Soil Extract Agar Research Articles

Laboratory Culture Media-Dependent Biocontrol Ability of Burkholderia gladioli strain B543

Cultivation of a biocontrol agent on a certain medium often results in reduced biocontrol efficacy and alters physiological state. In our previous study, Burkholderia gladioli strain B543 with long-term subculture on tryptic soy agar resulted in significantly reduced biocontrol ability against cucumber damping-off caused by P. ultimum. Therefore, we investigated the influence of laboratory culturing media on biocontrol activity and physiological state of Burkholderia gladioli strain B543 by using long-term repeated culture on a certain medium. When isolate B543 were successionally cultured on King`s B agar (KBA), tryptic soy agar, nutrient agar (NA), or soil extract agar more than 20 times, the isolate cultured on KBA or NA showed a significantly enhanced biocontrol efficacy and higher population density in the rhizosphere of cucumber compared to that of the others. However, the isolates cultured on KBA more than 20 times showed the lowest production of protease, siderophore, or antifungal substance(s), measured by skim milk agar, Chrome-Azurol-S agar, and potato dextrose agar amended with 10% of the culture filtrate, respectively. Our results suggest that adaptation to proper culturing medium can alter biocontrol ability and physiological state, and we must consider laboratory media in optimizing the use of biocontrol agents.

Isolation of novel bacteria and actinomycetes using soil-extract agar medium

Novel bacteria were discovered using an isolation technique consisting of (i) selection of microorganisms that grew on soil-extract agar medium, but not on conventional media, and (ii) detection of small microbial colonies with a microscope. Three bacterial strains thus isolated were provisionally designated Shinshu-th1, -th2, -th 3, and five actinomycete strains, Shinshu-MS-01, -02, -03, -04, -05, respectively. Sequence analysis of their 16S rDNA showed that th1 had 95--96% homology with three unculturable bacteria, and th2 had 96% similarity to Bradyrhizobium sp., one unculturable and one unidentified bacterial strain. A phylogenetic study indicated that both strains were alpha-Proteobacteria belonging to the order Rhizobiales and the family Bradyrhizobiaceae. Since they had low homology (96%) with their close relatives, it is possible that th1 and th2 belong to a new genus. The actinomycetes Shinshu-MS-02 and -03 had 95--96% homology with four strains of Actinomadura, -04 had 95--96% similarity to Streptosporangium and Microbispora, and -05 had 97--98% homology with three strains of Acrocarpospora, Herbidospora and Planotetraspora. According to the phylogenetic study, both 02 and 03 are possibly new species of Actinomadura, -04 of Streptosporangium, and -05 of Acrocarpospora. Shinshu-th 3 and -MS-01 were identified as Mycobacterium cookii and Frankia sp., respectively, having 99% homology with these species.

Populations and functional diversity of bacteria associated with barley, wheat and canola roots

We investigated the effects of field pea (Pisum sativum)-based crop rotations on endophytic bacteria in roots and surrounding soil of cereal and oilseed crops. Barley (Hordeum vulgare), wheat (Triticum aestivum) and canola (Brassica rapa) were each grown (a) following peas inoculated with Rhizobium leguminosarum bv. viceae, (b) following uninoculated peas or (c) in monoculture. At flagleaf (cereal) or flowering (canola) growth stage, populations of soil-extract agar (SEA)-culturable bacteria ranged from log10 7.12 to log10 7.82 cells g-1 soil dry weight in the bulk soil, log10 7.15 to log10 8.12 cells g-1 soil in the rhizosphere, log10 7.77 to log10 10.39 cells g-1 root dry matter (DM) on the rhizoplane, and log10 5.56 to log10 7.63 cells g-1 root DM in root interiors (endophytic bacteria). Differences between treatments in populations and functional diversity of bacteria depended on where the bacteria were sampled in the continuum from bulk soil to root interiors. This affected correlations with crop N and yield because these crop parameters were lowest in monoculture. Thus, populations of endophytic bacteria and functional diversity of rhizospheric bacteria were negatively correlated with crop N accumulation and yields. However, the diversities of endophytic and bulk soil bacteria were positively correlated with crop N and yields. Key words: Community-level physiological profile, crop rotation, substrates, endophytic bacteria, microbial functional diversity, rhizosphere

Morphology of Verticillium dahliae and V. tricorpus on semi-selective media used for the detection of V. dahliae in soil

