Improving Plant Performance Through Microbiome Manipulation: The Potential Role of Current Bioengineering Approaches

Abstract

In the recent past, microbiome manipulation has emerged as a promising approach to improve plant growth performance by exploring the deep insight of plant–microbe interactions. The exploration of a plant microbiome either present on an ectosphere or endosphere can provide a far better understanding about the potential application of plant-associated microbes for the improvement of plant growth, protection from pathogen invasion, and tolerance to environmental stresses of a diverse nature. In this context, next-generation sequencing methods, omics approaches, and synthetic biology have made significant progress in plant microbiome research and are being frequently used to explore the intriguing role of plant-associated microorganisms. Despite the successfulness of conventional approaches, the incorporation of CRISPR/Cas9, RNA interference technology, rhizosphere engineering, microbiome engineering, and other manipulation techniques appear to be a promising approach to enhancing plant performance, and tolerance against biotic and abiotic stress factors. The present review presents the significance of plant microbe interaction, vital functional aspects, collaborative action, potential constraints, and finally the latest developments in bioengineering approaches destined for microbiome modulation with an objective to improve the performance of a host plant challenged with environmental stressors.

1. Introduction

Currently, agricultural productivity is facing challenges to meet the escalating food demand of a continuously rising global population not only because of declining cultivable land areas, but also due to unfavorable climate change. According to an estimate, agricultural productivity needs to be enhanced by 60% by the year 2050 [1]. Noteworthy is that climate change and a limited availability of natural resources such as agricultural land and water availability limits agricultural productivity [2]. During the past two decades, a substantial progress in agricultural practices has been marked; nevertheless, abiotic environmental stressors such as drought, salinity, flood, nutrient deficiency, soil degradation, indiscriminate application of hazardous agrochemicals, and biotic stress factors including resistance resurgence in pathogens have resulted in compromised crop productivity, suggesting the development of environmentally sound strategies to fulfil the food demand.

Plants and microbes are considered to be continuously co-evolved by forming a complex ecosystem, thereby considerably influencing the growth as well as the survival of each other. The concept of “holobiont” has come into existence to reveal the interaction between the host and associated microorganisms in a given environmental complex with plausible implications in biology, ecology, and the evolutionary process of the host plant. Furthermore, the “holobiont” framework comprising of the host and associated microorganisms is suggested to evolve synergistically for mutual cross-talk and co-signaling [3,4]. The diverse microbiome existing in the rhizosphere, phyllosphere, and endophytically in different plant parts with the characteristics ability to augment host plant growth are referred to as “plant-growth promoting microorganisms” (PGPMs) [5,6]. The PGPMs comprising of different bacteria, fungi, etc. are acknowledged to enhance agricultural productivity [1,7,8,9].

Recently, microbiome manipulation has been carried out to improve agricultural productivity or maintain the plant health via different processes such as tillage practices, synthetic microbial consortia, microbial inoculation, rhizosphere engineering, and microbiome engineering [10,11] (Figure 1). Although these approaches are dependent upon various factors such as the host plant, associated microbial communities, soil, and other environmental factors, manipulation of these factors directly or indirectly influence plant growth or developments.

Therefore, there is a need to explore various aspects of plant microbe research, microbiome functioning, and how the microbial communities influence plant growth [12,13]. This review aims to provide an overview of the latest developments in microbiome manipulation. Additionally, various aspects of plant–microbe interactions, traditional approaches of microbiome manipulation, and how plant performance can be improved using the latest emerging approaches such as CRISPER/Cas9, RNAi, or microbiome engineering, etc. are discussed.

