Mass Production of Arbuscular Mycorrhizal Fungi on the Sorghum Plants Inoculated with Burkholderia arboris Using Soybean Mill Waste and Vermicompost-Amended Soil-Sand Substrate.

Arbuscular mycorrhizal (AM) fungi are being used as a new generation of biofertilizers to increase plant growth by improving plant nutrition and bio-protection. However, because of the obligatory nature of the plant host, large-scale multiplication of AM propagules is challenging, which limits its applicability. This study evaluates the ability of Burkholderia arboris to increase AM production in soybean mill waste and vermicompost amended by soil-sand mixture planted with sorghum as a host plant. The experiment was conducted in a nursery using a completely randomized design with four inoculation treatments (B. arboris, AM fungi, B. arboris + AM fungi, and control) under sterilized and unsterilized conditions. AM production was investigated microscopically (spore density and root colonization), and biochemically (AM-specific lipid biomarker, 16:1ω5cis derived from neutral lipid fatty acid (NLFA), and phospholipid fatty acid (PLFA) fractions from both soil and roots). Integrating B. arboris with AM fungi in organically amended pots was found to increase AM fungal production by 62.16 spores g-1 soil and root colonization by 80.85%. Biochemical parameters also increased with B. arboris inoculation: 5.49 nmol PLFA g-1 soil and 692.68 nmol PLFA g-1 root and 36.72 nmol NLFA g-1 soil and 3147.57 nmol NLFA g-1 root. Co-inoculation also increased glomalin-related soil protein and root biomass. Principal component analysis (PCA) further supported the higher contribution of B. arboris to AM fungi production under unsterilized conditions. In conclusion, inoculation of AM plant host seeds with B. arboris prior to sowing into organic potting mix could be a promising and cost-effective approach for increasing AM inoculum density for commercial production. Furthermore, efforts need to be made for up-scaling the AM production with different plant hosts and soil-substrate types.

Evaluating coal char as an alternative to biochar for mitigating nutrient and carbon loss from manure-amended soils: Insights from a greenhouse experiment.

Animal manure has been increasingly adopted as a more sustainable substitute for synthetic fertilizers but might result in increased dissolved organic C (DOC) and phosphate (PO4 3- ) leaching and elevated greenhouse gas emissions from soil. Biochar may reduce nutrient loss from manure-amended soils, but large-scale application has been hindered, in part, by its high cost. Minimum cost alternatives, such as incomplete coal combustion residue (char), may provide a more viable option to farmers, but char needs to be analyzed in comparison to high-temperature pine biochar before recommendations can be made. We valuated losses of soil C, N, and P, as well as plant yields and changes in microbial biomass, in two contrasting soils amended with dairy slurry or swine lagoon wastewater and with biochar or coal char over 105 d. Dissolved organic C leaching decreased with addition of biochar or char (0.6-27% or 1.6-36%), independent of soil texture and manure type. Leaching of PO4 3- was reduced by biochar (15-24%) and char (38-50%) in the silt loam. Soil N leaching increased after char application (likely due to our high application rate) but was unaffected by biochar. Char reduced CO2 emissions from the sandy loam by 9.7-54%, whereas both biochar and char increased CO2 emissions in the silt loam by 38-48% during plant root senescence. Depending on soil characteristics, char may outcompete biochar with respect to reduction of PO4 3- and DOC leaching. Unlike biochar, some char-N is available, and this should be accounted for when considering application rates.

A new method for detecting micro-fragments of biodegradable mulch films containing poly(butylene adipate-co-terephthalate) (PBAT) in soil.

Biodegradable mulch (BDM) is a potentially sustainable alternative to polyethylene plastic mulch film for intensive crop production. Certified BDMs must demonstrate >90% biodegradation in compost and agricultural soil, but the environmental fate of BDM micro-fragments is usually not measured. While using fatty acid methyl ester (FAME) analysis to study changes in soil microbial communities under different BDMs and management conditions, two peaks were detected by gas chromatography that were derived from a BDM containing poly(butylene adipate-co-terephthalate) (PBAT). The largest peak was identified as 1,4-benzenedicarboxylic acid, dimethyl ester, or dimethyl terephthalate (DMT). The smaller peak was hexanedioic acid dimethyl ester or dimethyl adipate. From this observation we hypothesized that the FAME method could be used to detect and quantify micro-fragments (<5 mm in length) of PBAT-containing BDM in soil. To test this, fragments of two commercially available BDMs were added to soil and extracted for FAME analyses. Linear relationships were observed between DMT and mulch mass added to soil for both BDMs when the initial mass of fragments was <3.5 mg (r2   > .99). Based on our findings, the FAME method could be redeployed to study the environmental fate and possible accumulation of BDM micro-fragments in soil over time.