The morphology of two soil-borne Verticillium species, V. dahliae and V. tricorpus, was studied on two semi-selective agar media, in the absence and presence of soil. Morphology of the fungi differed considerably between the media, with respect to presence and shape of microsclerotia, dark hyphae (i.e. short melanised hyphae attached to the microsclerotia) and dark mycelium (i.e. melanised mycelium throughout the colony). On modified soil extract agar (MSEA), a pectate based agar, V. dahliae always had globose to elongate microsclerotia, without dark hyphae or dark mycelium, whereas V. tricorpus always had dark hyphae or dark mycelium, and microsclerotia, whenever present, were globose to irregular in shape. On ethanol agar (EA), V. dahliae had large microsclerotia and abundant dark hyphae, whereas V. tricorpus did not form microsclerotia, but always abundant dark mycelium. For the first time we observed the formation of dark hyphae by V. dahliae to a great extent. In the presence of soil, most characteristics were less pronounced, and V. dahliae microsclerotia were smaller, but V. tricorpus produced large microsclerotia, even when they were absent in pure culture. Morphological characteristics suitable for discrimination between the two species on MSEA plates in the presence of soil were selected and tested with fresh isolates from agricultural fields. The two fungi could be distinguished using qualitative characteristics and microsclerotial size. Molecular analysis and morphology on potato dextrose agar confirmed all identifications made on soil dilution plates.

Accuracy and precision of population estimates of Verticillium dahliae on growth media in quantitative soil assays

Verticillium dahliae Kleb. is a serious pathogen of many plant species. Growth media used to measure population density of V. dahliae in soil were evaluated for high recovery of the pathogen, as well as accuracy and precision of population estimates from naturally and artificially infested sandy loam soil using the soil dilution method. Recovery of V. dahliae from naturally infested field soil was highest on soil pectate Tergitol agar (SPT), soil extract agar + sodium polypectate (SEAP), modified pectate agar (MPA), potato dextrose agar + streptomycin sulphate (PDAS), and Talboys' prune lactose agar (TPA); however, PDAS and TPA were overgrown with contaminating fungi, making enumeration difficult. Use of SPT medium resulted in the most precise population estimate with a standard error (SE) of 12.6% of the mean followed by use of pectate agar (PA) (SE = 14.8%) and SEAP (SE = 19.1%). Ethanol, biotin, and Dox salts enhanced recovery of V. dahliae from naturally infested soil, but combining Dox salts with ethanol and biotin significantly reduced population density. Soil extract had no significant effects on population density. Accuracy of V. dahliae population estimates from sterile artificially inoculated soil was highest on modified soil extract agar (MSEA) (64%) followed by SPT (58%). However, accuracy of V. dahliae population estimates from nonsterile artificially inoculated soil was highest on SPT (36%). Soil extract is not an essential ingredient and biotin may increase recovery of V. dahliae from soil.Key words: Verticillium dahliae, verticillium wilt, population density, recovery, accuracy, precision.

Influence of soil sample sizes on the assessment of bacterial community structure

When assessing bacterial community structure in soil it is important to establish a satisfactory procedure for sampling. The influence of sample sizes of a forest soil on the assessment of bacterial community structure was investigated. Four sample sizes (0.01, 0.1, 1.0, and 10.0 g) were evaluated. Time of colony appearance on a nutrient-limited soil extract agar was used to characterise the culturable heterotrophic and Pseudomonas communities. Genetic community structure was assessed with denaturing gradient gel electrophoresis (DGGE) of 16S rDNA using a bacterial primer set. The largest variation in the heterotrophic community structure for bacteria was seen when comparing the 0.01 g replicates. Variation was also seen for the 0.1 and 1.0 g replicates. However, there was no significant difference between the 10.0 g replicates. The 0.01 and 0.1 g replicates showed variation in genetic community structure within the sample sizes, whereas variation between the replicates within the larger sample sizes (1.0, 10.0 g) was negligible. Variation in the heterotrophic community structure for Pseudomonas was seen between replicates of all sample sizes. Hence, the size of soil samples influenced the bacterial community structure observed for bacteria, whereas chance seemed to play an important role when looking at a more narrow community structure.

Contribution of cytophaga-like bacteria to the potential of turnover of carbon, nitrogen, and phosphorus by bacteria in the rhizosphere of barley (Hordeum vulgare L.).

