Molecular Mechanisms Of Temperature Adaptation In The Diffusion And Prevalence Of Colletotrichum Fructicola

Camellia oleifera(Abel.)is a unique woody oil plant in southern China.Due to its wide distribution,Camellia oleifera exhibits varying climates and phenology across different regions.Anthracnose in C.oleifera,caused by Colletotrichum spp.,spreads extensively across regions throughout the year,resulting in severe production reduction and huge economic losses.Our research group previously identified Colletotrichum fructicola as the primary pathogen of C.oleifera anthracnose in China.As the plantation area expands and the seedling trade increase,the Camellia oleifera anthracnose fungus,which was originally active in different climates,is more likely to undergo genetic exchange in the sense of population,which increases the potential for mutations in genes related to virulence and adaptability.To clarify the population relationship and genetic characteristics of anthracnose pathogens from various regions,and to explore the influence of climate on the transmission traits of anthracnose pathogens and their mechanisms,this study selected seven origins of C.oleifera with varying climate characteristics and identified key climatic factors.We studied the genetic relationship of geographical populations of C.fructicola strains from these seven origins,compared the genetic differentiation of growth and sporulation traits among the C.fructicola populations,and analyzed the differential expression mechanisms of the genetic differentiation of these traits.The study hopes to make it clear that the climatic characteristics of areas where C.fructicola mainly distribution,elucidating the genetic structure of C.fructicola populations under different climatic conditions,exploring the adaptive differentiation of growth traits and phenotypes of C.fructicola from different geographical sources,and analyzing the ecological adaptation mechanism of the spread and prevalence of C.fructicola in Camellia oleifera under the background of climate change.The following results were obtained:(1)Screening of key climate factors affecting the spread and prevalence of Colletotrichum fructicolaClimate data from seven geographical sources of C.fructicola between 2001 and 2021 were analyzed,and the characteristic climate factors of each geographical source were extracted through trend testing,coefficient estimation,and principal component analysis.The analysis revealed a correlation between climate trends and latitude,with temperature and sunshine hours showing similar trends,while precipitation exhibited an opposite pattern.Clustering analysis of climatic factors revealed distinct temperature conditions in Guangnan,Yunnan,and Wuzhishan,Hainan,compared to other regions;in terms of comprehensive sunshine conditions,the clustering of temperature is closest to the clustering of comprehensive factors,while the clustering of precipitation and wind speed is very different from the comprehensive factors.Furthermore,the geographic clustering of major climate factors closely aligns with the annual average variation trend.Principal component analysis revealed that the top three components accounted for over 85%of the cumulative variance,with temperature-related parameters making the highest contribution,such as the average annual temperature,the median annual temperature,the annual temperature range,and the coefficient of variation of the annual average temperature,is the highest.Temperature emerged as a crucial climatic factor influencing the geographical origin of the seven anthracnose pathogens,warranting further research and analysis.(2)Genetic diversity and differentiation of Colletotrichum fructicola from different geographical sourcesWe re-sequenced 21 strains of C.fructicola from 7 distinct geographical sources and analyzed their SNP diversity and genetic structure.Our study revealed that mutations in exons and intergenic sequences accounted for over 90%of total mutations across all strains.Notably,nearly 50%of mutations were nonsense mutations,while approximately 40%were sense mutations.The predominant types of mutations were conversions and transversions.Interestingly,the number and patterns of SNPs remained relaatively stable among the tested strains.Cluster analysis based on SNP information grouped the 21 strains into two main clades.Within these clades,strains from Yunnan and Hainan exhibited closer genetic relationships,distinct from strains originating from other sources.These clustering results were consistent with the analysis of population structures.Further analysis of the genetic diversity coefficient(Fst)among populations based on SNP information revealed moderate genetic differentiation(Fst between 0.05 and 0.15)among strains from Henan,Hubei,Hunan,Guizhou,and Jiangxi.In contrast,strains from Yunnan and Hainan displayed a higher degree of differentiation(Fst above 0 25)compared to other geographical populations.Our findings indicate that the 21 strains of C.fructicola exhibited genetic differentiation based on geographical origin,showcasing rich genetic diversity and a high potential for mutation.