The research on relationship between rhizosphere microbes and root exudates has a great significance on discussion of interaction between rhizosphere microbes and plants, as well as control of soil-borne diseases and insect pest. GC-MS was used to analyze changes of tobacco root exudates under the antagonistic action of tobacco bacterial wilt and black shank. It turned out that when pathogens of tobacco bacterial wilt and black shank in tobacco root microorganisms increase, tobacco root exudates augmented rapidly among of which organic acids have the biggest growth, followed by amines. When the pathogens of tobacco bacterial wilt and black shank are inhibited by the active substance of antagonistic antibacterial, 20 - 23 kinds of root exudates are added; besides, the content of 7 substances was reduced to 0. Another inter-esting finding was that the fluctuations of phthalic acid, isophthalic acid and benzoic acid, which have caused continuous cropping obstacles, were very dis-tinct. The results of this study have provided novel clues for the exploration of continuous tobacco cropping obstacles and soil-borne diseases.
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 [
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 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.
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.
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 [
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.
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.
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 (
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
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 (
Components of tobacco root exudates | Peak area (%) | |||
---|---|---|---|---|
Molecular Formula | Control group | Pathogen of bacterial wilt | Pathogen of bacterial wilt + antagonist | |
Methylamine | C7H21N | 8.9354 ± 0.29 | 9.1213 ± 0.26 | 9.5606 ± 0.25 |
Tyramine | C8H11NO | (-) | 2.1331 ± 0.30 | 1.2131 ± 0.32 |
Silanamine | C7H18N2 | (-) | 5.4464 ± 0.22 | 6.5480 ± 0.34 |
Bis(trimethylsilyl)carbodiimide | C7H18N2 | 1.2014 ± 0.35 | 1.6521 ± 0.29 | 1.2091 ± 0.25 |
N-Methyltrifluoroacetamide | C3H4F3NO | 0.1541 ± 0.10 | 0.2514 ± 0.05 | 0.2618 ± 0.06 |
Hexanol | C12H28O | 4.4790 ± 0.24 | 3.5823 ± 0.23 | 3.5856 ± 0.35 |
Ethylene glycol | C8H22O2 | 1.6186 ± 0.22 | 1.6312 ± 0.32 | 1.4583 ± 0.23 |
Diethylene glycol | C10H26O3 | 1.1490 ± 0.22 | (-) | (-) |
Phenylethyl alcohol | C8H10O | 1.1589 ± 0.15 | (-) | (-) |
Glycerol | C12H32O3 | 2.6808 ± 0.40 | 2.7021 ± 0.19 | 3.8914 ± 0.39 |
Thiodiglycol | C4H10O2S | 0.1238 ± 0.06 | 0.2156 ± 0.06 | (-) |
D-Pinitol | C7H14O6 | (-) | (-) | 0.1423 ± 0.04 |
D-Mannitol | C24H62O6 | (-) | 1.1671 ± 0.16 | 2.1651 ± 0.20 |
Myo-Inositol | C6H12O6 | (-) | 0.5698 ± 0.14 | 0.5412 ± 0.11 |
Silanol | C13H14O | (-) | 0.1296 ± 0.09 | 0.2315 ± 0.08 |
1-Butanol | C4H10O | (-) | 2.5128 ± 0.39 | (-) |
2,3-Butanediol | C10H26O2 | (-) | 0.7250 ± 0.11 | 0.5283 ± 0.02 |
1-Monopalmitin | C19H38O4 | (-) | 1.3210 ± 0.21 | 1.1593 ± 0.23 |
Isopropyl alcohol | C3H8O | (-) | (-) | 1.1452 ± 0.17 |
Triethylene glycol | C8H18O4 | 2.1423 ± 0.17 | (-) | (-) |