The functional potential of bacteria isolated from the rhizosphere of barley (Hordeum vulgare L.) in May, July, and August and cultivated on nutrient-rich substrate (1/10 TSBA) and nutrient-poor substrate (cold soil extract agar) was determined. There was no significant difference in numbers of CFU when counted on nutrient rich or poor substrate. Bacterial numbers increased approximately 3-fold in the rhizosphere soil from May to August but was unchanged in bulk soil over the same period. A total of 4474 randomly isolated bacteria were screened for enzymatic activities involved in carbon turnover (amylase, cellulase, mannanase, xylanase, and chitinase), nitrogen turnover (protease, nitrate and nitrite reductase), and phosphate turnover (phosphatase). In the rhizosphere soil, bacteria carrying C and P turnover enzymes were not stimulated by the growing plant whereas protease and nitrate and nitrite reductase were stimulated by the growing plant. No changes were observed in the bulk soil. Two taxonomic groups were followed: Cytophaga-like bacteria (CLB) and fluorescent pseudomonads, the latter being abundant in the rhizosphere and important contributors to the cycling of organic matter in soil. Unexpectedly in the spring samples, CLB were around 25% of all bacteria isolated, whereas fluorescent pseudomonads made up less than 10%. The relative proportion of these bacterial groups then decreased during the plant growth season but at all times showing a clear rhizosphere effect. Furthermore, up to 70% of the isolates carrying enzymes involved in the turnover of carbon, in the May sample, were identified as CLB, indicating the importance of this group in early colonization of the rhizosphere. The fluorescent pseudomonad group contributed less than 3%.

Evidence that tropical soil environment can support the growth of Escherichia coli

Concentrations of faecal coliforms and Escherichia coli in environmental waters have historically been used to establish recreational water quality standards. When these bacteria are used as indices of water quality, it is assumed that there are no significant environmental sources of these bacteria which are unrelated to direct faecal contamination. However, we have previously reported that in tropical island environments such as in Hawaii, these faecal indicators are consistently found at high concentrations in all streams and the source of these faecal bacteria is the soil. To become so well established in soil we hypothesized that these faecal bacteria must have the ability to multiply in the natural soil environment at ambient temperature (23–25°C). Three lines of evidence support this hypothesis: (1) E. coli was shown to grow on 10% soil extract agar, (2) populations of faecal coliforms and E. coli from sewage were shown to immediately increase by about three logs when simple nutrients (glucose and salts) were added to natural soil and (3) faecal coliforms and E. coli increased by two logs within 24 h when a minimal amount of sewage was added to cobalt-irradiated soil. These results indicate that tropical soil environments provide sufficient means to support the growth of faecal coliforms and E. coli. However, under natural soil conditions, indigenous soil microorganisms are much more efficient in obtaining nutrients and we hypothesize that faecal bacteria grow sporadically in response to available nutrients.

Evidence that tropical soil environment can support the growth of Escherichia coli

Concentrations of faecal conforms and Escherichia coli in environmental waters have historically been used to establish recreational water quality standards. When these bacteria are used as indices of water quality, it is assumed that there are no significant environmental sources of these bacteria which are unrelated to direct faecal contamination. However, we have previously reported that in tropical island environments such as in Hawaii, these faecal indicators are consistently found at high concentrations in all streams and the source of these faecal bacteria is the soil. To become so well established in soil we hypothesized that these faecal bacteria must have the ability to multiply in the natural soil environment at ambient temperature (23–25°C). Three lines of evidence support this hypothesis: (1) E. coli was shown to grow on 10% soil extract agar, (2) populations of faecal coliforms and E. coli from sewage were shown to immediately increase by about three logs when simple nutrients (glucose and salts) were added to natural soil and (3) faecal coliforms and E. coli increased by two logs within 24 h when a minimal amount of sewage was added to cobalt-irradiated soil. These results indicate that tropical soil environments provide sufficient means to support the growth of faecal coliforms and E. coli. However, under natural soil conditions, indigenous soil microorganisms are much more efficient in obtaining nutrients and we hypothesize that faecal bacteria grow sporadically in response to available nutrients.

Interactions of asparagns root tissue with soil microorganisms as a factor in early decline of asparagus

Sterilized root residues of asparagus added at a rate of up to 20gkg‐1 fresh soil did not influence severity of root and crown rot caused by Fusarium oxysporum f.sp. asparagi (Foa). Root residues accumulated in field soil during asparagus growing for 10 years did not influence disease severity either. Inoculation of this soil with laboratory‐prepared Foa after treatment at 65°C (30min), at which the indigenous pathogen was killed but toxic substances present in asparagus root residues were left undamaged, led to the same disease severity as inoculation of similarly‐treated fresh soil.On soil extract agar, aqueous root extracts of asparagus but not those of other crops retarded growth of 31 out of 112 fungal isolates from a range of taxa. Sensitive fungi included Gliocladium spp. and Trichoderma harzianum, but not Foa.Colonization of Foa‐infested soil by Fusarium species was greatly enhanced by addition of root material from asparagus, Brussels sprouts, and chicory, but not by that from strawberry and perennial rye grass. As the fraction of Foa amongst the Fusarium population was small, it is concluded that competitive saprophytic ability of the pathogen is far less than that of the nonpathogenic Fusarium species. Fungistasis to Foa was not or was only slightly reduced in soils amended with root residues.In contrast to data reported in the literature, the present results do not suggest an appreciable increase of Foa root rot., or of the Foa population in soils, due to substances present in root residues.

