Identification And Phylogeny Of The Pathogens From Carya Cathayensis Root-Rot

The root-rot disease of Carya cathayensis,just like Carya cathayensis canker,is another important disease which affecting the growth and yield of Carya cathayensis trees in recent years.Recently,there are many studies on Carya cathayensis canker,but the research on Carya cathayensis root-rot is less.In this study,we got the management method of Carya cathayensis forest in the past three years,the disease indexes of Carya cathayensis root-rot and Carya cathayensis canker in July2018,and used SPSS for data analysis to explore the factors that related to the disease index of Carya cathayensis root-rot;the pathogens were isolated by tissue separation method from the root samples of Carya cathayensis root-rot disease,and identified by morphological method,molecular biology method and pathogenicity experiments;it were identified by sequence alignment and phylogenetic trees that the taxonomic status of major pathogens,the phylogenetic relationships between major pathogens and other Fusarium species,and the phylogenetic relationships between different subgroups within the major pathogens species.The results were as follows:1.The Carya cathayensis root-rot disease index and the Carya cathayensis canker disease index were obtained from the Carya cathayensis production areas in Hangzhou Lin’an(286 research points),and the correlation analysis was performed using SPSS software.The results showed that at the 0.01 level,there were a significant positive correlation between Carya cathayensis root-rot disease index and Carya cathayensis canker disease index(correlation coefficient R=0.593).Where Carya cathayensis canker was severer,the Carya cathayensis root-rot was more serious.2.According to the independence test,Carya cathayensis root-rot disease index was significantly correlated with herbicides usage(chi-square value was 32.726,p<0.01);Carya cathayensis root-rot was more serious in the samples which used herbicides in the past three years.There was no correlation between the Carya cathayensis root-rot disease index and the usage of chemical fertilizer(the chi-square value is 2.037,p=0.361>0.05);There was no significant difference in the Carya cathayensis root-rot disease index between the chemical fertilizered areas and non-chemical fertilizered areas.3.Five Fusarium spp.strains(TK1,TK2,LG,DS,DXG)were isolated by tissue separation from Carya cathayensis rot-root samples collected from Tuankou,Longgang,Daoshi,and Daxiagu.The pathogens were identified by morphological identification and molecular biological identification(ITS1/ITS4,β-tubulin F/R,EF-1αF/R PCR)that strains TK1,LG,DS,DXG are Fusarium oxysporum,strain TK2 is Fusarium asiaticum(Fusarium graminearum lineage 6).This is the first report of Fusarium graminearum from the root-rot diseased Carya cathayensis.The SCAR(sequence-characterised amplified region polymorphism)type analysis was performed for the TK2 strain using the Fusarium graminearum-specific primers Fg16F/Fg16 R.The SCAR type of the TK2 strain was determined to be group 5 by multiple sequence alignment.4.The neighbor-joining phylogenetic tree based on Fusarium ITS and EF-1α sequences showed that Fusarium oxysporum was be divided into three parts(i,ii,iii).Fusarium oxysporum(i)had highhomology with Fusarium verticillioides,Fusarium oxysporum(ii)had high homology with Fusarium proliferatum,Fusarium oxysporum(iii)had high homology with Fusarium solani.The TK1 strain isolated in this experiment belonged to Fusarium oxysporum(i).The neighbor-joining phylogenetic tree based on Fusarium ITS,β-tubulin and EF-1α sequences indicated that Fusarium graminerum complex had high homology with Fusarium culmorum and Fusarium lunulosporum.5.The neighbor-joining phylogenetic tree based on Fusarium oxysporum EF-1α sequences indicated that there was a low level of diversity in the EF-1α sequence of Fusarium oxysporum strains;most of the Fusarium oxysporum strains,including all forma specialis in the tree,were cluttered into one group which belonging to Fusarium oxysporum(i).EF-1α sequences can not effectively distinguish the forma specialis of Fusarium oxysporum.The maximum parsimony and bayesian inference phylogenetic tree based on Fusarium graminerum complex EF-1α sequences indicated that Fusarium graminerum complex could be divided into nine lineages(lineage 1~9),and lineage 1,2,8,9 had high homology,lineage 5,6 had high homology,lineage 3,4,7 had high homology.The TK2 strain isolated in this experiment belonged to Fusarium graminerum lineage 6(Fusarium asiaticum).6.The TK1 strain could infect Lin’an Carya cathayensis and caused root-rot by root-wound method and spore suspension inoculation method in the pathogenicity experiments.the TK1 strain(Fusarium oxysporum)was one of the pathogens of Carya cathayensis root-rot disease.The TK2 strain(Fusarium asiaticum)was difficult to infect the roots of Carya cathayensis by root-wound method,but it could infect the stems of Carya cathayensis seedlings through the wound and bring an obvious black infestation spot at the wound.