We thank funding from FAPESP (2015/26479-6) and CNPq (447453/2014-9). JLSM thanks CNPq for productivity grants (303664/2020-7). MPP thanks Capes (master&#39;s degree scholarship, process 88887.600591/2021-00) and CNPq.

<ol>
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The authors have nothing to disclose and no conflict of interest.

The superficial disinfestation of plant samples is one of the most critical stages in the presented protocol. No contamination in the PDA dishes with drops from the last wash are highly desirable. Bacteria are frequently observed as contaminants in the isolation dishes, usually more than airborne sporulating fungi, considering endophytic bacteria are also common within plant tissues3,11. Thus, the addition of antibiotics in the culture medium when installing the ...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65135">Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants</a>. The Authors section was updated from:
Juliana Lishcka Sampaio Mayer
to:
Juliana Lischka Sampaio Mayer

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65135">Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants</a>. The Authors section was updated.

Erratum: Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Endophytic fungi are, by definition, those that inhabit the interior of plant organs and tissues in inconspicuous infections (i.e., without causing harm to their host)1,2. These fungi can neutrally or beneficially interact with host plants, may confer resistance to pathogens and unfavorable environmental conditions, and may contribute to the synthesis of beneficial compounds for the plant (e.g., growth factors and other phytohormones)1,3. Mycorrhizal endophytes are fungi that establish mycorrhizal associations with the plant, taking part in nutrient transfer4. In Orchidaceae, the interaction with mycorrhizal endophytes is fundamental for seed germination in the vast majority of species, and seedling establishment in all the plants in the family5. In such contexts, mycoheterotrophic orchids represent a case of total dependence regarding their mycorrhizal partners, as they depend on mineral nutrients and carbon compounds transference by these fungi during their whole life cycle6. Therefore, the isolation and identification of associating fungi is a fundamental base when investigating mycoheterotrophic life strategies. Moreover, little is known about the roles of fungal endophytes in mycoheterotrophic plants or even the real diversity of these fungi7,8.
The investigation of endophytic fungi may be conducted via different techniques, traditionally described as culture-independent or -dependent, for instance: (a) direct observation, (b) fungal isolation and morphological and/or molecular identification, and (c) total DNA extraction of plant tissues and molecular identification9. In direct observation (a), endophytic fungi may be investigated while still in the interior of plant cells and tissues by light or electron microscopy9, as different microscopy protocols are detailed by Pena-Passos et al.10. By isolation methods (b), fungal endophytes can be characterized according to their colonies, hyphae, and reproductive or resistance structure morphology. Also, via isolation techniques, it is possible to conduct the molecular identification of isolates through DNA extraction, amplification of molecular identification sequences (barcodes or fingerprints), and sequencing11. The latter technique (c) enables the molecular identification of endophytic fungi per DNA extraction while in the interior of plant tissues (metabarcoding), followed by library preparation and sequencing12.
Moreover, fungal isolates may be applied in symbiotic germination trials, using seeds from autotrophic or mycoheterotrophic orchids. An example of such an application is the investigation conducted by Sisti et al.13, describing the germination and initial stages of protocorm development in Pogoniopsis schenckii, a mycoheterotrophic orchid, in association with some of its isolates, comprising non-mycorrhizal endophytic fungi. The applied symbiotic germination protocol is detailed and presented in a video by Pena-Passos et al.10. Isolating fungi in association with different plant organs allows diverse investigation focuses regarding the nature of plant-fungal interactions (e.g., to comprehend either ecological or physiological aspects of the association, as well as inquiries into the nutrient transference from fungi to the plant)9.