Evaluation of Bacillus Strains for Plant Growth Promotion and Predictability of Efficacy by In Vitro Physiological Traits.

Bacilli are commonly used as plant growth-promoting agents but can be limited in effectiveness to certain crop and soil environments. The objectives of this study were to (1) identify Bacillus strains that can be consistent in promoting the growth of corn, wheat, and soybean and (2) determine whether physiological traits expressed in vitro can be predictive of growth promotion efficacy/consistency and be used for selecting effective strains. Twelve Bacillus strains isolated from wheat rhizospheres were evaluated in greenhouse pot tests with nonsterile soil for their effects on the growth of corn, soybean, and wheat. The strains also were assessed in vitro for multiple physiological traits. All 12 strains increased corn growth significantly compared to the controls. The four most efficacious strains on corn-Bacillus megaterium R181, B. safensis R173, B. simplex R180, and Paenibacillus graminis R200-also increased the growth of soybean and wheat. No set of traits was a predictor of growth promotion efficacy. The number of traits expressed by a strain also was not an indicator of efficacy as strain R200 that was positive for only one trait showed high growth promotion efficacy. Effective strains can be identified through pot tests on multiple crop plants, but in vitro physiological assays are unreliable for strain selection.

Tetracycline and Sulfonamide Antibiotic Resistance Genes in Soils From Nebraska Organic Farming Operations.

There is widespread agreement that agricultural antibiotic resistance should be reduced, however, it is unclear from the available literature what an appropriate target for reduction would be. Organic farms provide a unique opportunity to disentangle questions of agricultural antibiotic drug use from questions of antibiotic resistance in the soil. In this study, soil was collected from 12 certified organic farms in Nebraska, evaluated for the presence of tetracycline and sulfonamide resistance genes (n = 15 targets), and correlated to soil physical, chemical, and biological parameters. Tetracycline and sulfonamide antibiotic resistance genes (ARGs) were found in soils from all 12 farms, and 182 of the 196 soil samples (93%). The most frequently detected gene was tetG (55% of samples), followed by tet(Q) (49%), tet(S) (46%), tet(X) (30%), and tetA(P) (29%). Soil was collected from two depths. No differences in ARGs were observed based on soil depth. Positive correlations were noted between ARG presence and soil electrical conductivity, and concentrations of Ca, Na, and Mehlich-3 phosphorus. Data from this study point to possible relationships between selected soil properties and individual tetracycline resistance genes, including tet(O) which is a common target for environmental samples. We compared organic farm results to previously published data from prairie soils and found significant differences in detection frequency for 12 genes, eight of which were more commonly detected in prairie soils. Of interest, when tetracycline ARG results were sorted by gene mechanism, the efflux genes were generally present in higher frequency in the prairie soils, while the ribosomal protection and enzymatic genes were more frequently detected in organic farm soils, suggesting a possible ecological role for specific tetracycline resistance mechanisms. By comparing soil from organic farms with prairie soils, we can start to determine baseline effects of low-chemical input agricultural production practices on multiple measures of resistance.

Changes in soil microbial communities after 10 years of winter wheat cultivation versus fallow in an organic-poor soil in the Loess Plateau of China.

