In Situ Analysis And Function Of Allelochemicals/chemical Signals In Pepper Root System

The root exudates of plant were the main source of plant allelopathy,which was of great significance to maintain the stability of soil ecosystem.It was a challenging task to obtain the root exudates of plants efficiently and accurately and complete the quantitative analysis.In this paper,a new undisturbed and in situ sampling device with capillary effect was designed,which was combined with chromatographic analysis methods（GC-MS、HPLC）to obtain pepper root exudates in soil under undisturbed and in situ state.The root exudates of pepper were analyzed qualitatively and quantitatively,so as to study allelopathy.The new in situ sampling and analysis method was used to further study the chemical signal transmission produced by the interaction between pepper root exudates and Phytophthora capsici.The new in situ sampling device was characterized.X-ray diffraction results showed that the chemical composition of the undisturbed in situ device was no different from those of soil,and the composistion was oxide crystal of magnesium,aluminum and silicon.The particle surface of the in situ sampling device was an irregular convex-concave structures and the pipe wall of the in situ sampling device could form capillary structures.Compared with farmland soil,the bulk density and density of in situ sampling device were similar to those of farmland soil.The capacity of capillary water holdup,capillary porosity and permeability of in situ sampling device were significantly higher than that of soil.The ability of collecting pepper root exudates of undisturbed and in situ sampling device was better than those of the traditional methods.Therefore,the new undisturbed in situ sampling device could simulate the soil environment and be used for in situ collection of root exudates.The plant root exudates were collected and analyzed by the combination of undisturbed and in situ sampling device and various analysis methods.The collected pepper root exudates contained 28 chemicals.Among them,14 compounds have high similarity with known compounds.2,4-di-tert-butylphenol（2,4-DTBP）,Diisobutyl adipate（DIBA）,Dibutyl phthalate（DBP）and Limonene were the main components of root exudates.When 2,4-DTBP was artificially added to the soil at a concentration of 22.4 mg·kg^-1,the concentration of 2,4-DTBP detected by in situ sampling analysis method was 23.6 mg·kg^-1,and the recovery rate reached 105.35%.The maximum adsorption capacity of in situ sampling device for 2,4-DTBP was 199.6 mg·kg^-1.This proved that the in situ sampling device has the ability to collect allelochemicals.Combined with GC-MS and HPLC,the in situ sampling device could accurately deduce the true concentration of some pepper root exudates in soil.The allelopathic activities of the above allelochemicals that identified by using the undisturbed and in situ sampling device combined with a variety of analytical methods were further determined by bioassay of sensitive plants.The results showed that the low concentration of 2,4-DTBP(less than 50 mg·L^-1)could improve the germination rate of lettuce seeds,and the high concentration of 2,4-DTBP(more than 300 mg·L^-1)had obvious inhibitory effect on lettuce seeds and inhibited their germination.In the orthogonal experiment,A₃B₄C₁D₄(2,4-DTBP:40 mg·L^-1,DIBA:75 mg·L^-1,DBP:0 mg·L^-1,Limonene:20 mg·L^-1)had the strongest allelopathic inhibition.This study found that pepper has obvious autotoxic effect of continuous cropping.The analysis of root exudates of continuous cropping pepper showed that the content of 2,4-DTBP increased with the increase of continuous cropping years,which is23.6 mg·kg^-1 for one year（Y1）,63.5 mg·kg^-1 for two years（Y2）and 153.2 mg·kg^-1 for three years（Y3）,respectively.The autotoxicity of pepper continuous cropping could significantly reduce the photosynthesis and the root activity of pepper,increased the content of MDA and the ROS level.Which seriously affected the normal growth of pepper plants.Pepper continuous cropping soil had obvious allelopathy inhibition on cucumber.The increase of 2,4-DTBP in soil significantly decreased root activity of cucumber,slowed down the growth rate of cucumber,significantly reduced the leaf area of cucumber and inhibited the photosynthesis of cucumber.Therefore,it was confirmed that the 2,4-DTBP was the main allelochemicals in the root exudates of pepper and the main factor of continuous cropping obstacle of pepper.Meanwhile,the root exudates of P.capsici resistant 0504 and P.capsici susceptible 9813were collected and analyzed by using the above-mentioned in situ collection analysis method to further explore the chemical signal relationship between the interaction between pepper root and P.capsici.The results showed that there were significant differences in defense mechanisms between the resistant variety 0504 and the susceptible variety 9813.Compared with uninoculated,the contents of total phenols and OD phenols in and outside of roots of the two varieties increased significantly.The contents of total phenols and OD phenols in the roots of the resistant variety 0504 were higher than those of the susceptible variety 9813.On the contrary,the contents of total phenols and OD phenols in the root exudates of the susceptible variety 9813 were higher than those of the resistant variety 0504,especially in the later stage of inoculation.The activities ofβ-1,3-glucanase,peroxidase and phenylalanine ammonia lyase of the resistant variety 0504 were significantly higher than those of the susceptible variety 9813.But after inoculation,the change ofβ-1,4-glucanase activity of the two varieties showed that the enzyme activity of the susceptible variety 9813 was higher than that of the resistant variety0504.In summary,the resistant variety 0504 has a stronger resistance to P.capsici infection than the susceptible variety 9813.The resistance mechanism was different between the resistant variety 0504 and the susceptible variety 9813.After the comprehensive analysis of the changes of physiological indexes,morphological observation and multivariate statistical analysis results pepper plants after inoculation,it was determined that the sampling time nodes of signal substances secreted by pepper roots were 3^th,7^th and 10^th days after inoculation.The results showed that the main substances secreted by pepper roots after inoculation were organic acids,alcohols and phenylpropanoids.In the early and middle stage of inoculation,the species and content of organic acids in the susceptible variety 9813 were relatively high,which reduced its ability to resist P.capsici.Among the chemical substances secreted by the roots of the resistant variety 0504,5,7-dimethyldihydrocoumarin(1.43 mg·kg^-1)and 1-pentadecanol(2.66 mg·kg^-1)and the combination of the two components could inhibit the infection of P.capsici.The combination of 5,7-dimethyldihydrocoumarin,1-pentadecanol and the two components can reduce the damage of root cell membrane,reduce the damage of intracellular oxidants and improve the activity of resistant enzymes.Among them,the combination of the two components has the best effect.The 5,7-dimethyldihydrocoumarin and 1-pentadecanol may be the key signal substances to resist P.capsici infection.