Soil DNA Research Articles

Impact of elevated atmospheric CO2 on the wheat rhizomicrobiome under the additional influence of warming, drought, and nitrogen fertilization

The rhizomicrobiome is an important component of agricultural soil that may face vulnerability due to global change. Here, we focused on its response in a 2-year cropland free air carbon dioxide enrichment (FACE) experiment on winter wheat in combination with warming (+3 °C) and drought. To characterize bacterial abundance and prokaryotic diversity, 16S rRNA gene PCR amplicons obtained from soil DNA were analysed. Elevated CO2 (e[CO2]) increased under conventional water supply bacterial abundance, along with higher above-ground plant biomass, but had no tangible effect on the overall diversity and community composition. In contrast, drought reduced bacterial abundance under e[CO2] despite higher plant biomass, while it did not affect bacterial abundance at ambient CO2. Warming and N-fertilizer supplies increased bacterial abundance and community composition, but these responses were not modified by CO2. Compared to the wheat growth stage and variation in N-supply, the effect of CO2 on the overall community composition was minor, explaining 2.2% of its variation. While 7 taxa, representing only 0.3% of relative abundance and most of them affiliated with Acidobacteria, changed with the CO2 level, many more responded to growth stages and nitrogen supply. Fewer growth stage and N supply-responsive taxa were detected with e[CO2]. Our results provide strong evidence that wheat plant responses to e[CO2] transfer to the rhizomicrobiome affecting the abundance composition of their constituting microbes. Additional drought conditions can strongly change the direction of these responses and should thus be considered for predicting implications of global change for the cropland soil microbiomes.

Soil bacterial community structure and functioning in a long-term conservation agriculture experiment under semi-arid rainfed production system.

Soil microbial communities are important drivers of biogeochemical cycling of nutrients, organic matter decomposition, soil organic carbon, and Greenhouse gas emissions (GHGs: CO2, N2O, and CH4) and are influenced by crop and soil management practices. The knowledge on the impact of conservation agriculture (CA) on soil bacterial diversity, nutrient availability, and GHG emissions in semi-arid regions under rainfed conditions is vital to develop sustainable agricultural practices, but such information has not been systemically documented. Hence, studies were conducted for 10 years in rainfed pigeonpea (Cajanus cajan L.)-castor bean (Ricinus communis L.) cropping system under semi-arid conditions to assess the effects of tillage and crop residue levels on the soil bacterial diversity, enzyme activity (Dehydrogenase, urease, acid phosphatase, and alkaline phosphatase), GHG emissions, and soil available nutrients (Nitrogen, phosphorus, and potassium). Sequencing of soil DNA through Illumina HiSeq-based 16S rRNA amplicon sequencing technology has revealed that bacterial community responded to both tillage and residue levels. The relative abundance of Actinobacteria in terms of Operational Taxonomic Unit (OTUs) at phyla, class as well as genera level was higher in CA (NTR1: No Tillage + 10 cm anchored residue and NTR2 NT + 30 cm anchored residue) over CT (conventional tillage without crop residues). CA resulted in higher enzyme activities (dehydrogenase, urease, acid phosphatase, and alkaline phosphatase) and reduction in GHG emissions over CT. CA recorded 34% higher and 3% lower OC, as compared to CT, and CTR1, respectively. CA recorded 10, 34, and 26% higher available nitrogen, phosphorus, and potassium over CT and CTR1, respectively. NTR1 recorded 25 and 38% lower N2O emissions as compared to CTR1 and CTR2, respectively. Whereas only NT recorded 12% higher N2O emissions as compared to CT. Overall, the results of the study indicate that CA improves the relative abundance of soil bacterial communities, nutrient availability, and enzyme activities, and may help to contribute to the mitigation of climate change, and sustainability in rainfed areas.