The methodologies presented in section 1 are based on a collection of subterranean organ samples, as these organs present the most difficulties in collection, and they are of major interest since mycorrhizal endophytes colonize them. However, both included protocols (steps 1.1 and 1.2) may be applied to other mycoheterotrophic plant organs (e.g., rhizomes, floral stems, and fruits). The collection methodology described in step 1.1 is designated for isolating endophytic fungi (section 2) for morphological characterization (sections 4 and 5) and/or total DNA extraction for isolate identification (section 6). On the other hand, the collection methodology described in step 1.2 is exclusively assigned to total DNA extraction of plant tissues for metabarcoding techniques (section 7). In section 3, four methods for filamentous fungi storage and preservation are presented, two for short-term storage (3-6 months) and the other two adequate for long-term storage (&#62;1 year). The morphological characterization (sections 4 and 5) may be associated with molecular identification to reinforce it and provide important information on fungal macro- and micromorphology. Figure 1 summarizes the collective methodologies described thereafter.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65135/65135fig01.jpg" /> 
Figure 1: Schematic summarization of the presented methods. Plant collection and fungal isolation, preservation, and molecular identification by culture-dependent and -independent methodologies. <a href="https://www.jove.com/files/ftp_upload/65135/65135fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Adhesive tape</td><td></td><td></td><td>(from any company, for adhesive tape mount in micromorphological analyses)</td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A5354</td><td>(for installation of plant fragments; other antibiotics may be used - check step 2.2.1)</td></tr><tr><td>Autoclave</td><td></td><td></td><td>(from any company, for materials sterilization in many steps)</td></tr><tr><td>Bacteriological agar</td><td>Sigma-Aldrich</td><td>A1296</td><td>(for many steps)</td></tr><tr><td>C1, C2, C3, C4, C5, and C6 solutions</td><td>Qiagen</td><td>12888-50</td><td>(purchased with DNeasy PowerSoil kit)</td></tr><tr><td>Centrifuge&amp;nbsp;&amp;nbsp;</td><td>Merck/Eppendorf</td><td>5810 G</td><td>(for total DNA extraction from fungal isolates)</td></tr><tr><td>Centrifuge tubes</td><td>Merck</td><td>CLS430828</td><td>(for samples collection)</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>C2432</td><td>(for total DNA extraction from fungal isolates)</td></tr><tr><td>Congo red</td><td>Supelco</td><td>75768</td><td>(for hyphae staining)</td></tr><tr><td>Cryotubes</td><td>Merck</td><td>BR114831</td><td>(for many steps)</td></tr><tr><td>Ethanol</td><td>Supelco</td><td>100983</td><td>It will be necessary to carry out the appropriate dilutions (for many steps)</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>3609</td><td>(for total DNA extraction from fungal isolates)</td></tr><tr><td>Filter paper</td><td>Merck</td><td>WHA10010155</td><td>(for many steps)</td></tr><tr><td>Glass test tubes</td><td>Merck</td><td>CLS7082516</td><td>(for cryopreservation in unhulled rice grains)</td></tr><tr><td>Glass wool</td><td>Supelco</td><td>20411</td><td>(for cryopreservation in unhulled rice grains)</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td>Or dextrose (for cryopreservation in vermiculite)</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td>Or glycerin (for cryopreservation in vermiculite, for preparing LPCB)</td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>563935</td><td>(for total DNA extraction from fungal isolates)</td></tr><tr><td>Lactic acid</td><td>Sigma-Aldrich</td><td>252476</td><td>(for preparing LPCB - hyphae staining)</td></tr><tr><td>Lactophenol blue solution (LPCB)</td><td>Sigma-Aldrich</td><td>61335</td><td>(for hyphae staining)</td></tr><tr><td>Laminar flow hood</td><td></td><td></td><td>(class I, from any company, for many steps)</td></tr><tr><td>Light microscope</td><td></td><td></td><td>(from any company, for hyphae observation)</td></tr><tr><td>MB Spin