Plant-microorganism interaction maintains or dominates the ecological functions of terrestrial ecosystems. Microorganisms in soil environment are regarded as the largest reservoir of biological diversity in nature  . As many as 1011 microbial cells and 30,000 kinds of prokaryotes can be identified from one gram of rhizosphere soil   . Rhizosphere microbes  , considered as the second genome of plants  , alter the defensive ability of plants by regulating the host immune system   or recruit beneficial plant bacteria   , or inhibit the growth of plant pathogen by changing plant nutrition supply  . Therefore, rhizosphere microbes played a vital role in plant growth, health, yield and other aspects of regulation  . However, root exudates are the potential and important intermediates in the interaction between rhizosphere microorganisms and plants   . The study of the interaction between rhizosphere microorganisms and root exudates is helpful to the study of the interaction between rhizosphere microorganisms and plants. It is of great significance to evaluate plant growth and soil-borne diseases and insect pests.
Recently, the investigation of the relationship between rhizosphere microbes and root exudates mainly focused on the regulation of root exudates on the formation, metabolism, growth and diversity of rhizosphere microorganisms   . In fact, rhizosphere microorganisms can affect plant growth by absorbing a specific component of root exudates to stimulate changes in the abundance or quality of root exudates     . For example, inoculation with Bacillus subtilis B26 not only increased the yield of rape (B. distachyon), but also increased drought resistance  , even in the absence of nitrogen source. Both (Azospirillum) and (Herbaspirillum) of Vibrio herbaceus could promote the growth of Brassica napus  . These studies suggest that rhizosphere microbes play a potential role in plant root exudates. However, because of the complexity of soil microbes in heterogeneous environments, it is more challenging to explore how soil microbes directly affect root exudates.
In present study, the components of tobacco root exudates under the action of different rhizosphere microorganisms were detected and analyzed by gas chromatography-mass spectrometry (GC-MS), and the changes of tobacco root exudates were discussed. It provides effective information for the study of how microbes change cell membrane permeability and signal transduction, and provides a new clue for the prevention and treatment of tobacco soil-borne diseases.
2. Materials and Methods
Tobacco (Yunyan 87) was selected as culture material; Yunyan 87 was selected and bred by Yunnan Tobacco Science Research Institute and China Tobacco Breeding Research (South) Center. It was composed of Yunyan No. 2 as the female parent and K326 as the male parent. Besides, it was approved by the National Variety Approval Committee in December 2000. The main agronomic traits of Yunyan 87 were extremely stable, and its coefficient of variation was smaller than that of the control k326. The growth was uniform and widely adaptable in the field. The blade has the similar thickness, and the layer is yellow and easy to bake. It has an average yield about 2613 kg/hm2. The comprehensive evaluation index is superior to the main production variety k326. Yunyan 87 has high quality, stable production and wide adaptability, strong resistance to stress, easy to bake and so on. Tobacco bacterial wilt pathogen (Ralstonia solanacearum) and its antagonistic bacteria (Bacillus amyloliquefacien YH22); black shank pathogen (Phytophthora parasitica var. nicotianae) and its antagonistic bacteria (Bacillus amyloliquefacien TU1). All of the above bacteria were preserved in our laboratory.
2.1. Tobacco Cultivation and Root Exudate Extraction
First, Tobacco seedling transplanted to peat soil after the Great Cross (1 kg peat soil was added with bacterial wilt pathogen and black shank pathogen bacteria solution 50 ml, 1.0 × 109 CUF/mL), then sampling after 20 days of cultivation. The culture temperature was 30˚C, watering every 5 days and ensure adequate lighting. The remaining samples were supplemented with antagonistic bacteria against tobacco bacterial wilt and black shank (50 ml, 1.0 × 109 CUF/mL), sampling after 20 days of cultivation too. Collection of tobacco root exudates has adopted the method reported in the literature  ; repeating 3 times for each experiment and 3 parallels at a time. The collected root exudates were extracted with ethyl acetate: soak each uprooted tobacco with 100 ml distilled water, store in the dark for 48 hours, centrifuge the leachate and then leave the supernatant for suction filtration and adjust pH of the solution to acidity (pH2), alkalinity (pH11), and neutral (pH7) separately, with the same volume of ethyl acetate, extracting the solution. Then concentrated the extract under reduced pressure to 1 mL (45˚C), filtered through a 0.45 μm filter, and finally collected the organics three times. The solution was transferred to a rotary evaporation flask, and evaporated to dryness at 50˚C. The residuals were stored at low temperature (−80˚C).