Agricultural management methods, such as cultivation or fallowing, have led to significant changes in soil fertility and hence, crop yield. Such changes may have stemmed from changes in soil microbial communities and associated biogeochemical processes. This phenomenon is particularly true in organic-poor soil in the Loess Plateau of China. In this study, we examined three existing soil management regimes as part of a 10-year field experiment and evaluated their effects on fungal and bacterial community structures by performing high-throughput 454 pyrosequencing. These management regimes were (i) fertilized winter wheat (Triticum aestivum L.) (FW), (ii) continuous natural fallow with weeds but without crop grown (NF), and (iii) continuous bare fallow without weeds or crop grown (BF). After 10 years, soil organic carbon (SOC), microbial biomass carbon (MBC), and available potassium (K) concentrations were highest in NF. Soil N behaved differently, with BF obtaining the highest nitrate nitrogen (N). Meanwhile, slight differences in total N (TN) were observed among FW, NF, and BF. Available phosphorus (P) was highest and available K was lowest in FW. Microbial communities were dominated by Ascomycota (59.1% of fungal sequences), and Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria (75.7% of bacterial sequences) in FW, NF and BF at the phylum level. Soil management regimes did not affect the fungal and bacterial richness and diversity but significantly modified their community compositions. Compared with FW, the abundances of Ascomycota (fungi phylum) and Alternaria, Gibberella, and Emericella (fungi genus) were increased by NF, whereas the values of Chaetomium, Humicola, and Cryptococcus (fungi genus) were decreased by BF. The abundances of Verrucomicrobia (bacteria phylum), and Steroidobacter (bacteria genus) were increased by NF, and Bacteroides (bacteria genus) was increased by BF. Canonical correspondence analysis showed that SOC, available P, and TN might be the key factors in community formation. Therefore, the decadal absence of plants (BF) affected soil fertility by increased available K and nitrate N, whileas natural fallow (NF) affected soil fertility by increased SOC, available K, and MBC, and they all changed fungal and bacterial community compositions.

Molecular diversity and distribution of indigenous arbuscular mycorrhizal communities colonizing roots of two different winter cover crops in response to their root proliferation.

A clear understanding of how crop root proliferation affects the distribution of the spore abundance of arbuscular mycorrhizal fungi (AMF) and the composition of AMF communities in agricultural fields is imperative to identify the potential roles of AMF in winter cover crop rotational systems. Toward this goal, we conducted a field trial using wheat (Triticum aestivum L.) or red clover (Trifolium pratense L.) grown during the winter season. We conducted a molecular analysis to compare the diversity and distribution of AMF communities in roots and spore abundance in soil cropped with wheat and red clover. The AMF spore abundance, AMF root colonization, and abundance of root length were investigated at three different distances from winter crops (0 cm, 7.5 cm, and 15 cm), and differences in these variables were found between the two crops. The distribution of specific AMF communities and variables responded to the two winter cover crops. The majority of Glomerales phylotypes were common to the roots of both winter cover crops, but Gigaspora phylotypes in Gigasporales were found only in red clover roots. These results also demonstrated that the diversity of the AMF colonizing the roots did not significantly change with the three distances from the crop within each rotation but was strongly influenced by the host crop identity. The distribution of specific AMF phylotypes responded to the presence of wheat and red clover roots, indicating that the host crop identity was much more important than the proliferation of crop roots in determining the diversity of the AMF communities.

Systemic and local regulation of phosphate and nitrogen transporter genes by arbuscular mycorrhizal fungi in roots of winter wheat (Triticum aestivum L.).

Previous studies have reported that the expression of phosphate (Pi) or nitrogen (N) transporter genes in roots of plants could be regulated by arbuscular mycorrhizal (AM) fungi, but little is known whether the regulation is systemic or not. The present study investigated the systemic and local regulation of multiple phosphate and nitrogen transporter genes by four AM fungal species belonging to four genera in the roots of winter wheat. A split-root culture system with AM inoculated (MR) and non-inoculated root compartments (NR) was used to investigate the systemic or local responses of phosphate and nitrogen transporter genes to colonization by four AM fungi in the roots of wheat. The expression of four Pi transporter, five nitrate transporter, and three ammonium transporter genes was quantified using real-time PCR. Of the four AM fungi tested, all locally increased expression of the AM-inducible Pi transporter genes, and most locally decreased expression of a Pi-starvation inducible Pi transporter gene. The addition of N in soil increased the expression of either Pi starvation inducible Pi transporters or AM inducible Pi transporters. Inoculation with AM fungi either had no effect, or could locally or systemically down-regulate expression of nitrogen transporter genes depending on gene type and AM fungal species.

A long-term nitrogen fertilizer gradient has little effect on soil organic matter in a high-intensity maize production system.

