Integrated Biological Control of Tobacco Bacterial Wilt (Ralstonia solanacearum) and Its Effect on Rhizosphere Microbial Community

Biological control agents and soil amendments have been applied to control tobacco bacterial wilt, but the mechanism is not well-known. In the present study, a field experiment was performed to investigate the soil physicochemical properties, disease index (DI) and disease incidence of tobacco bacterial wilt, and rhizosphere microbial community. The results show that the control efficacy of single application of antagonistic bacteria and calcium cyanamide was 46.43% and 51.92%, respectively. While the combined control efficacy of antagonistic bacteria and calcium cyanamide was 65.79%. Besides, the combined application of antagonistic bacteria and calcium cyanamide could increase soil pH, total N alkaline N, and exchangeable Ca, which were negatively associated with the microbial diversity, soil-borne pathogenic microorganisms, and incidence of tobacco bacterial wilt. Additionally, the combination of antagonistic bacteria and calcium cyanamide can improve the proportion of some antagonistic microbial species, and these antagonistic microbial species were inversely associated with the DI of tobacco bacterial wilt. In conclusion: The integrated measure may influence soil microbial communities through enhancing soil physicochemical properties and rebuild healthy soil microbial community structure to mitigate tobacco bacterial wilt. The current study presented valuable insights into the mechanisms enhancing soil health in the integrated measure. *These authors contributed equally: Yun Hu and Chunli Li. #Corresponding author. How to cite this paper: Hu, Y., Li, C.L., Yang, X.Q., Feng, J., Wang, L., Chen, S.W., Li, Y.Y. and Yang, Y. (2021) Integrated Biological Control of Tobacco Bacterial Wilt (Ralstonia solanacearum) and Its Effect on Rhizosphere Microbial Community. Journal of Biosciences and Medicines, 9, 124-142. https://doi.org/10.4236/jbm.2021.93012 Received: January 30, 2021 Accepted: March 28, 2021 Published: March 31, 2021 Copyright © 2021 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
Tobacco bacterial wilt is a soil-borne bacterial infection triggered by Ralstonia solanacearum, which seriously damages the growth of tobacco [1] [2]. Nowadays, tobacco bacterial wilt is distributed in almost all the flue-cured tobacco growing areas in the world, especially in tropical and subtropical tobacco areas. It has been a major devastating disease threatening world tobacco production, which in turn causes great economic loss [3].
Tobacco bacterial wilt has received extensive attention, and many research studied have been reported about how-to restraint the tobacco bacterial wilt in breeding resistant varieties, agricultural control, and chemical control. However, these conventional approaches have a partial effect and are associated with many problems, such as lack of resistant varieties, easy loss of resistance, and the poor control effect of chemical agents, environmental pollution, and so on [4].
The occurrence of tobacco bacterial wilt is not only related to high temperature and high humidity climate but also related to soil types, soil nutrients and continuous cropping [5] [6]. It was reported that continuous harvesting obstacles lead to the imbalance of the number and proportion of microbial populations in the soil and the relative increase of R. solanacearum [7]. Besides, the incidence of tobacco bacterial wilt shows a significant positive correlation with pH values of the soil [8] [9]. Therefore, tobacco bacterial wilt is difficult to be effectively controlled, thus, new methods should be developed for effective control. In recent years, biological control technology has become a research hotspot because of its environmental and ecological safety and its ability to achieve sustainable development of tobacco [10]. Studies show that culturing of antagonistic into bioorganic fertilizers has helped in control of tobacco bacterial wilt. It can not only increase the population of beneficial microorganisms in soil and enhance the disease resistance of tobacco plants but also improve the rhizosphere micro-ecological environment, thus promote root growth and nutrient absorption [11] [12]. Besides, soil amendments can change the physicochemical properties of soil, improve and repair soil ecological environment, affect soil microbes, and reduce the number of pathogens [13] [14]. Due to the complexity of the environment and the limitation of single control methods, the prevention, and control effect of the disease-affected field is not an ideal strategy. Therefore, an integrated approach based on the combination of biological control and soil amendments was proposed as a new method to control tobacco bacterial wilt.
In this study, the antagonistic bacteria and calcium cyanamide were used to repair the tobacco field from bacterial wilt. An effective control mechanism of tobacco bacterial wilt was explored by studying the physicochemical properties

Incidence and Disease Index of Tobacco Bacterial Wilt
The disease incidence and DI of bacterial wilt in the BA, CC, and BC groups were found remarkably lower than those in the CK group. Moreover, the incidence and DI in the BC group were also found remarkably lower than those in the BA and CC groups ( Figure 1). The control efficacy of the BC group (65.79%) was observed higher than the BA group (46.43%) and CC group (51.92%). These outcomes showed that merging the antagonistic bacteria and calcium cyanamide could control tobacco bacterial wilt more effectively than a single application of antagonistic bacteria and calcium cyanamide.