Factors for Conversion of the Content of Double-Stranded DNA to Carbon of Soil Microbial Biomass

The relationship between the concentration of soil DNA and microbial biomass, determined by the method of substrate-induced respiration was studied in a wide range of soils differing in particle size distribution, acidity, organic carbon content, microbial biomass, and type of human activity in antiquity and present time. Calcaric Leptosols and Leptic Phaeozems of medieval agricultural lands in the Central Caucasus, Stratozems of agricultural terraces of the late Middle Ages – Modern times in the middle mountain zone of the Eastern Caucasus, as well as Kashtanozems and Solonetzes with different grazing intensity in the dry steppe zone (Rostov region) were chosen as the key sites. It has been shown that the determination of soil dsDNA concentration is a reliable and simple method for determining microbial biomass in soils with a loam texture, organic carbon content less 2%, and microbial biomass less 700 µg C/g of dry soil. The conversion factor FDNA in such soils varied in a narrow range from 5.24 to 5.41. In soils with a high content of organic carbon, an increase of FDNA (6.56 and 10.56) was observed due to the presence of recalcitrant e-xtracellular DNA. Agristratified soil of sandy loam texture was characterized by a lower degree of preservation of dsDNA, which resulted in a decrease in the determined microbial biomass (FDNA = 4.22). A reduced conversion factor FDNA (4.78) was also found in the soils of pastures in the dry steppe zone, which confirms the known limitations of using the substrate-induced respiration method in alkaline soils. Human activity does not significantly affect the relationship between the amount of soil DNA and microbial biomass.

Urban land uses shape soil microbial abundance and diversity

Soil microbial biodiversity provides many useful services in cities. However, the ecology of microbial communities in urban soils remains poorly documented, and studies are required to better predict the impact of urban land use. We characterized microbial communities (archea/bacteria and fungi) in urban soils in Dijon (Burgundy, France). Three main land uses were considered – public leisure, traffic, and urban agriculture – sub-categorized in sub-land uses according to urban indexes and management practices. Microbial biomass and diversity were determined by quantifying and high-throughput sequencing of soil DNA. Variation partitioning analysis was used to rank soil physicochemical characteristics and land uses according to their relative contribution to the variation of soil microbial communities. Urban soils in Dijon harbored high levels of microbial biomass and diversity that varied according to land uses. Microbial biomass was 1.8 times higher in public leisure and traffic sites than in urban agriculture sites. Fungal richness increased by 25 % in urban agriculture soils, and bacterial richness was lower (by 20 %) in public leisure soils. Partitioning models explained 25.7 %, 46.2 % and 75.6 % of the variance of fungal richness, bacterial richness and microbial biomass, respectively. The organic carbon content and the C/N ratio were the best predictors of microbial biomass, whereas soil bacterial diversity was mainly explained by soil texture and land use. Neither metal trace elements nor polycyclic aromatic hydrocarbons contents explained variations of microbial communities, probably due to their very low concentration in the soils. The microbial composition results highlighted that leisure sites represented a stabilized habitat favoring specialized microbial groups and microbial plant symbionts, as opposed to urban agriculture sites that stimulated opportunistic populations able to face the impact of agricultural practices. Altogether, our results provide evidence that there is scope for urban planners to drive soil microbial diversity through sustainable urban land use and associated management practices.

Response of Bacterial Communities to Heavy Metal Contamination in an Abandoned Chromate Factory

The chromium slags left by industry emission has caused serious damage to the local ecological environment, and microorganisms were sensitive to the variation in the surrounding environmental factors. In this work, 14 soil samples around an abandoned chromate factory were collected. We utilized the 16S rRNA regions from soil DNA to explore the possible effects of environmental factors on soil bacterial communities. High-throughput DNA sequencing results suggested that bacterial communities varied greatly from different soil samples, but Actinobacteria and Proteobacteria were predominant in all samples in the phylum level. In addition, Pseudarthrobacter, Thiobacillus, Paenisporosarcina, Sphingomonas, and Bacillus were abundant at a more refined species level. Based on redundancy analysis (RDA) and variation partitioning analysis (VPA), the results revealed that pH value, soil organic carbon (SOC), total nitrogen (TN), total phosphorus (TP), chromium (Cr) and lead (Pb) could obviously affect the bacterial community structure. Heavy metals, soil nutrients, and pH could explain 37.69%, 13.61%, and 31.41% of bacterial community variation, respectively. This study would provide a theoretical basis for future remediation of local Cr-contaminated soil.