Global maize production alters an enormous soil organic C (SOC) stock, ultimately affecting greenhouse gas concentrations and the capacity of agroecosystems to buffer climate variability. Inorganic N fertilizer is perhaps the most important factor affecting SOC within maize-based systems due to its effects on crop residue production and SOC mineralization. Using a continuous maize cropping system with a 13 year N fertilizer gradient (0-269 kg N ha(-1) yr(-1)) that created a large range in crop residue inputs (3.60-9.94 Mg dry matter ha(-1) yr(-1)), we provide the first agronomic assessment of long-term N fertilizer effects on SOC with direct reference to N rates that are empirically determined to be insufficient, optimum, and excessive. Across the N fertilizer gradient, SOC in physico-chemically protected pools was not affected by N fertilizer rate or residue inputs. However, unprotected particulate organic matter (POM) fractions increased with residue inputs. Although N fertilizer was negatively linearly correlated with POM C/N ratios, the slope of this relationship decreased from the least decomposed POM pools (coarse POM) to the most decomposed POM pools (fine intra-aggregate POM). Moreover, C/N ratios of protected pools did not vary across N rates, suggesting little effect of N fertilizer on soil organic matter (SOM) after decomposition of POM. Comparing a N rate within 4% of agronomic optimum (208 kg N ha(-1) yr(-1)) and an excessive N rate (269 kg N ha(-1) yr(-1)), there were no differences between SOC amount, SOM C/N ratios, or microbial biomass and composition. These data suggest that excessive N fertilizer had little effect on SOM and they complement agronomic assessments of environmental N losses, that demonstrate N2 O and NO3 emissions exponentially increase when agronomic optimum N is surpassed.

Does inorganic nitrogen fertilization improve soil aggregation? Insights from two long-term tillage experiments.

The relationship between inorganic fertilization and soil aggregation is not well understood. We studied cumulative nitrogen (N) fertilization impacts on aggregation, soil organic C (SOC), pH, and their relationships under irrigated and rainfed experiments in Nebraska after 27 and 28 yr, respectively. The dominant soil series were Crete silt loam at the irrigated site, and Coleridge silty clay loam at the rainfed site. We studied irrigated continuous corn ( L.) in chisel plow (CP) and ridge till (RidgeT) receiving 0, 75, 150, and 300 kg N ha yr and rainfed continuous corn and corn-soybean [ (L.) Merr.] in moldboard plow (MP), reduced till (RT), and no-till (NT) with corn receiving 0, 80, and 160 kg N ha yr. Fertilization altered soil aggregation in all tillage systems under continuous corn. Mean weight diameter of water-stable aggregates (MWDA) increased in the upper 7.5-cm depth in NT but decreased in the 7.5- to 60-cm depth by 1.5 times with N application. Fertilization reduced pH but had little or no effect on SOC. Both MWDA and pH ( = 0.47***) decreased under irrigated corn, particularly in the 7.5- to 30-cm depth. No-till and RT had two to five times greater near-surface MWDA than MP. Continuous corn had greater MWDA than corn-soybean in the upper 30-cm depth except in MP. Long-term N fertilization improves near-surface soil aggregation in NT continuous corn but reduces aggregation in the subsoil. Results also suggest that, if fertilizers are applied at rates of about 80 kg N ha, deterioration of soil aggregation would be minimal.

Arbuscular mycorrhizal fungi differ in their ability to regulate the expression of phosphate transporters in maize (Zea mays L.).

Previous studies have found that some phosphate (Pi) starvation inducible transporter genes are downregulated and arbuscular mycorrhizal (AM) inducible Pi transporter genes are upregulated in maize roots associated with the fungus Glomus intraradices. However, little is known about the functional diversity of different AM fungal species in influencing the expression of Pi transporters in maize roots. Here, we studied the expression of two Pi transporter genes ZEAma:Pht1;3 (Pi starvation inducible) and ZEAma:Pht1;6 (AM inducible) in maize root colonized by different AM fungal inoculants. Non-mycorrhizal maize, maize colonized by Glomus deserticola (CA113), Glomus intraradices (IA506), Glomus mosseae (CA201), Gigaspora gigantea (MN922A) and the co-inoculation of all four species were established. The expression patterns of the two genes were quantified using real-time, reverse transcription polymerase chain reaction. The expression level of ZEAma:Pht1;6 was 26-135 times higher in AM plants than in non-mycorrhizal maize roots, whereas the expression level of ZEAma:Pht1;3 was five to 44 times lower in AM plants than in non-mycorrhizal plants. Expression of the two genes differed with inoculation treatment, and increasing the diversity of AM fungi in maize roots led to greater expression of ZEAma:Pht1;6 as well as Pi uptake in shoots. The expression of ZEAma:Pht1;6 was significantly positively correlated with AM colonization rate, concentration of AM biomarkers in maize roots, Pi uptake and dry weight of shoot, but negatively correlated with the expression of ZEAma:Pht1;3. Addition of Pi fertilizer at a low concentration significantly increased the expression of ZEAma:Pht1;6 but had no effect on the expression of ZEAma:Pht1;3.

