Study On The Relationship Between The Main Diseases And Quality Of Paris Polyphylla Smith Var.chinensis(Franch.) Hara

Paris polyphylla var.chinensis was also called Qiyeyizhihua which belongs to Paris of Liliaceae.In the Chinese folk,it was also called“Zaoxiu”,“Ziheche”,“Sancengcao”,etc.P.polyphylla var.chinensis was first collected in Shennongbencaojing and it was the original plant of every version of Chinese Pharmacopoeia,which was collected in Edition 2015 of Chinese Pharmacopeia（Volume One）.It has the functions of clearing away heat,detoxifying,reducing swelling,relieving pain,cooling the liver and calming the nerves,etc.The P.polyphylla var.chinensis was used for curing scabies,sore throat,poisonous snake bites,bruises,and cold wind convulsion,etc.More than ten years ago,most of the P.polyphylla var.chinensis resources were wild,however,due to the long growth period,generally 6～8 years,and the large market demand,both of which has stimulated farmers to over-exploitation to some extent,resulting in increasingly scarce wild resources,and in short supply.At the same time,as the market price of P.polyphylla var.chinensis has soared in recent years,the price of P.polyphylla var.chinensis has reached as high as 950～1250 yuan/kg,which has made the P.polyphylla var.chinensis become rare,endangered and precious medicinal material.（1）In the past ten years,in order to alleviate the shortage of wild P.polyphylla var.chinensis resources,the production mode of artificial large-scale breeding and planting of P.polyphylla var.chinensis was constantly emerging.The growth state of P.polyphylla var.chinensis has changed from a symbiotic state of multiple plant species to a single plant population state.Almost all kinds of pathogens were rare or light in the original ecology,after large-scale planting,many P.polyphylla var.chinensis planting bases encountered irreversible serious diseases almost every year,which caused a large area of P.polyphylla var.chinensis roots,flowers and fruits rot,resulting in no harvest of fruits and seeds and no yield of rhizome in that year,causing huge direct or indirect losses,which seriously affected the competitiveness of P.polyphylla var.chinensis by artificial planting in the market.Therefore,safe and effective disease control technology was of great significance to ensure the quality of PARIDIS RHIZOMA and promote the sustainable and healthy development of P.polyphylla var.chinensis industry.At present,most of the research reports on the control of diseases and insect pests in P.polyphylla var.chinensis were concentrated in China,the number was very limited,and there were few reports abroad.This project carried out field investigations and sample collections on disease of P.polyphylla var.chinensis grown in artificial breeding bases in Baishi Village,Shuimo Town,Wenchuan County,Southwest Minzu University.It was found that the main diseases of P.polyphylla var.chinensis were leaf spot and soft rot and these two diseases have greatly harmed the growth,yield and quality of the P.polyphylla var.chinensis in the base.Therefore,this project intends to take these two diseases of P.polyphylla var.chinensis as the core,to isolate and identify the two pathogens,and screen the corresponding pesticides in the field.At the same time,The differences in microstructure,thin layer chromatography,and the contents of Moisture,ash,Alcohol soluble extracts,total saponins,Polyphyllin I,Polyphyllin II,Polyphyllin V,Polyphyllin VI,Polyphyllin VII,Polyphyllin H,Gracillin between diseased and healthy P.polyphylla var.chinensis plants were compared.In addition,a comparative study on the acute toxicity,hemostatic time,and clotting time of diseased and healthy plants was conducted.It was looked forward to find a biological control for leaf spot and soft rot of P.polyphylla var.chinensis,and discuss the relationship between the disease and its quality.The main results were as follows:Based on the field investigation,the leaf spot samples of P.polyphylla var.chinensis were collected and the pathogen causing the leaf spot of P.polyphylla var.chinensis was identified as Alternaria tenuis Nees（sp.）by“tissue isolation method”and its mycelium was yellowish brown to tan.The morphology of Alternaria tenuis Nees（sp.）spores was observed and it was found that one end of the spore had a bud tube,both the spore and mycelium had septum,and the number of spores was more.The pathogenicity of Alternaria tenuis Nees（sp.）was determined by Koch’s Rule.After 8 days of inoculation,it was found that there were water spots on the edge of the P.polyphylla var.chinensis