Physiological restriction of the L‐lactic acid production by Rhizopus arrhizus

AbstractThe influence of basic physiological factors on the quality of inocula and L(+)‐lactic acid production by Rhizopus arrhizus CCM 81 09 were studied.The most effective preparation of the spores (5 × 107 spores/ml) and subsequent good lactate production was achieved on the agar medium with soil extract and malt agar. The optimum initial amount of active spores for inoculation was 103–104 spores/ml. The preparation of inoculum required intensive stirring with lower aeration and pH maintained in the range from 4.8 to 6.0 by the addition of CaCO3. The maximum yield of lactic acid production was achieved by using 5% (v/v) of 24‐h‐old inoculum. The intensity of lactic acid production in the inoculum was proportional to its production in the subsequent steps of fermentation and can be used as a fast control of the physiological state of the producers.

Fruiting of Lyophyllum tylicolor in plate culture on Soytoneglucose agar and urea-treated soil extract agar

Lyophyllum tylicolor, which forms mycelial basidia (and basidiospores), produced fruit-bodies when cultivated at 20°C under continuous illumination of 400–700 lux on agar plates containing Bacto-Soytone and glucose or an extract from urea-treated soil. Under these conditions, mycelial basidia were also observed on the Soytone-glucose agar, but not on the soil extract agar. In darkness, fruit-bodies and mycelial basidia were not observed on either medium. In culture on the soil extract agar, fruit-body primordia were produced at the position of the margin of the colony when it was transferred from darkness to continuous light; stipes did not elongate under illumination of ca. 2000 lux; and mycelial basidia and basidiospores, but not fruit-bodies, developed when glucose concentration in the medium was as high as 1% (w/v).

Germination of Teliospores of Tilletia bardayana, the Causal Agent of Kernel Smut of Rice, in Relation to Some Physical Factors

AbstractTeliospores of Tilletia harclayana germinated equally well on the surface of 2 % water agar, soil extract agar and in cavities made in water agar. Maximum germination occurred at 29 °C under 12 h photoperiod with artificial daylight (cool fluorescent light). There was marked increase in germination when the teliospores in bunted grains were pre‐treated for 30 min at 60 °C. Seventeen‐week‐old tehospores did not germinate while 73 week‐old‐tehospores showed retention of good germinability. Teliospores buried at 2 mm depth showed no germination whereas those at the moist surface germinated to produce sporidia. Implications of these results in the disease development are discussed.

An evaluation of straw-extract agar media for the growth and sporulation of Madurella mycetomatis

Four new media, namely Wheat straw extract agar, Bajra straw extract agar, Jowar straw extract agar and Paddy straw extract agar, were evaluated for their potential to stimulate the growth and sporulation of Madurella mycetomatis in comparison with the conventional Sabouraud dextrose agar and Soil extract agar. Vegetative growth of M. mycetomatis on the four types of Straw extract agars was superior to that obtained on Sabouraud dextrose agar. Isolates of M. mycetomatis sporulated better and faster on the Straw extract agars than on the Sabouraud dextrose agar and Soil extract agar. Straw extract agar is recommended as a sporulation medium for M. mycetomatis. It may prove useful especially for studies of the conidium ontogeny of the fungus for elucidating its taxonomic status.

Biodegradation of Dinoseb (2- sec -Butyl-4,6-Dinitrophenol) in Several Idaho Soils with Various Dinoseb Exposure Histories

We examined the ability of native microorganisms in various Idaho soils to degrade dinoseb and studied some physical and chemical soil characteristics which might affect the biodegradation process. Dinoseb biodegradation rates were higher in silt-loam soils than in loamy-sand soils. Biodegradation rates were not influenced by previous exposure of the soils to dinoseb. Bacterial numbers, measured by standard plate counts on soil extract agar, were the best predictors of biodegradation rates, accounting for 53% of the variability between soils. Soil nitrate-N inhibited dinoseb biodegradation and accounted for 39% of the variability. Sorption of dinoseb to soil surfaces also appeared to influence biodegradation rates. No other soil parameter contributed significantly to the variability in biodegradation rates. Persistence of dinoseb in one soil was due to inhibition of biodegradation by nitrate, while in another soil persistence appeared to be due to lack of native degradative microorganisms.

