Effects Of Potassium On The Growth And Development And Its Transcriptional Regulation Mechanism Of Valsa Mali

Apple Valsa canker（AVC）caused by Valsa mali is one of the most serious diseases in apple production.The process of the disease is complex and has the characteristics of latent infection.Studies had shown that there is a significant negative correlation between the AVC and potassium content in apple tree.Previous studies have suggested that the increase of potassium content in apple trees can enhance the resistance against AVC by enhancing the tree vigor and increasing the thickness around the bark.However,little study on the effect of potassium against V.mali has been applied.In this study,the effects of potassium on the growth rate,dry weight,mycelial morphology and spore germination of V.mali were evaluated.Based on transcriptome sequencing and bioinformatics analysis,the transcriptional regulation mechanism of V.mali under the influence of potassium ion was illuminated.This study laid a foundation for further clarifying the mechanism of AVC control by increasing potassium fertilizer,revealing the molecular mechanism of V.mali in response to potassium ion stress,and providing a new theoretical basis for the control of AVC by increasing potassium application.The main results are as follows:1.Effect of potassium on mycelial growth and spore germination of V.maliThe effect of potassium ion on the mycelial growth of V.mali was characterized by low concentration promotion and high concentration inhibition.Mycelial growth rate method and dry weight method were used to evaluate the effect of different concentrations of pure potassium on the mycelial growth of apple canker.The results showed that when the pure potassium content（PPC）in PDB/PDA media was lower than 9.01 g/L,the mycelial growth of V.mali was promoted.However,when PPC was higher than 9.01 g/L,the mycelial growth of V.mali was inhibited.When PPC was 3.37 g/L,the mycelial growth rate of V.mali was the highest,32 mm/d.Potassium ion had a significant effect on the colony morphology and mycelial growth of V.mali,and the influence intensified with the increase of potassium content.When PPC was lower than 3.37 g/L,there was no significant change with the colony morphology and hypha thickness.While,the number of branches increased significantly,and the distance between branches shortened and became dense,which corresponded to the growth rate of the colony.However,when PPC was higher than 3.37 g/L,the colony irregular edge and layers appeared,and the hypha branches number decreased and immature.With the increase of PPC,the colony edge showed irregular snowflake shape and oily surface;the hypha became obviously thin and the surface was rough and curved deformity,the number of branches was a few,and most branches could not develop normally.Potassium did not show any promotion on spore germination of V.mali.While,when PPC was less than 9.04 g/L,the spore germination rate was more than 80%.With the PPC increased,the spore germination rate decreased correspondingly,and the correlative equation is y=-4.2732x+104.81（R2=0.944）.When PPC was 22.9 g/L,spore germination was almost inhibited.2.Study on the effect of potassium on the pathogenicity of Valsa maliAfter a certain concentration of potassium treatment,the pathogenicity of V.mali decreased.Mycelial discs were picked on V.mali colony cultivated on PDA contained with different concentrations of potassium.And then,the discs were inoculated on the needle injured apple leaves.The disease rates of inoculated leaves were examined to evaluate the change of pathogenicity of V.mali.The results showed that when PPC was less than 4.63 g/L,the pathogenicity of the V.mali was higher,the disease rates were more than 50%.When PPC was higher than 4.63 g/L,the pathogenicity decreased correspondingly,and the correlative equation was y=-0.153x2-0.5715x+57.508（R2=0.9677）.3.Transcriptome of Valsa mali treated with high concentration potassiumThe author sequenced the transcriptome of V.mali using Illumina RNA-Seq sequencing technology.The fungus was treated with high concentration potassium,and the transcriptome of V.mali cultured in PDB without potassium used as control.The author identified 1236 differentially expressed genes（DEGs）during the phase potassium pressing.One thousand and twenty six of these DEGs were up-regulated,and another 210 genes were down-regulated.GO and KEGG enrichment analyses these DEGs,seven among the top 40 GO enrichment gene family were categorized into ion transmembrane transport,and another 12 were related to secondary metabolism and mycotoxin.The author identified 30 signal pathways with significant difference by KEGG database analysis（P value<0.05）,and the most genes were mapped to metabolic pathway,and the second was biosynthetic pathway of secondary metabolites.These results showed that the high potassium stress may have a strong impact on the cell metabolism and pathogenicity of V.mali.The author analyzed GO enrichment sets related to ion transport,four DEGs were found in seven ion transport GO enrichment sets.All these four genes involved in the mTOR signaling pathway.These four genes are vacuole calcium transporter gene VM1G₀6906,sodium/calcium exchange protein gene VM1G₀9085 and VM1G₀9086,and stress activated MAP kinase interaction protein gene VM1G₀0479.There were 15 DEGs in the intersection of two or more GO sets and KEGG pathways,including four cytochrome P450 family genes and five GMC oxidoreductase family genes.In particular,the proteins encoded by the five GMC oxidoreductase genes belong to the proteins with FAD domain.4.qRT-PCR analysis of gene expressionThe author analyzed four mTOR pathway genes and two cytochrome P450 enzyme genes at different time points by qRT-PCR.It was found that each gene expression varied with time in the culture containing 12.82 g/L potassium.The vacuolar calcium transporter gene,sodium/calcium exchange protein gene and cytochrome P450 gene expression all reached three peaks at 3 hpi,12 hpi and 72 hpi respectively,and the highest expression level was found at 72 hpi.The expression of stress activated MAP kinase interacting protein VM1G₀0479 was not affected in 12 hpi,while up-regulated at 24 hpi,and reached the highest expression level at 72 hpi.