During the review process, we carried out a bibliometric data analysis to evaluate the significance of topic microbiome manipulation in improving plant performance. We retrieved 68,401 articles published between 2000 and 2025, from the PubMed database by searching for the keywords “Microbiome Manipulation” OR “Microbial Modulation” And “Plant health” OR “Plant performance” OR “Microbiome engineering” OR “Rhizosphere engineering” OR “CRISPER/Cas” or “RNAi” OR “Bioengineering approaches” OR “Holobionts” OR “omics” OR “crop rotation” OR “plant—microbe interactions” OR “tillage intensity” OR “Conventional Agriculture “ OR “Biotic stress” OR “abiotic stress ” (data accessed on 15 February 2025). Out of 15,462 keywords, 277 were selected with a cutoff of 15 occurrences and we downloaded 10,000 papers in a tab-delimited format and created bibliometric analysis (Figure 2) using VOS viewer processing software (v1.6.9). The different colors present in the network show linked terms; however, the size of the labels showing the number of publications and the distances present between two nodes represent the degree through which they are associated.

2. Plant–Microbes Interactions and Their Significance

Plants interact with a plethora of microbial species, in which some of them directly or indirectly play a significant role in plant growth and developments [13]. The plant rhizosphere is considered as a hotspot of microbial communities and is defined as a thin layer of soil that surrounds and is influenced by plant roots. The plant root secretes an array of exudates constituted of amino acids, flavonoids, lipids, organic acids, and other secondary metabolites, and these biochemical components act as an energy source or signaling molecules for the microbial species [14,15,16,17]. The amount and the nature of exudates varies with internal or external surrounding factors such as host genotype, seasons, temperature, nature of the soil, etc., which also influence the composition of host-associated microbial communities [18,19]. For example, during the seedling stages, the nature and amount of sugar molecules is different than at the harvest stages; similarly, draught or other stress factors also influence the amount and nature of exudates, which ultimately affect the microbial community structures [20].

Each of the plant organs such as the root, stem, leaves, flowers, etc. have some specific microbial composition which may vary with changing environmental conditions, host genotypes, plant developmental stages, seasons, or stress factors [13]. The microbial communities structure of the plants are generally dominated by the bacterial phyla Proteobacteria, Bacteroidetes, Acidobacteria, and Actinobacteria [21,22,23] and by the fungal phyla Ascomycota and Basidiomycota. In the recent past, various authors studied the microbial communities structure of different plant species. For instance, Fu et al. [24] reported Proteobacteria and Actinobacteria as the most prominent bacterial phyla in the semiarid alpine grassland. Nan et al. [25] reported the dominance of bacterial phyla Actinobacteria and Proteobacteria in the rhizosphere of three species of Stipa. Xu et al. [26] in the global study of citrus rhizosphere also reported Proteobacteria, Actinobacteria, and some other bacterial group like Acidobacteria and Bacteroidetes as the prominent one, while Ascomycota and Basidiomycota were the dominant fungal phyla.

It has been observed that the endosphere contains lesser microbial species than the ectosphere or rhizospheric soil. For example, an abundance of bacterial population in the rhizospheric soil was reported in the range of 10⁶–10⁹, while only 10⁴–10⁸ CFU (colony forming unit) per gram of root tissues was observed in the plant endosphere [27,28,29]. In studies of the plant endosphere, a dominance of Proteobacteria was also reported followed by Actinobacteria. Singh et al. [30] also reported an abundance of phyla Firmicutes and Proteobacteria as endophytic in the Momordica charantia L. Goulart et al. [31] observed a higher abundance of Proteobacteria in the vegetative and reproductive stage of Passiflora incarnata. Other bacterial phyla that were dominant in the vegetative growth were Firmicutes followed by Actinobacteria and Bacteroidetes, while Firmicutes (28.5%) was the second dominant group in the reproductive stages.

3. Functional Aspect of Plant Microbiomes

The host plants are considered to offer a habitat for a multitude of microbial communities. The intimate association of microorganisms with plants has been acknowledged to facilitate plant growth [32]. The extensive investigation on the microbiome associated with plants has helped in understanding the intricate communications [33]. The employment of a beneficial microbial population has demonstrated a characteristic role in augmenting crop productivity under changing climatic conditions, diversified stress factors, and a deteriorating soil environment [34,35,36].

