| Pestalotiopsis species exist extensively in tropical and subtropical area,most of which are thryptophytes causing leaf blights,fruit rot and other post-harvest diseases,some of which are saprobes and some of which are a kind of important endophytic fungi in woody plants.The species diversity of endophytic Pestalotiopsis in plant is abundant.In this study,taxonomy,molecular systematics and DNA barcoding of Pestalotiopsis were studied with some Pestalotiopsis strains isolated from the stems and leaves of different plants in Guangxi and Zhejiang,some Pestalotiopsis strains were donated from Zhejiang Academy of Agricultural Sciences and Guizhou University,and relationship between endophytic and pathogenic Pestalotiopsis species on Myrica rubra was analysed based on molecular phylogenetic method with four DNA sequences.The main results were s?mmarized as follows:1.Nine hundred and ninty-five Pestalotiopsis strains isolated and collected were identified as 13 taxa based on the combination of morphological characteristics and sequence data of ITS(internal transcribed spacer)and ?-tub(beta-tubulin gene),which were Pestalotiosis versicolor,P.dissmeninta,P.microspora,P.oxyanthi,P.vismiae,P.longisetula,P.clavispora,P.batatae,P.macrochaeta,P.pauciseta,P.theae,P.karstenii and P.hangzhougensis,respectively.2.A survey on diversity of endophytic Pestalotiopsis of 16 plant species belonging to Caesalpiniaceae,Magnoliaceae and Myrtaceae in Nanning Liangfengjiang Forest Park of Guangxi were carried out.The result showed that 89 endophytic Pestalotiopsis isolates obtained from 16 plant species were identified as 11 Pestalotiopsis species based on morphological characters,of which eight,six,eight and six endophytic Pestalotiopsis species were respectively obtained from Melaleuca leucadendra(Myrtaceae),Syzygi?m polypetaloide?m(Myrtaceae),Peltophor?m tonkinense(Caesalpiniaceae),and Saraca chinensis(Caesalpiniaceae),in which the most endophytic Pestalotiopsis species colonized,among which the highest isolation frequency was 2.86%from Peltophor?m tonkinense.It was also found that colonization frequencies of endophytic Pestalotiopsis were higher on twig tissues than leaf tissues for most plant species and higher in aut?mn than in other seasons.In comparison,Shannon-Wiener's diversity index of endophytic Pestalotiopsis was the highest in Caesalpiniaceae(2.2353),the second in Myrtaceae(2.1635)and the lowest in Magnoliaceae(1.8637).3.The phylogenetic analysis of Pestalotiopsis based on single and multiple DNA fragments was carried out and results showed that the phylogenetic relationship of Pestalotiopsis species was well revealed on the phylogenetic tree of EF1-?,the combination of B-tub and EF1-?,the combination of ITS,?-tub and EF1-?,and the combination of the four DNA fragments of ITS,?-tub,EFl-? and GPDH,respectively.By comparison,there was best correlationship between morphological characters and phylogenetic tree generated with combination of ?-tub and EF1-? DNA fragments because the phylogenetic tree was divided into two major clades which reflected two type of the intermediate coloured cells of conidia:light(Brown to olivaceous)and dark(?mber or fuliginous to brown)and multiple subclades which reflected different groups included apical appendage knobed at the extremities,the n?mber of apical appendage and the range of length and width of conidia.However,basal appendage presence or absence and basal appendage branched or unbranched were not reflected on phylogenetic tree of Pestalotiopsis.4.Molecular barcodes of Pestalotiopsis species were screened from four candidates of DNA segments,i.e.ITS,?-tub,EF1-? and GPDH.The availability of the four candidate of DNA segments from high to low were ITS(100%)= EF1-?(100%)>?-tub(97.3%)>GPDH(81.1%)based on the success rate of PCR and sequencing.Average intra-specific similarity(AS)and average inter-specific similarity(ASi)of every molecular phylogenetic tree were calculated with BioEdit software and it meant that different species could be distinguished better with the DNA sequence if the average intra-specific similarity was higher and the average inter-specific similarity was lower.The result showed that the sequence of average intra-specific similarity of four candidate was B-tub(AS =0.994)>EF1-?