Short-term responses of plant growth-promoting bacterial community to the herbicides imazethapyr and flumioxazin

Imazethapyr and flumioxazin are widely recommended herbicides for soybean fields due to their broad-spectrum effects. However, although both herbicides present low persistence, their potential impact on the community of plant growth-promoting bacteria (PGPB) is unclear. To address this gap, this study assessed the short-term effect of imazethapyr, flumioxazin, and their mixture on the PGPB community. Soil samples from soybean fields were treated with these herbicides and incubated for 60 days. We extracted soil DNA at 0, 15, 30, and 60 days and sequenced the 16S rRNA gene. In general, the herbicides presented temporary and short-term effects on PGPB. The relative abundance of Bradyrhizobium increased, while Sphingomonas decreased on the 30th day with the application of all herbicides. Both herbicides increased the potential function of nitrogen fixation at 15th days and decreased at 30th and 60th days of incubation. The proportions of generalists were similar (∼42%) comparing each herbicide and the control, while the proportion of specialists increased (varying from 24.9% to 27.6%) with the application of herbicides. Imazethapyr, flumioxazin and their mixture did not change the complexity and interactions of the PGPB network. In conclusion, this study showed that, in the short term, the application of imazethapyr, flumioxazin, and their mixture, at the recommended field rates, does not negatively affect the community of plant growth-promoting bacteria.

Staircase Wetlands for the Treatment of Greywater and the Effect of Greywater on Soil Microbes

Fresh water is an increasingly scarce resource in both urban and rural development. As a response to this challenge, non-potable water reuse is on the rise. This research explored a potential off-grid system for water purification, consisting of a staircase wetland with terracotta pot plants working as a filter for greywater. This study further investigated the physicochemical properties of greywater and the soil before and after wetland purification. The removal of total suspended solids, total coliforms, fecal coliforms, etc., was always between 90 and 99%. Results show that the filtered water satisfied all requirements for water reuse, e.g., a pH of 7–7.5 and a turbidity < 5 NTU. This research then uniquely investigated the effect of greywater on soil microbes and soil biomass using soil DNA extraction and the tea bag index testing method. The filtered greywater absorbed by soil decomposed the soil faster (66% for green tea) and stabilized it better compared to tap-water-absorbed soil or unfiltered greywater. DNA generation sequencing revealed no significant differences in alpha diversity between the control and treatment samples. The beta diversity differences were significant. This nature-based solution can lead to reduced loads on the sewage system, resulting in less wastewater generation.

Integration of multiomics with precision nutrition for gestational diabetes: Study protocol for the Westlake Precision Birth Cohort

Integration of multiomics with precision nutrition for gestational diabetes: Study protocol for the Westlake Precision Birth Cohort

Impact of Chronic Crude Oil Pollution on Nitrification in Edaphic Systems

Nitrification plays a key role in regulating soil fertility, concentration of soil inorganic nitrogen and production of the highly potent greenhouse gas, nitrous oxide, from soil. This study investigated the effect of long-term crude oil pollution on the activity, abundance and diversity of autotrophic nitrifying bacteria in impacted soils from Bomu and Abonema in the Nigerian Niger Delta compared to unpolluted systems. Nitrifier activity was investigated by determining the soil oxidation potentials while the most probable number microtechnique using Winogradsky media was used to ascertain nitrifying bacteria abundance and soil DNA analysis alongside plate cultures defined nitrifying bacterial diversity. Nitrifier activity was found to be higher in the polluted soils than in the unpolluted soils while quantification of the nitrifying bacteria revealed that polluted samples had higher nitrifier counts. Statistically significant differences existed amongst nitrifier counts and activity (p≤0.05) between polluted and unpolluted systems with positive correlation between nitrifying bacteria counts and oxidation potential. The results highlighted the dominance of cultivable Nitrosomonas and Nitrobacter in both polluted and unpolluted soils while DNA analysis revealed that Nitrospira was the dominant nitrifier genus in unpolluted soils and Nitrobacter in polluted soils. Nitrospira was not detected the polluted ecosystems. No ammonia oxidisers were detected via metagenomics analysis. The ammonia oxidisers proved to be more sensitive to chronic crude oil pollution than the nitrite-oxidising bacteria. Long term crude oil exposure produced a 3 – 5 times increase in the activity of nitrifying bacteria in polluted soils with approximately twice the abundance when compared to the unpolluted systems.