Boric acid | BC9H27O3 | 8.4826 ± 0.24 | 8.5092 ± 0.23 | 9.5069 ± 0.33 |
Oxalic acid | C2H2O | (-) | 4.2156 ± 0.43 | 3.5623 ± 0.35 |
Propionic acid | C3H6O2 | (-) | 3.5986 ± 0.38 | (-) |
Palmitic acid | C19H40O2 | (-) | 1.1267 ± 0.22 | 3.1092 ± 0.37 |
Hydrocinnamic acid | C9H10O2 | (-) | 0.0186 ± 0.02 | 0.8218 ± 0.19 |
Stearic acid | C21H44O2 | 5.4667 ± 0.14 | 4.0571 ± 0.26 | 7.0821 ± 0.42 |
Isophthalic acid | C8H6O4 | (-) | 2.5862 ± 0.24 | (-) |
Terephthalic acid | C8H6O4 | 0.7792 ± 0.25 | 1.5281 ± 0.25 | 0.8589 ± 0.18 |
Myristic acid | C14H28O2 | (-) | 0.5836 ± 0.15 | (-) |
Itaconic acid | C5H6O4 | (-) | 0.0782 ± 0.02 | 0.1290 ± 0.04 |
Benzimidate hydrochloride | C9H12NClO | (-) | 0.0576 ± 0.02 | (-) |
Sebacic acid | C10H18O4 | (-) | 0.3276 ± 0.04 | (-) |
Lactic acid | C9H22O3 | 7.1991 ± 0.29 | 5.3126 ± 0.24 | 6.4286 ± 0.29 |
Benzoic acid | C7H6O2 | 1.5823 ± 0.23 | 3.8569 ± 0.43 | 1.2982 ± 0.28 |
Acetic acid | C10H12O2 | 2.6790 ± 0.26 | 3.4087 ± 0.28 | 4.4589 ± 0.13 |
Octanoic acid | C8H16O2 | 1.5820 ± 0.30 | 1.5692 ± 0.23 | 1.8263 ± 0.26 |
---|---|---|---|---|
3,4-Dimethoxybenzoic acid | C9H10O4 | (-) | (-) | 0.0350 ± 0.01 |
Dehydroabietic acid | C20H28O2 | (-) | (-) | 1.0464 ± 0.13 |
Caffeic acid | C9H8O4 | (-) | (-) | 0.0805 ± 0.04 |
3-Hydroxybutyricacid | C4H8O3 | (-) | (-) | 0.0428 ± 0.01 |
Sucrose | C12H22O11 | (-) | (-) | 0.0025 ± 0.0009 |
D-Trehalose | C12H22O11 | (-) | 1.3090 ± 0.17 | 0.1690 ± 0.13 |
Galactopyranose | C6H12O6 | (-) | (-) | 0.0158 ± 0.006 |
D-(+)-Mannose | C6H12O6 | (-) | 0.0431 ± 0.01 | 0.0543 ± 0.01 |
Phenethyl acetate | C10H12O2 | 5.4376 ± 0.26 | 5.0627 ± 0.25 | 5.0289 ± 0.21 |
Valtrate | C22H30O8 | (-) | (-) | 0.5030 ± 0.07 |
Glycerol monostearate | C21H42O4 | (-) | (-) | 0.3082 ± 0.03 |
Dodecanoic acid | C24H48O2 | (-) | (-) | 0.0150 ± 0.03 |
Propyl heptanoate | C10H20O2 | (-) | 0.0591 ± 0.008 | 0.0523 ± 0.007 |
Cyclopropane | C3H6 | (-) | 0.0112 ± 0.009 | (-) |
Urea | CH4N2O | 0.0039 ± 0.001 | 0.0036 ± 0.0009 | 0.0153 ± 0.001 |
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 (
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 (
Components of tobacco root exudates | Peak area (%) | |||
---|---|---|---|---|
Formula | Control group | Pathogen of black shank | Pathogen of black shank + antagonist | |
Methylamine | C7H21N | 8.9354 ± 0.58 | 8.6354 ± 0.43 | 8.1213 ± 0.46 |
Tyramine | C8H11NO | (-) | 2.0031 ± 0.36 | 1.6131 ± 0.30 |
Silanamine | C7H18N2 | (-) | 4.3898 ± 0.27 | 5.3218 ± 0.33 |
Bis(trimethylsilyl)carbodiimide | C7H18N2 | 1.2014 ± 0.22 | 1.1304 ± 0.34 | 1.9807 ± 0.42 |
N-Methyltrifluoroacetamide | C3H4F3NO | 0.1541 ± 0.16 | 1.3543 ± 0.31 | 0.3318 ± 0.19 |
Hexanol | C12H28O | 4.4790 ± 0.42 | 3.5823 ± 0.52 | 4.5998 ± 0.35 |
Ethylene glycol | C8H22O2 | 1.6186 ± 0.31 | 1.1563 ± 0.25 | 1.2380 ± 0.32 |
Diethylene glycol | C10H26O3 | 1.1490 ± 0.43 | (-) | (-) |
Phenylethyl alcohol | C8H10O | 1.1589 ± 0.44 | (-) | (-) |
Glycerol | C12H32O3 | 2.6808 ± 0.68 | 2.0565 ± 0.41 | 1.9967 ± 0.11 |
Thiodiglycol | C4H10O2S | 0.1238 ± 0.08 | 0.1615 ± 0.11 | (-) |
D-Pinitol | C7H14O6 | (-) | (-) | 0.1120 ± 0.04 |
D-Mannitol | C24H62O6 | (-) | 1.6160 ± 0.28 | 2.0615 ± 0.33 |