leaves,the spots was tan,and then the spots gradually expanded,and the spots were connected into flakes.At the same time,there came spots on the back of the leaves,which was similar to the phenomena of leaf spot in the field.The soft rot samples of P.polyphylla var.chinensis were collected and the pathogen causing the soft rot was identified as Botrytis cinerea Pers.by“tissue isolation method”and its aerial hyphae were fluffy,its color was beige to light brown,and the appearance of its conidia was grape spike,and they were monospora,ellipse and they were colorless or light colored.The pathogenicity of Botrytis cinerea Pers.was determined by Koch’s Rule.After 7 days of inoculation,it was found that the disease spread and the rot symptom appeared,which was consistent with the field soft rot symptoms.（2）Six kinds of common biological insecticides were selected and their virulence against the leaf spot of P.polyphylla var.chinensis was determined by mycelial growth rate method,the results showed that 10%polyoxin WP,68%Ningnanmycin AS,80%ethylicin Ec have good antibacterial effect and its EC₅₀values were 0.283 mg/L,1.938 mg/L and 2.804 mg/L,respectively.The field control experiment was carried out with these three reagents and the results showed that the control effect of 10%polyoxin WP on leaf spot of P.polyphylla var.chinensis was the best,the control effect was 76.98%.Combined with the indoor toxicity test and the field control effect,we can concluded that 10%polyoxin WP could be used as the best agent for controlling leaf spot of P.polyphylla var.chinensis widely.Ten kinds of common biological insecticides were selected and their virulence against the soft rot of P.polyphylla var.chinensis was determined by mycelial growth rate method and the results showed that 10%polyoxin WP,68%Ningnanmycin AS,chitosan AS,2%Kasugamycin AS,3%medium bioactinomycin WP have good antibacterial effect and its EC₅₀values were 1.738 mg/L,3.419 mg/L,25.126 mg/L,25.405 mg/L,29.436 mg/L.Field trials showed that the control effect of 10%polyoxin WP on soft rot of P.polyphylla var.chinensis.was the best,the control effect was 66.46%.we can concluded that 10%polyoxin WP could be used as the best agent for controlling soft rot of P.polyphylla var.chinensis widely.In addition,the meteorological factors such as the maximum temperature,the minimum temperature,the maximum humidity,the minimum humidity,the weather and the rainfall during the field experiment were measured.Through this data,it could be concluded that the overall rainfall was large,the humidity was high and the climate was humid during the disease epidemic.（3）According to the provisions of identification and inspection items in the quality standard of P.polyphylla var.chinensis in Chinese Pharmacopoeia（2015Edition）,the differences in microstructure,thin layer chromatography,and the contents of Moisture,ash,Alcohol soluble extracts between disease and healthy P.polyphylla var.chinensis were compared.The result showed that:1)There was no significant difference between the diseased and healthy powder of P.polyphylla var.chinensis and both of them contained acicular crystal,clusters of calcium oxalate,scalariform vessel,reticulate vessel and starch grain,which was consistent with the identification results of P.polyphylla var.chinensis powder stipulated in Chinese Pharmacopoeia（2015 Edition,Volume I）.2)The results of TLC identification showed that the root,stem,leaf of the diseased and healthy P.polyphylla var.chinensis all have the same fluorescent spots on the corresponding positions,indicating that both diseased and healthy P.polyphylla var.chinensis contained Polyphyllin VII.3)The content of moisture in diseased P.polyphylla var.chinensis was between 6.2%～9.05%,the content of total ash was between 3.35%～6.45%,the content of acid insoluble ash was between 0.28%～1.25%,the content of physiological ash was between2.21%～5.66%,and the content of alcohol soluble extract was between17.36%～34.88%;The content of moisture in healthy P.polyphylla var.chinensis was between 6.83%～8.51%and the content of total ash was between 3.39%～6.59%,the content of acid insoluble ash was between 0.22%～1.21%,the content of physiological ash was 2.48%～6.25%,the content of alcohol soluble extract was between14.57%～26.95%.The results of variance analysis showed that there was no significant difference in the contents of moisture,ash and alcohol soluble extract between the diseased and healthy root,stem and leaf of P.polyphylla var.chinensis which indicated that the disease had little or no effect on the contents of moisture,ash and alcohol