Rhizospheric Soil Physicochemical Properties in Different Treatments
The physicochemical properties of rhizospheric soil were analyzed (Table S1). The pH, alkaline N, available K, exchangeable Ca, and exchangeable Mg of rhizospheric soil in the BA, CC, and BC groups were found remarkably higher (p < 0.05) than that in the control group. The total N of rhizospheric soil in the CC and BC groups was also observed higher (p < 0.05) than that in the control group. However, the organic matter, available P of rhizospheric soil in the CC, and BC groups were observed lower (p < 0.05) than those in the control group. These results show that the soil physicochemical properties were significantly affected by a single application of antagonistic bacteria, calcium cyanamide, and a combination of the application of antagonistic bacteria and calcium cyanamide. Moreover, applying calcium cyanamide could effectively increase the pH, total N, alkaline N, available K, exchangeable Ca, and exchangeable Mg of rhizospheric soil.

Tobacco Rhizosphere Microbial Community Richness and Diversity
In total, 6,168,190 fine raw sequences with an average length of 390 bps for bacteria and 350 bps for fungi were obtained after quality filtering (Table S2). The difference between OTUs, Sobs, Chao1, and Shannon index of microbial richness and diversity were analyzed ( Table 1). As for the bacteria, the Chao1 and Sobs index from the rhizosphere soil of the BC group was observed higher (p < 0.05) than those from other groups (BA, CC, and CK). OUT numbers from the rhizosphere soil of the BC and CC group showed no significant difference, but all significantly higher (p < 0.05) than CK and BA groups. The same trend was observed for the Shannon index. OUT numbers and Sobs, chao1, Shannon index from the bacterial rhizosphere soil of the BC group was higher than other groups. As for the fungal community, the four indexes from the rhizosphere soil of the BC group were the highest. OUT numbers and Sobs, chao1, Shannon index from the fungal rhizosphere soil of the BC group was higher than other groups. These results suggested that the effects of antagonistic bacteria combined with calcium cyanamide were higher than the single application of antagonistic bacteria or calcium cyanamide.