Columns</td><td>Qiagen</td><td>12888-50</td><td>(purchased with DNeasy PowerSoil kit)</td></tr><tr><td>Methyl blue (cotton blue)</td><td>Sigma-Aldrich</td><td>M5528</td><td>(for preparing LPCB - hyphae staining)</td></tr><tr><td>Microcentrifuge tube (1.5 mL)</td><td>Merck</td><td>HS4323</td><td>(for total DNA extraction from fungal isolates)</td></tr><tr><td>Microcentrifuge tube (2 mL)</td><td>Merck</td><td>BR780546</td><td>(for many steps)</td></tr><tr><td>Mineral oil</td><td></td><td></td><td>(for preservation of fungal isolates)</td></tr><tr><td>Paper bags</td><td></td><td></td><td>Average size 150 mm x 200 mm (for samples collection)</td></tr><tr><td>Petri dish (Glass, 120 mm x 20 mm)</td><td>Merck/Pyrex</td><td>SLW1480/10D</td><td>(autoclavable, for fungi slide culture, prefer higher ones)</td></tr><tr><td>Petri dish (Glass, 50 mm x 17 mm)</td><td>Merck/Aldrich</td><td>Z740618</td><td>(for purification of fungal isolates); alternatively: polystyrene petri dishes (sterile, &amp;gamma;-irradiated, non-autoclavable)</td></tr><tr><td>Petri dish (Glass, 80 mm x 15 mm)</td><td>Merck/Brand</td><td>BR455732</td><td>(for installation of plant fragments); alternatively: polystyrene petri dishes (sterile, &amp;gamma;-irradiated, non-autoclavable)</td></tr><tr><td>Phenol</td><td>Sigma-Aldrich</td><td>P1037</td><td>(for total DNA extraction from fungal isolates, for preparing LPCB)</td></tr><tr><td>Porcelain mortar</td><td>Sigma-Aldrich</td><td>Z247464</td><td>(for total DNA extraction from fungal isolates)</td></tr><tr><td>Porcelain pestle</td><td>Sigma-Aldrich</td><td>Z247502</td><td>(for total DNA extraction from fungal isolates)</td></tr><tr><td>Potato dextrose agar (PDA)</td><td>Millipore</td><td>P2182</td><td>(for many steps)</td></tr><tr><td>PowerBead tubes</td><td>Qiagen</td><td>12888-50</td><td>(purchased with DNeasy PowerSoil kit)</td></tr><tr><td>Rapid mounting medium (Entellan)</td><td>Sigma-Aldrich</td><td>1.0796</td><td>(for fungi slide culture)</td></tr><tr><td>Silica gel</td><td>Supelco</td><td>717185</td><td>(for cryopreservation in unhulled rice grains)</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S9888</td><td>(for total DNA extraction from fungal isolates)</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Sigma-Aldrich</td><td>L3771</td><td>Lauryl sulfate sodium salt (for total DNA extraction from fungal isolates)</td></tr><tr><td>Sodium hypochlorite (w/ 2% active chlorine)</td><td></td><td></td><td>(commercial product, for superficial desinfestation)</td></tr><tr><td>Soil DNA extraction kit (DNeasy PowerSoil kit)</td><td>Qiagen</td><td>12888-50</td><td>(for total DNA extraction from plant organs)</td></tr><tr><td>Spectrophotometer - Nanodrop 2000/2000c</td><td>ThermoFisher Scientific</td><td>ND2000CLAPTOP</td><td>(for total DNA extraction from plant organs)</td></tr><tr><td>Stereomicroscope</td><td></td><td></td><td>(=dissecting microscope, from any company, for macromorphological analyses)</td></tr><tr><td>Tetracycline</td><td>Sigma-Aldrich</td><td>T7660</td><td>(for installation of plant fragments)</td></tr><tr><td>Thermoblock</td><td>Merck/Eppendorf</td><td>EP5362000035</td><td>(or from other companies)</td></tr><tr><td>Tissue homogenizer and cell lyzer</td><td>SPEX SamplePrep</td><td></td><td>2010 Geno/Grinder - Automated Tissue Homogenizer and Cell Lyzer (for total DNA extraction from plant organs)</td></tr><tr><td>Toluidine blue O</td><td>Sigma-Aldrich/Harleco</td><td>364-M</td><td>(for hyphae staining)</td></tr><tr><td>Trehalose</td><td>Sigma-Aldrich</td><td>T9531</td><td>(for cryopreservation in vermiculite)</td></tr><tr><td>Tris Base Solution (Tris)</td><td>Sigma-Aldrich</td><td>T1699</td><td>(for total DNA extraction from fungal isolates)</td></tr><tr><td>Unhulled rice grains</td><td></td><td></td><td>(for cryopreservation)</td></tr><tr><td>U-shaped glass rod</td><td></td><td></td><td>(or an adaptation - check step 5.4.1, for fungi slide culture)</td></tr><tr><td>Vermiculite</td><td></td><td></td><td>Fine granulometry (for cryopreservation in vermiculite)</td></tr><tr><td>Vortexer</td><td>Sigma-Aldrich/BenchMixer</td><td>BMSBV1000</td><td>(for total DNA extraction from fungal isolates)</td></tr><tr><td>Yeast extract</td><td>Sigma-Aldrich</td><td>Y1625</td><td>(for cryopreservation in vermiculite)</td></tr></tbody></table>

