Potential Of Phoma Herbarum As Mycoherbicide For Commelina Communis Control And Security For Environmental Organisms

The SYAU-06 strain of Phoma herbarum which was high pathogenic to dayflower (Commelina communis L)and safe to most of crops was obtained from diseased leaf in different area of liaoning province.The potential of SYAU-06 strain as mycoherbicide for dayflower was evaluated systematically at the two aspect s of living microorganism and fungi toxin.The research included host range,biological characteristics,infection process and infection influence factors of SYAU-06 strain as well as the herbicidal activity,extraction technology,action mechanism,biological safety,separation and purification technology and structure elucidation of phytoxin produced by the strain.The experiment provided a new method to biocontrol dayflower.The pathogenic fungi from diseased dayflower leaf were selected.Seven strains were thoroughly observed which were collected from the lesion spots of dayflower leaves in different areas of Liaoning province.These strains were identified by morphological characteristic as Phoma herbarum,Curvularia lunata,Bipolar is sp.,Exserohilum turcicum, Alternaria alternarta,Colletotrichum gloeosporioides and pyricularia grisea.The test of pathogenicity to dayflower and safety to crops indicated that the pathogenicity of Phoma herbarum was most of which diseased rate and disease index was 95.67%and 76.42 respectively.Furthermore,the strain was secure to most of crops besides slight injury to crop seedling.The pathogenicity of Curvularia lunata,Bipolaris sp.,Exserohilum sp., Colletotrichum gloeosporioides and pyricularia grisea was low of which incidence of leaf was lower than 50%and disease index was less than 20.The diseased rate of dayflower inoculated with Alternaria alternaria was 60%and disease index was 21.14,but the strain infection rice and corn.After assaying the sequence of 28S and sequence alignment,it was found that the similarity of four strain of P.herbarum with that of high pathogenic strain was over 98%,thus confirming from molecular level that strain belonged to Phoma herbarum.The experiment compared the difference of 11 strains of P.herbarum obtained from different area by single spore isolation in cultural characteristics,pathogenicity,and ability of secreting toxin and also analyzed their genetic difference by RAPD method.The strain SYAU-06 from Benxi was superior to others in growth speed,spore yield and toxin-producing capability.The test of host range showed SYAU-06 strain was safe to eighteen species of crops and vegetable besides corn and sorghum had slight injury response.Among twenty-seven species weeds from fourteen family the dayflower was most sensitive to the fungi,redroot amaranth and emarginated amaranth was slightly sensitive as well as goosefoot. Considering the pathogenity and safety SYAU-06 strain was screened as candidate microherbicide to control dayflower.The experiment researched the biological characteristics of SYAU-06 strain and the compatibility with pesticides.It found that the optimal conditions for hypha growth were as below:the optimal medium was dayflower juice added glucose,the optimal carbon source was lactose and glucose,the optimal nitrogen source was yeast extract,the optimal temperature was 25-30℃,the optimal pH was 8.The test established optimal conditions for producing pycnidium that optimal medium was corn powder,optimal temperature was 25-30℃,optimal pH was 7-8.UV irradiation induced the forming of pycnidium.The conidia germination was best when temperature was 25-30℃and pH was 7.Some nutrient substance such as glucose and sucrose could promote conidia germination.Biological compatibility of 12 pesticides with P.herbarum was investigated with mycelium growth rate method and conidia germination method.Among pesticides insecticides exhibited best compatibility,then herbicides,the last was fungicides.The process of mycelium of SYAU-06 strain infection to dayflower leaf was observed under light and electronic microscope.The cell division of mycelium did not begin 2h after inoculation.Branch appeared at end of new mycelium 6h after inoculation and appressorium formed 8h after inoculation.The mycelium invaded leaf tissue through epidermal cell, intercellular space and stomata 12h after inoculation.Massive mycelium was observed under leaf epidermal cell 24h after inoculation.According to diseased leaf rate,disease index and