Soil DNA Samples Research Articles

Environmental Detection of Parasites in the Marginalized Paiute Reservations Compared to a Nearby Area.

The amounts of parasite DNA in soil samples from different playgrounds and other public areas can help identify areas of possible microbe transmission and give indications of the possible occurrence of parasite infection in nearby communities. We collected 207 soil samples from parks in Paiute indigenous tribal areas in southwestern Utah and from the higher income city of St. George, Utah, and tested them for 11 parasites that can cause human disease. Molecular tests revealed an elevated odds ratio (OR) of 3.072 (range, 1.114-8.065) for detecting the helminth Trichuris trichiura and an elevated OR of 3.036 (range, 1.101-7.966) for any protozoa (not including Acanthamoeba) in the tribal land playgrounds compared with St. George parks. These findings support previous studies showing that areas in lower socioeconomic communities, especially marginalized communities, tend to have more parasites in the soil, which may lead to higher disease prevalence rates.

Dead or Alive, that Is the Question: Development and Assessment of Molecular Synchytrium endobioticum Viability Tests

Potato wart disease caused by the obligate biotrophic fungus Synchytrium endobioticum is a devastating disease that can result in significant crop losses. Resting spores of this pathogen can remain viable and infectious in soil for decades. The detection of viable resting spores using conventional methods such as bioassays and direct microscopic examination are challenging and time-consuming and require specific expertise and facilities. Molecular methods, such as real-time PCR, have been shown to be effective in detecting the presence of S. endobioticum DNA in soil samples but cannot differentiate between viable and nonviable spores. In this paper, we present three novel mRNA-based molecular tests to potentially detect viable S. endobioticum resting spores. The tests are specific to the transcribed mRNA and do not detect the genomic DNA of the target genes. We demonstrate the analytical sensitivity using synthetic constructs of the target mRNAs. The tests were found to be able to repeatedly detect 10 target copies per reaction. Soils and waste of potato processing industries free from S. endobioticum were used to assess the exclusivity of the tests. The biological relevance of mRNA detection was determined in the context of replicated bioassays. Applications of the tests to facilitate collection management, assessment of the effects of treatments on presumed viability of S. endobioticum resting spores, and the potential use in descheduling of previously infested plots are discussed. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

An improved protocol for metagenomic DNA isolation from low microbial biomass alkaline hot-spring sediments and soil samples.

High-quality, humic-acid-free pure DNA is a prerequisite for functional and sequence-based approaches of metagenomics. In the present investigation, an improved extraction buffer was developed by making a combination of powdered activated charcoal (2%; w/v), polyvinyl poly pyrrolidone (2%; w/v), and CaCl2 (2%; w/v). This trio significantly improved the purity and yield of the metagenomic DNA from the hot spring's hot and alkaline soil. The quality of extracted metagenomic DNA was successfully validated by PCR amplification and restriction enzymes. Besides, the thermophilic amylase encoding genes were also retrieved from these soil DNA samples. Extreme habitats I harbour low microbial biomass and, therefore, demand in-situ lysis of the microbial cells to access their genomes. The protocol can potentially extract DNA from geothermal spring habitats where the count of microbial cells is low.

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.

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.

Combined assembly of long and short sequencing reads improve the efficiency of exploring the soil metagenome

BackgroundAdvances in DNA sequencing technologies have transformed our capacity to perform life science research, decipher the dynamics of complex soil microbial communities and exploit them for plant disease management. However, soil is a complex conglomerate, which makes functional metagenomics studies very challenging.ResultsMetagenomes were assembled by long-read (PacBio, PB), short-read (Illumina, IL), and mixture of PB and IL (PI) sequencing of soil DNA samples were compared. Ortholog analyses and functional annotation revealed that the PI approach significantly increased the contig length of the metagenomic sequences compared to IL and enlarged the gene pool compared to PB. The PI approach also offered comparable or higher species abundance than either PB or IL alone, and showed significant advantages for studying natural product biosynthetic genes in the soil microbiomes.ConclusionOur results provide an effective strategy for combining long and short-read DNA sequencing data to explore and distill the maximum information out of soil metagenomics.

Highly comparable metabarcoding results from MGI-Tech and Illumina sequencing platforms.

