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Here we describe a suite of methods to identify potential taxonomic and functional soil microbial indicators of soil quality and plant health in biofuel crops in various areas and land types. This approach draws on tools to assess microbial diversity, greenhouse gas fluxes, and soil physicochemical properties in bioenergy cropping systems. Integrative statistical models are then used to identify potential microbial indicators for sustainable management of bioenergy crops.The genus Streptomyces constitutes approximately 50% of all soil actinomycetes, playing a significant role in the soil microbial community through vital functions including nutrient cycling, production of bioactive metabolites, disease-suppression and plant growth promotion. Streptomyces produce many bioactive compounds and are prime targets for industrial and biotechnological applications. In addition to their agrobiological roles, some Streptomyces spp. can, however, be phytopathogenic, examples include, common scab of potato that causes economic losses worldwide. Currently used chemical control measures can have detrimental effect to environmental and human health as a result alternative methods to chemical disease control are being investigated. [https://www.selleckchem.com/products/irak-1-4-inhibitor-i.html https://www.selleckchem.com/products/irak-1-4-inhibitor-i.html] One alternative is the use of streptomycete specific phages to remove this pathogenic bacterium before it can cause the disease on potatoes. However, due to co-existence of non-common scab-causing species belonging to the genus Streptomyces, phage treatment is likowever, when phages were added into the pots, the growth of wheat was detrimentally impacted. This finding might suggest that the reduced presence of antifungal streptomycetes via phage-induced lysis might encourage opportunistic fungal infections in plants.The rhizosphere microbiome of plants is essential for plant growth and health. Recent studies have shown that upon infection of leaves with a foliar pathogen, the composition of the root microbiome is altered and enriched with bacteria that in turn can systemically protect the plant against the foliar pathogen. This protective effect is extended to successive populations of plants that are grown on soil that was first conditioned by pathogen-infected plants, a phenomenon that was coined "the soil-borne legacy." Here we provide a detailed protocol for soil-borne legacy experiments with the model plant Arabidopsis thaliana after infection with the obligate biotrophic pathogen Hyaloperonospora arabidopsidis. This protocol can easily be extended to infection with other pathogens or even infestation with herbivorous insects and can function as a blueprint for soil-borne legacy experiments with crop species.Studying the plant phyllosphere to understand inhibition patterns to the growth of fungal foliar pathogens by using the Arabidopsis thaliana pathosystem offers unique opportunities for evaluating strategies for plant protection against foliar diseases. The wide array of bacteria inhabiting the phylloplane of plants has been researched to a much lesser extent compared to the bacteria in the rhizosphere. This difference is especially evident as bacteria derived from the aerial section of plants are rarely used in formulations of foliage sprays against pathogens and pests. In this chapter we outline easy and reliable methods for sample preparation to profile phyllosphere bacteria using high throughput amplicon sequencing and isolate/characterize potentially beneficial phyllosphere bacteria from Arabidopsis thaliana that inhibit in vitro the growth of foliar pathogens such as Alternaria brassicicola. The use of the described methods for profiling and screening phyllosphere bacteria may provide tangible progress on the discovery of new potential biological control agents against agriculturally important pathogens.Bacillus spp. have great agricultural potential as a plant growth promoter and biocontrol agent. However, little is known concerning the bacterial molecular basis for the improvement of plant fitness. Thus, it is highly desirable to develop techniques that can contribute to the elucidation of the genetic basis for the mechanisms involved in beneficial bacterium-plant interactions. In this context, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 is a powerful tool based on programmable molecular scissors that perform precise incisions in any DNA sequence. CRISPR-Cas9 can alter gene sequences and constitutes a cutting-edge tool to elucidate the role and function of bacterial genes associated with the benefits of plant interactions. The method described here uses a feasible CRISPR-Cas9 system in a double plasmid, one plasmid harboring the Cas9 endonuclease and the other the sgRNA, to promote gene knockout/editing in the Bacillus genus. This approach favors high efficiency in generating mutants for one or more genes in continuous or multiplex editing. Additionally, due to its universality, it can be applied to genera other than Bacillus.Network analysis facilitates examination of the interactions between different populations in a community. It can provide a range of metrics describing the social characteristics of each population and emergent structural properties of the community, which may be used to address novel ecological questions. Using a publicly available dataset, this chapter provides point-by-point code and instructions to infer and analyze a SPIEC-EASI (SParse InversE Covariance Estimation for Ecological Association Inference) network using free, open source software (R and Gephi).DNA sequencing has become a common tool in environmental microbial ecology, facilitating characterization of microbial populations as well as complex microbial communities by circumventing culture bottlenecks. However, certain samples especially from host-associated environments (rhizosphere, human tissue) or complex communities (soils) can contain a high degree of DNA sequences derived from hosts (plants, human) or other organisms of non-interest (arthropods, unicellular eukaryotes). This chapter presents a simple in silico method to remove contaminating sequences in metagenomes based on aligning sequences to reference genomes of the target organism.Quantitative-PCR (qPCR) enables the quantification of specific DNA targets, such as functional or phylogenetic marker genes associated with environmental samples. During each qPCR cycle, the number of copies of a gene (or region) of interest in DNA samples is determined in real time using a fluorescence-based label and compared to a standard serial dilution. Here, we describe a qPCR method to quantify the ammonia oxidizing bacteria involved in the first step of nitrification, using the amoA gene as a proxy of their abundance. The preparation of the standards from environmental samples and qPCR is presented in detail for specifically quantifying microbial abundance in environmental samples such as soil.High-throughput sequencing of universal bacterial 16S rRNA gene (16S rDNA) amplicons is a routine method for characterizing bacterial diversity in a range of environments. For eukaryotic host-associated communities, however, plastid and mitochondrial genes are often co-amplified with, and greatly outnumber, bacterial 16S rDNA. This makes it difficult to obtain sufficient numbers of target 16S rDNA sequences to characterize the diversity of endophytic bacterial communities. This chapter describes a method that improves the amplification of bacterial 16S rDNA from plant tissues by using a peptide nucleic acid (PNA) PCR clamp. The PNA clamp selectively binds to a targeted region of the plant genome and inhibits its amplification during PCR. PNA clamps are especially useful for characterizing bacterial communities on plant tissues with lower levels of microbial colonization such as the root tips and leaves.Assessment of endophytic and saprotrophic microbial communities from wood-extracted DNA presents challenges due to the presence of surface microbes that contaminate samples and plant compounds that act as inhibiting agents. Here, we describe a method for decontaminating, sampling, and processing wood at various stages of decay for high-throughput extraction and purification of DNA.Plants harbor a large reservoir of fungal diversity, encompassing endophytic, epiphytic, phytopathogenic, and rhizosphere-associated fungi. Despite this diversity, relatively few fungal species have been characterized as sources of bioactive secondary metabolites. The role of secondary metabolites is still not fully understood; however, it is suggested that these metabolites play important roles in defense mechanisms and fungal interactions with other organisms. Hence, fungal secondary metabolites have potential biotechnological applications as prototype molecules for the development of therapeutic drugs. In this chapter, we describe the main methods used for routine fungi isolation, production of crude fungal extracts, and chemical characterization of bioactive compounds. In addition, explicative notes about the steps described are provided to explore the diversity of the endophytic, phytopathogenic, epiphytic, and rhizosphere fungi and to evaluate the biotechnological potential of each group.Protists are mostly unicellular eukaryotes. Some protists are beneficial for plants, while others live as endosymbionts and can cause severe plant diseases. More detailed studies on plant-protist interactions exist only for plant pathogens and parasites. A number of protists live as inconspicuous endophytes and cause no visible disease symptoms, while others appear closely associated with the rhizosphere or phyllosphere of plants, but we still have only a vague understanding on their identities and functions. Here, we provide a protocol on how to assess the plant-associated protist community via Illumina-sequencing of ribosomal marker-amplicons and describe how to assign taxonomic affiliation to the obtained sequences.Plant Growth Promoting Bacteria (PGPB) are a group of beneficial microorganisms that can positively influence plant fitness and development by improving nutrient acquisition, influencing global plant hormone levels (direct effect), or by reducing the detrimental effects of various pathogens on plant development (indirect effect). The use of PGPB in agriculture as formulated bioinoculants is a potential approach to reduce the negative environmental impacts caused by the continuous application of chemical fertilizers and pesticides. The evaluation of a great number of bacteria in the laboratory for key traits involved in the improvement of plant fitness is a suitable strategy to find prospective candidates for bioinoculants. This chapter presents the main methods described in the literature to quickly screen potential candidates from a bacterial collection to directly and indirectly promote the plant growth.Beneficial plant-microbe interactions are important and desirable for sustainable intensification of agriculture. Here, we describe methods to isolate microbes from the roots of field-grown wheat plants. This includes the rhizosphere and rhizoplane soil, as well as the root endosphere. We also describe a method to test for endosphere competence of putative endophytes.Arbuscular mycorrhizal fungi (AMF) are an important element of the plant microbiome as they establish an endosymbiotic relationship with the roots of most plant species. This association enhances access to nutrients and water for plants, and provides the fungus with plant-derived organic carbon. In this chapter, I describe a range of methods to work with AMF including soil sampling; isolation of AMF propagules (spores, sporocarps, roots, and mycelium) by a wet sieving and centrifugation in a sucrose solution; trap (from field soil with AMF spores) and one-species pot cultures (from AMF spores divided into morphotypes); staining of mycorrhizae in plant roots; and production of diagnostic slides. These methods are widely used in taxonomic and ecological studies to characterize the morphology of AMF.