Reclassification And Genetic Diversity Analysis Of Rubber Tree Powdery Mildew Fungus In China

Powdery mildew is a foliar disease of rubber trees.It is one of the most important leaf diseases in rubber plantations worldwide.The causal agent of rubber tree powdery mildew,Oidium heveae,has been considered to be reclassified under Erysiphe,Golovinomyces or Podosphaera.The classification of this fungus consequently is still controversial.Thus far genetic diversity have not been studied.This study aimed to reclassify and explored genetic diversity of rubber tree powdery mildew fungus in China.In order to understand the biological evolution and the establishment of new strategy for controlling of powdery mildew,we also investigate the other phyllospere microorganisms community might be interact with different severities of powdery mildew.We reclassified rubber tree powdery mildew fungus in China and determine the relationship between classification and ecological environmental factors,morphological and molecular identification methods were used.In addition,we have also applied DNA barcoding.For exploring its genetic diversity,haplotype identification,multilocus phylogenic analysis,RFLP and ISSR molecular marker system were used.Furthermore,Microbial diversity and community structures on rubber tree with different regions of powdery mildew was studied using high throughput sequencing technology.The obtained results are summarized bellow:1.57 specimens were collected between 2013 to 2017 from different ecological areas in Hainan,Yunnan,and Guangdong provinces,which are the main rubber tree planting regions in China.With the support of morphological observation by light microscopy(LM)and scanning electron microscope(SEM),pathogenicity identification,molecular identification,and haplotype identification,Erysiphe quercicola is suggested as the only causal agent of rubber tree powdery mildew in China.With different geographical locations,different cultivar types,and different plant phenologie,the genotype of rubber tree powdery mildew fungi collected in China was found to be similar.Even though differences were observed in the size of conidia on the specimen,all powdery mildew fungi collected were determined to be in the same branch of Erysiphe quercicola clade,by the phylogenetic analysis.However,alignments of multiple internal transcribed spacer(ITS)and 28S ribosomal DNA(rDNA)sequences showed various base deletions and changes among different specimens.Haplotype analysis showed the close genetic relationship and significant rich haplotype diversity between rubber tree powdery mildew fungi in China and several other countries.2.For rapid and accurate identification of the rubber tree powdery mildew,we have sampling the rubber tree powdery mildew and the others Erysiphales host plants as materials.Then we tested the universality of powdery mildew DNA barcodes including ITS,28S,18S,Rpb4,TUB2,GAPDH and CUT,and examined their abilities of species identification and phylogenetic construction in genus of Erysiphe.The result revealed,ITS,28S,Rpb4 and CUT genes are suitable for a DNA barcode for rubber tree powdery mildew.Evolution and origin of rubber tree powdery mildew were analysed using multi-gene sequence analysis.And using multiple gene sequences,we calculated that rubber tree powdery mildew existed about 43 millins of years ago.3.Intergenic spacer(IGS)and 28S gene were amplified for 18 strains of rubber tree powdery mildew fungus using universal primers,which collected from various regions of Hainan and Yunnan provinces.IGS-RFLP and 28S-RFLP digested by Aus I,Msp I and Hinpl I showed that digest sites varied from the strains with profile polymorphism.The IGS-RFLP and 28S-RFLP profile could become distinctive DNA fingerprint for the strains.On the basis of IGS-RFLP and 28S-RFLP data analyzed,the similarity coefficient varied from 0.54 to 0.88,the isolates were clustered into 4 groups at the similarity coefficient of 0.67.HN-401 and HN-402 were in the first group;HN-403,HN-504,HN-204,HO-73,YN-201,YN-206,YN-202 and YN-203 were in the second group;HN-404,HN-405,HN-408,HN-501,YN-205,HN-506 and HN-507 in the third group;HN-502 separated from all the populations in the dendrogram.No obvious association was found between the genetic diversity and any other of parameter tested:host species,host phenophase or geographical position.4.Genetic diversities and phylogenetic relationships of 4 populations of 15 strains of rubber tree powdery mildew fungus from various regions of Hainan and Yunnan provinces were analyzed by inter simple sequence repeat(ISSR)molecular makers.The results showed that 8 primers were selected from 21 primers which had clear and stable polymorphic bands.A total of 145 loci were obtained,on average,amplification site of each primer 18.1 and polymorphic loci was 100%.In the level of populationg,Nei's gene diversity and Shannon information index were 0.1629-0.1944 and 0.2447-0.3402.The coefficient of gene differentiation(Gst)was 0.3194,while gene flow among populations(Nm)was 1.0655.Analysis of molecular variance(AMOVA)demonstrated that the genetic differentiation among populations was relatively high.The genetic variation among and within populations were 15%and 85%respectively.Genetic similarity coefficient(GS)ranged from 0.7739 to 0.9381 among populations.With 0.90 as the lowest GS,the 4 populations were clustered into three groups:YN-2 population was in one group,HN-2 and YN-1 populations were in another group,HN-1 population separated from all the populations in the dendrogram.Rubber tree powdery mildew fungus has a higher genetic diversity level.The geographical position and environment were not directly related to the cluster results.5.The rubber leaves with severities level 3 and level 0 of powdery mildew were collected from Danzhou,Baisha,Wanning and Wuzhishan in Hainan.Then,the bacterial community structures and diversities were analyzed by high-throughput DNA sequencing.At the phylum leve,the dominants of 4 areas were Cyanobacteria,Proteobacteria and Actinobacteria under all 2 severity levels.At the genus level,the dominants in Baisha were Cyanobacteria,Mitochondria and the dominants in Danzhou were Cyanobacteria,Mitochondria and Curtobacterium;the dominants in Wanning were Cyanobacteria,Pseudomonas and Pantoea;the dominants in Wuzhishan were Cyanobacteria,Pantoea and Acidobacteria.At the phylum level and genus level,the community structures and relative abundance of different regions are significantly different.Powdery mildew could be slightly affected the bacterial community structures on rubber leaves,but it has a moderate influence on relative abundance.6.The rubber leaves with severities level 3 and level 0 of powdery mildew were collected from Danzhou,Baisha,Wanning and Wuzhishan in Hainan.Then,the fungal community structures and diversities were analyzed by high-throughput DNA sequencing.At the phylum level,the dominants of 4 areas were Ascomycota,Fungi_unclassified and Basidiomycota under all 2 severity levels.At the genus level,the dominants in 4 areas were Erysiphe,Cladosporium and Fungi_unclassified,while they have respective dominant genera in different areas.At the phylum level and genus level,the community structures and relative abundance of different regions are significantly different.Powdery mildew could be slightly affected the fungal community structures on rubber leaves,but it has a moderate influence on relative abundance.