With the developments in DNA nanoball sequencing technologies and the emergence of new platforms, there is an increasing interest in their performance in comparison with the widely used sequencing-by-synthesis methods. Here, we test the consistency of metabarcoding results from DNBSEQ-G400RS (DNA nanoball sequencing platform by MGI-Tech) and NovaSeq 6000 (sequencing-by-synthesis platform by Illumina) platforms using technical replicates of DNA libraries that consist of COI gene amplicons from 120 soil DNA samples. By subjecting raw sequencing data from both platforms to a uniform bioinformatics processing, we found that the proportion of high-quality reads passing through the filtering steps was similar in both datasets. Per-sample operational taxonomic unit (OTU) and amplicon sequence variant (ASV) richness patterns were highly correlated, but sequencing data from DNBSEQ-G400RS harbored a higher number of OTUs. This may be related to the lower dominance of most common OTUs in DNBSEQ data set (thus revealing higher richness by detecting rare taxa) and/or to a lower effective read quality leading to generation of spurious OTUs. However, there was no statistical difference in the ASV and post-clustered ASV richness between platforms, suggesting that additional denoising step in the ASV workflow had effectively removed the ‘noisy’ reads. Both OTU-based and ASV-based composition were strongly correlated between the sequencing platforms, with essentially interchangeable results. Therefore, we conclude that DNBSEQ-G400RS and NovaSeq 6000 are both equally efficient high-throughput sequencing platforms to be utilized in studies aiming to apply the metabarcoding approach, but the main benefit of the former is related to lower sequencing cost.

Influence of Trichoderma asperellum and Bacillus subtilis as biocontrol and plant growth promoting agents on soil microbiota

PurposeThe manipulation of soil microbiota can involve changes in microbial diversity and microbial activities, and it is carried out for practical purposes. The microbial diversity can be modified by the inoculation of beneficial microorganisms into soil or by agricultural management practices. This study provides information on the influence of introducing beneficial soil microorganisms on soil indigenous microbiota. The aim of this study was to determine how biocontrol and plant growth promoting agents Trichoderma asperellum and Bacillus subtilis and their consortium affect indigenous soil microbiota without placing emphasis on the plant as a determinant of change.MethodsExperimental soil samples were treated with B. subtilis and T. asperellum and their consortium. The shift of number of bacterial and fungal CFUs in soil was determined. Biolog EcoPlate assay demonstrated the metabolic activity of microorganisms in soil. The concentration of Trichoderma genus, Firmicutes, Gammaproteobacteria and Acidobacteria DNA in soil samples was determined by molecular methods. A correlation analysis was performed between microbiological and molecular data.ResultsSoil treatment with T. asperellum, B. subtilis and the plant pathogenic fungus Botrytis cinerea changed the number of CFUs and amplified DNA fragments of certain taxa. The study showed that added microorganisms did not significantly affect the metabolic diversity of the community and Shannon-Wiener biodiversity index but change the utilization of carbohydrates, complex carbon compounds and organic phosphorus compounds.ConclusionIntroduced biocontrol and plant growth promoting agents T. asperellum and B. subtilis survive in soil during a 60-day experiment and influence composition and functionality of indigenous populations. Whereas B. subtilis and T. asperellum and their consortium are inhibitors of the pathogenic fungus B. cinerea, this could further positively affect specific crops.

Phytophthora austrocedri in Argentina and Co-Inhabiting Phytophthoras: Roles of Anthropogenic and Abiotic Factors in Species Distribution and Diversity

This work reports the first survey of Phytophthora diversity in the forests soils of Andean Patagonia. It also discusses the role of anthropogenic impact on Phytophthora distribution inferred from the findings on Phytophthora diversity and on the distribution of Phytophthora austrocedri-diseased forests. Invasive pathogen species threatening ecosystems and human activities contribute to their entry and spread. Information on pathogens already established, and early detection of potential invasive ones, are crucial to disease management and prevention. Phytophthora austrocedri causes the most damaging forest disease in Patagonia, affecting the endemic species Austrocedrus chilensis (D. Don) Pic. Sern. and Bizzarri. However, the relationship between anthropogenic impacts and the disease distribution has not been analyzed enough. The aims of this work were: to evaluate Phytophthora diversity in soils of Andean Patagonia using a metabarcoding method, and analyze this information in relation to soil type and land use; to assess the distribution of Austrocedrus disease over time in relation to anthropogenic and abiotic gradients in an area of interest; and to discuss the role of human activities in Phytophthora spread. High throughput Illumina sequencing was used to detect Phytophthora DNA in soil samples. The distribution of Austrocedrus disease over time was assessed by satellite imagery interpretation. Twenty-three Phytophthora species, 12 of which were new records for Argentina, were detected. The most abundant species was P. austrocedri, followed by P. × cambivora, P. ramorum and P. kernoviae. The most frequent was P. × cambivora, followed by P. austrocedri and P. ramorum. Phytophthora richness and abundance, and Austrocedrus disease distribution, were influenced by land use, anthropogenic impact and soil drainage. Results showed several Phytophthoras, including well-known pathogenic species. The threat they could present to Patagonian ecosystems and their relations to human activities are discussed. This study evidenced the need of management measures to control the spread of P. austrocedri and other invasive Phytophthora species in Patagonia.