soluble extract of P.polyphylla var.chinensis,While there were significant differences in the contents of total ash and acid insoluble ash between the diseased and healthy leaf（n=9,P<0.01）)and the content of total ash in healthy P.polyphylla var.chinensis was less than that of diseased P.polyphylla var.chinensis while the content of acid insoluble ash was the opposite.4)The content of total saponins in roots,stems and leaves of healthy and diseased P.polyphylla var.chinensis were determined by UV spectrophotometry and the results showed that the contents of total saponins in diseased P.polyphylla var.chinensis was between0.14%～1.68%and that in healthy P.polyphylla var.chinensis was between0.35%～4.89%.The results of variance analysis showed that there were significant differences in the contents of total saponins between the roots,stems and leaves of the healthy and diseased P.polyphylla var.chinensis（P<0.01）which indicated that the content of total saponins in P.polyphylla var.chinensis decreased significantly after the disease happened.5)The IR fingerprint was used to compare the differences of absorption peaks in roots,stems and leaves of healthy and diseased P.polyphylla var.chinensis and the results showed that the component of steroidal saponins which was the main active component in P.polyphylla var.chinensis had an obvious characteristic absorption peak in the healthy P.polyphylla var.chinensis root（S10）while it was absent in the diseased P.polyphylla var.chinensis root（S1）,which indicated that the contents of steroidal saponins in diseased plant were affected;Absorption peaks of S1(near 2 942.64 cm^-1)and S7(near2939.99 cm^-1)were C-H antisymmetric telescopic vibration of polysaccharides and lipids,while healthy plants did not have absorption peak at corresponding position,and it illustrated that the diseases induced changes in other chemical constituents of P.polyphylla var.chinensis.At the same time,the spectral characteristics of different plant parts（roots,stems and leaves）of the healthy or diseased P.polyphylla var.chinensis also have great differences,which suggested that the chemical composition and content of different plant parts（roots,stems and leaves）from the same origin and the same plant were different.6)A method of UPLC-ELSD was established to determine the contents of Polyphyllin I,Polyphyllin II,Polyphyllin V,Polyphyllin VI,Polyphyllin VII,Polyphyllin H,Gracillin in roots,stems and leaves of healthy and diseased plants,and their contents in different plant parts（roots,stems and leaves）of the diseased and healthy plants were evaluated quantitatively.The ACQUITY UPLC?BEH C18column（2.1 mm×50 mm,1.7μm）was adopted,the ELSD detector drift tube temperature was 55°C and the Nitrogen pressure was 40 psi,gain 500;The mobile phase was acetonitrile and water in gradient elution mode.The column temperature was 30°C with a flow rate of 0.2 m L·min^-1（0-3 min,10%→51%C;3-20 min,51%→80%C）and the injection volume of 3μL.The content of Polyphyllin I in the diseased P.polyphylla var.chinensis was between 0.023%～0.283%while in that of healthy P.polyphylla var.chinensis was between 0.045%～0.248%.The content of Polyphyllin II in the healthy and diseased P.polyphylla var.chinensis was very low,which was not detected in most of the samples;the content of Polyphyllin V in healthy and diseased P.polyphylla var.chinensis was close,which was between0.021%～0.186%;the content of Polyphyllin VI in healthy and diseased P.polyphylla var.chinensis was between 0.011%～0.687%,which was close;the contents of Polyphyllin VII and Polyphyllin H in P.polyphylla var.chinensis were close and the contents were between 0.118%～0.687%and between 0.009%～0.373%respectively;the content of Gracillin was between 0.052%～0.195%and the total content of seven kinds of Paris Polyphylla saponin ranges from 0.272%to 1.542%.The results of variance analysis showed that there was significant difference in the content of Polyphyllin H in roots and stems of healthy and diseased P.polyphylla var.chinensis（P<0.05）and their content trend of roots and stems was opposite.The contents of the other six saponins and their total saponins in the same parts of healthy and diseased P.polyphylla var.chinensis were not significantly different,indicating that the occurrence of disease had little effect on the content of these components.The contents of Moisture（X1）,total ash（X2）,acid insoluble ash（X3）,physiological ash（X4）,Alcohol soluble extracts（X5）,total saponins（X6）,Polyphyllin I（X7）,Polyphyllin II（X8）,Polyphyllin V（X9）,Polyphyllin VI（X10）,Polyphyllin VII（X11）,Polyphyllin H（X12）,Gracillin（X13）and