Stresses either of biotic and abiotic negatively influence crop productivity worldwide. Noteworthy is that abiotic stress factors have emerged as a major constraint that limits agricultural productivity. The microbial association with the host plant helps alleviate the repercussions of abiotic stressors through the biosynthesis of polymeric substances including exopolysaccharides serving as a protective barrier in the root zone, in addition to the release of osmolytes, including sugar and amino acids as illustrated in the strains of Acinetobacter, Pseudomonas, and Pantoea recovered from drought-resistant and salt-tolerant plants [37]. Additionally, phytohormones and volatiles released by host-associated microbiome triggers induced systemic tolerance (IST). The microbiological biosynthesis of enzyme ACC deaminase lowers the ethylene level. For instance, Achromobacter piechaudii ARV8 equipped with the ability to produce ACC deaminase has been suggested to provide tolerance against drought in Capsicum annuum L. and Solanum lycopersicum L. [38]. Salinity has been envisaged as an important stressor limiting crop productivity substantially. Leveraging salt-tolerant microorganisms could be a sustainable option to mitigate the challenges of agricultural productivity. A recent investigation by Ning et al. [39] explored the feasibility of leveraging halotolerant bacterial strain Enterobacter asburiae D2, endowed with a multitude of growth promotion attributes, to elevate the challenges of salinity stress in Oryza sativa L. Khumairah et al. [40] and Sarker et al. [41] reported on halotolerant rhizobacteria, which after inoculation significantly improved the salinity tolerance and enhanced plant growth [40,41].

PGPMs and their products such as biofilm, exopolysaccharides, and biosurfactants are frequently utilized to mitigate the challenges of heavy metal accumulation in the soil. In addition to mitigation processes of heavy metals, PGPMs also help in growth promotion of plants [42]. For example, treatment with Klebsiella sp. M2 led to an increase in dry weight ranging from 14.3 to 35.9% and the release of metabolic constituents responsible for growth promotion in Triticum aestivum L. Heavy metal–tolerant microbial strains of endophytic nature and those residing in the rhizospheric zone can be exploited for the amelioration of heavy metal toxicity in crop plants. Previous authors reported various different bacterial species such as Enterobacter, Psudomonas, Bacillus, and Klebsiella having the capability to accumulate excess heavy metals [43].

A microbiome can be harnessed for encountering the rising threats of crop damage caused by different phytopathogens. The microbial species having the ability to protect the plants from pathogen invasion or plant diseases are termed as “microbial biocontrol agents.” The microbial biocontrol agents inhibit the phytopathogen growth by secreting antimicrobial metabolites, antibiotics, siderophores, volatile bioactive compounds, etc. or by host immune response or by inducing systemic response [44]. The phenomenon of induced systemic response (ISR) directs the expression of key genes related to defence, such as phenylalanine ammonia lyase (PAL), peroxidase, chitinase, etc. [1]. Also, the release of secondary metabolic products like quorum sensing biomolecules that aggravate the event of ISR. Furthermore, different microbial species can exert various direct as well as indirect effects involving key strategies like enzymes secretion, which causes cell wall degradation, and volatile components restricting the metabolic activities with the resultant inhibition of phytopathogens multiplication [33].

Quorum sensing (QS) is a type of microbial communication mechanism in which bacteria communicate and work together through various chemical cues referred to as “auto-inducers” to govern populations and sustain the equilibrium of a certain process. Additionally, QS modifies the gene expression patterns in response to cell density, which leads to the modification or activation of various transcriptional factors in bacteria [45]. QS also showed direct involvement in the regulation of various functions such as biofilm formation and colonization within the bacterial populations, which plays a significant role in plant growth promotion even under stress conditions [46]. However, the QS molecules like N-acyl homoserine lactones (AHLs) induce the systemic resistance in plants, making them resistant against the pathogens [47].

However, the full potential of a microbiome with reference to sustainable agriculture under diverse climatic conditions has to be explored. The search for newer microbial species, their application as soil or plant inoculants, and the creation of synthetic microbial consortia to control the phytopathogen growth needs to be studied more extensively. The following section deals with the collaborative action of a plant microbiome and strategies being employed for microbiome manipulation using conventional and modern bioengineering approaches.