The Composition and Structure of Tobacco Rhizosphere Microbial Community
The leading bacterial phyla found in every treatment were Proteobacteria (37.80% -72.28%), Actinobacteria (6.15% -16.18%), firmicutes (2.07% -11.714%), Acidobacteria (7.75% -15.70%), Gemmatimonadetes (2.90% -7.91%), Chloroflexi (3.23% -5.19%), Bacteroidetes (1.01% -3.96%), and Nitrospirae (0.71% -2.50%) (Figure 2(a)). The relative population of Gemmatimonadetes, Acidobacteria, firmicutes, Actinobacteria, and Proteobacteria made up almost 81.51% -91.16% amid the 10 predominant bacterial phyla. The relative population of Proteobacteria in the BA, CC, and BC groups was higher than that in the CK group, while the relative population of Actinobacteria, firmicutes, and Gemmatimonadetes in the BA, CC, and BC groups were lower than that in the CK group, demonstrating that single application of antagonistic bacteria, calcium cyanamide and the combination of antagonistic bacteria and calcium  cyanamide affected the bacterial community structure. The top 6 fungal phylum accounted for 68.79% -87.06% of the fungal community, and Ascomycota (36.40% -60.98%), Mortierellomycota (6.21% -48.60%), Basidiomycota (0.25% -9.07%), Mucoromycota (4.23% -3.62%), Chytridiomycota (0.25% -0.63%) and Rozellomycota (0.04% -0.35%) were the 6 dominant fungal population (Figure 2(b)). The relative population of Basidiomycota in the CC and BC groups was found higher than those in the CK and BA groups. The relative population of Ascomycota in the CC and BC groups was observed higher than those in the BA group but lower than those in the CK group. Moreover, the relative population of Mortierellomycota in the CK group was significantly higher than that in CC and BC groups, but lower than those in the BA group. The findings demonstrate that the application of antagonistic bacteria combined with the calcium cyanamide influenced the composition of the fungal community. The different distributions of the microbial community at the genus level be-Journal of Biosciences and Medicines tween the other treatment groups and CK group are illustrated in Figure S1.
Terracidiphilus was one genus that found a significant difference between the BA and the CK groups ( Figure S1(a)). There were 44 significantly different genera between the CC and the CK groups, and 42 significantly different genera between the BC and the CK group ( Figure S1(b) and Figure S1(c)). The abundance of antagonistic bacteria Bacillus, Paenibacillus, Lysobacter, and Streptomyces in the BC and CC groups were higher than that in the CK group. while the abundance of nitrogen cycling bacterial Gaiella, Microvirga, and Nitrosospira in the BC and CC groups were higher than that in the CK group. Moreover, the population of increasing plant health and growth bacteria Novosphingobium in BC and CC groups was higher than that in the CK group. The population of carbon cycling bacteria Steroidobacter and Solirubrobacter in BC and CC groups were both higher than that in the CK group, but the population of Chthonomonas in CC and BC groups was significantly lower than that in the CK group ( Figure S1(b) and Figure S1(c)). At the genus level of fungal community, there was no significant difference of fungal proportions between the BA and CK groups, while Aspergillus, and Trichoderma as antagonistic fungal, were more abundant in the BC and CC groups than in the CK group ( Figure S1(d) and Figure S1(e)). For further exploring the distribution of the pathogenic and antagonistic microorganisms in different treatments, the abundances of these three types of microorganisms were calculated based on the absolute taxa abundance ( Figure 3). Four pathogenic microorganisms were found in all the treatments, three of them were fungal pathogens, including Alternaria, Fusarium solani, and Fusarium oxysporum, and one was bacterial pathogens (R. solanacearum). The population of F. oxysporum, F. solani, and R. solanacearum were higher (p < 0.05) in the CK group than that in other treatments. Moreover, the population of pathogenic microorganisms in the BC group was the lowest of all treatments (Figure 3(a)).
Twelve genera of antagonistic bacteria, including Streptomyces, Paenibacillus, Sphingomonas, Stenotrophomonas, Flavobacterium, Bacillus, Actinospica, Catenulispora, Haliangium and Lysobacter, and two genera of antagonistic fugal, including Aspergillus and Trichoderma were found in all treatments ( Figure   3(b)). The population of most antagonistic microorganisms in the CK group was significantly lower (p < 0.05) than other treatments, excepting Flavobacterium, and Catenulispora. Therefore, the population of most antagonistic microorganisms was improved by loading antagonistic bacteria, calcium cyanamide, and combination of antagonistic bacteria and calcium cyanamide. Especially, Sphingomonas was significantly improved by loading antagonistic bacteria, and Streptomyces, Flavobacterium, Aspergillus, and Trichoderma were significantly improved by loading calcium cyanamide.

The Correlation among the Microbial Community, Disease Index, and Soil Physicochemical Properties
The Spearman correlation among the soil physicochemical, DI, and microbial  Values are means of SD (n = 3). Bars with different letters are significantly different at p < 0.05 by LSD test.
community were measured to evaluate association amid them (