Comparative Insights into the Microbial Diversity and Community Structure of Striga hermonthica-Infested Maize Rhizosphere

The damaging competition between crops and parasitic weeds has a negative impact on agricultural productivity; however, the impact of disturbance on the soil’s microbial community has received less attention. Hence, this study investigates the microbial composition and diversity of the maize rhizosphere infected with Striga hermonthica using a shotgun sequencing approach from two maize-growing fields (Eruwa, Nigeria and Mbuzini, South Africa). The rhizosphere soil DNA was extracted from infested soil using a Nucleospin soil genomic DNA extraction kit and sequenced on an Illumina platform. The dominant phyla were Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Acidobacteria, Chloroflexi, Cyanobacteria, Planctomycetes, Verrucomicrobia, Chlorobi, Proteobacteria, Firmicutes, Nitrospirae, Thermotogae, Synergistetes, Ascomycota, Euryarchaeota, and Crenarchaeota. Bacteria phyla were observed to be of higher proportion in the rhizosphere soil samples obtained from Striga-infested maize field in Eruwa (Es) than those recovered from Mbuzini (Ms). The alpha diversity of microbial communities indicated insignificance differences (p > 0.05) between the five taxonomical groups (phylum, class, order, family, and genus), while the beta diversity produced a significant (p = 0.01, R = 0.52) difference in the microbial diversity of the infested soil. In summary, the study sheds light on the diversity and composition of the microbiome of Striga hermonthica-infested soil, which influences the microbial functions in the management and sustenance of plant health against parasitic weeds.

Plant residues do not have an immediate impact on soil bacterial community composition and abundance

AbstractPlant residues are often used as soil amendments in laboratory experiments, but they can reportedly release compounds interfering with soil DNA extraction and subsequent molecular biological analyses. Theoretically, for accurate comparison of microbial community composition in soils with and without added plant residues after a period of incubation, no significant difference at the beginning of the experiment is required between the amended and unamended control soils. We mixed plant residue into soil and immediately (within 10 min) commenced DNA extraction, and then performed 16S rRNA gene sequencing and quantitative PCR (qPCR) to determine bacterial community composition and abundance. Soil without plant residue addition served as a control. Five commonly used DNA extraction kits, 16S rRNA gene primer pairs, and soils, and two types (rice straw and alfalfa shoots) and three addition rates (2%, 4%, and 6%; w/w) of plant residue, were tested. In all cases, we found no significant difference in measured bacterial community composition or abundance between the treatments with and without added plant residue.

Earthworms shape prokaryotic communities and affect extracellular enzyme activities in agricultural soil

Earthworms are known for their important role in soil ecosystem functioning and are used as indicators of ecosystem health. Earthworms and soil microorganisms are major players in soil ecosystem processes. However, understanding of their interactions is limited. Using microcosms, we studied the effect of earthworms on soil microorganisms in entire soil mesocosms by comparing soil with and without earthworms. Soil microbial activity was determined by an extracellular enzyme activity assay, while soil DNA was used to determine prokaryote abundance by quantitative PCR targeting 16S rRNA genes and community composition and diversity by amplicon sequencing of 16S rRNA genes. The microbial activity showed an indication of increase of chitinase, α-glucosidase, β-glucosidase and endo-β-glucanase during incubation with a specific increase in endo-β-glucanase activity in the presence of earthworms. Importantly earthworms decreased species richness (p = 0.002) and were a significant factor (p = 0.008) in shaping soil prokaryotes community structure. Moreover, our results revealed enrichment of bacterial phyla of Bacteroidetes and Proteobacteria, as well as reduction in relative abundance of the archaeal phylum Thaumarchaeota, suggesting that the presence of earthworms favors specific microbes in soil. Further, differential abundance analysis showed strong correlations between enzymatic activities (all tested except phosphomonoesterase) and relative abundances of specific bacterial OTUs. Our findings suggest that earthworms influence the soil microbial communities and their activity in soil, and hence earthworm-prokaryote interactions should be incorporated in future soil microbiome studies.