It is well established that plant-associated microbial species play a significant role in improving plant performance via enhancing plant growth or controlling phytopathogen growth. However, in natural and engineered ecosystems, millions of microbial species reside or co-exist and each possess specific functions and metabolic capabilities [9]. These microbial species perform some specific community’s level performance that contribute a significant role in maintaining plant stability and health during normal or stress conditions [12]. In general, both an energy source and nutrient requirements are fundamental needs for the survival of microorganisms. Although within the microbial community the requirement of an energy resource and the nutrient supplements depends upon the reciprocal exchange of metabolites within the different microorganisms present in the community [12].

4. Dysbiosis in Plant Microbiomes

Dysbiosis signifies an alteration within a microbial community that negatively affects the health of particular plants. Some environmental factors like stress, nutrient content in the soil, and different types of pathogens can throw off the balance within microbes [48]. For example, the increased presence of pathogenic microorganisms within the mycorrhizosphere may suppress the recruitment of good mycorrhizae, thereby reducing plant health and productivity [49]. In addition, certain plants may produce allelochemicals that modify the makeup of their associated microbiomes, resulting in poor interactions with plants, which may lower plant performance [50]. The consequences of dysbiosis are not limited to individual organisms but rather whole ecosystems. The alterations of the diversity of microbes have consequent effects on the microbially mediated processes of nutrient cycling, the health of soils, and the composition of plant communities.

5. Approaches for Microbial Manipulation

In the recent past, a number of studies have suggested different approaches for the manipulation of the microbiome in order to improve plant fitness and agricultural productivity so as to meet the rising food demand for an incessantly growing human population. Some of the commonly used methods such as crop rotation, organic fertilizer input, tillage intensity, and microbiome reorganization are presented in the following sections.

5.1. Crop Rotation

Crop rotation is a common traditional approach practiced to maintain soil productivity and modulate the native microflora of the agricultural soil. It has been observed that repetitive cropping of the same crop can lead to a loss of moisture content, organic matter, and native microbiota [51]. Therefore, maintaining the nutrient status and composition of native microbiota rotation in the cropping system has been preferred for a long time. In a recent study, Yang et al. [52] acquired six years of data and reported a significance of diversified crop rotations and observed that an addition of sweet potato and legumes plants viz. peanut and in the traditional wheat–maize rotation significantly enhanced the soil microbial diversity and agricultural output up to 38%. Additionally, it reduced the emission of N₂0 by 39% and improved the greenhouse gas balance by 88% in the North China plain. Similar types of observations like growing maize after the cultivation of rice can help with nutrient acquisition and the assembly of beneficial microbial communities studied and reported by various authors in the previous studies [53,54].

5.2. Organic Fertilization

Currently, a huge amount of chemical fertilizers and pesticides are being used to enhance agricultural productivity, but their continuous utilization adversely affects the texture and productivity of plants, native microflora, and human health [6]. Therefore, organic amendments, or bio-organic fertilization, are now being used as efficient methods for maintaining soil productivity or native microflora [55,56].

Soil health and the functioning of an ecosystem largely dependent upon the interactions between the soil organic matter and the native microflora. The presence of a higher content of organic matter positively modulates the growth and metabolism of microbes and also shapes the microbial community structure and diversity [56,57]. The presence of a diverse microbial community not only helps in maintaining soil fertility but also helps in degrading complex environmental contaminants. Delgado-Baquerizo et al. [57] reported that the presence of a diverse microbial community modulates the breakdown of litter and other complex organic matter. Similarly, Sun et al. [58] reported that nutrient-rich leaf litter is more easily decomposed than lignin-rich woody litter, which can constrain microbial growth if not adequately supplied [58].