Discussion
The knowledge about the mechanism and action of integrated biological control of bacterial wilt will help out the restraining of soil-borne disease in agriculture. Current study amid to investigate the microbial community, the occurrence and DI of tobacco bacterial wilt, and physicochemical properties of rhizosphere soil. The single application of antagonistic bacteria, calcium cyanamide, and the combination of antagonistic bacteria and calcium cyanamide were confirmed to have an effect on the microbial community and successfully control the soil-borne disease. Additionally, the combination of antagonistic bacteria and calcium cyanamide showed to be more effective than the single application of antagonistic bacteria or calcium cyanamide, suggesting the integrated biological control method may be an applied approach for restraining tobacco bacterial wilt. fungal and bacterial communities in the combined application of antagonistic bacteria and calcium cyanamide treatment group were remarkably higher than that in the single application of antagonistic bacteria and calcium cyanamide treatment groups. Additionally, the microbial diversity had a negative relationship with DI of bacterial wilt. This is similar to a previous report [24]. The results indicated that the integrated measure has a significantly positive effect on the diversity and richness of the microbial community of rhizospheric soil, thus reducing tobacco bacterial wilt. Moreover, it was also found that the integrated measure influences the structure of the microbial community. In the present investigation, compared with the control group, certain genera of microorganisms Gaiella, Microvirga, Nitrosospira, Steroidobacter, Solirubrobacter, and Novosphingobium were found in a larger population in rhizospheric soil subject to the combined application of antagonistic bacteria and calcium cyanamide. Previously reports showed that these genera have mostly beneficial effects on recycling nutrients in the soil, the genera Gaiella, Microvirga, and Nitrosospira played an important role in nitrogen cycling [9] [25]. Some genera of Chthonomonas, Steroidobacter, and Solirubrobacter were found to participate in organic carbon cycling in the soil [26]. Besides, Novosphingobium was reported to assist plant growth by increasing plant health and growth [27]. In the current study, these beneficial genera had a significantly negative relationship with DI of tobacco bacterial wilt. These genera can directly and effectively control soil-borne diseases by competing for nutrients, space, and induced systemic resistance [28].
Therefore, the integrated measure can affect soil microbial community and rebuild healthy soil microbial community structure to mitigate tobacco bacterial wilt.
The antagonistic bacterial species such as Streptomyces, Paenibacillus, Sphingomonas, Stenotrophomonas, Bacillus, Actinospica, Haliangium, and Lysobacter are well recognized as useful rhizosphere microorganisms and can mitigate many soil-borne diseases and promote plant growth and health [29] [30]. These were present abundantly in the combined application of antagonistic bacteria and calcium cyanamide treatment group. The antagonistic fungal Aspergillus and Trichoderma have been reported to interact directly with roots to produce bioactive substances that help in the growth of plants and fight against abiotic and biotic stress [31] [32]. The abundance of the above antagonistic fungal strains was higher in the single application of calcium cyanamide treatment group and the combination of antagonistic bacteria and calcium cyanamide treatment group. Furthermore, these antagonistic microbial showed a significantly negative relationship with the DI of tobacco bacterial wilt. Therefore, the proportion of antagonistic microbial was improved by the integrated measure treatment group, which is conducive to recruiting antagonistic adherents of the soil microbiome to shield tobacco plants from disease. Studies have reported that diverse microbial communities tend to be less vulnerable to pathogens attack than a simpler microbial community [33]. Our study showed that the abundances of pathogens R. solanacearum, F. oxysporum, and F. solani were signifi- cantly lower in the combined application of antagonistic bacteria and calcium cyanamide treatment group than the control group. Besides, these pathogens had significantly correlated with soil physicochemical properties and positive correlation with the DI of bacterial wilt. Consistent with the above findings, the combination of antagonistic bacteria and calcium cyanamide treatment group can control tobacco bacterial wilt through increasing the microbial community diversity and resist the invasion of pathogens.

Field Experiment
The

Rhizosphere Soil Samples and Basic Parameter Calculation
Rhizosphere soil samples were collected at 68 d post-transplantation by the five-spot-sampling method and transported to the lab, then stored at −80˚C for microbial community analysis, and at 25˚C for physicochemical properties analysis. To study the soil characteristics and available nutrients, collected soil specimens were air-dried and mechanically crushed into less than 2 mm mesh sized powder. Exchangeable magnesium (Mg) and calcium (Ca), available potassium (K), available phosphorus (P), alkaline nitrogen (N), total nitrogen (N), organic matter, and pH were determined by atomic absorption spectrophotometry, flame spectrophotometer, sodium bicarbonate method, alkali solution diffusion method, Kjeldahl method, potassium dichromate volumetric method, and potentiometric method, respectively [35].

Incidence and Disease Index Calculation
The symptoms of tobacco bacterial wilt of all 60 plants in each section were monitored at 15 d post-transplantation. The severity scale from 0 to 9 was used as reported in an earlier study [14]. Disease incidence (I) and disease index (DI)

Extraction and Purification of Soil Microbial DNA
For microbial DNA extraction, 0.5 g soil was weighed precisely from each sample stocked for genome analysis. The FastDNA Spin Kit (MP Biomedicals, USA) was used to extract the soil microbial genomic DNA following the prescribed procedures and specifications. The 1% agarose gel electrophoresis was employed to measure the integrity of DNA samples. Subsequently, the Nanodrop was used to evaluate the purity and concentration of DNA.

PCR Amplification and Sequencing
The microbial DNA isolated from the rhizosphere soil samples was subjected to use as a template for replication of ITS rRNA and 16S rRNA genes. The DNA samples were diluted to 1 ng·μL −1 with millipore water. The primers 806R

Sequence Data Analysis
The FLASH (V1.2.7) software was employed to merge the paired-end reads and assigned to the corresponding samples using the unique barcodes. The QIIME (V1.7.0) software was used at specific filtering parameters to filter the raw tags and gain the fine clean tags.

Statistical Analysis
The

Conclusion
The findings of the current study suggest that the integrated measure can regulate soil physicochemical properties, and effectively reduce the incidence of tobacco bacterial wilt, thus obviously increase the diversity and richness of soil microorganisms. It can further effectively resist the invasion of pathogens, and promote the transformation of diseased soil ecosystem into healthy and sustainable development through prevention and control of tobacco bacterial wilt.

Conflicts of Interest
The authors declare no conflict of interest. Journal of Biosciences and Medicines