for inducing mass sporulation and for the study of morphological characters of fruiting structures in a number of seed-borne fungi has been worked out. Young cultures are grown on autoclaved leaves of Pennisetum glaucum (pearl millet) planted on the surface of water agar plates. Sporulation of a wide range of fungi was observed within a short period of incubation. The extent of sporulation was enhanced by incubating the cultures under near u.v. radiation but different fungi showed diverse requirements for initiation of sporulation. The advantage of the technique for mass sporulation of different fungi and its application are discussed.

Morphological and pathogenic variations in the isolates of <i>Rhizoctonia solani</i> in Bangladesh

Rhizoctonia solani isolates were collected from soil of different agro-ecological zones of Bangladesh and also from infected plant parts of different crops and grasses. Collected isolates were classified into five different cluster groups on the basis of morphological and cultural characters. Five isolates taking one from each of the five different cluster groups were selected to study their pathogenicity and host range on 35 different crops. Pathogenicity and host range of the isolates were determined by planting the seeds in water agar plate infested with R. solani isolates and incubated at 25°C temperatures. After analyzing the morphological and cultural characters of the isolates, it was found that there was no relations between the isolates with respect to their origin from where they were collected. It indicated that the diversity among the isolates was not correlated with their origin. In case of host range and pathogenicity among the five selected isolates of different cluster groups, the isolate JES-16 was an avirulent isolate. The isolate SYL-30 had narrow host range and a low virulent isolate. The isolates DIN-8 and GAZ-18 possessed wide host range and might be considered as virulent isolates. The isolate GAZ-9 was a highly virulent isolate with a wide host range. Keywords: Rhizoctonia solani; morphological and pathogenic variations; isolates. DOI: 10.3329/bjar.v35i3.6443Bangladesh J. Agril. Res. 35(3) : 375-380

Isolation of amoebae from the air.

Summary In preliminary studies in a hospital in Manchester in which tissue culture was used for cultivation, Hartmannella castellanii was isolated from the air on two occasions and from surface samples on one. Water agar plates with a lawn of Klebsiella aerogenes were exposed in a slit sampler, or as settle plates, in and outside a laboratory in London and the following amoebae were isolated: 12 strains of H. castellanii, 7 of H. leptocnemusjagricola, 5 of H. glebae, 1 of H. agricola, 3 of Naegleria sp. and 1 of Schizopyrenus sp. In outdoor air an average of one amoebic cyst of any species was found in 322 cu. ft (9-1 m3) and one cyst of H. castellanii in 644 cu. ft (18·2 m3). All the isolates of H. castellanii damaged HeLa cells. Four that grew poorly or not at all at 37°C showed a marked cytopathic effect at 33°C. An identification key is given.