the total content of seven Paris Polyphylla saponin（X14）was used as 14 evaluation indexes and the Spss19.0 was used for principal component analysis and cluster analysis.The difference between diseased and healthy P.polyphylla var.chinensis was evaluated by calculating the comprehensive score ranking.According to the results of comprehensive scores,the healthy leaves（S16,S17）,healthy stems（S13）,healthy roots（S10,S11）,diseased stems（S4,S5,S6）,diseased leaves（S7）ranked in the top 9,and the rest of the samples ranked behind,which showed that there was no regular difference between diseased and healthy P.polyphylla var.chinensis.The quality of the samples was also evaluated by the total content of Polyphyllin I,Polyphyllin II,Polyphyllin VI and Polyphyllin VII by the provisions of Chinese Pharmacopoeia.The ranking results of the two evaluation methods showed that S16,S10 and S11 were all ranked in the top 3,and no uniform rule was found in the ranking of the rest samples.The results of the system cluster analysis were consistent with the comprehensive score results of the principal component analysis,and the samples with similar ranking were clustered into one group.（4）The influence of the disease on the coagulation and hemolysis of P.polyphylla var.chinensis was evaluated by measuring the hemostasis and hemostasis of the healthy and diseased P.polyphylla var.chinensis.The rhizome powder of diseased P.polyphylla var.chinensis was diluted by double distilled water and the high,middle and low dose group,Yunnan Baiyao group and physiological saline group were set up and the acute toxicity,hemostasis time,APTT and PT of the diseased rhizome in mice were measured.The results of acute toxicity showed that:compared with the control group,there was no obvious abnormality in the mice of the medicated group,suggesting that the powder of the diseased P.polyphylla var.chinensis did not cause damage to the heart,liver,spleen,lung,kidney and stomach of the mice;the high,medium and low dose groups of the diseased P.polyphylla var.chinensis powder could shorten the bleeding time of the mice,and significantly reduced the PT and APTT of the mice.（preliminary experiment）.The rhizome powder of the diseased and healthy P.polyphylla var.chinensis was diluted by double distilled water and the high,middle and low dose group,Yunnan Baiyao group and physiological saline group were set up.The hemostasis time,APTT,PT,TT and FIB of the diseased and healthy P.polyphylla var.chinensis rhizome in rat were measured.The results showed that the bleeding time of rats in the high,medium and low dose groups of the diseased P.polyphylla var.chinensis could be shortened（P<0.05）,and the hemostasis effect in the high dose group of diseased P.polyphylla var.chinensis was the best;The bleeding time of rats in the high,middle and low dose groups of healthy P.polyphylla var.chinensis could also be shortened significantly（P<0.01）,and the hemostasis effect of the high dose group was the best;the hemostasis effect of the high dose group of healthy P.polyphylla var.chinensis was better than that of the diseased P.polyphylla var.chinensis.On the whole,the hemostasis effect of the healthy P.polyphylla var.chinensis was better than that of the diseased;Yunnan Baiyao control group and high,medium and low dose groups of diseased and healthy P.polyphylla var.chinensis could significantly shorten the PT,APTT and TT and raised the FIB of the test rats;both healthy and diseased P.polyphylla var.chinensis have strong coagulation and hemostasis effects and the effect of healthy P.polyphylla var.chinensis was better than that of the diseased one.The main diseases of P.polyphylla var.chinensis were leaf spot and soft rot.The occurrence of the diseases has changed the growth state of P.polyphylla var.chinensis,and the long-term disease stress has greatly reduced the yield of P.polyphylla var.chinensis.The disease significantly reduces the types of chemical components and content of steroidal saponins,the main effective component of P.polyphylla var.chinensis,which directly affects the quality of the herbal medicine,and its effects of coagulation and hemostasis were significantly decrease.So,it was suggested that timely and effective use of biodiversity and a small amount of low-grade pesticides were to control the disease of P.polyphylla var.chinensis,which may cause loss to the minimum.It was also the key measure to ensure the quality of P.polyphylla var.chinensis,from the source in order to alleviating the current situation of the shortage of medicinal materials resources of P.polyphylla var.chinensis.