reduction in fresh weight,the influence factors of SYAU-06 strain infection dayflower were studied from three aspects of biocontrol fungi,host developmental stages and environment condition.At the same inoculation concentration mycelial suspensions incited significantly more severe disease than conidia and chlamydospore.In order to control dayflower application SYAU-06 strain successfully required mycelial suspensions concentration of 1.0×10⁶ mL^-1,7-9 days of culture time,3 leaf stag of host,28-32℃of temperature,over 80%relative humidity and at least 48h dew duration.The pathogenicity of strain greatly increased with the addition of tween80 and glucose.The illumination in dew duration affected disease severe and short-illumination benefited the disease development.The herbicidal activity was assayed with the measured parameters of seed germination, radicle and germ elongation,plant growth and damage responses on leaf.A highest concentration of 500μg·mL^-1of toxin incited inhibition rate of seed germination,radicle and germ elongation was 17.89%,37.26%and 28.37%respectively,reduction 15.5%in plant fresh weight and 6.53mm² lesion area in leaf.The experiment identified weed control spectrum of toxin and sensitivity to main crops.The result showed that dayflower and goosefoot was most sensitive to toxin which injury response reached to level 4,then redroot pigweed and eragrostioid which injury response was level 3 and level 2 respectively.Among main crops corn and sorghum were susceptible to toxin while wheat,soybean and peanut were insensitive to toxin.The extraction methods,extraction condition and toxin stability was researched systematically.The result indicated that the toxin secreted by P.herbarum not only was in hypha but also in culture solution and the extraction effect of ethyl acetate was the best.The soybean median and PSK media can be respectively used as solid and liquid culture media to produce toxin and grow mycelium.The optimal cultural conditions for producing toxin were temperature 32℃,cultured period 14d,cultured ways shaking of 150r·min^-1.The stability bioassay result showed toxin was steady to storage time and illumination but susceptible to temperature.The action mechanism of phytoxin from P.herbarum to plant was studied.The influence of toxin on cell membrane permeability and MDA content was carried out.The result indicated that toxin increased permeability of cell membrane.The relative conductivity increased with increased of toxin concentration and treatment time,Na⁺and K⁺leakage were higher than those of control.The content of MDA increased with over-oxidation of cell membrane action.At the same time content of chlorophyll in plant leaf treated by toxin decreased and respiration appeared abnormal.The toxin inhibited the activity of CAT,POD and APX related to resistance in dayflower leaf tissue.The safety of crude phytoxin from P..herbarum on environmental organisms was evaluated.The result showed crude toxin was low poison to antheraea pernyi which LC₅₀of contact toxicity was 58775.07 mg·L^-1and LD₅₀of stomach toxicity was 2359.78μg·g^-1.The toxin also displayed secure to bee(Apis mellifera Apis mellifera),trichogram matids (Trichogramma dendrolimi Matsumura),and earthworm(Eisenia foelide).The LC₅₀of toxin to Daphina pulex after 12h and Chlorella pyrenoidosa after 96h was 3771.97 mg·L^-1and 14214.68 mg·L^-1respectively,indicating low toxicity.The number of fungi,bacteria and actinomycetes in the soil showed no significant difference between treatment and control.The separation and purification technology of phytoxin was researched and the preliminary structure of purified toxin was definited.The appropriate developing solvent for TLC of toxin was screened as n-hexane:ethyl acetate:formic acid(5:1:0.1)or(10:1:0.1)by fluorescent microscopy at 254 nm and 365nm as well as iodine fumigation.The pure toxin which purity achieved 97.89%by HPLC detection was obtained through two silica gel column chromatography and a alumina column chromatography.The U V spectrum of purified toxin showed absorption peak was at 235nm,in mass spectrograph the peak of 237.7 (m/z)was stable deducing the molecular weight of pure toxin was 238.The result of IR showed that toxin component had hydroxyl,methyl,carboxy and nitro but also had the character of substituted benzene ring on ortho position.According to UV and IR the structure of substance with herbicidal activity was inferred to be o-nitrophenyl acid.