Pathogen Identification,Toxin Analysis Of The Pathogen And Control Test Of Ring Spot Of Kadsura Coccinea

Kadsura coccinea is used as traditional Chinese medicinal in the treatment of disease in China.It has the functions of detumescence,detoxification,anticancer,and treatment of bruises and injuries.In recent years,with the deepening of the research on K.coccinea,new value of K.coccinea has been continuously discovered,and the market demand for K.coccinea has been expanding.In order to meet the market demand,large-scale cultivation of K.coccinea has been carried out in Guangxi,Hunan and other places.With the large-scale cultivation of K.coccinea,the disease has also increased year by year.After investigated the disease of K.coccinea grown in Guangxi,It was found that a ring spot on K.coccinea was a serious new disease.In this study,diseased leaves with ring spot from the K.coccinea plantation were collected,pathogen isolation,pathogenicity test and pathogen identification were carried out,toxins of the pathogen were analyzed,endophytic fungi of K.coccinea which antagonize the pathogen of ring spot on K.coccinea were isolated and molecular structure of antagonists were clarified,control experiment of ring spot on K.coccinea was carried out.The main results of this work were as follows:1.Pathogen identification of ring spot on K.coccineaOn the basis of the lesions of ring spot on K.coccinea were sectioned and observed under microscope,it was found that there were a large number of conidiomata of Neopestalotiopsis sp.and preliminarily judged that Neopestalotiopsis sp.probably was the pathogen of the disease.After pathogen isolation,three kinds of dominant fungi were obtained:Neopestalotiopsis sp.,Colletotrichum sp.and Phyllosticta sp.Pathogenicity of representative strains was tested and re-isolation was carried out to complete Koch’s postulates.Finally,it was determined that HP2 strain was pathogenic fungus.Based on morphological and molecular characters of HP2 strain,the pathogen was identified as Neopestalotiopsis clavispora.2.Isolation and component analysis of toxins produced by HP2 strainHP2 strain was cultured on PDA medium（30L）for 21 days.A total of11.6875 g crude extract was obtained by organic solvent soaking extraction,ethyl acetate extraction and evaporated to dryness by rotary evaporator.Three toxins were isolated by means of activity tracing,normal silica gel column,Sephadex gel column（Sephadex）and thin layer chromatography（TLC）,which components were determined as afritoxinone B,afritoxinone A and oxysporone,among which content of oxysporone was the most.The activities of the three toxins on the host（K.coccinea）and non-host（Ipomoea batatas,Mangifera indica and Bauhinia purpurea）were measured by acupuncture.It was found that oxysporone was the most poisonous to the leaves of K.coccinea,because each concentration of oxysporone caused necrotic lesion on the leaves of K.coccinea and area of necrotic lesion was 21.54-84.33 mm²;afritoxinone B was weaker poisonous to the leaves of K.coccinea because area of necrotic lesion was 4.52-77.62 mm²;and afritoxinone A was the weakest poisonous to the leaves of K.coccinea,because not only part concentration caused necrotic lesion on the leaves of K.coccinea,also area of necrotic lesion was merely 2.13-36.92 mm²after nine days of treatment.These three toxins also caused necrotic lesion on the leaves of three non-host plants.The results showed that oxysporone was the main pathogenic factor of N.clavispora（strain HP2）and all of three toxins were Non-Host-specific toxins（NHST）.3.Screening of endophytic fungi from K.coccinea against Neopestalotiopsis clavispora（HP2 strain）and identification of the antagonistsHealthy leaves,stems and roots of K.coccinea were collected,698 pieces of healthy tissue were isolated and 220 strains of endophytic fungi were obtained,among which 15 strains with strongly antagonistic effect on HP2 strain were screened by the method of plate confrontation.KCR23 strain,an endophytic fungus,was selected for identification,isolation and activity determination of the active compounds based on extraction of antagonists and re-screening by the method of inhibition zone.Based on morphological character and ITS sequence,KCR23 strain was identified as Fusarium sp.A total of 34.8289 g crude extract was obtained by organic solvent soaking extraction,ethyl acetate extraction and evaporated to dryness by rotary evaporator.And three antagonists which were identified as cyclosporin A,cyclosporin B and cyclosporin C were isolated by means of activity tracing,normal silica gel column,Sephadex gel column（Sephadex）and Thin layer chromatography（TLC）after KCR 23 strain was cultured on PDA medium（30L）for 21 days.The results showed that cyclosporin A had the best antagonistic effect,which EC₅₀value was 0.0323μg/m L,followed by cyclosporin B,which EC₅₀value was 0.1767μg/m L,the activity of cyclosporin C was the weakest,which EC₅₀value was 0.3026μg/m L based on antagonistic effect of these three compounds against HP2 strain determined by inhibiting mycelium growth rate method.Excavation of endophytic fungi and their antagonists provides a new material basis for prevention and control of the ring spot on K.coccinea.3.Control experiment of fungicides against ring spot of K.coccineaSix fungicides,which were 80%mancozeb（WP）,70%thiophanate methyl（WP）,25%azoxystrobin（SC）,45%prochloraz（EW）,40%difenoconazole（SC）and 25%propiconazole（EC）,were selected against ring spot of K.coccinea.Inhibiting effect of six fungicides against HP2 strain was tested by means of inhibiting mycelium growth rate method and result showed that prochloraz had the best inhibiting effect,which EC₅₀was 0.0251μg/m L;azoxystrobin was moderate,which EC₅₀was 0.6224μg/m L and mancozeb was the weakest,which EC₅₀was 7.2665μg/m L.Inhibiting effect of the mixtures of azoxystrobin mixed with propiconazole and prochloraz mixed with difenoconazole in different ratio was tested by means of inhibiting mycelium growth rate method and result showed that all mixtures of azoxystrobin and propiconazole showed synergism and the best proportion was one azoxystrobin:nine propiconazole,and only mixture of one prochloraz and nine difenoconazole showed synergism,other mixture of prochloraz and difenoconazole showed negative synergism.Three fungicides with better indoor toxicity were applied to control the disease in the field and result showed that azoxystrobin（1000×）had the best control efficiency（87.67%）,propiconazole（1000×）had moderate control efficiency（54.46%）and prochloraz（1000×）had the lowest control efficiency（16.96%）at 30th day after spraying fungicides.In summary,N.clavispora was the pathogen of ring spot on K.coccinea,After infecting the leaves of K.coccinea,the pathogen mainly produced three kinds of toxins:afritoxinone B,afritoxinone A and oxysporone.The oxysporone was the main pathogenic factor of N.clavispora.And all of three toxins were NHST.KCR23 strain,an endophytic fungus of K.coccinea,with strongly antagonistic effect on HP2 strain.Cyclosporin A produced by KCR23 strain which had the best antagonistic effect,the EC₅₀value was 0.0323μg/m L.Among the six fungicides,prochloraz had the best indoor toxicity,but the field control effect was the lowest control efficiency,while the indoor toxicity of azoxystrobin is general,but the field control effect is the best.