2.2. Derivatization of Root Exudates and GC-MS Detection Conditions
Add mixture of 40 μL 20 mg/mL methoxyamine salt pyridine and 10 μL 5% phenylacetate to the freeze-dried root exudates; then incubating in a 60˚C incubator for 60 min. Finally, 50 μL of the silylating reagent N,O-Bis(trimethylsiyl) acetamide (BSTFA) was added and incubated at 70˚C for 60 min. Add mixture of 40 μL 20 mg/mL methoxyamine salt pyridine and 10 μL 5% phenylacetate to the freeze-dried root exudates; then incubating in a 60˚C incubator for 60 min. Finally, 50 μL of the silylating reagent N,O-Bis(trimethylsiyl)acetamide (BSTFA) was added and incubated at 70˚C for 60 min, cooled to room temperature naturally. GC-MS was analyzed by Thermo Scientific Gas Chromatography/Mass Spectrometer (DSQII). GC condition: the capillary column is TG-5MS (30 m × 0.25 mm × 0.25 μm), the inlet temperature is 250˚C, and the split ratio is 50:1. The heating program of column temperature: from 50˚C to 250˚C at a rate of 3˚C/min, continue to rise to 250˚C at a rate of 25˚C/min, remaining for 4 min. The carrier gas is He (99.999%), the flow rate is 20 psi, and the injection volume is 1 μL. MS condition: ionization energy is 70 eV, ion source temperature is 300˚C, scanning range is 30 - 600 m/z and transmission line temperature is 280˚C.
2.3. Data Statistics and Analysis
Processing and analyzing data with Origin 8.0 and SPSS 16.0, and GC-MS data was analyzed by Trace Finder 4.0. Statistics was obtained by Trace Finder Data Library. The relative content of root exudates was calculated by peak area normalization method.
3. Results and Discussion
1) Analysis of tobacco root exudates after treating by pathogen of tobacco bacterial wilt and its antagonist.
The composition of the root exudates of Yunyan 87 was analyzed by GC-MS after irrigating pathogen of bacterial wilt and its antagonist. The main components in the control group were amines, alcohols, acids, lipids, sugars and esters, with the corresponding percentages of 15.78%, 36.84%, 36.84%, 0%, 5.26% and 5.26%. Under the function of pathogen of bacterial wilt, the contents of amines, alcohols, acids, lipids, sugars and esters were 18.42%, 31.57%, 44.73%, 5.26%, 5.26% and 5.26% respectively. While by the antagonistic effect of pathogen of bacterial wilt, the contents of amines, alcohols, acids, lipids, sugars and esters were 11.92%, 23.81%, 38.09%, 9.52%, 11.91% and 4.76% accordingly (Figure 1). There were more than 23 types of root exudates of Yunyan 87 compared with the control group, mainly including 3 kinds of amines, 6 kinds of alcohols, 10 kinds of acids, 2 kinds of sugars, 1 kind of esters and cyclopropene respectively (Table 1). There are the same components of 5 types with contents decrease and 9 types with contents increase. It was noteworthy that the most remarkable increase was terephthalic acid (96.11%) and benzoic acid (143.75%). After the addition of antagonistic bacteria of bacterial wilt, there were 28 kinds of components increased among of which 9 were newly appeared and 12 of them decreased among of which 8 were completely disappeared, namely thiodiglycol, 1-butanol, and C. acid, isophthalic acid, myristic acid, ethyl benzoate, azelaic acid and propylene.