isolation, characterization, and total dna extraction to identify endophytic fungi in mycoheterotrophic plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

In the isolation protocol, considering there is contamination from the water used on the last wash and the contamination is also detected in the Petri dishes with inoculated fragments, different actions may be taken, depending on the type of contaminant (Table 1). This procedure must be repeated from the beginning in case of highly sporulating fungal contaminants, which also present accelerated growth, and intense-multiplying bacteria, resistant to the chosen antibiotics....

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Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...

isolation-characterization-total-dna-extraction-to-identify

Research

JoVE Journal

Biology

2.6K Views.  State University of Campinas (UNICAMP). De hier gepresenteerde technieken zijn nuttig voor het onderzoeken van de endofytische schimmelgemeenschap die verband houdt met verschillende plantenorganen. Isolatie van endofytische schimmels maakt hun identificatie mogelijk en is waardevol in andere technieken zoals symbiotische ontkieming. Bereid om te beginnen acht tot 10 centimeter PDA-kweekmedium petrischalen aangevuld met drie milliliter per liter antibioticum.Controleer op verontreiniging door het kweekmedium petrischaaltjes ...

Video: Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Genetics

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control

Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in protecting plants from herbivory and disease. This protocol demonstrates two inoculation methods to establish B. bassiana endophytically in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control. Plants are grown from surface-sterilized seeds for two weeks before receiving a B. bassiana treatment of 108 conidia/ml (or water) applied either as a foliar spray or a soil drench. Two weeks later, the plants are harvested and their leaves, stems and roots are sampled to evaluate endophytic fungal colonization. For this, samples are individually surface sterilized, cut into multiple sections, and incubated in potato dextrose agar media for 20 days. The media is inspected every 2-3 days to observe fungal growth associated with plant sections and record the occurrence of B. bassiana to estimate the extent of its endophytic colonization. Analyses of inoculation success compare the occurrence of B. bassiana within a given plant part (i.e. leaves, stems or roots) across treatments and controls. In addition to the inoculation method, the specific outcome of the experiment may depend on the target crop species or variety, the fungal entomopathogen species strain or isolate used, and the plant's growing conditions.

This protocol demonstrates two inoculation methods to introduce the fungal entomopathogen Beauveria bassiana as an endophyte in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control.

 Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in ...

Bioengineering

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Environment

Quantification of the Potential Impact of Glyphosate-Based Products on Microbiomes

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway, which is virtually universal in plants. The inhibition of the enzyme stops the production of three essential amino acids: phenylalanine, tyrosine, and tryptophan. EPSPS is also present in fungi and prokaryotes, such as archaea and bacteria; thus, the use of GBP may have an impact on the microbiome composition of soils, plants, herbivores, and secondary consumers. This article aims to present general guidelines to assess the effect of GBP on microbiomes from field experiments to bioinformatics analyses and provide a few testable hypotheses. Two field experiments are presented to test the GBP on non-target organisms. First, plant-associated microbes from 10 replicated control and GBP treatment plots simulating no-till cropping are sampled and analyzed. In the second experiment, samples from experimental plots fertilized by either poultry manure containing glyphosate residues or non-treated control manure were obtained. Bioinformatics analysis of EPSPS protein sequences is utilized to determine the potential sensitivity of microbes to glyphosate. The first step in estimating the effect of GBP on microbiomes is to determine their potential sensitivity to the target enzyme (EPSPS). Microbial sequences can be obtained either from public repositories or by means of PCR amplification. However, in the majority of field studies, microbiome composition has been determined based on universal DNA markers such as the 16S rRNA and the internal transcribed spacer (ITS). In these cases, sensitivity to glyphosate can only be estimated through a probabilistic analysis of EPSPS sequences using closely related species. The quantification of the potential sensitivity of organisms to glyphosate, based on the EPSPS enzyme, provides a robust approach for further experiments to study target and non-target resistant mechanisms.

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. In this article, we introduce general guidelines to quantify the effect of GBP on microbiomes, from field experiments to bioinformatics analyses.

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium).

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. 
We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. 
The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...

Exploring Entomopathogenic Fungi Resources from Forest Wood Borers

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits.
Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs.

Author Spotlight: Evaluation of Entomopathogenic Fungi in Wild Monochamus alternatus Populations for Biocontrol Applications in Forest Wood Borers

Here we present a protocol for obtaining entomopathogenic fungi from a forest wood borer and a substitutive way to evaluate their entomopathogenic activities using a Coleopteran model insect. This method is efficient and convenient for exploring entomopathogenic fungal resources from wood-boring insect pests in natural forests.

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

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