Quantification of Pythium populations in ginseng soils by culture dependent and real-time PCR methods

Ginseng (Panax quinquefolius L.) is widely cultivated in North America as a medicinal herb. However, yields are often reduced by various root pathogens, including Pythium species, which cause damping-off in young plants. In order to improve the prediction of disease risk, real-time PCR assays were developed and used in conjunction with dilution plating on selective media to quantify populations of Pythium irregulare Buisman and Pythium ultimum Trow directly from soil. The assays were tested on artificially infested soils and then used on a variety of naturally infested, ginseng-cultivated soils in south-western Ontario. Data on P. ultimum DNA concentrations were positively correlated with the number of P. ultimum viable propagules on selective media. However, in the case of P. irregulare, the presence of cryptic species resulted in incongruent relationships between the dilution plate and real-time PCR data. We therefore sequenced the ITS region of a large number of P. irregulare isolates in order to determine the proportion that would be detected by the real-time PCR assay. The ability to quantify pathogen populations directly from soils using real-time PCR (calibrated with data on inoculum potential) will improve disease risk assessment and lead to a reduction in pesticide application.     

The ecology of a Pythium community in relation to the epidemiology of carrot cavity spot

Carrot cavity spot (CCS) is characterised by the appearance of small sunken elliptical lesions on the tap root. It is caused by a complex of Pythium species, but the species diversity and interactions within the complex have never been studied for modelling CCS epidemics. The diversity of a pathogenic Pythium community was assessed during 3 consecutive years in a field experiment after an initial artificial soil infestation with P. violae. 1241 lesions were examined, yielding 728 Pythium isolates. Conventional microbiological methods and restriction polymorphism of the internal transcribed spacer regions of the rDNA of 209 representative Pythium isolates allowed us to identify 655 isolates as belonging to six Pythium species, including P. violae and five indigenous species (P. sulcatum, P. intermedium, P. sylvaticum/irregulare, P. coloratum, and P. ultimum). Biological traits, such as pathogenicity, optimum temperature for mycelial growth and saprophytic survival of the inoculum, explained the fluctuations in the composition of the complex over 17 successive samplings during the 3-year period, most notably the prevalence of first P. violae and then P. sulcatum. P. violae and P. sulcatum were occasionally isolated in mixture from single lesions (10.4% and 9.6%, respectively). Other species were more frequently isolated in mixture: 30.8% for P. intermedium, 33.8% for P. sylvaticum/irregulare, 42.9% for P. ultimum, and 66.7% for P. coloratum. A contingency analysis allowed us to define ‘major’ and ‘minor’ species on both pathological and ecological criteria (frequency of occurrence in the complex, pathogenicity and ability to induce lesions by themselves), and demonstrated that infection by one ‘major’ pathogen species (P. violae or P. sulcatum) is not positively correlated with the presence of a second Pythium species. The ratio between ‘observed’ and ‘expected’ mixed infection frequency under the assumption of independent infection (mir) was less than 1 for P. violae, P. sulcatum, P. intermedium, and P. sylvaticum/irregulare (P < 0.05). For all Pythium species, there was a negative linear relationship between mir and pathogenicity (R² = 0.638): the less a Pythium species was pathogenic on carrot, the more often it was isolated from a CCS lesion in mixture with at least one other species. The non-significance of interactions between species during the infection phase suggests that CCS epidemics can be analysed as if they were caused by a single Pythium species.     

Re-establishment of suppressiveness to soil- and air-borne diseases by re-inoculation of soil microbial communities