In conventional practices, nutrient-rich agricultural residues, organic manures, biocomposts, or vermicompost have been used to improve and maintain the soil texture or productivity [59]. Previous authors reported the use of bio-organic fertilization in improving plant growth [60,61,62,63]. For example, Lahbouki et al. [64] used horse manure, and Arbuscular Mycorrhizal Fungi (AMF) to amend the soil and enhance tomato productivity. Li et al. [65] used different bio-organic fertilizers to evaluate the impact on Dendrocalamus farinosus growth and found that the utilization of bioorganic fertilizers enhances the growth via restructuring the soil microbiome and metabolome. Wang et al. [66] reported the impact of bio-organic fertilizers on the yield enhancement of pear, which also shapes the rhizospheric microbiome. Xu et al. [60] reported that organic fertilizers like black soldier fly frass derived from different organic wastes can significantly benefit plant growth by improving soil fertility and microbial diversity. Similarly, a combination of sheep manure and chemical fertilizers showed a beneficial role in plant growth promotion [61]. Furthermore, the application of organic manure in Qingke cultivation has been found to influence the rhizosphere microbiota composition and plant growth [62]. More field studies, however, are required to translate the advantageous actions of the microbiome for augmenting the productivity of a plant challenged with diverse abiotic and biotic factors.

5.3. Tillage Intensity

Tillage intensity refers to the degree and frequency of soil disturbance caused by agricultural practices. It ranges from intensive to no tillage intensity, which involves significant soil disruption, to minimal or no disturbance in soil. In tillage practices, the relocation of crop residues has been carried out, which affects and redistributes moisture, the available carbon, and oxygen availability to the soil [67,68]. Currently, different types of tillage intensity such as conventional tillage, reduced tillage, or no tillage has been practiced that significantly affect soil structure, organic matter distribution, and the soil microbiome [69,70,71]. In previous studies, authors reported on different tillage practices and their impact on maintaining soil or plant health. Dewi et al. [69] reported on long-term no-tillage and integrated the use of biochar to improve soybean production. Hu et al. [70] observed that a reduced tillage system improved carbon, nitrogen, and phosphorous in the soil system.

5.4. Reconstructing the Existing Microbiome by Microbial Inoculation

In sustainable agricultural practices, the screening and characterization of a specific microbial strain is one promising approach [72]. Advancement in the latest next-generation sequencing technology, metagenomics, metabolomics, and transcriptomics study enhanced the understanding of microbial community structure and their functioning. These studies also help in characterizing the microbial strains, having better suitability and capability to use as soil or plant inoculants to enhance agricultural productivity [73]. Although for successful inoculants, the strains must have the capability to survive under field conditions and have the potential to be outcompeted by the native microbiome after the introduction [74]. The transition of microbial strains or consortia from the laboratory to the field condition is one of the most challenging tasks. It has been observed that transition sometimes diminishes the efficacy and survivability of microbial strains [75].

In contrast, in recent times, inoculation of two or more than two microbial species consortia either of the same genera or different genera, such as bacteria–bacteria, bacteria–fungi, bacteria–fungi–yeast, have been formulated to create a diverse functional genetic pool, having the potential to act multidimensionally against environmental challenges or stresses [76]. The synthetic consortia of different microbial strains shows potential to resist against the multiple constraints, where the single strain only has limited options. For instance, under low-phosphorus condition, inoculation of phosphate-solubilizing bacteria along with a synthetic consortia of multiple strains showed a superior effect in the crop growth [74]. These consortia effectively provide sufficient solubilized phosphorus, demonstrating their potential to enhance plant growth in nutrient-deficient soils. Wang et al. [77] identified and characterized 23 strains to create a potential synthetic consortia of different strains to promote rice growth. Manjunath et al. [78] reported two different synthetic consortia, Pseudomonas putida P7 + Paenibacillus favisporus B30 and Pseudomonas putida P45 + Bacillus amyloliquefaciens B17, to successfully enhance the growth of maize plants by 27.78 and 23.21%. Similarly, Olanrewaju and Babalola [79] used different microbial strains singly or in combination to evaluate their potential on maize seed growth and found that a mixture of consortia was more potent than a single inoculation. Similarly, Yin et al. [80] created 10 synthetic consortia of 14 different bacterial strains in which nine syncoms showed an antagonistic role against the wheat pathogen Rhizoctonia solani AG8.

6. Engineering Approach to Improve Plant Performance

Despite the successfulness of conventional approaches suggested for microbiome modulation, the introduction of current bioengineering approaches is considered to have promising potential in improving plant fitness under stressed conditions. Some methods of microbiome manipulation including rhizosphere engineering, deliberated alteration in microbiome characteristics, CRISPR/Cas9, and RNA interference with a profound effect in offering tolerance to host plants under environmental stress are presented in the next section.

