Introduction

Tobacco bacterial wilt, caused by Ralstonia solanacearum E.F. Smith f. sp. nicotianae, is one of the most destructive bacterial soil-borne diseases and affects tobacco production in Guizhou Province, China (Liu et al. 2015). Many traditional strategies such as tillage management are not always effective, since R. solanacearum can survive in soils for a long time (King et al. 2008). Crop rotation and disease-resistance breeding are proved effectively in controlling soil-borne disease. However, crop rotation is often unrealistic in China due to the limited amount of land available for tobacco growing, and the breeding of disease-resistant varieties often leads to low yield and quality of tobacco leaves (Liu 2014). In addition, the application of traditional chemicals (bactericides) to control bacterial wilt has been shown in some cases to be minimally effective and at the same time has resulted in a negative impact on the environment health risks to tobacco consumers (Liu et al. 2013). More recently, the Chinese government proposed a “double reduction policy”; therefore, biocontrol and bioremediation have been considered a promising management strategy (Zhang et al. 2011; Wang et al. 2013a). At present, research on the use of antagonistic bacteria to control bacterial wilt is mainly confined to the laboratory, and poor control effects have been examined in pot or field conditions in practical production (Jiang et al. 2017).

Antagonistic bacteria rarely colonize the root after soil application because native microbes aggressively outcompete invasive microbes and the applied strains are poorly adapted to the soil environment. Thus, it is crucial for biocontrol to increase the colonization ability of antagonistic bacteria. Bioorganic fertilizer, in which antagonistic bacteria are secondarily fermented with organic fertilizer, can control bacterial wilt by improving soil microbial ecology, suppressing pathogens and increasing plant resistance (Abawi and Widmer 2000; Li et al. 2009). Organic fertilizers are one of the most effective soil amendments because they not only improve soil quality, but also provide the antagonistic microbes with nutrients, thus ensuring their survival and allowing them to reach a population number sufficient to disease control. Zhao et al. (2014b) found that Paenibacillus polymyxa SQR21 enhanced bioorganic fertilizer (BIO), decreased the population of Fusarium oxysporum in soil rhizosphere, and effectively controlled watermelon Fusarium wilt to a certain extent. Nevertheless, the agricultural environments are variable and complex. Single measure like microbial agent application and soil pH regulation can only change a single element in a short time among many pathogenic factors. Due to the unique conditions of agricultural environments and the limitation of single prevention technology, there is no single measure for bioremediating severely diseased field soil until now (Chen 2014). Wei et al. (2017) suggested that a single biocontrol agent performed unstable under field conditions. The integrated measures for biocontrol can eliminate the cask effect caused by a single prevention technology and become an effective approach to the prevention of soil-borne disease (Wang et al. 2013b; Liu et al. 2014).

In this field trial, integrated control measures including soil pretreating with lime or application of bioorganic fertilizer were applied in a severely tobacco bacterial wilt–infected field for 3 years in Guizhou Province, Southwest China. We explored the effect and soil microbiota in the rhizosphere soil. The most significant effect was observed after 3 years of bioremediation. The combined use of lime and bio-organic fertilizer can obtain the best disease control efficacy with the delayed occurrence simultaneously, as well as the highest tobacco yield and output value. The results of this research can lead to a better understanding of the biocontrol of tobacco bacterial wilt and provide a foundation for effective biocontrol mechanisms.

Materials and methods

Development of disease-control bioorganic fertilizer

The antagonistic strain LX5 (Bacillus licheniformis) was isolated from previously healthy tobacco rhizosphere soil and stored in the China General Microbiological Culture Collection Center as CGMCC No. 8266 (Li et al. 2011).