The aim of this study was to investigate the potentials and limitations in restoring soil suppressiveness in disturbed soils. Soils from three sites in UK and Switzerland (STC, REC, THE) differing in their level of suppressiveness to soil-borne and air-borne diseases were γ-irradiated and this soil matrix was re-inoculated with 1% (w/w) of either parent native soil or native soil from the other sites (‘soil inoculum’). Suppressiveness to air-borne and soil-borne diseases was quantified by means of the host-pathogen systems Lepidium sativum (cress)-Pythium ultimum, an oomycete causing root rot and seedling damping-off, and Arabidopsis thaliana-Hyaloperonospora parasitica, an oomycete causing downy mildew. Soil microbial biomass, activity and community structure, as determined by phospholipid fatty acid (PLFA) profiles, were measured in native, γ-irradiated, and re-inoculated soils. Both, L. sativum and A. thaliana were highly susceptible to the pathogens if grown on γ-irradiated soils. Re-inoculation completely restored suppressiveness of soils to the foliar pathogen H. parasitica, independently of soil matrix or soil inoculum, whereas suppressiveness to P. ultimum depended on the soil matrix and, to a lesser extent, on the soil inoculum. However, the soil with the highest inherent suppressiveness did not reach the initial level of suppressiveness after re-inoculation. In addition, native microbial populations as defined by microbial biomass, activity and community structure, could not be fully restored in re-inoculated soils. As for suppressiveness to P. ultimum, the soil matrix, rather than the source of soil inoculum was identified as the key factor for re-establishing the microbial community structure. Our data show that soils do not or only slowly fully recover from sterilisation by γ-irradiation, indicating that agricultural soil management practices such as soil fumigation or heat treatments frequently used in vegetable cropping should be avoided.     

Hyperparasitism of oospores of Phytophthora megasperma var. sojae

Hyperparasites of oospores of Phytophthora megasperma Drechs. var. sojae Hildb. were present in each of 15 field soils tested. Maximum numbers of oospores parasitized ranged from 42.5 to 87.5% for flooded soils, and from 25.5 to 73.0% for soils adjusted to 50% water holding capacity; the mean for all soils was 51.5%. The frequency of hyperparasitism was not correlated with the disease potential soils for Phytophthora root-rot of soybean as determined in seedling tests on flooded soil samples. Of eight isolated hyperparasitic fungi tested in steamed soil, the most efficient parasites were Hyphochytrium catenoides, Humicola fuscoatra, and Pythium monospermum, each of which parasitized at least 76% of oospores during 3 weeks. Hyphae were not parasitized by any of the eight fungi. Parasitism by H. catenoides in sterilized soil increased as soil temperature increased from 16° to 28°C. Parasitism by P. monospermum was maximum at 20°–24°C. Oospores of P. meyasperma var. sojae race 7 were more resistant to infection by hyperparasites than were oospores of races 1 and 3. Oospores produced in culture were slightly more susceptible to hyperparasitism in soils than were oospores produced in soybean seedlings.     

Resistance of microbial populations in DDT-contaminated and uncontaminated soils

One DDT-contaminated soil and two uncontaminated soils were used to enumerate DDT-resistant microbes (bacteria, actinomycetes and fungi) by using soil dilution agar plates in media either with 150 μg DDT ml⁻¹ or without DDT at different temperatures (25, 37 and 55°C). Microbial populations in this study were significantly (p<0.001) affected by DDT in the growth medium. However, the numbers of microbes in long-term contaminated and uncontaminated soils were similar, presumably indicating that DDT-resistant microbes had developed over a long time exposure. The tolerance of isolated soil microbes to DDT varied in the order fungi>actinomycetes>bacteria. Bacteria from contaminated soil were more resistant to DDT than bacteria from uncontaminated soils. Microbes isolated at different temperatures also demonstrated varying degrees of DDT resistance. For example, bacteria and actinomycetes isolated at all incubation temperatures were sensitive to DDT. Conversely fungi isolated at all temperatures were unaffected by DDT.     

Influence of temperature on solvent-pesticide interaction effects towards fungi

The effect of temperature on interactions between combinations of the solvent acetone and the fungicide captan was determined using the fungi Pythium uhimum, Sclerotinia homeocarpa, and Pestalotia sp. Seven concentrations of acetone, ranging from 0.1 to 3.0% (v/v), were interacted with four concentrations of captan, ranging from 1.0 to 10.0 ppm (mg L^?1 ). This procedure was repeated at 15, 20, 25, and 30 °C using potato dextrose agar at pH 5.5. Acetone and captan interacted synergistically towards P. ultimum and S. homeocarpa, and antagonistically towards Pestalotia sp., regardless of the temperature. The exact temperature response was dependent upon both the captan level and culture used. As the temperature increased from 15 to 30 °C, the toxicity of captan decreased by up to 40% with P. ultimum, and from < 10 to 20 with S. homeocarpa and Pestalotia sp. With only a few exceptions, the magnitude of interactions observed generally decreased as the temperature was increased from 15 to 30 °C. This was most pronounced with P. ultimum. Generally, the lowest interaction magnitudes were recorded at 20 °C for P. ultimum, 20 to 25 °C for S. homeocarpa, and 30 °C for Pestalotia sp. The greatest interaction magnitudes were usually obtained at 15 or 30 °C with P. ultimum, 15 °C with S. homeocarpa, and 25 °C with Pestalotia sp.     