Ten years of green manuring and biofumigation alters soil characteristics and microbiota

Green manure and biofumigant crops are common within cropping rotations. These practices aim to maintain or increase the yield of agricultural soils by preventing degradation and protecting important ecosystem services, and their benefits to organic carbon levels, soil aggregation, erosion prevention and overall soil fertility are well documented. However, most studies have only examined the short term (one to three year) impacts of green manures and biofumigants, and information on their long term effects is lacking.This study examines the effects of 10 to 13 years of continuous ryegrass (Lolium multiflorum, var. Tetila) green manuring and brassica biofumigation (Brassica juncea, var. Caliente 199) on the soil's chemical, physical characteristics, microbial communities and resulting yields of a fertile, clay rich soil (Red Ferrosol) within an intensive vegetable cropping rotation. The aim was to assess the treatments' efficacy for improving or altering the forementioned parameters to improve sustainable yields, and to examine how the treatments affected the soil's microbial ecology. Soil aggregate stability was assessed at cash crop maturity, and soil chemistry, soil microbiota, and vegetable yields were sampled after the cash crop harvest for three consecutive years. To determine how the soil microbial communities had changed in response to the treatments soil DNA extracts were analysed by high throughput amplicon sequencing.Ryegrass green manuring and brassica biofumigation both altered the soil microbial communities and appeared to favour copiotrophic, organic matter degrading bacteria and fungi. Both treatments increased the relative abundance of arbuscular mycorrhizal fungi (AMF) by 800 % to 1400 % relative to the fallow in two of the three years. Soil aggregate stability was consistently higher in the ryegrass and biofumigant plots and a 20.4 % increase in soil carbon was observed in the treated plots on one side of the trial site. A 20 % increase in cash crop yield (carrots) was recorded in one of the three years.

Genomic microbiome analyses of surface sand samples from the Kyzyl-Kum Desert (Uzbekistan): characterization and comparative study.

The Kyzyl-Kum Desert extends over an area of 300,000 Km2, in the region bordering Kazakhstan, Uzbekistan and Turkmenistan and is mainly covered by sand dunes. The Kyzyl-Kum desert is also known for its large deposits of minerals of economic interests, the exploitation of which is affecting the local ecosystem and increasing the desertification. We examined the bacterial biodiversity of surface sand samples from several sites from the Kyzyl-Kum desert using pyrosequencing of PCR amplified bacterial 16S rRNA genes from total extracted soil DNA. We also examined several physicochemical parameters of the sand samples to investigate any possible correlations between bacterial community structure and environmental drivers. The predominant bacterial phyla present in the samples were found to belong to members of the Actinobacteria, Proteobacteria and Bacteroidetes. The most abundant genera in our samples were found to belong to the Arthrobacter, Adhaeribacter and Roseomonas genera. We found that the relative abundance of members belonging to the Actinobacteria phylum, commonly found in desertic areas, increase in abundance in sites with higher content of organic matter and sulfur, whereas members of the Proteobacteria and Bacteroidetes phyla seems to diminish in abundance in coarse silt and fine-grained soils and those rich in magnesium, suggesting that those parameters might influence the bacterial community composition in this desertic area. This study is the first to provide new insights into the prokaryotic community composition from this unusual desert site.

The utility of dust for forensic intelligence: Exploring collection methods and detection limits for environmental DNA, elemental and mineralogical analyses of dust samples

Environmental DNA (eDNA), elemental and mineralogical analyses of soil have been shown to be specific to their source material, prompting consideration of using the airborne fraction of soil (dust) for forensic intelligence work. Dust is ubiquitous in the environment and is easily transferred to items belonging to a person of interest, making dust analysis an ideal tool in forensic casework. The advent of Massive Parallel Sequencing technologies means metabarcoding of eDNA can uncover bacterial, fungal, and even plant genetic fingerprints in dust particles. Combining this with elemental and mineralogical compositions offers multiple, complementary lines of evidence for tracing the origin of an unknown dust sample. This is particularly pertinent when recovering dust from a person of interest to ascertain where they may have travelled. Prior to proposing dust as a forensic trace material, however, the optimum sampling protocols and detection limits need to be established to place parameters around its utility in this context. We tested several approaches to collecting dust from different materials and determined the lowest quantity of dust that could be analysed for eDNA, elemental composition and mineralogy, whilst still yielding results capable of distinguishing between sites. We found that fungal eDNA profiles could be obtained from multiple sample types and that tape lifts were the optimum collection method for discriminating between sites. We successfully recovered both fungal and bacterial eDNA profiles down to 3 mg of dust (the lowest tested quantity) and recovered elemental and mineralogical compositions for all tested sample quantities. We show that dust can be reliably recovered from different sample types, using different sampling techniques, and that fungi and bacteria, as well as elemental and mineralogical profiles, can be generated from small sample quantities, highlighting the utility of dust for forensic intelligence.

