Isolation And Identification Of Pathogens Fusarium Fujikuroi From Daylily Leaf Blight And Resistance Evaluation Of Different Varieties

Hemerocallis is a perennial herb,widely used in landscaping and other fields.Hemerocallis is not only of high ornamental value,but also rich in nutrition.Hemerocallis is of high edible value.Hemerocallis plant extract has anti-depression and other medicinal effects.During the growth of hemerocallis,some biological stresses are often encountered,especially fungi,which are easy to cause various diseases,which seriously affect the growth and development of hemerocallis,and then affect its ornamental value and application value.In this study,plant samples with possible leaf blight were collected from the germplasm bank of hemerocallis in Shanghai,and a single strain of pathogen was obtained by tissue separation.Morphological and molecular biological methods were used to identify the pathogen.The control effect of five fungicides on the pathogen was evaluated,and resistance experiments were conducted on twenty-two different varieties of hemerocallis.The main results of this study are as follows:1.The pathogenic bacterium HK1 was isolated and purified by tissue separation from the leaves of hemerocallis hemerocallis with suspected leaf blight.The observation showed that the mycelia of HK1 was white in the early stage,and in the culture plate,the colony gradually spread from the center to the periphery in a circular shape,the colony center was white,and the outward diffusion was pale pink.The outermost layer of the colony was white,with an erect posture and dry surface,and the mycelia was compact and opaque.Its edges are irregular and free of exudate.The back culture plate is light orange,surrounded by yellow rings,and the color gradually diffuses from the center to the periphery,gradually becoming lighter.A small number of cultures were selected and observed under a microscope.The spores were sickle-shaped,slender and slightly curved,usually with 3 to 5 transverse compartments.The apex cells were curved and gradually tapered,and the spore size was about(3 to 12)μm×(1 to 3)μm.The mycelium was septate and slender.2.The pathogen HK1 was inoculated on healthy and disease-free leaves of Hemerocallis fulva,and the leaves were treated with scraping,clipping,stabbing and no-injury respectively.After 7 days of inoculation,all the leaves inoculated with HK1 showed the symptom of black spot,while the healthy leaves in the blank treatment group showed no symptom of black spot.The diseased leaves of the treatment group and the control group were isolated and purified,and the infected tissues were cultured to obtain the same strain as HK1 in colony and spore morphology,these results indicated that HK1 was the pathogen of Hemerocallis fulva leaf blight.3.The internal transcribed spacer(ITS)of HK1 was amplified by PCR with ITS1/ITS4 primers.The sequencing results showed that the ITS sequence of HK1 was 563 bp in length,the phylogenetic tree was constructed by Neighbor-Joining method of MEGA7.0 software.The results showed that the isolates were located in the same branch as Fusarium fujikuroi.Based on the sequence alignment analysis and the results of morphological,physiological and biochemical identification,the strain HK1 was identified as Fusarium fujikuroi.4.The bactericidal efficacy of common fungicides HK1 was determined by bacteriostatic method,including 20% moguanidine · Copper acetate,10% difenoconate ring file,80%allicin,50% Kejundan and 50% chlorobromoisocyanuric acid.The results showed that HK1 could not grow in the medium containing 2.5 mg/L,5 mg/L,10 mg/L,20 mg/L,50 mg/L difenoconate and allicin,and the inhibition rate was 100%.HK1 could be grown in the medium containing Kechundan,chlorobromoisocyanuric acid and mancoguanidine-copper acetate,but it had some inhibitory effects.The inhibitory effect of chlorobromoisocyanuric acid was the weakest,only 12.998%.By calculating the inhibition rates of three fungicides(Kejundan,chlorobromoisocyanuric acid,moguanidine-copper acetate)to Fusarium fujikuroi and fitting the data,the regression equations of the toxicity of three fungicides to HK1 were obtained,the correlation coefficients(R)of the three regression equations were 0.9548,0.9851 and 0.8321,respectively.The fitting degree of the regression equations of Kejundan and chlorobromoisocyanuric acid were good and reached significant level.The half-lethal concentration(EC50)of 50% chlorobromoisocyanuric acid to HK1 was 156.6150 mg/l.compared with the other two fungicides,50% Capectan and 20% cupric mancoganate showed moderate inhibition.5.The 22 hemerocallis varieties were inoculated with HK1 scratches in vivo and without HK1 scratches in vivo.The identification results showed that there were 6 varieties immune to HK1,which were ’Shenai’,’Shangying Chuangfei’,’Shangying Hongchen’,’Christmas baby’,’Zibei Bei’,’Hailanhong’,and 5 varieties with high resistance.They are ’Da Hong Pao’,’Orange spirit’,’Flamingo’,’Red Charm’ and ’Pink beauty’.There are 4 varieties of medium resistance,which are ’Shangying Window’,’Sunshine Boy’,’Orange Sea’ and ’Bow Tie’.There are 5 varieties of medium sensitivity,which are ’Golden treasure’,’Tranquility Morgan’,’Xianhu baby’,’Pink Palace’ and ’Xianhu Star’.There were two susceptible varieties,namely’Indian Sky’ and ’Water Dragon’,and none of the highly susceptible varieties.