Survey of extreme solvent tolerance in gram-positive cocci: membrane fatty acid changes in Staphylococcus haemolyticus grown in toluene.

We exploited the unique ecological niche of oil fly larval guts to isolate a strain of Staphylococcus haemolyticus which may be the most solvent-tolerant gram-positive bacterium yet described. This organism is able to tolerate 100% toluene, benzene, and p-xylene on plate overlays and saturating levels of these solvents in monophasic liquid cultures. A comparison of membrane fatty acids by gas chromatography after growth in liquid media with and without toluene showed that in cells continuously exposed to solvent the proportion of anteiso fatty acids increased from 25.8 to 33.7% while the proportion of 20:0 straight-chain fatty acids decreased from 19.3 to 10.1%. No changes in the membrane phospholipid composition were noted. Thus, S. haemolyticus alters its membrane fluidity via fatty acid composition to become more fluid when it is exposed to solvent. This response is opposite that commonly found in gram-negative bacteria, which change their fatty acids so that the cytoplasmic membrane is less fluid. Extreme solvent tolerance in S. haemolyticus is not accompanied by abnormal resistance to anionic or cationic detergents. Finally, six strains of Staphylococcus aureus and five strains of Staphylococcus epidermidis, which were not obtained by solvent selection, also exhibited exceptional solvent tolerance.

Alicyclobacillus vulcanalis sp. nov., a thermophilic, acidophilic bacterium isolated from Coso Hot Springs, California, USA.

A thermo-acidophilic Gram-positive bacterium, strain CsHg2T, which grows aerobically at 35-65 degrees C (optimum 55 degrees C) and at pH 2.0-6.0 (optimum 4.0), was isolated from a geothermal pool located in Coso Hot Springs in the Mojave Desert, California, USA. Phylogenetic analysis of 16S rRNA gene sequences showed that this bacterium was most closely related to the type strains of Alicyclobacillus acidocaldarius (97.8 % identity) and Alicyclobacillus sendaiensis (96.9 %), three Japanese strains denoted as UZ-1, KHA-31 and MIH 332 (96.1-96.5 %) and Alicyclobacillus genomic species FR-6 (96.3 %). Phenotypic characteristics including temperature and pH optima, G+C composition, acid production from a variety of carbon sources and sensitivity to different metal salts distinguished CsHg2T from A. acidocaldarius, A. sendaiensis and FR-6. The cell lipid membrane was composed mainly of omega-cyclohexyl fatty acid, consistent with membranes from other Alicyclobacillus species. Very low DNA-DNA hybridization values between CsHg2T and the type strains of Alicyclobacillus indicate that CsHg2T represents a distinct species. On the basis of these results, the name Alicyclobacillus vulcanalis sp. nov. is proposed for this organism. The type strain is CsHg2T (ATCC BAA-915T = DSM 16176T).

TNT biotransformation and detoxification by a Pseudomonas aeruginosa strain.

Successful microbial-mediated remediation requires transformation pathways that maximize metabolism and minimize the accumulation of toxic products. Pseudomonas aeruginosa strain MX, isolated from munitions-contaminated soil, degraded 100 mg TNT L(-1) in culture medium within 10 h under aerobic conditions. The major TNT products were 2-amino-4,6-dinitrotoluene (2ADNT, primarily in the supernatant) and 2,2'-azoxytoluene (2,2'AZT, primarily in the cell fraction), which accumulated as major products via the intermediate 2-hydroxylamino-4,6-dinitrotoluene (2HADNT). The 2HADNT and 2,2'AZT were relatively less toxic to the strain than TNT and 2ADNT. Aminodinitrotoluene (ADNT) production increased when yeast extract was added to the medium. While TNT transformation rate was not affected by pH, more HADNTs accumulated at pH 5.0 than at pH 8.0 and AZTs did not accumulate at the lower pH. The appearance of 2,6-diamino-4-nitrotoluene (2,6DANT) and 2,4-diamino-6-nitrotoluene (2,4DANT); dinitrotoluene (DNT) and nitrotoluene (NT); and 3,5-dinitroaniline (3,5DNA) indicated various routes of TNT metabolism and detoxification by P. aeruginosa strain MX.