The effect of ph on fungitoxic interactions between a solvent and pesticide

The effect of pH on interactions between combinations of the solvent acetone and the pesticide captan was determined using the fungiPythium ultimum, Sclerotinia homeocarpa, andPestalotia sp.. Seven concentrations of the solvent acetone, ranging from 0.1 to 3.0 % (v/v), were interacted with four concentrations of the fungicide captan, ranging from 1.0 to 10.0 ppm (mg L^?1). This interaction procedure was repeated at pH 4.5, 5.5, 6.5, and 7.5, using a temperature of 30 °C. Acetone and captan interacted synergistically towardsP. ultimum andS. homeocarpa, and antagonistically towardsPestalotia sp., regardless of the pH. However, the solvent concentration at which synergism or antagonism was first observed usually decreased as pH increased. The actual pH response obtained was dependent upon both the captan level and culture used. As pH increased from 4.5 to 7.5, the toxicity of captan decreased by up to 40% withS. homeocarpa andPestalotia sp., and 80% withP. ultimum. WithS. homeocarpa andPestalotia sp., the magnitude of synergism or antagonism increased as the captan concentration was raised from 1.0 or 2.5 ppm up to 7.5 or 10.0 ppm. With P. ultimum, the degree of synergism decreased at pH 4.5 and 5.5, but increased at pH 6.5 and 7.5, as the captan concentration was raised from 2.5 to 10.0 ppm. The lowest interaction magnitudes were recorded at pH 4.5 forP. ultimum, but was variable for the other cultures. The greatest interaction magnitudes were obtained at pH 4.5 forS. homeocarpa, 5.5 forPestalotia sp., and 6.5 or 7.5 forP. ultimum.     

Suppression of damping-off of cucumber caused by Pythium ultimum with live cells and extracts of Serratia marcescens N4-5

Environmentally friendly control measures are needed for the soil-borne pathogen, Pythium ultimum. This pathogen can cause severe losses to field- and greenhouse-grown cucumber and other cucurbits. Live cells and ethanol extracts of cultures of the bacterium Serratia marcescens N4-5 provided significant suppression of damping-off of cucumber caused by P. ultimum when applied as a seed treatment. Live cells of this bacterium also suppressed damping-off caused by P. ultimum on cantaloupe, muskmelon, and pumpkin. Culture filtrates from strain N4-5 contained chitinase and protease activities while ethanol extracts contained the antibiotic prodigiosin, the surfactant serrawettin W1, and possibly other unidentified surfactants. Production of prodigiosin and serrawettin W1 was temperature-dependent, both compounds being detected in extracts from N4-5 grown at 28 °C but not in extracts from N4-5 grown at 37 °C. Ethanol extracts from strain N4-5 grown at 28 °C inhibited germination of sporangia and mycelial growth by P. ultimum in in vitro experiments. There was no in vitro inhibition of P. ultimum associated with ethanol extracts of strain N4-5 grown at 37 °C. Prodigiosin, purified from two consecutive thin-layer chromatography runs using different solvent systems, inhibited germination of sporangia and mycelial growth of P. ultimum. Another unidentified compound(s) also inhibited germination of sporangia but did not inhibit mycelial growth. There was no in vitro inhibition associated with serrawettin W1. These results demonstrate that live cells and cell-free extracts of S. marcescens N4-5 are effective for suppression of damping-off of cucumber caused by P. ultimum possibly due in part to the production of the antibiotic prodigiosin.     

Effects of solarization of soil on nematode and fungal pathogens at two sites in victoria

The effect of covering soil with transparent polyethylene sheets, known as soil solarization, on the viability of plant pathogens was determined. The treatment was tested in mid-summer on sandy loams in N.W. and S. Victoria. Columns of moist soil were inoculated with one of a variety of pathogens, viz. Fusarium oxysporum, Pythium irregulare, Plasmodiophora brassicae, Sclerotium cepivorum, S. rolfsii, Sclerotinia minor, Verticillium dahliae and the nematodes Macroposthania xenoplax, Meloidogyne javanica, Pratylenchus penetrans and Tylenchulus semipenetrans. Columns were placed vertically in soil, and then treated either for 4 weeks in N.W. Victoria, or 6 weeks in S. Victoria.Preliminary laboratory tests showed that pathogens were killed by temperatures within the range 38–55°C. The relative sensitivities of pathogens to fluctuating soil temperatures were similar at both sites. The most sensitive were the nematodes, and the fungi V. dahliae, S. cepivorum and S. minor, while F. oxysporum, P. irregulare and P. brassicae were the least sensitive. In N.W. Victoria treatment effects were apparent to 26 cm and most pathogens were not recovered from 0 to 11 cm. In S. Victoria treatment effects were apparent to a depth of 16cm and most pathogens were not recovered from 0 to 6cm.     

