Physiochemical Properties and Microflora of the Rhizosphere Soil of Tobacco Plants with and without Bacterial Wilt

Abstract

Bacterial wilt is a destructive soilborne disease caused by Ralstonia solanacearum, posing a severe threat to plants in the Solanaceae family. It impacts on tobacco productivity worldwide. This study was conducted to analyze the changes in the soil’s physical and chemical properties, the number of microbes, and the bacterial diversity of the rhizosphere soil before and after the wilt disease. The rhizosphere soil of healthy and diseased tobacco plants was collected from Pucheng, Nanping, Fujian Province, Southern China. The results revealed significant differences in the trends of physical and chemical properties of the soil of healthy and diseased plants. The soil pH, available potassium (K), available phosphorous (P), and organic matter contents (SOM) were lower in the rhizosphere soil for healthy plants than for pre-diseased plants (HW). Only the available P, among all physical and chemical properties in the rhizosphere of diseased plants (HS), was significantly lower than those for pre-diseased plants (HW), changing from 149.59 mg/kg to 59.19 mg/kg. The order of numbers of the three main microbes in the rhizosphere soil for healthy plants (HC) and pre-diseased plants was the following: bacteria > actinomycetes > fungi. The number of actinomycetes in the soil of the diseased tobacco plants increased significantly. A comparison of the rhizosphere soil of diseased and healthy tobacco plants showed that the relative abundance of the bacterial community in the rhizosphere soil of the pathogenic tobacco plants changed significantly. The community diversity was increased, and the Pseudomonadaceae, to which the bacterial pathogen of bacterial wilt belonged, rose to a certain extent. Both pre-diseased and healthy plants showed changes in the physical and chemical properties, microbial quantity, and microbial diversity, thus proving that tobacco disease was closely related to the soil’s ecological environment.

1. Introduction

Tobacco is an important commercial crop in China, and the tobacco industry plays a positive role in promoting farmers’ employment and improving agricultural production technology [1]. However, tobacco diseases have perplexed the development of tobacco planting for a long time. Among them, bacterial wilt caused by Ralstonia solanacearum has become the leading infectious disease affecting the growth and quality of tobacco [2], causing significant losses to the tobacco planting industry. R. solanacearum can survive in the soil for a long time and invade through the wounds of tobacco roots or stems. Bacterial wilt is a disease mainly transmitted through soil, and the soil is the basis of tobacco growth and the main ecological factor affecting tobacco quality [3]. Therefore, the soil for tobacco growing has been gradually highlighted in the research on controlling and preventing tobacco bacterial wilt [4].

Rhizosphere soil microorganisms are regulators of nutrient transformation and transport between soil and plants. Studies have shown a strong correlation between soil microorganisms and soil physicochemical properties [5], which can reflect the soil fertility and the changes in the number of rhizosphere soil microorganisms before and after the bacterial wilt to a certain extent. Since only a small number of microorganisms in soil can be cultivated under laboratory conditions, microbial research methods without cultivation have been gradually used in microbial ecological research [6]. However, the culturable microorganisms in soil have higher biomass and metabolic activity, and the changes in the culturable microbial flora can provide information closely related to ecological functions.

Soil microbial diversity is used to characterize the variation of soil microbial community structure, complex interactions, nutrient levels, and quantitative changes in the composition. It can reflect the changing process of soil ecology earlier and plays an important role in plant growth, plant development, and community-structure succession [7]. Currently, the studies mainly focus on the impact of tobacco bacterial wilt, the control measures on the quality of tobacco leaves, and some soil properties in disease prevention and control [8,9]. However, comparing the physicochemical properties and microflora of rhizosphere soil between healthy and diseased tobacco plants before and after the onset of bacterial wilt has not been well reported.

In this study, the tobacco plants in Pucheng county, Nanping city, Fujian province, Southern China, were taken as the research object; the physicochemical properties and microbial flora of the rhizosphere soil of healthy and diseased tobacco plants with bacterial wilt (pre-onset, post-onset, and healthy soil samples) were analyzed, and the differences in the rhizosphere soil microecology were investigated. This study provides a reference and basis for controlling tobacco bacterial wilt in the future by regulating the microecology of the rhizosphere soil.

2. Materials and Methods

2.1. Study Area

Pucheng county, Nanping city, Fujian province, Southern China, was chosen as the study area (Figure 1). Pucheng has an area of 3383.02 square kilometers in the Wuyi Mountains, which separate Fujian and Jiangxi provinces. In Pucheng, the summers are hot, oppressive, and mostly cloudy; the winters are short, cold, and partly cloudy; and it is wet year-round. Over the year, the temperature typically varies from 36 °F to 92 °F and is rarely below 26 °F or above 96 °F. The climatic parameters are shown in detail in Figure 2. The soil type of the study area is ultisol.

