Diagnosing Brown Leaf Blotch Of Spider Lily (Hymenocallis Littoralis) And Screening Effective Fungicides For The Disease Control

Spider lily(Hymenocallis littoralis(Jacq.) Salisb.) is a perennial bulbous plant belonging to Amaryllidaceae family and originated from tropical America.It is currently widely distributed in China and is planted for ornamental and medical purposes.A new disease,brown leaf blotch,was found on this pecious plant in Chongqing and a number of other Southern China provinces and there has not been any report or record of this disease in literatures.The objectives of this study were 1) to diagnose and describe the disease,2) to identify the pathogenic organisms,3) to investigate the major factors affecting the growth,sporulation and infectivity of the pathogens,and 4) to screen fungicides for controlling the disease in the laboratory and in the fields.These would be of great importance in further studies of the disease and its pathogens and would provide useful infrmation and experimental data for desgning disease management strategies in spider lily cultivation practice.1.The symptoms of the spider lily brown leaf blotch and the isolation of the pathogenThe typical symptoms of the disease were characterized the initial small circular purple spots on leaves,which developed to oval spots and may join together to form large irregular spots.The spots were brown at the center,deep purple at the border and surrounded by a yellow halo of newly-infected tissue.Two isolates(SLD1 and SLD2) of fungi were shown responsible for the disease infections through the Koch's Postulate steps.They induced blotch symptoms on leaves after separate and combined inoculations;the inoculated pathogens were reisolated from the diseased tissues.Pure cultures of SLD1 and SLD2 were obained via isolating from diseased leaves with tpical blotch symptoms.2.The morphological and molecular identifications of pathogenic strainsThese acervuli of SLD1 on under epidermis are scattered or clustered on leaves and they are glossy with brown setae.The conidiophores arearranged in the form of the palisade at the base of acervuli;they are short,colorless or olive brown,septate and smooth.Conidia are hyaline,oval to cylindrical,single-celled and 16.0-24.5×4.8-6.0μm in size.On potato dextrose agar plates(PDA), the fungal colony is round,white initially and turned to slightly reddish later because of the production of conidial spores or spore masses.The hyphae are septate,colorless or tranparent, measured 1.2-2.4μm in diameter.The rDNA ITS sequence of SLD1 was obtained via PCR amplication(GenBank accession NO GQ119342).BLAST analysis showed that SLD1 was a species of the Colletotrichum genus and shared 99%of 'maximum identity' with the species C.gloeosporioides.The acervuli of SLD2 are cup-shaped,disperse or aggregated,grew underleath the epidermis first and then protuberated outside.The base of the acervulus is dark-brown and formed from thick-sheathed,spindle-shaped cells.The conidiophores are priduced at the base portion of the acervulus;they are colorless or light brown,having septae and irregular branches.Conidia are fusiform or clavate-shaped,15.6-27.5×4.5-7.6μm,straight or slightly curly.Each conidium has four septae dividing it into five cells;the three cells at the middle are olive-brown and cylindrical while the other two cells at the ends are colorless,subuliform and smaller than the middle cells. There are two or three(mostly three) colorless appendages on the top end and one appendage at the bottom end of the conidium.The colony on PDA plate is nearly round,primarily white and changed to gray or black colored later because of the productions of pigments and pycnidia.The mycelia are hyaline or colorless,septate,and 1.5-2.5μm in diameter.Based on the morphology.The rDNA ITS sequence of SLD2 was also obtained through PCR amplication(GenBank accession NO GQ119343),BLAST analysis showed that SLD2 was a species of the Pestalotiopsis genus and shared 100%of 'maximum identity' with the species P.vismiae.3.The biological characteristics of SLD1 and SLD2For SLD1,the best medium was potato dextrose agar(PDA),the suitable temperature for its mycelium or colony growth was 25-30℃and the optimum acidity was pH8.Of the tested carbon and nitrogen resources,the fungus grew normally on media with glucose,maltose,sucrose,beef extract or peptone.The amount of sporulation of SLD1 on PDA medium and PSA agar medium was more than other media;the most suitable temperature for sporulation was at 25℃,pH ranged from 7 to 8;and light treatment has negative effect to sporulation.The conidia can germinate well in the ranged of pH 4-12,and pH10 is best pH condition for germination.One of the most optimum media for SLD2 growth was potato dextrose agar(PDA),the most suitable temperature for colony growth was 20-25℃and the best acidity was pH6.Of the tested carbon and nitrogen resources,SLD2 grew and sporulated normally on media contained glucose,maltose,soluble starch,beef extract or peptone.The fungus produced significantly higher aount of spores on PDA or PSA containing spider lily leaves extract liquid.The best temperature for sporulation was at 25℃and best pH value was pH 6,but different light treatments had no effect to sporulation.Conidia of SLD2 germinated normally at the range of pH4-12 but pH6.5 was most favorable tbr germination.4.Effects of fungicides on spider lily brown leaf blotch and screened chemicals for disease controlLaboratory bioassay results showed that the seven fungicides could inhibit the growth of the two strains except strephtomycin sulphateand Validamycin,but the concentration of inhibition and the effect were different.The inhibition effect of carbendazim and mildothane were the best among five fungicides of the SLD1,effectives concentrations(EC₅₀) of them for inhibiting the growth hyphae were 0.33917and 0.82777μg/mL,respectively.The inhibition effects of wuyiencin, chlorothalonil and mancozeb decreased gradually in the order given;and the EC₅₀ of them were 7.93466,46.8933 and 261.82750μg/mL.The inhibitive effects of these fungicides on the mycelial growth of SLD2 were lowered in the order from carbendazim,mildothane,wuyiencin, chlorothalonil to mancozeb;the effective concentrations(EC₅₀) of them were 0.16058,0.39071, 3.65280,29.09667 and 55.54383μg/mL The field experiment showed that the disease control effect of 50%carbendazim and 70%mildothane applied at 1:1000 were better than that of 1%wuyiencin at 1:1000.The field control effect of these three fungicides were 68%-76.6%,67.3%-76.2%and 50.0%-64.1%at 20 days after spray.The applications of three fungicides resulted in significantly lower control efficiency when they were sprayed at 1:2000 than at 1:1000.Therefore, chlorothalonil and mancozeb were suggested for applications in controllng spider lily brown leaf blotch disease in the field cultivation practices.