Bacterial wilt disease caused by soilborne pathogenic bacterium Ralstonia solanacearum is a serious threat to flue-cured tobacco (Nicotiana tabacum L.) production worldwide, including China (Cai et al., 2021). Ralstonia solanacearum is widely distributed in tropical and subtropical regions of the world and has a broad host range that infects more than 250 plant species (Paudel et al., 2020). It is a serious threat to important field crops of the Solanaceae family including tomato, potato, tobacco, ginger, eggplant, and pepper, with average yield losses that range from 10 to 55% (Kim et al., 2016).

Ralstonia solanacearum infects all tobacco plant parts (roots, stalk, and leaves) and generally produces symptoms of yellowing and wilting of leaves, discoloration of xylem vessels, and black necrotic spots on the stem. It multiplies systemically in the xylem vessels followed by death of whole plant (Cai et al., 2021). Infected soil acts as a primary source of inoculum, and the pathogen oozes out in the rhizosphere soil of diseased plants from the roots upon completion of life cycle (Wu et al., 2020). The bacterium survives in soil, water, and plant residues for a longer period as saprophytism in the absence of a specific host plant (Li et al., 2014). It spreads from diseased to healthy plants through rain splashes, irrigation water, and mechanical operations (Qi et al., 2020).

As far as losses caused by this disease are concerned, in China, incidence and yield losses vary from region to region, host to host, climatic condition, and pathogenic strain (Jiang et al., 2017). However, incidence and yield losses are recorded between 15 to 35% but can reach up to 75% and 50–60%, respectively, when disease is present with root rot pathogen Phytophtora nicotianae (Jiang et al., 2017). In high humidity and mono-cropping regions, it occurs in epidemic form, and yield losses reach up to 100%. Soil physicochemical properties, climatic conditions, locations, and rhizosphere microbial diversity play an important role on the occurrence of soilborne diseases (Cai et al., 2021).

The plant rhizosphere is considered as one of the most complex ecosystems on earth and hot spot habitat for diverse microbes (Raaijmakers, 2015). Most disease-resistant and developmental mechanisms in plants are directly related to the diversity of rhizosphere microbes (Mendes et al., 2013; Dong et al., 2019). Plant genotype and soil type are the two main factors that are responsible for assembling a healthy rhizosphere microbiome (Dong et al., 2019). Advancements in science and modern sequencing tools made the study of host–microbe interaction easier (Govindasamy et al., 2014; Mhlongo et al., 2018). However, knowledge gaps are still present between host–microbe interactions and their underlying mechanism, which need to be filled (Bulgarelli et al., 2013).

Nowadays, biological control via disease suppressive specific endophytes and rhizobacteria such as Bacillus, Lysobacter, Streptomyces, and Pseudomonas is considered as practical approach to suppress the incidence of many soilborne diseases including bacterial wilt, Fusarium wilt, and clubroot by the mechanism of direct antagonism, reshaping the rhizospheric microbial diversity, and the production of metabolites (Ma et al., 2018; Wu et al., 2020; Zhang et al., 2020a; Wei et al., 2021). The application of bioorganic fertilizer and biochar along with biocontrol agents significantly suppresses the incidence of tobacco bacterial wilt disease (Liu et al., 2013; Zhang et al., 2017; Li et al., 2022). Because of the broad host range, species complexity, wide geographical distribution, and persistent nature, no effective control exists to date against this devastating disease, and it is difficult to completely control the incidence of tobacco bacterial wilt disease (Li et al., 2014; Shen et al., 2018).

Flue-cured tobacco is a major cash crop in Yunnan Province, China. Yunnan is well-known for its unique environment and climatic conditions. It produces high-quality flue-cured tobacco famous for its pure taste, fragrant aroma, and golden color (Li et al., 2019). Yunnan produces about 50% of China’s total tobacco leaf yield, with an annual production of around 750,000 tons and 320,000 ha of agricultural land under tobacco cultivation (Tang et al., 2020). However, R. solanacearum poses significant yield losses every year. Thus, to successfully mitigate the bacterial wilt pathogen, it is necessary to understand the population dynamics and distribution of the microbiome in tobacco plants. Therefore, the present study aims to explore the core microbiota (bacteria and fungi) associated with different locations (Qujing, Shilin, and Wenshan), plant components (rhizosphere soil and roots), and nature of plant (healthy and diseased). We hypothesized that this study helps us to establish a model for studying the naturally occurring flue-cured tobacco microbiome to mitigate the incidence of tobacco bacterial wilt disease.