(AS=0.990)>GPDH(AS = 0.932)>ITS(AS = 0.877),and the sequence of average inter-specific similarity of four candidate was ?-tub(ASi =0.781)<EF1-?(ASi=0.860)<ITS(ASi =0.884)<GPDH(ASi=0.957);the average intra-specific similarity of ITS+?-tub combination(AS = 0.994)was close to that of ITS+EF1-? combination(AS?0.992),and the average of inter-specific similarity of ITS+B-tub combination(ASi =0.863)were slightly higher than that of ITS+EF1-? combination(ASi = 0.848);the average intra-specific similarity of ITS+?-tub+EF1-? combination(AS= 0.994)was close to that of ITS+?-tub+EF1-?+GPDH combination(AS = 0.993),but the average of inter-specific similarity of ITS+?-tub+EF1-? combination(ASi = 0.768)was lower than ITS+?-tub+EFl-?+ GPDH combination(ASi = 0.783).There were a relatively clear boundary between average intra-specific similarities and average inter-specific similarities of ?-tub,ITS+?-tub combination,ITS+EF1-? combination and ITS+?-tub+EF1-?combination,while average intra-specific similarities and average inter-specific similarities of ITS,EF1-?,GPDH and ITS+?-tub+EFl-?+GPDH combination were overlapped,which meant that ?-tub,ITS+?-tub combination?ITS+EF1-? combination and ITS+?-tub+EF1-?combination could be suitable candidates of molecular barcodes on Pestalotiopsis genus,and ITS,EF1-?,GPDH and ITS+?-tub+EF1-?+ GPDH combination were not so suitable candidates of molecular barcodes on Pestalotiopsis genus.Based on above,six Pestalotiopsis species were differentiated and seven Pestalotiopsis specieswere not differentiated with ITS;ten Pestalotiopsis specieswere differentiated and three Pestalotiopsis specieswere not differentiated with ?-tub;.nine Pestalotiopsis specieswere differentiated and four Pestalotiopsis specie were not differentiated with EF1-?;Five Pestalotiopsis species were differentiated and seven Pestalotiopsis species were not differentiated with GPDH(GPDH of P.batatae was lack);the sequence for species identification rate were ?-tub(76.9%)>EF1-?(69.2%)>ITS(46.2%)>GPDH(38.5%).Among combinations of DNA sequences,eleven Pestalotiopsis specieswere differentiated and two Pestalotiopsis specieswere not differentiated with ITS+?-tub combination;nine Pestalotiopsis specieswere differentiated and four Pestalotiopsis species were not differentiated with ITS+EF1-? combination;eleven and nine Pestalotiopsis species were differentiated with ITS + ?-tub combination and ITS+EF1-? combination,respectively;100%and 92.3%Pestalotiopsis species were differentiated with ITS+?-tub+EF1-? and ITS+?-tub+EF1-?+GPDH,respectively.On the basis of the success rate of PCR and sequencing,average intra-specific similarity and average inter-specific similarity of every molecular phylogenetic tree,differentiation rates of four candidaes and current situation of GenBank in which the dada of ITS sequences is plentiful and the data of other three sequences is relatively lack,it was proposed that ITS could be chosen as the main barcode,?-tub could be used as auxiliary barcode and ITS+?-tub+EF1-? combination could be used when a Pestalotiopsis strain was very obscure on identification.5.The relationship between endophytic and pathogenic Pestalotiopsis species on strawberry(Myrica rubra)was investigated based on molecular phylogenetic analysis of ITS,?-tub,EF1-?,and GPDH gene sequences,respectively.It was found that Pestalotiopsis isolates of the same species,regardless of if they were endophytic or pathogenic on strawberry,they clustered together in all four phylogenetic trees.The results also revealed that Pestalotiopsis species were not generally specific to plant parts,which Pestalotiopsis versicolor and Pestalotiopsis microspora were endophytic and pathogenic on tranks,twigs and leaves of strawberry;Pestalotiopsis theae was endophytic on twigs and leaves of uncultivated strawberry. |
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