Diagnosis Of Water Hyacinth Leaf Blight And Examination Of The Biological And Pathological Characteristics Of Its Pathogen (Alternaria Sp.)

Water hyacinth,Eichhornia crassipes(Mart.)Solms,is one of the most damaging weeds in the world and one of the most important alien invasive plant species in China.Through wide survey and sampling of diseased water hyacinth plants,isolation and diagnosis,three fungal strains were obtained from Eichhornia crassipes.Alternaria sp.APT001 strain was shown highly pathogenic to the weed and it was investigated in some details in the present study.1.Three fungal strains(APT001,FPT001 and PPT001)were obtained from Eichhornia crassipes and they were preliminarily idendified as Alternaria sp.,Fusarium sp.and Pestalotiopsis sp.respectively.The results of pathogenicity tests indicated that Alternaria sp. stain(APT001)was the most virulent or pathogenic to the weed plants;it induced spot and blight symptom on leaves of the weeds and the disease was named leaf blight of water hyacinth based on the symptoms.The mean disease severity(DS)on leaves was up to 6.83(Charudarran scale)in four weeks after inoculation with mycelium of Alternaria sp.All the inoculated leaves were killed within five weeks,and the whole mean DS on leaves was 7.97.2.On potato sucrose agar(PSA)plates,the APT001 fungus grew fast and formed blue-black colonies which were covered with white aerial mycelia and measured approximately 70.1 mm in diameter in 5 days of incubation at 25℃.Conidiophores chains(normally 7-12 spores)they were brownish to brown with 1-3 transsepta and 0-4 longiseptae,measured 24.7 (12.5-37.5)×10.1(7.5-12.5)μm,with or without pseudobeak which were.8.96(5-16.25)×5.42(3.75-5)μm Conidia were straight or curved,septated,branched or unbranched,colorless to brownish.Based on these morphological characteristics and the production of black pigments and toxins in culture,this pathogenic stain was identified primarily as a member of the Alternaia tenuissima group and nomenclatured preliminarily as Althernaria sp.It was also thought of as a new Alternaria species morphologically,but this needs to be verified by further investigation with molecular identification techniques.3.After a preliminary experiment which indicated no significant effects of light,aeration and wetness,further investigations were conducted to examine effects of nutrient media, temperature and acidity/alkalinity(pH)on growth and sporulation of this fungus.Fungal colony could grow normally at 15℃-30℃and the best growth was observed at 25℃.Eight media were tested at 25℃and pH7.0.Very significant differences(P＜0.01)in colony growth were detected between different media.AFT001 grew best on PSA and the poorest on WA and the growth of the fungus was normal on other media tested.When varied the pH values of PSA medium plates from 6.5 to 10.0,AFT001 could grow normally at 25℃,and the best growth was at pH9.0.Poor or abnormal fungal growth was resulted in at pH4.0 and pH12.0.The temperature and pH tests were then done using PSA plates(for colony growth)or PSA slides and results indicated that the two factors had extremely significant effects(P＜0.001)on the fungus.In conclusion,nutrient media,temperature and pH value had striking impacts on and were thus the key factors affecting, Alternaria.sp;the best medium,temperature and pH value were,respectively,PSA,25℃and pH9.0 for growth.UV light(12L/12D)was needed for fungal sporulation,the spore production reached to 3.44×10~5/per dish after 20 days which was 2.53 times of that treated with fluorescent light.4.Temperature affected the pathogenicity of Alternaria sp.significantly.The highest disease intensity on the primary leaves reached 7.77 10 days after inoculation and incubation at 30℃.Wetness duration was shown an important factor affecting infectivity of the pathogen, 100%inoculated leaves were infected and diseased when they were maintained for more than 24 hours of wetness at 25℃.The disease severity reached 6.54 in the 48-hours-wetness treatment.Light intensity was shown marked inhibitive effect on conidial germination and infection of the pathogen and thus resulted in lower disease severity on the plants.5.The fungal pathogen was cultured in potato sucrose(PS)liquid medium for 20 days under light and darkness and the filtrates of the culture media were highly toxic to the weed plants.All leaves were yellowed and some tiny black spots were seen on the leaves a days after treated separately with the culture filtrates.Treatments by soaking,spraying and pricking all induced almost the same intensity of toxic symptoms and wetness had no effect on the toxicity of the culture filtrates against water hyacinth leaves.6.Row toxin complex of Alternaria sp.was obtained by extracting with ethyl acetate and with chloroform.The complex was assayed for toxicity on water hyacinth and the results showed that both the extracts induced disease symptoms on hyacinth leaves, but the recovery rate of row toxin complex by ethyl acetate is highest,reached to more than 90%.The extract by petroleum ether did not have any toxicity to water hyacinth.When the solutions of toxin extracts were adjusted to different pH values from 4.0 to 11.0,disease symptoms on water hyacinth leaves developed normally within pH4.0-11.0.Pathogenicity of the toxin extract remained unchanged after being treated at=120℃for 1 hour.The toxin extracts were treated under ultraviolet light for 2 hours and disease symptom developed normally on hyacinth leaves after the ultraviolet-treated toxin extract was applied.7.The pathogenicity of both the fungus and its culture filtrates were examined on 16 common and important crops from 6 different plant families.There was no disease symptom observed on leaves of all the 16 crop plants even until 20 days after inoculation with the conidiospore suspension and after treatment with the fungal culture filtrates.These results indicated that both the pathogen and its toxin were not toxic or pathogenic to these crops and would thus be safe if they are going to be applied to control hyacinth in the crop fields.