2.2. Experimental Design

In this test, three kinds of tobacco soil treatments were designed: (1) soil samples (HW) of tobacco plants before the onset of bacterial wilt (rhizosphere soil of pre-onset tobacco plants after 70 days of transplanting), (2) soil samples (HS) of tobacco plants after the onset of bacterial wilt (rhizosphere soil of the post-onset tobacco plants after 90 days of transplanting), and (3) soil samples (HC) of the healthy tobacco plants (rhizosphere soil of the healthy tobacco plants after 90 days of transplanting). Three tobacco plants with similar growth status and the same health level were selected for each treatment.

2.3. Experimental Methods

Tobacco plants in good shape were randomly selected according to the five-point sampling method, and the topsoil at the root of the tobacco plant was removed. The tobacco plant was uprooted, the soil around the root system was shaken off, and the root (5–20 cm away) from the foundation was intercepted. The soil samples near the root surface were taken as rhizosphere soil and stored in a sealed bag with a number. Afterwards, the sealed bags were placed in an ice box and taken to the lab for follow-up testing.

The pH value of the soil was determined by the potentiometric method (the standard method recommended by the Ministry of Rural Agriculture of China: NY/T 1121.2-2006). The contents of soil-available nitrogen (N), available phosphorus (P), available potassium (K), and organic matter were determined by the alkali hydrolysis diffusion method (LY/T 1229-1999), acid–ammonium fluoride extraction molybdenum–antimony anti-colorimetry (NY/T 1121.7-2006), flame spectrophotometer (NY/T 889-2004), and the potassium dichromate volumetric method (GB/T 50123-1999), respectively.

The dilution plate method determined the number of cultivable soil microorganisms [10]. The beef extract peptone agar was used as a bacterial-isolation medium, the potato sucrose agar as a fungal-isolation medium, and the Gauss No. 1 agar as an actinomycete-isolation medium. Total soil DNA was extracted from 0.5 g of soil using a Power Soil^® DNA isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA, United States) as instructed by the manufacturer. The microbial diversity was determined by Sangon Biotech (Shanghai, China) Co., Ltd.

Different diversity indexes reflect different aspects of microbial community diversity. The Shannon index measures the heterogeneity of the community. The larger the Shannon index is, the higher the microbial diversity in the soil and the richer the species are. The Chao index is an index for predicting the total number of bacteria in the soil environment. The larger the Chao index, the more species there are. The coverage index refers to the coverage of the sample library. In general, the higher the value, the less likely it is that the sequence is not detected in the sample.

The indices of bacterial diversity were calculated as follows:

The Chao index was used to estimate the index of OTU numbers contained in the sample to reflect community richness [11].

$$S_{chao}=S_{obs}+\frac{n_1\left(n_1-1\right)}{2\left(n_2+1\right)}$$

The Shannon index was employed to estimate the microbial diversity index in the sample [12].

$$H_{shannon}=-{{\displaystyle \sum }}_{i=1}^{S_{obs}}\frac{n_i}{N}\ln \frac{n_i}{N}$$

The coverage index refers to the coverage of each sample library, which informs whether the sequencing results reflect the actual situation [11]. It was calculated as follows:

$$\mathrm{C}=1-\frac{n_i}{N}$$

In the above equations, S_obs is the number of OTUs actually measured, N is the number of all sequences, and n_i is the number of OTUs containing ‘i’ sequences.

2.4. Data Analysis

The data were calculated and statistically analyzed using Microsoft Excel^TM and SPSS, version 19.0 (SPSS Inc., Chicago, IL, USA). The statistical significance of the results was determined by performing Fisher’s protected least significant difference (LSD) test (p ≤0.05). A rarefaction curve can be used to compare species richness, homogeneity, or diversity across samples with varying levels of sequencing data. In addition, it can be used to clarify the rationale for the volume of sequencing data. In this study, random sampling of the sequence was used, and the selected sequence number, Chao index, and Shannon index were used to construct the rarefaction curves.

3. Results

3.1. Physicochemical Properties of Rhizosphere Soil of Tobacco Plants

As the tobacco plant grew and matured, the pH value and contents of available K, P, N, and the organic matter in the rhizosphere soil of healthy tobacco plants (HC) increased compared to pre-disease tobacco plants (HW). However, the content of available N in the rhizosphere soil of the diseased tobacco plant (HS) was 150.50 mg/kg, which was higher than that of HW. The pH value and the contents of available K and available P in HS were lower than those in HW (

Table 1. Changes in the physicochemical properties of the rhizosphere soil of tobacco. Values are mean ± SE.