A Biochemical Method for Estimating Viability of Teliospores ofTilletia controversa

(...) Lipase activity was detected consistently in extracts from viable teliospores by a fluorescein diacetate (FDA) assay. No fluorescence activity was observed in extracts from autoclaved spores. By comparison, lipase detection was inconsistent when 4-methyl-umbelliferyl-palmitate was used as substrate. Extracts of teliospores that did not germinate on soil extract agar exhibited no lipase or glucosidase activity. Viability of individual teliospores could not be assessed by lipase assay because spore coats were impermeable to FDA. The FDA assay reduced the time needed to assess the average viability of a teliospore population from 2 mo to 1 hr

Factors Influencing Teliospore Germination inTilletia fusca

The influence of cold temperature storage and exogenous nutrients on teliospore germination of 11 isolates of Tilletia fusca was assessed. Storage at 5 C stimulated germination of some isolates when they were incubated at 5 C, both in light and in dark. The relation of cold temperature and light activation to dormancy mechanisms and the significance of dormancy in the life cycle of T.fusca was discussed. Isolates of T.fusca varied in germination responses to water agar, soil extract agar, and media that contained different concentrations of mineral salts, asparagine, and sucrose. (...)

Pseudomonas Solanacearum-suppressive soils in Taiwan

Among five streptomycin-supplemented media tested, modified soil extract agar was most effective in the recovery of streptomycin-resistant Pseudomonas solanacearum added to soil. P. solanacearum formed creamy fluidal colonies with transparent margins on modified soil extract agar and was easily distinguishable from colonies of indigenous soil bacteria. Soils collected from different areas in Taiwan were tested for suppressiveness toward P. solanacearum. Suppressiveness was measured by inoculating soils with a known quantity of P. solanacearum and measuring recovery rate by soil dilution plating after 3 days exposure in soil. About 25% of the soil samples tested were suppressive to P. solanacearum, reducing the number of the bacteria added to 30% or less of the original in 3 days. In general, low pH soils in Taiwan tend to be less favorable for survival of P. solanacearum than high pH soils. Among the 10 soil samples suppressive to P. solanacearum tested, 4 were strongly suppressive and 1 moderately suppressive to tomato wilt caused by the pathogen. The remaining 5 soils were conducive to the disease. Among the 10 pathogen-conducive soils tested, 1 was moderately suppressive and the rest were conducive to tomato wilt caused by the pathogen.

EFFECT OF MEDIA ON GROWTH AND INTERACTIONS BETWEEN A RANGE OF SOIL‐BORNE GLASSHOUSE PATHOGENS AND ANTAGONISTIC FUNGI

SummaryThe growth of and interactions between the glasshouse pathogens, Rhizoctonia solani Kühn, Fusarium oxysporum Schlect., Pyrenochaeta lycopersici Schneider & Gerlach, Phomopsis sclerotioides van Kesteren, Sclerotinia sclerotiorum (Lib.) de Bary and Botrytis cinerea Pers. and the antagonistic fungi, Trichoderma harzianum Rifai, Trichoderma viride Pers., Coniothyrium minitans Campbell, Gliocladium roseum (Link.) Bain and Pythium oligandrum Dreschler were examined on tap water agar, soil extract agar and potato dextrose agar. The medium had a significant effect on the growth rates of the fungi as well as the production of, and response to, volatile and non‐volatile antibiotic compounds and hyphal interactions. Responses to the media appeared to be related both to inherent properties of the fungi and to their natural ecological behaviour. The significance of in vitro assays in screening systems for biological control agents of soil‐borne plant pathogens is discussed.

Effect of pH, temperature and nutrients on the germination of a vesicular-arbuscular mycorrhizal fungus,Glomus fasciculatum in vitro

The effect of various pH (5·5, 6·5, 7·5 and 8·5) and temperature (20, 25 and 30°C) on germination of spores ofGlomus fasciculatum on 1% water agar was studied. Maximum germination (81·3%) occurred at pH 6·5 at 25°C. Spore germination was also studied with other nutrient media such as corn meal agar, modified Hepper medium, charcoal agar (0·01%) and soil extract agar. A significant increase was observed in charcoal agar and soil extract agar (+13·4% and +10·9% respectively).