Identification And Biological Characteristics Of The Pathogen Of Wisteria Leaf Spot Disease

Wisteria(Wisteria sinensis(Sims)Sweet.)belongs to wooden vine of bean Wisteria,is a famous green ornamental garden flower.Wisteria owns ornamenta value,medical value and environmental protection value of three,it has a very high potential research value.As the growth of the wisteria acreage,many fungal diseases occur on Wisteria under natural habitats.Especially the Wisteria leaf spot dieases occur in common,seriously lower the ornamental value of Wisteria,It can lead the whole plant leaves wither and even bring devastating harm on wisteria when it badly spread.But few report about research of Wilteria leaf spot diesease both in China and abroad.In order to effectively control the expansion and spread of the disease,to guarantee the sustainable development of ornamental plants and landscape industry,it urgent need to conduct a comprehensive investigation and identification of wisteria leaf spot disease.This research firstly collected the leaves with typical disease spot from Chizhou city and Huoqiu county in Anhui province,next separated and purified pathogenic bacteria from the typical diseased leaves,then identified which kind the true pathogenic fungi belong to from pathogenic determination and morphology and molecular biology identification.After that,studied the biology characteristic of the pathogens and do the fungicide toxicity measurement,to look forward to lay theoretical basis for disease prevention and control.The main results were as follows:1.Separation of pathogenic fungi and pathogenic determinationThis study collected the leaves with typical disease spot from Chizhou city and Huoqiu county in Anhui province in August 2014.according to the conventional separation meathod,2 fungi strains were discovered and both of them have same colony morphology,the two fungi strains were respectively numbered ZT-1 and ZT-2.This research selected for ZT-1 strain to do the pathogenic determination.The results showed that both mycelia block and conidial suspension inoculated on heathy leaves(premise that make wound on the blade)can produce typical symptoms on the leaves.And the same fungus strain can be isolated from the diseased inoculated leaves,which can prove that the fungi strain ZT-1 is the pathogenic fungi of Wisteria leaf spot disease.2.Identification of the pathogenBased on the observation of pathogenic spores phenotype and the morphology morphology of condia and colony,analysed with ITS?gpd and TEF three gene sequences of ZT-1 strain and with DNAMAN software to construct phylogenetic tree according to theresult of Genbank Blast after PCR amplification and sequencing.The results showed that Alternaria alternata owns the highest homology with ZT-1 for only this kind appears in the three phylogenetic trees at the same time.According to morphological observation: on the filter paper with PCA,25 ?,12 h light and 12 h dark cultivating 5 days,sporulation patterns appears in short tree,every produced conidum chain always includes 1-5 conidia and deffierent condium chains can be discovered in the base and sides of the conidium;Conidia like down rod,inverted pear-shaped,ovoid,broadly elliptic,body of condium 22-51×8-13?m,with 1-5 diaphragm and 0-3 vertical or inclined diaphragm.And ZT-1 has the same morphological features with Alternaria alternata.So the pathogen lead to the leaf spot disease is Alternaria alternata(Fr.: Fr.)Keissler,the disease was named Wisteria leaf spot diease.3.Biological characteristics of pathogenHyphae of pathogen ZT-1can grow in the range of 10-35 ?,the optimum growth temperature is 28 ?,lethal temperature is 55 ?,10 min.Hyphae can grow under the condition of pH4-10,the optimum pH for the growth is pH6.Hyphae of pathogen grow faster under continuous illumination conditions than no light or lack of light.It can grow in the absence of carbon and nitrogen sources.But when glucose and starch as carbon source,or protein building and beef extract as nitrogen source,hyphae have a better growth than other carbon sources or nitrogen sources.The ammoniacal salt have inhibitory effect on mycelial growth.Conidia of ZT-1 are able to germinate in range of 10-35 ?.With the increase of temperature between 5-29 ?,the conidia germination rate is gradually increasing,the optimum germination temperature is 29 ?,lethal temperature is 55 ?,10 min.It can germinate within the scope of pH3-10,the optimum pH is 6.2.Conidia appear higher germination rate under the condition of continuous illumination than no light or lack of light,the optimal carbon sources is sucrose,maltose,fructose and glucose.4.Fungicides toxicity function for pathogenic fungusThe 10 kinds of fungicides selected for inhibiting the growth of pathogenic bacteria colony have different inhibitory effects.Pyraclostrobin has the strongest inhibitory effect on the growth of mycelium,its EC50 is 0.0777 ?g/ml,the smallest one of 10 fungicides.The worst inhibition effect on the pathogen is azoxystrobin and carbendazim,both of their EC50 values are more than 1000? g/ml.The 10 kinds of fungicides selected for inhibiting conidial germination of pathogen has different inhibitory effect.Difenoconazole appears the best inhibitory effect,its EC50 values is 0.3513?g/mL,carbendazim's inhibiting effect is the worst,the EC50is16270.5916? g/mL.