Myo-Inositol | C6H12O6 | (-) | 0.2298 ± 0.17 | 0.0982 ± 0.06 |
Silanol | C13H14O | (-) | 0.2629 ± 0.14 | 0.3234 ± 0.08 |
1-Butanol | C4H10O | (-) | 2.5342 ± 0.38 | (-) |
2,3-Butanediol | C10H26O2 | (-) | 0.0830 ± 0.05 | 0.5429 ± 0.19 |
1-Monopalmitin | C19H38O4 | (-) | 1.0321 ± 0.29 | 1.1421 ± 0.29 |
Isopropyl alcohol | C3H8O | (-) | (-) | 1.0852 ± 0.27 |
Triethylene glycol | C8H18O4 | 2.1423 ± 0.34 | (-) | (-) |
Boric acid | BC9H27O3 | 8.4826 ± 0.31 | 8.5014 ± 0.37 | 9.5421 ± 0.25 |
Oxalic acid | C2H2O | (-) | 4.6245 ± 0.53 | 3.5460 ± 0.54 |
Propionic acid | C3H6O2 | (-) | 1.5436 ± 0.28 | (-) |
Oleic acid | C21H42O2 | (-) | 0.0743 ± 0.04 | 0.0628 ± 0.05 |
Palmitic acid | C19H40O2 | (-) | 1.2187 ± 0.23 | 3.0752 ± 0.29 |
Hydrocinnamic acid | C9H10O2 | (-) | 0.0567 ± 0.04 | 0.1822 ± 0.14 |
Stearic acid | C21H44O2 | 5.4667 ± 0.36 | 3.9579 ± 0.29 | 5.0162 ± 0.37 |
Isophthalic acid | C8H6O4 | (-) | 2.5536 ± 0.30 | (-) |
Terephthalic acid | C8H6O4 | 0.7792 ± 0.36 | 1.2501 ± 0.27 | 0.8705 ± 0.35 |
Myristic acid | C14H28O2 | (-) | 1.5432 ± 0.29 | (-) |
ITACONIC ACID | C5H6O4 | (-) | 0.1482 ± 0.10 | 1.1209 ± 0.30 |
Benzimidate hydrochloride | C9H12NClO | (-) | 0.0942 ± 0.05 | (-) |
Sebacic Acid | C10H18O4 | (-) | 0.8764 ± 0.24 | (-) |
Lactic Acid | C9H22O3 | 7.1991 ± 0.42 | 3.2412 ± 0.26 | 2.4098 ± 0.26 |
Benzoic acid | C7H6O2 | 1.5823 ± 0.34 | 2.2651 ± 0.34 | 1.0092 ± 0.15 |
Acetic acid | C10H12O2 | 2.6790 ± 0.28 | 3.8057 ± 0.35 | 4.4490 ± 0.27 |
---|---|---|---|---|
Octanoic acid | C8H16O2 | 1.5820 ± 0.29 | 1.9920 ± 0.74 | 1.8836 ± 0.34 |
3,4-Dimethoxybenzoic acid | C9H10O4 | (-) | (-) | 0.6530 ± 0.31 |
Dehydroabietic acid | C20H28O2 | (-) | (-) | 1.0472 ± 0.09 |
Caffeic acid | C9H8O4 | (-) | (-) | 0.1054 ± 0.03 |
3-Hydroxybutyricacid | C4H8O3 | (-) | (-) | 0.0855 ± 0.07 |
Sucrose | C12H22O11 | (-) | (-) | 0.0355 ± 0.003 |
D-Trehalose | C12H22O11 | (-) | (-) | (-) |
Galactopyranose | C6H12O6 | (-) | (-) | 0.0238 ± 0.002 |
D-(+)-Mannose | C6H12O6 | (-) | 0.1233 ± 0.08 | 0.0945 ± 0.30 |
Phenethyl acetate | C10H12O2 | 5.4376 ± 0.68 | 5.3637 ± 0.32 | 5.3490 ± 0.29 |
Valtrate | C22H30O8 | (-) | (-) | 0.5789 ± 0.26 |
Glycerol monostearate | C21H42O4 | (-) | (-) | 0.8582 ± 0.25 |
Dodecanoic acid | C24H48O2 | (-) | (-) | 0.0952 ± 0.03 |
Propyl heptanoate | C10H20O2 | (-) | 0.0589 ± 0.04 | 0.1509 ± 0.03 |
Urea | CH4N2O | 0.0039 ± 0.001 | 0.0136 ± 0.002 | 0.0134 ± 0.003 |
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 (
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 (
Plant-root exudates-rhizosphere microbes form a cyclical interaction [
There are some differences in the number of tobacco root exudates under different treatments. The proportion of the amount is shown in
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).
The authors declare no conflicts of interest regarding the publication of this paper.
Li, C., Yu, J., Gan, L., Sun, J.G., Wang, C.J., Wang, Q., Chen, S.W. and Yang, Y. (2018) Effects of Tobacco Pathogens and Their Antagonistic Bacteria on Tobacco Root Exudates. Open Journal of Applied Sciences, 8, 518-531. https://doi.org/10.4236/ojapps.2018.811042