Detection and spread of Phytophthora austrocedri within infected Juniperus communis woodland and diversity of co‐associated Phytophthoras as revealed by metabarcoding

AbstractPhytophthora austrocedri is a recently invasive soilborne pathogen which is causing widespread mortality of Juniperus communis in northern Britain. The pathways by which a single genotype of P. austrocedri has spread to infect such a geographically dispersed range of woodland sites within a relatively short timeframe are unknown. This study examined the detectability of P. austrocedri in soil and water within infected J. communis woodland using qPCR to gain a better understanding of the pathogen's key mechanisms of spread. A Phytophthora metabarcoding method was also applied to investigate the wider diversity of Phytophthora species present in water at one of the sites. qPCR analyses of P. austrocedri in soil samples at a J. communis woodland exhibiting low‐to‐moderate levels of disease suggested a slow natural spread of the pathogen in soil, requiring high moisture conditions. However, the ubiquity of P. austrocedri DNA in soil samples collected across a heavily infected J. communis site suggests that once established at a site the pathogen can be spread readily in soil locally, most likely vectored by animal movements and/or human activities. The hypothesis that P. austrocedri is aerially transmitted in rainwater was not adequately proven, and an alternative hypothesis for the widespread distribution of the pathogen on J. communis in northern Britain is presented. Metabarcoding identified DNA from a diverse range of Phytophthora species in river and rainwater samples although the main target pathogen, P. austrocedri, was not amplified which disagreed with some of the qPCR findings. Possible reasons for this are discussed.

Molecular detection of Paracoccidioides in soil from an urban area of southern Brazil

INTRODUCTION: Previous studies that detected Paracoccidioides spp. DNA in soil taken from rural areas have shown this to be a valuable tool for ecological and epidemiological studies. This study reports the detection of Paracoccidioides spp. DNA in soil samples from an urban area of southern Brazil. METHODS: Sixteen soil samples were submitted to nested-PCR and the amplicons of a representative number of positive samples were sequenced. RESULTS: Paracoccidioides spp. DNA was found in 44% of samples. Four DNA amplicons were sequenced, showing 100% homology with P. brasiliensis. CONCLUSIONS: The southern Brazilian urban population is commonly exposed to the Paracoccidioides fungus.

Prevalence, risk factors and genotype distribution of Toxoplasma gondii DNA in soil in China.

In the present study, we performed a cross-sectional survey to determine the occurrence and genotype distribution of T. gondii DNA in soil samples collected from different sources from six geographic regions in China. Between March 2015 and June 2017, 2100 soil samples were collected from schools, parks, farms and coastal beaches, and examined for T. gondii DNA using three PCR assays targeting 529-bp repeat element (RE) sequence, B1 gene and ITS-1 gene sequences. Also, we investigated whether geographic region, soil source and type, and sampling season can influence the prevalence of T. gondii DNA in the soil. Soil samples collected from farms and parks had the highest prevalence, whereas samples collected from school playgrounds and coastal beaches had the lowest prevalence. PCR assays targeting 529-bp RE and ITS-1 gene sequences were more sensitive than the B1 gene-based assay. Positive PCR products were genotyped using multi-locus PCR-RFLP, and ToxoDB #9 was the predominant genotype found in the contaminated soil samples. Multiple logistic regression identified factors correlated significantly with the presence of T. gondii DNA in the soil to be the source of the soil, including farms (odds ratio 3.10; 95% confidence interval [CI], 1.52 to 6.29; p=0.002) and parks (2.59; 95% CI 1.28 to 5.27; p=0.009). These results show that Chinese soil hosts T. gondii of the most prevalent genotype in China (ToxoDB#9) and that the soil type influences infection patterns.

A rapid and sensitive method to detect Toxoplasma gondii oocysts in soil samples

Documenting the extent of soil contamination by Toxoplasma gondii oocysts is a key issue to prevent the worldwide infection caused by this protozoan. Our aim was to improve the practicability and sensitivity of a low-cost method to detect T. gondii DNA in soil samples developed a few years ago. Various parameters of the reference protocol were modified to determine their effect on the detection of T. gondii DNA in soil samples (“natural soil” and “sand”) spiked with oocysts. We tested i) filtration using stomacher bags, ii) Tween 80, Tween 20, SDS and Triton X100 as dispersion solutions, iii) sucrose solution, zinc chloride solution, Optiprep and Percoll as density gradients, iv) freeze/thaw versus mechanical grinding as lysis methods, and v) Qiagen versus Fastprep as extraction kits The optimized protocol is quicker and easier to use than the previous one, and includes the following items: 0.1% Tween80/PBS for dispersion, sucrose solution for flotation, mechanical grinding, and FastDNA spin kit for extraction. It accurately detects T. gondii DNA in both fresh and frozen soil samples and displays a detection limit below 1 oocyst/g of fresh soil.