Soils and the conditional allelopathic effects of a tropical invader

Allelopathy may contribute to the formation of mono-dominant stands of exotic species, but the effects of allelochemicals can be highly conditional. We explored variation in the production of phenolics in leaves, accumulation of phenolics in soils, and the inhibitory effects of soils under an aggressive invader Prosopis juliflora across a range of invaded sites and potential mechanisms by which soils alter the effects of P. juliflora leaf litter. For eight sites in Northwest India we compared the concentration of total phenolics and the seedling growth of Brassica campestris in soils from beneath P. juliflora to that in soils collected away from P. juliflora canopies. We then explored these effects in detail in soils from two sites that differed substantially in texture by germinating seeds of B. campestris in these soils amended with P. juliflora macerated leaf leachate. Finally, we tested the effects of l-tryptophan in soils from these two sites on the seedling growth of B. campestris. Across all sites soil beneath P. juliflora contained higher levels of total phenolics and suppressed the growth of B. campestris than soil that was not under P. juliflora. We observed much variation among P. juliflora-invaded sites in the total phenolic levels of soils and the degree to which they suppressed B. campestris and the concentration of phenolics in soils significantly correlated with the root length of B. campestris when grown in these soils. Soil from two sites amended with P. juliflora macerated leachate suppressed seedling growth of B. campestris, with the effect being higher in sandy soil than sandy loam soil. In soil amended with leachate the strong suppression of B. campestris corresponded with much higher total phenolic and l-tryptophan concentrations. However, in other tests l-tryptophan did not affect B. campestris. Our results indicate that the allelopathic effects of P. juliflora can be highly conditional and that variation in soil texture might contribute to this conditionality.     

Microbial community shifts in Pythium ultimum-inoculated suppressive substrates

In this study, the role of compost amendments for the biocontrol of Pythium ultimum was evaluated in bioassays with cucumber (Cucumis sativa L. variety “Chinesische Schlangen”). The addition of compost to the peat-based growing substrates resulted in a significant reduction of disease symptoms of cucumber plants in the presence of P. ultimum compared to pure substrate. Microbial community composition of compost-amended substrates and with different levels of P. ultimum inoculum (0, 5‰) was analyzed by polymerase-chain-reaction-based techniques. To detect and compare dominant bacterial and fungal representatives of suppressive substrate mixes with different pathogen inoculum, 16S and 18S rRNA clone libraries were established. Phylogenetic analysis of the 16S rRNA clones revealed Actinobacteria and α-Proteobacteria to be the prominent classes in the presence of P. ultimum, which are not part of the dominant microflora in the mixes without the pathogen. 18S rRNA sequences for the Pythium-inoculated compost supplemented samples were dominated by Chytridiomycota and Sordariomycetes, whereas in uncontaminated soil–compost mixes, a large part of the sequences were related to Homobasidiomycetes. Thus, it is assumed that the presence of P. ultimum induces distinct shifts in microbial communities favoring to groups known to comprise potential biocontrol agents.     

The invasive plant Solidago canadensis L. suppresses local soil pathogens through allelopathy