Treatment	pH	Available K (mg/kg)	Available P (mg/kg)	Available N (mg/kg)	Organic Carbon (g/kg)
HW	5.22 ± 0.031	196.22 ± 9.33	149.59 ± 6.39	105.70 ± 3.32	27.31 ± 0.89
HC	5.52 ± 0.030	261.07 ± 11.36	159.19 ± 2.61	152.60 ± 4.01	29.20 ± 0.94
HS	4.77 ± 0.018	137.45 ± 6.58	102.16 ± 5.05	150.50 ± 4.46	25.03 ± 1.02

).

The changing trend of soil physicochemical properties varied with the health, pre-onset, and post-onset states of the tobacco plants. It indicated an essential correlation between tobacco plant disease and soil physicochemical properties. The contents of available N measured in the rhizosphere soil of diseased and healthy tobacco plants (HS and HC) were similar, possibly due to the reduction in nitrogen source utilization after a long period of vigorous tobacco planting.

3.2. Changes in Culturable Microorganism Quantity in Tobacco Rhizosphere Soil

As shown in

Table 2. Tobacco rhizosphere soil microbial quantity changes.

Treatment	Bacteria (×106 cfu/g)	Fungus (×105 cfu/g)	Actinomyces (×105 cfu/g)
HW	6.73 ± 0.16	2.13 ± 0.05	3.73 ± 0.09
HC	6.84 ± 0.15	1.53 ± 0.04	1.95 ± 0.05
HS	4.07 ± 0.09	2.56 ± 0.05	7.06 ± 0.14

, between HW and HC, the number of bacteria in the rhizosphere soil was almost the same, and the number of fungi and actinomycetes in HC was less than that in HW. Comparing HW and HS, the number of bacteria in HW was significantly larger than that in HS, while the number of fungi in HW was slightly smaller than in HS. Moreover, the number of actinomycetes in HS was also significantly higher than in HW, with an increase of 89.28%. Compared with HC, the number of bacteria in HS was less, and that of fungi and actinomycetes was higher.

3.3. Changes in Bacterial Community Structure in Tobacco Rhizosphere Soil

The dominant bacteria of HW and HC were different from those of HS. According to the relative abundance, the top four dominant bacteria in HW were Chitinophagaceae, Sphingomonadaceae, Oxalobacteraceae, and Flavobacteriaceae; the top four dominant bacteria in HC were Sphingomonadaceae, Chitinophagaceae, Oxalobacteraceae, and Bradyrhizobiaceae; and the dominant bacteria in HS were Chitinophagaceae, Pseudomonadaceae, Sphingomonadaceae, and Planctomycetaceae (Figure 3).

There were considerable differences in the relative abundances of bacterial communities in the soils of HC, HW, and HS. For example, the relative abundance of Sphingomonadaceae, Oxalobacteraceae, and Caulobacteraceae in HS was significantly smaller, while the relative abundance of Planctomycetaceae and Rhodocyclaceae was considerably larger. Besides, the relative abundance of Flavobacteriaceae was smallest in HC (0.26%), slightly higher in HS (1.27%), and most extensive in HW (4.35%). R. solanacearum, the pathogen of bacterial wilt, belongs to the Pseudomonadaceae. The relative abundance of this bacteria was 2.96% in HC and 4.94% in HS. The pathogen of bacterial wilt in the rhizosphere soil had increased to a certain extent after the onset (Figure 3).

3.4. Changes in Bacterial Diversity in the Rhizosphere Soil of Tobacco Plants

As shown in

Table 3. Bacterial diversity indexes in the rhizosphere of tobacco plants.

Treatment	Shannon	Chao1	Coverage
HW	7.43	13,413.82	0.93
HC	8.02	15,708.13	0.91
HS	7.68	15,062.57	0.91

, the coverage values of HW, HS, and HC rhizosphere soil are higher than 0.9. It indicates that the sequence coverage obtained by sequencing is high, reflecting that the sample library can truly represent the diversity of soil bacteria in this region. HC has the highest Chao index, followed by HS, and HW has the smallest one. In other words, the total amount of bacteria in healthy tobacco plants is higher than before and after the onset of disease, indicating that the richness of bacteria in the rhizosphere soil is higher in healthy tobacco plants.

For the Shannon index, HC has the largest value, followed by HW, and the smallest value is for HS. It indicates that after the onset of the tobacco plant disease, the diversity of the rhizosphere-bacterial community and the evenness and richness of the community-species composition are the highest. The Shannon index of HS is higher than that of HW, indicating that the rhizosphere soil microflora induced by R. solanacearum in the diseased tobacco rhizosphere soil has changed accordingly.