6.1. Rhizosphere Engineering

Rhizosphere engineering is an emerging approach through which plant performance has been improved following some key strategies like modification in the root architecture to enhance the root absorption area, modifications in the amount and nature of root exudates to attract some specific microorganisms, plant engineering to overexpress or knockout the genes to attract specific microbial taxa, and soil amendments to promote the growth of some specific plant growth–promoting bacteria [81,82,83].

In previous studies, authors reported on various approaches of rhizosphere engineering. In most of the studies, during root engineering, the primary focus is to knockout the genes that are responsible to check the growth and developments of the root system [84,85] and the functional aspect of the root system can be modulated by modifying the gene expression pattern [86]. Modification in the root architecture is another strategy of rhizosphere engineering to improve the plant performance. In this approach, the architecture of the root has been modified in such a way that the root can hold or absorb a better amount of water and nutrients [84,87,88].

The composition, amount, and the nature of the root exudates affect interactions patterns between the host and microbes and are influenced by the plant development stages, plant genotypes, soil conditions, or the surrounding environmental factors [89]. These root exudates act as chemo attractants or chemo repellents for the microbial species. Modifications in the composition or specific molecules in the root exudates are one of the strategies of rhizosphere engineering, through which specific microbial species enriched in the plant rhizosphere enhance and modulate microbial growth [90].

Inoculation of the soil with beneficial microbial strains is used to improve plant growth and productivity [91,92]. Although environmental conditions and plant species are vital for the success of rhizosphere engineering, which is based on selection and adaptation of microbial strains, recent improvements in high-throughput sequencing and metagenomics have supported the characterization and recognition of beneficial microbial strains from different environmental conditions [91]. For example, unique strains of Bacillus and Paenibacillus were recruited from the rhizosphere of drought-tolerant plants and these were later utilized to improve drought resistance in new crops [92]. Likewise, another suggested track to recover plant performance is the insertion of specific genes into microbial genomes, to progress the microbial performance. For example, Pseudomonas fluorescens was obtained to produce the plant hormone indole-3-acetic acid (IAA), subsequent in progressed root growth and nutrient exploitation in wheat, which demonstrated the microbial functions [93].

6.2. Microbiome Engineering

Microbiome engineering is receiving accelerated attention in view of improving crop productivity and encountering the hurdles of climate change [94,95,96,97]. Although different plant parts are known to harbor specific microbial communities and are endowed with the potential to influence the host biology [98], during the process of microbiome engineering, the microbial communities structure of the plant has been modified or optimized (Figure 3). This involves various processes viz. modification of indigenous microbial strains with the help of genetic engineering approaches like CRISPER/Cas9, RNAi interference, etc.; transplantation of beneficial microbiome from the healthy to the target sites; or the development or inoculation of syncoms. However, the primary goal of microbiome engineering is to improve the functioning of microbes in such a way that shows an improved stress tolerance potential, improved nutrients utilization patterns, etc. [99,100].

The identification of genes or alleles responsible for the pathogenicity, synergistic, or antagonistic effect is a prime concern in microbiome engineering and their modifications either by enhancing protein expression or by knocking out the gene is a common practice. To complete this process, various techniques such as CRISPR/Cas9, RNA interference have been performed [101,102].

6.2.1. CRISPER/CAS

CRISPR/Cas9 is one of the precise and efficient tools used in gene modification, especially for the knockout or knockdown process of a particular gene [103]. The first-generation CRISPER gene technology has been utilized for the site-specific cleavage of double-standard DNA [104]. The second generation leads to precise conversion of or replacement of single base into another without cleaving double-standard DNA [105].