Recent studies suggest that invasive plants pose a significant effect on local soil pathogens, which in turn affects on the plant invasion. However, the mechanisms by which invasive plants affect soil pathogens were less well known. We conducted four experiments to test the hypothesis that the invasive plant species Solidago canadensis L. may affect soilborne pathogens through exudation of allelochemicals. Two common soilborne pathogens Pythium ultimum and Rhizoctonia solani were used in the study. Tomato (Lycopersicon esculentum Mill) variety Qianhong No.1 which is sensitive to soil pathogens P. ultimum and R. solani was used to indicate pathogenic activity (in terms of seedling mortality and damping-off). Extracts from root and rhizome of S. canadensis significantly suppressed the growth and pathogenic activity of both pathogens under Petri dish culture and sand culture (experiments 1 and 2), providing direct evidence that S. canadensis exerts allelopathic effects on these pathogens. Subsequently, a pathogen inoculation experiment under sand culture showed that pathogenic activity of both P. ultimum and R. solani was lower under the soil with S. canadensis compared to that under the soil with a common native plant Kummerowia striata (Thunb.) Schindl (experiment 3), implying that invasive S. canadensis had but native K. striata did not have allelopathic effects on soil pathogens through root and rhizome exudation. Finally, results from field soil tests showed that mortality and damping-off rate of tomato seedlings were significantly lower under the soils collected from the fields dominated by S. canadensis than that dominated by native plants at both sampling sites, suggesting that suppression of pathogens also occurs in the field. From the present experimental results we suggest that invasive S. canadensis may acquire spreading advantage in non-native habitat by using “novel weapons” to inhibit not only local plants but also soilborne pathogens.     

Mycoparasitism by Pythium oligandrum and P. acanthicum

Isolates of the reported mycoparasites Pythium oligandrum, P. acanthicum and P. periplocum markedly reduced growth and cellulolysis by Botryotrichum piluliferum, grew rapidly across agar plates precolonized by Phialophora radicicola var radicicola (sensu Deacon) and, where tested (not P. periplocum), were non-pathogenic towards higher plants. Isolates of P. echinulatum, P. mamillatum. P. megalacanthum, P. spinosum, P. ultimum and one isolate of P. acanthicum behaved differently from the mycoparasites and could, themselves, be placed in two groupings in these tests. It is suggested that the ability or otherwise to grow on Phialophora-precolonized agar plates may help to distinguish broad biological groupings within the genus Pythium, but these groupings may cut across conventional taxonomic ones.One isolate of P. acanthicum was tested for its effects on a range of cellulolytic fungi: it reduced their growth to different extents, as did P. oligandrum.Plates of potato-dextrose agar precolonized by Phialophora radicicola were used to isolate selectively P. oligandrum and similar fungi from soils, but the use of hemp seed baits in conjunction with precolonized plates was less selective for these fungi.Straw pieces precolonized by P. oligandrum and buried in soil decomposed at the same rate as virgin straws or those precolonized by P. ultimum or Mucor hiemalis. Subsequently, Stachyholrys atra appeared to colonize straws more frequently from soil, and Fusarium spp. less so, in the presence of P. oligandrum than in its absence. In the laboratory, P. oligandrum was antagonized by Slachyholrys, whereas Fusarium spp. were frequently overgrown by the Pythium.     

Quantitative PCR assays to enumerate Rhizobium leguminosarum strains in soil also target non viable cells and overestimate those detected by the plant infection method

The plant infection method is commonly used to estimate the Most Probable Number (MPN) of soil rhizobia. Here, a qPCR method was set-up and validated with newly developed ANU (strain specific) and RHIZ (more general) primers to quantify the specific Rhizobium leguminosarum bv. trifolii ANU843 strain or general R. leguminosarum strains. Detection limits of qPCR protocols in soil were 1.2 × 10⁴ (ANU) and 4.2 × 10³ (RHIZ) cells per g soil. The qPCR assay appears robust and accurate in freshly inoculated soils but overestimated MPN for indigenous soil rhizobia. An incubation experiment showed that qPCR detected added DNA or non viable cells in soils up to 5 months after addition and incubation at 20 °C in moist conditions.     

Effect of nitrogen and phosphate fertilisers on microbial and nematode diversity in pasture soils