Immobilization of bacterial mixture of Klebsiella variicola FH-1 and Arthrobacter sp. NJ-1 enhances the bioremediation of atrazine-polluted soil environments.

In this study, the effects of the immobilized bacterial mixture (IM-FN) of Arthrobacter sp. NJ-1 and Klebsiella variicola strain FH-1 using sodium alginate-CaCl2 on the degradation of atrazine were investigated. The results showed that the optimal ratio of three types of carrier materials (i.e., rice straw powder, rice husk, and wheat bran) was 1:1:1 with the highest adsorption capacity for atrazine (i.e., 3774.47 mg/kg) obtained at 30°C. On day 9, the degradation efficiency of atrazine (50 mg/L) reached 98.23% with cell concentration of 1.6 × 108 cfu/ml at pH 9 and 30°C. The Box-Behnken method was used to further optimize the culture conditions for the degradation of atrazine by the immobilized bacterial mixture. The IM-FN could be reused for 2-3 times with the degradation efficiency of atrazine maintained at 73.0% after being stored for 80 days at 25°C. The population dynamics of IM-FN was explored with the total soil DNA samples specifically analyzed by real-time PCR. In 7 days, the copy numbers of both PydC and estD genes in the IM-FN were significantly higher than those of bacterial suspensions in the soil. Compared with bacterial suspensions, the IM-FN significantly accelerated the degradation of atrazine (20 mg/kg) in soil with the half-life shortened from 19.80 to 7.96 days. The plant heights of two atrazine-sensitive crops (wheat and soybean) were increased by 14.99 and 64.74%, respectively, in the soil restored by immobilized bacterial mixture, indicating that the IM-FN significantly reduced the phytotoxicity of atrazine on the plants. Our study evidently demonstrated that the IM-FN could significantly increase the degradation of atrazine, providing a potentially effective bioremediation technique for the treatment of atrazine-polluted soil environment and providing experimental support for the wide application of immobilized microorganism technology in agriculture.

Dung Beetle Activity Is Soil-Type-Dependent and Modulates Pasture Growth and Associated Soil Microbiome

The introduction of numerous exotic dung beetles across southern Australia in regions where native dung beetles are not generally efficient in processing livestock dung has resulted in significant reductions in the quantity of such dung on the soil surface in recent years. However, the direct impacts of such ecosystem services on pasture quality and soil nutrient mobility have not yet been investigated in the Riverina region of New South Wales (NSW), an area recognised for prime cattle and sheep production in Australia. Utilising 48 soil columns for lysimetry, we quantified the impact of a common introduced dung beetle (Bubas bison) in this region on water quality after permeation through four different soil types sown to winter annual pastures. Dung beetle treatments included dung plus dung beetles, dung alone and no dung beetles, and no dung and no beetles as a control. Dung beetles and soil type impacted on the performance of improved overseeded annual pastures as measured by biomass accumulation over a four-month growing season. The four soil types, namely, Chromosol, Kandosol, Rudosol, and Vertosol, differed considerably with respect to their water-holding capacity and nutrient profiles, as assessed by initial soil testing and soil leachate evaluation following rainfall plus simulated rainfall events. The concentration of Escherichia coli resulting from cattle dung, cattle dung plus beetles, and the control soils without dung or beetles was assessed in collected leachates over a three-month period. E. coli numbers were significantly increased following B. bison activity, when compared to the dung-only and control treatments. Evaluation of the soil microbiome, by assessing genomic DNA in soils sampled 10 cm below the soil surface where dung beetles remained active following tunnelling, revealed significant differences among soil types with respect to bacterial and fungal communities. Within each soil type, dung beetle activity impacted the fungal community structure, but not the bacterial community. Pasture performance as assessed by biomass accumulation was significantly improved following dung beetle activity in later stages of pasture growth, while E. coli numbers and total coliforms appeared unaffected by beetle presence.

Biocontrol of the causal brown patch pathogen Rhizoctonia solani by Bacillus velezensis GH1-13 and development of a bacterial strain specific detection method.

