The Systemicity In Tobacco Plants And Fungicidal Activities Of Phenazine-1-carboxylic Acid-valine Conjugate

Tobacco is one of the most important economic crops in China and all over the world.Tobacco in growing stage often subjects to different pathogen's infection.soil-born diseases are the most serious among them,such as tobacco black shank,tobacco sore shin and other rhizome diseases.It has become a major problem in China's tobacco production and has caused huge economic losses,but its comprehensive control strategy has been a nodus and hot spots in academic research.With the increasing demand of pesticides with green,high efficiency and low toxicity,the demand for biological pesticides has shown a trend of dramatic increase.As a new microbial source fungicide,Phenazine-1-carboxylic acid?PCA?is mainly used to prevent and control various plant pathogens,such as Rhizoctonia solani,Fusarium oxysporum and Phytophthora capsici.It has the characteristics of high efficiency,low toxicity and good good compatibility with the environment,but it does not have phloem mobility,so it has a few limitations in controlling soil-born diseases.In order to improve the phloem mobility,our research group has conjugated it with amino acids to obtain a series of PCA-amino acid conjugates.Because of the highest phloem mobility and moderate fungicidal activities among these PCA-amino acid conjugates,PCA-L-valine conjugate?PCA-L-Val?was selected for further study of its fungicidal activities against tobacco soil-born diseases,its systemicity and distribution in tobacco plants,and the differences between PCA-D-valine conjugate?PCA-D-Val?.In this paper,the fungicidal activities of PCA-L-Val and PCA-D-Val against R.solani and Phytophthora parasitica were studied in vitro and in greenhouse.The EC₅₀values of PCA against P.parasitica and R.solani are 79.142 and 52.333?M,respectively,while the EC₅₀values of PCA-L-Val and PCA-D-Val against P.parasitica are 383.004 and 369.995?M,and the EC₅₀values of them against R.solani are 139.274and 96.937?M,respectively.According to the results of in vitro,the control efficiency of test compounds against tobacco sore shin were tested by smearing leaves,spraying and irrigating roots.The results showed that the control efficiency of PCA-L-Val against tobacco sore shin was higher than that of PCA-D-Val and PCA by smearing leaves and spraying at the concentration of 100,200,400?M.However the control efficiency of PCA aganist tobacco sore shin was higher than PCA-L-Val and PCA-D-Val by irrigating roots.At the concentration of 400?M,PCA-L-Val and PCA-D-Val showed about 80%and 65%control efficiency against tobacco sore shin by the three treatments,respectively.PCA displayed about 50%control efficiency against tobacco sore shin by smearing leaves and spraying,but the control efficiency of PCA is above 80%by irrigating roots.Based on the results of fungicidal activities,it is speculated that the compounds can be transported in the phloem to achieve the purpose of preventing and controlling diseases.Therefore,the systemicity and distribution of the compounds in plants are further to studied.In this paper,methanol was used as extractant,liquid-liquid acid-base extraction and C₁₈solid phase extraction were used to purify.The content of compounds in tobacco samples was detected by liquid chromatography-mass spectrometry.The results showed that the concentration of the test compounds in the tobacco root,stem and leaf samples had a linear relationship with the peak area.Within the concentration range,the recovery of the addition in various parts of tobacco is in a range of 81.81%?94.60%.The standard deviation is between 1.53 and 8.30,and the coefficient of variation is in the range of 1.23 to 8.23,which meets the national pesticide residue analysis and detection standards.The method detection limit and method determination limit of PCA are 0.0005 and 0.0002?M,and the method detection limit and method determination limit of PCA-L-Val and PCA-D-Val are 0.001 and 0.002?M,respectively.The leaf dish method was used to detect the content of test compounds in the tobacco leaves.The results showed that the absorption of the compounds in the leaves was time-and concentration-dependent,The results showed that the amount of compound absorption in the leaves was time and concentration dependent.When the concentration is higher than 0.5?M,the growth rate of the compound content in the leaf is fast,and when the concentration is lower than 0.5?M,the growth rate of the compound content in the leaf is slower.With the increase of time,the content of medicament in the leaves has increased to a certain extent.Meanwhile the content of PCA-L-Val in leaves was higher than PCA-D-Val and PCA.Then the content of test compounds in each part of the plant was detected by smearing different concentrations of test compounds on middle leaves of the tobacco plant.PCA-L-Val and PCA-D-Val can be detected in the roots,stems,and leaves of tobacco plants at concentrations of 200and 400?M.The content of PCA-L-Val the parts of plants were significantly higher than PCA-D-Val and PCA.At the concentration of 400?M,the content of PCA-L-Val and PCA-D-Val in lower leaves were 3.22 and 0.24?mol/kg,respectively.PCA was not detected in the lower stem,lower leaves and roots of tobacco plants.The content of test compounds in each part of the plant peaked after 12 and 18 h of treatment,and then began to decline,indicating that the absorption value of the compounds reached its peak between 12 and 18 h.After immersing the roots of tobacco plants in the solution containing a 100?M concentration of the test compounds for 6 h,the content of the three compounds can be detected in the root,stem,leaf and apical growth points,and the content of them in the leaves is the highest.The contents of PCA,PCA-L-Val and PCA-D-Val in the leaves were 2.89,4.03,and 13.59?mol/kg.Meanwhile the content of PCA-L-Val in all parts of tobacco plant was significantly higher than that of PCA-D-Val and higher than that of PCA.In addition,the parent compound PCA was released when PCA-L-Val and PCA-D-Val were transported in the phloem.of treatment,After PCA-L-Val treatment,the content of PCA in the lower leaves was the highest,the content of the parent compound PCA in the lower leaves was as high as 11.57?mol/kg after 6 hours,even higher than that of the smeared middle leaves,followed by the upper leaves and the lowest content was found in stem and root.While after PCA-D-Val treatment,When the concentration was 400?M,the content of PCA-D-Val in the lower leaves was 3.16?mol/kg after 18 hours.And the content of PCA-D-Val in each part of tobacco plant was lower than that of PCA-L-Val.The above results show that the conjugates of valine and PCA not only improve its systematic property and phloem mobility,but also have ambimobility activity.In addition,it is decomposed to release the parent molecule during transportation,which can recover PCA's fungicidal activities.And the fungicidal activity of PCA-L-Val against tobacco sore shin by smearing leaves and spraying was similar to that of PCA by irrigation treatment,which indicated that soil-borne diseases could be controlled by spraying stems and leaves by improving the phloem mobility.At the same time,the fungicidal activity and systemic mobility of valine conjugates in L configuration were significantly higher than those in D configuration.These results provide an important basis for the further research and practical application of PCA-L-Val to control plant diseases.