In the recent past, in the changing climatic conditions eruptions of novel pathogens or virulent strains and their sustainable management is immediate need for optimum agricultural productions [106,107,108]. CRISPR/Cas9 is increasingly recognized as a promising tool for managing plant diseases. Additionally, it has been applied to genetically modify microorganisms to boost their beneficial effects on crops [109,110]. Previously, authors utilized CRISPER/Cas9 technology to control phytopathogens. For example, knockdown of the OsERF922 gene, which is known as an “ethylene responsive factor”, reduced the chance of infection of Magnaporthe oryzae compared to the wild type [111]. Similarly is the knockdown of ERF922 genes, which are upregulated during the M. oryzae infection [111]. The editing in the ERF genes help with resistance in pathogen infections [112]. One study reported a significance of CRISPER/Cas9 technology in generating downy mildew–resistant grapes by editing the MLO-7 gene, which has been upregulated during infection of Erysiphe necator [113]. Paula de Toledo Thomazella et al. [114] reported the significance of CRISPER/Cas9 technology in editing the SlDmr6-1 gene in tomato, which is responsible for providing resistance against different phytopathogens. Further, Wang et al. [115] reported on the knockout of the VvWRKY52 gene through CRISPER/Cas9 technology, which provided resistance against Botrytis cinerea in grapes. Hu et al. [116] reported on the knockout of the CsLOB gene, responsible for bacterial canker diseases in grapefruit. Similarly, knockdown of CsLOB1 in Wanjincheng Orange provided resistance against bacterial canker diseases. Prihatna et al. [117] utilized CRISPER/Cas9 technology to inhibit the Solyc08g075770 (CYCLOPS) gene, which after inhibition enhanced the resistance against the fusarium wilt in tomato. Similarly, Paula de Toledo Thomazella et al. [114] reported broad spectrum disease resistance in tomato via CRISPER-cas9–mediated mutagenesis of DMR6 ortholog.

6.2.2. RNA Interference (RNAi)

RNAi is a gene-silencing approach followed to inhibit or silence the gene regulation by inhibiting the functional aspect of target mRNA. During this process, ds RNA is processed into small interfering RNA (siRNA), which along with some specific proteins bind with the target mRNA and cleaves that specific viral or pathogenic mRNA to control the pathogenicity [118,119,120]. Although for the phytopathogen control, different gene-silencing approaches such as host-induced [121] or spray-induced [122] has been frequently followed.

In the recent past, the scientific community successfully utilized this RNAi technology to inhibit the pathogenicity or protect the crop from phytopathogens including fungi, virus, nematodes, etc. Previous authors have reported on the role of RNAi technology in controlling or inhibiting pathogenicity. Baum et al. [123] reported RNAi in the insect by feeding or injecting ds RNA. However, some of the authors targeted insect genes by expressing dsRNA in the transgenic plants [124,125]. Koch et al. [122] used RNAi technology to control the infection of Fusarium graminearum in barley by spraying CYP3-dsRNA. Mao et al. [126] enhanced the resistance in cotton plant against the cotton bollworm by expressing CYP6AE14 dsRNA. However, utilization of RNAi technology has certain limitations as a gene-silencing approach; for example, the RNAi tool may not be able to target nucleic noncoding RNA or the non-coding RNA present in the nucleus [127]. In addition to this, the RNAi tool may not be able to differentiate the target mRNA or not target the mRNA having very few similarities with the target RNA [128].

7. Future Prospects and Recent Developments

Microbiome engineering is a constantly developing field and is receiving attention from multiple disciplines thereby incorporating novel approaches. However, to engineer the microbiome, multiple hurdles have to be encountered based upon the feasibility and scale-up caliber of the approach. The following section has tried to uncover the newer approaches and their limitations in microbiome engineering.

7.1. Role of Exudate Metabolites

Plants and microbes have co-evolved. The reciprocal benefit to plant and microbes is mediated by means of secondary metabolites. Plants secrete a number of volatile compounds, phytohormones, signaling molecules, extracellular enzymes, and surface receptors in their root exudate, possessing the potential to interfere with their microbial interaction in a positive direction [129]. The potential of root exudates can be harnessed to engineer a rhizospheric microbiome and ultimately increase plant productivity. The selected addition of root exudate can specify the microbiome growth and nutrition acquisition direction [130]. In their study, Jin et al. [131] deciphered that maize seedling exudates promoted growth and reduced oxidative stress in PGPB Pseudomonas fluorescens. However, this approach is associated with certain limitations including difficulty in scale-up and identification of specific metabolites in the root exudate.