(3)Adaptive differentiation of Colletotrichum fructicola traits to temperature from different geographical sourcesAt 28℃ as the control,three different culture environments were provided for 21 C.fructicola strains:35℃ for high temperature,10℃ for low temperature,and a temperature range of 10℃ to 37℃ for fluctuating conditions.The growth and sporulation changes of the strains were observed for 96 hours under different temperature conditions.Single-factor analysis of variance(ANOVA)was conducted on the collected trait data to calculate the quantitative trait diversity coefficient Qst.The Qst-Fst relationship was analyzed using a mixed linear regression model.It was found that,except for the Wuzhishan strain,the growth Qst of other strains increased by 3 to 5 times compared to the control under high temperature and fluctuating conditions,but showed no significant change under low temperature.The sporulation Qst responded to all temperature conditions,but significant differences were observed among strains.The changes in growth and sporulation of the strains under different temperature lacked obvious correlation and consistency.Mixed linear regression results of growth rate and sporulation growth rate found that the regression t values for the independent variables of temperature parameters were all within 0.05,indicating that temperature significantly influenced the phenotypic differentiation of growth traits in C.fructicola strains.Analysis of Qst-Fst revealed that the Qst-Fst value of strains from Xinyang,Henan,Macheng,Hubei and Wuzhishan,Hainan was above 0 under some temperature treatments,indicating adaptive genetic differentiation due to temperature selection pressure.Conversely,other populations exhibited opposite performance under the same treatments,suggesting that temperature pressure is not a universal determinant of genetic differentiation in growth traits of C.fructicola.This study found that temperature significantly influenced the phenotypic differentiation of growth and sporulation traits in C.fructicola but did not significantly affect its genetic differentiation.The temperature-adaptive differentiation of C.fructicola growth and sporulation traits is caused by the plasticity of temperature on phenotype and the influence of other genetic factors on genes.(4)Differential expression of genes in temperature adaptation of Colletotrichum fructicolaStrains from Xinyang,Henan,and Wuzhishan,Hainan were selected for transcriptome sequencing and differential expression analysis following exposure to different temperature treatments.Compared to the control group cultured at 28℃,Xinyang strains showed differential expression of 6558 genes at 35℃,while Wuzhishan strains exhibited differential expression of 7036 genes.Under the temperature difference of 10℃ to 37℃,Xinyang strains displayed differential expression of 160 genes,whereas Wuzhishan strains showed differential expression of 5851 genes.Following treatment at 35℃ and temperature difference,genes expressed by Xinyang and Wuzhishan strains were predominantly enriched in 322 metabolic pathways across 28 metabolic networks.Among these,22 metabolic networks were associated with the metabolism of carbon sources,amino acids,and lipids,2 were related to gene repair,and 4 were linked to cellular processes and signal transduction.Through enrichment screening of gene expression levels in 28 metabolic networks,63 differentially expressed genes were functionally annotated.Specifically,genes associated with histone deacetylation were upregulated,while those related to sterol synthesis were downregulated under 35℃ treatment In response to temperature fluctuation,Xinyang strains exhibited fewer specifically upregulated genes(10)compared to Wuzhishan strains(15).However,the overall expression levels of these genes in Xinyang strains were higher than in Wuzhishan strains.Therefore,it is believed that in environments with pronounced temperature fluctuations,Xinyang strains,experiencing milder climates,require longer response times and mobilize more genetic resources to adapt to these fluctuations.The research findings contribute to a deeper understanding of the dissemination and prevalence mechanisms of C.fructicola in southern China,aiding in regional anthracnose disease prediction,analysis,and control strategy formulation.Additionally,they provide theoretical supplementation for the establishment of spread and prevalence models of pathogenic fungi in woody plants.