Soil microbial and nematode populations, soil microbial community structure, and microbial and nematode functional diversity were studied in two fertiliser trials on perennial pasture at three sampling times. The N trial involved the application of 0, 200 and 400 kg N ha⁻¹ y⁻¹ in the form of urea. The P trial involved the application of 0, 30, 50 and 100 kg P ha⁻¹ y⁻¹ as superphosphate. The purpose of this study was to determine biological characteristics that may be used as indicators of soil quality as affected by fertiliser inputs.The N or P treatments had no effect on total bacteria, cellulolytic microbes, or the fluorescein diacetate hydrolysis. The fungus Fusarium culmorum was found only in the 200 kg N treatment (P<0.01). Gliocladium roseum declined in isolation frequency with increasing N (P<0.05,) while other Gliocladium spp. increased (P<0.01).The microbial community structure, ecophysiological index (EP), and colony-development index (CD) were determined using: colony development rates in 1/10 tryptose soy agar (TSA), a Pseudomonas medium, and a nutrient poor medium. These parameters were not affected by the addition of the N or P fertilisers. In the N trial, the functional diversity of soil microbes, as determined by Shannon Diversity Index (H) and average well colour development (AWCD) (using Biolog gram negative microplates) was higher in the unfertilised than fertilised treatments. The values for H and AWCD were 4.2 and 0.78 in the unfertilised compared to 4.0 and 0.53 in fertilised treatment (P<0.01, 48 h, mean for both N treatments), respectively. There were no significant differences in these values in the P trial.Populations of the plant feeding nematodes Pratylenchus and Paratylenchus were greater (P<0.05) whereas those of Meloidogyne were lower (P<0.001), in soils fertilsed with N than in unfertilised soils. The genera Aporcelaimus, Dorylaimellus, and Tylencholaimellus were found only in control plots and their loss paralleled faunal changes resulting from pasture improvement reported elsewhere. Nematode Maturity Index (MI) values were 1.78, 1.85, and 1.53 for the N fertiliser treatments (P<0.05) suggesting a reduction at 400 kg N. The MI was not affected by the application of P (mean, 2.01), however, but all values in the P trial were greater than in the N trial. In the N and P trials an average of 29 and 35 nematode taxa were discriminated. The ratio of bacterial-feeding nematodes to bacterial-feeding plus fungal-feeding nematodes was similar across all treatments of the N (0.90–0.92) and P (0.84–0.90) trials, suggesting no relative change in the importance of bacterial- and fungal-mediated decomposition pathways in these soils as a result of fertiliser application.The finding that most microbiological characteristics did not respond to many years of fertiliser treatments suggests that the microbial community in the soils are similar and fertiliser amendments are insufficient to induce changes (either direct or indirect due to plant effects) in these communities. However, the consistent decrease in functional diversity of soil microflora and nematode populations with the application N, but not P, indicates that the N application can impact on community structure.     

Susceptibility of Pythium spp and other fungi to antagonism by the mycorparasite Phytium Oligandrum

Phytium spp and isolates within species differed in susceptibility to the mycoparasite Pythium oligandrum Drechs., as evidenced by their degree of inhibition by it on cellulose and ability to support its growth across their colonies on agar. Yet no Phythium sp. was highly susceptible to it, and P. graminicola Subramanian was highly resistant.No evidence was found that P. oligandrum produces toxins active against other fungi. In liquid culture, P. oligandrum grew on undisturbed colonies of Phialophora sp. (highly susceptible) but not P. ultimum Trow or Fusarium culmorum (W. G. Sm.) Sacc. (moderately resistant) and not Rhizoctonia solani Kuhn (highly resistant). It grew in culture filtrates of Phialophora sp., P. ultimum and F. culmorum, utilizing organic nitrogen and thiamine released by these fungi, but not in culture filtrates of R. solani. It grew on mycelial macerates of all these fungi, though poorly on those of R. solani. Resistance to parasitism by P. oligandrum seems to reside, at least partly, in low levels of nutrient release from host hyphae.     

Phosphorus addition enhances loss of nitrogen in a phosphorus-poor soil

Plants and microbes have limited stoichiometric flexibility to take up and store nitrogen (N) and phosphorus (P). Variation in the relative availability of N and P to plants and microbes may therefore affect how strongly N and P are held in terrestrial ecosystems with important implications for net primary productivity and carbon sequestration. We hypothesized that an increase in P availability in a P-poor soil would increase N uptake by plants and microbes thereby reducing N loss. We grew mixtures of the C3 grass Phalaris aquatica L. and the legume Medicago sativa L. in mesocosms with soils low in P availability and then used a novel technique by adding a ¹⁵N tracer with and without 1 g P m⁻² to soil with different moisture and available N conditions, and measured the ¹⁵N recovery after 48 h in microbes, plants and soil. In contrast to our hypothesis, we found that P addition reduced ¹⁵N in microbes without water stress by 80% and also reduced total¹⁵N recovery, particularly without water stress. Water stress in combination with N addition further showed low total ¹⁵N recovery, possibly because of reduced plant uptake thereby leaving more ¹⁵N in the soil available for nitrification and denitrification. Our results suggest that P addition can result in large gaseous N loss in P-poor soils, most likely by directly stimulating nitrification and denitrification.     