The soil bioremediation, consisting of liming and bioorganic fertilizer, has effectively controlled various soil-borne diseases (Trillas. et al. 2006). In this trial, compared with the T1 treatment (CK), the control efficacies of the bacterial wilt in the T4 treatment (integrated control measures) reached 75.2% (the second year) and 61.3% (the third year). On one hand, soil pH correction through liming to neutralize the acidity to levels amenable for plant growth is an important agricultural practice for improved productivity. On the other hand, bioorganic fertilizer, the antagonistic bacteria added with a nutrient carrier, consequently prevents R. solanacearum from colonizing root niches in the early stage of the tobacco growing season (El-Abyad et al. 1993). However, a limited effect on efficiently controlling tobacco bacterial wilt has been achieved either by single liming or by bioorganic fertilizer application. The integration of liming and bioorganic fertilizer in tobacco production not only improves soil texture, thus facilitating deep rooting and enhancing stress resistance (Jiang et al. 2017), but also decreases the pathogen population (Wang et al. 2013b).

Improve soil microbial community structure and function to remediate soil

The efficient achievements of disease control mainly depend on the microbial community structure. Different patterns of community structure were observed between control soil and bioremediated soil in our study. In particular, high relative abundances of Ralstonia, Candidatus_Solibactor, Stenotrophomonas, and etc. were observed in control, while Chitinophaga, Nitrospira, etc. were richer in T4. With a bacterial wilt disease incidence of 100% in the field, the abundance of Ralstonia is highest in control among treatments. Ralstonia is the pathogen that caused Solanaceae crop’s bacterial wilt. After soil remediation, the improvement of soil microbial community structure, especially the abundance of Ralstonia, was significantly reduced, so that the dominant position of pathogenic bacteria was reduced, and the pathogen population was less than the tip** point inducing wilt disease (Li et al. 2021). Otherwise, if the microorganisms are associated with a variety of physical and chemical traits, forming an association network, then the microbial ecosystem will not be fragile and keep stable (Wang et al. 2017). Our results were similar with some reports that showed several soil properties such as soil available P, available K, and TN and the C/N ratio were significantly correlated with abundant phyla (Zhao et al. 2014a). This finding may be a good explanation for soil microbial balance, which was also proved by other research that the application of integrated bioorganic fertilizers effectively improved the soil microbial balance and restored the soil ecosystem, resulting in a complex and healthy soil microbial system (Liu et al. 2013). As such, we hypothesize that soil well-structured bacterial community can improve physical–chemical properties. These findings support the previously tested measures for controlling soil-borne diseases (Zhang et al. 2008).

In our study, we found that the bacterial communities of the T3 and the T4 treatments were clustered together based on the principal component analysis, indicating a similar soil microbial structure and functional diversity. In contrast, the T1 treatment was separated from the T4 treatment in the PCA, indicating distinct bacterial communities. These results were consistent with other studies performed by Zhang et al. (2008) and Lang et al. (2012). Most of the variation that determine the community group can be explained by the interactions among soil properties, such as total nitrogen, total phosphorus, total potassium, and available nitrogen and organic matters. As previously reported, when the soil properties, the microorganism ecology, and the microorganism activity are improved, the soil microbial structure and functional diversity support healthier plants (Zhang et al. 2012; Luo et al. 2015). As for the interactions of species in the co-occurrence networks, we were able to find that the microbiomes in T4 were associated with large number of nodes and edges compared to that in T1 treatment, indicating that the soil was transitioned from diseased soil to healthy soil (Wei et al. 2019). After the soil remediated, the keystone species play different roles compared with the original soil, thus signifying the complexity of multi-species interactions and achieving a closely relevant micro-system, which was ecologically meaningful to the environment (Wang et al. 2017). The linked members in a module were functionally associated taxa that work together to achieve a distinct function or an ecological process. In this research, the integrated control measures, comprising a combination of bioorganic fertilizer application and liming, not only can effectively control tobacco bacterial wilt and significantly improve the flue-cured tobacco yield and output value but can also in general assist in recovering the microbial community, representing a promising application whose utility might be extended to other crops. It is a breakthrough that role-shifts prevailed among the network members. Microbes were unipathically associated with variables in control but multiplex in bioremediated soil. These measures can act to transform a wilt-inducing soil to a healthy and fertile soil, thus efficiently enabling the biocontrol of tobacco bacterial wilt in severely affected fields.