Ralstonia solanacearum is a serious threat to flue-cured tobacco production worldwide, including China. It is estimated that approximately 47,133 hm^–2 lands are affected by this pathogen, resulting in millions dollars losses every year (Cai et al., 2021). Many integrated disease management strategies are adopted to control the incidence of bacterial wilt pathogen (Jiang et al., 2017). In recent years, biological control via disease suppressive biocontrol agents is considered a promising approach to reduce the incidence of many field crop diseases (Munir et al., 2021; Ahmed et al., 2022). Studies have proven that soil health and rhizosphere microbial diversity are the key factors responsible for soilborne disease occurrence and plant health (Köberl et al., 2013; Dong et al., 2018; Hao and Ashley, 2021).

Soil sickness affects both crop quality and yields when the same crop or its relative species are continuously grown in the same soil under a monocropping system (Zhang et al., 2020b). The rhizosphere microbiome acts as the first line of defense against soilborne pathogen infection and abiotic stress (Bulgarelli et al., 2013; Mendes et al., 2013). In previous studies, flue-cured tobacco bacterial wilt diseases associated with bacterial and fungal communities were studied by direct isolation and high-throughput sequencing technique. However, most studies performed on rhizosphere microbial diversity aimed at bacterial communities with little knowledge about fungal communities of flue-cured tobacco grown on a specific location in pots and field experiments after artificial inoculation of R. solanacearum combined with other biocontrol agents.

In this study, we provided a comprehensive view of the naturally occurring core microbiome of healthy and bacterial wilt–infected (diseased) flue-cured tobacco plants. We presented an in-depth appraisal of bacterial and fungal communities associated with different locations, sample types, and plant components through 16S and ITS1-5f high-throughput sequencing analysis. Our results support the concept that distinct plant components have a significant impact on bacterial and fungal communities, irrespective of sample types and location.

Different plant components have been observed to study the impact of rhizospheric and endophytic bacterial communities on the growth and health of plants with no specific assemblage pattern (Compant et al., 2008; Lundberg et al., 2012; Lebeis et al., 2015). The plant’s rhizosphere acts as the primary host, hot spot habitat, and passage for colonization of microbes into different plant components through roots, especially for bacterial communities (Bulgarelli et al., 2013; Kaushal et al., 2020). In our study, samples were collected from fields where flue-cured tobacco has been continuously grown for the past 10 years as an annual crop for commercial production. Therefore, infected soil and tools used for mechanical operations are primary carriers of bacterial wilt pathogen from one field to another.

Our study demonstrated that rhizosphere soil greatly influences microbial diversity and host more bacterial and fungal communities than roots. Most of the communities present in roots are also present in rhizosphere soil in all locations. The results suggest the microbial communities present in roots were transferred and colonized from the rhizosphere soil. Studies have proven that host plants selectively promote colonization of specific bacterial and fungal communities in roots from rhizosphere soil (Turnbaugh et al., 2007; Lundberg et al., 2012). It was found that rhizosphere soil showed a high microbial diversity than roots. However, healthy tobacco plant rhizosphere soil and root samples host a high diversity of bacterial and fungal communities than bacterial wilt–infected tobacco plant samples. This may be due to disease stress, which is directly linked to the decreased value of available carbon for microbes in the rhizosphere and regulating specific microbe’s growth (Kaushal et al., 2020). Flue-cured tobacco plant resistance and severity of R. solanacearum in different plant components also contribute to the prevalence of microbial communities. Bacterial and fungal OTU count was recorded maximum in all locations and plant components in rhizosphere soil samples than in the root samples.