Brown patch caused by the basidiomycete fungus Rhizoctonia solani is an economically important disease of cool-season turfgrasses. In order to manage the disease, different types of fungicides have been applied, but the negative impact of fungicides on the environment continues to rise. In this study, the beneficial bacteria Bacillus velezensis GH1-13 was characterized as a potential biocontrol agent to manage brown patch disease. The strain GH1-13 strongly inhibited the mycelial growth of turf pathogens including different anastomosis groups of R. solani causing brown patch and large patch. R. solani AG2-2(IIIB) hyphae were morphologically changed, and fungal cell death resulted from exposure to the strain GH1-13. In addition, the compatibility of fungicides with the bacterial strain, and the combined application of fungicide azoxystrobin and the strain in brown patch control on creeping bentgrass indicated that the strain could serve as a biocontrol agent. To develop strain-specific detection method, two unique genes from chromosome and plasmid of GH1-13 were found using pan-genome analysis of 364 Bacillus strains. The unique gene from chromosome was successfully detected using both SYBR Green and TaqMan qPCR methods in bacterial DNA or soil DNA samples. This study suggests that application of GH1-13 offers an environmentally friendly approach via reducing fungicide application rates. Furthermore, the developed pipeline of strain-specific detection method could be a useful tool for detecting and studying the dynamics of specific biocontrol agents.

Effect of Keratin Hydrolysates Obtained from Feather Decomposition by Trichophyton ajelloi on Plant Germination, Growth and Biological Activity of Selected Arable Soils under Model Conditions

The use of fertilizers based on organic waste as a result of microbial biodegradation and biotransformation is becoming increasingly common practice in plant cultivation. This is to limit the chemicals used in agriculture and thus protect the environment and consumer health. The aim of this study was to evaluate a hitherto unexplored effect of hydrolysates obtained after biodegradation of chicken feather waste by a soil strain of the keratinolytic fungus Trichophyton ajelloi on germination, early growth, and development of plants, in particular with high sulfur requirements, and to verify, in model conditions, their impact on soil biological activity and the total pool of soil DNA. Undiluted and diluted keratin hydrolysate generally stimulated seed germination as well as root and shoot growth of the Lepidium sativum L. (garden cress) and Brassica napus L. var. napus (oilseed rape) on sandy soil (Cambisol I), loamy soil (Cambisol II), and Chernozem. In the model experiment, in the variants with and without oilseed rape on sandy soil and Chernozem, the introduction of keratin hydrolysate generally increased the total abundance of microorganisms. In sandy soil, feather hydrolysate mostly increased respiratory activity, dehydrogenase activity, and alkaline phosphatase activity by an average of approx. 38% and the total DNA pool by 15% and 23% in the variant with and without plants. The activity of protease and acid phosphatase increased by an average of 4% and 6% only in the variant without oilseed rape. Respiratory and enzymatic activity in Chernozem, in the variants with and without oilseed rape, tended to show a downward trend, despite periodically recorded increases. The total DNA pool has increased by 8% in the oilseed rape variant. Oilseed rape biomass was almost two-fold higher after adding feather hydrolysate to both soils, and its yield was higher by 98% in Chernozem than in sandy soil. The results have demonstrated that keratin hydrolysate can be used as a biofertilizer.

Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material

Tungiasis is a neglected tropical disease caused by skin-penetrating female Tunga penetrans fleas. Although tungiasis causes severe health problems, its ecology is poorly understood and morphological descriptions of the larvae are unavailable. To identify T. penetrans immature stages and sites where they develop, diagnostic PCRs are required. However, flea larvae feed on soil organic matter rich in PCR inhibitors. Here, three DNA preparation methods, including a soil DNA kit that removes inhibitors, a simple ammonium acetate precipitation approach (AmAcet) and a crude lysate of larvae (CL), were combined with amplification by the highly processive FIREPol® Taq or the inhibitor-resistant Phusion® polymerase. Independent of the polymerase used, the frequency of successful amplification, Cq values and PCR efficacies for the low-cost CL and AmAcet methods were superior to the commercial kit for amplification of a 278 bp partial internal transcribed spacer-2 (ITS-2) and a 730 bp pan-Siphonaptera cytochrome oxidase II PCR. For the CL method combined with Phusion® polymerase, the costs were approximately 20-fold lower than for the methods based on the soil DNA kit, which is a considerable advantage in resource-poor settings. The ITS-2 PCR did not amplify Ctenocephalides felis genomic or Tunga trimammilata ITS-2 plasmid DNA, meaning it can be used to specifically identify T. penetrans.