According to the analysis, we found that the content of propionic acid, isophthalic acid, isophthalic acid, terephthalic acid, myristic acid, ethyl benzoate, benzoic acid and sebacic acid showed the greatest volatility, the content in the control group was 0 (except for terephthalic acid, the content was 0.7792). The treatment group of pathogen of bacterial wilt reached the highest value, while the content decreased to 0 after the addition of antagonistic bacteria (except for terephthalic acid, from 1.5281 to 0.8589, with a decrease of 77.91%). Totally, amines were significantly decreased by loading B. amyloliquefacien YH22 (T-test, p = 0.045), alcohols content were continuously and significantly decreased by loading R. solanacearum and B. amyloliquefacien YH22 (T-test, 0.01 < p < 0.05), the content of acids and lipids were alwalys significantly increased
Figure 1. Total ion current of GC-MS with (a) treatment of pathogen of bacterial wilt; (b) treatment of pathogen of bacterial wilt and its antagonistic bacteria.
by loading these two kinds of microbes, the loading of R. solanacearum did not change the content of sugars, but significantly increasedby the loading of B. amyloliquefacien YH22 (T-test, p = 0.03), the loading of microbes did not obviously change the content of esters in three treatments (Figure 2).
Table 1. The content of tobacco root exudates under Ralstonia solanacearum and Bacillus amyloliquefacien YH22.
Figure 2. Changes of tobacco root exudates after treatment of bacterial wilt pathogen and its antagonist ((a), (b), (c) represent significant difference).
2) Analysis of tobacco root exudates after treating by pathogen of black shank and its antagonist.
The composition of the root exudates of Yunyan 87 was analyzed by GC-MS after irrigating pathogen of black shank and its antagonist (Figure 3). Under the condition of the pathogen of black shank, the contents of amines, alcohols, acids, lipids, sugars and esters were 14.28%, 28.57%, 48.57%, 2.85%, 2.85% and 2.85% respectively. By the treatment of antagonistic bacteria of black shank, the contents of amines, alcohols, acids, lipids, sugars and esters were 11.36%, 22.72%,
Figure 3. Total ion current of GC-MS with (a) treatment of pathogen of black shank; (b) treatment of pathogen of black shank and its antagonistic bacteria.
36.36%, 6.81%, 11.36% and 11.36% separately. There were more than 20 types of root exudates of Yunyan 87 compared with the control group, mainly including 2 kinds of amines, 5 kinds of alcohols, 11 kinds of acids, 1 kind of sugars, 1 kind of esters respectively (Table 2). There are the same components of 8 types with contents decreased and 4 types with contents increased. It was noteworthy that
Table 2. The content of tobacco root exudates under Phytophthora parasitica and Bacillus amyloliquefacien TU1.
the most remarkable increase was terephthalic acid (60.43%) and benzoic acid (43.15%). After the addition of antagonistic bacteria of black shank, there were 24 kinds of components increased among of which 2 were newly appeared and 13 of them decreased among of which 7 were completely disappeared, namely thioethylene glycol, 1-butanol, propionic acid, isophthalic acid, myristic acid, ethyl benzoate and azelaic acid (Figure 4).
According to the analysis, we found that the content of propionic acid, isophthalic acid, myristic acid, ethyl benzoate and azelaic acid showed the greatest volatility, the content in the control group was 0. The treatment group of pathogen of black shank reached the highest value, while the content decreased to 0 after the addition of antagonistic bacteria, followed by terephthalic acid and benzoic acid, which increased from 0.78 to 1.53 and 2.27 respectively, and decreased to 0.86 and 1.01 after adding antagonistic bacteria. Totally, amines were significantly decreased by loading B. amyloliquefacien TU1 (T-test, p = 0.048), alcohols content were continuously and significantly decreased by loading P. parasitica var. nicotianae and its antagonistic bacteria Bacillus amyloliquefacien TU1 (T-test, p = 0.03), the content of acids and lipids were alwalys significantly increased by loading these two kinds of microbes, the loading of P. parasitica var. nicotianae did not change the content of sugars, but significantly increasedby the loading of B. amyloliquefacien TU1 (T-test, t = 0.02), the loading of microbes did not obviously change the content of esters in three treatments (Figure 4).