Host plant environment is known to support or suppress the colonization of certain bacterial and fungal genera with different plant components. Thus, bacterial and fungal communities in these specific plant components are either boosted or exhausted (Kaushal et al., 2020). We observed the core stable bacterial and fungal communities in all sample types that were colonized in different plant components with respect to OTUs distribution. In all rhizosphere soil and root samples for bacterial community composition phylum, Proteobacteria and Cyanobacteria were found in high relative abundance, respectively. Whereas for fungal community composition, phyla Ascomycota and Basidiomycota were found in high relative abundance in both rhizosphere soil and root samples. Results showed that bacterial wilt pathogen has a great impact and significantly influences the bacterial communities compared with fungal communities.

Most of the diverse bacteria and fungi can enhance plant growth, suppress disease incidence, and confer other biological functions that benefit the plants (Santoyo et al., 2016; Gouda et al., 2018). A fraction of core bacterial communities identified in this study are known as plant growth promoters, enhance resistance in plants, and suppress the incidence of disease via production of a specific antibiotic, volatile organic compounds, secondary metabolites, and nitrogen fixation in many fields crops. Bacterial genera such as Amycolatopsis, Bacillus, Streptomyces, Ensifer, Sphingopyxis, Neorhizobium, Pseudonocardia, Sphingomonas, Haliangium, Bradyrhizobium, Arthrobacter, Bryobacter, and Lysobacter were found in high relative abundance, which contribute to plant growth promotion, nutrition acquisition, suppression of soilborne diseases and other plant pathogens. However, a minor fraction of fungal communities such as Gibberella and Purpureocillium was found as plant growth promoters and biocontrol agents, whereas others are saprophytic and pathogenic and cause disease in many important field crops.

Bacterial wilt pathogen R. solanacearum was highly abundant in diseased rhizosphere soil and root. A high abundance of the bacterium was found with nitrogen-fixing ability, producing secondary metabolites, and bacterial predator of bacteria in the soil such as Neorhizobium galegae, Lysobacter dokdonensis, and Ensifer adhaerens. Plants pathogenic fungi such as Plectosphaerella cucumerina, Alternaria alternata, Thanatephorus cucumeris, and Fusarium sp. were found in high relative abundance diseased rhizosphere soil and root. Thus, we conclude that these beneficial bacterial communities are positively related to flue-cured tobacco plant health, whereas the pathogenic fungal communities enhance the population of R. solanacearum and the incidence of bacterial wilt disease. Our results are in accordance with the study of Jiang et al. (2017), who reported that the incidence of tobacco bacterial wilt disease increases when it prevails with other root rot pathogen P. nicotiana. Analysis of co-occurrence networks showed that R. solanacearum has a significant impact on the bacterial community structure compared with fungal communities. Fewer negative correlations were found between bacterial communities in healthy plants compared to diseased plants.

In this study, we conclude that flue-cured tobacco plants host a range of bacterial and fungal communities. Plant components significantly impact bacterial and fungal communities. Rhizosphere soil is enriched with bacterial and fungal communities compared to roots, whereas high diversity was found in healthy plants rather than diseased plants. By comparing the microbial communities with other crops, we suggest that several bacterial communities discovered in the flue-cured tobacco microbiome aid in plant growth promotion and disease suppression. A fraction of fungal communities pathogenic to flue-cured tobacco was found in diseased plants, which correlates with the occurrence of tobacco bacterial wilt disease. However, future work should focus on studying the microbiome of flue-cured tobacco cultivars with resistance to bacterial wilt pathogen both as naturally occurring and artificial inoculation. This will provide experimental and theoretical information, that microbiome of flue-cured tobacco can be engineered to manage the bacterial wilt disease for better yield and quality production.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

ZZ and GJ designed the experiment. WA, QL, SL, and JZ visited the tobacco-growing areas and collected samples. WA, ZD, and JY analyzed data and drew figures. WA, SM, and SK wrote the initial draft of manuscript. WA, SM, GJ, and ZZ wrote, reviewed, and edited final draft of the manuscript. All authors contributed to the final draft of the manuscript and agreed to the published version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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