Global distribution of Trebouxiophyceae diversity explored by high-throughput sequencing and phylogenetic approaches.

Trebouxiophyceae are a ubiquitous class of Chlorophyta encountered in aquatic and terrestrial environments. Most taxa are photosynthetic, and many acts as photobionts in symbiotic relationships, while others are free-living. Trebouxiophyceae have also been widely investigated for their use for biotechnological applications. In this work, we aimed at obtaining a comprehensive image of their diversity by compiling the information of 435 freshwater, soil and marine environmental DNA samples surveyed with Illumina sequencing technology in order to search for the most relevant environments for bioprospecting. Freshwater and soil were most diverse and shared more than half of all operational taxonomic units (OTUs), however, their communities were significantly distinct. Oceans hosted the highest genetic novelty, and did not share any OTUs with the other environments; also, marine samples host more diversity in warm waters. Symbiotic genera usually found in lichens such as Trebouxia, Myrmecia and Symbiochloris were also abundantly detected in the ocean, suggesting either free-living lifestyles or unknown symbiotic relationships with marine planktonic organisms. Altogether, our study opens the way to new prospection for trebouxiophycean strains, especially in understudied environments like the ocean.

Paracoccidioides spp. in Soil from the Pampa Biome in Southern Brazil.

High rates of paracoccidioidomycosis disease and infection have been recently reported in southern Brazil. However, the knowledgment about the ecoepidemiology of Paracoccidioides spp. in the region is limited, therefore, this study aimed to evaluate the presence of Paracoccidioides spp. DNA in soil samples from a rural area in Southern Rio Grande do Sul, Brazil. Thirty pools of soil samples from Bagé, RS (31º19'53"S 54º06'25"W) were submitted to physicochemical analysis, and to fungal DNA extraction by Norgen Biotek® Kit (Thorold, Canada), followed by Nested PCR technique with ITS4 and ITS5 as external primers, and PBITS-E and PBITS-T as internal primers. DNA amplification products of about 424bp compatible with Paracoccidioides spp. were detected in eight (26.7%) of the 30 pools of samples, being three were sequenced and identified as P. brasiliensis. Positive soils were characterized by high levels of humidity, organic matter, basic saturation, and pH. This study shows for the first time the presence of Paracoccidioides spp. DNA at soils from the Brazilian Pampa Biome, proving that people living in those areas are exposed to the main agent of paracoccidioidomycosis.

Effects of elevated ground-level ozone on paddy soil bacterial community and assembly mechanisms across four years

It is well known that elevated ground-level ozone (eO3) poses a threat to the ecosystem. Little knowledge about the underground variables, especially on soil microorganisms, however, has been revealed. Such knowledge will tremendously help to advance our understanding of the correlation between ecosystems and climate change, as well as our ability to predict future trajectory. For this purpose, we have collected soil DNA samples (eO3 vs. Ambient, each having 36 samples) over four years. Our results have verified the temporal responses and the underlying assembly mechanisms of the paddy bacterial community to eO3. Contrary to the widespread consensus, it was found that eO3 stimulated bacterial alpha diversities. The higher complexity and the centralization of the co-occurrence network of the bacterial community suggested that this stimulation was due to a microbial survival strategy in response to the limited resources, which led to the instability of the community. Furthermore, the observed slower temporal turnover of the bacterial community composition in response to eO3 was due to the decreased deterministic processes derived from plants, which implied that eO3 disrupted the coordination between soil microorganisms and rice crop. All above phenomena provided novel insights into the adverse influences of eO3 on the soil microbial community. If O3 concentration increases continuously, the adverse effects will be aggravated and harm the related ecological functions.

Anthropogenic disturbance equalizes diversity levels in arbuscular mycorrhizal fungal communities.

