Identification And Basic Research Of Raspberry New Diseases Caused By Pestalotiopsis And Alternaria

With the increase of raspberry planting in China,the occurrence of raspberry disease is becoming more and more serious.In 2016 to 2017,Diseases survey on raspberry were conducted in liaoning province.Four new diseases were found according to compare with previous studies.In this study,the pathogens of four new diseases was isolated,purified,pathogenicity tested and identified respectively.Furthermore,biological characteristics of the pathogens,fungicide screening and cultivar susceptibility to the pathogens were done.The results were as follows:1.Four new raspberry diseases were identified to be caused by Pestlotiopsis adusta,Neopestalotiopsis megna,Alternaria sp.,and A.tenussima.The results of isolation and purification of four new diseases showed that the isolates with the same colony phenotype were obtained for each disease sample.Among of them,4 isolates as the representative were selected and gave the code.Strain SNHS03 Kd mainly infected raspberry leaves,with irregular dark brown and about 0.3-1.2cm in size.The colour of its colony(PDA,25 ?,10 d)was white to orange,no obvious circle ripple.The conidina is 16.7-22.4 ?m ×4.7-6.6 ?m in size with 2-3 apical appendage(7.8 ?m-13.2 ?m in length).Phylogenetic analysis was performed using combination of ITS,-tubulin and tef-1 genes(the international general molecular identification method for species level of the genus Pestalotiopsis.According to the results of morphological and molecular identification,strain SNHS03 Kd was identified as Pestalotiopsis adusta.Strain SNHS12 B mainly infected raspberry leaves,a nearly oval shape,The colour of its colony(PDA,25 ?,10 d)was white,have circle ripple.The conidina size is 39.7~44.9×9.1~11.6 ?m in size with 2-3 apical appendage(7.8 ?m-13.2 ?m in length).The conidina is 16.7-22.4 ?m ×4.7-6.6 ?m in size with 2-3 apical appendage(7.8 ?m-13.2 ?m in length).Phylogenetic analysis was performed using combination of ITS,-tubulin and tef-1 genes(the international general molecular identification method for species level of the genus Pestalotiopsis.According to the results of morphological and molecular identification,strain SNHS12 B was identified as Neopestalotiopsis megna.train SNHS06 A mainly damages the leaves of raspberry,with the shape of disease spot nearly oval,brown and yellow.Its colonies(PDA,25 ?,7 d)white,the conidina size is 24 ?m~39 ?m×7.1 ?m~12.6 ?m.Through NCBI comparative analysis of ITS genes of the strain,it was found that ITS similarity with A.tenussima was up to 98%,and it was determined to be the species of microspore chain lattice spore.Histone-3 gene was used to differentiate A.tenussima related species,and the results showed that there was cross phenomenon between species.SNHS06 A strain was required further studies.Strain SNHS21 B mainly damages the new shoot of raspberry,with fusiform disease spot,reddish-brown edge,gray white center.,the conidia size is 13 ?m~45 ?m×6.4 ?m~10.8 ?m.Through NCBI comparative analysis of ITS genes of the strain,it was found that ITS similarity with a.tenussima was up to 100%,and it was determined to be the species of microspore chain lattice spore.Histone-3 gene was used for phylogenetic analysis of the strain and A.tenussima related species.According to the results of morphological and molecular identification,The strain SNHS21 B was identified as A.tenussima.2.Biological characteristics research of pathogensIn this study,the biological characteristics of 4 tested pathogens were determined under different trails(5 carbon sources,5 nitrogen sources,5 culture media,6 temperatures and 3 light conditions).The results showed that the optimum carbon source,optimum medium,optimum temperature and illumination conditions of the four representative strains were consistent.The optimum carbon source is maltose.The optimum medium was PDA.The optimal temperature is 25 ?;Not sensitive to light conditions.The optimal nitrogen source conditions of the four representative strains were different.The optimal nitrogen source of P.adusta and N.megna were glycine,however,SNHS06A(Alternaria sp.)was Na NO3 and SNHS21B(A.tenussima)was cysteine.3.Inhibition test to pathogens of six fungicidesIn this study,six fungicides(Thiophanate-methyl,Iminoctadine trialbesilate,Pyraclostrobin,Iprodione,Fludioxonil and Difenoconazole-azoxystrobin)were tested on target pathogens.The results showed that Fludioxonil and Difenoconazole-azoxystrobin had the well efficacy to 4 pathogens with EC50 less than 0.15 ?g/ml.Pyraclostrobin and Iprodione had good efficacy on Pestalotiopsis,but poor efficacy on Alternaria.Thiophanate-methyl and Iminoctadine trialbesilate can play the role to P.adusta,while less effect to other 3 strains.4.Resistance difference of 10 raspberry cultivars to pathogensIn this study,10 cultivars of raspberry(Qiuping,Qiufu,Jinqiu Dingkang,Dingfeng,H1,75,0,76,77)were evaluated for their disease resistance.4 cultivars against SNHS03Kd(P.adusta)were Dingkang,H1,Jinqiu and 0,as well as 4 cultivars susceptible to SNHS03Kd(P.adusta)were Dingfeng,Qiuping,76 and 77.3 cultivars against SNHS12B(N.megna)were 75,76 and jinqiu,as well as 2 cultivars susceptible to SNHS12B(N.megna)were H1 and dingfeng.2 cultivars against SNHS06A(Alternaria sp.)were dingkang and jinqiu,as well as 2 cultivars susceptible were 76 and 77.In summary,Jinqiu and Dingkang were resistance cultivars to tested pathogens.