Tool and techniques study to plant microbiome current understanding and future needs: an overview

Figures & data

Figure 1. Role of the plant microbiome.

Table 1. Major tools and techniques for studying of plant microbiome and their merits and demerits [96].

S. No.	Tools/techniques	Merits	Demerits	Major drawbacks	Explanations
1.	Microbial cultivation by plating methods	A simple approach enables a more in-depth analysis of colonies, such as species identification and metabolic features.	It’s tough to distinguish between morphological colonies because the resolution capability is smaller and there aren’t any images.	Only culturable bacteria are detectable.	It’s a crucial technique for molecular-based identification analysis.
2.	Electron microscopy-SEM-TEM	It’s a valuable approach for studying the surface of diverse microorganisms and determining the location of ecological niches, as well as learning about bacteria’ cytotoxic pathogenic capabilities.	These are huge pieces of equipment that need time-consuming and expensive sample preparation. It necessitates an isolated location with a steady voltage. It generates graphics in black and white.	The main disadvantage of this method is that the sample size must be restricted to withstand the high vacuum pressure, electron transmission, and fit inside the analyzer.	Images are created by the interaction of electrons with the material in these procedures.
3.	Nucleic acid extraction/DNA sequencing	For searching genes in the soil microbial community that produces reliable and quick results. The scientific community prefers this strategy the most.	Incomplete and partial sampling is common. It is a more costly method that necessitates a lower throughput and post-PCR analysis.	The consistency of the chemical and the purity of the sample may limit the scope of the investigation.	It is based on a molecular approach for analyzing nucleic acid extraction biases, which must be reduced.
4.	PCR/qPCR	PCR is a procedure that is quick, simple to use, sensitive, repeatable, and culturally independent. The method is low-cost, time-saving, requires little input, and has a high throughput. It is common to practice using routine procedures to detect and quantify microorganisms.	It necessitates a high level of skill and technical support for settling, as well as expensive equipment costs, the use of primer dimers, and non-specific amplification.	Microbes in the range of 0.1 to 1% can be detected.	It is a common method for detecting microbial species from soil or leaf samples using molecular techniques.
5.	Molecular finger printings techniques (T-RFLP, DGGE, TGGE, SSCP, RISA, LH-PCR).	It can easily compare samples, increasing the odds of discovering various fingerprints from the same sample.	A maximum of 1000 members of the target community are contacted.	The nature of separation processes is a key flaw in molecular fingerprints, and interpretations must be done with caution.	Used to collect data on the microbial population through comparison research, has evolved into routing fingerprinting.
6.	RAPD	It’s a really simple, low-cost, and time-consuming procedure. Any DNA sample can be used with RAPD. This method does not necessitate the use of target genomic data.	High molecular weight and pure genomic templates are required.	The fundamental disadvantage of this procedure is its lack of repeatability.	RAPD is a form of PCR reaction in which small portions of primers are duplicated in random order.
7.	Clone genomic libraries	It’s a simple way to look into any gene in a community or at the diversity level.	Sample preparation is a time-consuming process. Because of its huge genome, it does not produce adequate results in eukaryotic creatures.	The most significant disadvantage of this method is identifying a clone from the library that scrambles a certain gene or gene of interest.	It is a really good approach, however, it does not give a fair impression of the targeted gene.
8.	Stable isotope probing	It has a positive impact on the dynamic community. It establishes a link between community structure and function.	Difficulties of opportunistsConcealing the information.	Microorganism activity is quite low, which makes probing difficult.	It is a procedure that is used in situ and is widely acknowledged in the scientific community.
9.	Microarrays	It is highly sensitive, low-cost, and provides direct information on species sequencing. It has an extremely high throughput. It does not necessitate the use of any special equipment.	It only produces chipped genes, is more time consuming, and expensive. When closely related members of the same gene family are tested, the results are negative.	Cross hybridizations with minimally homologous sequences are the most significant disadvantage.	It allows for high-throughput analysis across multiple locations.
10.	Next generation technique (NGS)–metagenomics-transcriptomic	It is an effective method for conducting comparative investigations. It has the ability to examine everything at once and with a high throughput.	It is more costly and time demanding. In NGS, method error is a big issue.	Researchers make poor interpretations due to flaws (method/technical procedure).	It can be utilized in a variety of investigations, but the data must be interpreted carefully.

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