Quantification of Escherichia coli O157:H7 in soils using an inhibitor-resistant NanoGene assay

Humic acids are ubiquitous and abundant in terrestrial environments; therefore, they are often co-extracted with nucleic acids and interfere with quantitative PCR (qPCR) assays. In this study a recently developed NanoGene assay that is resistant to interference by humic acids was evaluated for gene detection in soil samples. The NanoGene assay utilizes a combination of magnetic beads, dual quantum dots labels, and DNA hybridization in solution. Seven soil samples containing different amounts of organic matter were tested to compare NanoGene and qPCR assays for their respective ability to detect a bacterial pathogen. We spiked the soils with Escherichia coli O157:H7, extracted genomic DNA, and conducted NanoGene and qPCR assays targeting the E. coli O157:H7-specific eaeA gene. To prevent the inhibition of PCR that is common when using DNA extracted from soils, we used a range of template DNA concentrations and BSA addition in the qPCR assay. Compared to the qPCR assay the NanoGene assay was significantly more resistant to the inhibitory effect of humic acids, successfully quantifying the eaeA gene within a linear (R² = 0.99) range of 10⁵ through 10⁸ CFU/g soil for all seven soil samples tested. In contrast, the qPCR assay was significantly inhibited using the same template DNA isolated from soils containing a range of organic content (2.0%–12%). Interestingly, the qPCR assay was still inhibited despite additional purification steps, suggesting that humic acids were still associated with DNA at a level that was inhibitory to qPCR. This study demonstrated that the NanoGene assay is suitable for quantitative gene detection in diverse soil types and is not susceptible to inhibition by humic acids and other organic compounds that commonly lead to false negative results in qPCR assays.     

Long-term suppression of Pythium abappressorium induced by Brassica juncea seed meal amendment is biologically mediated

Evidence indicates that seed meal (SM) of Brassica juncea is an effective biofumigant against Pythium spp., an important biological component contributing to apple replant disease. However, the ability of this seed meal to provide disease control even after termination of allyl isothiocyanate (AITC) emission suggested that unidentified mechanisms are also involved in suppression of certain pathogens in B. juncea SM-amended soil. When soils were infested with Pythium abappressorium 2–12 weeks after SM was applied, disease suppression was consistently observed in SM-treated soil. Bagging of soil for the initial 48 h after SM application, to simulate tarping of soil in the field, enhanced disease control. Application of SM either as coarse or fine particles produced similar effects on disease suppression. B. juncea SM amendment also suppressed the proliferation of P. abappressorium observed in Brassica napus SM-treated soils at a time point well after AITC emission from soils was no longer detected. Pasteurization of SM-amended soil eliminated soil suppressiveness toward this pathogen, demonstrating the important contribution of the soil microbiota to the disease control attained in AITC evacuated soil. Terminal-Restriction Fragment Length Polymorphism profiles obtained for 18S rDNA from fungal communities associated with SM-amended and non-amended soil demonstrated distinct variation in terms of composition. Visible changes in fungal community composition in SM-treated soils were also observed, and analyses indicated the preferential proliferation of Trichoderma spp. in SM-treated soils. These findings suggest that modification of the resident fungal community in SM-amended soil may contribute to the observed long-term suppressiveness of B. juncea SM-amended soils toward apple root infection by P. abappressorium.     

Filamentous actinobacteria of the saline soils of arid territories

A large number of actinomycetes (hundreds, thousands, and millions of CFU/g of soil) were isolated from saline soils in the territory of the landscapes of the Ber hillocks. A significantly smaller quantity of actinomycetes was isolated from the soda saline soils and sor saline soils that formed at the bottom of the ephemeral salty lakes in Buryatiya and in the estuary of the Syr-Darya River. Actinomycetes were represented in the studied soils by the Streptomyces, Micromonospora, Actinomadura, and Nocardiopsis genera. Among streptomycetes, the species of the Albus section and the Albus series predominated. The activity of the consumption of substrates by cultures of actinomycetes was largely influenced by conditions of preincubation. Obviously, the influence was related to the alteration in the metabolism of actinomycetes as one of the mechanisms of adaptation to the increased osmolarity of environment. The alcalotolerance, thermotolerance, and xerotolerance of halotolerant actinomycetes of the saline soils of arid territories were experimentally revealed.