As shown in Figure 4, the rarefaction curve of Chao is steep, indicating that the distribution of species in HC, HW, and HS soil samples is uneven. The Shannon index rarefaction curves tend to be flat, meaning that the sequencing data are sufficient to capture most of the microbial diversity information in the samples. HC has the most extensive community-species diversity, followed by HS, and HW has the smallest one.

4. Discussion and Conclusions

R. solanacearum, a soilborne pathogen that lives in soil and tobacco plant residues, causes tobacco bacterial wilt. After invading through the wound of the root and stem of the tobacco plant, it multiplies in the vascular bundle-threaded duct, destroys the transport organs, and makes the stems and leaves wither and die without water [13]. In the existing research, it was generally believed that bacterial wilt was related to soil type, fertility level, climatic conditions, the selection of continuous cropping, and the damage conditions of the roots and stems of tobacco plants [14]. However, in tobacco planting, it was found that the occurrence of tobacco bacterial wilt in the same plot had considerable randomness [15]. This was mainly related to the individual tobacco plants and the soil microecology constructed with tobacco rhizosphere soil and related microorganisms [16].

Studies have shown that bacterial wilt is more likely to occur in acidic soil [17]. The results of this study show that the soil pH of healthy tobacco plants is higher than that of diseased tobacco plants, which is consistent with the existing research results. It was reported that increasing the pH of the rhizosphere soil could effectively prevent and control bacterial wilt [18,19]. This research indicated that the occurrence of tobacco bacterial wilt had an important relationship with soil pH. Therefore, the prevention and control of bacterial wilt could be achieved by regulating soil pH. The organic matter content in the rhizosphere soil of healthy tobacco plants was higher than that of the diseased tobacco plants. This was because healthy tobacco plants have developed active roots that secrete and spill more microbes into the soil, thus affecting the amount of carbon entering the soil, the decomposition rate in the soil, the size of the organic carbon pool, and the carbon distribution in the components [20].

The content of available K in the soil of healthy tobacco plants was high, which was consistent with the research results obtained by the authors of [21]. Improving K nutrition was generally conducive to enhancing the disease resistance of host plants [21]. Previous studies have suggested that the content of available K in soil was affected by temperature, fertilization, soil pH, and soil organic matter content [22,23]. The content of available P in the rhizosphere soil decreased after tobacco plant disease, which was consistent with the analysis results of the authors of [22,23]. After the infection of tobacco plants, the pH value and the contents of available K, P, and N, as well as organic matter, were lower than those of healthy tobacco plants. The possible reasons are that in the early stages of bacterial wilt infection, the rapid growth of R. solanacearum consumed a lot of nutrients in the soil, leading to a rapid increase in the soil nutrients, K and P. In contrast, the lack of available P and available K in the rhizosphere soil of tobacco plants caused a further decline in the resistance of tobacco plants to bacterial wilt. At the same time, the reduction in pH and other changes facilitated the growth and reproduction of bacterial wilt and eventually led to the rapid infection of tobacco plants and the aggravation of the disease.

Compared with healthy tobacco plants, the number of bacteria in diseased tobacco plants was smaller, and the number of fungi and actinomycetes was more significant. This showed that the incidence of tobacco bacterial wilt was related to the decrease in bacteria and the increase in fungi and actinomycetes in the rhizosphere soil. In future preventive measures, the control effect of tobacco bacterial wilt could be improved by adding soil flora regulators and strengthening the construction of microbial flora [24,25]. The stability of the whole microbial community in the soil could be expressed by soil microbial diversity, which reflects the soil ecological mechanism and the impact of soil environmental stress on the microbial community.

In general, the microbial diversity in a healthy rhizosphere soil ecosystem was higher than that in a diseased rhizosphere soil ecosystem. The results of this experiment confirm this point of view. By comparing HC, HS, and HW, it can be found that the relative abundance of bacterial communities has changed. For example, the relative abundance of Flavobacteriaceae was the smallest (0.26%) in HC and the highest (4.35%) in HW. This shows that the growth and onset of bacterial wilt in tobacco plants will change the structure of bacterial communities and the number of bacteria. However, only from the perspective of community structure and relative abundance, it is difficult to find the actual difference in soil bacterial communities under different conditions (such as that between healthy and diseased tobacco plants). In future research, the significance of inter-group differences in bacterial community structure and the microorganisms with significant differences between different sample groups should be further analyzed.

The physicochemical properties and microflora of the rhizosphere soil of healthy tobacco plants and diseased tobacco plants with bacterial wilt (pre-onset, post-onset, and healthy soil samples) were very different. The pH values and the contents of available K, P, and N, as well as organic matter, in the rhizosphere soil of healthy tobacco plants were higher than those of diseased tobacco plants. In the rhizosphere soil of the healthy plants, the microflora was mainly comprised of bacteria, followed by fungi and actinomycetes.