Figure 4. Changes of tobacco root exudates after treatment of black shank pathogen and its antagonist ((a), (b), (c) represent significant difference).
Plant-root exudates-rhizosphere microbes form a cyclical interaction  , and rhizosphere microorganisms convert organic nutrients of root exudates into inorganic nutrients to facilitate plant utilization, thus affecting comprehensive indicators of plants     . By analyzing the changes of root exudates of tobacco under the effect of different microbes, it was found that when the pathogenic microorganisms in the rhizosphere microorganisms increased, the root exudates of tobacco augmented evidently. When the abundance of pathogens of bacterial wilt in the rhizosphere increased, the organic acids, amines and lipids increased by 32.78%, 13.35% and 15.76% respectively among which organic acids displayed the greatest rise, increased by 10.11%; followed by amine, increased by 6.09%. When the pathogen of bacterial wilt was inhibited by the active substances of its antagonistic bacteria, the root exudates changed significantly, and there were newly 19 kinds of elements added, but 1-butanol, propionic acid, isophthalic acid, myristic acid, phenethyl imine acid, sebacic acid and cyclopropene disappeared. Although the mechanism of effect of propionic acid, myristic acid, ethyl benzoate and sebacic acid was unclear, studies have confirmed that terephthalic acid, isophthalic acid and benzoic acid are allelopathic substances to many crops. They are crucial substance that cause continuous cropping obstacles      . When the abundance of pathogen of bacterial wilt and its antagonist changed in the rhizosphere, the changes of root exudates were similar to the treatment of bacterial wilt and its antagonist.
There are some differences in the number of tobacco root exudates under different treatments. The proportion of the amount is shown in Figure 2 and Figure 4, but the types of secretions are mainly amines, alcohols, organic acids and sugars. In the control group of tobacco root exudates, there was no carbohydrate, but it was detected in the treatment group of pathogen of bacterial wilt. After adding antagonistic bacteria, the types of carbohydrates increased and the change of organic acids was the most obvious: the control group was 36.84%, and the treatment group of bacterial wilt pathogen was 44.74%. Under the influence of pathogenic bacteria and antagonist, the types of organic acid increased to 51.61%, with a rise of 21.44% and 15.35% respectively. The change rule of root exudates of black shank pathogen and its antagonist was similar to that of bacterial wilt pathogen and its antagonist. However, the types of organic acid increased to 47.22% and 53.33%, with a rose of 28.18% and 12.93%. Data has shown that through the treatment of bacterial wilt, black shank pathogen and their antagonistic bacteria, the content of propionic acid, isophthalic acid, myristic acid, ethyl benzoate and azelaic acid fluctuated abundantly, while the control group was 0. The treatment group of bacterial wilt pathogen reached the highest its peak, and the content decreased to 0 after the addition of antagonistic bacteria, followed by terephthalic acid and benzoic acid, which increased from 0.78 to 1.53 and 2.27 respectively, yet decreased to 0.86 and 1.01 separately after adding antagonistic bacteria. The results showed that propionic acid, isophthalic acid, myristic acid, ethyl benzoate and azelaic acid were direct inducers of bacterial wilt and black shank pathogens, and their antagonistic bacteria could alleviate harmful bacteria that induced tobacco to produce allelochemicals (terephthalic acid, isophthalic acid and benzoic acid). The dramatical increase of the abundance of pathogen in soil was a vital factor which led to continuous cropping obstacles. It was speculated that the possible mechanism was due to the increase of the abundance of pathogen  , which induced crops to produce allelopathic substances, thus allelochemicals provide nutrients for the colonization of harmful bacteria. It formed a vicious circle that eventually resulted in continuous cropping obstacles. Although this conjecture needs further confirmation, the study provides new clues for exploring tobacco continuous cropping disorders and soil-borne diseases.
We wish to thank State Key Laboratory of Biocatalysis and Enzyme Engineering for providing the Laboratory space and equipments required for the research work. This work also has been supported by China National Tobacco Corporation, Grant (110201502018, 110201502014 and 027Y2014-013).
*These authors contributed equally to this work.
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