The arbuscular mycorrhizal (AM) symbiosis is a key plant-microbe interaction in sustainable functioning ecosystems. Increasing anthropogenic disturbance poses a threat to AM fungal communities worldwide, but there is little empirical evidence about its potential negative consequences. In this global study, we sequenced AM fungal DNA in soil samples collected from pairs of natural (undisturbed) and anthropogenic (disturbed) plots in two ecosystem types (10 naturally wooded and six naturally unwooded ecosystems). We found that ecosystem type had stronger directional effects than anthropogenic disturbance on AM fungal alpha and beta diversity. However, disturbance increased alpha and beta diversity at sites where natural diversity was low and decreased diversity at sites where natural diversity was high. Cultured AM fungal taxa were more prevalent in anthropogenic than natural plots, probably due to their efficient colonization strategies and ability to recover from disturbance. We conclude that anthropogenic disturbance does not have a consistent directional effect on AM fungal diversity; rather, disturbance equalizes levels of diversity at large scales and causes changes in community functional structure.

Differences in soil fungal assemblages associated with native and non-native tree species of varying weediness

Here we characterize and compare the diversity of belowground fungal communities of maples (Sapindaceae: Acer) varying in both nativity and weediness, and interpret our findings in the context of multiple non-exclusive theories on tree invasions and fungal associations. We made our fungal community comparisons based on high-throughput Illumina sequencing of the internal transcribed spacer region of fungal ribosomal DNA of soil samples associated with the roots of different species of maple collected from six sites throughout Central Pennsylvania. In our system, we found that weedy species, regardless of nativity, had the greatest soil fungal richness and that the nonnative invasive Norway maple had the highest abundance of mycorrhizal mutualists. Despite that much of the fungal community variability in our system was attributable to inter-site variability, we found that the core fungal communities associated with nonnative tree species were an inclusively larger set that included nearly all of those associated with native trees in addition to many not found with the natives, and the core communities of non-weedy species were largely a subset of those associated with weedy maples. In addition to confirming the strong influence that site variation has on soil fungal communities, our findings are also largely consistent with positive feedback from native fungal communities, possible co-invasion by fungal associates that are only associated with the nonnative trees, and generally add to the growing number of studies that have observed a greater abundance of mutualists associated with invasive trees that interact with arbuscular mycorrhizal fungi.

A re-evaluation of dilution for eliminating PCR inhibition in soil DNA samples

Quantitative real-time PCR (qPCR) analysis for accurate quantification of targeted microbial genes is compromised by the presence of co-extracted inhibitors from soil samples. Dilution of DNA extracts is a commonly-used method to reduce levels of inhibition. However, the applications of dilution method are mostly empirical, and need to be further elaborated. Here, we propose a dilution model to re-evaluate dilution as a method to eliminate qPCR inhibition. We found that DNA extracts without dilution or with a minor dilution (e.g., 10-fold) resulted in qPCR inhibition for most of studied soils. However, excessive dilution (e.g., 200- or 400-fold) caused an overestimation of the quantified gene copy numbers. Only under a moderate dilution range could qPCR inhibition be efficiently eliminated, which has been well captured by our proposed dilution model. The pre-testing of qPCR inhibition for determining the appropriate dilution range for extracted DNA samples aids accurate quantification of nucleic acids in soils.

Soil pretreatment and fast cell lysis for direct polymerase chain reaction from forest soils for terminal restriction fragment length polymorphism analysis of fungal communities.

Humic substances in soil DNA samples can influence the assessment of microbial diversity and community composition. Using multiple steps during or after cell lysis adds expenses, is time-consuming, and causes DNA loss. A pretreatment of soil samples and a single step DNA extraction may improve experimental results. In order to optimize a protocol for obtaining high purity DNA from soil microbiota, five prewashing agents were compared in terms of their efficiency and effectiveness in removing soil contaminants. Residual contaminants were precipitated by adding 0.6 mL of 0.5 M CaCl2. Four cell lysis methods were applied to test their compatibility with the pretreatment (prewashing + Ca2+ flocculation) and to ultimately identify the optimal cell lysis method for analyzing fungal communities in forest soils. The results showed that pretreatment with TNP + Triton X-100 + skim milk (100 mM Tris, 100 mM Na4P2O7, 1% polyvinylpyrrolidone, 100 mM NaCl, 0.05% Triton X-100, 4% skim milk, pH 10.0) removed most soil humic contaminants. When the pretreatment was combined with Ca2+ flocculation, the purity of all soil DNA samples was further improved. DNA samples obtained by the fast glass bead-beating method (MethodFGB) had the highest purity. The resulting DNA was successfully used, without further purification steps, as a template for polymerase chain reaction targeting fungal internal transcribed spacer regions. The results obtained by terminal restriction fragment length polymorphism analysis indicated that the MethodFGB revealed greater fungal diversity and more distinctive community structure compared with the other methods tested. Our study provides a protocol for fungal cell lysis in soil, which is fast, convenient, and effective for analyzing fungal communities in forest soils.