Identifiation Of Partial Alternaria By Morphology And Multiple-gene Analysis

Alternaria is the important pathogen of crops.The big-spored Alternaria can cause black spot on many important crops and cause a great worldwide lose.The molecular research of Alternaria identifiation is not consummate.Through the collection and indentification of Alternaria in this study,we can have a profound understanding of the existence status and find more new big-spored Alternaria resources.This study will lay the foundation for the big-spored Alternaria classification and utilization research in the future.More than 1000 plants herbarium were collected from Dali,Kunming,Xishuangbanna on Yunnan province,Xining on Qinghai province,Lanzhou,weiyuan on Gansu province,Xian on Shanxi province,Zhengzhou,Lingbao,Jiaxian on Henan province,et al.We got 117 small-spored isolates and 8 large-spored isolates.We had25 large-spored Alternaria which included 17 laboratory strains.Based on the morphological characteristics,ZMJ30 was A.zinniae,ZMJ73 was A.daucicola,ZMJ72 was A.panax,ZMJ10 was A.cinerariae,ZMJ81 was A.plantaginis,ZMJ80 was A.ranunculi,ZM140768-1 was A.macrospora,ZM140773 was A.helianthinficiens,ZM131190 was A.helianthi,ZM141147 was A.tagetica,ZM140227 was A.tageticola,ZM141179 was A.dauci,ZM12272-1 was A.porri,ZM140831 was A.brassicae,ZM030316 was A.pilosa,ZM030296 was A.tribuli,ZM030201 was A.humuli-scandens,ZM160038 was A.solani,ZM160039 was A.macrospora,726546 was A.petasitis,726547 was A.Petasitis.ZMJ76,ZM12268-a,ZM12260-b,ZM12250-a were Unknown species.In this study,3 new species,2 new record species in China were described.New species were A.plantaginis,A.tageticola,A.pilosa.New record species were that A.macrospora caused leaf spot on tomato(Lycopersicon esculentum Mill.)and A.cinerariae caused leaf spot on Petasites japonicus.In the classification of fungi,nine genes,eg.ITS,EF-1?,HIS,RPB2,ACT,OPA2,APN2,Gpd,were commonly used.According to the amplification rate and whether a single amplified product,namely agarose gel electrophoresis bands whether single,we finally screened out five amplification rate higher,more single strip of genes,eg.ITS,EF-1?,HIS,RPB2,ACT,from those nine commonly used genes.Amplified fragment size were: 573 bp,339bp,426 bp,951bp,436 bp.Based on the sequences of ITS,EF-1?,HIS,RPB2,ACT genes and downloaded Alternaria related gene sequences,we constructed phylogenetic five trees.In addition,we stitched those five sequences together and constructed another phylogenetic tree.ACT and ITS gene were conserved.Only large-spored specie and small-spored species were obviously divided into two parts,the strain ZMJ72 was identified as A.panax,strain fanqiezaoyi identified as A.solani,and other big spore strains can not be identified to species.EF gene was too conservative,can not identificated large-spored species from small-spored species.Despite the higher amplification rate and single band,but the amplified fragments were in different sizes,so,EF gene is not suitable for Alternaria genus classification and identification.HIS gene was also relatively conservative,it can not separate the large-spored and small-spored species.RPB2 gene not only can distinguished large-spored and small-spored species,but also can identified the strain ZM141179 as A.dauci,ZMJ72 as A.panax.Multiple gene maked Alternaria classification more chaotic.large-spored and small-spored species could not separated,and large-spored species' identification were less credible.In surmise,Multipled-gene could not used for Alternaria classification.