7.2. Use of Soil Probiotics

Various research is continuously being conducted to understand the impact of probiotics on the human gut microbiome [132]. Similar studies on the impact of external microbial inoculation on the soil and plant microbiome is warranted. PGPM inoculation is a traditional method to increase plant productivity and control plant pathogens [133]; however, their substantial role in microbiome modulation has not been studied much. Recently, Liu et al. [134] demonstrated that inoculation of Rhodobacter sphaeroides and Bacillus amyloliquefaciens in oilseed rape rhizosphere increased the growth of bacteria belonging to Pseudomonadaceae and Flavobacteriaceae, leading to better soil nutrient cycling. Similarly, the inoculation of Burkholderia vietnamiensis modified soil microbiota and controlled root-knot nematode infestation in watermelon [135]. Nevertheless, this approach is also associated with traditional challenges of competition from the native microbiota. Alternatively, combined approaches of root exudate and PGPR inoculation can be used for a better adaptation to a localized niche.

Increasing agriculture productivity under the pressure of climate change has asked to add another dimension in microbiome modulation. A prompt understanding between synthetic biology, microbiome engineering, and ecology will not only help in climate conservation, but will also increase plant productivity. Harnessing cultured and non-cultured resilient microbes combined with climate smart agriculture can help in combating emerging climatic biotic and abiotic stresses [135]. Epstein et al. [136] explained the role of microbiome modulation in harboring climate resilience in corals, providing some inputs for microbiome engineering in the soil and plant rhizosphere.

7.3. Optimization of Inoculant Size and Growth Conditions Under In Situ Conditions

Microbiome manipulation is directly correlated with the inoculum size and factors encountered in the specialized niche [9]. Whether artificial microbial consortia, traditional soil amendments, microbiome breeding [137] and transplantation of the microbiome, or modification of root architecture and exudates, every approach needs to be optimized for successful application [138,139,140]. Seed bio-priming [141], seed coating, encapsulation, nanoformulations [142], and in situ microbiome engineering are some of the latest approaches used for sustained delivery of microbial formulations [143,144]. Nevertheless, further research is required for homogenous optimization of microbial inoculation.

7.4. Role of NGS, Metagenomics of Uncultured Microbiome, and Use of Genetic Tools

Recent advancements in molecular biology can offer important strategies in microbiome modulation. The firm knowledge of microbiome molecular ecology and interactions is necessary for successful manipulation. Next-generation sequencing (NGS) for studying microbial transcriptome, proteome, and metabolome has revealed dynamic changes during plant–microbe interactions [145,146,147]. High-throughput sequencing of PGPB Pseudomonas putida RA-responsive microRNAs and Bacillus amyloliquefaciens revealed their role in the growth and development of Arabidopsis and the growth of rice under stress [147,148]. Some of the microbes are difficult to culture under laboratory conditions. Metagenomics approaches including high-throughput sequencing, marker gene tags (iTAG), amplicon sequencing, and in situ metagenomics engineering can help in understanding the complexity level and intra-community level interactions [94,149]. Further, genetic editing tools such as miRNA, siRNA-based tools, RNAi, CRISPR/CAS9, and multiomics approaches can be combined with microbiome engineering for increasing plant productivity.

8. Conclusions

In recent decades, to ensure food security for the rising global population, enhancement in agricultural productivity and improved plant performance under stress conditions are some of the main goals. The better understanding of plant–microbe interactions can be a vital option to use the beneficial microbial species as plant or soil inoculants or their use to modulate rhizosphere functioning. Although the isolation, characterization, and selection of beneficial microbial species is complex process, the latest omics and technology make this hurdle easier and help researchers in the identification of microbial communities and their hidden potential. But survivability of the microbial strains in the field trail is one of the challenging tasks. The identification of key genes, microbial engineering, and rhizosphere engineering could be emerging as one of the sustainable solutions to enhance plant performance.